Innate and adaptive immune responses in the lungs ...418702/FULLTEXT01.pdf · persistent bacterial infections which escape the innate immune system are eliminated by the adaptive

Licentiate thesis from the Department of Immunology,

Wenner-Gren Institute, Stockholm University, Sweden

Innate and adaptive immune responses in the lungs.

Contribution to protection against mycobacterial infections

Olga Daniela Chuquimia Flores

Stockholm 2011

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SUMMARY

Host defense against Mycobacterium tuberculosis (Mtb) is mediated by a combination of

innate and adaptive immunity. In this thesis we investigated the role of components of innate

system such as TLR2 signalling and alveolar epithelial cells type II (AEC II) in the immune

responses against mycobacterial infections.

Since TLR2 has been shown to be important in the defense against mycobacterial infections;

in paper I we investigated the role of TLR2 to generate acquired immune responses. We

compared both humoral and cellular immune responses in TLR2-/- and WT (wild type) mice

immunized with the mycobacterial antigens 19kDa (TLR2 ligand) or Ag85A (non-TLR2

ligand). We did not find any differences in the humoral responses in both mouse strains.

However, we found some deficiencies in the T cell memory compartment of TLR2-/- mice

immunized with 19kDa. In addition, the antigen presenting cells (APC) compartment in

TLR2-/- mice, for instance bone marrow derived macrophages (BMM) and pulmonary

macrophages (PM) in this study, has also shown deficiencies. This effect was more evident

when PM were used as APC. We next evaluated the responses in both BMM and PM upon

stimulation with anti-CD40 and TLR ligands where PM were the low responders to TLR2

ligand and to anti-CD40 both in the production of different cytokines and in the up-regulation

of the co-stimulatory molecules. Together, our results have demonstrated the importance of

TLR2 in the generation of specific immune responses.

In paper II, we investigated the role of AEC II in the defense against mycobacterial

infections. AEC II have been suggested to play an important role in the local immune

responses to inhaled pathogens. First, we compared murine AEC II with PM in their ability to

take up and control mycobacterial growth and their capacity as APCs. AEC II were able to

internalize and control bacterial growth as well as presenting antigen to memory T cells. In

addition, both cells types were compared in their capacity to produce cytokines, chemokines

and other factors where AEC II exhibited a different pattern of secretion than PM. Also, a

more complete profile of AEC II responses reveled that AEC II were able to secrete different

factors important to generated various effects in others cells. The major finding in this study

was that upon TNF, AEC II produced MCP-1 a chemokine involved in the recruitment

monocytes/macrophages to the sites of infection. Since TNF is predominantely produced by

macrophages, we speculate that both cell types may communicate and influence each other.

In conclusion, our results provide more evidence of the important role of AEC II in the

immune responses in the respiratory tract.

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LIST OF PAPERS

This thesis is based on the following original manuscript, which will be referred to by their

roman numeral in the text.

I. Muhammad J. Rahman, Olga D. Chuquimia, Dagbjort H. Petursdottir,

Natalia Periolo, Mahavir Singh and Carmen Fernández. Contribution of TLR2

signalling to the specific immune response. Deficiencies in T-cell memory and

antigen presenting cell compartments in the TLR2 knockout mice. Manuscript.

II. Olga D. Chuquimia, Dagbjort H. Petursdottir, Muhammad J. Rahman,

Katharina Hartl, Mahavir Singh and Carmen Fernández. The role of alveolar

epithelial cell type II in initiating and shaping pulmonary immune responses.

Communication between the innate and adaptive immune systems. Submitted.

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TABLE OF CONTENTS Page

SUMMARY 2

LIST OF PAPER 3

TABLE OF CONTENTS 4

LIST OF ABBREVIATIONS 5

INTRODUCTION 6

Mucosal immunity in the respiratory tract 6

Innate and adaptive responses in the lung 7

Tuberculosis 10

Mycobacterium tuberculosis 10

Pathogenesis of tuberculosis 11

Immune responses against tuberculosis 12

Innate immune cells 12

Adaptive immune cells 16

Role of TLRs in mycobacterial Infection 18

Cytokines and chemokines in TB infection 19

Pro-inflammatory Cytokines 19

Anti-inflammatory cytokines 22

Chemokines 23

GM-CSF 25

PRESENT STUDY 26

Aims 26

General aim 26

Specific aims 26

Materials and Methods 26

Results and Discussion 27

Paper I 27

Paper II 31

Concluding Remarks 35

Future Plans 35

ACKNOWLEDGEMENTS 36

REFERENCES 37

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LIST OF ABBREVIATIONS

AEC II Alveolar epithelial cells type II

AM Alveolar macrophages

APC Antigen-presenting cell

BCG Bacillus Calmette-Guérin

CT Cholera toxin

DC Dendritic cells

DC-SIGN Dendritic cell-specific intracellular adhesion molecule-3-grabbing non-integrin

ELISA Enzyme- linked immunosorbet assay

HIV Human immunodefiency virus

HK-BCG Heat- killed BCG

IFN-α Interferon alpha

IFN-γ Interferon gamma

IL Interleukin

i.n. Intranasal

IP-10 Interferon gamma-induced protein 10 kDa

i.v. Intravenous

kDa KiloDalton

KC keratinocyte-derived chemokine

LAM Lipoarabinomannan

LM lipomannan

Lys-BCG BCG lysate

LPS Lipopolysaccharide

MALT Mucosa-associated lymphoid tissue

MCP-1 Monocyte-chemotactic protein-1

MHC Major Histocompatibility complex

MIP-2 Macrophage-inflammatory protein-2

MMP-9 Matrix metallopeptidase-9

Mtb Mycobacterium tuberculosis

Pam3 Lipopeptide tripalmitoyl-S-glycerylcysteine

PM Pulmonary macrophages

s.c. Subcutaneously

TB Tuberculosis

TCR T-cell receptor

TGF-β Transforming growth factor beta-β

TLRs Toll-like receptors

TNF Tumor-necrosis factor

WT Wild type

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INTRODUCTION

Mucosal immunity in the respiratory tract

The mucosal surface is a major portal of entry for pathogens in the respiratory,

gastrointestinal, urogenital tracks and other moist surfaces in the lining of reproductive track.

The mucosal surfaces have the task of providing protection of the mucus membrane against

infection as a natural barrier, preventing the uptake of antigens (microorganisms and foreign

particles). Another important task is the preservation of mucosal homeostasis with the

production low immune responses to harmless antigens (mucosal tolerance).

In the respiratory tract, the mucosal (local) immune system and systemic immune

system are involved in the defense and protection against inhaled microorganisms (control of

pathogen movement and infection levels). However, the innate branch of the mucosal

immune system is critical for controlling infection in the early stages of exposure to inhaled

microorganisms. Once the inhaled bacteria arrive to the mucosa surface, they are trapped by

mucus and removed toward the pharynx and swallowed. Antimicrobial peptides are produced

and secreted by the surface epithelium of the respiratory tracks to kill many microorganisms

that have penetrated the mucous layer. Those bacteria that are resistant to antimicrobial

peptides are killed by a variety of reactive oxygen species produced by phagocytes. Finally,

persistent bacterial infections which escape the innate immune system are eliminated by the

adaptive immune system (1,2).

An important function on the respiratory tract is the maintenance of local

immunological homeostasis and therefore the integrity of gas exchange surfaces related to the

discriminatory functions of the immune system to their limits. The incoming antigens in the

respiratory tract are dominated by highly immunogenic but harmless proteins of plant and

animal origin. The host could undergo premature death from chronic airway inflammation if

these harmless proteins could induce strong adaptive immune responses. For that, it is

imperative that the respiratory mucosal immune system discriminates the background

antigenic noise from the much rarer signals transmitted by pathogen associated antigens to

produce an immunological balance in the air ways. Memory T-cell responses have to be

tightly regulated to minimize damage in the local epithelial cell surfaces, particularly for the

alveolar gas exchange surfaces, as these tissues contain the largest vascular bed in the body

and function as a magnet for circulating memory (3).

