Download Survival strategies of inside the human macrophage Mycobacterium tuberculosis Amanda Welin

Linköping University Medical Dissertations
No. 1223
Survival strategies of
Mycobacterium tuberculosis
inside the human macrophage
Amanda Welin
Division of Medical Microbiology
Department of Clinical and Experimental Medicine
Faculty of Health Sciences
Linköping University
SE-58185 Linköping, Sweden
Linköping University 2011
© Amanda Welin, 2011
All rights reserved.
Papers I & II are reprinted with permission from the American Society for Microbiology.
ISBN: 978-91-7393-251-6
ISSN: 0345-0082
Printed by LiU-Tryck, Linköping 2011
Klotet är bundet i sin bana
materien är frusen energi
den upplyste bunden i Nirvana
men forskaren är fri
rörelsen kniper som en tång
i regelbunden reguladetri
formerna är gjutna i betong
men forskaren är fri
Kjell Höglund
Supervisor
Maria Lerm, Linköping University
Co-supervisor
Olle Stendahl, Linköping University
This work was supported financially by the Swedish Research Council (Projects 2003-5994,
2005-7046, 2006-5968, 2007-2673, 2009-3821), the Bill & Melinda Gates Foundation, the
King Gustav V 80-year Memorial Foundation, the Swedish Heart-Lung Foundation,
SIDA/SAREC, Ekhaga Foundation, Carl Trygger Foundation, County Council of
Ostergotland, Clas Groschinsky Foundation, and Söderbergs Foundation.
Table of contents
ABSTRACT........................................................................................................................................................... 1
SUMMARY IN SWEDISH / SAMMANFATTNING PÅ SVENSKA ............................................................... 3
LIST OF ORIGINAL PAPERS ........................................................................................................................... 5
ABBREVIATIONS................................................................................................................................................ 7
BACKGROUND ................................................................................................................................................... 9
TUBERCULOSIS ........................................................................................................................ 9
TB – a global emergency.................................................................................................... 9
TB epidemiology............................................................................................................... 10
The disease ....................................................................................................................... 11
Diagnosis and chemotherapy ........................................................................................... 13
MYCOBACTERIUM TUBERCULOSIS ......................................................................................... 14
The bacterium................................................................................................................... 14
Cell wall ........................................................................................................................... 14
ESAT-6 ............................................................................................................................. 15
Mycobacterial strains....................................................................................................... 16
IMMUNE RESPONSE AGAINST MTB ......................................................................................... 17
Innate immune response................................................................................................... 17
Adaptive immune response............................................................................................... 18
The granuloma ................................................................................................................. 20
MACROPHAGES ..................................................................................................................... 22
Function and activation ................................................................................................... 22
Macrophage polarization................................................................................................. 22
Pathogen recognition receptors ....................................................................................... 24
Cytokine production ......................................................................................................... 26
Phagocytosis..................................................................................................................... 26
Antimicrobial properties of the macrophage ................................................................... 28
Phagosomal maturation ...................................................................................................................... 28
Antimicrobial mechanisms in the phagosome ................................................................................. 30
Enhancing the antimicrobial properties of the phagosome............................................................ 33
Human and mouse macrophages ..................................................................................... 34
MTB INTERACTION WITH MACROPHAGES .............................................................................. 34
Recognition and ingestion................................................................................................ 34
Complement receptor .......................................................................................................................... 35
Macrophage mannose receptor ......................................................................................................... 35
Fcγ receptor .......................................................................................................................................... 36
Other receptors..................................................................................................................................... 36
TLRs....................................................................................................................................................... 36
NLRs ...................................................................................................................................................... 37
Cytokine response ............................................................................................................ 38
Inhibition of bactericidal mechanisms ............................................................................. 38
Inhibition of phagosomal maturation ................................................................................................. 39
Significance of phagosomal maturation components ..................................................................... 41
Escape from the phagosome ............................................................................................................. 42
Other survival strategies ..................................................................................................................... 44
Controlling intracellular Mtb growth .............................................................................. 44
Cell death during Mtb infection ....................................................................................... 45
Apoptosis............................................................................................................................................... 47
Necrosis................................................................................................................................................. 49
Inflammasome-related cell death pathways..................................................................................... 50
Other types of cell death ..................................................................................................................... 51
Missing pieces of the puzzle ............................................................................................. 51
AIMS .................................................................................................................................................................... 53
RESULTS AND DISCUSSION ........................................................................................................................ 55
PAPER I: ................................................................................................................................ 55
PAPER II: ............................................................................................................................... 57
PAPER III:.............................................................................................................................. 60
PAPER IV: ............................................................................................................................. 62
CONCLUDING REMARKS .............................................................................................................................. 65
REFERENCES ................................................................................................................................................... 67
ACKNOWLEDGEMENTS................................................................................................................................. 83
Abstract
Mycobacterium tuberculosis (Mtb) is the bacterium responsible for tuberculosis (TB). For
decades, it was believed that TB was a disease of the past, but the onset of the HIV epidemic
resulting in a greatly increased number of TB cases, the emergence of antibiotic resistant Mtb
strains, and the relative ineffectiveness of the BCG vaccine have put TB back on the agenda.
With almost two million people being killed by TB each year, the World Health Organization
has declared it a global emergency. TB is an especially big issue in low-income countries,
where crowded living conditions accelerates spread of the disease, and where access to health
care and medication is problematic. Mtb spreads by aerosol and infects its host through the
airways. The bacterium is phagocytosed by resident macrophages in the lung, and when
successful is able to replicate inside these cells, which are actually designed to kill invading
microbes. Mtb is able to evade macrophage responses in part by inhibiting the fusion between
the phagosome in which it resides and bactericidal lysosomes, as well as by dampening the
acidification of the vacuole. The initial macrophage infection results in a pro-inflammatory
response and the recruitment of other cells of the innate and adaptive immune systems, giving
rise to the hallmark of Mtb infection – the granuloma. It is believed that in up to 50 % of
exposed individuals, however, the infection is cleared without the involvement of the adaptive
immune system, indicating that the innate immune system may be able to control or clear the
infection if activated appropriately. This thesis focuses on the interaction between the host
macrophage and Mtb, aiming to understand some of the mechanisms employed by the
bacterium to evade macrophage responses to enable replication and spread to new host cells.
Furthermore, mechanisms used by the macrophage to keep the infection under control were
studied, and a method that could be used to measure the replication of the bacilli inside
macrophages in vitro in an efficient way was developed. We found that a mycobacterial
glycoprotein, mannose-capped lipoarabinomannan (ManLAM), which is shed from the bacilli
during phagocytosis by macrophages, integrates into membrane raft domains of the host cell
membrane via its GPI anchor. This integration leads to an inhibition of phagosomal
maturation. Subsequently, we developed a luciferase-based method by which intracellular
replication of Mtb as well as viability of the host macrophage could be measured in a rapid,
inexpensive and quantitative way in a 96-well plate. This method could be used for drug
screening as well as for studying the different host and bacterial factors that influence the
growth of Mtb inside the host cell. Using this method, we discovered that infection of
1
macrophages with Mtb at a low multiplicity of infection (MOI) led to effective control of
bacterial growth by the cell, and that this was dependent on functional lysosomal proteases as
well as phagosomal acidification. However, we found no correlation between controlled
bacterial growth and the translocation of late endosomal membrane proteins to the
phagosome, showing that these markers are poor indicators of phagosomal functionality.
Furthermore, we discovered that infection of macrophages with Mtb at a higher MOI led to
replication of the bacilli accompanied by host cell death within a few days. We characterized
this cell death, and concluded that when replication of Mtb inside macrophages reaches a
certain threshold and the bacteria secrete a protein termed ESAT-6, necrotic cell death of the
host cell occurs. However, although the bacilli activated inflammasome complexes in the host
cell and IL-1β was secreted during infection of macrophages, Mtb infection did not induce
either of the recently characterized inflammasome-related cell death types pyroptosis or
pyronecrosis. Thus, we have elucidated some of the strategies that Mtb uses to be able to
survive and replicate inside the macrophage and spread to new cells, as well as studied the
conditions under which the host cell is able to control infection. This knowledge could be
used in the future for developing drugs that boost the innate immune system or targets
bacterial virulence factors in the macrophage.
2
Summary in Swedish / Sammanfattning på svenska
Tuberkulos orsakas av en bakterie som heter Mycobacterium tuberculosis. I Sverige ansågs
sjukdomen länge vara ett överspelat problem, men sedan AIDS-epidemins utbrott har
tuberkulos åter hamnat i rampljuset. AIDS-sjuka har lättare att utveckla tuberkulos än andra
personer. Dessutom har bakteriestammar som är resistenta mot de antibiotikapreparat som
finns tillgängliga utvecklats under senare år, och vaccinet som finns mot tuberkulos (BCGvaccinet) ger inget bra skydd. Tuberkulos är ett idag ett väldigt stort problem, särskilt i fattiga
länder där låg levnadsstandard gör att smittspridningen är stor och där inte alla människor har
tillgång till sjukvård. Nästan två miljoner människor dör av tuberkulos varje år vilket har gjort
att Världshälsoorganisationen har påkallat särskilda insatser för att få bukt med denna globala
hälsokris. Tuberkulosbakterien smittar genom luftvägarna, oftast när en infekterad person i
omgivningen hostar. Om man andas in bakterierna så äts de upp av speciella vita blodkroppar
i lungan som kallas makrofager och är en sorts ätarcell. Bakterien hamnar inuti en blåsa i den
vita blodkroppen som kallas fagosom. Ätarcellerna är designade att döda mikroorganismer,
men tuberkulosbakterien har utvecklat sätt att komma undan de skadliga ämnen som cellen
producerar och kan därför växa till inuti cellen. Den infekterade cellen skickar ut signaler till
resten av immunförsvaret vilket leder till att fler vita blodkroppar rekryteras till lungan. Dessa
bildar ett så kallat granulom, eller tuberkel, kring de infekterade cellerna, och kapslar därmed
in infektionen så att personen inte blir sjuk eller smittsam. Ifall personens immunförsvar
försämras, vilket kan bero på t.ex. hög ålder eller AIDS, så använder bakterierna dock
granulomet till sin fördel. Granulomet går då sönder och smittsamma bakterier strömmar ut i
luftvägarna. Man blir därmed sjuk i tuberkulos och kan också smitta andra personer. I vissa
individer kan det nedärvda immunsvaret, vilket ätarcellerna tillhör, döda bakterierna utan att
granulom bildas. Det är dock inte känt vilka betingelser som ger cellen förmågan att döda
bakterierna. I detta arbete ville vi studera hur tuberkulosbakterien lyckas överleva inuti
ätarcellen trots alla skadliga ämnen som cellen producerar. Dessutom ville vi ta reda på om vi
kunde få makrofagerna att hindra tillväxten av bakterier. Vi fann att när bakterierna äts upp av
en makrofag så överförs en molekyl från bakterien till cellens yttre membran. När molekylen
sitter i membranet så hindrar den blåsan där bakterien vistas från att rekrytera olika ämnen
som är skadliga för bakterien. På så vis kan bakterien överleva inuti ätarcellen. Sedan
utvecklade vi en metod för att mäta tillväxten av tuberkulosbakterier inuti ätarcellerna.
Metoden var snabbare och billigare än tidigare alternativ, och den kan användas för att studera
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vilka betingelser som gör makrofagen kapabel att hämma bakterietillväxten. Dessutom kan
den användas för att hitta nya läkemedel. Med hjälp av denna metod upptäckte vi att
makrofagerna kunde hämma bakterietillväxten om man endast tillsatte en bakterie per cell,
men inte om man tillsatte tio bakterier per cell. Cellen hade möjlighet att kontrollera tillväxten
på grund av att pH-värdet i blåsan där bakterien vistades kunde sänkas och ämnen som var
skadliga för bakterien kunde aktiveras. Vidare fann vi att om vi tillsatte tio bakterier per cell
så dog makrofagerna, men om vi bara tillsatte en bakterie per cell så kunde makrofagerna
överleva länge. Vi tittade närmare på hur makrofagerna dog, och fann att de dog genom så
kallad nekros, en typ av celldöd som inte är planerad och som leder till inflammation.
Bakterien dödade cellen genom att utsöndra ett ämne som kan förstöra membran. Detta arbete
har lett till ny kunskap om hur tuberkulosbakterien lyckas överleva trots den fientliga miljön i
ätarcellen, hur den dödar värdcellen för att sprida sig till nya celler och om hur cellen kan
hämma bakterietillväxten. Denna information kan användas för att utveckla läkemedel som
riktar in sig på bakteriens samspel med makrofagen.
4
List of Original Papers
This thesis is based on the following publications and manuscripts, referred to in the text by
their Roman numerals:
Paper I
Welin A, Winberg ME, Abdalla H, Särndahl E, Rasmusson B, Stendahl O & Lerm M (2008).
Incorporation of Mycobacterium tuberculosis lipoarabinomannan into macrophage membrane
rafts is a prerequisite for the phagosomal maturation block. Infection and Immunity 76(7):
2882-87.
Paper II
Eklund D*, Welin A*, Schön T, Stendahl O, Huygen K & Lerm M (2010). Validation of a
medium-throughput method for evaluation of intracellular growth of Mycobacterium
tuberculosis. Clinical and Vaccine Immunology 17(4): 513-17.
*These authors contributed equally
Paper III
Welin A, Raffetseder J, Eklund D, Stendahl O & Lerm M (2011). Importance of phagosomal
functionality for growth restriction of Mycobacterium tuberculosis in primary human
macrophages. Manuscript under revision.
Paper IV
Welin A*, Eklund D*, Stendahl O & Lerm M (2011). Human macrophages infected with
virulent Mycobacterium tuberculosis undergo ESAT-6-dependent necrosis, but not pyroptosis
or pyronecrosis. Manuscript under revision.
*These authors contributed equally
5
6
Abbreviations
Apaf-1, apoptosis protease activating factor-1
ASC, apoptosis-associated speck-like protein containing a CARD
AM, acetoxymethyl
BCG, Bacillus Calmette-Guérin
BH3, Bcl-2-homology 3
CaMKII, Ca2+/calmodulin-dependent kinase II
CARD, caspase recruit domain
CFP-10, 10 kDa culture filtrate protein
DC, dendritic cell
DC-SIGN, DC-specific intercellular-adhesion-molecule-3-grabbing non-integrin
DISC, death-inducing signalling complex
DOTS, Directly Observed Treatment and Short-course drug therapy
EEA1, early endosomal antigen 1
ER, endoplasmic reticulum
ESAT-6, 6 kDa early secreted antigenic target
ESX-1, ESAT-6 system 1
HMGB1, high-mobility group box 1 protein
Hsp72, heat shock protein 72
iNOS, inducible nitric oxide synthase
IPAF, ICE-protease activating factor
IκB, inhibitory κB
LAM, lipoarabinomannan
LAMP, lysosomal-associated membrane protein
LM, lipomannan
LRR, leucine rich repeats
ManLAM, mannose-capped LAM
MOI, multiplicity of infection
Mtb, Mycobacterium tuberculosis
NLR, nucleotide-binding domain, leucine-rich repeat containing protein / NOD-like receptor
MR, mannose receptor
NLRP, pyrin domain-containing NACHT, LRR and PYD domains-containing protein
NRAMP1, natural resistance-associated macrophage protein 1
PAMPs, pathogen-associated molecular patterns
PILAM, phospho-myo-inositol-capped LAM
PIM, phosphatidyl-myo-inositol mannosides
PRR, pathogen recognition receptors
PYD, pyrin domain
RD1, region of difference 1
RILP, Rab-interacting lysosomal protein
RIP, receptor interacting protein
RNI, reactive nitrogen intermediates
ROS, reactive oxygen species
TB, tuberculosis
TEM, transmission electron microscopy
TGN, trans Golgi network
TIR, Toll/IL-1 receptor
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Treg, regulatory T cells
WASP, Wiskott-Aldrich syndrome protein
WHO, World Health Organization
XIAP, X-linked inhibitor of apoptosis
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Background
Tuberculosis
TB – a global emergency
Man has been in constant battle with tuberculosis (TB) since ancient times. Mycobacterium
tuberculosis (Mtb) DNA has been discovered in Egyptian mummies from 2000 B.C. [1] and
TB was described by Hippocrates as early as 400 B.C. [2]. Historical texts identify the disease
as “consumption,” “wasting away,” “king’s evil,” “lupus vulgaris,” “the white plague” or
“phthisis” based on its clinical manifestations [2, 3]. TB is transmitted by aerosols containing
Mtb, released from the lungs of an infected individual through coughing and subsequently
infecting a new host through the airways. The Industrial Revolution during the 18th and 19th
centuries in Europe led to crowded living conditions in urbanized areas, which provided
optimal conditions for spread of TB. This resulted in epidemic levels, with 20-30 % of all
deaths being caused by the disease [3, 4]. During the second half of the 19th century, however,
deaths from infectious diseases including TB drastically decreased in Europe, as housing,
diet, education, and sanitation improved, and with the launch of sanatoria where patients were
exposed to fresh air and a healthy diet. The decline in TB incidence in Europe occurred before
the discovery of antibiotics, stressing the importance of living conditions and social factors in
containing TB, and highlighting some of the difficulties that are still faced in low-income
countries today [5].
