Download The Role of Nitric Oxide in Host Defence Against Mycobacterium

Linköping University Medical Dissertations
No. 1304
The Role of Nitric Oxide
in Host Defence Against
Mycobacterium tuberculosis
Clinical and Experimental Studies
Jonna Idh
© Jonna Idh
Paper I-III are reprinted with permission from the respective publishers.
Cover: A serving of peanuts, by Emelie Algotsson.
Illustrations are created by the author and produced by Per Lagman, LiU Tryck.
ISBN 978-91-7519-911-5
ISSN 0345-0082
Printed in Sweden by LiU Tryck
Linköping 2012
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This thesis is dedicated to all whom lost their health, their love, or their life due to TB. 3
Supervisors Thomas Schön, Kalmar County Hospital and Linköping University
Olle Stendahl, Linköping University
Financial enclosure The work included in this thesis was supported by the Swedish Research Council, the
Swedish Heart and Lung Foundation, King Oscar II Jubilee Foundation,
SIDA/SAREC, Minor Field Studies (MFS/SIDA), European and Developing
Countries Clinical Trials Partnership, (EDCTP, European Union), the Research
Council of Southeast Sweden (FORSS), the Swedish Society of Medicine, the Lion
Research Foundation, Knut and Alice Wallenberg Foundation and the Swedish
Society of Tropical Medicine and International Health.
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Table of Contents Supervisors ....................................................................................................................................................... 4 Financial enclosure ...................................................................................................................................... 4 ABSTRACT ......................................................................................................................................... 1 SAMMANFATTNING PÅ SVENSKA .............................................................................................. 3 LIST OF ORIGINAL PAPERS .......................................................................................................... 5 Paper I ................................................................................................................................................................ 5 Paper II .............................................................................................................................................................. 5 Paper III ............................................................................................................................................................. 5 Paper IV ............................................................................................................................................................. 5 INTRODUCTION ............................................................................................................................... 7 ABBREVIATIONS .............................................................................................................................. 8 BACKGROUND ............................................................................................................................... 11 TUBERCULOSIS ................................................................................................................................................ 11 A historical view ......................................................................................................................................... 11 Epidemiology ............................................................................................................................................... 11 Transmission ................................................................................................................................................ 13 Risk factors for tuberculosis .................................................................................................................. 15 Nutrition and tuberculosis ..................................................................................................................... 16 Diagnosis ........................................................................................................................................................ 17 Clinical presentation ................................................................................................................................. 19 Treatment ...................................................................................................................................................... 20 Vaccine ............................................................................................................................................................ 23 The genus Mycobacterium ..................................................................................................................... 24 Mycobacterium tuberculosis ................................................................................................................. 24 HOST IMMUNITY IN TUBERCULOSIS ............................................................................................................. 26 Innate immune responses ....................................................................................................................... 26 Within the macrophage .......................................................................................................................... 28 T cell mediated response ......................................................................................................................... 29 The granuloma ............................................................................................................................................ 31 NITRIC OXIDE ................................................................................................................................................... 33 Biochemistry ................................................................................................................................................. 33 Nitric oxide in health and disease ....................................................................................................... 34 1
Nitric oxide in tuberculosis .................................................................................................................... 35 SURVIVAL STRATEGIES OF M. TUBERCULOSIS ............................................................................................ 37 AIMS .................................................................................................................................................. 39 MATERIALS AND METHODS ...................................................................................................... 43 Study site ........................................................................................................................................................ 43 Study subjects (Paper I-­‐III) .................................................................................................................... 44 Chemotherapy and nutritional supplementation (Paper II) ................................................... 44 Follow-­‐up ....................................................................................................................................................... 44 Chest X-­‐ray .................................................................................................................................................... 45 Laboratory analyses (Paper II and III) ............................................................................................. 45 Measurement nitric oxide in exhaled air and nitric oxide metabolites in urine ............. 45 Drug susceptibility testing and spoligotyping of M. tuberculosis (Paper III) .................. 45 Susceptibility testing to nitric oxide (Paper III) ........................................................................... 46 Luciferase-­‐based viability assay (Paper IV) ................................................................................... 46 Infection of macrophages (Paper IV) ................................................................................................ 46 Statistics (Paper I-­‐IV) ............................................................................................................................... 47 Ethical consideration ............................................................................................................................... 47 RESULTS AND DISCUSSION ....................................................................................................... 51 Systemic levels of nitric oxide in pulmonary TB ........................................................................... 51 Local levels of nitric oxide in pulmonary TB .................................................................................. 52 Treating TB with peanuts – are you nuts? ...................................................................................... 53 Nitric oxide – does it kill the bug? ....................................................................................................... 55 Nitric oxide resistance – from bench to bedside ........................................................................... 56 Antibiotic resistance associated to nitric oxide susceptibility – a novel finding ............ 57 Survival of M. tuberculosis within the macrophage .................................................................... 59 CONCLUDING REMARKS ............................................................................................................. 61 CONCLUDING FIGURE .................................................................................................................. 62 FUTURE AREAS OF INTEREST .................................................................................................. 63 REFERENCES .................................................................................................................................. 67 ACKNOWLEDGEMENTS .............................................................................................................. 85 2
Abstract Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), responsible for
significant morbidity and mortality worldwide, especially in low-income countries.
Considering aggravating factors, such as HIV co-infection and emerging drug resistance, new
therapeutic interventions are urgently needed. Following exposure to M. tuberculosis,
surprisingly few individuals will actually develop active disease, indicating effective defence
mechanisms. One such candidate is nitric oxide (NO). The role of NO in human TB is not
fully elucidated, but has been shown to have a vital role in controlling TB in animal models.
The general aim of this thesis was to investigate the role of NO in the immune defence against
M. tuberculosis, by combining clinical and experimental studies. In pulmonary TB patients,
we found low levels of NO in exhaled air, and low levels of NO metabolites in urine. HIV coinfection decreased levels of exhaled NO even further, reflecting a locally impaired NO
production in the lung. Low levels of exhaled NO were associated with a decreased cure rate
in HIV-positive TB patients. Household contacts to sputum smear positive TB patient
presented the highest levels of both urinary NO metabolites and exhaled NO. Malnutrition, a
common condition in TB, may lead to deficiencies of important nutrients such as the amino
acid L-arginine, essential for NO production. We therefore assessed the effect of an argininerich food supplement (peanuts) in a clinical trial including pulmonary TB patients, and found
that peanut supplementation increased cure rate in HIV-positive TB patients.
We also investigated NO susceptibility of clinical strains of M. tuberculosis, and its
association to clinical outcome and antibiotic resistance. Patients infected with strains of
M. tuberculosis with reduced susceptibility to NO in vitro, showed a tendency towards lower
rate of weight gain during treatment. Moreover, there was a clear variability between strains
in the susceptibility to NO, and in intracellular survival within NO-producing macrophages. A
novel finding, that can be of importance in understanding drug resistance and for drug
development, was that reduced susceptibility to NO was associated with resistance to firstline TB drugs, in particular isoniazid and mutations in inhA.
Taken together, the data presented here show that NO plays a vital role in human immune
defence against TB, and although larger multicentre studies are warranted, arginine-rich food
supplementation can be recommended to malnourished HIV co-infected patients on TB
treatment.
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Sammanfattning på svenska Tuberkulos orsakas av bakterien Mycobacterium tuberculosis och så sent som för hundra år
sedan var tuberkulos en av de vanligaste dödsorsakerna i vår del av världen. Med bättre
levnadsvillkor och antibiotika minskade förekomsten av tuberkulos och behovet av
tuberkulosforskning och vaccinutveckling likaså. Intresset för sjukdomen har återigen ökat till
följd av spridningen av antibiotikaresistenta tuberkelbakterier. I länder med låg
levnadsstandard är tuberkulos ett fortsatt stort hot mot folkhälsan och under 2010 beräknades
1,4 miljoner människor ha dött, och 9 miljoner nyinsjuknat i tuberkulos. Tuberkelbakterien
sprids via luftvägarna och kan i lungan orsaka den klassiska lungtuberkulosen, men kan även
spridas till andra organ. I lungan tas tuberkelbakterien upp av immunceller som gemensamt
försöker förgöra bakterien, alternativt kapsla in den i ett så kallat granulom. En av
immuncellernas försvarsmekanismer mot bakterier är kväveoxid (NO). Huvudsyftet för denna
avhandling var att studera betydelsen av NO i immunförsvaret mot tuberkulos. Arbetet har till
största delen genomförts i Gondar, i norra Etiopien där tuberkulos är mycket vanligt. Vi fann
att tuberkulospatienter hade låga nivåer av NO både lokalt i lungan och systemiskt i kroppen.
Låga nivåer av NO i lungan var kopplat till sämre utläkning hos tuberkulospatienter med
samtidig HIV-infektion. Personer som bodde tillsammans med en smittsam tuberkulospatient,
men som inte själva blivit sjuka, hade däremot höga nivåer av NO. Om höga nivåer av NO
minskar risken att utveckla tuberkulos i senare i livet vet vi ännu inte. Tuberkulos och
undernäring är nära associerade och kan leda till brist på viktiga näringsämnen. Aminosyran
L-arginin, substratet för NO, är ett sådant näringsämne. Jordnötter innehåller mycket Larginin och finns lokalt i Gondar. Vi initierade därför en studie där tuberkulospatienter fick
äta extra jordnötter, eller ett födotillskott med lågt arginin-innehåll, som tillägg till gängse
antibiotikabehandling. Tillägg av jordnötter visade sig öka utläkningen av tuberkulos, men
endast hos de patienter som också var HIV-infekterade. För att studera hur effektivt NO kan
avdöda tuberkelbakterien, exponerade vi bakterierna för NO. Känsligheten för NO varierade
mellan olika stammar och var associerad till hur väl bakterierna överlevde i immunceller.
Känsligheten för NO var även associerad till om bakterierna var antibiotikaresistenta, framför
allt om de var resistenta till en av de viktigaste tuberkulosmedicinerna, isoniazid. Detta har
inte visats tidigare och kan vara viktigt för framtida antibiotikaforskning.
Sammantaget tyder våra resultat på att kväveoxid spelar en viktig roll vid tuberkulos och att
tillskott av L-arginin är gynnsamt för immunförsvaret mot tuberkelbakterien.
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List of original papers This thesis is based on the following papers, which will be referred to in the text by their
Roman numerals.
Paper I Idh J, Westman A, Elias D, Moges F, Getachew A, Gelaw A, Sundqvist T, Forslund T,
Alemu A, Ayele B, Diro E, Melese E, Wondmikun Y, Britton S, Stendahl O, Schön T. Nitric
oxide production in the exhaled air of patients with pulmonary tuberculosis in relation to HIV
co-infection. BMC Infectious Diseases 2008; 8:146.
Paper II Schön T, Idh J, Westman A, Elias D, Abate E, Diro E, Moges F, Kassu A, Ayele B, Forslund
T, Getachew A, Britton S, Stendahl O, Sundqvist T. Effects of a food supplement rich in
arginine in patients with smear positive pulmonary tuberculosis - a randomised trial.
Tuberculosis 2011; 91:370-7.
Paper III Idh J, Mekonnen M, Abate E, Wedajo W, Werngren J, Ängeby K, Lerm M, Elias D,
Sundqvist T, Aseffa A, Stendahl O, Schön T. Resistance to first-line anti-TB drugs is
associated with reduced nitric oxide susceptibility in Mycobacterium tuberculosis. Accepted
for publication in PLoS ONE.
Paper IV Idh J, Lerm M, Raffetseder J, Eklund D, Larsson M, Pienaar E, Tony Forslund, Werngren J,
Juréen P, Ängeby K, Sundqvist T, Stendahl O, Schön T. Susceptibility of clinical strains of
Mycobacterium tuberculosis to reactive nitrogen species in activated macrophages.
Manuscript.
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INTRODUCTION Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains a major
worldwide health problem. Even though antibiotics can cure TB, the disease causes
approximately 1.4 million deaths annually. The efforts to control TB are mainly hampered by:
the prolonged time from initial symptom to diagnosis, logistic challenges in reaching TBendemic areas, the extended time of treatment to eliminate the infection, the immunological
impact of HIV infection, increasing drug resistance, and the fact that the vaccine against
M. tuberculosis (BCG vaccine) has limited efficiency. To overcome these challenges, the
development of new drugs and vaccines, more accurate and rapid diagnostic tools as well as
research on therapeutic strategies that target the immune system, are urgently needed. This
thesis focuses primarily on the role of nitric oxide (NO) in the immune defence against
M. tuberculosis. Results presented here can be applied in the area of drug development, by
investigating the ability of M. tuberculosis to withstand the toxic effects of NO, but also in the
area of immunotherapy and vaccine development, by assessing aspects of NO-mediated
immunity in the host defence against TB.