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Innate and adaptive responses in the lung

The lung consists of two major anatomic compartments: the vascular and the airway

compartment. Endothelial cells in the arteries, veins, and capillaries are the cells most

actively involved in an inflammatory response in the vascular system. Epithelial cells

therefore could have similar function in the respiratory compartment. Distal airway-epithelial

cells and alveolar epithelial cells are vital for maintenance of the pulmonary air-blood barrier.

The alveolar epithelium is composed of Type I Alveolar epithelial cells or membranous

pneumocytes and Type II Alveolar epithelial cells (AEC II) or granular pneumocytes. Type I

epithelial cells are squamous, large thin cells that cover 90-95% of the alveolar surface and

are essentially involved in gaseous exchange. AEC II are cuboidal cells that constitute 15%

of total parenchymal lungs cells and cover about 7% of the total alveolar surface. AEC II

contain characteristic lamellar inclusion bodies, the intracellular storage of pulmonary

surface-active material (surfactant) (4,5). They are also considered as progenitor cells capable

of proliferating and differentiating into type I cells. Recent evidence suggests that airway

epithelial cells might also act as immune effector cells in response to harmful exogenous

stimuli. Several studies have shown that airway epithelial cells express on their surface

adhesion molecules and secrete various immune molecules such as cytokines, chemokines

and other factors (6-11). Through the expression and production of these inflammatory

mediators, not only the vascular but also the airway epithelium is thought to play an

important role in the initiation and exacerbation of an inflammatory response within the

airways.

Also, in the lungs the presence of biosensors as pattern recognition receptors (PRR) is

important in the lung immune responses. The toll-like receptors (TLRs) are able to induce a

signalling pathway with activation of kinases and nuclear factors, resulting in transcription of

inflammatory mediators such as tumor necrosis factor (TNF) or members of the interleukin

family (12). In addition, the leukocyte homing to sites of acute inflammation is a crucial step

during an inflammatory response. Adhesion molecules play a major role in the inflammatory

process by mediating adherence of leukocytes to the endothelium and initiating extravasation

of these cells. The intercellular adhesion molecule-1 (ICAM-1), a member of the

immunoglobulin superfamily, is a cell surface glycoprotein and a ligand for the β2-integrins

CD11a/CD18 and CD11b/CD18 on leukocytes. It is up-regulated by a variety of

inflammatory stimuli such as endotoxin and different cytokines. The ICAM-1 is expressed by

endothelial and epithelial cells but its functional role could be different in both cells.

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In terms of the different immunological functions, the lung has also been divided into two

compartments: the conducting airways overlayed by mucosal tissue, and the lung

parenchyma Figure 1, which comprises thin-walled alveoli that are specialized for gas

exchange.

The basic morphology of the conducting airways is similar and consists of a surface

epithelium composed largely of ciliated and secretory cells overlying subepithelial tissue that

consists predominantly of connective tissues and glands. The proportion and type of these

elements vary at different levels of the conducting system. The cells in the conducting

airways with the production of locally secreted immunoglobulin (Ig)A provide mechanisms

for mucociliary clearance of inhaled antigens. The cells of the immune system are present

Figure 1: Local immune cells in the two lung compartments.

(Nature rev.2008; 8:142-152, reprinted with permission from NPG)

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within the epithelium of all conducting airways. Dendritic cells (DC) and macrophages have

dense networks in the epithelium. The major population of DC is composed of myeloid DC

subset but also the plasmacytoid DC can be found in the mucosa airway. Resident airway

mucosal DC are specialized in immune surveillance with a high capacity for antigen uptake,

but a reduced ability to stimulate T cells. They are strategically positioned for antigen uptake

both within and directly under the surface epithelium and continuously sample incoming

airborne antigen by extending their dendrites through the intact epithelial layer into the

airway lumen (13,14).

Lymphocytes can be found either singly or in clusters in the airway lamina propria and

in the submucosa. Effector and memory CD4+ and CD8

+ T cells (defined by their expression

of CD45RO), as well as B cells are also present in the airway mucosa (in the intraepithelial

and within the underlying lamina propria) and may play a role in the constitution of

bronchial-associated lymphoid tissue (15) which has been suggested to have a significant role

in local immunological homeostasis in the respiratory tract early in life. Most intraepithelial T

cells express CD8+, whereas CD4

+ T cells are more frequently found in the lamina propria

(16,17). B cells might be contributing to local antigen presentation in the lymph nodes that

drain the lungs. Plasma cells are in the lamina propia and the role of these cells is mainly the

production of polymeric IgA but also IgM to clear inhaled pathogens (18). Other types of

cells such as mast cells, basophiles, eosinophils and neutrophils have also been found in the

lamina propria.

The lung parenchyma consists of alveoli that are separated by fine vascularised

interstitial tissue. Lung parenchymal DC, macrophages, and T cells arise in the alveolar

space, the alveolar-epithelial layer and the interstitium. In the steady-state conditions the

alveolar space (as reflected by broncho-alveolar lavage fluid composition) consists of 80-

90% macrophages, the remainder being T cells and DCs. However, it has been found a large

sequestered T-cell population in the lung parenchyma but its role in the local mucosal

homeostasis is still unclear (18-24). The lung parenchyma also contains B cells and mast

cells, but no plasma cells.

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Tuberculosis

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis

(Mtb), which most commonly affects the lungs. It is transmitted from person to person via

droplets from the throat and lungs of people with an active respiratory disease. In 2009, there

were an estimated of 9.4 million cases of TB globally (equivalent to 137 cases per 100 000

population). Of this incident an estimated 1.1 million (12%) were HIV-positive. These

numbers are slightly lower than those reported in previous years, reflecting better estimates as

well as reductions in HIV prevalence in the general population. Of these HIV-positive TB

cases, approximately 80% are in the African region (24).

Bacillus Calmette-Guérin (BCG) is the unique available vaccine against TB, BCG is

prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus,

Mycobacterium bovis. The vaccination programs with BCG in new-borns and infants protect

and reduce risk against childhood TB meningitis and milliary TB (vaccinated near 90%).

However, its efficacy diminishes with time and it affords only variable protection against

pulmonary disease. Consequently, to find a more effective TB vaccine is considered a global

priority (25-27). Over one hundred TB vaccine candidates (DNA and subunit vaccines) have

been developed, using different approaches to induce protective immunity (27). Until now,

there is not a new vaccine able to achieve a level of protection better than BCG. However,

models of vaccination using priming with BCG and boosting with mycobacterial antigens

strategy have been used previously to achieve high levels of protection (28). We have

demonstrated that BCG priming and HBHA (heparin-binding hemagglutinin, a mycobacterial

antigen) boosting in neonatal mice induce protective immune responses (29).

Mycobacterium tuberculosis

Mtb is a fairly large non motile rod-shaped bacterium distantly related to the

Actinomycetes. Many nonpathogenic mycobacteria are components of the normal flora in

humans, found most often in dry and oily locals. The rods are 2-4 µm in length and 0.2-0.5

µm in width. The tubercle bacilli are obligatory aerobic intracellular pathogens with

predilection for the lung tissue rich in oxygen supply. Mtb has a slow generation time, 15-20

hours; this physiological characteristic may contribute to its virulence (30). The diagnostic of

TB is based on the determination of TB in sputum samples, physical examination,

radiography, PCR and other types of studies such as culture of mycobacteria in the

Löwenstein-Jensen (LJ), Kirchner, or Middlebrook media (7H9, 7H10, and 7H11) (30).