With the discovery of streptomycin in 1943, TB became a medically treatable disease, and
incidence continued to plummet in industrialized countries throughout the 20th century. This
fact, together with the hubris of the eradicationist era due to a combination of the success in
eradicating smallpox, the effectiveness of DDT in eliminating malarial mosquitoes, and an
immense faith in science and technology, led to infectious diseases being neglected by
governments and public health agencies until the end of the 20th century. All the while, TB
continued to take its toll on poor and vulnerable populations in low-income courtiers, as well
as marginalized populations in high-income countries [5]. However, the HIV epidemic, the
emergence of new tropical diseases, and antibiotic resistance have again made infectious
disease into a recognized global threat. The evolution of extensively drug-resistant TB, the
increased susceptibility of HIV-positive persons with weakened immune systems to TB,
increased mobilization of people due to globalization, and the relative ineffectiveness of the
9
Bacillus Calmette-Guérin (BCG) vaccine have put TB back on the agenda during the past
twenty years [6, 7]. The BCG vaccine, a live attenuated variant of M. bovis, has been used
since the early 20th century. However, it has proven rather unsuccessful, especially in
preventing adult pulmonary disease, and a more effective vaccine is sorely needed [7].
With nearly two million people dying from the disease annually, TB is truly a global
emergency. Public health and financial efforts including improved access to health care, better
control of transmission, improved and more available diagnostics, and increased treatment
and cure rates are urgently needed. New scientific knowledge about the basic mechanisms
underlying TB and how the host can overcome it is also imperative, in order to develop a
new, improved vaccine and new drugs that tackle the emergence of antibiotic resistance [5, 7].
TB epidemiology
About 10 million new cases of TB are registered in the world annually; 30 % of these can be
found in India and China and 80 % in the 20-25 highest-burden countries, preferentially in
Africa, South America, and Asia [4]. Nearly two million persons die from TB each year, and
it is estimated that one third of the world’s population is infected with Mtb [5-7]. Most
individuals who are exposed to the pathogen, however, do not develop disease. It is believed
that these individuals (up to 50 % of those exposed) clear the infection through a robust innate
immune response. It is difficult to firmly establish this number, however, as these individuals
do not show signs of disease and have no immunological memory against the pathogen,
giving a negative result in diagnostic tests based on the presence of memory T cells. On the
other hand, clearance through an adaptive immune response or the establishment of a latent
TB infection, which occurs in the remaining 50 % of individuals, will leave primed T cells
behind, whose response can be detected. This means that it is difficult to distinguish latent TB
infection from a resolved infection using a test based on immunological memory.
Furthermore, only 5 % of latently infected individuals go on to develop active TB within five
years, while the remaining 95 % contain their latent TB throughout their lifetime; only
developing active TB if immunocompromised, e.g. through simultaneous HIV-infection,
treatment with immunosuppressive drugs, old age, or through re-infection [7]. The notion that
many individuals are able to clear the infection through an effective innate immune response
indicates an important role for this part of the immune system in achieving sterilization of
infection, and may provide a hint as to how we can boost the immune response to clear Mtb.
10
The disease
Transmission of Mtb occurs by inhalation of contaminated droplets released from the lungs of
an infected individual, typically through coughing. The infection process and formation of the
granuloma are illustrated in Fig. 1. Upon inhalation, the bacterium is ingested by means of
phagocytosis by resident alveolar macrophages and tissue dendritic cells (DC), which are
designed to kill pathogens but inside which Mtb can subvert the killing mechanisms to allow
replication. The initially infected cells release pro-inflammatory cytokines which leads to
recruitment of more DC, monocytes and neutrophils from the blood stream and the infected
DC become activated and migrate to the local lymph node where they activate specific T
cells. The cytokines IL-12 and IL-18 from the infected cells induce NK cell activity, and the
NK cells in turn produce IFN-γ, which activates the macrophages to produce TNF-α and
microbicidal substances [8, 9]. Through cytokine and chemokine signalling, other immune
cells are recruited and the pathological hallmark of TB, the granuloma, is formed.
In the granuloma, macrophages differentiate further into epitheloid cells or foamy
macrophages, or fuse to form giant cells, and become surrounded by lymphocytes and an
outer cuff of fibroblasts and extracellular matrix proteins. Thereby, the bacilli are contained
until the granuloma fails due to immunosuppression [10, 11]. For a long time, the granuloma
was viewed as beneficial only for the host – it coincided with the onset of adaptive immunity
and reduction of bacterial growth in the lung – but recent studies in zebrafish embryos
infected with a close relative of Mtb, Mycobacterium marinum, have indicated that
mycobacteria also use the granuloma for their benefit upon initial infection, recruiting new
macrophages to allow spread between host cells [12]. The granuloma centre becomes caseous
in active disease, containing necrotic macrophages which in advanced TB form cavities in the
lung. Spillage of infectious bacilli into the airways occurs when the structure ruptures, and
this allows spread to new individuals [10, 11].
TB of the lungs, resulting in symptoms such as chronic bloody coughs, night sweats and
weight loss, is the most common clinical manifestation of Mtb infection. However, any organ
in the body can be affected by spread of bacteria through the lymphatics, causing
disseminated, or extra-pulmonary, TB [2, 3]. Extra-pulmonary TB can manifest itself as
pericarditis, meningitis, or spinal TB, for example [13]. In latent TB infection, on the other
hand, the bacilli are thought to be contained in the granuloma or in other tissue, remaining in
11
a dormant non- or slowly-replicating state for decades, waiting for an opportunity to start
replicating when the host is immunocompromised and unable to prevent growth [14].
Figure 1. The infection route of Mtb in a human host leading to the formation of a
granuloma. Resident alveolar macrophages (Mø) phagocytose inhaled bacteria. This leads to
a pro-inflammatory response and recruitment of cells of the innate and adaptive immune
systems, and the formation of a granuloma. The bacilli can be contained within the structure
for long periods of time, but if immune control fails, the bacilli will commence replication,
and a necrotic granuloma core develops. The granuloma then ruptures and Mtb is spilled into
the airways [10].
12
Diagnosis and chemotherapy
Diagnostic methods for TB include chest x-rays, sputum spear microscopy, Mtb-specific
PCR, immunological memory-based tests including the less specific tuberculin skin test and
more specific IFN-γ release assays, phage amplification assays, solid culture and automated
liquid culture, as well as several tests for antibiotic resistance. Although dependable and
reasonably fast in high-income countries, reliable and rapid diagnosis of TB is still a major
problem in resource-poor settings and there is an urgent need for faster and less expensive
tests to confirm TB cases and find drug resistant strains [15]. The World Health Organization
(WHO) estimates that 61 % of all TB cases are diagnosed, which is approaching the goal of
70 %, but there is still a long way to go before 100 % of TB patients are detected. In 2008, 85
% of the reported cases were treated and cured [4].
Established cases of TB are treated with a combination of four first line antibiotics to which
the strain is likely to be susceptible, for two months, followed by two drugs for four months
[16]. First-line drugs include rifampicin, isoniazid, pyrazinamide, and ethambutol. These are
effective mainly against actively replicating bacilli rather than the non- or slowly-replicating
ones present in latent TB infection, which results in the long treatment time required to
remove both replicating and dormant bacilli [14]. If the strain is resistant to multiple antibiotic
types, the treatment involves more than four drugs, selected from the five lines of available
substances, for up to 18 months. Surgery to remove infected tissue may also be necessary if
treatment fails due to drug resistance [16]. Chemotherapy is administered through Directly
Observed Treatment and Short-course drug therapy (DOTS) programs, where patients are
observed when they take their medication to ensure compliance, as non-compliance is a major
contributor to the development of antibiotic resistance [4, 17].
Thus, although progress has been made since TB was put back on the global health agenda in
the early 1990’s, there is a vital need for better TB control as well as new vaccines and drugs
as TB is still the leading cause of death from infectious disease in the world [2]. The
development of these is dependent on an understanding of the basic mechanisms of Mtb
virulence, and this thesis focuses on this issue, specifically on the ways by which the
bacterium is able to survive and replicate inside host macrophage by subverting the immune
response. This type of knowledge is pivotal in determining how to modify current vaccine
strains to elicit an efficient immune response, and what should be the targets of new
antibiotics. The development of a method to screen for drugs that act as immunomodulators
13
on the macrophage or virulence blockers interfering with the Mtb virulence factors on the host
cell is also an important step in drug discovery, and this thesis includes the validation of such
a method.
Mycobacterium tuberculosis
The bacterium
Robert Koch identified the microbe responsible for TB in 1881 by culturing crushed
granulomas. Mtb is classified as a Gram positive bacterium although it stains poorly with
crystal violet because of its unique cell wall composition [2]. The bacterium is rod-shaped and
is classically defined as non-sporulating, although recent reports point to the formation of
spores in aged mycobacterial cultures [18]. It does not have a flagellum or a capsule. The
complex, waxy cell wall gives the bacillus its acid-fast property, meaning that it is resistant to
decolourization by acids during staining procedures. Mtb replicates very slowly, with a
doubling time of about 24h. The bacterium measures around 0.5 µm in diameter and 1-4 µm
in length, and is an aerobic intracellular pathogen [2].
Cell wall
As illustrated in Fig. 2, the uniquely composed cell wall of Mtb largely consists of long-chain
fatty acids termed mycolic acids linked to arabinogalactan, which is attached to the
peptidoglycan. In addition, the cell wall contains several lipoglycans including
lipoarabinomannan (LAM), its precursors lipomannan (LM), and phosphatidyl-myo-inositol
mannosides (PIM). These components are non-covalently attached to the plasma membrane
through their GPI anchors, and they extend to the exterior of the cell wall [2, 19]. LAM
consists of a phosphatidyl-myo-inositol anchor, a D-mannan polymer attached to the inositol
ring, D-arabinose chains, and capping motifs at the end of the arabinose residues [20]. LAM
acts as a virulence factor of Mtb, contributing to the inhibition of macrophage functions
important for killing the pathogen by inhibiting phagosomal maturation and interfering with
cell signalling and shifting the cytokine response from pro- to anti-inflammatory [19, 21-23].
Virulent, slow-growing mycobacteria like Mtb harbour mannose-capped LAM (ManLAM) in
their cell wall, while rapidly growing non-virulent species of mycobacteria such as M.
smegmatis harbour non-capped AraLAM or phospho-myo-inositol-capped LAM (PILAM),
and the type of capping is important for virulence [24]. The cell wall of Mtb also contains a
19-kDa lipoprotein of unknown function which has been implicated in virulence through a
14
role in host cell death and manipulation of bactericidal mechanisms [25]. The 19-kDa
lipoprotein of Mtb, as well as LM, and AraLAM from rapidly growing mycobacteria, provoke
an inflammatory response in the host by binding to Toll-like receptors (TLR) on the host cell
surface [26, 27].
Figure 2. Schematic representation of the complex Mtb cell wall. Arabinogalactan is attached
to the peptidoglycan. Mycolic acids and glycolipids extend through the cell wall [28].
ESAT-6
An Mtb virulence factor that has received great attention in recent years is the 6 kDa early
secreted antigenic target (ESAT-6). ESAT-6 is secreted in a 1:1 heterodimeric complex with
10 kDa culture filtrate protein (CFP-10) by a secretion system called the ESAT-6 system-1
(ESX-1) or type VII secretion system. The system is encoded by the region of difference 1
(RD1) of the mycobacterial genome, and is conserved in several mycobacterial species
including M. marinum and M. bovis. However, repeated passage of M. bovis to obtain the
vaccine strain BCG led to deletion of the RD1, resulting in attenuation [29, 30]. ESAT-6, as
well as the previously mentioned LAM and 19-kDa lipoprotein, elicit a specific immune
response in an infected host, and are therefore major antigenic determinants of Mtb [29].
Since ESAT-6 is present in Mtb but absent in BCG, a specific IFN-γ response against it is
indicative of Mtb exposure and the protein is thus used in diagnostic IFN- γ release assay tests
[31]. ESAT-6 is also under investigation for vaccine use in recombinant BCG strains [32].
15
ESAT-6 has multiple virulence mechanisms, but the best studied is its role in plasma
membrane lysis, which is thought to play a role in the spread of Mtb from one macrophage to
another [33-36]. In M. marinum-infected macrophages, it is known that ESAT-6 can lyse the
phagosomal membrane, allowing escape of the bacillus into the cytoplasm of the macrophage
and subsequent pore formation in the cell membrane leading to spread [37, 38].
Mycobacterial strains
Human TB is most often caused by an Mtb strain, but M. africanum and M. bovis infection
can also lead to the development of TB [2]. Mtb strains vary in phenotype and virulence, with
for example the Beijing strain being particularly virulent, developing drug resistance and
causing extra-pulmonary TB more often than other strains [39]. H37 is a laboratory strain that
was isolated from a 19-year old pulmonary TB patient in 1905, and later dissociated into a
virulent strain (H37Rv) and an avirulent strain (H37Ra), based on virulence in guinea pigs
[40]. Although both strains can be cultured in suitable medium in the laboratory, only the
H37Rv strain is capable of replication inside human macrophages [41]. It has recently been
described how H37Rv and H37Ra differ genetically and phenotypically, and the major
difference lies in a mutation in the phoP gene, which is necessary for adaptation to the
intracellular environment [42-44]. PhoP forms a two-component regulatory signal
transduction system together with PhoR, where PhoP acts as a transcriptional regulator. The
system is important for sensing and adapting to environmental stimuli [45]. Studies have
shown that mutations in the phoP gene lead to a defect in the secretion of ESAT-6, which can
be synthesized but not released from the bacillus [46, 47].
The H37-strains, as well as BCG and different clinical isolates, are commonly used to study
the pathogenesis of mycobacteria in different in vivo and in vitro systems. M. marinum is also
commonly used, as it has many of the features of Mtb and is practical to handle since it is less
prone to cause disease in humans than Mtb and can be used to infect zebrafish embryos and
the amoeba Dictyostelium [48-50]. It can also be used to model tuberculous lesions by
infection of mouse tails [38]. However, the extrapolation of data obtained with mycobacteria
that are not pathogenic to humans should be done with great care, as many mechanisms are
specific for Mtb and dependent on for example a functional ESX-1 region and PhoP/PhoR
regulatory system. Thus, the data presented in this thesis is mainly based on work performed
with live, virulent Mtb infecting primary human macrophages.
16
Immune response against Mtb
Innate immune response
The innate immune response plays an important role in the protection against TB as it
provides the first line of defence that the invading pathogen meets, and since the innate
immune system is thought to be able to clear the infection in many cases, if activated
correctly. However, because Mtb has evolved strategies to manipulate the macrophage,
allowing intracellular survival and replication, the innate immune system is also a prerequisite
for mycobacterial pathogenesis. The innate immune system is comprised of anatomical
barriers such as the skin as well as the complement system and several types of innate
immune cells. Mtb interacts with a number of these cells, and binds to receptors on their cell
surface. These receptors include TLR, complement receptor (CR) 3, mannose receptor,
scavenger receptors, and DC-specific intercellular-adhesion-molecule-3-grabbing nonintegrin (DC-SIGN), and engagement of these leads to the induction of an inflammatory
response that can lead to clearance of the bacilli or drive granuloma formation [7, 12]. The
alveolar macrophages that first ingest the bacilli and arriving monocytes from the bloodstream
provide the bacteria with its niche, but may also be able to disarm the pathogen if stimulated
correctly. The DC that ingest Mtb can also provide a replication niche, simultaneously being
essential for antigen presentation to T cells in the draining lymph node [7, 8]. On the other
hand, Mtb has developed mechanisms to prevent both DC migration and antigen presentation
[51]. This highlights the complexity of the interaction between Mtb and the innate immune
system. Apart from macrophages and DC, neutrophils and NK cells have been implicated in
the immune response against Mtb.
Neutrophils are among the first cells to respond to inflammatory stimuli by migrating to the
infection site, and Mtb infection is accompanied by massive influx of neutrophils [8].
Conflicting evidence on the role of neutrophils in Mtb infection exists. It has been shown that
neutrophils can be activated in response to Mtb and kill the pathogen using its range of
antimicrobial molecules contained in their granules, including defensins, lactoferrin,
cathelicidin, and lysozyme, which is transferred into the Mtb-containing phagosome upon
fusion with granules [8, 52, 53]. Neutrophils also exert efficient killing of microbes through
the assembly of the NADPH oxidase in the phagosomal membrane, which leads to the
generation of superoxide followed by reactive oxygen species (ROS) in the phagosome [52]
and have been shown to be able to kill Mtb in a Ca2+-dependent manner [54]. Furthermore,
17
neutrophils are able to activate macrophages through release of granule proteins [55] and heat
shock protein 72 (Hsp72). Hsp72 is released from apoptotic neutrophils, which have recently
been found to be able to induce macrophage activation in addition to having an inflammationresolving role [56]. However, in vivo-studies give conflicting evidence as to whether
neutrophils have a protective or tissue-damaging effect during Mtb infection [7, 8, 53].
