Download Macrophage programming and host responses to bacterial infection Xiao Wang 王潇

Macrophage programming and host
responses to bacterial infection
Xiao Wang 王潇
Doctoral thesis in Molecular Genetics
Department of Molecular Biosciences, The Wenner-Gren Institute
Stockholm University, 2016
Cover illustration: H&E staining picture of murine lungs infected with Mycobacterium.bovis
BCG and fluorescence microscopic picture of macrophage fusion are integrated into a swedish
dala horse.
©Xiao Wang, Stockholm University 2016
ISBN: 978-91-7649-426-4
Printed in Sweden by Holmbergs, Malmö 2016
Distributor: Department of Molecular Biosciences, the Wenner-Gren Institute
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“ Everything you see exists together in a delicate balance. ”
-- Mufusa, The Lion King (1994)
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SAMMANFATTNING
Makrofager är dynamiska och heterogena immunceller som spelar en viktig
roll i immunförsvaret vid bakteriella infektioner. Olika bakteriella patogener,
såsom Neisseria meningitidis och Mycobacterium tuberculosis, kan ändra
värdens immunrespons genom att interferera med makrofagers differentiering och polarisering.
Målet med den här avhandlingen har varit att förstå makrofagers roll i sjukdomsprocessen vid bakteriella sjukdomar.
I artikel 1, fann vi att NhhA, ett membranprotein som återfinns på ytan av
meningokocker, kan aktivera makrofager genom TLR4-beroende och TLR4oberoende mekanismer. I artikel 2, beskrev vi hur NhhA aktiverar monocyter genom receptorn TLR2 och stimulerar produktion av cytokinerna IL10 och TNF. Detta sker genom att aktivera signalvägarna ERK samt JNK
och resulterar i differentiering av monocyterna till makrofager med hög expression av CD200R. Dessa makrofager var associerade med immunhomeostas och asymptomatisk kolonisation i nasofarynx. I artikel 3, undersökte vi
det humana cellytsproteinet CD46 och dess roll i regleringen av apoptos,
differentiering och polarisering av makrofager. Vi fann att makrofager som
uttrycker CD46 har fenotypen M1. Dessa M1-makrofager producerade
övervägande proinflammatoriska cytokiner såsom IL-6, TNF och IL-12 vid
meningokockinfektion eller stimulering med lipopolysackarid. Genom att
använda en experimentell djurmodell för meningokocksepsis kunde vi
bekräfta en roll för dessa makrofager vid uppkomsten av septisk chock. M.
tuberculosis, en gram-positiv bakterie, orsakar många dödsfall framförallt i
utvecklingsländer. I artikel 4 fann vi att makrofager som uttrycker CD46 har
förbättrad överlevnadsförmåga, hög förmåga att döda bakterier och tenderar
att forma gigantiska flerkärniga celler vid kronisk tuberkulosinfektion. En
ökad förståelse för fysiopatologin vid bildningen av granulom kan bidra till
utvecklingen av nya läkemedel mot tuberkulos.
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SUMMARY
Macrophages are dynamic, plastic, and heterogeneous immune cells that
play an important role in host immune defense against bacterial infection.
Various bacterial pathogens, such as Neisseria meningitidis and Mycobacterium tuberculosis, can modulate host immune responses by interfering with
macrophage differentiation and polarization.
The focus of this thesis was to understand the role of macrophages in the
pathogenesis of bacteria-induced diseases, which has important implications
in the search for novel therapeutic strategies to control those infectious diseases.
In Paper I, we found that NhhA, a conserved meningococcal outer membrane protein, can activate macrophages through both Toll-like receptor 4
(TLR4)-dependent and -independent pathways. In Paper II, we demonstrated that NhhA activates monocytes through TLR2 and triggers autocrine IL10 and TNF production through the ERK and JNK pathways, which skew
monocyte differentiation into CD200Rhi macrophages. These immune homeostatic macrophages are associated with nasopharyngeal carriage of meningococci. In Paper III, we examined the role of human CD46, a ubiquitous
transmembrane protein, in regulating macrophage apoptosis, differentiation,
and functional polarization. We revealed that macrophages expressing CD46
exhibit an M1 phenotype and are prone to generate proinflammatory cytokines, such as IL-6, TNF, and IL-12, upon lipopolysaccharide challenge or
meningococcal infection. The important role of these macrophages in the
development of septic shock was further confirmed by in vivo studies using a
CD46 transgenic mouse disease model. M. tuberculosis, a gram-positive
bacterium, remains an important cause of death in developing countries. In
Paper IV, we reported that murine macrophages expressing human CD46
exhibit enhanced viability and bactericidal capacity and are prone to form
granulomas following chronic mycobacterial infection. Increased understanding of host factor roles in the physiopathology of tuberculosis is critical
for the design of effective vaccines and new drugs.
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LIST OF PAPERS
The thesis is based on the papers listed below, which will be referred to by
Roman numerals in the text.
I.
Sjölinder M*, Altenbacher G*, Wang X, Gao Y, Hansson G and
Sjölinder H. The meningococcal adhesin NhhA provokes proinflammatory responses in macrophages via TLR4-dependent and independent pathways. Infect Immun 2012;80:4027–33.
* MS and GA contributed equally.
II.
Wang X, Sjölinder M, Gao Y, Wan Y and Sjölinder H. Immune homeostatic macrophages programmed by the bacterial surface protein
NhhA potentiates nasopharyngeal carriage of Neisseria meningitidis.
mBio 2016;7:e01670-15.
III.
Wang X, Ding Z, Sjölinder M, Wan Y and Sjölinder H. CD46 accelerates macrophage-mediated host susceptibility to meningococcal
sepsis. (Manuscript under revision)
IV.
Wang X, Sjölinder M, Wan Y, De Rovere M, Petursdottir D, Fernández C and Sjölinder H. Protective role of CD46 against mycobacterial infection through functional modulation of macrophages.
(Manuscript under revision)
Paper not included in this thesis:
Chen Y, Sjölinder M, Wang X, Altenbacher G, Hagner M, Berglund
P, Gao Y, Lu T, Jonsson AB and Sjölinder H. Thyroid hormone enhances nitric oxide-mediated bacterial clearance and promotes survival after meningococcal infection. PLoS One. 2012;7:e41445-14.
Permission of reprints was obtained from the publishers.
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ABBREVIATIONS
aHUS
AP-1
BCG
CCL
CD46
CXCL
DC
Erk
G-CSF
GM-CSF
Hsp
IRF
IL
IRAK-4
JNK
LTA
LOS
LPS
MARCO
M-CSF
MV
MyD88
NF-κB
NhhA
NLR
PAMP
PRR
RIP
SCR
TB
TGF-β
TIRAP
TLR
TNF
TRAM
TRIF
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Atypical hemolytic uremic syndrome
Activator protein-1
Bacillus Calmette–Guérin
Chemokine (C-C motif) ligand
Cluster of differentiation 46; a membrane cofactor protein
Chemokine (C-X-C motif) ligand
Dendritic cell
Extracellular-signal-regulated kinases
Granulocyte colony-stimulating factor
Granulocyte-macrophage colony-stimulating factor
Heat shock protein
Interferon regulatory factor
Interleukin
IL-1 receptor-associated kinase-4
c-Jun N-terminal kinase
Lipoteichoic acid
Lipooligosaccharide
Lipopolysaccharide
Macrophage receptor with a collagenous structure
Macrophage colony-stimulating factor
Measles virus
Myeloid differentiation primary response gene (88)
Nuclear factor kappa-light-chain-enhancer of activated B cells
Neisseria hia/hsf homologue A
Nucleotide oligomerization domain (NOD)-like receptor
Pathogen-associated molecular pattern
Pattern recognition receptor
Serine/threonine kinase receptor interacting protein
Short consensus repeats
Tuberculosis
Transforming growth factor beta
TIR domain-containing adaptor protein
Toll-like receptor
Tumor necrosis factor
TRIF-related adaptor molecule
TIR domain-containing adaptor protein inducing IFN-β
CONTENTS
SAMMANFATTNING ................................................................................iv
SUMMARY.................................................................................................... v
LIST OF PAPERS ...................................................................................... vii
ABBREVIATIONS ................................................................................... viii
CONTENTS ..................................................................................................ix
INTRODUCTION ......................................................................................... 1
Chapter 1 Macrophages and bacterial infection ................................ 1
1.1 Bacterial recognition...................................................................... 1
1.2 Macrophage polarization ............................................................... 2
1.3 Resident and inflammatory macrophages...................................... 3
1.4 Factors governing functional differentiation of macrophages ....... 4
1.5 Macrophage cell death in bacterial infections ............................... 5
Chapter 2 CD46 ..................................................................................... 7
2.1 CD46 as a complement factor ........................................................... 7
2.2 CD46 as a pathogen receptor ............................................................ 8
2.3 CD46 as an immune regulator ........................................................... 9
2.4 CD46+/+ transgenic mice as an animal disease model ..................... 10
Chapter 3 Neisseria meningitidis ........................................................ 12
3.1 Carriage and invasive disease ......................................................... 12
3.2 Virulence factors ............................................................................. 13
3.2.1 LOS ..................................................................................................... 13
3.2.2 NhhA ................................................................................................... 14
3.3 Macrophage activation upon meningococcal infection ................... 14
Chapter 4 Mycobacteria ........................................................................ 17
4.1 Carriage and disease ........................................................................ 17
4.2 Mycobacteria ................................................................................... 18
4.3 Mycobacteria and macrophage interaction ..................................... 19
4.3.1 Bacterial recognition and internalization ............................................ 19
4.3.2 Intracellular survival of M. tuberculosis in macrophages ................... 20
4.3.3 Cell death of macrophages during M. tuberculosis infection .............. 21
4.3.4 Granuloma formation .......................................................................... 23
AIMS OF THE STUDY .............................................................................. 25
RESULTS AND DISCUSSION ................................................................. 26
FUTURE PERSPECTIVE ......................................................................... 32
ACKNOWLEDGEMENTS ........................................................................ 34
REFERENCES ............................................................................................ 36
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INTRODUCTION
Chapter 1 Macrophages and bacterial infection
Bacteria, as the dominant life form on Earth, inhabit every ecological niche
including the human body [1]. Although most bacteria are harmless or beneficial commensals co-evolving with the host, some are pathogenic [2].
Mammalian innate immunity mediated by phagocytes such as neutrophils,
macrophages, and dendritic cells (DCs) provides the first barrier protecting
the host from bacterial infection [3]. Macrophages were first described by
Elie Metchnikoff in 1893 for their phagocytic feature during tissue inflammation [4]. Today there is a broad understanding that macrophages are heterogeneous populations with versatile tissue-specific or niche-specific functions [5]. These functions range from maintaining dedicated tissue homeostasis through the clearance of apoptotic cell debris or iron processing to
responding to infection through ingesting and eliminating pathogens or
modulating inflammation by cytokine production [4]. In addition to their
central roles in innate immunity, macrophages bridge innate and adaptive
immunity by collecting and presenting antigens [6]. In response, bacteria
manipulate host defense mechanisms to benefit their own survival, replication, or transmission [5].