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Pathogenesis of Tuberculosis

Most frequently Mtb enter the body via the respiratory tract. Once Mtb gets in the

pulmonary alveoli, the TB infection (primary TB) begins with the invasion and replication of

the tubercle bacilli into the endosomes of macrophages. However, it has been described that

DC can take up and transport the bacteria from the site of infection in the lungs to the local

lymph nodes (31). Also, alveolar epithelial cells and other surrounding cells in the respiratory

tract have been reported to be invaded by Mtb (32-34). The invasion of Mtb is the first host-

pathogen interaction that decides the outcome of infection. The production and secretion of

IFN-γ (which can activate macrophages to destroy the intracellular bacteria) and other

cytokines and chemokines produced by CD4+ T cells are critical in the host is response to

Mtb. The infected DC are observed as the primary APC responsible for activating CD4+

T

cells as part of the adaptive immune response. It has been described that the modulation of

DC could lead to weak immunity against Mtb allowing a latent infection (35). CD8+ T cells

can also directly kill infected cells.

Within 2 to 6 weeks of infection, cell-mediated immunity develops, and there is an

influx of lymphocytes, fibroblasts and activated macrophages into the lesions resulting in

granuloma formation. The granuloma functions are overall to prevent the dissemination of

Mtb and to provide a local environment for communication between immune cells. However,

the granuloma formation does not always eliminate the mycobacteria. Thus, the bacteria can

become dormant giving rise to a latent infection. Another consequence of granuloma

formations is the development of cell death and tissue necrosis. Dead macrophages form a

caseum and there is an exponential growth of the bacilli contained in the caseous centers of

the granuloma. The bacilli may remain forever within the granuloma, get re-activated later or

may get discharged into the airways after an enormous increase in number, necrosis of

bronchi and cavitation. The tissue destruction and necrosis produce fibrosis, which represents

the last-attempt defense mechanism of the host when all other mechanism failed. The

secondary TB lesions start with the Mtb dissemination from the site of initial infection in the

lung through the lymphatic nodes or bloodstream to other parts of the lungs and in the body,

the apex of the lung and the regional lymph nodes being favored sites for the Mtb. Around

15% of TB patients develop extrapulmonary TB in the pleura, lymphatics, bones, genito-

urinary system, meninges, peritoneum, or skin. On the other hand, about 90-95% of the

people infected with Mtb have asymptomatic, latent TB infection, with only a 10% lifetime

chance that a latent infection will progress to TB disease. (30,35,36).

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Immune responses against Tuberculosis

Although, macrophages serve as the long-term host for mycobacteria, Mtb infects and

actives DC as well as other cell types in the lungs. However, activated macrophages can kill

intracellular bacteria and participate in a protective T helper cell type 1 (Th1) response. In

mycobacterial infection, Th1-type cytokines have been shown to be essential for protective

immunity (37). However, other factors are involved and can decide the outcome of the

disease. Both CD4+ and CD8

+ T cells provide protection against Mtb (38). However, T-cell

effector function can be achieved only after priming and differentiation has occurred.

Innate immune cells

Macrophages

In the lungs, macrophages are considered the first line of defense against inhaled

microorganism. Although, they are morphologically similar, it is possible that the function of

macrophages is regulated according to their localization in the lungs. In fact, they have been

considered to form several different subpopulations on the basis of their anatomic location

such as the airway macrophages, situated at or under the epithelial lining of conducting

airways, the alveolar interstitial macrophage, the alveolar surface macrophage, the

intravascular macrophages, located adjacent to the capillary endothelial cell and the pleural

macrophages resident in the pleura space (39). Normally, the principal function of

macrophages is phagocytosis and uptake of antigen from the immune system to defend local

tissues from the development of specific immune responses. Alveolar macrophages (AM)

have been shown to take up most of the particulate material that is delivered intranasally but

they do not migrate to regional lymphoid nodes. In addition, AM can self-regulate their

functions on request to mount an appropriate immune response, and they are not considered

to have a significant role in antigen presentation (40).

Mtb uses macrophages as its preferred habitat (30), which is important for its survival.

Macrophage Mtb interactions and the role of macrophage in host response can be

summarized under the following headings: surface binding of Mtb to macrophages;

phagosome-lysosome fusion; mycobacterial growth inhibition/killing; recruitment of

accessory immune cells for the local inflammatory response and presentation of antigens to T

cells for the development of acquired immunity (41). Complement receptors (CR1, CR2,

CR3 and CR4), mannose receptors and other cell surface receptor molecules play an

important role in binding the organisms to the phagocytes. The interaction between mannose-

receptors on phagocytic cells and mycobacteria seems to be mediated through the

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mycobacterial surface glycoprotein lipoarabinomannan (LAM) (42). Prostaglandin E2 and

IL-4, a Th2-type cytokine, up-regulate complement and mannose receptors functions. IFN-γ

decreases the receptor expression, resulting in diminished ability of the Mtb to adhere to

macrophages. According to some studies, prevention of phagolysosomal fusion is a

mechanism by which Mtb survives inside macrophages (40-43). The production of IFN-γ by

T cells is a consequence of antigen presentation from infected macrophages. TNF and IL-12

(43) are secreted by activated monocyte/macrophages as shown in murine studies (44,45). A

major effector function responsible for antimycobacterial activity induced by IFN-γ and TNF

is nitric oxide (NO), generation of reactive oxygen intermediates (ROI) and reactive nitrogen

intermediates (RNI) (46).

Dendritic cells

DC are APC specialized for T-cell activation. Immature DC are bone marrow-derived

cells present in most non-lymphoid tissues, where they act as sentinel cells against incoming

pathogens. The typical phenotype of DC in human lungs is the high expression of MHC class

II and CD205 (type I C-type lectin, that has been described as a DC-specific multilectin

receptor), together with low expression of CD8, CD40, CD80 and CD86. In this state, DC are

able to take up and process antigens. DC can also act as a potent APC in situ in some other

diseases eg. Asthma (20,21,23,47,48). The recruitment of airway mucosal DC in response to

bacterial stimuli is through the chemokine receptor (CCR)1 and CCR5. However, responses

to virus or protein recall antigens use alternative chemokine-ligand receptor combinations. In

addition, CCR7 is the principal molecule involved in the migration of antigen-bearing lung

DC to regional lymphoid nodes. Different subpopulations of DC have been identified in the

lungs (49,50). The myeloid DC subsets, in particular, have been found in the airway mucosa

as important subpopulations contributing to the local immunity. The plasmacytoid DC have

also been identified as another important subset in the tolerance to foreign antigens. A further

indication of the potentially important functions of the plasmacytoid DC subset is their

distinct pattern of TLR expression and the high capacity to produce interferon-(IFN)-α in

response to microbial stimuli (50-54). However, unlike myeloid DC, human plasmacytoid

DC have poor APC activity and there is no evidence for plasmacytoid DC migration out from

the lung (3,20).

In TB, DC can take up the Mtb through receptors such as TLR, DC-specific ICAM-3-

grabbing non-integrin (DC-SIGN) binding to mycobacterial mannose residues; the mannose

receptor; the DEC205 (CD205), the CR3 and scavenger receptors (53-57). Mtb affects

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antigen processing through inhibition of phagosome maturation and improved survival in

macrophages. The outcome of mycobacteria in DC is controversial with reports from no

growth only to survival or unrestricted growth (58-60). Following endocytosis of

mycobacteria, activated DCs migrate to draining lymph nodes, where they prime T cells (30).