NK cells are granular lymphocytes of the innate immune system that have cytotoxic functions
exerted through perforin and granzyme or granulysin [8], and provide the macrophage with a
stimulation signal through IFN-γ during Mtb infection [2, 57]. They are activated through
complex interactions between IL-12, IL-18, IFN-α, and a range of activating and inhibitory
receptors. Mtb-infected macrophages can be lysed directly by NK cells, and the NK cells
mount a pro-inflammatory response to Mtb, at least in vitro, restricting Mtb growth in an
apoptosis-dependent manner [8]. Furthermore, NK cells can kill regulatory T cells that are at
risk of dampening the immune response to Mtb [58]. However, the role of NK cells in human
Mtb infection is not completely clear [8], and an observed defect in the functionality of NK
cells in TB patients was found to be an effect rather than cause of disease [59].
This thesis focuses on the macrophage – the cell which both acts as an Mtb reservoir and an
effector cell of the innate immune system. The function of macrophages during Mtb infection
will be discussed further in upcoming chapters.
Adaptive immune response
After approximately two weeks of Mtb infection of an in-bred mouse, the adaptive immune
response has been mounted and this is accompanied by a drop in bacterial replication [2, 7].
Infected DC and macrophages present Mtb-antigens to T lymphocytes through MHC class I,
to CD8+ cytotoxic T lymphocytes, and through MHC class II, to CD4+ T helper cells, leading
to the activation and proliferation of the lymphocytes. Additionally, CD1-restricted T cells
can be activated through presentation of glycolipid antigens by DC, and γδ T cells through
presentation of phospholigands, and these contribute to protective immunity against TB by
producing IFN-γ or exerting cytotoxic activity [7, 59]. Memory T cells also form upon Mtb
infection. The infected macrophages and DC secrete cytokines including IL-12, IL-23, IL-7,
IL-15 and TNF-α, leading to attraction of more leukocytes to the infection site [7].
18
Depending on the cytokine environment, the CD4+ T cells can mount a Th1 response (IL-12,
IL-18, IFN-γ), or a Th2 response (IL-4, IL-5, IL-13). A Th1 response leads to the release of
pro-inflammatory cytokines including IFN-γ, which is thought to enhance killing of intramacrophage mycobacteria through NO and ROS production [7, 10, 60-62]. This has been well
characterized in mice, although the mechanism has not been fully elucidated in humans, and
is thought to be different [63, 64]. A Th2 response, on the other hand, leads to release of IL-4,
IL-5, IL-10 and IL-13, promoting B lymphocyte activation leading to an antibody response,
and promoting an anti-inflammatory macrophage response. Th17 cells, stimulated by IL-23,
IL-6, IL-21, and low TGF-β levels, are involved in recruitment of cells of the innate immune
system and Th1 cells, and secrete IL-17 [7]. Regulatory T cells (Treg), stimulated by IL-2 and
high TGF-β levels, can also be stimulated. Treg produce anti-inflammatory cytokines such as
IL-10 and can suppress microbicidal mechanisms in the macrophage, and the activity of these
cells is elevated in TB patients [7, 65].
Specific activation of CD8+ cytotoxic T cells can lead to killing of Mtb through a perforin and
granulysin-mediated pathway by which the infected macrophage undergoes cell death, or by
induction of apoptosis through the extrinsic pathway via Fas ligand [7, 66, 67]. It is known
that TB patients display a defect in the killing capacity of their cytotoxic T cells [68]. Thus, a
Th1/Th17 response, but also activation of cytotoxic T cells, is thought to be important aspects
of the adaptive immune response to Mtb infection [7].
The role of B lymphocytes and a humoral response in protection against TB is unclear, and
researchers have long dismissed their importance because of the intracellular localization of
Mtb [59]. However, evidence from experimentally infected animals suggests that an antibody
response can have an immunomodulating effect on cellular immunity through cytokine
signalling, as well as a protective role against infection by inhibiting bacterial replication,
neutralizing bacterial products, triggering of the complement system, and promoting
antibody-dependent cellular cytotoxicity. Perhaps most importantly, an antibody response
results in opsonisation of extracellular bacilli with IgG leading to phagocytosis by
macrophages and DC through FcγR. This is thought to result in a more robust macrophage
response, a stronger inflammatory response and more efficient antigen presentation than other
uptake routes [69-71].
19
Antigen presentation plays an important role in activating the adaptive immune response
against Mtb. Presentation of antigens from extracellular pathogens is usually achieved
through uptake by an antigen-presenting cell such as a DC, macrophage, or B cell, and
subsequent processing of phagosomal content for presentation to CD4+ T cells through MHC
class II, leading to activation of these cells and a cellular (Th1) and/or humoral (Th2)
response. Antigens from intracellular pathogens, on the other hand, are present in the cytosol
of the infected cell and are processed by a separate pathway and presented to CD8+ T cells
via MHC class I, so that the infected cell can be killed by cytotoxic T cells [66, 67, 72]. For a
long time, Mtb was believed to always be contained inside an impermeable phagosome in the
macrophage, giving rise to the question of how Mtb antigens can be presented via MHC class
I as well as MHC class II [73, 74]. However, recent evidence indicates that Mtb as well as M.
marinum can escape its vacuole and also reside in the host cell cytosol, giving an explanation
to this question [67, 75]. This knowledge has been used to design a BCG vaccine strain
expressing perfringolysin, which enables it to escape from the phagosome and trigger an
enhanced immune response through MHC class I [76]. However, an alternative explanation
for the presentation of Mtb antigens via MHC class I is an interaction between the
mycobacterial phagosome and the endoplasmic reticulum (ER) leading to proteasome
degradation and MHC class I presentation of antigens [77].
The granuloma
Upon Mtb infection, a granuloma forms through successive recruitment of innate followed by
adaptive immune cells, by means of complex cytokine and chemokine signals. The granuloma
represents the intersection of innate and adaptive immunity. A mature granuloma is highly
stratified, becomes vascularised and develops a fibrotic capsule [10]. In a granuloma that is
successful from the host’s point of view, cycles of immune activation and suppression are
thought to lead to both containment of bacilli and prevention of immunopathology [8].
However, the granuloma is also a prerequisite for the necrosis, tissue damage and spread of
the bacterium observed when containment fails [78], and recent evidence even points to a
favourable role of early granuloma formation for the pathogen [12]. On the other hand, this
has been questioned by a mouse study, where lack of granuloma formation correlated with
increased mortality [79]. Furthermore, the granuloma is thought to play a major role in
maintaining latent TB infection and avoiding reactivation of infection through complex
immune interactions. The hypoxic core of the granuloma is thought to induce a dormant
20
bacillary state where there is little or no replication of Mtb, and a cellular immune response is
thought to control bacterial growth during latent TB infection [2].
A dynamic interplay between dormant Mtb and cells of the host immune system, where cells
are continuously recruited into the granuloma, is necessary to prevent reactivation of the
bacilli and development of active TB [14]. However, it is becoming clear that latent TB is not
a static state with a homogenous population of non-replicating bacilli, and one piece of
evidence for this is the fact that treatment with isoniazid, which is only active against
replicating bacilli, for a prolonged period can have a sterilizing effect. One theory is that the
bacilli constantly re-infect their host through drainage of non-replicating bacilli into the
airways and formation of secondary granulomas and active disease if the bacilli manage to
reach the upper lobes of the lung [80, 81].
There are several reasons why mouse TB is not an ideal model for human TB, including
differences in mechanisms of microbial killing inside the macrophage, but also in terms of
granuloma development and structure. Granuloma centres in wild-type mice do not become
necrotic and caseate, the granulomas are not as stratified as in humans, and mice are generally
better equipped to handle high infectious doses with Mtb [10]. One study showed that
infection of mice deficient in iNOS production, on the other hand, did form necrotic
granulomas, providing a mechanistic explanation for the lack of mouse granuloma caseation
[82]. A contradicting theory is that mounting the amplitude of immune response that humans
do during caseation, leading to the necrotic tissue damage, would instantly kill a mouse
because of its small size [83]. Thus, additional models, such as Mtb infection of non-human
primates and M. marinum infection of zebrafish embryos, are useful in gaining insight into
granuloma formation and the role of the granuloma in Mtb pathogenesis that is difficult to
obtain from the mouse [48, 84]. Studies of zebrafish embryos have generated new knowledge
about the role of the innate immune system in granuloma formation, and it is now known that
epitheloid granulomas can be found long before the onset of adaptive immunity [85].
Additionally, these studies have generated the aforementioned evidence that at least in M.
marinum infection, granuloma formation is not merely a host strategy for entrapping the
infection. Granuloma formation is also beneficial for the bacterium in its initial spread, and is
dependent on secretion of the bacterial factor ESAT-6 [12]. It is, as mentioned, also the only
route by which Mtb can spread to new hosts.
21
In summary, it is becoming increasingly clear that the onset of adaptive immunity, which
coincides with inhibition of mycobacterial replication in mouse models, is not the only
decisive factor in determining the outcome of Mtb infection, despite the importance of a Th1
response for IFN-γ and NO-dependent killing of intra-macrophage Mtb in mice [10]. The
significance of a robust innate immune response is becoming equally evident, and knowledge
about how Mtb manages to survive, and how the host can overcome infection through a
strong innate response, is imperative to finding new drugs and vaccines against TB. It is still
not clear what the deciding host factor is for resistance or susceptibility to infection.
Macrophages
Function and activation
Macrophages are large mononuclear cells of the innate immune system which function as
professional phagocytes, meaning that they are capable of engulfing particles larger than 0.5
µm, including microbes. The word macrophage means “big eater” in Greek. In a resting state,
the role of the macrophage is to internalize debris and apoptotic cells in an non-inflammatory
manner [61]. During infection, their role is to ingest and destroy pathogens, recruit other cells
of the immune system, and present antigens from the microbe to cells of the adaptive immune
system. Resident macrophages are terminally differentiated and have a fixed location in the
body, at strategic points where infection can occur, e.g. alveolar macrophages are stationed in
the lungs, Kupffer cells in the liver and microglia in the nervous system. The precursor of
macrophages, the monocyte, circulates in the blood stream and is recruited into sites of
infection or tissue damage when stimulated. It then differentiates into a macrophage, with
increased phagocytic capacity and different morphology and adhesive properties.
Macrophages can become activated upon inflammatory or microbial stimulation. When a
macrophage is activated by microbial products, such as LPS, the cell acquires the
antimicrobial properties necessary for elimination of the invader, although some pathogens,
including Mtb, have found a way of circumventing this response [86, 87].
Macrophage polarization
Macrophages are functionally polarized; depending on how they are stimulated they can adopt
a pro- or anti-inflammatory profile and accordingly different effector functions. This is
illustrated in Fig. 3.
22
Figure 3. Functional polarization of macrophages. The stimuli that induce the respective
macrophage phenotype, the surface markers that are upregulated, and the cytokines and
receptor antagonists released by each phenotype are shown [86].
During the 1960’s and 1970’s, it was discovered that macrophages could be activated by
microbial products, but inactivated by IL-4 and IL-13 [88]. It was later discovered that the
latter stimuli were not simply inactivating the cells, but instead inducing a different type of
macrophage. Two types of macrophages were described – M1, or classically activated
macrophages, and M2, or alternatively activated macrophages. M1 polarization is induced by
pro-inflammatory or Th1 cytokines (IFN-γ, TNF-α, GM-CSF) and microbial products and M1
macrophages have microbicidal and inflammatory properties (secreting IL-1, IL-12, TNF-α,
IL-23, IL-6 and overexpressing the IL-1 receptor, MHC class II and TLR2 and -4). They
express inducible nitric oxide synthase (iNOS) and produce ROS. On the other hand, M2
polarization is induced by anti-inflammatory or Th2 cytokines (IL-4, IL-13, M-CSF, IL-10),
and M2 macrophages have regulatory properties and are not microbicidal. They express
arginase [86, 89].
More recently, it has been shown that macrophage polarization is highly plastic and
reversible, so that the same macrophage can partake in both induction and resolution of
inflammation [90]. Furthermore, the M2 macrophages have been further divided into three
subsets, all with different immunoregulatory properties, as well as roles in angiogenesis,
tissue remodelling and repair. M2a macrophages are induced by IL-4 or IL-13, M2b by
immune complexes and TLR agonists and M2c by IL-10 and glucocorticoid hormones. M2a
23
and M2c macrophages secrete anti-inflammatory cytokines such as IL-10 or TGF-β, as well
as different chemokines, and IL-1 receptor antagonist, and M2a also express decoy IL-1
receptor and MHC class II. M2b, on the other hand, display an intermediate phenotype
producing IL-10, IL-1, TNF-α, and IL-6, but contribute in immunoregulation despite
production of pro-inflammatory cytokines [86, 88, 89, 91]. Additionally, chemokines are
released and chemokine receptors expressed differentially on the different classes of
macrophages. It is becoming evident that these subtypes represent a continuum of differently
activated macrophage phenotypes [86]. Thus, the macrophage is a dynamic cell with multiple
complex roles in different immune processes, and the type of activation can have a great
impact on the outcome of infection.
Pathogen recognition receptors
Pathogen recognition receptors (PRR) are proteins expressed on the surface or in the
cytoplasm of innate immune cells that recognize a diverse range of bacterial products called
pathogen-associated molecular patterns (PAMPs), or stress signals termed danger-associated
molecular patterns [8]. Binding of PAMPs to PRR leads to either signalling events, if the
receptor is a signalling PRR, including TLR and nucleotide-binding domain, leucine-rich
repeat containing protein (or NOD-like receptor) (NLR), or to phagocytosis, if the receptor is
an endocytic PRR, such as the mannose receptor (MR) [92, 93].
One of the families of signalling PRRs, the TLRs, include 10 highly conserved
transmembrane proteins termed TLR1-TLR10. These proteins are usually located on the cell
surface and contain terminal leucine rich repeats (LRR) which recognize specific PAMPs, a
transmembrane domain, and finally a cytoplasmic signalling domain with great homology to
the IL-1 receptor. The latter is termed the Toll/IL-1 receptor (TIR) domain. The members of
the TLR family recognize distinct microbial products, e.g. LPS (TLR4), lipoproteins (TLR2),
flagellin (TLR5), unmethylated CpG sequences in DNA (TLR9), and single stranded (TLR7)
or double stranded (TLR3) viral RNA. Binding of PAMPs to their respective TLR leads to
signal transduction via TIR, interactions with adaptor proteins, and signalling cascades which
results in activation of the transcription factor NF-κB [94, 95]. In an unstimulated cell, the
heterodimer form of NF-κB, consisting of a p50 and p65 component, remains in the
cytoplasm because of its interaction with inhibitory κB (IκB) proteins. In the most common
scenario, a series of phosphorylation events follow TLR stimulation, and lead to
24
polyubiquitination and degradation of IκB, and NF-κB can move to the nucleus. This leads to
the binding of NF-κB to DNA and transcription of pro-inflammatory cytokine genes is
initiated. Thus, an appropriate immune response can be mounted, with interactions between
the macrophage and other cells of the innate and adaptive immune systems through cytokine
signalling and antigen presentation [61, 95].
The NLR family contains more than 20 different soluble proteins which serve as cytoplasmic
PRRs inside macrophages that sense danger signals, such as extracellular ATP and cell
disruption (K+ efflux), as well as microbial products [96, 97]. There are different theories as
to how NLRs recognize these widely different danger signals, and evidence points to the
involvement of a secondary messenger such as ROS [98]. The NLRs are central to a
functional innate immune system. NLR proteins contain a sensing LRR region, a central
oligomerization domain termed NACHT, and an effector domain (such as a pyrin domain
(PYD), caspase recruit domain (CARD), baculovirus inhibitor of apoptosis repeat domain or
transactivation domain) which determines the type and function of the NLR [96, 97]. There
are several subfamilies of NLRs, and the two main ones are the NACHT, LRR and PYD
domains-containing protein (NLRP) family and the NACHT, LRR and CARD domainscontaining protein (NLRC) family. The activation of NLRs can lead to NF-κB translocation to
the nucleus and transcription of proinflammatory cytokine genes. Additionally, NLRs
including NLRP1, NLRP3, and ICE-protease activating factor (IPAF/NLRC4) can form
molecular complexes termed inflammasomes together with CARD domain-containing
proteases known as caspases. In the case of NLRPs, this occurs with the help of scaffolding
proteins such as apoptosis-associated speck-like protein containing a CARD (ASC), which
through homotypic interaction acts as a bridge between the NLRP and the CARD domain of
the caspase. Subsequently, through inflammasome assembly, inflammatory caspases are
activated and the pro-inflammatory cytokine precursors proIL-1β and proIL-18 are cleaved
into their mature forms and released from the cell. The precursors are produced upon TLR
engagement and NF-κB translocation to the nucleus [96, 99]. Inflammasome activation is also
dependent on upregulation of the NLR gene, which occurs upon receptor ligation [100]. In
addition to cytokine production, activation of the NLRP3 inflammasome can lead to induction
of a type of cell death where the cell releases large amounts of pro-inflammatory cytokines,
and displays signs of necrosis with permeabilization of the plasma membrane. This cell death
pathway can be dependent on caspase-1, and is then termed pyroptosis, or independent of
caspase-1 but dependent on NALP3 inflammasome assembly, and is then termed
25
pyronecrosis. Inflammasome-related cell death can be beneficial for the host as it may kill the
microbe and leads to recruitment of other immune cells, but can also be thought of as an
“emergency exit” for a cell that is unable to handle an intracellular pathogen, as it inevitably
leads to excessive inflammation [97].