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ABBREVIATIONS AIDS
acquired immune deficiency syndrome
ART
anti-retroviral therapy
BCG
bacillus Calmette Guérin
BMI
body mass index
DC-SIGN
DC-specific intracellular-adhesion-molecule-3-grabbing non-integrin
DETA/NO
diethylenetriamine NONOate
DOTS
directly observed treatment, short-course
HIV
human immune deficiency virus
IGRA
interferon-gamma release assay
IFN
interferon
INH
isoniazid
iNOS
inducible nitric oxide synthase
MDR TB
multidrug-resistant tuberculosis
MOI
multiplicity of infection
NADPH
nicotinamide adenine dinucleotide phosphate
NO
nitric oxide
NO2
-
nitrite
NO3
-
nitrate
NOS
nitric oxide synthase
-
ONOO
O2
-
peroxynitrite
superoxide
PCR
polymerase chain reaction
PPD
purified protein derivative
RNS
reactive nitrogen species
ROS
reactive oxygen species
SIN-1
3-morpholino-sydnonimine
TB
tuberculosis
TLR
toll-like receptor
TNF
tumour necrosis factor
TST
tuberculin skin test
XDR TB
extensively drug-resistant tuberculosis
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A voice of TB “I had chest pain and dyspnoea but I didnt know that it was TB. When
the doctor told me I had TB I felt sad, because I then had to stay at
home from school. I come from the countryside, and I had a family
member with TB, I think I got it from that person. My parents are dead,
so now I live with my sister in town. She is not afraid of getting sick,
and we sleep in the same room. My school is in the countryside, but I
can´t stay there since I need to be in town to take my drugs. I have four
months left, and then I will return to school. I think I will be well after
treatment.”
Abera, 18-­‐year-­‐old young man, student at elementary school 9
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BACKGROUND Tuberculosis A historical view Tuberculosis (TB) is an infectious disease caused by the microbe M. tuberculosis [1]. It has a
history of thousands of years together with mankind, and was described by Hippocrates
(400 B.C.) and has been found evident in antique Egyptian mummies (2000-3000 B.C.) [2].
Many names have been used to describe the disease over the years, such as “white plague”,
“phthisis”, “wasting away”, “consumption”, “Pott’s disease”, “Gibbus deformity”, and
“scrofula” [3]. In late 17th century a French pathologist, Franciscus Sylvius, found pulmonary
nodules from TB patients and named these tubercula (small knots). He also observed how the
tubercula developed into lung ulcers (cavities). After some years it was suggested that TB
could be transmitted through the “breath” of a sick person [3] but it took nearly 200 years
before Robert Koch managed to isolate the causative agent of TB. Shortly after, the sanatoria
were introduced with a regimen of bed rest, preferably out in the open-air, and a nutrient-rich
diet [4]. Many poets and writers have described the life at sanatoria and the struggle against
TB. Examples from the Swedish literature are Edith Södergran [5], Sven Stolpe [6] and Olof
Lagercrantz [7], who all fell victims to TB.
Epidemiology The dynamics of TB in an epidemiological perspective depends greatly on socioeconomic
factors [8]. From the 16th to the 18th century TB was responsible for one in four deaths in
Europe, and reached a peak in the 19th century due to urbanization and crowded living
conditions [2]. As housing, diet and sanitation improved during late 19th century, the TB
incidence declined even before the discovery of antibiotics, stating the importance of social
factors and nutrition in containing TB [9]. After the introduction of chemotherapy in the
1940s, there was a steady reduction in disease burden for several decades. However, as a
consequence of the HIV/AIDS pandemic [10] and the development of drug-resistant strains,
TB has returned as one of the deadliest infectious diseases in the world [3, 11, 12].
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Despite treatment being available for more than 50 years, the World Health Organization
(WHO) declared TB a global public health emergency in 1993, and prioritized TB in the Stop
TB Partnership and Millennium Development Goals. Still, WHO estimated that TB caused
the death of about 1.4 million people in 2010, and that 9 million cases of TB were diagnosed
worldwide. Two-thirds of these TB cases occur in people between 15–59 years and are
devastating for the financial systems in high incidence countries, as well as for the 10 million
children that were estimated to be orphans as a consequence of parental death in TB (2009)
[13].
In Sweden, the TB incidence is low with 7.3/100 000, and 683 actual cases prevalent in 2010
[14], as compared to a high endemic areas like Ethiopia with an incidence of 261/100 000 and
a prevalence of 394/100 000 (figure 1). In a country the size of Ethiopia, this resulted in
absolute numbers of 220 000 new TB cases, 330 000 people living with TB and 29 000 deaths
due to TB (excluding HIV) in 2010.
Figure 1. Tuberculosis incidence rates per country in 2010 [13]. (Reprinted with permission from
WHO Press.)
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As a weak comfort to these significant numbers, WHO has presented a slow decrease in the
absolute number of new TB cases since 2006 [13], but this is not in agreement with others [8]
as it stands clear that to stop the TB epidemic there is a need for a more aggressive approach
against HIV [10, 15]. In 2010, 43% of TB patients in Ethiopia were tested for HIV and of
these 15% were HIV-positive [13]. Even though there have been improvements in screening
for TB in HIV-positive individuals, as well as initiation of TB preventive therapy for HIVpositive patients without active TB, only 34% of TB patients were tested globally in 2010
[13].
Transmission TB is almost entirely spread by air-borne droplets produced by a person with pulmonary TB,
where cavitary disease and the presence of bacteria in sputum (sputum smear-positive),
increase the risk of transmission [16]. Most people exposed to M. tuberculosis will not
develop active disease. As a result of a strong innate immune response, they will either clear
the infection with no signs of M. tuberculosis encounter, or they may contain the infection
with no signs of disease (latent TB). Due to immune suppression later in life, the latent
infection can reactivate to active disease [17] (figure 2). A latent TB infection can be detected
by immune reactivity against antigens of M. tuberculosis, with interferon-gamma release
assays (IGRA) and/or tuberculin skin test (TST) [18]. It is not possible to detect the presence
of the potentially well-contained bacteria themselves [1], but mycobacterial DNA has been
found in lung tissue from asymptomatic individuals in high endemic areas, who died from
causes other than TB [19].
There is a relatively high risk of transmission to close household contacts of TB patients [20,
21] and mathematical modelling of TB transmission and key factors in disease control, has
led to the findings that members of larger families are responsible for more disease
transmissions than those from smaller families [22]. However, in high endemic areas a
significant part of the transmission occurs outside the household, such as on public transports
[22], and the benefit of active case finding within the households in high-endemic areas has
not proven as obvious as in low-endemic areas [23].
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Figure 2. Transmission of M. tuberculosis. A strong innate immune response may eradicate
M. tuberculosis instantly, and there will be no immunological or radiological sign of M. tuberculosis
encounter. If the patient is infected, there are two possible scenarios. If the host manages to contain the
infection through effective cellular immune responses, there will be no symptoms of disease, and this
stage is called latent TB. Antigen recognition with tuberculin skin test (TST) and/or interferon-gamma
release assays (IGRA), is a sign of latent TB, but may not always indicate that viable M. tuberculosis
are present in the host. If the host is not capable of mounting an adequate immune response, bacteria
will continue to replicate and cause active disease. Such a scenario may take place in small children
and in late stage of HIV infection. If the immune response in latent TB is affected by factors such as
old age or chronic diseases, the latent infection can reactivate and develop into active TB, and spread
to a new host.
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Risk factors for tuberculosis There are known risk factors for TB, but even when taking those factors into consideration, it
is still not clear why the majority of individuals infected with M. tuberculosis will stay
healthy while others will develop disease. It is of great importance to understand the
underlying mechanisms behind the susceptibility or resistance to TB infection in order to
prevent infection and disease [24].
There is a sex-related difference in TB incidence [25]. In Ethiopia there was a male to female
ratio of 1.3 in 2010 [13]. The difference depends both on social structures and lower case
findings among women, but also on biological differences [25, 26]. Chronic diseases known
to be associated with increased risk of TB include diabetes [27-29], renal failure [30],
haematological malignancies [31] and solid-organ malignancies due to immune suppression
by the disease itself or by prescribed chemotherapy [32]. In recent years, increased use of
biological immune modulators such as tumour necrosis factor (TNF) antagonists for the
treatment of rheumatologic disorders is common, significantly increasing the risk of
reactivating latent TB [33]. Other risk factors for TB are smoking [34], indoor combustion of
biofuels [35], silicosis [36] and heavy alcohol consumption [1, 37].
The risk of developing TB after exposure to M. tuberculosis is greatest in the youngest
children [38] and decreases from the age of 5 until adolescence, when the risk again increases
with a peak in the age of 20-30 [25]. Elderly are at high risk due to dysfunction of the cellular
immune functions, similar to that seen in young children [25]. Immune dysfunction due to
HIV has lead to an epidemic of HIV-associated TB [10], where TB now is the leading cause
of death among HIV-positive individuals [1]. The two diseases accelerate one another, by
increased transcription of HIV in TB [39, 40] and decreased numbers of TB-protective T cells
in HIV [41]. The TB incidence ratio between HIV-negative and HIV-positive individuals is
around 20 to 37, depending on HIV prevalence [1]. Another co-infection, which could have
an impact on the development of TB, is chronic worm infection. In TB endemic areas there is
a high incidence of worm infection [42, 43] known to have immune modulatory effects, and a
subsequent negative impact on host response to TB [43, 44], and on immunity induced by
vaccination to protect against TB (BCG vaccination) [45].
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TB was previously thought to be an inherited disease and that has now been substantiated in
the last decades, since human genetics have shown to contribute to susceptibility to TB [46].
Variations in incidence of TB between regions may be explained by genetic differences [47].
On the other hand TB took the lives of millions of people during the pre-antibiotic era,
resulting in a natural selection of individuals with immunity strong enough to withstand the
infection or disease [24]. Excluding a few exceptions, one by one the genetic variants will not
have a big impact on the risk of TB, but together on a population level, the impact may be
profound [8]. Genetic identification of these risk factors could lead to individualized
treatment and novel vaccination strategies [46]. Genes confirmed to be important in host
defence in TB are the natural resistance-associated macrophage protein (NRAMP or
SLC11A1) [48], the vitamin D receptor (VDR), the nuclear factor kappa B (NFκB) pathways,
the human leucocyte antigen (HLA) region, pattern recognition receptors (PRR) such as DCSIGN and toll-like receptors (TLRs), as well as the IFN-γ pathways [49]. There is also a
genetic association between nitric oxide synthase (NOS2A) and tuberculosis confirming the
role of NO in human disease [46].
Nutrition and tuberculosis TB has been called “A poor mans disease” indicating risk factors for TB associated with
poverty, like crowded living conditions and malnutrition [50]. Previously TB went under the
name “Consumption” [51], describing the common symptom of weight loss seen in TB
patients. The weight loss is due to loss of appetite, but also to malabsorption and altered
metabolism [52]. Protein-energy malnutrition, micronutrients deficiencies and low body mass
index (BMI) increase the risk of TB [53-55] and malnutrition is associated with early death
during TB treatment [56]. Malnutrition affects the cell-mediated immunity (CMI), the
principle host defence against TB [57] and deficiencies of micronutrient such as vitamin D,
vitamin A and zinc reduce protective immune responses in murine TB models [58]. There is
also an association between human TB and micronutrient malnutrition with low levels of
substances such as vitamin A, vitamin D, iron, zinc, selenium, and plasma carotenoids [5962]. In the pre-antibiotic era cod liver oil, now known to be rich in vitamin D, was included in
the treatment of TB [63]. Phototherapy as sun exposure of the skin was also a common
treatment of TB [64], and the effect is thought to be due to the conversion of vitamin D to its
active form (1,25-dihydroxyvitamin D3) [65].
16
Several recent trials have investigated the issue of micronutrients in TB, and clinical effects of
multi-nutritional supplements and specific minerals or vitamins such as zinc, copper,
selenium, vitamin A, B complex, C, D, and E [66-72]. Epidemiological studies have found
lower levels of vitamin D in TB patients compared to controls [73], but supplementation with
vitamin D3 to patients treated for TB in Guinea Bissau showed no clinical effect [69]. In a
similar study in London there was a significant effect on time to sputum conversion, but only
in a subgroup of TB patients with a specific vitamin D receptor (VDR) polymorphism [74].
Despite that malnutrition may have a great impact on the TB epidemic, it has been difficult to
convincingly show a beneficial effect of supplementation in clinical studies, as described in a
recent Cochrane review [52]. This may be attributed to the relatively small sample sizes of
clinical studies performed so far [75].