1.1 Bacterial recognition
Host cells express a range of microbial sensors called pattern recognition
receptors (PRRs) to recognize pathogen-associated molecular patterns
(PAMPs), transduce signals, and activate the immune system [3]. Toll-like
receptors (TLRs) are phylogenetically conserved receptors usually expressed
on monocytes, macrophages, and DCs that recognize PAMPs [7]. Ten TLRs
have been identified in humans and 13 in mice [3]. TLR3, 7, 8, and 9 are
localized in the cytoplasm and mainly detect microbial nucleic acids. The
other TLRs reside on the surface of the plasma membrane; of these, TLR2
and TLR4 are the best defined [7]. TLR4 binds lipopolysaccharide (LPS) [8]
and heat shock protein (Hsp) 60 [9], whereas TLR2 recognizes a broad range
of PAMPs, including LPS, lipoteichoic acids (LTA), lipoproteins, lipomannans, liparabinomannans, and peptidoglycan [10]. By forming heterodimers
with other TLRs, TLR2 can extend the specificities in ligand recognition.
For instance, TLR2/1 dimers recognize triacylated lipoproteins, whereas
TLR2/6 dimers detect diacylated lipoproteins [10]. The other TLRs are believed to function as homodimers [11].
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Activation of TLR signaling is initiated with the recruitment of adaptor proteins via the cytoplasmic Toll/interleukin (IL)-1 receptor (TIR) domain. Four
adaptor proteins have been identified to be involved in TLR signaling, including myeloid differentiation primary response protein 88 (MyD88), TIR
domain-containing adaptor protein (TIRAP), TIR domain-containing adaptor
protein inducing IFN-β (TRIF), and TRIF-related adaptor molecule (TRAM)
[11]. The MyD88-dependent pathway is utilized by all TLRs expect TLR3
[12]. Upon stimulation with PAMPs, MyD88 associates with TIRAP and
recruits IL-1 receptor-associated kinase-4 (IRAK-4), which activates IRAK1 by phosphorylation [13]. Subsequently, IRAK-1 and IRAK-2 associate
with TRAF6 and promote its ubiquitination [14]. The ubiquitinated TRAF6
forms a complex with TAK1 and activates the IKK complex, leading to NFκB activation, or activates MAP kinase (JNK, p38, ERK) to trigger AP-1
activation [14]. Activation of these nuclear factors results in the production
of a series of proinflammatory cytokines such as TNF, IL-12, IL-23, and IL1 [11]. To achieve full ligand sensitivity, other co-receptors are sometimes
essential for TLR signaling activation. For instance, the recognition of LPS
by TLR4 requires LPS-binding protein (LBP) association with MD2 and
CD14 on macrophages [15]. In addition to the MyD88-dependent pathway,
TLR4 ligands can utilize the other two adaptors, TRAM and TIRF, to trigger
IRF3 activation and type I interferon production in a MyD88-independent
manner [16].
Apart from TLRs, many other PRRs play important roles in bacterial recognition. The mannose receptor (CD206) primarily presents on the surface of
macrophages and immature DCs, recognizes repeated mannose residues
presented on pathogens, and mediates phagocytosis [17]. Macrophage scavenger receptors (including SR-AI, SR-AII, MARCO, CD36, and CD163)
sense low-density lipoprotein, LPS, and LTA and promote removal of apoptotic cells by phagocytosis [18]. The nucleotide oligomerization domain
(NOD)-like receptors (NLRs) are intracellular PRRs containing leucine-rich
repeats in the C-terminal region. They recognize a range of pathogens in the
cytoplasm and trigger the production of inflammatory cytokines such as IL1β [19]. NOD1 and NOD2 are well-described NLRs that bind to the peptidoglycan components meso-diaminopimelic acid and muramyl dipeptide,
respectively, and synergize with TLR activation [20].
1.2 Macrophage polarization
Macrophages are highly heterogeneous immune cells. Traditionally, a binary
classification based on inflammatory states is used to define macrophage
subgroups when they are stimulated in polarizing conditions. Depending on
the distinct microenvironmental signals, macrophages can be polarized into
classically activated macrophages (M1) or alternatively activated macro2
phages (M2) [21]. M1, typically induced by IFN-γ or LPS, are efficient at
killing microbes by producing nitric oxide, reactive oxygen species, and
lysosomal enzymes [22]. They can also secret abundant proinflammatory
mediators such as TNF, IL-12, and IL-1, which is essential for controlling
acute infectious diseases [23]. Activated M1 become efficient APC cells
through expressing increased levels of MHC II molecules and costimulatory
molecules such as CD80/86 to trigger Th1 and Th17 cell differentiation. Th1
cell-attracting chemokines such as CXCL9 and CXCL10 are also expressed
by M1 [22]. M2 display anti-inflammatory functions and high phagocytic
capacity, which are associated with tissue remodeling and anti-parasite defense [24]. Correlates of the M2 phenotype include the production of IL-10,
arginase, CD206, CD163, YM1, FIZZ1 (in the mouse), CCL17, and CCL22
[24]. M2 are further classified into at least three polarization groups: M2a is
triggered by Th2 cytokines such as IL-4 and IL-13; M2b is induced by LPS
and immune complexes or IL-1R ligands; M2c is generated by stimulation
with IL-10, TGF-β, and glucocorticoid hormones [22]. In addition to M1 and
M2 subtypes, there are tumor-associated macrophages involved in either
pro-tumor or anti-tumor immunity [25]. Since the surface marker expressions display large overlaps, detecting specific gene expression profiles is a
useful approach to distinguish macrophage subsets. For instance, the transcriptional factor IRF5 directly activates IL-12 and inhibits IL-10 gene encoding, which is taken as a M1 marker [26]. In contrast, IRF4 competes with
IRF5 for binding to MyD88 and is required for regulating M2 polarization
[27].
However, although such dichotomous classifications might reflect the extreme states, they cannot represent the complex in vivo environment for wide
variations in the transition or distinct states. The real host environment contains a range of cytokines and growth factors, whose cooperation or antagonism could result in many more functional macrophage phenotypes. For
instance, several bacteria or bacterial components such as Haemophilus ducreyi [28], Helicobacter pylori [29], and meningococcal adhesin Neisseria
hia/hsf homologue A (NhhA) (Paper II) have been found to manipulate
macrophage polarization to a specific phenotype with a mixture of M1/M2
features. The transformation between M1 and M2 has also great impact on
the pathogenesis of many infectious diseases [30]. Endotoxin tolerance is a
representative example wherein the initial M1 phenotype in sepsis patients
changes to an immunosuppression or anti-inflammatory phenotype [31].
1.3 Resident and inflammatory macrophages
Macrophages are present in every organ system as resident tissue macrophages to maintain tissue homeostasis by surveiling signals from the local
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environment [4]. They are given specific names depending on the locations,
for instance, the Kupffer cells in the liver, alveolar and interstitial macrophages in the lungs, sinus histiocytes in the spleen and lymph nodes and
microglial cells in the central nervous system [32]. Transcriptional profiling
analyses showed that resident tissue macrophages have high heterogeneity
and minimal overlaps in transcriptional patterns, which are associated with
distinct functions [33]. Monocytes formed in the bone marrow and blood
circulation replenish a large pool of cells potentially differentiating to macrophages and DCs in response to environmental cues during infection or in
the steady state [4]. The general paradigm was that all tissue macrophages
are end cells derived from the monocytic progenitor in bone marrow. However, new studies proved that monocyte-differentiated resident macrophages
are only one of the lineages during homeostatic adaptions [32]. The major
tissue macrophage populations actually originate from the yolk sac. These
embryo-derived resident macrophages can persist throughout life and undergo proliferation [32].
In addition to their tissue-specific homeostatic functions, resident macrophages can contribute to initial inflammation in response to tissue injury or
infection [34]. For instance, during acute infection in the peritoneal cavity,
the resident peritoneal macrophages recognize the pathogens and produce a
series of cytokines and chemokines to help eliminate the invaders [34].
However, the numbers of those macrophages are rapidly lost thereafter,
which is referred as “disappearance reaction” [35]. Inflammatory monocytes
are then recruited from the circulation by CCL-2 and then differentiate into
M1-like macrophages, which are also called inflammatory macrophages
[36]. These processes were also observed in meningococci-infected murine
peritoneal cavity (Paper II). To neutralize the excessive inflammatory response, macrophages can undergo apoptosis or switch into immune homeostatic M2 phenotypes, which produce anti-inflammatory mediators and promote the clearance of apoptotic cells [37]. Intriguingly, the lost resident macrophages group can be restored gradually by the supplement of monocytederived cells, in situ proliferative cells, or cells derived from dedicated precursors [38].
1.4 Factors governing functional differentiation of macrophages
Monocytes are highly plastic and express various receptors that sense environmental stimuli and mediate their differentiation into macrophages or DCs
[39]. Generally, M1 and M2 can be preferentially derived from CCR2hi
Ly6C+ inflammatory monocytes and CCRlow Ly6C- resident monocytes, respectively [37]. The hemopoietic growth factors macrophage colonystimulating factor (M-CSF) and granulocyte-macrophage colony-stimulating
factor (GM-CSF) are the central factors driving monocyte/macrophage dif4
ferentiation and proliferation [40]. M-CSF is constitutively expressed and
released in the serum and recognizes the CSF-1 receptor (CSF-1R) presented
on monocytes or macrophages to induce their development and proliferation
[40]. GM-CSF plays an important role in monocyte/macrophage development, especially in the lung and peritoneal cavity in vivo [37]. GM-CSF
stimulation results in M1-like inflammatory polarization, whereas M-CSF
stimulation promotes macrophage differentiation into the homeostatic or
anti-inflammatory M2-like phenotype [41]. In the presence of IL-4, GMCSF can induce monocyte differentiate into DCs [42]. Recombinant M-CSF
and GM-CSF are hence extensively utilized in generating murine and human
monocyte-derived macrophages or DCs in vitro [41].
Many other cytokines have been shown to regulate macrophage differentiation. Endogenous IL-15 produced via TLR activation can promote monocyte
differentiation into macrophages [43]. IL-32 can trigger monocyte differentiation into macrophages with both M1 and M2 phenotypes or CD1b+ DC-like
cells [44,45]. IL-10 blocks the formation of DCs [46] and guides macrophage differentiation into an M2-like phenotype [47]. IL-6 can switch monocyte differentiation from DCs to macrophages via promoting the expression
of M-CSF receptors [48]. Furthermore, IL-12 combined with IL-18 induce
the formation of macrophages from monocytes [49].