Cytokine production
Newly synthesized cytokine-precursor proteins in the ER of macrophages are folded, qualitychecked and partially glycosylated, and then transported to the Golgi complex where they are
further processed and glycosylated, ending up in the trans Golgi network (TGN) [101]. There
are two major pathways for release of cytokines from the cell. The first one is a constitutive
secretory pathway where small vesicles called recycling endosomes sort and carry proteins
from the TGN to the cell surface in continuous small amounts, and this pathway can be
upregulated upon stimulation in macrophages to increase cytokine secretion. The second one
is a granule-mediated secretory pathway in professional secretory cells where proteins are
packed into granules for storage until degranulation is triggered, and the vesicle contents are
released through fusion with the plasma membrane. There are also cytokines, including IL-1β,
for which it is not known how secretion from the cytosol is achieved [101].
The timing of cytokine release from macrophages is tightly regulated to achieve proinflammatory conditions but subsequently also resolution of inflammation. The response can
be very quick, which can be illustrated by the TNF-α response to bacterial LPS. Upon
activation of macrophages with LPS, TNF-α can be detected both as a precursor in the TGN
and as a cleaved mature cytokine at the cell surface as early as 20 min after stimulation [102].
Thus, macrophages are major players in innate immunity and can be activated in different
ways to perform effector functions but also orchestrate other parts of the immune system.
Phagocytosis
Mammalian cells employ a range of endocytic mechanisms in order to take up extracellular
material, including pinocytosis (“cell drinking” in Greek) and phagocytosis (“cell eating” in
Greek). Endocytosis and membrane trafficking are pivotal to control the composition of the
cellular plasma membrane and regulation of most extranuclear cellular processes, as well as
being important in host defence. Some of the different types of endocytosis are clathrin-
26
mediated
endocytosis,
caveolae-/caveolin1-dependent
endocytosis,
flotillin-dependent
endocytosis, macropinocytosis, and phagocytosis. These endocytosis types differ in
membrane morphology during uptake, the type of cargo that can be taken up, as well as in
which proteins are implicated in the mechanism. The uptake can be receptor-mediated or nonreceptor-mediated [103].
Phagocytosis is a pathway by which phagocytes engulf large particles and microbes into
fluid-filled double-membrane vesicles (phagosomes) derived from the plasma membrane [87].
It is a receptor-mediated process dependent on dynamic remodelling of the actin network, to
form psuedopods or allow membrane invagination, recruit more membrane to the uptake site,
and close the phagosome. These events involve polymerization, contraction, and
depolymerisation of actin, which occurs in either a “zipper-like” or “sinking” way [104, 105].
Phagocytosis by macrophages occurs through an interaction between the cell and the
microorganism in a tightly regulated process. This can be either direct, when endocytic PRRs
on the cell surface such as the MR recognize PAMPs such as surface carbohydrates,
peptidoglycans or lipoproteins on the microbe, or indirect, when the microbe has been
opsonised by host factors such as IgG or components of the complement system. Encounter
between a macrophage and an opsonized microbe leads to binding of IgG to FcγR or of
complement components to CR3. The subsequent signalling events depend on the receptor
used for uptake, and are not fully understood [87]. Phagocytic uptake is always dependent on
actin dynamics, which are regulated in different ways depending on which receptor is
engaged, through different signalling pathways. In the case of FcγR-mediated phagocytosis,
the receptors accumulate at the binding site, initiating phosphorylation of their cytoplasmic
ITAM motifs by Src-family kinases. Subsequent phosphorylation events lead to Arp2/3
recruitment dependent of the Rho-GTPases Rac1, Rac2, and cell division control protein 42,
which in turn leads to actin polymerization [105, 106]. In CR3-mediated phagocytosis, actin
remodelling is dependent on the formin Dia1 [87, 107]. During phagocytosis, a substantial
part of the membrane is inevitably internalized and removed from the plasma membrane, and
this must be compensated for by exocytosis of endomembranes at the phagocytosis site [87].
The events following phagocytosis which normally lead to killing and elimination of the
microbe are also dependent on the receptors engaged in phagocytosis [108, 109], and
manipulation of uptake processes is a common mechanism for pathogens to escape an
27
effective immune response [103]. Autophagy is a separate pathway by which the cell ingests
and degrades its own components, and this mechanism has been implicated in more effective
delivery of material, including Mtb, to bactericidal phagolysosomes [61, 110].
Antimicrobial properties of the macrophage
Phagosomal maturation
Upon uptake of a pathogen by receptor-mediated phagocytosis into a macrophage, the
resulting phagosome undergoes a series of fusion and fission events with the endocytic
pathway, termed phagosomal maturation. The phagosome thus goes through several
maturation stages during which it interacts with endosomes and then lysosomes, and acquires
a wide range of antimicrobial properties designed to eradicate the invader. The phagosomal
maturation stages in macrophages include the early, intermediate, and late phagosome,
followed by the phagolysosome, and these can be identified based on the proteins present on
the phagosomal membrane, which correspond to the endosome type with which the
phagosome has interacted [87, 111]. The steps involved in phagosomal maturation are
schematically illustrated in Fig. 4.
Two theories of how the communication between the phagosome and the endosomal network
and lysosomes occurs have been postulated – the kiss and run-hypothesis where the vesicles
interact through transient fusion and fission events via a fusion-pore-like structure [112], and
the fusion hypothesis where the phagosome completely fuses with pre-existing endosomes
[87]. It has been argued that the kiss and run-hypothesis is more probable since phagosomes
do not acquire endosomal proteins and solutes simultaneously, and since molecules of
different size are recruited from the same endosomal type at different time points [104]. In
addition to acquisition through interaction with endosomes and lysosomes, phagosomes can
acquire proteins and lysosomal hydrolases from the TGN [61]. A microtubule-based transport
system is likely to be responsible for providing the scaffold for endosome movement [104,
105]. Membrane rafts, glycolipoprotein microdomains of the plasma membrane that
coordinate cellular signalling events, have been proposed to play a role in phagosomal
maturation [113, 114]. The described fusion and fission events eventually result in a
phagolysosome with a highly oxidative, acidic, and degradative environment designed to very
effectively eliminate invading microbes [87].
28
Figure 4. The steps involved in phagosomal maturation in the macrophage, from uptake of
phagocytic prey to degradation in the phagolysosome. Each stage is characterized by
different membrane composition and different abilities to communicate with the remaining
endosomal network (shown by double-headed arrows). An increasing number of vacuolar H+ATPases leads to the drop in pH [87].
The early phagosome has many of the characteristics of early endosomes, and are capable of
fusion with sorting and recycling endosomes, but not lysosomes [115]. Their accessibility to
the endosomal network is evidenced by their association with transferrin when this is added to
the extracellular medium [10]. The early phagosome has a low number of vacuolar H+ATPases pumping H+ into the phagosome, and thus a near-neutral pH of around 6.3, and
carries proteins such as early endosomal antigen 1 (EEA1) and the Rho-GTPase Rab5 [10,
87]. The role of the Rho-GTPases in phagosomal maturation is to direct endosomal trafficking
and mediate fusion between phagosomes and other organelles, and EEA1 is essential for
bridging membranes during the interactions through tethering of the fusion mediator syntaxin
6 [74, 116]. Thus, both Rab5 and EEA1 are necessary for phagosomal maturation to proceed.
The intermediate phagosome is a midway point between early and late phagosomes, with
retained Rab5 but loss of EEA1 [87]. The late phagosome, in turn, has undergone a drop in
pH caused by the pumping of H+ into the phagosome by the increasing number of vacuolar
H+-ATPases, resulting in a pH of about 5.5. The late phagosomal membrane has acquired
lysosomal-associated membrane protein (LAMP)-1, LAMP-2, LAMP-3/CD63, and Rab7,
from the TGN or from late endosomes, in a mannose 6-phosphate-dependent or independent
29
manner. Lysosomal hydrolases such as cathepsin D are cleaved into their mature form under
acidic conditions, and this commences in the late phagosome [10, 87, 117]. The late
phagosome is incapable of fusion with early endosomes [118].
Many of the fusion and fission events, including anchoring of EEA1 and other proteins to the
phagosomal membrane, are dependent on a PI3K which generates PI3P from PI3. The later
events resulting in the phagolysosome are dependent on Rab7A and Rab-interacting
lysosomal protein (RILP) for bridging the phagosomal membrane to microtubules, necessary
for movement within the cell and for membrane extensions [119]. All the events are
dependent on close apposition of the interacting membranes [87]. Furthermore, Ca2+ is
required for some of the phagosomal maturation events. Based on studies of the recruitment
of late endosomal LAMPs to the phagosome, it was previously thought that phagosomal
maturation was independent of Ca2+ in macrophages [120]. However, more recent work
shows that LAMP-1 is delivered to the phagosome via a PI3K-independent route, while Ca2+
only plays a role during PI3K-dependent pathways [121, 122].
The final stage of the maturation process, the phagolysosome, is a highly effective microbial
killer with active hydrolases and other microbicidal substances, a membrane protein
composition similar to that of lysosomes, and an acidic environment with a pH of about 4.5-5
due to the vast number of vacuolar H+-ATPases in the phagosomal membrane. The
phagolysosome can be distinguished from the late phagosome by its PI3P-, mannose 6phosphate receptor- and lysobisphosphatidic acid-poor internal membranes, and its elevated
content of mature cathepsin D [87, 117]. The antimicrobial properties that the phagolysosome
has acquired grants it the ability to completely degrade an ingested microorganism [104]. The
end-stage of phagosomal maturation is reached about 90 min post-internalization when an
IgG-covered particle is used as phagocytic prey [61].
Antimicrobial mechanisms in the phagosome
The main antimicrobial mechanisms of the mature macrophage phagosome are acidification
of the phagosome, activation of the NADPH oxidase NOX2, activation of iNOS, as well as
antimicrobial peptides and degradative proteins [87], as illustrated in Fig. 5.
30
Figure 5. The major antimicrobial mechanisms of the macrophage phagolysosome. The low
pH, RNI and ROS, antimicrobial peptides, damaging proteins and proteases, as well as irondepriving mechanisms contribute to the hostile environment in the mature phagosome [87].
To achieve acidification of the phagosome, a large number of vacuolar H+-ATPases are
required in the phagosomal membrane. The vacuolar H+-ATPase enzyme consists of a
cytoplasmic complex (V1) which hydrolyses ATP and transfers energy to a membraneembedded complex (V0) which is able to translocate H+ across the membrane. The complex is
central for phagosomal maturation, both for acidification as a means in itself to destroy
pathogens through an inhospitable environment where metabolism is difficult, as well as for
activating hydrolytic enzymes that in turn degrade pathogens, and finally as a controller of
membrane traffic [87, 123]. It is known that chemical inhibition of the vacuolar H+-ATPase
affects both acidification of the phagosome, fusion between phagosomes and lysosomes, and
the acquisition of hydrolytic activity [61]. In addition to these functions, the vacuolar H+ATPase pumps H+ in an electrogenic manner, which facilitates superoxide production as it
counteracts the negative charges transported by the NADPH oxidase, discussed below, and
31
the phagosomal H+ can be combined with products of the oxidase, generating more complex
ROS [87]. There are different theories as to where the vacuolar H+-ATPase is recruited from,
including the TGN [124, 125], endosomes [126], and tubular structures protruding from
lysosomes [127].
Macrophages use the NADPH oxidase NOX2 to generate ROS from O2 for killing
microorganisms, although this mechanism is more potent and better studied in neutrophils.
Upon activation, the enzyme complex subunits of the NADPH oxidase assemble in the
phagosomal membrane in a Rac1- or Rac2-dependent manner. The active NADPH oxidase
transfers cytosolic NADPH electrons to O2 in the phagosome, producing superoxide (O2-)
which forms hydrogen peroxide (H2O2) through dismutation in the phagosome. H2O2 in turn
further reacts with O2-, generating different ROS, which can kill the intraphagosomal
pathogen [87, 128].
iNOS, also termed NOS2 in phagocytes, is synthesised de novo upon microbial stimulation of
macrophages. It functions to produce nitrogen radicals on the cytoplasmic side of the
phagosome, which can then diffuse into the phagosome. iNOS has two subunits, which act in
concert to produce NO• and citrulline from L-arginine and O2. Upon reaction with oxygen
radicals produced by the NADPH oxidase, NO• is converted to reactive nitrogen intermediates
(RNI), which are very toxic to microbes in the phagosome, and can damage DNA, lipids and
proteins [87]. The importance of RNI in protection against microbial infection in mouse
macrophages has been well documented [129], and RNI are thought to play a role in human
macrophage infection as well, although this is more controversial [63, 130].
The mature phagosome contains many antimicrobial peptides and degradative proteins which
help in the destruction of phagosomal pathogens. Antimicrobial agents can either deprive the
microbe of nutrients or compromise its integrity by inducing membrane permeabilization.
Iron scavengers, such as lactoferrin, and iron exporters, such as natural resistance-associated
macrophage protein 1 (NRAMP1), remove iron thus depriving the microbe of an essential
factor required for DNA synthesis and mitochondrial respiration [87, 131]. Antimicrobial
peptides and proteins that more directly contribute to microbe killing by permeabilizing the
bacterial cell membrane include defensins, cathelicidins, lysozymes, lipases, proteases, and
hydrolases [87]. In neutrophils, these defence substances are packed in granules, while in
32
macrophages they can be expressed upon activation in a manner dependent on vitamin D
[132] or be acquired from other cell types [55, 133].
With their wide array of antimicrobial functions, it is evident that macrophages are key
players in the innate immune response. In addition to the mentioned mechanisms in the
phagosome, macrophages are capable of inducing programmed cell death through apoptosis
when infected with an intracellular pathogen, as a type of “emergency exit.” This can function
as a direct deprivation of the replication niche of the bacterium or virus, as well as a signal to
the remaining immune system [134]. Another mechanism is autophagy, a process by which
the cell degrades its own components through a pathway similar to that of phagosomal
maturation described above, which has been implicated in enhanced killing of intracellular
pathogens including Mtb [110, 135].
Enhancing the antimicrobial properties of the phagosome
Inflammasome activation is an antimicrobial mechanism in macrophages, where microbial
components activate caspase-1, which together with receptor signalling leading to
transcription of the pro-IL-1β gene results in cleavage of pro-IL-1β and release of the mature
cytokine from the cell. IL-1β signalling has in turn been implicated in improved phagosomal
maturation [136, 137]. Other cytokines have also been implicated in enhancing (IFN-γ, TNFα) or attenuating (IL-10) phagosomal maturation in human and mouse macrophages [62, 138140]. The link between macrophage activation and phagosomal maturation is not completely
clear, but it is known that NF-κB translocation, achieved by TLR or pro-inflammatory
cytokine receptor ligation, to the nucleus leads to transcription of genes encoding
antimicrobial proteins as well as cytokines and co-stimulatory molecules, and it has been
proposed that pro-inflammatory cytokine signalling has a direct effect on effector functions in
the phagosome or an effect on fusion and fission events between the phagosome and the
endocytic network [64, 138, 141, 142].
Conflicting evidence concerning the role of TLRs in phagosomal maturation exists. Blander
& Medzhitov showed that absence of TLR signalling leads to a significant impairment in the
acquisition of lysosomal markers and hypothesize that the accelerated phagosomal maturation
achieved by TLR ligation or uptake through FcγR creates a hostile environment for microbes
[143-145]. Conversely, Yates & Russell found that there was no difference in the maturation
33
of phagosomes containing uncoated silica beads and silica beads coated with the TLR ligands
LPS (a TLR4 ligand) and Pam3CSK4 (a TLR2 ligand) [146, 147]. The authors postulated that
although TLRs are enriched on the phagosome [142] and their ligation may slightly enhance
phagolysosomal fusion, ligation does not enhance the hydrolytic activity of the phagosome
and TLR ligation is not key to enhancing phagosomal maturation [146, 147].