Diagnosis Early diagnosis is a key strategy to control TB [76] but fast and accurate diagnostics are often
not available in high-endemic areas [13]. The diagnosis of TB relies on direct microscopy of
sputum smears, and where available, mycobacterial culture and polymerase chain reaction
(PCR)-based detection. Even if culture is time-consuming (3-6 weeks), and for safety reasons
a complicated procedure, it is the gold standard for diagnosis of TB [77]. For over a hundred
years, the sputum smear microscopy has been the primary tool for diagnosing TB, and is still
so in low income countries [78]. Typically a smear of a sputum sample is stained with ZiehlNeelsen stain (figure 3), which is inexpensive, fast, and specific in TB endemic areas, but
with low sensitivity (range 20 to 80%) especially in HIV co-infected TB patients [79].
Figure 3. Smear microscopy. Acid-fast bacilli shown by Ziehl-Neelsen staining, in a sputum smear
from a TB patient (×1000).
17
In order to increase the sensitivity, sputum samples may be processed by chemical or physical
methods [80] such as the concentration, sedimentation and the bleach method [81]. Staining
with auramine-fluorescent dye requires a fluorescence microscope, but can increase the
sensitivity further, even though still not satisfactory [82].
In 2010, 8 of the 22 high burden countries did not have even one laboratory with microscopy
service per 100 000 inhabitants. Among the 36 countries of high burden and high multidrugresistant (MDR) TB, less than 20 had one laboratory capable of performing culture and drug
susceptibility testing per 5 million inhabitants. Instead the clinician’s evaluation of symptoms,
sputum smear examination, and if available, chest X-ray is the most common way to diagnose
TB in low-income countries. In contrast, high-income countries with a low incidence of TB
have advanced techniques and sputum or tissue samples can be analysed with PCR within
hours to determine, not only if the patient is infected with M. tuberculosis, but also if the
strain is resistant to antibiotics [13].
In 1890, Robert Koch presented his findings of the liquid “tuberculin” that could be used as a
diagnostic tool [3], and is so still today. The tuberculin (or purified protein derivate, PPD) is
injected intradermally on the volar side of the lower arm (TST) [83]. An induration of more
than 10 mm after 48-72 hours is considered as positive, but there may be false negative results
due to anergy in a minority of patients with active TB, as well as false positive results due to
exposure to environmental mycobacteria and BCG vaccination [84]. The IGRAs have been
developed as an alternative to the TST for detection of latent TB. In contrast to TST, IGRAs
measure immune response to antigens such as ESAT-6, CFP-10 and TB7.7 (p4) expressed, by
members of the M. tuberculosis complex but only a very few other mycobacterial species, and
notably not M. bovis BCG [85]. Two commercial methods are available, the T-SPOT.TB test
(Oxford Immunotech, Abingdon, UK) and the QuantiFERON-TB Gold In-tube (Cellestis Ltd,
Carnegie, Australia) [86]. Both IGRAs and TST have a modest positive predictive value
around 3% [87]. Although IGRAs have no role in the diagnosis of active TB [88, 89], they
might reduce the number of people considered for preventive treatment in low-incident
countries [18, 86], due to their high negative predictive values [90]. In HIV-infected TB
patients, IGRAs perform similarly or slightly better than the TST [77].
18
Another alternative marker for TB is the chemotactic protein IP-10 (IFN-γ-induced protein
10), secreted from antigen presenting cells upon activation by T cells. IP-10-based tests
appear to perform comparably to the IGRAs, but may provide some additional information
for young children and HIV-infected individuals [91].
Antibody as well as antigen detection has been tried in TB diagnostics. There are antibodybased TB tests available in the market, but of little or no diagnostic value [92], but detection
of the TB antigen lipoarabinomannan (LAM) in urine of patients with TB has been a
contribution in HIV co-infected patients, even if sensitivity still is low [93].
Clinical presentation The lungs are the main site of disease for TB and are also the primary route of infection.
Patients with active TB present a wide range of symptoms from severely ill to minimal
complaints, which is also illustrated by the spectrum of host responses to the disease [94].
Common symptoms are cough, night sweats, weight loss and sometimes also haemoptysis,
fatigue and fever [95]. In post-primary (reactivating) pulmonary TB, a chest X-ray will most
commonly show an upper lobe infiltrate, or fibrosis with or without cavitation. It is also from
here that the bacteria are shed to new hosts by the infected patient expectorating airborne
droplets containing bacteria. Where resources allow, the chest X-ray may be complemented
by computed tomography and magnetic resonance imaging increasing the sensitivity [77].
HIV and TB co-infection may lead to atypical symptoms and disseminated disease causing
great difficulties in establishing a correct diagnosis [95]. For HIV-positive patients both chest
radiography and sputum examination, the common diagnostic tools in high endemic areas,
have an even poorer performance than in HIV-negative individuals. Other respiratory
infections may also be misinterpreted as sputum smear negative TB [96]. Chest X-rays of
HIV co-infected TB patients may show a pattern of primary TB with hilar lymphadenopathy
but less consolidation and cavitation [97] and the infiltrates are more often located to the
lower or middle lung fields than in HIV-negative TB patients [98] (figure 4).
19
A
C
B
Figure 4. Chest X-ray patterns of pulmonary TB. Minimal TB (A) with left upper lobe infiltrate in
an HIV-negative patient. Far advanced TB (B) involving the whole right lung and left upper lobe with
a cavity in the right upper lobe in an HIV-negative patient. Bilateral hilar enlargement (C) in an HIVpositive TB patient. (Patients from Gondar University Hospital, Ethiopia, examined by Dr Assefa
Getachew.)
Extrapulmonary TB is defined as TB in any other organ than the lung and includes lymphatic,
pleural, bone and/or joint, meningeal, visceral and genitourinary TB [99]. Extrapulmonary TB
constitutes a major portion of TB among HIV-positive, elderly and children [25, 99]. Miliary
TB is a severe form of extrapulmonary TB which may be associated with bacteraemia, fever,
fatigue and weight loss which is primarily diagnosed in immunocompromised persons such as
HIV-infected individuals or in young children [95].
From an era with no treatment available, time from symptoms to death or self-cure has been
estimated to be approximately 3 years for both smear-positive and smear-negative TB [100].
If untreated, the 10-year case fatality is reported to be around 70% (ranging from 53% to
86%) in smear-positive TB without HIV and approximately 20% in culture-positive smearnegative TB.
Treatment The drugs which are available today can lead to the cure of most TB patients harbouring
susceptible strains, but with emerging drug resistance this may soon no longer be the case,
and drug development is far behind the TB epidemic [13]. The discovery of drugs against TB
and the use of combination therapy instead of monotherapy in the 1940s and 1950s
dramatically reduced mortality in TB [13]. The first drugs in clinical use, around 1945, were
streptomycin (SM) and para-aminosalicylate (PAS) [101].
20
In order to reduce disease burden and transmission, TB drugs need to rapidly kill dividing
bacteria, prevent development of resistance and in the long run sterilize the tissue from slow
growing bacteria. The mechanisms of the drugs used today are combined to cover all these
tasks but unfortunately the time to achieve this goal is long and causes compliance problems
[101].
The present standard treatment consists of isoniazid (INH), rifampicin (RIF), pyrazinamide
(PZA) and ethambutol for two months, followed by INH and RIF for another four months
[102]. INH plays a critical role in the initial killing of replicating bacteria and has been in use
since 1952. It is a prodrug that needs activation by the bacterial catalase-peroxidase enzyme
(encoded by katG). The active form of INH targets mycolic acid synthesis, important for the
bacterial cell wall, and mutations in the gene target for this process (inhA) is the other
important factor in INH resistance [103]. RIF, introduced in 1966, acts by binding to bacterial
RNA polymerase (encoded by rpoB), blocking RNA synthase, and appears to be a more
effective sterilizing agent than INH. RIF discolours urine, faeces, sputum and tears in a redorange colour [101]. PZA is the third most important drug in TB but its role is mainly to
increase the sterilizing effect during the intensive phase (two first months). It has to be
cleaved into its active compound pyrazinoic acid and needs an acidic environment to kill the
bacteria by disruption of the cell wall. EMB was first reported in 1961 and is today used
primarily to prevent development of drug resistance to the other drugs used [101]. EMB
targets the synthesis of the bacterial cell wall components, arabinogalactan and
lipoarabinomannan, by inhibition of arabinosyltransferases (EmbA, EmbB, and EmbC) [104].
Since symptoms resolve within a few weeks after treatment initiation, patients may be
tempted to interrupt treatment leading to the survival of resistant subpopulations of the
bacteria [105]. This acquired resistance is the reason why compliance is a key factor in
reducing the burden of resistant TB [106]. To increase compliance and avoid development of
resistance, WHO launched the concept of direct observed treatment short course (DOTS) that
between 1995-2010 monitored 55 million TB patients around the world. Among new sputumpositive TB patients who were treated within the DOTS programme in Ethiopia in 2010, the
success rate was 84%, similar to worldwide results. Among relapse cases (n=4 898) in
Ethiopia 2010, 510 were tested for drug resistance and 121 were found to be MDR TB [13].
Besides improved case finding, DOTS is of major importance since treatment of patients with
sensitive strains of TB can increase the MDR TB cases if there is a low compliance [107].
21
Drug susceptibility testing is also detrimental to stop the TB epidemic, since patients with
resistant strains will otherwise be on ineffective treatment and transmission may continue
[105]. The use of rapid molecular detection of resistance in smear-positive specimens or
culture isolates has shown good accuracy. Mutations in rpoB, katG and inhA are used for
rapid PCR-based detection of resistance using the GenoType MTBDrplus assay [108-110].
Another widely spreading technique, because of its point of care strategy, is the Xpert
MTB/RIF assay (a PCR-based assay for use in a GeneXpert device). It enables detection of
TB and RIF resistance (rpoB) on sputum samples without culturing or safety laboratories,
facilitating rapid screening for TB and multidrug resistance. However false positive results for
RIF resistance in low endemic areas of MDR TB, as well as issues of high running cost have
been important concerns [111-113].
The last 20 years MDR TB, then extensively drug-resistant (XDR) TB [114], and recently
strains resistant to all TB drugs have emerged [115]. The definition of MDR TB is
M. tuberculosis resistant to at least INH and RIF, and XDR TB is MDR TB additionally
resistant to any fluoroquinolone (levofloxacin, ofloxacin or moxifloxacin) and one of three
injectable aminoglycosides (capreomycin, kanamycin, and amikacin) [107]. As soon as INH
and RIF cannot be used, the treatment duration will increase from 6 months to at least 18
months [101, 116]. Additionally, with the alternative drugs used in MDR TB and especially
XDR TB, the patients are left with insufficient treatment, significant side effects and
increased mortality rates, approaching 50-80% in some areas [117].
Even though no new molecule for treating TB have succeeded in reaching the market in the
past 40 years there are a number of TB drugs (new or repurposed) in preclinical and clinical
development. Bicyclic nitroimidazoles are drug candidates currently in phase II trials for the
treatment of tuberculosis [118, 119]. They are prodrugs, and one of the active metabolites
generates RNS thought to mediate the major part of the anaerobic effect that has been
observed [120, 121].
22
Vaccine Vaccination has long been considered the most cost-effective strategy against infectious
diseases [122]. In early 1900s, Albert Calmette and Camille Guérin isolated the causative
agent of bovine TB, M. bovis. At that time M. bovis was responsible for about 6% of all
human TB deaths in Europe, due to the use of unpasteurised milk [1]. Calmette and Guérin
observed that after growing the stain for some time they had selected an avirulent variant,
which gave immunological protection also against M. tuberculosis. After a decade they had
finally developed what we today call the BCG vaccine (bacille Calmette-Guérin) [3]. The loss
of the region of difference 1 (RD1) in BCG resulted in its attenuation [123], and also made it
possible to use the RD1-encoded 6-kDa early secretory antigenic target (ESAT-6) as an
antigen for differentiating between immunity to BCG vaccination and M. tuberculosis
exposure [124]. BCG is an intradermal vaccine, that it is cheap, safe and that protects children
efficiently against the early manifestations of TB [125]. Unfortunately, efficacy of the vaccine
against pulmonary TB in adults is highly variable and protection can decline fast. Large and
well-controlled vaccine trials have estimated the protection to range from 0 to 80% [1].
When developing new vaccines against TB it must be taken into account that, especially in
developing countries, the population can either be infected with M. tuberculosis but without
symptoms, or be fully naïve. Today, four strategies can be identified in vaccine development:
a replacement vaccine for BCG, a booster vaccine, a post-exposure vaccine or a therapeutic
vaccine [122]. A newly developed booster vaccine delayed and reduced clinical disease in
cynomolgus macaques exposed to M. tuberculosis, and also prevented reactivation of latent
infection [126]. The vaccine consists of an adjuvant, containing two of the M. tuberculosis
antigens secreted in the acute phase of infection (Ag85B and ESAT-6), and a nutrient stressinduced antigen (Rv2660c) [127]. Another promising approach is the recombinant live
vaccine (VPM1002) based on the properties of listeriolysin O (LLO), a haemolysin that forms
pores allowing the vaccine to translocate from the phagosome to the cytosol of the host cell,
and thereby induce a strong T cell response [128]. However, no vaccine candidate, that today
is in a Phase I or a Phase II trial, is estimated to be available earlier than 2020 [13].