1.5 Macrophage cell death in bacterial infections
In response to infectious stimuli, macrophages can be tightly regulated and
undergo programmed cell death such as apoptosis, which may restrict bacterial growth and eliminate the replicative niches for some intracellular pathogens [50]. However, the crafty pathogens can modulate macrophage cell
death pathways for their own benefit. Passive and uncontrolled cell death
such as necrosis can be pathogenic and lead to tissue damage and increased
bacterial dissemination [51]. Host cell death modes are hence important for
tissue homeostasis and immune regulation.
Apoptosis is a classical programmed cell death process, which occurs without triggering inflammatory responses [52]. The morphological features of
apoptosis include preserved plasma membrane integrity, cell shrinkage,
chromatin condensation, and nuclear fragmentation [52]. Apoptotic cells are
subsequently taken up by neighboring uninfected macrophages through efferocytosis [53], which avoids damage to surrounding cells by the leaked
apoptotic debris. Caspases, including the initiator caspases (capsase-2, -8, -9,
-10) and the effector caspases (caspase-3, -6, -7), are the central apoptotic
regulators [54]. Activation of a caspase cascade can be triggered by the
TNF- or FasL-mediated extrinsic pathways or the mitochondria-associated
intrinsic pathways [54].
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Necrosis is a different cell death mode associated with the loss of cell membrane integrity and uncontrolled excretion of inflammatory mediators [55]. It
is morphologically characterized by nuclear and cellular swelling and disorganized DNA fragmentation without chromatin condensation [56]. The necrotic process was conventionally thought to be passive and accidental;
however, recent evidence showed that necrosis could also occur in a programmed fashion. Necroptosis is caspase-independent programmed necrotic
cell death, which is mediated by the phosphorylation of RIPK1/3 and pseudokinase MLKL [57]. Signaling activation leads to calpain activation, lysosome destabilization, cathepsin release, and ROS production, which subsequently result in necrotic cell death [57]. In contrast to apoptosis, necroptosis
is highly inflammatory and leads to release of cellular content [55]. This cell
death mode can harm the host due to the failure in controlling bacterial dissemination, which has been shown in Salmonella enterica [58] and mycobacterial infection [59].
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Chapter 2 CD46
CD46, also known as the membrane cofactor protein (MCP), is a pleiotropic
multifunctional type I transmembrane protein ubiquitously expressed on all
nucleated human cells. It was primarily identified as co-regulator of complement activation [60] and later as a pathogen magnet for binding to a range
of microbes [61] and an immune regulator involved in modulating the adaptive immune response [62]. The broad spectrum of CD46 functions can be
attributed to the high structural heterogeneity of the protein [60] (Figure 1).
Various isoforms of CD46 resulting from alternative splicing have been
identified. As a transmembrane protein, CD46 has an extracellular domain
comprising four short consensus repeats (SCR1–4) and glycosylated region
B and/or C rich in serine, threonine, and proline (STP). One of two cytoplasmic tails, CYT-1 or CYT-2, is connected to the extracellular portion
through a transmembrane region [60]. CYT-1 and CYT-2 interact with effector proteins and appear to respond differentially to tyrosine phosphorylation
to mediate distinct T cell activations [63]. A variety of diseases from chronic
diseases such as multiple sclerosis [64], rheumatoid arthritis [65], and asthma [66] to acute infections such as bacterial sepsis [67,68] are associated
with deficiency or activation of CD46 function, indicating the importance of
CD46 in maintaining human health.
Figure 1. Structure of CD46. (Adapted from [60])
2.1 CD46 as a complement factor
The complement system is an ancient innate defense mechanism associated
with a biochemical cascade that assists in killing pathogens, tagging patho7
gens by opsonization, and recruiting inflammatory cells to eliminate the
marked microbes [69]. However, complement activation can cause host
damage if it fails to distinguish between self-cells and enemies. Hence, the
complement system must be tightly regulated, normally by a number of serum and membrane-bound complement regulatory proteins [70].
During the screen for novel C3b-binding proteins, CD46 was discovered as a
complement inhibitor in 1986 [71]. Indeed, individuals harboring mutations
in CD46 are predisposed to atypical hemolytic uremic syndrome (aHUS), a
disease associated with complement dysregulation [72]. The N-terminal
extracellular SCR domains of CD46 harbor the binding sites for complement
components. CD46 binds to C3b and C4b, leading to their proteolytic cleavage by serine protease factor I into the fragments C3bi and C4c/C4d, respectively [73]. The cleavage products are inactivated and incapable of continued
complement activation. This process irreversibly prevents the convertasemediated formation of C3 and C5 and protects host cells from autologous
complement attack [74]. This discovery has provided significant insights
into the treatment of patients with complement dysregulations.
2.2 CD46 as a pathogen receptor
Beyond complement regulation, CD46 also serves as an entry receptor for
various human bacterial and viral pathogens, whose ligations promote pleiotropic downstream signaling activations and actions [75]. For instance, the
viral hemagglutinin of measles virus (MV) binds the extracellular domain
SCR1 and SCR2 of CD46 [76], and the fiber knob of adenovirus interacts
with CD46 SCR2 [77]. SCR2 and SCR3 interact with human herpesvirus-6
(HHV-6) [78] and SCR3 is a binding site for Neisseria gonorrhoeae as well
[79]. M protein is a streptococcal ligand for CD46 [80]. In addition, complement-opsonized uropathogenic Escherichia coli exploit the C3b binding
site of CD46 to infect renal tubular epithelial cell [81]. Interactions between
CD46 and certain pathogens can modulate different cellular responses,
which will be discussed in the following section.
CD46 expression is tightly regulated, and several pathogens can downregulate cellular levels of CD46 via different mechanisms such as shedding or
internalization [68,82-84]. On epithelial cells [85] and T cells [86], the extracellular domain of CD46 can be cleaved by metalloproteinases and shed
into the surrounding milieu, and its intracellular tails can be cleaved by the
presenilin-gamma secretase enzymatic complex. Streptococcus pyogenes can
trigger CD46 shedding from the epithelial cell surface, which is associated
with apoptotic cell death [68]. Piliated Neisseria gonorrhoeae also binds to
epithelial cells and induces shedding of the CD46 ectodomain in a metalloproteinase-dependent manner [83], whereas cross-linking CD46 by MV in8
duces a macropinocytosis-like process, leading to the internalization and
degradation of CD46 in both lymphoid and non-lymphoid cells [87]. The
downregulation of CD46 may associate with enhancing complement sensitivity of infected host cells and affect CD46-dependent antigen presentation
and signal transduction [87].
2.3 CD46 as an immune regulator
CD46/CD3 costimulation regulates TCR signaling and induces T cell proliferation and activation, which was found as a breakthrough in 2001 [88].
Since then, extensive studies have focused on unraveling the role of CD46 in
T cell immunity. Subsequent work showed that CD46 functions as a costimulatory molecule of TCR, inducing differentiation or activation of IL10-secreting Tr1 cells, a subset of regulatory T cells (Tregs) [89]. The immunoregulatory property of CD46 has also been supported by several infection
studies. HHV-6 and MV interact with CD46 to suppress macrophage production of the Th1 cytokine IL-12 [90,91]. The MV-CD46 interaction also
leads to an immunosuppressive response of DCs with deceased IL-12 production and diminished antigen-presentation capacity [71]. Streptococcal M
protein activates CD46 and promotes granzyme B expression, leading to Tr1 like cell polarization [80]. The engagement of CD46 on CD4+ T cells also
leads to Tr-1 like regulation in response to mycobacterial infection [92].
However, these findings that CD46 negatively controls adaptive immunity
were unexpected because immunosuppression can harm the host during infection, which contradicts the protective role of CD46 as a complement
regulator. Subsequently, it was demonstrated that CD46 engagement via C3b
on CD4+ T cells during antigen presentation promotes an IFN-γ-secreting
Th1 response in the presence of low IL-2 concentration [93]. IL-2 concentrations in the milieu appear to be critical to the outcome of CD46-driven T cell
responses. CD46/CD3 activation can actually initially promote the generation of proinflammatory IFN-γ+ cells, and with high IL-2 concentration from
an exogenous source, Th1 cells can switch into IL-10-producing Tregs [65].
Jagged1, a Notch family member, was shown to be critical for driving Th1
immunity through the ligation with CD46 [94].
CD46 hence regulates both proinflammatory and immunoregulatory T cell
responses. Accordingly, patients deficient in CD46-dependent Th1 responses
suffer from recurrent infections [94], whereas impaired CD46-mediated Tr1
differentiation has been found in patients with autoimmune diseases such as
multiple sclerosis, asthma, and rheumatoid arthritis [65,66,95]. In addition,
around 30% of CD46-deficient patients develop a syndrome called common
variable immunodeficiency (CVID) [96]. These associations highlight the
important role of CD46 in regulating human adaptive immunity.
9
Despite the sporadic studies in macrophages and DCs mentioned above, the
role of CD46 in innate immunity remains ambiguous. IL-12 downregulation
has been observed in MV-infected monocytes and macrophages and in
HHV-6-infected macrophages [90,91]. However, enhanced IL-12 production
has also been reported on macrophages by crosslinking their CD46 with
several MV strains [97]. The distinct regulations might be dependent on the
macrophage maturation stages and the multiple signaling pathways of CD46.
Indeed, mouse macrophages transfected with human CYT-1 CD46 show
higher production of nitric oxide, an infection-fighting agent produced in
response to MV infection in the presence of IFN-γ, whereas macrophages
expressing CYT-2 CD46 show reduced nitric oxide production [98]. In addition, CD46 can induce autophagy of epithelial cells upon recognition of
pathogens including MV and group A Streptococcus via the CD46-Cyt-1GOPC pathway, which enables control of early pathogen infection [99].
The function of CD46 on neutrophils, monocytes, mast cells, and NK cells is
hitherto unknown. Crosstalks between CD46 and TLR, NLR, or RIG-1 likely exist but remains unexplored. Future studies are needed to fill these important gaps and expand our understanding of the precise and finely tuned
roles of CD46 as an immune regulator.
2.4 CD46+/+ transgenic mice as an animal disease model
Several pathogenic ligands of CD46 including MV and Neisseria are humanspecific; thus, studying their pathogenesis in vivo is challenging [100]. Although the murine CD46 protein exists, it is selectively expressed in spermatids and shares only 45% identity with human CD46 [101]. Mice express a
protein named crry/p65, which is a functional homologue to human CD46 in
some complement regulatory features but not in T cell activation [101].