Human and mouse macrophages
In Mtb-infected mouse and possibly human macrophages, phagosomal maturation as well as
RNI production and control of bacterial replication or bacterial killing can be enhanced
through stimulation of the cells with pro-inflammatory cytokines such as IFN-γ or TLR
ligands [60-62, 64, 148]. However, the discrepancies observed between human and mouse
cells, and the failure to see a reduction in bacterial killing in human macrophages where iNOS
activity has been blocked, indicate that different killing mechanisms are used by human and
mouse macrophages [63, 64]. On the other hand, induction of iNOS has been observed in
human granulomas [130, 149]. Our group works exclusively with human macrophages, to
ensure a relevance of the results for human TB. To sum up, macrophages, their PRRs,
microbicidal mechanisms and the cytokines they secrete play a central role in the innate
immune response to infection, and the pro- or anti-inflammatory response elicited is essential
in determining the outcome of infection.
Mtb interaction with macrophages
Recognition and ingestion
The macrophage is the main replication niche of Mtb, despite the bactericidal characteristics
and functions that this cell type normally has. The bacillus has evolved several strategies for
surviving in the otherwise hostile intracellular environment of the macrophage. Mtb interacts
with the macrophage through several different receptors, including CR, MR, and FcγR, which
leads to phagocytosis of the bacterium, and this is the route of entry employed by Mtb to gain
access to its replication niche. The receptor engaged for uptake depends on whether the
bacillus has been opsonized with complement or antibodies, or remains unopsonized, and can
be important in determining the subsequent events in the host cell as well as the outcome of
infection [71], although this has been debated [74]. Studies suggest that multiple receptors
may be engaged simultaneously during phagocytosis of Mtb [150].
34
Complement receptor
The complement system is a cascade of soluble proteins activated by antibody complexes or
microbes that functions to opsonize invading pathogens, as well as having roles in
chemotaxis, clumping of antigens, and lysis of bacterial cells. Macrophages harbour receptors
for complement on their cell surface, including CR1, which binds the complement
components C3b and C4b, and CR3 and CR4, which bind C3bi and glucan, enabling
recognition of complement-opsonized and sometimes unopsonized microbes [150]. Mtb
activates the complement system through the alternative pathway (without requiring an
antibody response), resulting in opsonisation with C3b and C3bi and uptake via CR.
Furthermore, Mtb actively recruits opsonically active C3b without triggering the complement
cascade, and some strains can also bind directly to the C3bi or β-glucan binding domain of
CR3 in the absence of opsonisation [151-155]. Mtb can thus be ingested by CR3 through both
opsonic and non-opsonic routes [156]. It has been shown that entry of Mtb into the
macrophage via the CR3 can be advantageous for the bacillus, leading to a prevention of
macrophage activation [106], and preferential entry via CR through actively recruiting
complement components may be a virulence mechanism of the bacillus [150]. However,
contradicting studies showed no effect of blocking CR3 on the replication kinetics of Mtb in
macrophages [157], and no effect of knock-out of the CR3 component CD11b on mouse
survival after Mtb infection [158, 159].
Macrophage mannose receptor
Differentiated macrophages, but not monocytes, harbour MR which binds glycosylated
molecules with terminal mannose, fucose or N-acetylglucosamine motifs, and this leads to
ingestion through phagocytosis or pinocytosis, depending on the cargo. The MR on
macrophages binds to mannosylated motifs of virulent Mtb, mediating phagocytosis.
However, presence of the MR is not sufficient for phagocytosis of particles, indicating that a
partner receptor is necessary for uptake to occur [160]. It is not clear how ligation of the MR
leads to phagocytosis, as the subsequent signalling events have been poorly characterized, but
it is known that MR ligation can lead to ROS production as well as phagocytosis. The MR is
downregulated by IFN-γ and it is therefore believed that the MR plays an important role early
during Mtb infection, before the onset of a Th1 response with high IFN-γ levels [150]. Only
virulent Mtb strains can bind the MR, and one reason for this is that virulent strains carry
mannose-capped ManLAM which can interact with the MR, but other factors may also be
35
involved [154, 161, 162]. One study showed that entry via the MR led to reduced phagosomal
maturation and less ROS production as compared to entry via a β-glucan receptor, and this
may represent another virulence mechanism of Mtb [108].
Fcγ receptor
A third receptor present on macrophages is the FcγR, which binds to IgG-opsonized Mtb in
the presence of a specific humoral response. Early experiments suggested that uptake of Mtb
via FcγR led to a higher level of phagolysosomal fusion in macrophages, as evidenced by
ferritin colocalization [163]. Later work supports this notion and additionally shows that
FcγR-mediated uptake of Mtb and other particles leads to ROS production and a proinflammatory response [69, 106], and that this uptake pathway differs from the CR-mediated
one in dependence on different Rho-GTPases [106]. It is known that FcγR-mediated
phagocytosis of IgG-coated latex beads results in rapid phagosomal maturation where a
phagosomal pH of about 5 is reached within 10-12 min [61]. Thus, FcγR-mediated
phagocytosis of Mtb may represent a pathway that is favoured by the host in mounting an
effective macrophage response, once adaptive immunity has been activated.
Other receptors
In addition to these three major receptor types, many other receptors and proteins can mediate
the interaction between Mtb and macrophages. Surfactant proteins in the lung can coat Mtb,
facilitating binding to surfactant protein receptors on the macrophage [164]. Furthermore,
surfactant proteins can interact with the macrophages to alter the uptake mechanism [165], as
well as affect MR activity [166]. Scavenger receptors on macrophages can bind to
sulpholipids on the Mtb surface, CD14 can bind LAM, and DC-SIGN, mannose-binding
lectin, and possibly dectin-1 can bind other Mtb motifs [150, 167]. The significance of these
interactions are not as well characterized, but studies have shown that binding of Mtb to DCSIGN leads to inhibition of DC functions [168].
TLRs
Apart from engaging endocytic PRRs to mediate phagocytosis, PAMPs on the Mtb surface
also bind signalling PRRs including TLRs. TLR2 forms a heterodimer with TLR1 or TLR6
36
on the macrophage cell surface, and together with CD36 recognizes different bacterial
structures, inducing a pro-inflammatory response [167]. TLR2 engagement by Mtb
components leads to vitamin D-dependent production of cathelicidin, and may play a role in
phagosomal maturation, as previously discussed [8]. Several Mtb components are recognized
by TLR2, including lipoproteins, the cell wall core structure, lipids, LM, PIM, and nonmannose-capped LAM. ManLAM from virulent Mtb, on the other hand, does not bind TLR2,
circumventing the TLR2-mediated pro-inflammatory response [169, 170]. The 19-kDa
lipoprotein of Mtb is also recognized by TLR1, and an Mtb extract can be recognized by
TLR6. TLR4, which is the receptor that together with CD14 binds LPS of Gram negative
bacteria, recognizes a yet undefined heat-labile Mtb component, and the intracellular TLR9
recognizes Mtb DNA in the phagosome [8, 169]. In vivo knock-out studies in mice have
shown a redundancy of TLRs when it comes to combating Mtb infection, and have given
contradicting results as to whether knock-out of TLR leads to increased susceptibility to Mtb.
However, it has been established that MyD88, an adaptor protein essential for NFκB
activation, is required for an effective immune response against Mtb [8, 167]. It has been
argued, however, that it is the vital role of MyD88 in IL-1R or IFN-γ signalling rather than in
TLR signalling and pathogen recognition that leads to its central role in Mtb immunity [171].
NLRs
As for NLR activation by Mtb and other mycobacterial species, it is known that the secreted
mycobacterial factor ESAT-6 is a potent activator of the NLRP3 inflammasome [35, 38, 172],
while a mycobacterial metalloprotease contributes to suppression of activation of the
IPAF/NLRC4 inflammasome [136]. Furthermore, IL-1β, produced upon inflammasome
activation, is essential for control of Mtb infection [173, 174]. However, the inflammasomes
do not seem to play a crucial role in vivo, since mice deficient in caspase-1, ASC [79, 174] or
NLRP3 [79, 175] produce equal levels of IL-1β as compared to wild type mice and display no
increase in bacterial load nor reduction in survival. The inflammasome scaffolding protein
ASC, on the other hand, may play a role in granuloma formation in a caspase-1-independent
manner [79]. Thus, the redundancy in IL-1β production means that NLRP3 inflammasome
activation is not essential for host defence against Mtb, at least not in mice. Activation of
other inflammasomes, as well as bacterial enzymes and other proteases than caspase-1,
including granzyme A, chymase, chymotrypsin, and matrix metalloproteases, are also able to
cleave pro-IL-1β into its mature form, and this may explain the redundancy [79].
37
Cytokine response
Pro-inflammatory cytokine signalling is crucial for an effective immune response against
intracellular pathogens, and mice lacking IL-1β, IFN-γ, IL-12, IL-6 or TNF-α quickly
succumb to infections [86]. The same cytokines play a great role in controlling human Mtb
infection, and their downregulation through IL-10 or TGF-β can be detrimental to the host,
despite the fact that these cytokines are crucial for limiting tissue damage [176]. Early during
Mtb infection of mice and humans, the macrophages are polarized towards an M1 phenotype,
although some individuals have a more M2-like profile which can be reversed by antibiotic
treatment [86, 177]. Macrophages from mouse bronchoalveolar lavage switch to a more M2like profile as Mtb infection progresses, with high levels of IL-4 and a loss of iNOS but
increase in arginase expression, while macrophages from inside mouse granulomae remain
more M1-like [177]. Th1 cytokines and an M1 macrophage response is thought to be essential
for control of human Mtb. Concurrent helminth infection, which leads to a switch to a Th2
response, increases morbidity in TB patients [178]. In addition, reactivation of latent TB can
be associated with a switch to a Th2 response [179]. Finally, Th2 cytokine treatment in Mtbinfected mice deprives the macrophages of their killing mechanisms, leading to increased
replication of Mtb through increased availability of iron [180].
Although pro-inflammatory cytokines are necessary for the host response to Mtb, they are
also responsible for tissue damage, again highlighting the importance of a tightly regulated
immune response against Mtb infection [38, 181]. In addition to the crucial cytokine response
to Mtb infection, chemokines including RANTES, MIP1-α, MIP2, MCP-1, MCP-3, MCP-5
and IFN-γ-induced protein 10 kDa are also released upon macrophage infection with Mtb, and
chemokine receptors including CCR5, RANTES receptor, MIP1-α receptor and MIP1-β
receptor are expressed. Some of these have been found to be essential for protection against
Mtb infection as well as for granuloma formation, although this response is less well
characterized [59, 176].
Inhibition of bactericidal mechanisms
Pathogens have evolved different mechanisms to allow survival inside the macrophage.
Listeria and Shigella spp. escape into the cytoplasm to avoid the degradative milieu of the
lysosome and have evolved mechanisms of surviving extraphagosomally. Coxiella and
Leishmania spp. can replicate inside phagolysosomes despite the hostile milieu. Legionella,
38
Brucella and Mycobacterium spp., in contrast, inhibit phagosomal maturation [61]. Mtb owes
its success as pathogen to its ability to interfere with the normally effective antimicrobial
properties of the macrophage. Thereby, the bacterium creates a niche that supports its
replication despite its localization inside the cell that was designed to kill microorganisms.
Inhibition of phagosomal maturation
There are several ways in which Mtb manipulates the macrophage to avoid killing and create
a favourable environment for replication, and the inhibition of phagosomal maturation is the
best characterized mechanism. This inhibition involves several of the aspects of phagosomal
maturation, including both fusion and fission events and recruitment of vacuolar H+-ATPases
allowing acidification, as illustrated in Fig. 6.
Figure 6. The Mtb phagosome and mechanisms of phagosomal maturation inhibition in
macrophages. LAM-mediated inhibition of the Ca2+ surge through sphingosine kinase
inhibition, and of the PI3K hVPS34, as well as SapM-mediated dephosphorylation of PI3P
and activation of p38MAPK by an unknown component leads to deficient recruitment of
EEA1 and function of Rab5, resulting in a lack of interaction with late endosomes and
lysosomes, as well as a lack of vacuolar H+-ATPase accumulation. This arrests the vacuole at
the early phagosome stage, with a relatively neutral pH [74].
39
An Mtb-carrying phagosome is a dynamic compartment, rather than a static one [74, 182,
183]. It retains the characteristics of an early phagosome in terms of pH (about 6.4), retention
of Rab5, and continued access to recycling endosomes [61, 184]. It also lacks mature
lysosomal hydrolases [74], and interactions between the phagosome and the TGN have been
blocked [20]. Several Mtb products are thought to be responsible for the inhibition in
maturation, including LAM, trehalose dimycolate and sulpholipids, as well as the phosphatase
SapM and the kinase PknG [61], and ESAT-6 has been implicated in inhibition of
phagosomal maturation during M. marinum infection of macrophages [185]. The mechanisms
behind the mycobacterial inhibition of phagosomal maturation have been partially elucidated.
Cholesterol, for example, is necessary both for the uptake of Mtb via CR and for the
inhibition of phagosomal maturation [186].
LAM, the abundant mycobacterial cell wall glycolipid, is shed by the bacterium upon entry
into the cell and can thereafter be found throughout macrophage membranes, in membrane
rafts of lymphocytes where LAM has been added experimentally, as well as exocytosed by
the host cell upon infection [187-189]. The molecule is thought to be a major player in the
inhibition of phagosomal maturation [124, 190]. It is not known how ManLAM integrates
into the cell membrane of the macrophage, or whether lipid rafts play a role, but one study
showed that ManLAM can physically obstruct membrane fusion, interfering with membrane
raft composition [191]. Furthermore, ManLAM can inhibit the recruitment of EEA1, which is
necessary for subsequent fusion and fission events leading to delivery of lysosomal
hydrolases and vacuolar H+-ATPases to the phagosome. The inhibition is achieved through
prevention of a Ca2+ surge in the cytoplasm which is necessary for calmodulin- and
Ca2+/calmodulin-dependent kinase II (CaMKII)-dependent delivery of EEA1 [121, 122, 190,
192]. Inhibition of the Ca2+ surge is thought to be preceded by an inhibition of a macrophage
sphingosine kinase [122]. A second mechanism that leads to inhibition of EEA1 recruitment
is blockade of PI3P, and this is thought to occur through ManLAM-mediated inhibition of the
PI3K hVPS34 as well as through SapM-mediated dephosphorylation of PI3P [20, 74, 190,
193]. A third, less well characterized mechanism of phagosomal maturation inhibition by Mtb
in macrophages is the phosphorylation of an unknown host substrate by the serine/threonine
kinase PknG, regulating phagosomal maturation [61], and another is activation of p38MAPK
by Mtb, leading to reduced Rab5 activity and thus reduced EEA1 on phagosomes [194, 195].
However, the role of ManLAM here is not clear [74]. A final mechanism of Mtb inhibition of
phagosomal maturation is through retention of a molecule called coronin 1, also involved in
40
Ca2+ signalling [71]. Thus, ManLAM and other Mtb components can block the
communication pathways between the phagosome and other endosomes in different ways,
resulting in a lack of recruitment of vacuolar H+-ATPases, as well as recruitment and
maturation of lysosomal hydrolases. This disruption is also thought to affect the
communication with the TGN [124]. The mechanisms described above result in a phagosomal
compartment that is deficient in microbial killing because of the lack of acidification and the
lack of mature lysosomal hydrolases.
Significance of phagosomal maturation components
As phagosomal maturation is inhibited by Mtb, it is generally accepted that maturation of the
phagosome would be harmful to the bacillus. However, the significance of each different
component of phagosomal maturation in inhibiting Mtb growth or killing the bacilli has not
been as well elucidated. There is a confusion in the field as most studies are based on mouse
macrophage models, which are readily activated by IFN-γ to enhance phagosomal maturation
leading to an increase in the production of RNI, and can thus mediate control of Mtb [61, 63,
64, 71]. Human macrophages, on the other hand, are more sensitive to Mtb infection, and it is
more difficult to induce bacterial killing through stimulation of these cells (our unpublished
results and [176]).
Mtb bacilli have been shown to be damaged by ROS and RNI in activated macrophages
[196]. However, Mtb has also been shown to be able to persist in this hostile environment due
to the production of the catalase-peroxidase KatG which can deactivate ROS and a resistance
mechanism against RNI which involves the mycobacterial proteasome [71]. From studies
using other bacterial infections to induce phagolysosome fusion, it has been postulated that
maturation of the Mtb-carrying phagosome does not lead to killing of the bacilli per se, and
growth in phagolysosomes has sometimes been observed [11]. Degradation of Mtb, on the
other hand, was rarely observed over a seven-day period [11, 197]. Another study showed that
phagolysosomal fusion and efficient acidification led to a reduction in growth rather than
killing of the bacilli when virulent Mtb was used [198].