23
The genus Mycobacterium Mycobacteria show high tolerance to environmental exposures and inhabit various reservoirs
such as water, soil, animals and humans and can be both commensals as well as highly
successful pathogens, such as M. tuberculosis, M. leprae and M. ulcerans [3]. The
M. tuberculosis complex includes strains of five species, M. tuberculosis, M. canettii,
M. africanum, M. microti, and M. bovis and the two subspecies, M. caprae and M. pinnipedii
[1, 129]. In contrast to M. tuberculosis, with an exclusive tropism for humans, M. bovis can
cause both bovine and human TB and is the cause of 5%–10% of human TB cases [130].
M. africanum is geographically limited to West Africa where it may be responsible for up to
50% of the pulmonary TB cases [131]. There are many different strains of M. tuberculosis,
but six main lineages associated with different geographical regions have been identified
[132]. It is suggested that the Beijing family of strains from Asia, and a strain family called W
and W-like are responsible for many drug-resistant cases and clonal clusters [133].
Mycobacterium tuberculosis M. tuberculosis is an intracellular pathogen infecting macrophages (figure 5) and other cells,
preferentially in tissues with high oxygen tension, such as the lungs [3]. The bacterium is nonmotile, rod-shaped, 0.3 to 0.6 µm wide and 1 to 4 µm in length, with a complex cell wall. It is
slow growing with a generation time of around 15-20 hours compared to Escherichia coli that
divides every 20 minutes. The prolonged time to divide and the ability to stay in a latent state
are some reasons for the extended treatment duration of both active and latent TB [101].
M. tuberculosis grows optimally under aerobe conditions but can switch to dormancy
programs and survive under anaerobic conditions as well. Under aerobic growth, ATP is
generated by oxidative phosphorylation from electron transport chains, while during
anaerobic conditions, alternative electron transport chains are present, such as nitrate
reductase [134].
24
Figure 5. M. tuberculosis within macrophages. Confocal microscopy analysis of the main effector
cell against M. tuberculosis, the macrophage (stained red with rhodamine-phalloidin). Macrophages
are designed to kill pathogens, but M. tuberculosis (green due to expression of the green fluorescent
protein, GFP) can evade the killing mechanisms and instead allow replication within the cells.
(Provided by Johanna Raffetseder, Linköping University.)
The sequencing of the whole genome of M. tuberculosis revealed 4,411,529 base pairs and
around 4,000 genes. It differs from other bacteria in the very large coding capacity for the
production of enzymes for lipogenesis and lipolysis. These enzymes are essential for lipolysis
of lipids from the host cell, for metabolism and bacterial cell wall synthesis of M. tuberculosis
[135]. The mycobacterial cell wall contains of long-chain fatty acids called mycolic acids,
linked to the polysaccharide arabinogalactan that is attached to peptidoglycans [3]. Due to the
lipid-rich cell wall, M. tuberculosis is nearly impermeable to basic dyes, and therefore
described as acid-fast [1]. The low permeability of the cell wall also results in a high degree
of intrinsic resistance to antibiotics [135].
25
Host immunity in tuberculosis Innate immune responses Epidemiological and experimental observations indicate effective innate immune mechanisms
against M. tuberculosis, which can be strong enough to avoid infection or induce a total
resolution of the infection in some individuals [136, 137].
Infection with M. tuberculosis is caused by inhalation of the bacteria in airborne droplets.
Once across the barrier of the epithelium in the airways, alveolar macrophages take up the
bacteria. Chemokines produced by infected epithelial cells and immune cells will recruit
neutrophils and monocytes to the site of infection [138, 139]. The monocytes will
differentiate into macrophages or dendritic cells within the tissue, and dendritic cells will
migrate to the draining lymph nodes, where mycobacterial antigens are presented to T cells
[1, 140, 141]. Activated T cells will proliferate and migrate into the infected tissue [142]. A
strong cytokine response will be mounted, with locally high levels of IFN-γ that activate
macrophages to produce antimicrobial substances [17, 137] (figure 6).
Neutrophils, recruited to the site of infection are initially beneficial for the host, but can also
cause tissue destruction [144]. In the neutrophil the bacteria are exposed to reactive oxygen
species (ROS) and antimicrobial molecules such as lactoferrin, defensins, lipocalin 2 and
cathelicidin LL-37 [145, 146]. Neutrophils are present in sputum and BAL from TB patients
and can be a niche for replication [147]. Even if neutrophils themselves cannot control the
infection, apoptotic neutrophils containing M. tuberculosis can activate macrophages and
dendritic cells [148, 149].
Subsets of NK cells and cytotoxic T cells (CD8+ T cells) are lymphocytes able to produce
granulysin, a small granule-associated peptide that may kill M. tuberculosis and can lyse
infected cells. Following delivery into the cell through the pore-forming perforin, granulysin
binds to the bacterial cell surface, and disrupts the membrane causing osmotic lysis of the
bacteria [146, 150].
26
Figure 6. Immune response in TB. Upon inhalation, M. tuberculosis is phagocytosed (1) by resident
alveolar macrophages (MΦ) in close vicinity to the small airways. The macrophage may instantly kill
the bacteria but in any case secrete pro-inflammatory cytokines (2) to recruit monocytes and
neutrophils from the blood stream (3) to the site of infection. In the tissue, monocytes will differentiate
into macrophages and dendritic cells (DC). Infected dendritic cells will migrate (4) to a regional
lymph node to present mycobacterial antigens to T cells that will proliferate (5), and in turn migrate
back into the infected tissue. A strong cytokine response of especially interferon-gamma (IFN-γ) (6)
will activate macrophages to produce TNF-α and antimicrobial substances, and as more immune cells
are recruited, a granuloma will form [17, 137, 143].
27
Within the macrophage After phagocytosis, the macrophage phagosome will normally fuse with a lysosome to form a
phagolysosome. However, M. tuberculosis is known to disturb this process [151]. The
phagolysosome is the compartment in which the macrophage can use its effector function and
still protect itself and surrounding cells from injury [152] (figure 7).
Figure 7. Antimicrobial mechanisms within the phagolysosome. The major defence mechanisms
within the macrophage phagolysosome include low pH, reactive oxygen species (ROS) produced by
NADPH oxidase, reactive nitrogen species (RNS) produced by inducible nitric oxide synthase (iNOS),
iron (Fe2+) scavengers and exporters, proteases and antimicrobial peptides such as cathelicidin [152].
28
Within the phagolysosome, mycobacteria are deprived of essential nutrients such as iron.
They are also exposed to anti-bacterial agents such as the vitamin D related peptide,
cathelicidin LL-37 [153-155], and to an oxidative burst caused by production of reactive
nitrogen species (RNS) and ROS [156]. The nicotinamide adenine dinucleotide phosphate
(NADPH) oxidase complex reduces molecular oxygen to ROS, toxic for many pathogens. It
is found both at the plasma membrane, producing extracellular ROS and within the
phagolysosome. The downstream superoxide products like hydrogen peroxide (H2O2),
hypochlorous acid (HOCl) or hydroxyl radical (OH•) are highly antimicrobial, even though
the effect on M. tuberculosis is believed to be limited [140, 146, 157].
T cell mediated response Macrophages and dendritic cells present M. tuberculosis antigen to T cells and B cells
initiating an adaptive immunity with IFN-γ production from T cells and antibody production
from B cells [17].
M. tuberculosis-specific lymphocytes appear approximately 2–3 weeks post-infection [158].
The fact that HIV-infected individuals, which exhibit decreased number and function of CD4+
T cells and defects in the IFN-γ signalling, are at high risk of TB, strongly suggests a vital
role of adaptive immunity in TB [142, 158].
CD4+ T cells can differentiate into T helper 1 (Th1), Th2, Th17 and Tregs depending on
stimuli. The Th1 response results in IFN-γ production and activation of macrophages, with
subsequent killing of intracellular bacteria by phagolysosomal fusion and effector
mechanisms of the macrophage. In contrast, the Th2 response produces B cell stimulating
factors (IL-4, IL-5, IL-10 and IL-13) and suppresses the Th1 response [3, 137]. The role of B
cells in TB is not clear, but has been recognized as potentially protective in TB [159, 160].
Depending on stimuli from T cells, macrophages polarize to M1 (classically activated) or M2
(alternatively activated) phenotype. M1 polarization is induced by Th1 cytokines and leads to
iNOS expression, ROS production and anti-microbial activity. M2 polarization is induced by
Th2 cytokines and has regulatory properties with up-regulation of arginase and limited
antimicrobial activity [161] (figure 8). Macrophages are also able to switch from one
activation state to another depending on stimuli [162].
29
A recently described subset of T cells, Th17 cells, recruits neutrophils and stimulates defensin
production through the cytokines IL-17, IL-21 and IL-22. Both Th1 and Th17 cells can be
downregulated by Treg cells, producing TGF-ß and IL-10. The precise role of Treg cells in
TB has not yet been elucidated, since inhibition of inflammation can both be beneficial and
detrimental [137]. Upregulation of Treg cells is found in BAL from latently infected
asymptomatic subjects, and may prevent further disease but also hinder complete sterilization
[163].
Helminth infection gives a dominant Th2 type immune responses, chronic immune activation
as well as up-regulated Treg activity [43]. By deworming before BCG vaccination, T cell
proliferation and IFN-γ production in peripheral blood mononuclear cells (PBMC) stimulated
with PPD could be increased [164]. Our group is now investigating the effect of deworming
TB patients with asymptomatic worm infections, based on the hypothesis that worm infection
impairs
effective
immunity
against
M. tuberculosis
(ClinicalTrials.gov
Identifier:
NCT00857116).
Figure 8. Macrophage polarization. Stimulation of macrophages with IFN-γ, TNF-α or IL-1β will
lead to the M1 phenotype, with up-regulation of iNOS, NO production and antimicrobial activity. If
stimulated with IL-4, IL-10 or TGF-β the phenotype will be a M2, with increased arginase activity
beneficial for tissue repair but not for bacterial killing [161, 165].
30
The granuloma The granuloma is the classical hallmark of TB. Initially thought to be exclusively beneficial to
the host, the role of the granuloma is now found to be more complex [166]. A granuloma is
constantly remodelled, due to the balance between pro- and anti-inflammatory immune
signals at the site of infection [158, 167, 168]. Varying proportions of macrophages,
multinucleated giant cells, foamy macrophages, epithelioid macrophages, dendritic cells, T
cells, B cells and neutrophils can be found within a granuloma, that can measure from just a
millimetre to > 2 cm [167] (figure 9) [143, 151]. At the onset of adaptive immunity, more
cells are recruited to the site of infection, extensively vascularised for this purpose. The
granuloma then acquires a more organized, stratified structure with a macrophage-rich centre
surrounded by lymphocytes that in turn may be covered with a fibrous cuff [158]. The
classically described caseous granuloma indicates a central necrotic region rich in foamy
macrophages [167] that may become hypoxic and induce a non-replicative state of the
bacteria [169].
Figure 9. The granuloma. The recruitment of immune cells to the site of infection leads to the
formation of a granuloma with varying cellular composition. To the left is a granuloma from a patient
with skin TB, showing a central accumulation of macrophages, surrounded by a border of
lymphocytes. To the right is a schematic illustration including macrophages, foamy macrophages,
neutrophils and dendritic cells, both infected and uninfected, surrounded by lymphocytes and a fibrous
cuff. [17, 143, 151, 167] (Left image provided by Thomas Schön, Linköping University and Kalmar
County Hospital.)
31
At old age, malnutrition, or in advanced HIV co-infection, containment of the infection within
the granuloma may fail and the capsule will rupture. Such extensive pathology may spill
bacilli into the airways resulting in transmission to new hosts [17]. In early HIV infection,
granulomas are still formed, but are found to be less organized as HIV proceeds [1]. The
granulomas in a TB patient are a perfect environment for HIV to spread to uninfected immune
cells [170]. M. tuberculosis itself also takes advantage of the high cell recruitment to the site
of infection and infects new cells, and the mounted immune response can turn out to be
favourable for both M. tuberculosis and HIV [170, 171].
It is difficult to study the phenomena of cavities during TB infection in humans, since
different species present a variety of responses. Cattle and guinea pigs form caseous
granulomas, but no cavities are seen in rabbits and non-human primates. Murine models (the
most common in vivo model for TB) only present granulomatous necrosis and fibrosis, but in
all species the granuloma formation is dependent on a cell-mediated response [172].