Upon Mtb exposure, engagement of TLRs on DC induces the secretion of cytokines IL-1β,

IL-12, IL-18, and IFN-α, which stimulates T cells to produce IFN-γ, a cytokine essential for

the bactericidal activity of DC through induction of reactive oxygen intermediates. Another

important task of DC is their role as key APC cells in TB since they express MHC class I and

II, CD1 (a MHC-like molecule) and co stimulatory molecules such as CD80, CD86 and

CD40 needed to prime naïve T cells. The mycobacterial T-cell inducing antigens are either

lipids presented on CD1 molecules, or proteins such as ESAT-6, CFP-10, Ag85, or

lipoprotein p19 (61). Several studies have also demonstrated that DC can induce protection

in mice infected with BCG (62)

Alveolar epithelial cells type II (AEC II)

The strategic location of AEC II in the interface between the outside and pulmonary

vasculature leads them to be considered as important immunologic modulators in the alveolar

space. AEC II may therefore be important in the first line of defense against inhaled

pathogens for instance Mtb. Ultra structural criteria used to identify AEC II are the presence

of lamellar bodies, apical microvilli and specific junctional proteins (47,48). These cells

perform different functions including the ion transport, alveolar repair in response to injury

and regulations of surfactant metabolism. AEC II are the source of lipid pulmonary

surfactants (SP-A, SP-B, SP-C and SP-D). SP-B and SP-C enhance the biophysical properties

of the lipid components of surfactant, including the lowering of surface tension, whereas SP-

A and SP-D are involved in innate immune defense enhancing the clearance of a variety of

lung pathogens by AM (63). In addition, AEC II secrete antimicrobial proteins, such as

lysozymes, and complement components (e.g., C2,C3,C4 and C5) and a variety of cytokines,

chemokines and diffusible factors that may be involved in the activation of AM and other cell

types during lung inflammation (64,65).

AEC II have also been implicated in the modulation of the innate and adaptive

immunity due to the expression of PRRs on their surfaces such as TLR2 and TLR4 (10,66).

Also, the constitutive expression of MHC II in AEC II (67) is consistent with the possible

function of AEC II as antigen presenting cells in the lungs. Other studies have suggested the

possible contribution of AEC II in T-cell tolerance to exogenous or innocuous antigens in the

15

lungs due to their lack of the expression of co-stimulatory molecules needed for the activation

of T cells (68). Moreover, AEC II were proposed to contribute in balancing inflammatory

and regulatory T cell responses in the lung by connecting innate and adaptive immune

mechanisms, and to establish peripheral T- cell tolerance to respiratory self-antigen (69).

The role of AEC II is still not clear in Mtb infection. However, previous studies have

demonstrated that Mtb is able to invade and replicate inside AEC II derived cell lines (70,71).

Also, the production of NO and IFN-γ by Mtb infected human AEC II cell line have

suggested the possible role of AEC II in the responses against Mtb (72,73). Furthermore, a

contribution of AEC II in the adaptive response against Mtb has been demonstrated when

AEC II present mycobacterial antigens to antigen-specific T cells (74). On the other hand,

there are many suggestions about the interaction of AEC II and macrophages due to their

close localization in the alveoli. One study has shown that bacterial growth was reduced in

Mtb or Mycobacterium avium-infected macrophages when these cells were co-cultured with

an AEC II-cell line (75). Also, AEC II presented an increase in the mitochondrial RNA

expression of surfactant proteins, cytokines, chemokines and GM-CSF, indicating a crucial

role of AEC II in the potentiation of macrophage-anti mycobacterial activity (10,75).

Neutrophils

Neutrophils are considered to be the earliest cells recruited to sites where antigen

enters in the body and/or inflammatory signals are triggered. They also have well-

characterized microbicidal mechanisms such as those dependent on oxygen and the formation

of neutrophil extracellular traps (76). In mice, the role played by neutrophils in TB is

controversial. These cells have been detected in the beginning of an infection as well as

several days after infection (77,78) , and they were thought to have an important role in the

control of mycobacterial growth. However, the capacity of neutrophils to kill mycobacteria is

still not fully understood. It might be that the major role of these cells is in granuloma

formation (79)

and of the transfer of their own microbicidal molecules to infected

macrophages (80).

Natural killer (NK) cells and Natural Killer T (NKT) cells

NK cells are a small fraction of lymphocytes, already specialized to display a

cytotoxic activity against certain types of target cells, especially, host cells that have become

infected with virus and host cells that have become cancerous. NK cells lack TCRs or BCRs.

The activation of NK cells is through the signals from activating and inhibitory receptors. In

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addition, NK cells can secrete different cytokines and chemokines such as IFN-γ, IFN-α and

IL-22 (81-83). NK cells improve also the function of γδ T cells, another type of lymphocytes

which play a role in the immune response against Mtb due to their capacities to act as

cytolytic cells and in their secretion of IFN-γ (84).

In TB, NK cells become activated during the early response to pulmonary TB with

large production of IFN-γ but their depletion does not markedly alter host resistance to Mtb

infection (41,57). However, the major contribution of NK cells in the defense against Mtb

could be the secretion of IL-22, an important cytokine involved in the promotion of

phagosome-lysosome fusion in macrophages (85).

In contrast to NK cells, NKT cells are T cells with αβ TCRs and with the expression

of some of the cell-surface molecules also present on NK cells. NKT cells recognize

glycolipid antigen presented by a MHC-like molecule called CD1d. Also, NKT cells are able

to secrete large amounts of either Type 1 cytokines such as IFN-γ or Type 2 cytokines such

as IL-4 and IL-13. Perhaps the function of NKT cells is to provide and early rapid help for a

cell-mediated immune response (IFN-γ) and or an antibody-mediated response (IL-4) distinct

from that seen by conventional T-helper cells which required several days to be activated. If

so, NKT cells would represent a link between innate and adaptive immunity. It has been

found that murine CD1d-restricted NKT cells mediate protection against Mtb in vivo (86).

Adaptive immune cells

CD4+ T cells

CD4+ T cells and their derived cytokines are crucial in the defense and protection

against Mtb. CD4+ T cells are recognising antigenic peptides in the context of gene products

encoded by the major histocompatibility complex (MHC) class II. The frequency of IFN-γ-

producing CD4+

T cells has been widely used as a correlation of protection against Mtb. The

role of IFN-γ in protection against TB has been clearly shown in mice with a disrupted IFN-γ

gene and in humans with mutations in genes involved in the IFN-γ and IL-12 pathways (38,

87-89). Also, mice deficient in either CD4 or MHC II molecules have shown an increase in

susceptibility to Mtb (90).

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CD8+ T cells

CD8+

T cells are also important for effective T-cell immunity against Mtb. CD8

+ T

cells are activated after interaction of the TCR with a processed antigen bound to MHC class

I. Also, CD8+ T cells have effector functions, such

as cytolysis and release of potent

cytokines such as IFN-γ and TNF (91). Mice

deficient in critical components of the MHC

class I processing and presentation pathway are more susceptible to Mtb infection (92). In

addition, CD1b (a human MHC-like molecule)-restricted CD8+ T cells are

able to inhibit the

growth of Mtb in vitro (93).

B cells

Classically, B cells and antibodies are thought to offer no significant contribution in

the protection against Mtb. However, the role for B cells in host immune response to Mtb,

has been suggested when mycobacteria-infected polymeric-Ig-receptor deficient mice display

a delayed immune response with an increase in the bacterial growth in the lungs, implicating

a role for secretory IgA in an optimal TB immunity (94). In addition, activated DC were able

to internalize more efficiently BCG when BCG were coated with specific antibodies (95).

Furthermore, the role of B cells as antigen presenting cell in TB has been suggested (96).

γδ T cells

γδ T cells may directly recognize small mycobacterial peptic antigens and non-protein

ligands in the absence of antigen-presenting cells. In mice, a single contact with Mtb

substantially increases the number of γδ T cells, but not the number of αβ T

cells (CD4

+ and

CD8+ T cells) in the draining lymph nodes.

In mice infected with Mtb, γδ T cells accumulate

at the site of infection and seem to be necessary for early containment

of mycobacterial

infections (97). Like γδ T cells, CD1-restricted T cells do not react with mycobacterial protein

antigens in the context of MHC class I or class II molecules. Instead, these

T cells react with

mycobacterial lipids or glycolipid antigens bound to CD1 on antigen-presenting cells.