It is known that phagosomal acidification and phagolysosomal fusion are affected when mice
deficient in different inflammatory cytokines are infected with mycobacteria [138]. As
mentioned, mycobacteria are able to exclude the vacuolar H+-ATPase from its phagosome
41
[199], and it has been found that cytokine activation of the Mtb-containing macrophages can
lead to enhanced acidification of the vacuole, at least in mice [62]. Another study showed that
the metabolic activity of Mtb is low in mature phagosomes, but high in immature phagosome,
which further shows how the bacilli actively reduce phagosomal maturation in order to avoid
the degradative milieu and may indicate that maturation of the phagosome decreases Mtb
metabolism [200]. However, it seems that it is difficult to induce killing of Mtb, especially
inside human macrophages in a model system. Acidification of the phagosome does seem to
play an important role in controlling mycobacteria in vivo, however, at least in mice, as one
study showed that mycobacterial mutants defective in the inhibition of phagosomal
acidification displayed reduced survival in mice [201]. Furthermore, it has been found that
Mtb can survive in acidic environments and the authors suggest that the maturation of
lysosomal hydrolases is more important for killing the bacilli than acidification per se [202].
Acidification is intimately related with other components of phagosomal maturation, as the
vacuolar H+-ATPase can play a role in the endosomal trafficking required both for fusion and
in the maturation of lysosomal hydrolases [123]. In addition, it is debated whether the
vacuolar H+-ATPases are recruited from lysosomes, meaning that recruitment is dependent on
phagolysosomal fusion, or directly from the TGN [124-127].
Routes of bacterial phagocytosis, induction of autophagy in the host cell, stimulation of the
cell with different cytokines, as well as other factors may also play a role in how bactericidal
mechanisms in the host macrophage including phagosomal maturation are induced and what
the outcome of infection is [11]. Thus, it has not been completely deciphered which
components of phagosomal maturation are necessary to kill the bacteria inside the human
macrophage to overcome the infection. Furthermore, there is ambiguousness as to whether
human macrophages can actually kill or merely control Mtb infection.
Escape from the phagosome
A long-standing consensus in the TB field has been that Mtb resides exclusively in
membrane-enclosed vacuoles within the macrophage, gaining access to nutrients through
endosomal trafficking. However, experiments in the 1980’s and 1990’s produced some
controversial images of Mtb that appeared free of a phagosomal membrane after a few days of
infection [203-205]. Since then, many groups have attempted to find such phagosome-less
bacilli. In these studies, Mtb-infected macrophages from mice or humans or granulomatous
42
tissue from infected animals or TB patients were analysed by transmission electron
microscopy (TEM). In the experiments, performed for up to nine days in vitro and longer in
vivo, no evidence of extra-phagosomal Mtb could be found [73, 126, 182, 188, 206-209]. In
one study, the accessibility of Mtb inside macrophages to Fab fragments that were injected
into the cytoplasm of the infected cell was assessed, and the authors concluded that the
bacteria were not accessible to the host cell cytosol [73]. Conversely, another study showed
that BCG are accessible to dextran molecules microinjected into the cytoplasm of
macrophages, and argued that this was a means of gaining access to cytosolic nutrients or for
the cell to present mycobacterial antigens via MHC class I [210]. In 2003, Brown and
colleagues discovered that M. marinum is able to escape its phagosome into the cytosol,
moving around by the propulsion of actin through Arp2/3 complex-mediated actin
reorganization in a manner dependent on activation of Wiskott-Aldrich syndrome protein
(WASP) [211, 212]. This feature was later found to be dependent on a functional RD1 region
[37].
The publications about M. marinum initiated a new debate about whether Mtb is also able to
also escape from the phagosome. In 2007, a paper was published showing by cryoimmunogold electron microscopy that Mtb could be found in increasing numbers in the
cytosol of DC and macrophages after at least two days of infection, and that escape from the
phagosome was dependent on ESAT-6 and led to increased replication rates in the cell
cytoplasm. The phenomenon was distinct from that observed in M. marinum as Mtb did not
form actin comet tails. The authors argued that this phenomenon was not merely due to the
bacteria outgrowing their phagosome and that it was not a result of membrane disruption
during apoptotic cell death. In this study, Mtb was found to colocalize with both LAMPs and
cathepsin D prior to escape, and escape was followed by cell death [75].
The contradicting results concerning escape from the phagosome may depend on the TEM
preparation techniques, the source of the primary cells or which cell line was used, the
multiplicity of infection (MOI), or the infection time. Varying differentiation protocols and
robustness of cell types, giving different permissibility to infection, are likely to contribute to
whether the bacteria are able to escape the phagosome, as the activation state of the
macrophage is a very important factor for controlling Mtb. Furthermore, the mycobacterial
cell wall can be mistaken for a phagosomal membrane due to its lipid-rich composition,
giving false-negative results [213]. As proposed in a review article, one explanation for the
43
presence of cytosolic Mtb in some preparations is that the phagosomal membrane is lysed in
the process of lysing the cell [11]. Cell lysis as well as escape from the phagosome are
dependent on ESAT-6 in the case of M. marinum infection [33, 34, 37]. According to this
theory, bacilli devoid of phagosomal membranes are on the way to escaping from the cell at
the moment when they are fixed for TEM [11].
Our unpublished data support the presence of cytosolic Mtb, but the bacilli devoid of
phagosomal membranes in our preparations are usually found in cells with a dispersed
cytoplasm that appear to be necrotic. Thus, it remains to be established whether phagosomal
escape is a strategy which the bacilli use to enable higher replication rates and access to
nutrients in the cytosol, or whether it is merely a consequence of general membrane lysis
during cell death induced by Mtb.
Other survival strategies
Apart from the strategies employed by Mtb to subvert macrophage microbicidal functions
described above, there are several others, including the inhibition of apoptosis, which will be
discussed in detail below. Another mechanism is the inhibition of the macrophage response to
pro-inflammatory cytokines including IFN-γ by interfering with signalling events downstream
of the IFN-γ receptor through the 19-kDa lipoprotein [214]. Furthermore, Mtb can suppress
the expression of MHC class II on macrophages to prevent antigen presentation to CD4+ T
cells by blocking transport and processing of the molecules, thereby avoiding an IFN-γ
response [215]. Another potential virulence mechanism is the disruption of actin, as actin is
necessary for the scaffolding of endosomes during phagosome-endosome interactions and a
correlation between the disruption of actin by Mtb and a delay in phagosomal maturation has
been observed [215-217]. Thus, Mtb has evolved an array of mechanisms to allow survival
within the macrophage, and it is not completely understood how the human macrophages can
be stimulated to produce the factors necessary for killing the pathogen, nor whether this is
possible at all in vitro or in vivo.
Controlling intracellular Mtb growth
As mentioned, it is generally very difficult to stimulate a macrophage to kill Mtb in vitro,
although a control of infection can sometimes be achieved [214]. Killing of Mtb inside human
macrophages has been attained using stimulation with bacterial lipoproteins (TLR ligands) in
44
one study [64]. In another study, IFN-γ or TNF-α activation of human macrophages
containing avirulent mycobacteria led to more effective phagosomal maturation [139]. Older
data gives conflicting views as to the effect of the pro-inflammatory cytokine IFN-γ on intramacrophage replication of Mtb in primary human cells, as it was shown that IFN-γ could
either inhibit [218] or promote [219] growth. Vitamin D has also been reported to be involved
in the killing of intra-macrophage Mtb in human cells through the induction of cathelicidin in
human cells. This was achieved upon TLR stimulation to upregulate the vitamin D receptor
and the vitamin D-1–hydroxylase genes, or through simultaneous stimulation with proinflammatory cytokines [132, 220-222]. However, we have not been able to reproduce these
results (our unpublished data).
It is known that TNF-α as well as IL-1β are crucial for control of human TB, as medications
that include antagonists of these can lead to reactivation of latent TB [173, 223]. IFN-γ is also
crucial as mutations in the IFN-γ receptor gene leading to defective signalling result in higher
susceptibility to Mtb infection [176]. However, it is still not known exactly what component
of the phagolysosome kills Mtb in human infection and how the functionality of the
macrophage can be enhanced and it is difficult to decipher how the phagolysosome can be
induced to kill the bacilli without knowing how or whether the cell can be stimulated to
achieve killing.
Cell death during Mtb infection
Cell death is important for many processes in the body, including homeostasis, development
and immune regulation. Cell death can be programmed, where the cell decides to die in a
tightly regulated manner, or more accidental in emergency situations such as tissue damage.
Different types of cell death are defined based on morphologic and biochemical features,
enzymologic criteria such as dependence on caspases, functional aspects such as whether
death is induced to kill a pathogen or occurs accidentally, and immunological characteristics
including whether death is accompanied by release of pro-inflammatory cytokines or occurs
in a quiescent or anti-inflammatory manner [224]. The major known cell death pathways that
macrophages can enter are shown in Fig. 7.
45
Figure 7. A simplified illustration of the major cell death pathways in macrophages,
including apoptosis, necrosis, pyroptosis and pyronecrosis. The cell death types are
characterized by morphologic, biochemical, enzymologic, functional and immunological
features [97, 134, 225].
46
In response to infection of a cell, inflammatory pathways are induced and the immune system
attempts to clear the invading pathogen through activation of complement, antimicrobial
mechanisms and cytokine release leading to immune cell recruitment. However, if the
infection remains unresolved, the infected cell can undergo programmed cell death in order to
deprive the microbe of its replication niche and expose it to extracellular immune defence
components. This is a typical response to viral infections, but cells can also undergo apoptosis
in response to bacterial infection, and many viruses and bacteria have evolved strategies to
circumvent this response [134]. Mtb is one of the pathogens that are able to manipulate the
host macrophage cell death response to its own advantage. For a long time, there was a
discrepancy in the results obtained concerning host macrophage death upon Mtb infection, as
some studies have shown Mtb to be pro-apoptotic [226-229], while others have shown the
bacterium to be anti-apoptotic in macrophages [230, 231]. However, it is becoming clear that
virulent Mtb is able to both inhibit apoptosis initially upon infection in order to retain its
replication niche, but also induce necrosis-like cell death in order to escape from its host cell
enabling infection of new macrophages [232]. It is still not completely clear, however, what
type of cell death is induced by Mtb and how this affects the immune response.
Apoptosis
Apoptosis is the classical programmed cell death type, with an important role in homeostasis
and embryonic development. Apoptosis is non-inflammatory as the cell contents are kept
inside the plasma membrane and the cell is rapidly taken up by neighbouring phagocytes by a
process named efferocytosis, and apoptosis is thus mainly considered to have an antiinflammatory and immunoregulatory role [233]. However, recent work points to a proinflammatory function of apoptotic neutrophils upon phagocytosis by macrophages [56].
Morphological features of apoptosis include plasma membrane blebbing, cell shrinkage,
nuclear condensation and fragmentation, and formation of apoptotic bodies, but the plasma
membrane remains intact. Biochemically, apoptosis is characterized by retention of highmobility group box 1 protein (HMGB1) in the nucleus and permeabilization of mitochondrial
membranes which leads to a decrease in mitochondrial membrane potential. The processes are
dependent on apoptotic caspases, and these are grouped into initiator caspases (caspase-2, -8,
-9, and -10) and executioner caspases (caspase-3, -6, and -7). Apoptosis can be induced
through the extrinsic or intrinsic pathway.
47
In a resting healthy cell, the pro-apoptotic Bcl-2 family proteins Bax and Bak are kept
inactivated through the anti-apoptotic Bcl-2 proteins, and the mitochondria remain intact. In
the intrinsic pathway, activated by some microbial species, DNA-damaging agents,
irradiation, growth-factor depletion, and some chemotherapeutics, Bax and Bak are activated
through inhibition of anti-apoptotic Bcl-2 by Bcl-2-homology 3 (BH3)-only proteins. Thus,
the outer mitochondrial membrane becomes compromised, and apoptogenic proteins,
including cytochrome c, SMAC/Diablo and HtrA2/Omi, are released from the mitochondrial
intermembrane space through a channel formed by the oligomerization of Bax and Bak [97,
134, 224, 232]. Recent work points to a direct effect of BH3 on Bax and Bak through a
conformational change leading to oligomerization and activation, as well as the indirect effect
through inhibition of anti-apoptotic Bcl-2 [234]. Upon mitochondrial membrane
permeabilization, cytochrome c forms the apoptosome complex together with apoptosis
protease activating factor-1 (Apaf-1), and this mediates the activation of caspase-9.
In the extrinsic pathway, binding of ligands (TNF, Fas ligand, and TRAIL) to death receptors
leads to the assembly of the death-inducing signalling complex (DISC), and activation of
caspase-8 and -10. Furthermore, the extrinsic pathway can be amplified by initiation of the
intrinsic pathway as well through caspase-8-mediated cleavage of Bid, which then acts on the
mitochondrion. The initiator caspases proteolytically activate the executioner caspases, and
the executioner caspases are liberated from their inhibitor X-linked inhibitor of apoptosis
(XIAP) by the mitochondrial death proteins SMAC/Diablo and HtrA2/Omi. The mature
caspases are then able to cleave several different substrates giving different effects, including
the inactivation of a DNA-repair enzyme and a DNase inhibitor, resulting in the mentioned
morphological features such as DNA fragmentation. Additionally, apoptosis can be induced
by granzyme B released from NK cells and cytotoxic T cells, and by a lysosomal pathway
following lysosomal membrane permeabilization and release of cathepsins, which in turn act
on mitochondria [97, 134, 224, 232].
Apoptosis can lead to killing of pathogens through removal of the replication niche of
obligate intracellular microbes, exposure to immune surveillance outside the cell, and through
recognition and uptake of apoptotic bodies leading to more efficient microbicidal mechanisms
in the phagocytosing macrophage as well as more effective antigen presentation [134].
Apoptosis has been assigned a role in the immune response against Mtb, as the bacterium has
been found to inhibit host cell apoptosis. This is achieved in part by the product of the gene
48
nuoG, as deletion of this gene from mycobacteria results in elevated levels of apoptosis [230,
231]. Furthermore, avirulent strains of Mtb have been found to be more potent inducers of
TNF-α-dependent apoptosis than their virulent counterparts [235], and apoptosis in response
to avirulent strains can be enhanced by addition of TNF-α while that in response to virulent
Mtb cannot [236]. Apoptosis exerts an antimicrobial effect on intracellular Mtb, as evidenced
by reduced bacterial viability upon infection of macrophages and subsequent chemical
induction of apoptosis [235]. Uptake of apoptotic macrophages containing Mtb by fresh cells
also results in reduced bacterial viability [227, 237]. Thus, prevention of macrophage
apoptosis by virulent Mtb appears to be a mechanism of virulence, and apoptosis is used by
the host as a defence mechanism and is detrimental to the bacilli. In neutrophils, on the other
hand, Mtb is a very potent inducer of apoptosis [56, 238].
Necrosis
Necrosis is a different type of cell death, which is usually not planned, and which occurs in
response to excessive stress, microbial invasion or tissue damage. Necrosis elicits
inflammation as the cell membrane is permeabilized and the cell contents are spilled into the
surrounding milieu. HMGB1 is released from the nucleus and this and other inflammatory
mediators and danger signals are spilled from the cell. Morphological features of necrosis
include organelle, nuclear and cell swelling, and DNA fragmentation in a disorganized
fashion due to hydrolysis, but no chromatin condensation. Necrosis occurs independently of
caspase activation, and was long thought to always be completely accidental and
uncontrolled. However, it is becoming evident that necrosis can also be the result of the
failure of death receptor ligation to induce caspase activation and thus apoptosis. This can
lead to activation of the serine/threonine kinases receptor interacting proteins (RIP) 1 and 3
and subsequent ROS production, calpain activation, destabilization of lysosomes, and release
of cathepsins. In large quantities, these mediators act as stress signals which can induce
necrosis [134, 224]. Necrosis can thus be considered an emergency cell death pathway which
is detrimental to the host as it results in excessive inflammation on the one hand, and a cry for
help that attracts the attention of surrounding immune cells in response to infection on the
other hand.
A large body of evidence is accumulating that virulent Mtb induces necrosis of the infected
macrophages upon infection and replication to a certain threshold in bacterial load. Different
49
reports give different views of the features of this necrotic cell death [232]. One study showed
high-MOI infection (MOI ≥ 25) to induce propidium iodide-positivity, indicating plasma
membrane permeabilization, of infected cells within 6 h of infection, as well as
externalization of phosphatidyl serine and nuclear condensation, in a manner independent of
caspases but dependent on cathepsins [227]. Another study found H37Rv to induce caspaseindependent plasma membrane permeabilization within 24 h after infection of macrophages at
MOI 10-20, and this was dependent on serine proteases [228]. A third study showed that
infection with H37Rv for 72 h at MOI 10 led to lysis of the host macrophages in a manner
dependent on mitochondrial membrane destabilization [226]. Furthermore, recent work shows
that Mtb is capable of inhibiting the production of the eicosanoid lipid mediator PGE2, which
plays a role in preventing mitochondrial damage as well as repairing the plasma membrane.
This leads to necrosis instead rather than apoptosis, a situation which is favoured by the
bacterium [239, 240]. Additionally, H37Rv can prevent completion of the apoptotic envelope
by removing a domain from annexin-1, and this is also thought to play a role in inducing
necrosis rather than the more hostile apoptosis, allowing dissemination in the lung [241]. A
mouse study using M. marinum also found the bacilli to be cytotoxic, and that this was a
prerequisite for bacterial spread to neighbouring cells and throughout the body [38]. Thus,
induction of necrosis is an important step in the pathogenesis of TB, allowing rupture of the
host cell and spread to other cells once the bacterial load is sufficiently high.