Hence, a transgenic mouse model universally expressing human CD46 was
generated to study the pathogenesis of MV infection in 1996 [102], and similar models have been developed subsequently to investigate a range of other
infections [65,68,103-105]. The CD46 transgenic mice are highly susceptible
to MV, Neisseria meningitidis, Streptococcus pyogenes, and Streptococcus
dysgalactiae [65,68,102,105]. In the case of meningococcal infection, CD46
transgenic mice show more severe bacterial sepsis with higher proinflammatory cytokine production and more efficient bacterial crossing of the bloodbrain barrier than wildtype (WT) mice [67,106].
We have discussed that the various isoforms of CD46 lead to different signaling pathways. The two CD46 cytoplasmic tails, CYT-1 and CYT-2, are
expressed in most human cell types, with the exception of preferential CYT2 expression in the brain, kidneys, and testes [65]. To study the functional
10
differences between the two intracellular tails of CD46, transgenic mice
specifically expressing either CYT-1 or CYT-2 were generated and used to
investigate the T-cell dependent inflammatory reaction [63]. The two cytoplasmic tails exhibited divergent roles: CD46 CYT-1 activation inhibits inflammation, whereas CYT-2 activation enhances it [63]. These transgenic
mouse models can also be useful tools in further studies, including examination of how the different isoforms of CD46 modulate innate immunity.
11
Chapter 3 Neisseria meningitidis
3.1 Carriage and invasive disease
The Genus Neisseria include at least 25 species identified based on 16S
rRNA technique [107]. Neisseria meningitidis (meningococcus) and Neisseria gonorrhoeae (gonococcus) are obligate human pathogens, whereas the
other strains are either opportunistic human pathogens or commensal species
colonize humans and other animals [107].
N. meningitidis is a gram-negative aerobic diplococcus that could be carried
asymptomatically in the nasopharynx by approximately 10% of the population [108]. Since humans are the only known hosts, carriers are thought to be
the major source of disease outbreak [108]. Once the bacteria penetrate the
mucosal membrane and enter the bloodstream, they can cause various diseases. The most common manifestation is meningitis, but meningococcal
septicemia has higher mortality rate [109]. N. meningitidis causes approximately 500,000 cases per year in the world and up to 50,000 deaths [110].
Without treatment, mortality can be as high as 70–90% among patients with
meningococcal disease [111]. Because of the risks of severe morbidity and
lethality, timely antibiotic therapy should be initiated in patients suspected of
having meningococcal disease. A third-generation cephalosporin, Penicillin
G, and chloramphenicol are the drugs of choice [107]. Despite the availability of antibiotics, case-fatality rates are still high at approximately 8–15%
due to the rapid onset of disease [112]. About 10–20% of survivors suffer
long-term sequelae including mental retardation, hearing loss, and loss of
limb use [111].
N. meningitidis is divided into 13 serogroups based on the immunological
reactivity of their capsular polysaccharides [113]. Serogroups A, B, C,
W135, X, and Y cause virtually all associated invasive diseases [113]. Most
cases in the western world are attributed to serogroups B and C, with
serogroup B dominant, whereas serogroup A contributes to the majority of
epidemic infection in the ‘African meningitis belt’ [114]. Effective polysaccharide vaccines are available against types A, C, W135, and Y but not type
B meningococci since the capsular polysaccharide of these bacteria contains
(2→8)-α-Neu5Ac, which is a self antigen [115]. Multicomponent meningococcal vaccine (Bexsero®) containing conserved protein components has
been developed in recent years and showed good protection against meningococcal B strains in Europe [116] [117]. The major components in these
novel meningococcal vaccines are Neisseria adhesion A (NadA), factor Hbinding protein (fHbp), Neisseria heparin-binding protein A (NhbA), and
outer membrane vesicles (OMVs) derived from a meningococcal strain
(NZ98/254) [117]. Despite great promise of new vaccines, cases of menin12
gococcal infection continue to be reported in both developed and developing
countries due to the lack of universal vaccine coverage and increasing antibiotic resistance [116].
N. meningitidis colonizes the mucosal surface via a wide range of adhesins
including pili, LOS, and Opa [107]. Around 50% of isolated carriage strains
lack a capsule, which might enhance bacterial colonization in the human
nasopharynx [118]. Biofilm formation is another strategy that protects bacteria from exposure to host bactericidal components such as IgA, IgG, and
antimicrobial peptides [119]. In addition, N. meningitidis can trick the human IgA-mediated defense by producing numerous OMVs [120]. Acquisition of nutrients, especially lactate and iron, is essential for bacteria to sustain colonization in the nasopharynx. Since the host environment lacks free
iron, N. meningitidis has developed a specific mechanism to obtain iron by
binding to host iron-transport proteins, thus allowing the bacteria to survive
in an optimized position [121].
Imbalance of immune homeostasis in the mucosal microenvironment leads
to increased virulence and provokes invasive growth of bacteria. For instance, it has been shown that N. meningitidis can uptake proinflammatory
cytokines IL-8 and TNF in a Type IV pili-dependent manner, which results
in upregulation of bacterial virulence factors [122]. Increased ambient temperature can also act as a “danger signal,” enhancing expression of virulence
genes and provoking invasive bacterial growth [123].
3.2 Virulence factors
N. meningitidis expresses a range of virulence factors that allow pathogenic
strains to colonize the nasopharyngeal mucosa, invade into the blood stream,
or cross the blood-brain barrier. These virulence factors include pili, capsular
polysaccharide, lipooligosaccharides (LOS), and a number of surface membrane proteins such as porins, opacity proteins, NadA, App, MspA, and
NhhA [124].
3.2.1 LOS
One of the most important virulence factors of Neisseria is endotoxin LOS.
LOS is composed of a membrane-bound lipid A, a core oligosaccharide, and
a polysaccharide O-antigen. It is referred as LOS since it lacks repeating
polysaccharide O-antigens in contrast to the LPS of enteric bacteria [125].
LOS initiates the inflammatory response by binding to TLR4 with the lipid
A moiety [126]. The outcome of meningococcal septic shock correlates with
the level of LOS in circulation [127]. In addition, LOS is involved in other
pathogenic processes such as cell adhesion, immune evasion, and serum
resistance [128,129]. Antigenic variation is commonly exhibited in LOS,
13
aiding bacterial evasion of the host immune response [130]. Modifications of
LOS with sialic acid in serogroups B, C, W-135, and Y allow the bacteria to
resemble host cell surfaces that also express sialic acid, resulting in higher
resistance to antibody, complement, or PMN-mediated killing [131,132].
3.2.2 NhhA
NhhA (57 kDa) is a multifunctional trimeric autotransporter adhesin expressed by N. meningitidis and N. lactamica but absent in N. gonorrhoeae
[133]. It was first identified for its 47% similarity to the adhesin AIDA-I of
E. coli and thereafter defined by its close homology to the Hia and Hsf adhesins of H. influenza [134]. It is also called meningococcal surface fibril
(Msf) based on its sequence similarity to Haemophilus surface fibril (Hsf)
[135]. As a typical autotransporter protein, NhhA contains three modular
parts: a C-terminal translocator domain, an N-terminal signal domain, and a
central passenger domain. The C-terminal 72 residues of NhhA are responsible for trimerization and translocation of the passenger domain on the bacterial surface [133].
NhhA plays a variety of roles during meningococcal infection including
interacting with activated vitronectin, which leads to enhanced bacterial
complement resistance [135,136]. Purified NhhA binds laminin and heparan
sulfate, components of extracellular matrix, which may enhance bacteriahost cell interaction [133]. A previous study in our group showed that NhhA
triggers macrophage apoptosis through caspase activation [137]. The role of
NhhA in mediating bacterial colonization of the nasopharyngeal mucosa,
evasion from phagocytosis, and complement-mediated killing has been
demonstrated in vivo in a mouse disease model [136]. NhhA is a conserved
protein expressed in the vast majority of disease-associated strains, although
its expression levels vary. Because it can induce bactericidal antibodies,
NhhA has been considered a potential vaccine candidate against meningococcal disease [138].
3.3 Macrophage activation upon meningococcal infection
Meningococci interact with a series of immune effector cells during colonization and invasion. Immune cells residing in the epithelium barrier of the
human nasopharynx represent the first line of host protection from bacterial
invasion [111]. Macrophages and DCs are the dominant cell types modulating the immune responses at this steady state [139,140]. Once bacteria enter
circulation, neutrophils become the predominant cell type that they encounter with, especially during the acute phase of infection [141]. Monocytes/macrophages play important roles in eliminating pathogens, maintaining environmental homeostasis, and initiating adaptive immunity because of
their functional heterogeneity [142].
14
Interaction between meningococci and the myeloid cells is a crucial step in
initiating immune activation. A recent study showed that whole N. meningitidis binds to galectin-3, increasing the interaction between bacteria and
monocytes/macrophages [143]. Scavenger receptors are involved in recognizing meningococci and mediating phagocytosis [144]. The Class A scavenger receptor on macrophages can bind to meningococcal proteins
NMB1220, NMB0278, and NMB0667 and enhance bacterial uptake [145].
Macrophage receptor with a collagenous structure (MARCO) is another type
of scavenger receptor that can bind N. meningitidis and induce innate activation [146]. Sialic acid-interacting Ig-like proteins expressed on myeloid cells
bind to meningococcal LOS, leading to enhanced macrophagocytosis of
bacteria [147]. Type IV pili binding to serum C-reaction protein results in
increased opsonin-dependent phagocytosis by macrophages and neutrophils
[148].
A large panel of surface components on N. meningitidis can activate macrophages to generate inflammatory mediators. Lipid A on LOS interacts directly with MD-2 to activate TLR4, leading to cytokine production by human
macrophages [149]. Although LOS is considered the major virulence factor,
an LOS-deficient N. meningitidis mutant can still induce the proinflammatory response in immune cells, suggesting that other surface proteins also play important roles during infection [150]. NadA targets human
monocyte/macrophages, induces formation of the Hsp90/Hsp70/TLR4 complex, and stimulates pro-inflammatory cytokine production [151]. PorB activates macrophages by directly binding to the TLR1/TLR2 complex in a
MyD88-dependent manner and inhibits apoptosis [152]. Capsular polysaccharides can trigger the immune response through TLR2- and TLR4-MD2
pathways in macrophages [153]. NhhA can induce a series of proinflammatory cytokines in macrophages through TLR4-dependent and -independent
mechanisms (Paper I). In addition to TLR2 and TLR4, TLR9 is an intracellular receptor that recognizes unmethylated CpG motifs on bacterial DNA
and contributes to the activation of bactericidal activity [154].