32
Nitric oxide Biochemistry In 1992, the gas nitric oxide (NO) was crowned as “molecule of the year” by Science
magazine, due to the finding that NO was an endothelium-derived relaxing factor, a mediator
of immune responses, a neurotransmitter, a cytotoxic free radical, and a signalling molecule
[173]. Nitric oxide (NO) is a small (30 Da) free radical, with diverse and opposing biological
activities. It is short-lived (lifetime of 1-1000 ms) and will quickly react with superoxide (O2-)
to form peroxynitrite (ONOO-). NO is soluble in both aqueous and hydrophobic
environments, and passes freely across cell membranes at a speed of 5-10 cell lengths in one
second [174].
NO is mainly generated by nitric oxide synthases (NOS). These large and complicated
enzymes catalyse the oxidation of L-arginine to NO and L-citrulline in a NADPH- and O2dependent manner, in the presence of five cofactors (haem, tetrahydrobiopterin (BH4), flavin
mononucleotide (FAD), flavin adenine dinucleotide (FMN) and Ca2+-calmodulin [175]
(figure 10). In absence of L-arginine, NOS instead acts as a superoxide generator [176].
HN
NH2
C
NH
NH
NOS
CH2
CH2
+ 2NADPH
+ 2O2
+ cofactors
CH2
CH2
NO
+ 2NADP
+ 2H2O
CH2
CH2
CH
CH
H 2N C
NH2
O
C
H 2N C
O
O
OH
OH
L-arginine
L-citrulline
Figure 10. Nitric oxide generation. Activated nitric oxide synthase (NOS) catalyses the formation of
NO and L-citrulline from L-arginine in the presence of nicotinamide adenine dinucleotide phosphate
(NADPH) oxygen (O2) and cofactors [175].
33
The amount and profile of NO production varies widely depending on from which of the three
NOS isoforms it is produced. Two of the isoforms are constitutive (NOS1 or neuronal, nNOS
and NOS3 or endothelial, eNOS) and one is inducible (NOS2 or iNOS) [177], although eNOS
is also able to change to an inducible form [178]. The main differences between the isoforms
are duration and magnitude of the production. The constitutive enzymes can produce short
bursts of NO while iNOS is permanently activated and can produce NO, for a prolonged
period [174]. If cofactors are available, production of NO in the microenvironment depends
on the presence of L-arginine and O2, localization of the NOS within the cell, arginase
activity and other consumptive mechanisms, such as presence of ROS, interaction with red
blood cells, and cellular metabolism [174].
Nitrite and nitrate were long thought to be end products of NO metabolism, but have been
shown to be able to be recycled back to NO under certain circumstances. If both L-arginine
and O2 are absent, NO can be produced by the acidification or reduction of nitrite, that in turn
can be produced by reduction of nitrate [179].
Nitric oxide in health and disease A lot of attention has been given NO in diverse aspects of health and disease. RNS such as
NO, nitrite, nitrate, peroxynitrite, S-nitrosothiols, nitrated fatty acids and N-nitrosamines are
continuously formed in the host. Effects range from important signalling events in the
function of regulatory proteins, cell survival and cell proliferation to toxic effects [174, 175,
179, 180]. RNS target crucial enzymes by reacting with haem, iron-sulphur (Fe-S) clusters,
cysteine and tyrosine residues, or with key metabolites such as the thiols in cysteine and
glutathione [165, 181].
The in vivo concentration of NO can vary from basal levels produced by epithelial cells
(< 2 nM) to that of an activated macrophage (> 1 µM) [174]. At lower concentrations, NO has
anti-inflammatory properties and modulate T cell functions, whereas high concentrations of
NO results in bacterial killing, T cell dysfunction as well as tissue injury [182]. In
macrophages, iNOS mediated NO production is enhanced by IFN-γ, TNF-α, bacterial
lipopolysaccharides (LPS), IL-1ß, IL-6, and IL-17, whereas transforming growth factor beta
(TGF-ß), IL-4, IL-10, IL-11, and IL-13 suppress the induction of iNOS in macrophages [165,
183].
34
In vivo production of NO can be identified by the presence of tyrosine nitration and the
relative stable metabolites of NO, nitrite and nitrate [180]. Endogenously produced NO will in
most situations react with red blood cells, be reduced to nitrate, transported to the kidneys and
excreted in the urine as such [184, 185]. Infected urine may contain considerable amounts of
nitrite as a result of bacterial nitrate reductase activity, and detection of nitrite in urine is
routinely used in the diagnosis of bacterial cystitis [186]. Acidification of urine results in NO
production from nitrite, which might explain why urinary acidification is effective in the
treatment of bacteriuria [187].
Nitric oxide can also be measured in exhaled air and is formed both in the upper and lower
airways [188-190]. Asthma [191], viral respiratory tract infections [192] and exposure to dust
[193] are associated with high levels of exhaled NO. Decreased levels of exhaled NO are seen
in patients with HIV [194] and cystic fibrosis [195]. Cigarette smoking results in reduced
levels of exhaled NO [196], but normalises after smoking cessation [197], and could be part
of the explanation of the increased risk of respiratory tract infections and chronic airway
disease seen in smokers [198, 199].
Low levels of the substrate for NO production, L-arginine, are found in patients with TB
[200, 201], malaria [202] and sepsis [73]. Clinical studies on administration of intravenous or
inhaled L-arginine have shown tumour reduction in breast cancer [203] improved endothelial
function in malaria, reduced symptoms in intermittent claudicatio, and improved pulmonary
function in cystic fibrosis [204, 205]. A diet with low L-arginine levels may impair NO
synthesis [206] and L-arginine supplementation is popular in the same way as nitrate, for
improving physical strength and to potentate sexual performance [179, 207, 208].
Nitric oxide in tuberculosis In murine models it has been shown that NO is essential for host defence against
M. tuberculosis, but the role of NO has not been fully established in man [209-211]. The
antimycobacterial effects of RNS were first shown experimentally in murine macrophages
infected with mycobacteria [212, 213]. Failure to express iNOS results in susceptibility to TB,
rapid proliferation of M. tuberculosis and early death in iNOS -/- mutated mice [211, 214216]. Murine latent TB infection can reactivate following treatment with an iNOS inhibitor
[215], and upregulation of iNOS can on the other hand result in accelerated clearance of
bacillary load [217].
35
At the site of TB infection in humans, the presence of iNOS and nitrotyrosine in macrophages
has been shown as indicators for NO production [218, 219] (figure 11). Alveolar
macrophages [220-222] as well as peripheral monocytes [223] from TB patients express
iNOS to a higher extent than controls. Levels of nitrite, IL-1ß and TNF-α were also elevated
from alveolar macrophages of TB patients compared to normal subjects, and a high
production of nitrite from alveolar macrophages was associated with increased resolution of
disease [224]. Increased levels of NO in exhaled air, and NO metabolites in urine from TB
patients indicate that the human immune defence is partly dependent on NO production in the
control of M. tuberculosis [221, 222].
Figure 11. Immunohistochemistry analysis of tissue samples from patients with TB. A caseous
granuloma (A), with surrounding inducible nitric oxide synthase (iNOS)-positive macrophages (brown
colour), from a patient with pleural TB. Alveolar macrophages (B) stain positive for nitrotyrosine in a
lung biopsy from a Swedish patient with pulmonary TB. (Provided by Thomas Schön, Linköping
University and Kalmar County Hospital.)
36
Survival strategies of M. tuberculosis M. tuberculosis has evolved to escape immune mechanisms and actually survives within
macrophages [225]. Mycobacteria owe their success to their ability to persist, without
symptoms, within the host for prolonged times, in an assumed viable but non-replicating
(dormant) state. Upon immune suppression they are able to replicate and cause disease [226].
Mycobacteria have been considered non-sporulating but recent findings suggest that
M. marinum and likely also M. bovis bacillus Calmette-Guérin can form spores as an
adaptation to a stressful environment [227, 228].
Other virulence properties of M.
tuberculosis include the ability to inhibit apoptosis, and induce necrotic cell death in
neutrophils and macrophages [229, 230]. In this way the bacteria escape the cells, evade the
host defences, and spread to uninfected cells [231] (figure 12).
Figure 12. M. tuberculosis trafficking within the macrophage. During an effective immune
response (left), phagocytosis of bacteria will lead to the formation of a phagosome followed by
acidification and several maturation steps. Finally the phagosome will fuse with lysosomes to form a
phagolysosome with a wide range of antimicrobial properties. However, M. tuberculosis can survive
(right) by preventing phagosomal maturation, acidification and fusion with the lysosomes, or may
escape into the cytosol [143, 151, 236].
By modulation of the inflammatory signal and by inhibition of phagosome-lysosome fusion,
M. tuberculosis survives within macrophages [156, 232]. Attached to the cell wall of
M. tuberculosis are lipoglycans including lipoarabinomannan (LAM) involved in this process
[233]. ESAT-6 is a virulence factor, encoded by RD1 found in several mycobacterial species
[234]. The virulence mechanisms for ESAT-6 include plasma and phagosomal membrane
lysis that release the bacteria from the phagosome into the cytosol [151, 235, 236].
37
To survive within the phagosome M. tuberculosis also inhibits transportation of iNOS to the
phagosome [237] as well as inhibits recruitment of H+-ATPase and acidification of the
phagosome, retaining a suitable pH [152]. M. tuberculosis also carries scavenger functions
against ROS and RNS in order not to face decreased growth rate, due to enzyme damage or
nutrient depletion [181, 238-240]. Pathogenic mycobacteria are inherently more resistant than
non-pathogenic to RNS and have several antioxidant systems [241-243] and clinical strains
vary in susceptibility to NO produced by acidified nitrite [241, 244, 245]. The catalase
peroxidase (katG), the alkyl hydroperoxide reductase subunit C (ahpC), superoxide dismutase
(SOD), haem proteins, bacterial proteases, thioredoxin and lipoamide dehydrogenase (Lpd)
can all scavenge ROS and RNS [246-251] (figure 13).
Figure 13. Survival strategies of M. tuberculosis. Bacterial defence mechanisms against the host
immune response include inhibition of phagosomal acidification, bacterial proteases, modification of
the bacterial cell wall to resist antimicrobial peptides, inhibition of the production of reactive oxygen
species (ROS) and reactive nitrogen species (RNS), as well as scavenger mechanisms (antioxidants) to
protect DNA and proteins from RNS and ROS [152].
38
Aims The overall aim of this thesis was to investigate the role of NO in host defence against
M. tuberculosis by combining clinical and experimental studies.
In the clinical setting our first aim was to investigate local and systemic levels of NO
during active pulmonary TB, and in household contacts to such infectious patients.
Our second aim was to assess the effect of an arginine-rich food supplement on NOproduction and clinical outcome in pulmonary TB patients during standard treatment.
Our final aim was to investigate the potential variability of clinical strains of
M. tuberculosis in susceptibility to NO, and if this variability correlated to antibiotic
resistance and ability of macrophages to control the infection, or to clinical manifestations
and treatment response in TB patients,
39
40
A voice of TB “Before the diagnosis I suffered from chest pain, loss of appetite and I
lost weight. When I heard I had TB I was afraid to transmit it to the rest
of my family. I was very sad and wondered how I got this disease. I
could not work properly because I was weak. When I came to the health
office I saw more patients with TB. I felt comfort that I would recover,
and now I´m not afraid of the disease.”
Demeku, 40-­‐year-­‐old, housewife 41
42
Materials and methods Study site Gondar College of Medicine, northern Ethiopia (figure 14), was the study site for the clinical
part of this thesis. With a population of approximately 83 million (2011), Ethiopia has the
second largest population in Africa. Apart from a few years occupation by Italy during the
Second World War, Ethiopia has never been colonised and today owns a rich and wellpreserved culture [252].
Figure 14. The Castle of Fasilades, Gondar town (left) and Simien Mountains National Park (right),
northern Ethiopia. (Photo by Jonna Idh.)
Gondar College of Medicine and Health Sciences, University of Gondar Hospital is a referral
hospital with 400 beds, serving approximately 6 million people and plays an important role in
teaching students within the medical field since decades.
HIV treatment has been available in Gondar since March 2005, profoundly affecting the
outcome of many patients including TB patients. In 2010, a TB isolation and treatment ward,
with 28 beds capacity, was built to improve the hospital TB infection control. The need is
much greater than the present beds available, but capacity building activities are noted,
including a new hospital construction with 400 beds.
The work with M. tuberculosis was taken place in BSL3 facilities at Armauer Hansen
Research Institute (AHRI), Addis Ababa, Ethiopia (Paper III) and at Linköping University
Hospital, Sweden (Paper IV).