CD1

molecules have close structural resemblance to MHC class

I but are relatively

nonpolymorphic. In mycobacterial infections, several different T-cell subsets have been

found to interact with CD1, including CD4

- CD8

- (double-negative) T cells, CD4

+ or CD8

+

single-positive T cells, and T cells. CD1-restricted T cells display cytotoxic activity and are

able to produce IFN-γ (97-101).

18

Role of TLRs in mycobacterial infection

The TLRs are probably the best studied group of PRRs. TLR as biosensors have a

critical role in the host defense recognizing conserved structures in bacteria and viruses to

induce innate immune responses and to prime antigen-specific adaptive immunity. In

humans, the Toll family comprises about 10 family members with a highly conserved

intracellular signalling domain that resembles the signalling domain found in the mammalian

IL-1 receptor. After activation of the receptor, this Toll/IL-1 receptor (TIR) domain interacts

with different adaptor molecules that through activation of NF-κB and/or IFN-regulatory

factors (IRF) leading to the transcription activation of a broad panel of genes. The homology

between Toll-like family members also extends to the extracellular part of the receptor.

Multiple leucine-rich repeats (between 19 and 25) and a single membrane proximal cysteine

motive are involved in specific binding to a wide variety of microbial and endogenous

ligands. Unclear is how such conserved domains in Toll-like members are able to recognize

different ligands specifically, also given that hydrophobic interactions seem to be a prominent

factor (102). In the respiratory tract, since the lung is continuously exposed to a wide variety

of airborne antigens and toxins, it is essential to have an appropriate faster and selective

immune response. This response requires precise regulation of both proinflammatory and

anti-inflammatory responses. Members of TLRs family in the initiate innate as well as

adaptive immune responses following their binding to pathogens associated molecular

patterns (PAMP). For example, the TLR2 binds to bacterial lipoproteins and lipoteichoic

(LTA), TLR4 recognizes LPS from most gram-negative bacteria. TLR5 recognizes bacterial

flagellin (monomer that makes up the filament of bacteria flagella), TLR7 and TLR8

recognise single stranded RNA from viruses and TLR9 mediates cellular response to DNA

containing unmethylated CpG motif present in bacterial DNA (102,103).

Innate immune responses after mycobacterial infection are initiated after recognition

of mycobacterial components by PRRs like TLRs. The immune-costimulatory activity of

mycobacterial DNA is attributed to the presence of palindromic sequences including the 5’-

CG-3’ motif “CpG motif” to bind TLR9 (104,105). The mycobacterial cell wall consists of

several glycolipids. Among these, lipoarabinomannan (LAM), lipomannan(LM) and

phosphatidyl-myo-inositol mannoside (PIM) are recognized by TLR2. The 19kDa lipoprotein

of Mtb also activates macrophages via TLR2 (102,106). The in vivo importance of the TLR-

mediated signals in host defense against Mtb was emphasised in studies using mice lacking

MyD88, a critical component in TLR signalling. MyD88-deficient mice are highly

susceptible to airborne infection with Mtb (85,107). In contrast to mice lacking MyD88, mice

19

lacking individual TLR are not dramatically susceptible to Mtb infection. Susceptibility of

TLR2-deficient mice to Mtb infection varies in different studies (108,109). BCG and Mtb

infected TLR2-/- and TLR4-/- mice were more susceptible to mycobacterial infections at

early stages of infections. Moreover, TLR2-/- but not TLR4-/-infected macrophages decrease

the antibacterial activity (110). In addition, in an in vitro study, the implication of TLR2 but

not TLR4 resulted in an impairment of IFN-γ mediated killing when macrophages were

stimulated with different TLR2 and TLR4 ligands (111). TLR4-deficient mice did not show

high susceptibility to Mtb infection (112,113). A report demonstrated that TLR9-deficient

mice are susceptible to Mtb infection and mice lacking both TLR2 and TLR9 are more

susceptible (114). These findings indicate that multiple TLRs might be involved in

mycobacterial recognition. However, mice deficient in TLR2, TLR4 and TLR9 express a

milder phenotype than MyD88 deficient mice in mycobacterial infection (115).

Cytokines and chemokines in Mtb infection

The immune response against Mtb is complex, involving many cytokines and

chemokines. Cytokines play an important role in the regulation of host immune response

against the mycobacteria by controlling effector functions of immune and non-immune cells.

Chemokines and chemokine receptors lead the cells to specific sites within the tissues; it is

probable that these cells participate in the granuloma formation seen in TB. The severity of

TB is defined by differences in the activation of immune regulatory chemokines and

cytokines.

Pro-inflammatory cytokines

IL-12

IL-12 is a key player in host defense against Mtb. IL-12 is produced mainly by

phagocytic cells such as DC and macrophages once Mtb is taken up. IL-12 has a crucial role

in the induction of IFN-γ production by T cells and NK cells (116). In TB, IL-12 has been

detected in lung infiltrates, in pleurisy, in granulomas, and in lymphadenitis. The expression

of IL-12 receptors is also increased in cells from bronchoalveolar lavage fluid from patients

with active pulmonary tuberculosis (117). The protective role of IL-12 can be inferred from

the observation that IL-12 deficient mice are highly susceptible to mycobacterial infections

(118). In humans

suffering from recurrent non-tuberculous mycobacterial infections,

deleterious genetic mutations in the genes encoding IL-12p40

and IL-12R have been

identified. These patients display a reduced capacity to produce IFN-γ (119). Apparently, IL-

20

12 is a regulatory

cytokine which connects the innate and adaptive host response

to

mycobacteria and probably it exerts its protective effects mainly through the induction of

IFN-γ.

TNF

Monocytes, macrophages, and DC infected with mycobacteria or in contact with

mycobacterial products induce the production of TNF, a prototype pro-inflammatory cytokine

(119). TNF plays a key role in granuloma formation, induces macrophage activation, and has

immunoregulatory properties in mice (120). TNF is also important for containment of latent

infection in granuloma in TB patients (121). Deficient mice which are unable to make TNF

or lack the TNF receptor p55 display an increased susceptibility for mycobacteria (121). In

human TB, no TNF gene mutations have been found and no positive associations have yet

been established between gene polymorphism for TNF and disease susceptibility (122).

IFN-γ

Protective anti-mycobacterial immune responses involve mainly IFN-γ secreted by T

cells to activate macrophages and do induce their microbicidal functions. However, other

cells such as NK cells and DC can produce IFN-γ in response to Mtb early during infection.

This will lead to the development of antigen-specific IFN-γ-producing CD4+T cells (123).

Also, it has been suggested that macrophages can produce IFN-γ in Mtb-infected mice (124).

However, Mtb has developed mechanisms to limit the activation of macrophage by IFN-γ

(111,121). In addition, it has been found in a human study, that the production of IFN-γ not

always correlates with mycobacterial inhibition (23). In line with this, it has been described in

mice that IFN-γ secretion from animals immunized with mycobacterial antigens does not

always correlate with protection (125). Thus, even if IFN-γ is important to generate immune

responses and protection against Mtb, it is not sufficient for eliminating these mycobacteria.

IL-18

IL-18, a pro-inflammatory cytokine which shares many features with IL-1, was

initially discovered as an IFN-γ-inducing factor, acting in synergy with IL-12. It has since

been found that IL-18 also stimulates the production of other proinflammatory

cytokines,

chemokines, and transcription factors. There is evidence for a protective role of IL-18 during

mycobacterial infections since IL-18 deficient mice are highly susceptible to

Mtb (126,127).

In mice infected with Mybobacterium leprae, resistance is correlated with a higher expression

21

of IL-18 (128). The major effect of IL-18 in this model seems to be the induction

of IFN-γ.