Appropriate amounts of pro-inflammatory mediators are beneficial for the
host through activation of neutrophils and attraction of Th1 and Th17 cells
during bacterial infection. However, excessive production of those cytokines
such as TNF and IL-1 can be pathogenic and lead to septic shock and organ
failure [34]. Several studies have shown that high levels of cytokines including IL-6, IL-8, TNF, and IFN-γ are associated with the severity and mortality
of meningococcal sepsis [155]. N. meningitidis adopts a nitric oxide detoxification mechanism to inhibit macrophage apoptosis, and the prolonged survival of macrophages might be associated with a high level of inflammatory
response and harm to the host [156]. Some host factors expressed on macrophages, including mannose-binding lectin (MBL) [157]and the scavenger
15
receptor SR-A [158], are reported to be host-protective during meningococcal septicemia, whereas human CD46 contributes to the susceptibility to
meningococcal infection (Paper III).
Although meningococci have a well-known capacity to induce the Th1-type
proinflammatory response [128,137,159], there is some evidence of negative
immune cell regulation by this bacterium. For example, CD200, an immune
inhibitory ligand expressed on macrophages, can be induced via the TLR
and NLR pathways upon meningococcal infection, thereby restricting macrophage activation and protecting the host from septicemia [160]. Nevertheless, how meningococci asymptomatically colonize the nasopharynx and
limit activation of the host inflammatory response remains largely unknown.
Toward addressing this lack, in Paper II, we demonstrated that the meningococcal adhesion NhhA programs a type of immune homeostatic macrophage, which potentiates meningococcal nasopharyngeal carriage.
16
Chapter 4 Mycobacteria
4.1 Carriage and disease
Tuberculosis (TB) is a bacterial infection caused by Mycobacterium tuberculosis that affects 9.6 million people and leads to 1.5 million
casualties annually, with most cases found in developing countries [161].
According to a WHO estimation, 28% of the world’s TB cases occur in Africa, and the three largest TB epidemics are in India, Indonesia, and China
[161]. HIV infection, diabetes, malnutrition, and smoking are the leading
risk factors for increased individual susceptibility to TB [162].
About one-third of the global population has been infected due to the highly
contagious feature of M. tuberculosis, but most people manage to mount a
sufficient immune response to eradicate the bacteria or keep the infection in
an asymptomatic state (also called latent TB infection, LTBI) [163]. Around
10% of exposed individuals will develop clinical disease during their lifetimes [162]. TB infection begins with the inhalation of the aerosol droplets
containing virulent mycobacteria [164]. Once the bacteria enter the lungs,
they primarily infect resident alveolar macrophages, confront the hostile
environment within the macrophages, and attempt to use them as survival
niches. After successful replication in the macrophages, M. tuberculosis can
trigger the host cell death to escape and infect newly recruited cells [164]. In
addition, infected DCs migrate to the peripheral lymph nodes to activate the
T lymphocyte response but simultaneously abet bacterial dissemination
[165]. M. tuberculosis most commonly infects the lungs and leads to clinical
symptoms such as night sweats, bloody coughs, and weight loss [162]. The
bacteria can also spread into extrapulmonary organs through hematogenous
transmission and cause other diseases including pericarditis, meningitis, or
spinal TB, especially in immunosuppressed patients and young children
[166].
M. bovis Bacillus Calmette–Guérin (BCG) is a live attenuated mycobacterial
organism that has been use as a TB vaccine worldwide since 1921 [167].
The effectiveness of the BCG vaccine varies from no protection to 70–80%
protection, with higher efficacy in children and lower efficacy in adults
[168]. Fortunately, TB is curable by using anti-TB drugs including isoniazid,
rifampicin, ethambutol, and pyrazinamide with a success rate of 85% or
more [161,169]. However, multidrug resistance has become an increasing
obstacle to progress in global TB control [169]. Therefore, TB remains a
threatening infectious disease with the highest mortality worldwide despite
the use of live attenuated vaccine and antibiotics. A fundamental understanding of TB pathogenesis and host-mycobacteria interaction is essential for
developing more efficient vaccines and anti-TB agents.
17
4.2 Mycobacteria
M. tuberculosis, the main etiological agent of TB, is an ancient enemy of
humans first discovered in 1882 by Robert Koch [170]. The bacterium is an
aerobic, non-motile, slow-growing (divide every 15–20 h) bacillus [60].
Mycobacteria have a thick cell wall with a high lipid content, conferring the
bacteria many unique clinical characteristics [171]. Mycolic acids are the
major and specific lipid components of the mycobacterial outer membrane
and key virulence factors crucial for survival and pathogenesis of M. tuberculosis [171]. The low impermeability of the mycobacterial cell wall, mainly
attributed to mycolic acids, provides the bacteria resistance to most chemotherapeutic agents and hydrophobic antibiotics [172]. The presence of mycolic acids also distances the bacteria from the acid produced by the host’s
immune system and enables survival within macrophages [172]. Due to the
unique cell wall structure, M. tuberculosis is impervious to the crystal violet
of Gram staining. Instead, the Ziehl-Neelsen stain for detecting acid-fast
bacillus is used for histological examination of mycobacteria because the
waxy, complex cell wall avoids the decolorization by acids during staining
procedures [173].
Figure 2. GFP-M.bovis BCG internalized by murine macrophages.
M. tuberculosis is genetically diverse, and variations in strain phenotypes are
associated with clinical isolates from different geographic regions. TB outbreaks are often induced by hypervirulent strains such as Beijing strains
[174]. In the laboratory, a virulent strain H37Rv and an avirulent strain
H37Ra are commonly used as model strains to investigate the pathogenesis
of TB [175]. Although M. tuberculosis is the primary culprit of TB infection
in human, there exist other TB-causing mycobacteria such as M. bovis, M.
africanum, M. canetti, and M. microti [176]. The latter three species are rare
and mainly found in Africa [176]. M. bovis is a causative agent of bovine TB
but can cross the species barrier to infect humans and other mammals [177].
The well-known M. bovis strain BCG is not only adopted globally as a TB
vaccine but also widely used as a model system mimicking attenuated M.
18
tuberculosis strains since BCG and M. tuberculosis share 99.9% genetic
identity [178,179] (Figure 2). Extensive laboratory TB studies have been
performed by intranasal administration of M. bovis BCG in mice models.
The attenuated virulence of M. bovis BCG strains is attributed to the lack of
an RD-1 region, which is found in all virulent M. tuberculosis and M. bovis
strains [180]. The Rv3875 gene encoding the 6-kDa early secretory antigenic
target (ESAT-6) within the RD-1 locus is thought to associate with bacterial
virulence [181]. In addition, RD-1-deficient mycobacteria fail to trigger rapid and continuous migration of macrophages, which results in less expansion
of the bacterial infection [182]. In some rare conditions such as Mendelian
susceptibility to mycobacterial disease (MSMD), the immunosuppressed
patients can still be predisposed to the disease caused by BCG infection
[183].
Besides those human-pathogenic strains, M. marinum, a natural pathogen of
ectotherms, can promote a systemic TB-like disease in zebrafish, which have
also been developed as a TB model system, especially for granuloma studies
[184]. However, any extrapolation of data obtained from the non-human
mycobacterial strains and animal models should include extra caution because of the lack of human-specific immune factors and the absence of M.
tuberculosis-specific virulence factors such as the ESX-1 region and
PhoP/PhoR system [185].
4.3 Mycobacteria and macrophage interaction
4.3.1 Bacterial recognition and internalization
Macrophages are the first line of human host defense against M. tuberculosis
invasion in the lungs and the primary targets for M. tuberculosis replication
niches [186]. As an intracellular pathogen, M. tuberculosis recognition by
various receptors on macrophages leads to its internalization and initiation of
the infection process. The mannose receptors (CD206) expressed on macrophages bind to mannosylated motifs of virulent mycobacteria, which promotes the phagocytosis of bacilli without activating the proinflammatory
response [187,188]. M. tuberculosis can also activate the complement system and be opsonized by C3b and C3bi, which bind to the complement receptor CR1, CR3, or CR4 [188,189]. FcγR is presented on macrophages and
binds to IgG-opsonized M. tuberculosis, resulting in uptake of mycobacteria
and production of ROS and proinflammatory cytokines [190]. FcγRmediated phagocytosis is also associated with enhanced phagolysosomal
fusion, which might benefit the host during M. tuberculosis infection [191].
Furthermore, many other receptors are involved in the interaction between
M. tuberculosis and macrophages, such as CD14, scavenger receptors, DCSIGN, mannose-binding lectin, and dectin-1 [192].
19
Apart from those endocytosis-related PRRs, TLRs play an important role in
M. tuberculosis infection by triggering the intracellular signaling pathways
and initiating cytokine production, which are essential for modulating the
macrophage bactericidal capacity and linking innate immunity to adaptive
immunity. TLR2-deficient mice fail to form granulomas, and both TLR2and TLR4-defective mice show higher susceptibility to mycobacterial infection than WT mice [193,194]. A range of extracellular mycobacterial TLR
ligands have been identified, such as lipomannan and lipoarabinomannan
(TLR2 ligands) [192,195]; diacylated lipoprotein (TLR1/2 complex ligand)
[196]; triacylated lipoprotein (TLR2/6 heterodimers ligand) [197]; and tetraacylated lipomannan, Hsp65, and 50S ribosomal protein (TLR4 ligands)
[192,198]. Furthermore, mycobacterial DNAs containing CpG motifs are
recognized by intracellular receptor TLR9, leading to the production of proinflammatory cytokines such as IL-12p40 and IFN-γ [199]. The interaction
between M. tuberculosis and cytosolic NOD2 receptor is also associated
with the secretion of cytokines against M. tuberculosis infection [200,201].
4.3.2 Intracellular survival of M. tuberculosis in macrophages
Macrophage phagosome maturation through fusing with lysosomes, resulting in an environment containing lysosomal enzymes, reactive oxygen intermediates, toxic peptides and acid PH, which is detrimental for bacterial
growth [202]. The vacuolar H+/ATPase in the phagosomal membrane is responsible for achieving acidification through hydrolysation of ATP and
translocating H+ across the membrane [203]. IFN-γ can activated macrophages and enhance phagosomal maturation, along with increased reactive
nitrogen intermediates (RNI) production, which improves bactericidal activity especially in murine macrophage models [204]. The ESAT-6 of M. tuberculosis triggers NADPH oxidase-mediated ROS generation, which is an
agent assisting in the control of bacterial growth [205].
However, M. tuberculosis has developed specific strategies that inhibit the
maturation of phagolysosomes to contend with the macrophage defense.
This process can be achieved by preventing the fusion of late endosomes and
lysosomes, repressing the recruitment of vacuolar H+/ATPase to raise the PH,
or detaining the early endosome markers and coronin-1 [206]. For instance,
the mannose-capped lipoarabinomannan (ManLAM) of M. tuberculosis,
which is a mannose receptor ligand, is thought to be a major player in blocking the fusion of phagosomes and lysosomes [187]. Cholesterol, an essential
factor for the uptake of M. tuberculosis through the complement receptor, is
also involved in the inhibition of phagosomal maturation [207]. In addition
to blocking phagosome maturation, M. tuberculosis employs other strategies
for its survival. To counteract bactericidal agents, catalase-peroxidase KatG
and the mel2 locus of mycobacteria confer their resistance to ROS [208], and
M. bovis BCG can prevent iNOS recruitment to macrophage phagosomes
[209]. Furthermore, M. bovis BCG or mycobacterial lipoprotein can block
20
the expression of MHC II induced by IFN-γ on macrophages, thus preventing antigen presentation to trigger type I T cell immunity [210].