43
Study subjects (Paper I-­‐III) Inclusion of TB patients and recruitment of household contacts took place at the DOTS clinic
within the Gondar Hospital, Ethiopia. Only outpatients fulfilling the inclusion criteria of
newly diagnosed sputum smear positive TB, age between 15 and 60, and willingness to take
part in the study were included (ClinicalTrials.gov identifier: NCT00857402). In total 317 TB
patients were eligible for inclusion (Paper I and II). Of the first 111 eligible patients, 95 were
included in Paper I. In total 180 patients were included in Paper II. Smear-positive TB was
defined as 2 of 3 morning sputum samples positive, or 1 of 3 positive with a chest X-ray and
clinical symptoms, suggestive of active pulmonary TB. Exclusion criteria were admission to
hospital, peanut allergy, pregnancy, previous treatment for TB, clinical signs or medical
treatment indicating any concomitant disease other than TB and HIV. Household contacts
(n=21) were defined as persons above 15 years of age, who continuously spend more than 12
hours per day together with a TB patient. The control group (n=63) was recruited from the
blood bank, and as for household contacts, the inclusion criteria were no symptoms of TB and
a chest X-ray without evidence of TB. Exclusion criteria were acute or chronic disease,
smoking and antibiotic or corticosteroid treatment.
Chemotherapy and nutritional supplementation (Paper II) The chemotherapy consisted of INH, PZA, RIF and ethambutol (EMB) during the intensive
phase of 2 months, followed by INH and EMB for an additional 6 months. At initiation of TB
treatment, patients were randomised (in blocks of six) to supplementation with 30 g of wheat
crackers called “daboqolo” (0.1 g L-arginine, 623 kJ) or 30 g peanuts (1 g L-arginine, 750 kJ)
daily for four weeks. Intake of all drugs and food supplementation was supervised daily. The
final outcome following treatment at eight months was registered according to the WHO
classification as cured, died, defaulted, treatment failure or transferred out [253].
Follow-­‐up AFB status by microscopy, erythrocyte sedimentation rate (SR or ESR), BMI, as well as
clinical symptoms such as haemoptysis, cough, and fever were monitored from the DOTS
centre throughout the treatment.
44
Chest X-­‐ray Chest X-ray grading was done according to the National Tuberculosis Association of the USA
in normal, minimal, moderately advanced and far advanced TB [254] and during follow-up,
the radiological response was classified as marked regression, regression, no change or
progress. The same radiologist, blinded for HIV status and treatment assignment, read all
chest X-rays.
Laboratory analyses (Paper II and III) The HIV status of the TB patients was analysed with Enzygnost Anti-HIV 1/2 Plus (DADE
BEHRING, Germany) and confirmed with Vironistika HIV Uni-Form II ag/ab (Biomérieux,
France). Serum levels of TNF-α, IL-10 and IL-12 were analysed using commercial ELISA
kits according to the instructions from the manufacturer. L-arginine was analysed by highperformance liquid chromatography (HPLC) using a modified version of the protocol
described by Carlberg [255].
Measurement nitric oxide in exhaled air and nitric oxide metabolites in urine NO in exhaled air was assessed using a chemiluminescence NO analyser (NIOX, Aerocrine
AB, Sweden), approved by the FDA [256] at a flow rate between 45–55 ml/s during 10 s with
dead space-time set to 0.5 s. The average of three measurements with the plateau
concentration within 2.5 ppb (parts per billion) or 10% was recorded. Urinary nitrite and
nitrate was measured with the Griess reaction after conversion of nitrate to nitrite by nitrate
reductase in the presence of NADPH, glucose 6-phosphate and glucose 6-phosphate
dehydrogenase, as described by Verdon et al [257].
Drug susceptibility testing and spoligotyping of M. tuberculosis (Paper III) Sputum specimens (n=50) from patients with active TB were processed and inoculated on
Lowenstein Jensen (LJ) slant media. M. tuberculosis was identified by a PCR-based approach
built on genomic deletion analysis as described elsewhere [258]. Drug susceptibility testing
for SM (2 mg/L), INH (1 mg/L), EMB (5 mg/L) and RIF (1 mg/L) was performed using the
WHO-recommended indirect proportion method on LJ media [258-260]. Spoligotyping was
performed with the oligonucleotides DRa and DRb to amplify the direct repeat (DR) regions
[261].
45
Susceptibility testing to nitric oxide (Paper III) Clinical isolates cultured on LJ medium were transferred to Middlebrook 7H9 broth and
grown until early log phase. A final concentration of 107 CFU/mL of bacteria was exposed in
duplicates to 1 mM diethylenetriamine NONOate (DETA/NO) or to PBS (used as control).
The antimicrobial effect of DETA/NO was determined by viable count (CFUs) in tenfold
dilution on Middlebrook 7H10 plates.
Luciferase-­‐based viability assay (Paper IV) To assess viability, a luciferase-based method has proven useful for M. tuberculosis [262]. In
principle, live bacteria carrying the pSMT1-plasmid encoding a hygromycin-containing gene
for selection and the gene for Vibrio harveyi luciferase, will emit flash luminescence upon
addition of the substrate n-decanal. The amount of light emitted can be measured by a
luminometer (GloMax® Multi-Detection System, Promega). For this purpose 7 clinical
strains of M. tuberculosis were transformed with the pSMT1-plasmid prepared from
recombinant E. coli DH5α as described by Garbe [263]. In addition, H37Rv harbouring the
pSMT1-plasmid was cultured on INH- and hygromycin-containing plates to select an INH
resistant mutant. All strains carrying the pSMT1-plasmid were then analysed for the presence
or absence of resistance mutations in rpoB, katG and inhA by GenoType MTBDRplus® (Hain
Lifescience) according to the instructions from the manufacturer. At a final concentration of
105 CFU/mL, strains were exposed to DETA/NO or peroxynitrite (3-morpholinosydnonimine or SIN-1 chloride) (0.1, 1 and 10 mM), with PBS used as the unexposed control.
Infection of macrophages (Paper IV) Murine macrophages (RAW 264.7; American Type Collection) were prepared for infection
by seeding in 96-well plates (25 000 cells/well) with or without stimulation (IFN-γ and LPS)
5 hours prior to infection. The macrophages were infected in triplicates in the 96-well plates
at a multiplicity of infection (MOI) of 5. Bacterial uptake was assessed by luminometry after
one hour of infection. Extracellular and intracellular growth was measured after two days, the
latter after lysis of the cells with distilled H2O. Nitrite and nitrate was measured in the
supernatants (as previously described) to detect activation of the macrophages and subsequent
NO production.
46
Statistics (Paper I-­‐IV) In Paper I data are presented as median and interquartile range (25–75%). To compare groups
the Mann-Whitney U-test was used and correlations were tested with Pearson’s correlation
test (r2). In Paper II effects of peanut supplementation, compared to daboqolo, were evaluated
by Chi-square test or Fisher’s exact test for discrete variables and Students t-test for
continuous variables. Continuous data in Paper II are expressed as means with 95%
confidence intervals. Kinetics of exhaled NO was evaluated by repeated measures ANOVA.
The experimental data in paper III are presented as median and range or first and third
quartiles (Q1-Q3) if not stated otherwise. Numerical data were analysed with Mann-Whitney
U-test, discrete data with Chi-square test and Fisher’s exact test. Multiple logistic regression
analysis with a stepwise correction was applied on variables with a p-value less than 0.1 in the
univariate analysis. In Paper IV, Students t-test was used for comparisons of two groups, a
one-way ANOVA for comparison of multiple groups and a two-way ANOVA for comparison
of multiple groups with different stimulations. A p-value of ≤ 0.05 was regarded as
statistically significant.
Ethical consideration All patients gave oral and written consent prior to inclusion. For children below 18 years,
informed and written consent was obtained from parents or guardians. The study including
Paper I, II and III was approved by the Ethics Committees at Gondar College of Medical
Sciences, Ethiopia (RPO/252/2005, GCMS), Ethiopian Science and Technology Commission
(RDHE/5-57/04, ESTC) and Karolinska Hospital, Stockholm, Sweden (KIFEK: 03-331). For
the sake of capacity building all laboratory analyses were initiated and performed at Gondar
College of Medicine (Paper I and II).
All patients included were treated according to the DOTS program [253] with addition of a
food-supplement rich in L-arginine (peanuts) or a wheat-based snack (daboqolo). L-arginine
has no known side effects in humans, but peanuts can cause allergic reactions and all study
subjects were asked about known allergy to peanuts before entering the study.
47
At the time of the study initiation, no ART (anti-retroviral therapy) was available in
government health services in Ethiopia, and none of the study participants received ART until
March 2005, when it became available at Gondar University Hospital. According to the
hospital routines the TB nurse and responsible doctor followed all patients. TB patients and
blood donors were offered pre- and post-test counselling, as recommended by WHO prior to
HIV testing.
There was no additional cost or economic incentive for the patient participating in the study.
Measurement of exhaled NO is a non-invasive and simple procedure with little discomfort.
The inconveniences for the patients regarding blood and urine sample collection were
regarded as small and in general patients found the close monitoring beneficial.
48
A voice of TB I had cough and high fever and I thought that I had TB. I think that I got
the disease as I was graining seeds at the mill. I am now unable to work
and I have to stay at home. I get food from money that I have saved. I
have one child and we stay in different rooms in the house, and the
rooms are well ventilated. We don´t sleep together. My husband is not
afraid to get TB, but I have my own cup and personal things. TB is a
difficult disease, but if you get an early diagnose, you will get well after
treatment.
Mitikie, 23-­‐year-­‐old, daily labourer 49
50
Results and discussion Systemic levels of nitric oxide in pulmonary TB To address the question of NO production during TB infection, we measured the NO
metabolites nitrite and nitrate in urine. We analysed the data with respect to HIV status, since
HIV per se is known to affect NO production. We found that HIV-negative TB patients had
significantly lower levels of nitrite and nitrate in urine than controls, and that household
contacts to TB patients had the highest levels (Paper I). Healthy blood donors also had higher
levels of nitrite and nitrate in urine, indicating that effective immune responses with NO
Nitrite/Nitrate (!M)
production might be protective against infection and active disease (figure 15).
3000
*
2500
*
*
*
2000
1500
1000
500
0
HIV-/TB
HIV+/TB
HC
BD
Figure 15. Levels of urinary nitrite and nitrate. HIV-negative TB patients (HIV-/TB, n=58), HIVpositive TB patients (HIV+/TB, n=35), household contacts to smear-positive TB patients (HC, n=12)
and blood donors (BD, n=45). Data are presented as median and interquartile range. * (p < 0.05).
Levels of nitrite and nitrate in urine from TB patients were previously found to be elevated
[222]. This discordance could partly be explained by higher levels of urinary nitrite and
nitrate in the control group in the present study. The significant increase in urinary nitrite and
nitrate found in HIV-positive compared to HIV-negative TB patients is in line with the
finding that HIV infection increases systemic production of NO [264, 265], and that
macrophages infected with HIV express iNOS and produce NO [266].
There can be profound effects of diet on urinary levels of nitric oxide metabolites [267]. After
a nitrate-rich meal, urinary levels return to baseline values within 24 hours with a maximum
concentration at 4-6 hours after intake [268, 269], leading to between-day variations [267].
Approximately 65-70% of the dietary nitrate is excreted in the urine [268] with some loss in
51
faeces and sweat [185]. We have tried to limit the influence of diet on our results, by adapting
an overnight fast and collecting the first morning urine. Although high outliers in nitrite and
nitrate may be due to intake of a nitrate-rich meal, it is unlikely to introduce significant bias
on comparisons at the group level.
Local levels of nitric oxide in pulmonary TB In order to more accurately assess the NO production from the site of infection in pulmonary
TB, we measured NO in exhaled air and found that HIV-positive TB patients had
significantly lower levels of NO in exhaled air compared to healthy household contacts and
controls. The same tendency was seen in HIV-negative TB patients (figure 16). This finding
is well in line with our hypothesis that NO is essential for protection against M. tuberculosis
infection.
30
*
*
25
FeNO (ppb)
20
15
10
5
0
HIV-/TB
HIV+/TB
HC
BD
Figure 16. Levels of exhaled NO (FeNO). HIV-negative TB patients (HIV-/TB, n=59), HIV-positive
TB patients (HIV+/TB, n=36), household contacts to smear positive TB patients (HC, n=17) and
blood donors (BD, n=46). Data are presented as median and interquartile range. ppb (parts per billion).
* (p < 0.05).