Indeed, in TB pleurisy, parallel concentrations of IL-18 and IFN-γ were found (129). Also,

Mtb-mediated production of IL-18 by peripheral blood mononuclear cells is

reduced in TB

patients, and this reduction may be responsible for reduced IFN-γ production (129). The role

of IL-18 in response against Mtb seems to be protective since lack IL-18 induces a decrease

of protective Th1 response, probably leading to mycobacterial propagation.

IL-1β

IL-1β is mainly produced by monocytes, macrophages and DC. In TB patients, IL-1β

is expressed in excess in the granulomatous lymph nodes from patients with tuberculosis

(130). Studies in IL-1α and -1β double-deficient mice suggest an important role of Il-1β in

TB (131). It has been found that IL-1R type I-deficient mice (which do not respond to IL-1)

display an increased mycobacterial outgrowth and also defective granuloma formation after

infection with Mtb (132). The major role of IL-1β in host defense against Mtb seems to be a

critical ligand to induce signalling in determining the MyD88 dependent phenotype (133).

Thus IL-1β is a critical component of innate resistant against Mtb.

IL-6

IL-6 is a pleiotropic cytokine which plays a major role in hematopoiesis, T- and B-

cell differentiation, and inflammation. IL-6 is secreted by T cells and macrophages as part of

the immune inflammation response to trauma such as burns or other tissue damage. IL-6,

which has both pro and anti-inflammatory properties, is produced early during mycobacterial

infection and at the site of infection. IL-6 may be harmful in mycobacterial

infections, as it

inhibits the production of TNF and IL-1β and promotes in vitro growth of M. avium (134).

However, it has been found that the secretion of IL-6 by infected Mtb macrophages in mice

may contribute to the inability of IFN-γ to eradicate Mtb infection (135). In addition, the

observations that IL-6-deficient mice display increased susceptibility to Mtb infection suggest

a protective role of IL-6 (136).

22

Anti-inflammatory cytokines

IL-10

This cytokine is produced by macrophages after phagocytosis of Mtb and after binding of

mycobacterial LAM (137). T lymphocytes, including Mtb-reactive T cells, are also capable

of

producing IL-10. In patients with TB, expression of IL-10 mRNA has been found in

circulating mononuclear cells, in pleural fluid,

and in alveolar lavage fluid IL-10 antagonizes

the proinflammatory cytokine response by down regulation of the production of IFN-γ, TNF

and IL-12 (138). IL-10 would be expected to interfere with host defense against Mtb.

Indeed,

IL-10 transgenic mice developed a larger bacterial burden upon mycobacterial infection (138,

139). In human TB, IL-10 production was higher in anergic patients, both before and after

successful treatment, suggesting that Mtb-induced IL-10 production suppresses

an effective

immune response (139).

Transforming growth factor (TGF)-β

The principal role of TGF-β is the control of proliferation and cellular differentiation

functions in most cells. Mycobacterial products induce production of TGF-β in monocytes and

DC. Interestingly, LAM from virulent mycobacteria induces TGF-β production (140). TGF-β

is produced in excess during human TB (141). TGF-β seems to neutralize protective

immunity in TB by suppressing T-cell mediated immunity, inhibiting proliferation and IFN-γ

production; in macrophages it antagonizes antigen presentation, proinflammatory

cytokine

production, and cellular activation (142). Naturally, inhibitors of TGF-β eliminate the

suppressive effects of TGF-β in mononuclear cells from TB patients and in macrophages

infected with Mtb. In the anti-inflammatory response, TGF-β and IL-10 seem to synergize.

TGF-β may also interact with IL-4. Paradoxically, in the presence of both cytokines, T cells

may be directed towards a protective Th1-type profile (143). However, the role of TGF-β is

maybe similar as the IL-10, probably inhibiting the activation of Mtb-reactive CD4+.

IL-4

In intracellular infections such as TB, IL-4 cytokine has been found to inhibit IFN-γ

production and macrophage activation. In mice infected with Mtb, progressive disease and

reactivation of latent infection are both associated with increased production of IL-4 (144).

However, this is not a consistent finding, and it still remains to be determined

whether IL-4

23

causes or merely reflects disease activity in human TB. Thus, the role of IL-4 in the

susceptibility to TB is not yet entirely resolved.

Chemokines

IL-8

In humans, IL-8 attracts neutrophils, T lymphocytes, and possibly monocytes. Upon

phagocytosis of Mtb or stimulation with LAM, macrophages produce IL-8. This production

is

substantially blocked by neutralization of TNF and IL-1β, indicating that IL-8 production is

largely under the control of these cytokines (145). Human pulmonary epithelial cells also

produce

IL-8 in response to Mtb (146). In TB patients,

IL-8 has been found in

bronchoalveolar lavage fluid, lymph nodes, and plasma. However, the central role of IL-8 in

the host immune response to Mtb seems to be the leukocyte recruitment to areas of

granuloma formation in TB (147).

Keratinocyte-derived (KC) chemokine (CXCL1) and macrophage-inflammatory protein

(MIP)-2 (CXCL2)

The murine chemokines KC and MIP-2 are the major chemoattractants responsible for

recruiting neutrophils. Both chemokines bind to chemokine receptor, CXCR2 (148). The two

chemokines are closely related (149). They are also considered homologs to the human GRO

chemokines that are functionally similar to the IL-8 CXC chemokine family (150,151). The

MIP-2 mRNA expression was induced in mice infected with different Mtb strains (151).

Also, Lipoarabinomannan (LAM), a cell wall component of Mtb resulted in a neutrophilic

cell influx into the bronchoalveolar lavage fluid and also induced increases in the lung

concentrations of MIP-2 and KC (152).

Monocyte chemotactic protein (MCP)-1 (CCL/21)

MCP-1 is produced by monocytes, macrophages and epithelial cells (153,154). Mtb,

preferentially induces production of MCP-1 by monocytes to the site of infection (155). In

murine models, deficiency of MCP-1 inhibits granuloma formation (156). Also, C-C

chemokine receptor 2-deficient mice, which fail to respond to MCP-1, display reduced

granuloma formation and suppressed Th1-type cytokine production (157) and

die early after

infection with Mtb (158). MCP-1 was found in elevated concentrations in alveolar lavage

fluid, serum, and pleural fluid from tuberculosis patients (159,160). Therefore, the role of

24

MCP-1 during Mtb infection is the recruitment of monocyte/macrophages and T cells to the

site of infection maybe to help in the granuloma formation.

Matrix metallopeptidase (MMP)-9

MMP-9, also known as 92 the kDa type IV collagenase, 92 kDa gelatinase or

gelatinase B (GELB), is an enzyme that in humans is encoded by the mmp9 gene. MMP-9 is

produced by monocytes, macrophages, neutrophils, keratinocytes, fibroblasts, osteoclasts and

endothelial cells, and is involved in inflammatory responses, tissue remodelling, wound

healing, tumor growth and metastasis (161,162). Enzymes of the matrix metalloproteinase

(MMP) family play a significant role in many biological activities including many aspects of

the granuloma formation (163,164). In general, MMPs are endopeptidases responsible for

degrading components of the extracellular matrix such as collagen and proteoglycans, and as

potent chemokine antagonists. They play an important role in leukocyte migration and tissue

remodelling (165). Some studies suggested that MMP-9 is up-regulated by Mtb and

associated with local tissue damage in TB meningitis (166). Pulmonary epithelial cells

potentially produce MMP-9 and an excess of MMP-9 due Mtb infection causes tissue

destruction (167). However, other studies have suggested that the early secretion of MMP-9

is required for recruitment of AM to induce tissue remodelling for allowing the formation of

tight well organized granulomas (168). Thus, the role of MMP-9 could be either helping in

the granuloma formation or producing tissue damage during Mtb infection.