Autophagy is a relatively newly identified host defense mechanism for controlling the intracellular survival of mycobacteria [211,212]. Type III PI3K
hVPS34 and PI3P are essential factors for both phagolysosome formation
[213] and the autophagy pathway [211]. Induction of autophagy by IFN-γ
promotes maturation of mycobacterial phagosomes in a PI3K-dependent
manner, thus inhibiting the bacterial survival within macrophages [211].
These initial findings in 2004 were confirmed by several subsequent studies
in different contexts. Downregulation of the long noncoding RNA (lncRNA)
MEG3 by IFN-γ can enhance autophagy and bacterial eradication by regulating the mTOR and PI3K-Akt signaling pathway [214]. Induction of autophagy also promotes the delivery of ubiquitin-derived bactericidal peptides to
the lysosome and enhances the killing capacity [215]. Furthermore, the mycobacterial lipoprotein LpqH has been shown to activate vitamin D3 and
cathelicidin-dependent anti-mycobacterial autophagy via TLR2/1/CD14
[216]. ATP-mediated elimination of intracellular mycobacteria is also associated with activation of autophagy [217]. In contrast, Th2 cytokines including IL-4 and IL-13 abrogate autophagy-mediated killing of intracellular mycobacteria in macrophages via Akt signaling [218].
Nevertheless, M. tuberculosis remains a highly successful pathogen that is
hardly eradicated but merely controlled [219]. To improve the prevention
and treatment of TB, further investigations are needed to fully decipher the
mechanisms of mycobacterial survival especially in human macrophages,
which are more susceptible than murine macrophage models.
4.3.3 Cell death of macrophages during M. tuberculosis infection
Once M. tuberculosis succeeds in intracellular replication, the bacteria escape from the macrophages and induce disseminated infection by triggering
host cell death [220]. Thus, the fate of macrophages during mycobacterial
infection is critical for pathogenesis of TB. Upon M. tuberculosis infection,
the major outcomes of macrophages are apoptotic cell death, necrotic cell
death, and survival[220]. Necrosis is characterized by plasma membrane
disruption, which fails to control mycobacterial replication and dissemination [221]. Apoptosis is associated with preserved plasma membrane integrity and efficient bactericidal capacity and is thus considered a host defense
mechanism [221]. The different cell death modes are challenging to distinguish because of the many factors involved including the heterogeneity of
bacterial virulence and macrophages, variation in the number of internalized
bacteria, and asynchronous infection [220]. Generally, virulent M. tuberculosis strains such as H37Rv prefer to induce necrotic cell death in macrophages by inhibiting apoptosis to enhance their contagiousness [222]. Attenuated strains including M. bovis BCG and M. tuberculosis H37Ra predomi21
nantly trigger apoptosis [221]. However, when macrophages are infected
with attenuated mycobacteria at high MOI, initial apoptotic cell fate can turn
into necrosis accompanied by bacterial spread [223].
Apoptosis triggered by mycobacteria can be achieved through extrinsic and
intrinsic pathways. The extrinsic pathway can be induced by TNF as a death
receptor ligand in an autocrine or paracrine manner, followed by assembly of
the death-inducing signaling complex (DISC) and caspase-8 activation
[221]. The intrinsic apoptotic pathway is functionally associated with mitochondrial outer membrane permeabilization (MOMP) governed by a series
of Bcl-2 family proteins [220]. Upon M. tuberculosis infection, the antiapoptotic Bcl-2 protein is inhibited, and the pro-apoptotic proteins Bax and
Bak are activated by the effect of Bcl-2-homology 3 (BH3) proteins. Activated Bax and Bak oligomerize and form a channel through the mitochondrial membrane, which allows the release of apoptogenic proteins including
cytochrome c, SMAC/Diablo, and HtrA2/Omi into the cytosolic compartment [224]. Cytochrome c and apoptosis protease activating factor-1 (Apaf1) form the apoptosome complex and trigger the activation of caspase-9.
Downstream, activation of both caspase-8 and caspase-9 leads to activation
of the executioner caspase-3, which directly triggers apoptotic cell death
[220]. Notably, the intrinsic pathway can be amplified by activation of the
extrinsic pathway, as the activated caspase-8 leads to BID cleavage and
BAX activation and further results in the release of cytochrome c and caspase-9 activation [221]. Furthermore, caspase-independent apoptosis induced
by mycobacteria has been observed; for example, macrophages infected with
M. tuberculosis at high MOI may undergo apoptosis via a cathepsinmediated lysosomal pathway [223].
The pathways of M. tuberculosis-induced necrosis are heterogeneous and
relatively less well understood. For instance, two forms of necrotic death,
pyroptosis and necroptosis, occur via caspase-1 and RIP1-RIP3, respectively
[220]. Excess TNF can induce mitochondrial ROS in mycobacteria-infected
macrophages via the RIP1-RIP3 kinase pathway, leading to necroptosis
without controlling the dissemination of mycobacteria [59].
Apart from apoptosis and necrosis, infected macrophages can also undergo
autophagic cell death in cases when engulfment by the autophagosomes is
excessive [55]. This type of cell death is thought to be beneficial for the host,
since it can efficiently control bacterial replication through enhanced bactericidal capacity and remove the bacterial survival niche in a quiescent manner [55]. In addition, autophagy inhibits apoptosis by suppressing Bid activation by Bectin 1 and degrading activated caspase-8 [225].
In summary, the different cellular fates of macrophages during mycobacterial infection can strongly influence the outcome of TB disease. A deeper un22
derstanding of how mycobacteria regulate macrophage cell death could improve approaches to therapeutic interventions for TB and vaccine efficacy.
4.3.4 Granuloma formation
Tuberculosis is characterized by the organized formation of immune cell
aggregates called granulomas [226]. The structure is initiated by the focal
accumulation of activated macrophages, which often undergo further changes including transformation to epithelioid form, fusion into multinucleated
giant cells (MGCs), differentiation into lipid-rich foam macrophages, or
formation of a caseum region due to necrosis [227]. The macrophage aggregates can further recruit other immune cells including DCs, neutrophils, NK
cells, T cells, B cells, and fibroblasts to form an organized complex [61].
In particular, MGCs formed by fused monocytes or macrophages are the
characteristic feature of granulomas [228]. The functional phenotypes of
MGCs triggered by mycobacteria can vary depending on the bacterial virulence. Generally, low-virulence species such as M. avium and M. smegmatis
tend to induce small MGCs with low numbers of nuclei per cell and retained
phagocytic activity; in contrast, highly virulent M. tuberculosis promotes
formation of large MGCs with impaired phagocytosis but strong antigen
presentation capability [229]. Macrophage fusion can be triggered by several
mediators including IL-4 [230], IFN-γ [231], GM-CSF [232], and CCL-2
[233]. The macrophage fusion receptor (MFR)-CD47 interactions [234] and
CD44 [235] are proposed to play central roles in cell-cell recognition during
MGC formation.
Granulomas can persist in the host for years and have been historically recognized as a protective structure for the host. They are thought to be important for maintaining latent TB infection by “walling off” the persisting
mycobacteria and protecting the neighboring tissue from infection [236].
The core of a matured granuloma is hypoxic, which restricts the replication
of M. tuberculosis and keeps them in a dormant bacillary state [60]. Indeed,
there are mouse studies showing that defective granuloma formation is associated with decreased survival during chronic M. tuberculosis infection [61].
Furthermore, many asymptomatic carriers of M. tuberculosis have evidence
of healed granulomas. However, some immunocompetent TB patients have
well-formed granulomas but fail to control infection, indicating whether
granuloma is protective largely depends on the context, such as the type and
activation states of cells in the structure [237]. Recently, dynamic imaging
studies of M. marinum-infected zebrafish embryos indicated that early granuloma formation might promote mycobacterial dissemination and benefit the
bacteria [182,236]. The granuloma-forming macrophages can move rapidly
and continuously recruit new cells that become infected again by uptaking
the dying infected cells [182]. These results provided a novel and more sophisticated view of the role of granulomas in TB pathogenesis, which could
23
have important therapeutic implications [236]. However, animal models are
never perfect since they lack human-specific immune factors that might play
important roles in the outcome the TB infection. For instance, human CD46
expression is protective for mice against M. bovis BCG infection, which is
associated with enhanced granuloma formation (Paper IV). Modulating the
pathways that benefit the host could lead to a new attractive approach to
combat TB, but more comprehensive studies of granulomas are needed to
provide essential information.
24
AIMS OF THE STUDY
The overall aims of the present study were to investigate the functional
modulation of macrophages upon infection with pathogenic bacteria including Neisseria meningitidis and Mycobacteria bovis BCG and to understand
the role of macrophages in the pathogenesis of those infectious diseases.
Specific aims
25
•
To investigate the effects of the meningococcal surface protein
NhhA on macrophage activation and the underlying signaling pathways. (Paper I)
•
To examine the role of NhhA in macrophage polarization and its
impact on the asymptomatic nasopharyngeal carriage of N. meningitidis. (Paper II)
•
To understand how the human transmembrane protein CD46 regulates macrophage function and sepsis reaction during meningococcal
or other gram-negative infection. (Paper III)
•
To characterize the effect of human CD46 on modulating macrophage polarization and cell death and host susceptibility to mycobacterial infection. (Paper IV)
RESULTS AND DISCUSSION
Paper I
Besides the major proinflammatory factor LOS, various meningococcal surface molecules such as PorB [152] and NadA [159] can serve as virulence
factors and induce proinflammatory innate immune responses, which are
essential for the innate host defense. Here we demonstrated that the purified
recombinant meningococcal outer membrane protein NhhA can provoke IL6 and TNF production in RAW 264.7 mouse macrophages in a time- and
dose-dependent manner. Using PCR-array screening followed by qPCR validation, we found that NhhA strongly stimulated the transcriptional upregulation of a number of proinflammatory mediators in RAW 264.7 macrophages. Among these cytokines, IL-1α, IL-1β, IL-6, G-CSF, and GM-CSF were
upregulated over 10,000-fold, whereas TNF was upregulated about 11-fold
in response to NhhA stimulation. The NhhA-dependent induction of these
six cytokines was corroborated in human THP-1 macrophages and human
primary monocyte-derived macrophages, although the human cells appear to
be less susceptible to NhhA stimulation than the murine macrophage cell
line.