In contrast to our finding, Wang et al [221] previously showed that pulmonary TB patients
presented higher levels of exhaled NO compared to healthy controls. In the study by Wang et
al, patients with poor nutritional status were excluded to avoid the influence of poor nutrition
on immunity. In our study population, HIV-positive as well as HIV-negative TB patients had
a median BMI of 16 kg/m2 with an estimated weight loss of 5-6 kg. Hence, they were in a
more deteriorated nutritional status, supporting the link between immune defence and
nutritional status. Wang et al also found that alveolar macrophages from patients with more
extensive disease had a lesser capacity to generate NO. This is in line with our finding that
52
patients with lower urinary nitrite and nitrate (< 1000 µM) had significantly higher levels of
IL-10 (known to downregulate iNOS) [270, 271], compared to patients with higher urinary
nitrite and nitrate (> 1000 µM) (Paper II).
Lower levels of nasal [272] and oral [194] exhaled NO have previously been shown in HIV
patients without TB, supporting the effect of HIV infection on the immune system’s capacity
to generate sufficient levels of NO in the lungs during TB infection. In line with this, there
was a higher rate of HIV-negative TB patients with exhaled NO > 25 ppb even though there
was no difference in median levels of exhaled NO between HIV-positive and HIV-negative
TB patients. There was also a trend towards higher levels of the pro-inflammatory cytokine,
IL-12 in HIV-negative patients (Paper I). Considering NO as protective against TB, this
supports the fact that HIV-positive individuals are at higher risk of M. tuberculosis infection,
or reactivation of infection, than HIV-negative individuals [1].
As described by Olivieri et al [273], it is difficult to determine reference values of exhaled
NO for a healthy population, since there are several factors affecting the measurable levels,
although a range from 2.6 to 28.8 ppb in men, and from 1.6 to 21.5 ppb in women has been
suggested [274]. Levels of exhaled NO reported in Paper I and II are within this range, even
though a well-defined reference range is more important in diagnostics or prognostics. In
measurements of exhaled NO we used a flow rate of 45–55 ml/s, which is recommended for
asthma [256]. As pulmonary TB infection is mainly localized to the lung parenchyma, a
higher expiratory flow rate [190, 275] may be needed to measure NO production optimally in
TB patients, although this might prove difficult in clinical practice since few patients may be
able to comply with such measurements in the acute stage of the disease.
Treating TB with peanuts – are you nuts? Malnutrition can lead to increased susceptibility to infections [54] and is known to be
associated with TB [57]. To further investigate the cause of low NO levels during TB, we
measured the precursor L-arginine in plasma from TB patients. We found no difference in
levels of plasma L-arginine between HIV-positive and HIV-negative TB patients, but all TB
patients had lower levels compared to a healthy cohort in the same area (60 µM vs. 119 µM)
[200]. The same was observed by Zea et al [201], where TB patients had lower levels of
plasma L-arginine than healthy controls.
53
Even though the contribution of dietary supplement must be seen in the perspective of the
effective TB-drugs available [58, 75], supplementation of nutrients may shorten duration of
treatment, as well as increase recovery in TB patients [54]. The hypothesis that
supplementation of L-arginine may be beneficial for TB patients had previously been
investigated by our group. Patients were then randomised to L-arginine or placebo and a
significant improvement (shorter duration of cough, increased weight gain and higher sputum
conversion rate) was seen in HIV-negative TB patients [276], thus we now wanted to
investigate the effect of a food supplement rich in L-arginine in addition to the standard TB
treatment.
Peanuts are known to be rich in L-arginine and are locally available at the study site in
Gondar, and were therefore used as the arginine-rich supplement. As a control we chose
another locally available snack of roasted wheat crackers called “daboqolo”. Our primary
outcome was cure rate, but as previously described, the effect of TB drugs is strong, with a
high cure rate by itself. Secondary outcomes were chosen to reflect a faster improvement of
symptoms and subsequently, an improved immune status. No effect of the arginine-rich food
was found either in primary or secondary outcome, but based on our previous study on Larginine supplementation [276], and other studies on micronutrient supplementation [67, 71],
HIV status is likely to have an impact. In the subgroup analysis of HIV status, HIV-positive
TB patients receiving the food supplement rich in L-arginine showed a cure rate of 84%
(31/37) compared to 53% (17/32) among the patients randomised to the low L-arginine
supplement (table 1).
In the previous arginine study, improvement of L-arginine was seen in the HIV-negative TB
patients instead of the HIV-positive TB patients. In the present study all patients received
nutritional supplementation and to elucidate the impact of the increased food intake itself, we
compared our findings to the follow-up of 60 smear positive TB patients, who did not receive
any food supplementation, and observed that the food supplementation per se had an overall
effect on sedimentation rate and the number of treatment failures.
54
Table 1. Effects of high (peanuts) versus low (daboqolo) L-arginine-containing food on clinical
outcome in TB patients.
HIV+/TB+
Peanuts
Primary outcome (8 months) % (n)
N 95% CI
Cured
83.8 (31) 37 71.0-96.2
Not cured
16.2 (6) 37 4.0-28.7
Treatment failure
5.4 (2)
37 0-13.2
Defaulter
2.7 (1)
37 0-8.2
Died
8.1 (3)
37 0-17.3
Transferred out
0
37
Secondary outcomes
Sputum smear conversion, 2m 88.9 (32) 36 78.0-99.7
Weight > 10 %, 2 m
44.4 (16) 36 27.0-61.5
Presence of cough, 2 m
50 (18)
36 33.0-67.2
Mean
N 95% CI
SR (mm/h), 2 m
57.9
36 49.0-66.7
CXR improvement, 2 m
1.9
29 1.7-2.1
eNO (ppb), 2 m
17.3
19 14.9-19.7
eNO (ppb), 5 m
21.5
18 16.3-6.7
uNO(!M), 2 m
2290
36 1726-2854
Daboqolo
% (n)
N 95% CI
53.1 (17) 32 34.8-71.4
46.9 (15) 32 28.6-65.2
12.5 (4) 32 4.0-24.6
15.6 (5) 32 2.3-28.9
15.6 (5) 32 2.3-28.9
3.1 (1)
32 0-9.5
0.006
0.005
NS
0.07
NS
NS
86.7 (26)
20.7 (6)
55.2 (16)
Mean
60.0
1.9
18.3
21.3
1753
NS
0.05
NS
p
NS
NS
NS
NS
NS
30
29
29
N
29
20
17
15
28
73.8-99.6
5.0-36.4
35.9-74.4
95% CI
51.3-68.4
1.5-2.2
14.5-22.1
16.6-25.9
1218-2289
p
!
Wheat in “daboqolo” is rich in the amino acids glutamine and glutamate, which can be
converted to L-citrulline in an intact intestinal mucosa, and in the kidney further to L-arginine
[206]. It is possible that this is what happens in HIV-negative TB patients, thereby masking
the effect of the arginine-rich food in this group. However in HIV-positive TB patients,
enteropathy with reduced levels of serum citrulline is common [277], indicating a reduced
conversion of glutamine in the intestine, thereby explaining the improvement seen after
arginine-rich food supplement in this sub-group.
Nitric oxide – does it kill the bug? Even though the host may be capable of fast and high production of NO, there is evidence
that M. tuberculosis can resist the toxic effects of RNS and ROS [239]. The influence of
strain variation on the outcome of infection with M. tuberculosis is an emerging area of
research [278]. Identifying such resistance mechanisms, directed towards the immune
responses, is of great interest when finding new drug targets and vaccines. To investigate this
we cultured clinical sputum specimens from pulmonary TB patients, and exposed each strain
to NO in vitro (Paper III).
55
All the isolates were identified as M. tuberculosis by PCR and by spoligotyping. Nine known
clusters and 4 unique clusters were identified. In fifty clinical strains exposed to NO there was
a time and dose-dependant susceptibility to NO. The median survival after 24 hours exposure
to 1 mM DETA/NO, was 10% (range 0-60%) (figure 17). Even though dormancy regions are
upregulated after NO exposure [240], suggesting that NO rather has a bacteriostatic than a
bactericidal effect on M. tuberculosis, no viable bacteria following exposure to 10 mM
DETA/NO were found even after 8 weeks of culture on solid media.
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Figure 17. Reduced NO susceptibility in spoligotype-based clusters of M. tuberculosis. Survival of
clinical isolates 24 hours after exposure to the NO donor DETA/NO. Presence of resistance to firstline TB drugs is indicated with circles; isoniazid (INH-R), streptomycin (SM-R) and rifampin (RIFR). Each point represents a mean value of duplicates and the dashed line is the median survival of all
50 isolates.
Nitric oxide resistance – from bench to bedside We hypothesised that with equal capacity to produce NO, a host infected with a NO tolerant
strain of M. tuberculosis would present a more severe disease or slower recovery compared to
a host infected with a NO susceptible strain.
We found no significant correlation between susceptibility of the strains to NO and treatment
outcome or sputum conversion, but the four patients who did not increase in weight were all
infected with NO tolerant strains. Three of these were HIV-positive, and HIV co-infection is
known to be associated with weight loss in TB patients [95], although there was no significant
56
difference in rate of weight gain between HIV-negative and HIV-positive TB patients in the
present study. In conclusion, we found no convincing impact of the level of NO tolerance of
the infecting strain on treatment response, but to be able to adjust for factors such as HIV
infection and antibiotic resistance larger sample sizes are needed.
Antibiotic resistance associated to nitric oxide susceptibility – a novel finding Resistance to first-line TB drugs (INH resistance (4/50), SM resistance (3/50) and RIF
resistance (1/50)) were distributed over 6 spoligotype-based clusters (figure 17). There was a
clear association between NO tolerance and resistance to first-line TB drugs, INH in
particular. INH-resistant strains showed a median survival of 53% (Q1-Q3, 32-57%) when
exposed to NO compared to 10% (Q1-Q3, 10-50%, p=0.006) in INH-susceptible strains.
Intrigued by the finding that reduced NO susceptibility could be linked to antibiotic
resistance, and in particular to INH resistance, we used a newly developed model with
luciferase-expressing M. tuberculosis to study bacterial viability upon NO exposure (Paper
IV). This model avoids time-consuming culturing, which may take several weeks before
reaching results, and enables direct measurement of viable M. tuberculosis [262]. Five INHresistant and 2 fully susceptible clinical strains as well as H37Rv were transformed with the
luciferase-expressing plasmid and exposed to NO. Again INH-resistant strains showed a
trend towards increased tolerance to NO (figure 18). An important observation was also the
variability in NO susceptibility among clinical isolates, and an increased tolerance to NO in
some of the clinical strains compared to H37Rv. This indicates that this laboratory strain,
widely used in TB research, may not reflect the phenotype of clinical isolates in terms of NO
susceptibility.
Since NO readily reacts with superoxide to produce peroxynitrite, we also exposed the strains
to this reactive compound (SIN-1). In our system NO was relatively more toxic than
peroxynitrite, although the comparison is difficult to fully assess in our experimental system,
considering the differences in chemical features and time of donation by the two compounds.
The half-life of the NO donor used is approximately 20 hours [279], while the peroxynitrite
generator will give a short burst of peroxynitrite for about 15-30 min [280]. M. tuberculosis
will thereby be exposed to NO over a longer period, probably more effective than a short
exposure time to SIN-1.
57
Among the 50 isolates from Ethiopian TB patients in paper III, there is no data on the exact
mutations responsible for antibiotic resistance, but the most common gene involved in INH
resistance in Ethiopia is katG [281]. Among the 7 luciferase-expressing strains, four strains
with an inhA mutation showed significant increase in tolerance to NO, compared to inhA
60
60
40
40
% survival
% survival
wild type (figure 18).
20
0
INH-susceptible
20
0
INH-resistant
inhA WT
inhA mutated
Figure 18. Survival of INH-resistant and inhA-mutated strains of M. tuberculosis exposed to
0.1 mM DETA/NO. Experiments were repeated three times and each point on the graph represents
the median of triplicates in one experiment. The horizontal lines represents is the median value of all
strains (n=9). Data are presented as % survival compared to unexposed control and a significant
difference (p ≤ 0.05) is indicated with *.
In a further attempt to investigate the association of INH resistance to NO susceptibility,
luciferase-expressing H37Rv was subcultured in INH to select a subset of the strain with
resistance to INH. This was successfully achieved, but with no significant effect on the NO
tolerance. No mutations were found in the katG or the inhA genes, suggesting that there are
other mutations responsible for the INH resistance [103] without any association to NO
tolerance.
INH is a prodrug that needs activation by the bacterial catalase-peroxidase enzyme (encoded
by katG) [282]. As katG mutations lead to reduced activity, or total loss of bacterial catalase,
they also increase the susceptibility of the bacteria to ROS and RNS [283]. This can be
compensated for by ahpC upregulation and increased tolerance to NO [284]. Other
mechanisms of INH resistance are mutations in ahpC, encoding for bacterial alkyl
hydroperoxidase, ndh genes encoding NADH dehydrogenase [103], and inhA encoding enoylacyl reductase. The latter is a NADH-dependent enzyme essential in mycolic acid
58
biosynthesis for the bacterial cell wall [285] and is inhibited by catalase-mediated formation
of an INH-NAD adduct [103, 286].