RANTES (CCL5)

RANTES or CCL5 is a chemokine that binds CCR1, CCR3, CCR4, and CCR5 and is

produced by epithelial cells, lymphocytes, and platelets, and acts as a potent chemoattractant

for monocytes, NK cells and memory T cells, eosinophils, DC and basophils. In addition,

RANTES and other chemokines can selectively activate their corresponding lymphoid cell

targets (169,170). RANTES has been shown to induce lymphocyte migration into the nasal

mucosa of allergic patients (169). In TB, RANTES seems to play a role in regulating

protective immune responses at the site of infection (171).

Interferon gamma-induced protein 10 kDa or IP-10 (CXCL10)

IP-10 is a chemokine secreted by several cell types such as monocytes, endothelial

cells and fibroblasts in response to IFN-γ. IP-10 has several roles such as chemoattraction for

monocytes/macrophages, T cells, NK cells, and dendritic cells, promotion of T cell adhesion

25

to endothelial cells, antitumor activity, and inhibition of bone marrow colony formation and

angiogenesis (172). It has been shown that Mtb inhibit the transcription of IP-10 however, in

presence of IFN-γ there is an induction of IP-10 protein which appears to be involve in novel

post-transcriptional events that incorporates non-canonical functions of NFκB and p38

(mapk) (173). Thus, the role of IP-10 during TB probably is probably similar to RANTES but

still is not clear.

GM-CSF

Granulocyte-macrophage colony-stimulating factor (GM-CSF) was first identified in

mouse lung tissue-conditioned medium following LPS injection into mice by its ability to

stimulate proliferation of mouse bone-marrow cells in vitro and generation of colonies of

both granulocytes and macrophages. GM-CSF can be produced by a wide variety of cell

types, including fibroblasts, endothelial cells, T cells, macrophages, mesothelial cells,

epithelial cells and many types of tumor cells (174). In these cells, bacterial endotoxins and

inflammatory cytokines, such as IL-1, IL-6, and TNF, are potent inducers of GM-CSF. GM-

CSF not only has the capacity to increase antigen-induced immune responses, but can also

alter the Th1/Th2 cytokine balance. It has recently been shown that mice lacking GM-CSF

die rapidly from severe necrosis when exposed to an aerosol delivered infection of Mtb

because of their inability to mount a Th1 response (175). GM-CSF over-expression, however,

failed to attract T cells and macrophages into the sites of infection, suggesting that

uncontrolled expression of GM-CSF can lead to defects in cytokine and chemokine

regulation. Therefore, excess GM-CSF does not induce an over Th1 response and very fine

control of GM-CSF is needed to fight Mtb infections (176).

26

PRESENT STUDY

Aims

General aim

The overall aim of this study was to determine the role of components in the innate immune

response against mycobacterial infection to maintain the mucosal response in the respiratory

tract, and the generation of protective immune responses in a mouse model.

Specific aims

- To determine the importance of TLR in recognition, processing and antigen

presentation in mycobacterial infections.

- To evaluate the interaction between pulmonary macrophages and non-hematopoietic

immune cells, in particular Type II alveolar epithelial cells in response to

mycobacterial responses.

Material and Methods

The materials and methods for these studies are described in the separate papers.

Briefly, the methods used in the papers are mentioned below,

- ELISA

- Mouse cytokine array panel A assay

- Flow cytometry

- Fluoresce microscopy

- Luminescence assay

All the in vivo studies were directed in mice according to the ethical guidelines available at

Stockholm University.

27

Results and Discussion

Paper I

Contribution of TLR2 signalling to the specific immune response. Deficiencies in T cell

memory and antigen presenting cell compartments in the TLR2 knockout mice.

TLRs have been found to be important in the host defense against invading microbial

pathogens playing an important role in immunity by mediating the secretion of various pro-

inflammatory cytokines along with other anti-bacterial effector molecules (106). Both, the

innate and acquired responses against Mtb infection depend to a large degree of pattern

recognition receptor such as TLRs and the common adapter MyD88. Many studies have

shown that deficiencies in TLR2, TLR4 and/or TLR9 result in increased susceptibility to Mtb

infections (110,111,114). Moreover, the absence of TLR2 in mice results in greatest

susceptibility to Mtb infection after high-dose aerosol while a low-dose resulted in slightly

increased bacterial growth (113,177,178). TLR2 has also been implicated in the recognition

of mycobacterial antigens and modulation of phagocytic functions. A prolonged recognition

of lipoproteins from Mtb by TLR2 has been found to limit the ability of macrophages to up-

regulate MHC II expression in response to IFN-γ. This effect is associated with a reduced

antigen presentation (179,180).

In this study, we evaluated the role of TLR2 in the recognition of mycobacterial

antigens. We compared the immune responses induced in wild type (WT) and TLR2

knockout (TLR2-/-) mice following immunizations with the 19kDa (TLR2 ligand) and the

Ag85A (non-TLR2 ligand) mycobacterial antigens.

Initially, we evaluated the humoral responses in both mouse strains. Previous reports

have shown that immunization of mice with recombinant Mycobacterium vaccae, which

express the 19kDa antigen, results in induction of a strong type 1 immune response to the

19kDa antigen. This response is characterized by IgG2a antibodies and IFN-γ production by

T cells (181). In addition, it has been reported that 19kDa induces the production of cytokines

such as IL-12 which is involved in promoting Th1 responses in macrophages (182). In our

study, we found that the antigen-specific antibody responses were comparable between WT

and TLR2-/- mice for both antigens. In addition, increased levels of IgG2a were detected

after 19kDa but not after Ag85A immunizations. These results confirmed and suggested that

28

immunization with 19kDa antigen induces Th1 responses while immunization with the

Ag85A antigen induced a more Th2 type of response.

We also evaluated cellular immune responses in both mouse strains. Our results have

shown that comparable levels of IFN-γ were produced by both mouse strains when spleen

cells from Ag85A immunized mice were re-stimulated with the same antigen in vitro. In

contrast, in the group of mice immunized with the 19kDa antigen, spleen cells from TLR2-/-

mice secreted significantly lower amounts of IFN-γ compared to spleen cells from WT mice.

We also evaluated whether immunization with the Ag85A was able to induce protection in

WT and TLR2-/- mice. We challenged the mice with BCG and the bacterial load was

determined in the lungs from WT and TLR2-/- mice immunized with Ag85A. We found that

mice immunized with the Ag85A from both mouse strains were protected to a similar

extends. These data are in line with previous reports of studying humoral and cellular

protection after the Ag85A immunization in mice (183).

The low levels of IFN-γ from TLR2-/- spleen cells after re-stimulation with 19kDa in

vitro led us to investigate whether T cells were not properly primed in vivo or if the antigen

presentation was not sufficient in vitro. To elucidate these questions, BMM from TLR2-/-

and WT, previously infected with BCG or pulsed with the antigens (19kDa or Ag85A) were

co-cultured with spleen cells from immunized mice with 19kDa or Ag85A from both mouse

strains in vitro. (Fig.2a, manuscript I)

We found that memory T cells were generated in vivo in both, TLR2-/- and WT mice

when spleen cells from both mouse strains were co-cultured with BMM from WT mice

pulsed with the 19kDa antigen or the Ag85A. The levels of IFN-γ were comparable in

response to both antigens. These results indicated that the 19kDa lipoprotein, a ligand for

TLR2, could be internalized, processed and presented in vivo to generate memory-T cells

even if the mice were deficient in TLR2.

We also found that the capacity of antigen presentation by TLR2-/- mice was independent of

TLR2 when BMM from TLR2-/- mice pulsed with the Ag85A were co-cultured with spleen

cells from WT and TLR2-/- mouse strains immunized with the Ag85A in vitro. However,

BMM from TLR2-/- mice (pulsed with 19kDa) were affected in their capacity to present

antigen to spleen cells from TLR2-/- but not with WT mice. These results suggested that the

presentation of the antigens was not dependent on the presence or absence of TLR2 in the

APC. However, it is clear that the presentation of antigens is affected if both, APC and T

cells are deficient in TLR2.