The signaling mechanisms involved in NhhA-induced cytokine release were
investigated using a set of pharmacological inhibitors and siRNA silencing.
NhhA-provoked IL-6 and TNF release were both associated with NF-κB
activation. However, IL-6 secretion was TLR4-MyD88-dependent, whereas
TNF production was TLR4- and MyD88-independent in murine RAW 264.7
macrophages. None of TLR1, TLR2, or CD14 was involved in NhhAtriggered production of IL-6 and TNF. Furthermore, in human monocytederived macrophages, NhhA promoted upregulation of G-CSF and IL-6
mRNA in a TLR4-dependent manner, whereas GM-CSF and TNF mRNA
were elevated via TLR4-independent pathways. The capacity of NhhA to
activate TLR4 signaling was further confirmed in the transfected HEK293
cells expressing TLR4. We found that NhhA significantly induced TNF
mRNA but not IL-6 mRNA. Together, these results suggest that NhhA simulates inflammatory responses in macrophages through both TLR4-dependent
and -independent pathways and that the involved signaling pathways can
vary by cell line or species.
These data provided evidence that the meningococcal outer membrane protein NhhA has immunostimulatory capacity by triggering production of cytokines in macrophages, which could contribute to a regulatory inflammatory milieu at the infection site. This knowledge can be integrated into developing potential complementary approaches to alleviate inflammatory reactions in meningococcal sepsis. Furthermore, NhhA has been suggested as a
26
vaccine or adjuvant candidate based on its ability to trigger anti-bactericidal
antibodies [238,239], so better understanding of the immunomodulation
function of NhhA is important for successful development of potential protein-based vaccines.
Paper II
In Paper I, we demonstrated that NhhA provokes inflammatory cytokine
production in macrophages. In the subsequent study, we observed that NhhA
could also trigger substantial production of inflammatory mediators in human peripheral monocytes. Prolonged NhhA treatment induced monocyte
differentiation into CD14+CD1a- macrophages (NhhA-MØ) instead of DCs
in a time- and dose-dependent manner, with the optimal effect observed
when monocytes were challenged with 50 nM NhhA for 3 days. NhhA containing the full passenger domain rather than the truncated NhhA protein is
required to efficiently trigger monocyte differentiation.
We investigated the signaling pathways underlying NhhA-provoked cell
differentiation. By using a series of specific inhibitory antibodies against
TLRs, we found that NhhA initiated monocyte activation through the
TLR1/TLR2 signaling pathways. Western blotting analysis revealed that
NhhA-triggered TLR1/2 activation on monocytes selectively led to MAP
kinase activation by the phosphorylation of ERK and JNK. In addition, NFκB was involved in the downstream signaling activation rather than AP-1. A
broad range of inflammatory mediators induced by the TLR1/2-NF-κB
pathways could be involved in promoting macrophage differentiation. Using
qPCR analysis and block assay, we found that NhhA induced the mRNA
production of IL-6, IL-12b, IL-10, and TNF in a TLR1/2-NF-κB-dependent
manner. To identify which cytokines were genuinely involved in the formation of NhhA-MØ, we took advantage of neutralizing antibodies or inhibitors against the selected cytokines. Endogenously produced IL-10 and
TNF were shown to participate in the action. In summary, NhhA programmed monocyte differentiation through TLR1/TLR2-activated ERK/JNK
MAP kinases and NF-κB signaling pathways in an IL-10/TNF-dependent
manner.
Subsequently, we attempted to characterize the phenotype of NhhA-MØ by
checking the surface marker expression, analyzing the cytokine profile, and
examining their bactericidal capacity. Surprisingly, NhhA-MØ displayed a
CD200Rhi CD206+ phenotype with low surface expression of M1-associated
HLA-DR, CD86, CD80, and TLR4. Consistent with the anti-inflammatory
features, NhhA-MØ was hyporesponsive to meningococcal infection, with
reduced induction of proinflammatory cytokines (IL-6, TNF, IL-12, IL-23,
and IL-1β) but enhanced levels of Treg or Th2 chemokines (CCL17, CCL18,
27
and CCL-22). In addition, NhhA-MØ could uptake and kill bacteria as efficiently as M1-like macrophages. Hence, NhhA promoted macrophage polarization into a specific immunotolerant and bactericidal phenotype rather than
the classical M1 or M2.
Figure 3. Schematic diagram illustrating the potential effects of NhhA-MØ on asymptomatic nasopharyngeal carriage of N. meningitidis.
NhhA-MØ is predicted to (1) prevent of bacterial dissemination by uptaking and eliminating
invaded bacteria and (2) exhibit hyporesponsiveness to meningococcal stimulation via generation of reduced amounts of inflammatory mediators but increased amounts of antiinflammatory factors. This homeostatic microenvironment might also alleviate tissue damage
and potentiate meningococcal persistence at the nasopharyngeal mucosa.
By using CD46+/+ transgenic mice, a conventional model of meningococcal
infection, the role of NhhA in reprogramming macrophages and its effect on
host-bacteria interaction during N. meningitidis infection was investigated in
vivo. We pretreated the mice intraperitoneally (i.p.) with NhhA for 3 days
followed by i.p. challenge with FAM20 (a serogroup C meningococcal
strain). A rapid influx of immune cells predominantly contributed by the
F4/80hiCD11bhi infiltrating macrophages was observed in the peritoneal cavity of NhhA-treated mice but not in the heat-inactivated NhhA-treated control
mice. Accordingly, peritoneal macrophages isolated from the NhhA-treated
mice displayed anti-inflammatory phenotypes with enhanced CD206 and
Arg1 production, attenuated proinflammatory reaction in response to meningococcal stimulation, and increased killing capacity. The intrinsic effect of
NhhA on macrophage programming in vivo was further shown using NhhAdeficient meningococci (ΔNhhA). As expected, CD46+/+ mice i.p. infected
with ΔNhhA produced fewer inflammatory macrophages in their peritoneal
cavity than those infected with WT meningococci, and the ΔNhhA-polarized
28
macrophages produced elevated IL-12 but diminished IL-10 and iNOS. Attributed to the specific functional phenotypes of NhhA-MØ, mice pretreated
with NhhA efficiently controlled bacterial dissemination and survived meningococcal infection, providing strong support for the role of NhhA in macrophage programming in vivo.
The mechanism by which meningococci asymptomatically colonize nasopharyngeal mucosa in healthy individuals remains a long-standing mystery.
We hypothesized that the anti-inflammatory and bactericidal NhhA-MØ
might modulate the nasal microenvironment and favor commensal bacterial
colonization. The notion was supported in an intranasal infection model. We
observed that intranasal NhhA pretreatment in mice downregulated mucosal
immune activation, which was associated with enhanced bacterial colonization in the nasopharynx. A schematic diagram summarizing the role of
NhhA in nasopharyngeal carriage of meningococci is shown in Figure 3.
Thus, fine-tuning monocyte activation might be a strategy exploited by N.
meningitidis to trigger immune ignorance and maintain commensal colonization.
Paper III
Transgenic mice expressing human CD46 (CD46+/+ mice) have been used as
a model system to investigate the pathogenesis of several human-specific
pathogens including N. meningitidis [67,68,102] . CD46+/+ mice were found
to be more susceptible to meningococcal systemic infection and exhibited
enhanced proinflammatory response and higher mortality than the WT mice
[67,106]. The increased susceptibility of CD46+/+ mice to all other pathogens
such as group A streptococcus and MV was apparently correlated to the
function of CD46 as a receptor, whereas the role of CD46 in meningococcal
adhesion is debated [75,240]. Thus, the underlying mechanism by which
CD46 contributes to the severity of meningococcal sepsis remains largely
unknown.
Our results broadened the understanding of the disposition of CD46+/+ mice
to bacterial sepsis, suggesting that it is not restricted to the infection of meningococci or other pathogens as CD46 ligands but might be generally applicable to gram-negative sepsis. A non-lethal dose of LPS for WT mice (20
mg/kg, i.p. injection) caused an intensive inflammatory response and increased mortality in the CD46+/+ mice. We found that the susceptibility of
CD46+/+ mice was largely attributed to the greater amounts of M1-like macrophages in their peritoneal cavity than that for WT mice. CD46 signaling
improved monocyte-derived macrophage differentiation triggered by M-CSF
or GM-CSF. Moreover, in the presence of CD46, macrophages expressed
elevated levels of CCL2, a chemokine associated with the recruitment of
29
monocytes, which might also contribute to the increased number of peritoneal macrophages in the CD46+/+ mice. Functionally, CD46+/+ macrophages
displayed enhanced production of pro-inflammatory cytokines including IL6, TNF, IL-12, and IL-1b compared with the WT macrophages in response
to LPS challenge or meningococcal infection both in vitro and in vivo. These
cytokines are all clinically correlated to the severity of bacterial sepsis.
CD46+/+ macrophages also showed enhanced bactericidal capacity relative to
WT macrophages but underwent rapid apoptosis during LPS or meningococcal infection, which might lead to the uncontrolled bacterial dissemination in
vivo. Consistently, adoptive transfer of CD46+/+ macrophages into the peritoneal cavity of WT mice augmented the production of pro-inflammatory mediators and increased mortality in WT mice during the infection of N. meningitidis. Conversely, depletion of macrophages partially attenuated the septic responses in the CD46+/+ mice upon meningococcal infection. These results further indicated the role of CD46 in affecting the outcome of bacterial
sepsis in a macrophage-dependent manner.
In conclusion, our study first proposed that human CD46 signaling is involved in modulating the development, polarization, and cell death of macrophages, which could greatly influence the resolution of septic shock. In
addition, these findings increased knowledge about the CD46+/+ transgenic
mouse as an animal disease model. Understanding the role of host factors
such as CD46 has great implications in the search for new therapeutic targets
to combat the life-threatening bacterial sepsis.
Paper IV
Our results presented in Paper III showed that CD46 participated in the
modulation of macrophage polarization and apoptosis upon acute gramnegative infection. Therefore, we aimed to determine how CD46 performs
immunoregulation during chronic infectious diseases such as TB. CD46+/+
transgenic mice intranasally challenged with M. bovis BCG displayed reduced bacterial dissemination, less influx of immune cells, and better granuloma formation than the WT mice, indicating a potential protective role of
CD46 in mycobacterial infection.