Since RNIs are potent lipid peroxidising and nitrating agents, affecting cell wall lipids [287],
it could be speculated that the variation of NO susceptibility in inhA-mutated strain could be
due to modifications of the cell wall, and compensatory upregulation of alternative defence
mechanisms. This merits further investigation since the understanding of interactions between
different drug resistance mutations and compensatory mutations are important to be able to
make predictions about global epidemics of MDR TB and XDR TB [288], as well as for
identification of new drug targets.
Survival of M. tuberculosis within the macrophage To investigate if NO susceptibility had any impact on the ability of macrophages to control
the infection, 7 clinical strains and H37Rv were investigated in activated macrophages. Even
if human macrophages produce NO at the site of infection, it is not yet possible to explore this
mechanism in vitro [289]. Thus we used the murine cell line (RAW 264.7) with the capacity
to generate NO after stimulation with IFN-γ/LPS (figure 19).
35
Ratio D2/D0
30
25
20
15
10
5
E3
C 94
D
C 2
BT 155
1
B
0
BT 8-7
5
B
BT 08
7
B
0 6
BT 2-1
3
B
02 4
-1
41
E1
15
5
-R
R
IN
37
37
R
v
H
H
H
v
0
Unstimulated
IFN-γ/LPS
Figure 19. Effect of IFN-γ/LPS stimulation on growth control of clinical strains of
M. tuberculosis in macrophages. The overall capacity to control M. tuberculosis infection in
unstimulated and IFN-γ/LPS stimulated macrophages (RAW 264.7). Data are presented as fold change
of total bacterial load (intra- and extracellular) day 2 divided by day 0 (ratio D2/D0) and bars represent
median with error bars at min and max from three experiments run in triplicates.
59
There was variability in the macrophage capacity to phagocytose the different strains, as well
as differences in their efficiency to achieve growth control of the different strains. However,
there was a general agreement with the cell-free system, that IFN-γ/LPS-stimulated
macrophages (producing NO) controlled the infection more effectively. A limitation of our
study is that we did not include an iNOS inhibitor to quantify at which level the reduction in
growth within the macrophages was strictly NO-dependent. Preliminary experiments with
H37Ra (an avirulent laboratory strain of M. tuberculosis) showed that macrophages treated
with iNOS inhibitors were clearly less effective in controlling bacterial growth than NOproducing cells, which has been shown also with virulent strains of M. tuberculosis [213].
The inability to identify a significant correlation between survival to NO in a cell-free system
with survival in activated macrophages, most probably reflects that macrophages do not only
use NO, but several other bactericidal systems, such as ROS production and cathelicidin, to
control infection [290, 291] and that the microenvironment where the NO exposure takes
place affects the potency of NO [181].
60
Concluding remarks This thesis provides insight into the role of nitric oxide in the immune defence against
M. tuberculosis by applying preclinical findings in clinical studies.
We found that household contacts to TB patients had high levels of nitric oxide, and that
active TB was associated with low nitric oxide levels, indicating a protective role of nitric
oxide in TB. In HIV-positive TB patients, low levels of exhaled nitric oxide were associated
with reduced cure rate, and an arginine-rich food supplement increased cure rate among these
patients. We also found that nitric oxide is toxic for M. tuberculosis both in vitro and in
activated macrophages, and that reduced nitric oxide susceptibility is associated with
isoniazid resistance.
The novel finding of an association between reduced susceptibility to nitric oxide and
isoniazid resistance in clinical strains of M. tuberculosis, could be of importance in
identifying new therapeutic targets. Although new vaccines and antimicrobial drugs are
urgently needed to combat TB, complementary ways to strengthen the host response is
beneficial. Increasing nitric oxide production in macrophages at the site of infection could be
one strategy to reach this goal, both in terms of protection against developing infection, as
well as for increasing cure rates in those that already suffer from active disease (figure 20 and
21). Although larger multicentre studies are warranted, arginine-rich food supplementation
can be recommended to malnourished HIV co-infected patients on TB treatment.
Take-­‐home message Nitric oxide kills M. tuberculosis and peanuts can contribute to the treatment of TB. 61
Concluding figure Figure 20. A model of the role of NO, generated by the work included in this thesis. Nitric oxide
(NO) in the host is part of the immune defence in tuberculosis (TB). High levels of NO (↑↑↑) upon
exposure to M. tuberculosis may clear the bacteria even before infection occurs. Intermediate levels of
NO (↑↑) may lead to latent infection, where the cell-mediated immunity (CMI) and NO control the
infection for a lifetime. In case of insufficient levels of NO production (↑↓) by a poor CMI, or because
of NO resistance or other virulence factors in the bacterial strain, bacterial replication will continue
and cause active disease. In latent disease, malnutrition may lead to deficiency of L-arginine, the
substrate for NO production, and reduced levels of NO, with subsequent loss of control of the
infection and development of active disease.
62
Future areas of interest Figure 21. Future areas involving NO-based therapies. The outcome of the M. tuberculosis-human
encounter depends both on host and bacterial characteristics, and can result in bacterial clearing or
persistent infection. Four main areas can be identified as targets for new NO-based interventions.
Immunization of the host (1) to prepare for an early and strong NO response upon M. tuberculosis
encounter. Therapeutic strategies (2) to increase NO output post-exposure, either by L-arginine
supplementation or iNOS up-regulation. The bacterial defence mechanisms (3) against NO and other
anti-bacterial agents are potential targets for new drugs. Immune modulatory therapies (4), unleashing
contained bacteria by inhibition of cell mediated immunity (CMI) of NO production, could be
combined with new drugs to shorten treatment and to reach sterilization.
63
64
A voice of TB “When I heard I had TB I knew that it was curable so I was not afraid. I
don´t know if I had been in contact with any person with TB. In order to
get the diagnosis I had to go to many health centres, but finally a private
clinic and an X-ray confirmed my TB. The disease does not affect my life
much since I work as usual with no difficulties and I am sure I will be
well after the treatment.”
Hlmarim, 51-­‐year-­‐old, governmental employee 65
66
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84
Acknowledgements I am sincerely grateful to everyone who has contributed to this thesis, and I would especially like to
mention the following people:
My SUPER supervisor Thomas Schön for your never-ending energy, enthusiasm and knowledge (it
has amazed me from day one, and still does), and for your support both when things go well and when
life is tough.
Professor Olle Stendahl, my supervisor, for welcoming me to the department despite my more
adventurous than scientific attitude (I hope the latter has improved a bit), for trust in independence and
support when asked for (from wherever in the world you happened to be).
Professor Tommy Sundqvist, the ideas man, for an endless stream of hypothesis, for teaching me
statistics and nitric oxide chemistry, and for finding something interesting in ever so boring data.
Ebba Abate, my co-pilot in this work and my new brother, you are the best!
Professor Sven Britton, the founder of the fruitful Swedish-Ethiopian collaboration, for the admirable
attitude of not accepting things as they are but instead do all your best to change it. Daniel Elias, for
introducing me to the Ethiopian culture and for continuing to take part in all the work despite your
move to Denmark. Anna Westman, Karolinska Hospital, for your encouraging and warm attitude and
for all the work you did during the start-up of the clinical studies. Mekidim Mekonnen, for your
impressive and accurate works in the lab, I wish you all the best. Wassihun Wedajo, for contributing
with your excellent lab skills and for a lot of good fun. Dr Abraham Aseffa, director of AHRI, for
your dedication to science and to Ethiopia.
My friends and colleges at Gondar College of Medicine. Dr Feleke Moges, for taking excellent care
of me my first time in Gondar. Dr Assefa Getachew, for examining hundreds of X-rays, but most for
your hospitality, teaching attitude and positive spirit. Nurse Meseret Senbeto, for long and reliable
work at the DOTS and for wonderful times with your family. Nurse Tezera Jemere, for excellent
monitoring of patients at the DOTS. Nurse Meseret Atale, for being an extraordinary research nurse,
for doing your very best during all circumstances and for being a true caring and responsible person.
Assistant Saba Ekuby, for your extended service at the DOTS, helping out with all kinds of things,
for your lovely way and for always opening your door for my family and me. Mrs Sabash, for inviting
me to your home and for your way of communicating about serious things with no common language
us between. Atanaw Kassahun, for not giving up your search for patients lost to follow-up. Yeshi
Kassahun and Abebech Molla, for entering endless amounts of data. Belay Anagaw, for support in
the lab and for always cheering me up. Aschalew Gelaw, my Ethiopian lab mate, for the all your work
in the lab and for your wonderful sense of humour. Martha Alemayehu for excellent contributions in
the lab and for being a very nice friend. Lamesgin Muluken for your sharp way and accurate work in
the lab. Tigist Feleke and Alemnesh Kehali, for support with samples and lab work and for being so
friendly. Dr Ermias Diro, for taking medical responsibility at the study site and for interesting
discussions in the field of research, medicine and life in general. Dr Shitaye Alemu, for your friendly
attitude and your courage, inspiring many women around you. Addis Alemu, Endalkachew Melese,
Yared Wondikun, Belete Ayele, Afework Kassu, Yohannes Asfawossen, Yohannes Mikena,
Alemnigus Yigzaw, Yenew Kebede, for all contributing to the clinical part of this thesis.
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My friends and colleges at the Department of Medical Microbiology. Katarina Tejle, for helping me
out with just about everything, lending me materials and sharing your skills in the lab, but most of all
for becoming a close friend. Tony Forslund, for admirable patience in teaching me the basics in lab
work and supporting me at the lab. Daniel Eklund, for sharing methods and skills and for your
relaxed and friendly way. Robert Blomgran, for making long working hours in the office not so bad
after all, and for correcting and encouraging me during the write-up of this thesis. Maria Lerm, with a
capacity greater than most of us, for your open mind and valuable inputs. Johanna Raffetseder for
invaluable support in the lab and for your positive, friendly and quick way. Elsje Pienaar, for your
calm and sharp appearance, your genuine helpfulness, and for introducing me to Craig and his family.
Clara Braian, for your friendly way and for proofreading this thesis, even though you were on
maternity leave. Amanda Welin, for introducing me to macrophages and mycobacteria, for making
complicated things seem easy and for you colourful and happy appearance. Alexander Persson, for
fun times at work and at conferences, and for all the chitchatting when things were slow. Martin
Winberg, for being such a nice roommate, we miss your singing. Hanna Abdalla for your
friendliness and incredible ability to stay strong far from your family. Mary Esping, and previously
Ingegerd Wranne, for your incredible capacity to keep papers and figures in order for us with absent
minds. Kristina Orselius, for sharing your experience of laboratory work on nitric oxide. Lena
Svensson, for the friendly and relaxed atmosphere that you bring, and for loving dogs. Elisabeth
Hollén for supporting me during my first small steps into science and nitric oxide, for good talks and
great music. Henrik Andersson, for sharing your extensive knowledge in immunology and for your
friendly and helpful attitude. Christina Samuelsson, for excellent technical assistance and for making
it more cosy at work than at home. Medical students Märta Kroon, Petra Axenram, Anna Bornefall
and Sofia Forsgren, for being so nice and contributing to the overall work on TB. TB lab personal:
Britt-Marie, Sara, Ann-Marie, Eva, Slavica and Helena for coping with us around, and Michaela
Nordvall, for efficient technical support. Marie Larsson, for your friendly way and for supporting me
in the TB lab, I wish I had your lab skills. Jacob Paues, Lennart Persson and Sven-Göran
Fransson, for providing a clinical connection to the TB research also in Sweden. Lars Brudin, for
statistical support. Professor Pia Forsberg, my mentor, for support in both private and occupational
matters, and for being an extraordinary person. Kristian Ängeby, Jim Werngren and Pontus Juréen,
for providing strains, and for sharp reflections on results and methods. TB lab personal at
Karolinska Hospital, for being so friendly during my stay time. Susanna Brighenti, for enthusiasm
and fruitful discussions on TB.
My extended family of close and loved friends, especially Emma and Charlotte, Pia-Maria,
Emma H, Joachim, Cecilia, Andreas, Anna-Kajsa, David, Ellinor, Jesper, Ulf, Åsa, Carl and
Henrietta, Helena E, Helena R, the family Modin, Katarina, Marie and Lisa with families, MajBritt, the Kindgren family, the Lundberg family, the Lindstein family, the Hegethorn family, the
Wareborn family, the Blomqvist family, and the extended Idh family, for your sweet support,
excellent crisis management and for putting things in perspective.
Most of all, I want to thank my family. Pappa B-G, for love, understanding and for not giving up.
Mamma Maria, for your love and encouragement. Lillasyster Yster (Nina), for unconditional love
and for keeping me down on earth.
Last but not least, thank you Rickard, for taking my hand,
and heading out on a joint journey through life.
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