Inflammasome-related cell death pathways
Pyroptosis and pyronecrosis are two types of inflammasome-related cell death types which
are sometimes induced by intracellular infection. These modes of cell deaths are proinflammatory, and are thus a resource for the cell in recruiting more cells of the immune
system when unable to control infection by itself, but can also be detrimental to the host when
it elicits excessive inflammation. In addition to the aforementioned processing of proIL-1β
and proIL-18, inflammasome activation can also lead to induction of cell death, with many of
the characteristics of necrosis including loss of plasma membrane integrity and the release of
pro-inflammatory and danger signals. Plasma membrane integrity loss is preceded by the
formation of pores of 1-2 nm size in the plasma membrane. This type of cell death can be
dependent on caspase-1, ASC, and different NLRs depending on the microbe, in which case it
is termed pyroptosis, or independent of caspase-1 but dependent on cathepsin B, ASC and
NLRP3, in which case it is termed pyronecrosis. During inflammasome-related cell deaths,
50
the plasma membrane is permeabilized, and DNA fragmentation can occur [97, 134, 225].
Although it is known that Mtb can activate the NLRP3 inflammasome through ESAT-6 [35,
38, 172], it has never been investigated whether Mtb can induce one of the inflammasomerelated cell death types in its host macrophage. However, a study using M. marinum
investigated a similar question in mice, and found that cell death induced by ESX-1-secreted
proteins was independent of inflammasome components, but that it led to IL-1β secretion that
was detrimental to the host [38].
Other types of cell death
Apart from the types of cell death described above, macrophages can also lose viability
through autophagic cell death, which can occur upon autophagy of vesicles containing the
microbe and subsequent delivery to mature phagosomes and killing of the microbe, in cases
where engulfment of the cell’s own cellular material is excessive. This type of cell death is
generally beneficial for the host as it allows removal of the cell in a quiescent manner,
simultaneously to promoting the antimicrobial functions of the cell [242], and autophagy has
been shown to play a role in the effective macrophage response to Mtb infection [61, 110,
135]. It is also likely that future cell biology studies will lead to the discovery of yet more
modes of cell death.
In summary, cell death has been implicated both in Mtb virulence and in the effective immune
response against the infection, and deciphering the type and role of cell death induced by the
bacilli in different contexts is an important step in understanding how Mtb survives in and
subverts the antimicrobial functions of the host macrophage.
Missing pieces of the puzzle
From the above review of the literature, it is evident that the interaction between the Mtb
bacillus and the host macrophage is essential in determining the outcome of infection.
However, the way in which the bacteria subvert the response of the macrophage originally
designed to kill is still not completely understood. There are gaps in the knowledge, including
how the bacteria inhibit phagosomal maturation and how the bacilli induce cell death.
Furthermore, although a lot of effort has gone into studying phagosomal maturation in the
context of Mtb, it is still not clear what determines the outcome of infection. The deciding
51
factors for overcoming the block in phagosomal maturation, the crucial determinants in
controlling the bacilli and disabling their replication, and the activation factors necessary to
enhance macrophage function to remove the infection are not fully known. The dogma that
inhibition of phagolysosomal fusion is the determinant factor that allows mycobacterial
growth has not been well substantiated in a human system. Furthermore, methods designed to
study these factors are needed to enable a better understanding. Many pieces of information
come from studies of mouse or other animal models, and are based on results obtained with
avirulent species of mycobacteria and the extrapolation of these to the human in vivo situation
is often difficult.
52
Aims
The general goal of this thesis was to understand the mechanisms by which Mtb is able to
survive and replicate inside the human macrophage. The focus of each paper was as follows:
Paper I:
The aim was to elucidate mechanisms responsible for the inhibition of mycobacterial
phagosome maturation. Specifically, it was investigated whether the inhibitory effect of the
Mtb glycolipid ManLAM on phagosomal maturation was due to incorporation of ManLAM
into membrane rafts of human macrophages.
Paper II:
The aim was to develop a robust and sensitive method that could be used to efficiently
measure the growth of Mtb inside macrophages over several days of infection as well as
monitor the viability of the host cells, to be used for drug screening and for studying how
macrophages can be stimulated to control Mtb growth.
Paper III:
The aim was to investigate which components of phagosomal maturation are important for the
ability of the macrophage to control intracellular Mtb growth. It was specifically investigated
whether translocation of lysosomal markers to the phagosome, phagosomal acidification, or
other phagosomal functions were responsible for restricting Mtb growth in a situation where
little change in bacterial numbers occurred.
Paper IV:
The aim was to characterize the host cell death observed as virulent Mtb replicate within
human macrophages, in order to elucidate whether Mtb could induce the inflammasomerelated cell death modes pyroptosis or pyronecrosis in human macrophages.
53
54
Results and Discussion
All experiments were performed using human monocyte-derived macrophages (hMDMs).
These were prepared by separating monocytes from whole blood obtained from healthy
donors and allowing these to differentiate into hMDMs adhered to the bottom of a cell culture
flask in the presence of 10 % normal human serum for about one week. In order to study the
interaction between hMDMs and Mtb, infection with live virulent Mtb (H37Rv) or avirulent
Mtb (H37Ra) or incubation with purified mycobacterial cell wall components was performed.
Culture of H37Rv and subsequent hMDM infections and analyses were performed in the
BSL3 facility at Linköping University Hospital. A complete description of the materials and
methods used in these studies can be found in Papers I-IV.
Paper I:
LAM is a glycolipid present in abundance in the cell wall of mycobacteria, and the molecule
is attached to the plasma membrane by a GPI anchor [19]. LAM interacts with host
macrophages during infection, and is thought to act as a virulence factor for Mtb. The capping
motifs of the molecule are important for virulence, and mannose-capped ManLAM is present
in virulent species, allowing interaction with the macrophage MR and DC-SIGN and resulting
in an anti-inflammatory response thought to be favoured by the bacterium [154, 161]. Upon
infection of macrophages or DC with Mtb, ManLAM is shed from the bacterium and can be
detected throughout the cell, exerting effects on the cell including interference with signalling
events and cytokine production leading to a suppression of the pro-inflammatory response
[21, 22, 187, 189, 208]. The best characterized function of ManLAM is its ability to interfere
with phagosomal maturation events, in part by preventing a Ca2+ surge in the cytoplasm and
blocking PI3K, both necessary for the recruitment of EEA1 and subsequently lysosomal
hydrolases and vacuolar H+-ATPases to the maturing phagosome [122, 190]. Previous studies
show that ManLAM is incorporated into the cell membrane of lymphocytes upon incubation,
and localizes to membrane raft domains, which are cholesterol- and glycosphingolipid-rich
regions of the plasma membrane that act as signalling platforms, thus interfering with cell
signalling events [187, 243]. However, this has not been studied in human macrophages or in
the context of phagosomal maturation. We aimed to investigate whether the inhibitory effect
of ManLAM on phagosomal maturation was due to incorporation of the molecule into
membrane rafts of human macrophages.
55
In order to mimic shedding of mycobacterial lipids from the bacterium during infection,
hMDMs were incubated with ManLAM from virulent Mtb, PILAM from an avirulent
mycobacterial species, PIM, a mycobacterial glycolipid that resembles the GPI anchor of
LAM, or PIM followed by ManLAM. Immunofluorescent staining of LAM and confocal
microscopy analysis, as well as dot blot analysis of cell fractions from the hMDMs, was
performed. The experiments showed that ManLAM was incorporated into the membrane raft
fractions of the hMDM plasma membrane upon incubation, in a manner dependent on the GPI
anchor since pre-incubation with PIM competitively inhibited ManLAM incorporation. This
was consistent with the previous studies on lymphocytes [187, 243].
Subsequently, the effect of the incorporation of ManLAM on phagosomal maturation, in
terms of CD63 translocation to the phagosome and phagosomal acidification, was analysed
using immunofluorescent staining or LysoTracker staining of acidified organelles. We found
that ManLAM, but not PILAM, reduced the maturation of phagosomes containing zymosan
or latex bead particles. This effect was also dependent on insertion of the GPI anchor of LAM
into the cell membrane as PIM abrogated the effect. Finally, we studied whether the
mechanism of phagosomal maturation inhibition by LAM involved interference with TLR
signalling, as TLR signalling has been implicated in phagosomal maturation [144] and since
ManLAM has been shown to interfere with TLR signalling in mouse macrophages by
upregulating the negative TLR regulator IL-1 receptor-associated kinase (IRAK)-M [21].
Furthermore, we looked for evidence of activation or inhibition of p38MAPK, which has been
suggested to be an effect of LAM and to affect phagosomal maturation [74, 194, 195].
However, LAM was not found to influence these signalling factors, and we suggest that other
mycobacterial components are responsible for activating p38MAPK, at least in macrophages.
The conclusion of this study was that the effect of ManLAM on phagosomal maturation is
preceded by the insertion of the GPI anchor of the glycolipid into rafts of the host cell
membrane. This gives insight into one of the ways in which Mtb can manipulate the host cell.
A question that arose from this study was how the observed inhibition of phagosomal
maturation affects the intracellular survival of Mtb.
A study published around the same time as Paper I using live M. marinum and BCG mutants
showed that the type of capping present on the LAM molecule had a small effect on
phagosomal maturation in macrophages, but did not affect the survival of the bacteria in vitro
56
or in vivo [244]. This may suggest a redundancy in the immunosuppressive function of
ManLAM, with other mycobacterial components also being able to inhibit phagosomal
maturation and the pro-inflammatory macrophage response. Alternatively, phagosomal
maturation inhibition may be the initial part of a greater survival strategy. The relationship
between different components of phagosomal maturation and Mtb replication was studied
further in Paper III.
Paper II:
The emergence of antibiotic resistant strains of Mtb poses a major threat to the cure of current
and future TB patients. Thus, finding new antimicrobials that target Mtb replication or
metabolism is imperative for improving treatment [245]. Traditionally, drug discovery is
based on incubation of broth cultures of Mtb with drug candidates and subsequent
determination of the number of viable bacilli by viable count-based methods. Viable count,
also called CFU plating, involves serially diluting lysed infected cells and subsequently
plating them on agar plates in several replicates, followed by manual enumeration of the
number of CFU. Since Mtb is a very slow-growing bacterium, analysis of results cannot be
performed until colonies appear on the plates, which takes 2-3 weeks. As Mtb is an obligate
intracellular pathogen, finding alternative methods to screen for drugs that include the host
cell and are more rapid and cost-effective would be very useful. Such a method could be used
to identify immunomodulators, which act on the host cell to enhance killing of the
microorganism, or virulence blockers which interfere with the different intracellular survival
strategies used by the bacterium, as well as traditional antibiotics that act directly on the
bacterium. Furthermore, this type of method takes into account the ability of the compound to
cross the cell membrane, which is essential for reaching the bacterial target.
In experimental studies on the interaction between Mtb and the host macrophage, a common
question is whether the cell can be manipulated to induce killing or growth control of the
intracellular bacterium. This also requires viable count-based methods where the infected
cells are lysed and CFU plating is performed. Furthermore, a problem with such assays is that
they rarely take into account whether the infected cells are alive or dead, which can influence
the result as lysed macrophages tend to detach from the surface and are thus not included in
the cell lysate that is analyzed. We wanted to develop a method by which we could rapidly
measure the number of living bacteria in our infected macrophages, while simultaneously
57
monitoring extracellular growth of bacilli as well as viability of the host cells. The aim of this
study was thus to validate the use of a luciferase-based medium-throughput method for the
analysis of intra-macrophage growth of Mtb against the CFU plating assay.
Fig. 8 demonstrates the principles of the method presented. It is based on infection of hMDMs
seeded in triplicate in 96-well plates with H37Rv harbouring the pSMT1 plasmid, which
carries the gene for Vibrio harveyi luciferase under the control of the constitutively expressed
hsp60 promoter. This strain has previously been used to study the growth of Mtb in mice
[246-248]. Upon addition of a substrate, n-decanal, flash luminsescence is emitted and can be
measured by a luminometer. Only viable bacteria emit light as the reaction is dependent on
the cofactor reduced flavin mononucleaotide (FMNH2) which is only present if the bacterium
is alive. Luminescence in cell lysates and the culture supernatant is monitored over several
days, and a calcein acetoxymethyl (AM)-based viability assay is also carried out on each day.
Figure 8. The principles of the method used to evaluate intra-macrophage replication of Mtb.
hMDMs (Mø) seeded in a 96-well plate are infected with Mtb. At the desired time points, the
cells are either lysed and subjected to luminometry (left), or their viability is evaluated using
calcein AM (right). Luminescence and fluorescence corresponds to the number of bacteria or
live macrophages, respectively.
58
hMDMs infected with H37Rv at MOI 10 for up to three days were lysed and subjected to
both viable count (adopted for the 96-well plate) and the luciferase-based method. Both
methods were able to detect bacterial growth over time. Interassay (between different donors)
and intraassay (within the triplicates) variability was compared between the two methods.
Interassay variability was high for both methods, due to the differences between donors in
ability to handle Mtb infection, but intrassay variability was lower for the luciferase-based
method. There was a linear relationship between the number of viable bacteria in the sample
and emitted luminescence, down to at least 5000 bacteria. Thus, the CFU-based method was
more sensitive as a small number of bacilli could be detected, but had a smaller range which
depended on the dilutions used. In order to monitor intracellular growth, it is important to
make sure that the bacteria measured are actually intracellular or cell-associated. The data
obtained from the lysates corresponded well to the number of GFP-expressing bacilli
observed per cell by fluorescence microscopy, confirming that the lysates indeed represented
cell-associated bacilli. In previous studies of intracellular mycobacterial growth, extracellular
bacilli have been removed by using the antibiotic amikacin [211]. However, we monitored
both intra- and extracellular H37Rv over three days with and without amikacin, and
concluded that amikacin was having an effect on both portions. We therefore decided to
monitor both extracellular and cell-associated bacilli, in order to monitor the entire system.
In concert with the calcein AM method showing whether the hMDMs were viable or not, the
luciferase-based method was of use in evaluating the ability of Mtb to grow intracellularly
under different conditions. The ability of the assay to detect differences in growth was
demonstrated by adding antibiotics which disabled growth of H37Rv, and by comparing the
growth of the virulent H37Rv to the avirulent H37Ra and the vaccine strain BCG. The latter
were not able to grow intracellularly, in contrast to H37Rv. The high interassay variability
due to differences between donors or different pre-treatments could be accounted for by
normalizing all values against the corresponding phagocytosis (Day 0) value, giving foldincreases from day to day that could be easily compared. The viability assay revealed that
cells infected with H37Rv at MOI 10 lost viability over a few days of infection.
We conclude that the luciferase-based method is useful for monitoring intracellular replication
of Mtb in a more efficient, cost-effective, and less labour-intensive way as compared to CFU
plating. We believe that the method can be applied in screening for drugs against Mtb in its
natural niche, which can be very beneficial in finding good antimicrobials that work alone or
59
in synergy with existing antibiotics. Furthermore, the method can be used to study how the
inhibition of macrophage functions correlates to the ability of the bacteria to replicate
intracellularly (Paper III). Finally, the observation that the host macrophage died during
infection at MOI 10 led to the work presented in Paper IV.
Paper III:
In the literature, it is almost always assumed that infection of macrophages with Mtb leads to
ineffective phagosomal maturation in terms of translocation of lysosomal contents to the
phagosome and acidification, and that this is the factor that determines whether the bacteria
can replicate effectively. It is believed that this can be overcome by stimulating the cells with
pro-inflammatory cytokines, leading to phagolysosomal fusion, RNI production and killing of
the bacilli [11, 117, 139, 249]. However, these assumptions are mainly based on studies
performed on mouse cells which are readily stimulated to express iNOS, and which are better
able to handle infection with virulent Mtb in vitro than human cells [60-62]. Furthermore,
they are often based on studies that use avirulent mycobacteria, and it is conceivable that it is
easier to induce killing of bacteria that are incapable of intracellular replication [126, 199].
There are publications showing that human macrophages can be stimulated to control or kill
Mtb [64, 218], but others claim that this is very difficult if not impossible [176], and there is
even an older study showing that IFN-γ has the opposite effect on Mtb growth in human cells
[219]. Our unpublished data indicate that overnight stimulation with IFN-γ, LPS, or
Pam3CSK4 does not lead to higher phagolysosomal fusion or phagosomal acidification upon
infection with H37Rv nor decreased intracellular replication of the bacilli. Although a lot of
time and effort has gone into characterizing and explaining the phagosomal maturation block,
the effect of different components of the microbicidal lysosome on the bacilli has been largely
overlooked, and it is not really known whether phagosomal maturation directly correlates
with growth restriction of virulent mycobacteria in human macrophages. The aim of this study
was to investigate whether phagolysosomal fusion, acidification, or other functional aspects
of the mature phagosome correlated with growth restriction of Mtb within unstimulated
human macrophages in vitro. The bulk of the analyses were based on the observation that
hMDMs could control H37Rv infection at a low MOI (1), but not at a high MOI (10), and we
wanted to investigate the deciding factors necessary to control infection at the low MOI.