Macrophages are the first line of immune defense against M. tuberculosis in
the lung but can also be exploited by the bacteria as a survival niche; hence,
macrophages play critical roles in the pathogenesis of TB [241]. We isolated
macrophages from both CD46+/+ and WT mice and compared their functional phenotypes in response to BCG infection ex vivo. The expression of human CD46 on macrophages significantly strengthened their capacity to control the intracellular growth of BCG, which was attributed to the higher production of RNI and enhanced autophagy. The engagement of CD46 also
30
inhibited the mycobacteria-triggered cell death of macrophages. We found
that inhibition of apoptotic cell death occurred via both the extrinsic and
intrinsic pathways. TNF, as a stimulus of the extrinsic pathway, was produced at a lower level in CD46+/+ macrophages relative to WT macrophages
upon BCG infection. In the intrinsic pathway, CD46+/+ macrophages expressed decreased levels of pro-apoptotic factors such as Bad and Bid but
higher levels of anti-apoptotic factors including Bcl-2 and Bcl-xL. Dampened caspase-3/7 activities in response to BCG challenge were detected in
CD46+/+ macrophages compared to the WT macrophages, which could be
involved in both extrinsic and intrinsic pathways. Additional anti-apoptotic
factors such as IL-6 and CCL-2, which inhibit apoptosis through upregulation of Bcl-2 and Bcl-xL [242], were expressed at higher levels in BCGinfected CD46+/+ macrophages, supporting our observations above. The prolonged lifespan and enhanced bactericidal capacity of CD46+/+ macrophages
might be associated with reduced bacterial dissemination detected in the
lungs.
Granuloma formation is a hallmark of TB, and the formation of MGCs is the
characteristic feature of granulomas [236]. Consistent with the enhanced
granuloma formation in the lungs of CD46+/+ mice, we observed that
CD46+/+ macrophages had a higher fusion capacity to form MGCs in response to BCG challenge in vitro. Consistently, the fusion factors including
CCL2, IFN-γ, and IL-4 were produced at higher levels in CD46+/+ macrophages upon BCG infection. Whether the granulomas play a host-protective
role in TB diseases remains controversial [237]. Most studies have used preclinical animal models such as mice and zebrafish, which do not express
human-specific immune factors. Our study demonstrated that in the presence
of human CD46, the formation of granulomas upon BCG infection was associated with controlled mycobacterial burden. In addition, we first showed
that BCG downregulates the surface expression of CD46, which could be
exploited by the mycobacteria as a survival strategy. Thus, we revealed that
human CD46 expression in mice played a host protective role against BCG
infection, which provides important implications for the development of
more effective vaccines and drugs against TB.
31
FUTURE PERSPECTIVE
The common situation in scientific research is “old problem, new findings,
yet more questions.” Herein, we addressed some of the questions raised during this thesis work, which could be interesting to explore in future studies.
In Paper I and Paper II, we revealed a dual role of NhhA as a virulence
factor triggering intensive pro-inflammatory responses and as an immune
regulator maintaining the homeostatic environment during the bacterial carriage state. The distinct functions of NhhA might depend on the different
activation stages of monocyte/macrophage linages. NhhA activates monocytes through TLR1/2 and macrophages via TLR4-dependent/-independent
signaling pathways, respectively, indicating the complexity of the surface
receptors involved in NhhA-induced signaling. In Paper II, NhhA-activated
Akt/PI3K in monocytes was found to be TLR1/2-independent, suggesting
that identification of additional receptors for NhhA is required for a complete understanding of related mechanisms.
NhhA is considered as a vaccine candidate against N. meningitidis infection
[238,239]. However, certain sequence variations of NhhA exist among species [243]. Further studies for both Paper I and Paper II are essential to
investigate whether the role of NhhA is strain-specific. In addition, how the
innate immunomodulation by NhhA is functionally linked to adaptive immunity remains unexplored but important for providing a foundation for new
vaccine development.
The mechanisms by which meningococci asymptomatically colonize the
nasal mucosa are largely unknown. We revealed that NhhA-modulated macrophages potentiate the immune homeostatic environment for commensal
carriage (Paper II). Cytokine patterns are critical to maintain the balanced
environment. In particular, IL-10 is beneficial for the colonization of several
bacteria including group B Streptococcus [244], Staphylococcus aureus
[245], and Bordetella bronchiseptica [246]. Elucidation of the role of IL-10
or other cytokines in meningococcal colonization would be worthwhile in
subsequent studies.
By using the CD46+/+ transgenic mice model, we unraveled the distinct roles
of CD46 in acute meningococcal and chronic mycobacterial infection in
Paper III and Paper IV. CD46 engagement leads to increased apoptosis of
macrophages upon LPS or meningococci-infection but increased macrophage viability in response to BCG infection. In addition, meningococci
trigger elevated TNF secretion, whereas BCG induces lower levels of TNF
production in CD46+/+ macrophages than in WT macrophages. This func32
tional discrepancy might arise from the high heterogeneity of CD46 structure,
which leads to distinct signaling pathways [247]. The two cytoplasmic tails,
CYT-1 and CYT-2, are associated with promoting and decreasing inflammatory reactions in T cells, respectively [63]. The availability of transgenic
mice specifically expressing CYT-1 and CYT-2 could enable a deeper understanding of CD46 signaling in innate immune regulation. The function of
CD46 also appears to be tightly regulating by its crosslinking with other host
receptors such as TLRs, which is worth investigations in future studies. Furthermore, how the CD46-modulated macrophages interact with T cell immunity during sepsis or TB will be interesting to explore.
A series of pathogens have been shown to downregulate CD46 expression on
host cells [68,82,83]. We reported for the first time that CD46 surface expression could be downregulated by the mycobacterial BCG in Paper IV,
although whether the process is mediated by internalization or shedding remains unknown. Further investigations are necessary to determine the underlying mechanism of BCG-triggered CD46 downregulation and its role in the
pathogenesis of TB.
Whether granuloma formation in TB benefits the host or bacteria is debated
[248]. Conclusions from work in animal models are complicated by the absence of human-specific factors. By introducing the human host factor
CD46, we provided new evidence that granulomas might be protective
against BCG infection in Paper IV. However, the avirulent mycobacterial
strain used in our study lacks RD-1, a critical factor for the motility of macrophages that contributes to infectivity [182]. Further studies employing
CD46+/+ virulent mycobacterial strains are required. Meanwhile, it is important to test these results in human systems and examine potentially supporting clinical evidence.
33
ACKNOWLEDGEMENTS
This work was carried out during the years 2012-2016 at the former Department of Genetics, Microbiology and Toxicology and the present Department
of Molecular Biosciences, the Wenner-Gren Institiute, Stockholm University.
I owe my deepest gratitude to my supervisor Hong for all the patience and
effort she has paid, for her guidance and support during every step of my
study and for her continued kind help with my future career.
I would like to acknowledge my previous supervisors Prof. Göran Akusjävi
(Master’s project) and Prof. Hongjun Li (Bachelor’s project) for bringing
me into the science world. Many thanks to my co-supervisor Prof. Ann-Beth
Jonsson for always being considerate and supportive to me. I am also grateful to Sushil for all the valued suggestions and always being nice. Thank
Helena and Klas for being kind and helpful. Thank Prof. Ulrich Theopold,
Maria Lerm and Andor Pivarcsi for joining my Licentiate committee and
providing profound comments on my projects.
My sincere thanks also go to our co-authors and collaborators. Mikael, for
all the insightful opinions and helping me with the Swedish summary.
Yumin, for helping me perform the NhhA experiments and being a good
companion during the first year. Ding, it is so nice to have your assistance in
the animal room. Marco, for being positive and helpful and brightening up
my days, although you always put the cell culture stuff on the top shelf.
Thank Prof. Carmen Fernández, for kindly providing me the powerful
BCG strain and useful suggestions, which was a great help to my last project. Dagbjört, for generously sharing the recipes, protocols and experiences
in mycobacterial and animal work.
I feel fortunate to work in such a friendly and supportive atmosphere of
MBW department, especially of our F5 corridor. Great thanks to my current
and former colleagues in ABJ-group, Helena-group, Sushil-group and
later-joined Klas-group. I really enjoyed the Monday meetings with you.
Nele, you were the one who kindly opened the door for me when I first entered the department and then witnessed and accompanied in my whole PhD
life. Raquel, thanks so much for being such a caring and perceptive friend.
Sunil, your positive attitude and laugh are super contagious. Sara.E, thanks
for being a nice companion on many late-working days in the office. Miriam, your rigorous attitudes to science inspired me a lot. Lisa, thanks for
encouraging me to investigate more on CD46 mice, which now became my
Paper III. Cecilia, also a nice companion in the old office, thanks for guiding
me the right way to use knife and fork. Sara.S, it was a pleasure teaching
together with you and also thanks for helping me with the microscope. Han34
na, for being nice and introducing the Ethiopian culture to us. Gabriela, for
being kind and for the interesting stories about Holland and middle-east.
Jakob, for the pleasant talk during Fika time and some dinners. Pilar, I am
grateful for the thoughtful discussions on macrophages. Linda, thanks for
encouraging me to be stronger. Niklas, it is so nice to have someone exchanging birthday greetings every winter and thanks for all the novel stories
and ideas you have brought to me. Katarina, thanks for being positive,
funny and wise. Mattias, it was great to teach together with you and with
your tasty cakes. Fredrik, you are also a good teaching companion and nice
colleague. Mano, thanks for being funny during the dinners and friendly all
the time. To my office colleagues during my third and last year, Gabor,
Spycros, Eva, Ymke, Espranza and Shad, thank you so much for your
companionship. Also, thanks Lucille for generously sharing your experience
in BCG and mice work. Yeneneh, for always being friendly since we met in
the introductory course.
I am so grateful to have many amazing friends during my seven years in
Sweden. To our lovely members of ‘Dr.Her Club’, Yueshi, Simei and
Xiaojing, thank you for your warmest companionship with Girl power.
Hongwei, for being helpful with my car and on the course. Kun, Xiongzhuo
and Chen Hou, for sharing numerous enjoyable moments and always being
by my side. Yunpo, for being funny during lunch break and helping me
with the IHC phototaking. Thank Liqun and Xin Li for your warm friendship and all the joy brought by Muchen. Bojing, you are such a reliable
friend, I will miss the days driving in the dark to the stables with you. Li Yu
and Wei Yuan, you knew you are the ‘stockhom yellow page’ and super
helpful friends. Many thanks also go to the ‘Laowuzei’s (the old friends in
Uppsala) named Jinzhi Hu, Shuang Gao, Weilin, Xue Li, Chengxi Shi, Li
Li, Liang Tian, Caihong and Chengjun Wu for all the help and all the
memorable time.
Biao Wang, thanks for introducing me to the exciting world of techno startups. Special thanks to Yang Sun for all the meaningful discussions about
science and life. Maria, I really appreciate your friendship which transcends
country, age and field. Johnny, my best sharer ever, I am truly grateful for
your listening and support all through these years. Du Yeye, my respectable
life mentor, thank you for motivating me with your great wisdom.
To my beloved Kearon, thank you for coming, for sharing life with me now
and in the future. To my dearest family and friends in China, in particular
my parents and grandparents, thanks for giving me respect, freedom, support and unconditional love in my growth.
35
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