60
In order to address these issues, hMDMs were infected with H37Rv or H37Ra at two different
MOIs, and different components of phagosomal maturation as well as the growth of the
bacilli under different conditions were studied. First of all, we established that H37Ra could
not replicate inside hMDMs, and that H37Rv could replicate upon infection at MOI 10, but
not MOI 1. Infection of unstimulated hMDMs at MOI 1 led to a balance between the bacteria
and the cells for at least one week, where the cells were kept alive and there was no net
change in bacterial numbers beyond the first few days of infection. Immunofluorescence
staining and confocal analysis showed that both H37Rv and H37Ra at MOI 10 were able to
inhibit the translocation of the lysosomal marker CD63 (LAMP-3) to the phagosome, as
compared to phagosomes containing opsonized zymosan particles or killed H37Rv. These
observations show that our assay was able to detect differences in CD63 translocation, and the
data were consistent with previous studies [23, 42, 122, 182, 194]. We then compared the
translocation of CD63 to the phagosome at MOI 10 and 1, and found that infection at both
MOIs inhibited the translocation of CD63 to a similar extent. Furthermore, enumeration of the
number of H37Rv bacilli that were associated with the lysosomal markers CD63 or LAMP-1
showed that as the bacteria divided in the hMDM, the number of bacilli in compartments
positive for the markers increased, which may indicate replication in these compartments.
These results led us to conclude that lysosomal markers such as CD63 are not good indicators
of whether Mtb subversion of the macrophage response has been effective, and prompted us
to further investigate which factors could lead to control of H37Rv infection inside hMDMs.
The acidification of phagosomes containing Mtb at the two MOIs was compared using
LysoTracker staining, revealing that a higher proportion of acidified compartments were
observed at MOI 1 than at MOI 10. This led us to believe that acidification of the phagosome
was directly or indirectly controlling growth of the bacilli at the low MOI, consistent with
mouse studies showing that Mtb mutants with defects in acidification inhibition are less able
to survive [201]. However, acidification of the phagosome has multiple effects on endocytic
trafficking as well as on the maturation of lysosomal hydrolases, and there are indication that
low pH per se does not harm Mtb [123]. Thus, we investigated which functional aspects of
the phagosome were contributing to controlling bacterial replication at the low MOI, by using
inhibitors of the the vacuolar H+-ATPase, cathepsin D, or the NADPH oxidase. We found that
inhibition of both the vacuolar H+-ATPase and cathepsin D resulted in a significantly
increased growth rate of H37Rv infecting hMDMs at MOI 1, highlighting the importance of
these functional aspects of the phagosome for effective Mtb control.
61
Phagolysosomal fusion is one of the components of phagosomal maturation that Mtb has
evolved an ability to inhibit, as studied in Paper I, and this factor is likely to be important for
preventing delivery of lysosomal components to the bacterium and thereby for virulence of
Mtb in vivo. However, the conclusion of this study was that the translocation of LAMPs to the
phagosome is not a good indicator for an effective macrophage response against the
bacterium in human macrophages. Instead, functional aspects of phagosomal maturation such
as activity of the vacuolar H+-ATPase and functional lysosomal proteases such as cathepsin D
are responsible for controlling growth of the bacilli in unstimulated hMDMs at the low MOI.
Apart from providing insight into the importance of different functional aspects of the
phagosome in controlling Mtb growth, this study generated a low MOI-model which is
currently under evaluation for use as a tool for studying macrophage function-induced
dormancy of Mtb. Furthermore, this study and Paper II showed that infection of hMDMs at
MOI 10 led to host cell death, and this was further scrutinized in Paper IV.
Paper IV:
It is becoming increasingly clear that upon infection of macrophages, Mtb has both antiapoptotic and pro-necrotic properties. Inhibition of apoptosis initially upon infection is used
by the bacillus to avoid the microbicidal effects of apoptosis, while lysis of the host cell when
reaching a certain number of bacilli per cell is used as a means of spreading to adjacent
macrophages [231, 232]. In Paper II, we observed decreased viability of macrophages
throughout infection with H37Rv at MOI 10 as the bacteria replicated. This prompted us to
investigate the type of cell death that was induced by the bacilli in our system. Furthermore,
mycobacteria have been shown to activate the NLRP3 inflammasome through the secreted
protein ESAT-6, but it has not been investigated whether this activation can induce one of the
inflammasome-related cell death types pyroptosis or pyronecrosis [35, 38, 172], which is the
case for some other bacterial pathogens [250-252]. ESAT-6 has membrane-damaging
properties and is thought to participate in the escape of mycobacteria from the phagosome
[30, 33, 34, 37, 75], and it would therefore be of interest to study ESAT-6 in the context of
cell death induced by live virulent Mtb. The aim of this study was thus to investigate whether
virulent Mtb could induce the inflammasome-related cell death modes pyroptosis or
pyronecrosis in human macrophages.
62
First of all, the uptake and replication of H37Rv in hMDMs at MOI 10 and 1 were studied
during the first two days of infection, in order to compare the bacterial load at the two MOIs.
We observed initial replication at both MOIs (although this vanished during longer incubation
at the low MOI), and the bacterial load at MOI 1 was about 1/20 of that at MOI 10. Infection
at MOI 10 was accompanied by almost complete loss of hMDM viability after three days of
infection. As inflammasome-related cell death coincides with release of the cytokine IL-1β
due to inflammasome and caspase-1 activation, cell culture supernatants from H37Rvinfected hMDMs were assayed for IL-1β. Infection at MOI 10 resulted in large amounts of
IL-1β being released from the cells in a caspase-1-dependent manner, while infection at MOI
1 did not induce an IL-1β response. In order to elucidate what type of cell death was induced
by H37Rv, three assays looking at different features of cell death were performed. Inhibitors
of different cellular enzymes implicated in cell death pathways were included to determine the
induction route of cell death. H37Rv infection at MOI 10, but not MOI 1, over two days gave
rise to extensive DNA fragmentation, mitochondrial membrane permeabilization, and loss of
plasma membrane integrity. None of these effects were dependent on the cellular enzymes
tested, including caspase-1, cathepsin B and other caspases. The morphological features
together with the failure of these inhibitors to counteract cell death showed that the cell death
type induced was not pyroptosis, pyronecrosis, or apoptosis. The features were, instead,
consistent with necrosis. We postulate that Mtb induces necrosis when it reaches a certain
threshold in bacterial burden, and that this is important for spread to neighboring cells. This is
consistent with ideas presented in other studies [226, 232, 239, 241].
In addition to bacterial load, we hypothesized that secretion of ESAT-6 was important for the
induction of cell death. To test this, hMDMs were infected with H37Rv with a deleted ESAT6 gene or RD1 region. Deletion of these genes led to impaired ability to induce host cell
death, and complementation of ESAT-6 restored the death-inducing capability. Additionally,
CFU plating showed that there was a defect in growth of the mutant bacilli inside the cell, and
based on a previous study [34] this was attributed to the disability of the mutant bacteria to
spread to new cells upon replication. We also found that the mutant strains failed to induce a
strong IL-1β response, probably due to the lack of inflammasome activation in the absence of
ESAT-6. A recent study suggested that ESAT-6 secretion leads to pore formation in the host
cell membrane, which is followed by a K+ efflux leading to inflammasome activation and IL1β release [253], consistent with this notion. Thus, the necrosis observed here was dependent
on ESAT-6 secretion. This is in agreement with previous data on M. marinum [12, 38], which
63
additionally shows that mycobacteria induce IL-1β secretion but that ESAT-6-dependent
necrosis is independent of caspase-1 [38].
The conclusion of this study was that infection of hMDMs with H37Rv leads to ESAT-6dependent necrosis when the bacterial numbers reach a certain threshold. However, infection
does not lead to pyroptosis or pyronecrosis, although cell death coincides with caspase-1
activation and IL-1β release. The lysis of host cells is likely to be important for cell-to-cell
spread as well as for formation of the necrotizing centres of human granulomas. We
hypothesize that the bacilli inhibit apoptosis when bacterial numbers are low, to avoid the
harmful effects, but when replication is successful and the number of bacilli in the cell
reaches a sufficiently high level, ESAT-6 secretion leads to lysis of the phagosomal
membrane and subsequently the macrophage plasma membrane. The inflammasome is
activated in the process, increasing the pro-inflammatory response which can lead to either an
enhanced immune response or immunopathology. This study provided new knowledge about
how Mtb induces host cell death at different bacterial load. The low MOI infection had little
effect on the host cells, and did not elicit an inflammasome response. Future characterization
of this model may provide insight into the bacterial strategies employed to ensure survival at
low bacterial numbers, but also further into how the cell controls bacterial growth and how
this can be stimulated to enhance the immune response and clear Mtb infection.
64
Concluding Remarks
The work presented in this thesis provides insight into the mechanism responsible for the
phagosomal maturation inhibition exerted by Mtb, the significance of different phagosomal
components in controlling Mtb replication inside the host cell, and the mechanism employed
by the bacterium to kill the host cell, allowing spread of infection. Furthermore, a method for
measuring intracellular growth of Mtb is presented, which can be used for experimental
research and potentially for identifying novel antimicrobials. These results are important in a
clinical context as they provide new knowledge about how Mtb subverts the macrophage
response, and this type of knowledge can be used in the future in developing ways to
stimulate the innate immune response to enhance killing of the bacteria.
More specifically, the work provided evidence that mycobacterial ManLAM contributes to
the inhibition of phagosomal maturation by inserting its GPI anchor into membrane rafts of
the macrophage cell membrane. Furthermore, it showed that the luciferase-based method for
measuring intra-macrophage growth of Mtb that was developed was a reliable tool for
studying the effect of different growth conditions on mycobacterial replication. The method
was used to study what factors could affect macrophage control of Mtb replication, and it was
discovered that infection of non-activated hMDMs at a low MOI led to control of the
infection. The results showed that LAMP translocation to the phagosome was not a correlate
of an effective macrophage response. Instead, functional aspects of the phagolysosome were
shown to be important for controlling growth of Mtb at a low MOI. Finally, the work
provided evidence that Mtb does not induce pyroptosis or pyronecrosis in hMDMs, but rather
necrosis in an ESAT-6-dependent manner that coincides with IL-1β secretion.
The hypothesis generated by the presented work is shown in Fig. 9. According to this model,
infection of human macrophages with virulent Mtb leads to shedding of ManLAM into the
host cell plasma membrane. Once incorporated into membrane rafts via the GPI anchor,
ManLAM inhibits endosomal trafficking to prevent recruitment of the vacuolar H+-ATPase
and lysosomal components, thus protecting itself from the normally microbicidal functions of
the cell. The subsequent events are dependent on whether the bacteria are present at
sufficiently high numbers, whether ESAT-6 secretion is effective, and whether the
macrophage has been activated and mounts a strong immune response. In a clinical context,
65
this represents active disease versus clearing the infection or developing latent TB. If the host
cell is weak, e.g. when the host is immunocompromised due to HIV co-infection, or the
bacterial load is high, the bacteria can inhibit the acidification of the compartment in which
they reside, and start to replicate. If ESAT-6 is present, the phagosomal membrane and
subsequently the plasma membrane are lysed, and the bacteria can spread, forming a necrotic
granuloma core. The inflammasome is activated as a consequence, by K+ efflux or bacterial
components, causing IL-1β release leading to inflammation. If the host cell is strong, e.g. has
been pre-stimulated by Th1 cytokines, or the bacterial load is low, the robust macrophage
response is able to suppress Mtb replication. This leads to a very low concentration of ESAT6 in the phagosome, and both the phagosome and cell membrane are kept intact. Additionally,
the inflammasome is not activated. This may lead to a higher rate of bacterial killing, or to the
induction of a dormant state of the bacilli, with little or no replication.
Figure. 9. The hypothesis generated by the work presented in this thesis. Two outcomes of
macrophage (Mø) infection with Mtb are presented, one which is suggested to occur if the
host immunity is weak (left), and the other if host immunity is strong and the macrophage
appropriately pre-activated (right).
66
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Acknowledgements
I would like to express my great appreciation to everyone who has contributed along the way
during the past five years. To all brilliant colleagues, friends and family, THANK YOU!
I would especially like to mention the following people:
My supervisor Maria Lerm, for giving me the opportunity to work in your group and for
always believeing in me and inspiring me to try new ideas. Your encouragement never fails to
motivate me, and your enthusiasm never ceases to amaze me!
My co-supervisor Olle Stendahl, for providing support, sharing your knowledge and always
trying to put the research into context.
My lab and office companion and friend Danne Eklund, for making the endless hours in the
TB lab so much more enjoyable, for challenging my opinions, for all the discussions about
work, life and everything and for your support. You saved my lab life!
My other past and present office mates, Robert Blomgran, for helping me out when I was a
lost newbie, Veronika Patcha-Brodin, for lots of laughs, Johanna Raffetseder, for helping
me finish the microscopy project and for your enthusiasm, and Elsje Pienaar, for providing
us with new research perspectives and for your positive laid-back attitude.
Hana Abdalla, for kindness and help during my first year in the lab.
My informal mentor since the Biomedical Research School times, Kajsa HolmgrenPeterson, for always taking time to ask me how things are going and for lots of laughs.
Kalle Magnusson, for always showing an interest in what I’m doing and taking time to help
out with confocal microscopy and other matters, big or small.
All the lab members at the Department of Chemistry at Bose Institute in Calcutta, for
welcoming me to the lab and for all your help with my constructs. A special thank you to Dr.
Joyoti Basu and Dr. Manikuntala Kundu for taking such good care of me while in India.
The FORSS group, and especially Thomas Schön, for interesting discussions and for always
reminding me of the clinical side of things. Also thanks to Marie Budai, for kindness and
help in the TB lab, and to Petra at Lungkliniken, for a nice collaboration.
Mary Esping, for superb administrative service and help with all types of queries.
Christina Samuelsson, for excellent technical assistance, for always being so helpful and for
taking care of last-minute orders.
The Masters students we have supervised over the years for help with the projects, including
Elmira, Atem, Sadaf, Olena and Carmen. Thanks also to all other students and visitors at
Medical Microbiology that have come and gone for fun times at and outside work.
All the other people who have helped out with my projects, including B-A Fredriksson, for
help with electron microscopy, Annette Molbaek, for help at the core facility, and of course
83
all the ladies in the TB lab: Britt-Marie, Ann-Marie, Sara, Eva, Slavica and Helen, for
putting up with us and being so friendly.
All fellow Ph.D. students at Medical Microbiology, old and new, who have made the past five
years so much more fun! A special thank you to Alexander Persson, for always being there
when I needed help during my first few years and for good memories walking in “this general
direction” at conferences, Martin Winberg, for your involvement in the LAM project as well
as engouraging chit-chat and fun Friday games during the first few years, Jonna Idh, for
companionship and encouragement on the road to the Ph.D. and for your never-ending
energy, Thommie Karlsson, for always providing help and support even though you don’t
really have time, Johan Nordgren, for always putting a smile on my face, Eyler Ngoh, for
interesting discussions about the Swedish ways, Deepti Verma, for support and kindness
from the very beginning, Asya Bolshakova, Magalí Martí Generó, Anders Carlsson, Peter
Wilhelmsson, and all the others, for fun times and for creating a great atmosphere at work.
All past and present staff at Medical Microbiology, especially Anita Sjö, for always being
helpful and for nice times during student labs, Katarina Tejle, for help with orders and blood
issues and for being so friendly, Kristina Orselius, for kindness and help in the lab during the
first few years, Tony Forslund, for always providing support in technical matters, Vesa
Loitto, for teaching me all about molecular biology, Blanka & Henrik Andersson, for
interesting discussions, Jonas Wetterö and Elena Vikström, for always lending a hand in lab
matters, Maggan Lindroth, Elisabet Hollén, Lena Svensson, and all the others, for kindness
and nice times during coffee breaks.
A big thank you to my amazing Linköping friends, including Ia & Emil, for all the evenings
before and mornings after, for always caring and helping me believe in myself and reminding
me what’s really important, Tove, for all the wonderful times and for your inspirational
attitude towards work and life in general, Ida, for great times swimming in Viggeby and
chasing thieves in Thailand and for making my first few years in Linköping so much more
enjoyable, and to Lena, Sofia, and Micke, for support along the way and for all the great
memories from the BMF days onward.
All my old friends, especially Anja, Sanita and Gustav, for always being there and for
having no idea what I do at work.
My family, with outposts in Sjövik, Björboholm and Krokslätt, for being the way you are and
for always providing well-needed refuge. Also thanks to the Skärblacka/Bergen crew for
being so welcoming.
Last but certainly not least, thank you Mattias, min livskamrat och bästa vän, for all your
love and support. I can’t wait for our upcoming adventures!
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