29

Since Mtb uses macrophages as its preferred habitat in the lung, we asked whether the

processing and antigen capacity of pulmonary macrophages (PM) could be affected by TLR2

and whether PM could also accomplish a similar pattern as BMM. It has been found that

local soluble factors in the lung environment could determine the behavior and phenotype of

AM and other cells (184). Therefore, we were aware that the environment in the lung could

determine the behavior and phenotype of the resident macrophage population in this tissue.

To answer these questions, spleen cells from TLR2-/- and WT mice immunized with the

19kDa antigen were co-cultured with PM from TLR2-/- and WT mice previously pulsed with

19kDa in vitro.

We found a different pattern of PM in the capacity of antigen presentation compared

to BMM. Moreover, PM from TLR2-/- were clearly affected in the antigen presentation to

both mouse strains. In addition, TLR2-/- memory-T cells were not generated in TLR2-/- mice

(Fig.2b, manuscript I).

The different patterns in antigen presentation between BMM and PM led us to study

the behavior of these two types of macrophages in response to different stimuli. LPS and

Pam3Cys-Ser-(Lys)4trihydrochloride (Pam3) were used as TLR4 and TLR2 ligands,

respectively. Anti-CD40 was used to activate macrophages via CD40 ligation. PM and BMM

from WT mice were stimulated with the stimuli mentioned above in vitro and the

supernatants were collected. Kinetics of the IL-10, IL-6, IL-12 and TNF production were

measured by ELISA. In addition, un-stimulated and stimulated PM and BMM were

analyzed by FACS for measuring the expression of MHC II, co-stimulatory molecules such

as CD40, CD80, CD86 and F4/80 (mouse mature macrophage marker) expression.

We found that PM and BMM had similar pattern in the production of cytokines.

However, LPS induced an early response (4 h) while Pam3 produced a late response (24 h) in

the production of TNF and IL-12 by both cell types. Nevertheless, PM were more affected in

the production of TNF upon Pam3 stimulation.

The results from FACS analysis have shown that BMM were able to up-regulate the

expression of MHC II, CD80, CD86 and CD40 upon TLRs and anti-CD40 stimulation.

However, MCH II expression by PM was almost twofold stronger than BMM. In addition,

PM exhibited a high expression of CD80. The expression of CD86 by PM was not up-

regulated. We also evaluated other functional responses between PM and BMM such as

mycobacterial uptake and intracellular growth control. We found that BMM displayed a

stronger capacity to internalization and control of bacterial growth compared with PM (data

not shown).

30

All these results together suggested that BMM might be considered as naive macrophages

with a full expression of the large macrophage capacities whereas the growth, survival,

phenotype and behavior of PM in the lungs might depend in part on locally produced factors

(growth factors, cytokines or surfactant factors). Interestingly, the TLR2 ligand used in this

study Pam3 induced low TNF response production in PM.

In summary, in this study we suggest that in the absence of TLR2, immune responses

could be generated. However, our results suggest that might be PM are more restricted to

produce a successful antigen presentation in the lungs due to the influence of local factors.

The functional activity and phenotype of BMM and PM were also evaluated in this study. We

found that BMM were more efficient than PM in the internalization and growth control of

mycobacteria. In addition, we found a different phenotype of co-stimulatory molecules and

MHC II expression between PM and BMM. However, both cell types, BMM and PM were

able to produce similar patterns of cytokines in response to TLRs and anti-CD40 stimulation.

We hypothesized that the activation, behavior and expression of molecules involved in

antigen presentation are influenced by the local tissue environment, for instance the lungs.

31

Paper II

The role of alveolar epithelial cell type II in initiating and shaping pulmonary immune

responses. Communication between the innate and adaptive immune systems.

The role of macrophages and DC responses to mycobacterial infection has been long

studied before and these cells are considered key players in the defense against mycobacterial

infections. However, other cell populations in the lungs have been proposed to play important

roles in the pathogenesis and defense against Mtb. Due to their strategic localization,

expression of immune markers such as TLR and MHC II and close interaction with other

cells, especially with macrophages in the lungs, AEC II have been also considered to play an

important role during mycobacterial infections. In addition, AEC II secrete a variety of

antimicrobial products, cytokines and chemokines to induce different responses in the innate

and adaptive system in the lungs.

In paper II we compared AEC II with pulmonary macrophages (PM) in their ability to

generate immune responses against mycobacterial products and BCG. We first compared the

ability to take up and the capacity of intracellular growth control upon BCG infection in vitro

in both cell types isolated from mouse lungs. Our data showed that even if PM were more

efficient in both capacities, AEC II were also able to take up and control BCG growth. These

results from primary cells were in line with previous reports of Mtb infection and replication

inside AEC II cell lines (70,71).

We also performed an in vitro experiment to compare the capacity of primary AEC II as APC

with a professional APC, for instance PM. Previous reports have shown that AEC II express

constitutively MHC II (67) and also that murine AEC II can present mycobacterial antigens

to T cells (74). Our findings showed that AEC II pulsed with the 19kDa antigen

(mycobacterial antigen) were clearly able to stimulate spleen cells from mice immunized with

the 19kDa antigen. However, the magnitude of the response was low compared with pulsed

PM. These results confirmed the capacities of AEC II to take up, process and present

antigens. Therefore, we and others suggested a possible role of AEC II in the adaptive

responses as APC to mycobacterial infection.

However, the specific role as specialized APC in an in vivo situation in the lungs

might be secondary. It is important to consider the localization in separated compartments of

AEC II and T cells. Consequently, AEC II have to promote the migration of T cells from the

peripheral blood and other compartments to the lung to generate a successful antigen

32

presentation. Also, another crucial factor to remark is expression of co-stimulatory molecules

for a successful antigen presentation to T cells. In human and mouse studies AEC II have

been reported to express a low grade or lack of expression of classical co-stimulatory

molecules (68,185). The lack of co-stimulatory molecules in AEC II has been suggested to

induce T-cell tolerance to suppress inflammatory responses in the lungs against harmless

antigens (68). Moreover, one study has shown that AEC II are able to induce regulatory

peripheral T cells inducing tolerance against self-antigens in the lungs through the expression

of factors such as TGBβ (186). Thus, it might be more likely that the participation, in the

adaptive system, of AEC II is through to the secretion of factors modulating the activation

and function of different cell types present in the lungs.

In the lungs, the production of cytokines, chemokines and other factors by local cells

decides the outcome of inflammatory responses in this tissue. Although, immune cells such

as macrophages, DC are secreting many of these factors; AEC II and other non-immune cells

in the lungs are able to produce many factors constitutively or upon different stimuli (73,75).

To gain a better understanding of the role of AEC II in the production of factors against

mycobacteria, we compared the production of MCP-1, MIP-2, KC, TNF, MMP-9 and IL-12

in primary AEC II with that of PM upon different stimuli in vitro. We used as stimuli: heat

killed (HK)-BCG and BCG lysate (Lys-BCG) as mycobacterial products, cytokines such as

TNF and IFN-γ were used due to their importance in the responses to mycobacteria and LPS

was used as a TLR4 ligand. We found a different pattern of cytokine and chemokine

production in both cell types. MCP-1 chemokine was mostly secreted by primary AEC II and

PM were the main producers of MIP-2 (homologue in mouse of human IL-8). Since

macrophages secrete TNF and MCP-1 can activate macrophages, the possible influence of

PM on AEC II and vice versa was suggested when primary AEC II secreted high amounts of

MCP-1 upon TNF stimulus. We also found that the major ligands may be present in the BCG

cell wall due to the fact that Lys-BCG was not as good stimulator compared with HK-BCG.

TNF and IL-12 were only produced by PM upon LPS stimulation (data not shown). We also

investigated the role of MMP-9, a molecule involved in the granuloma formation for

controlling mycobacterial infections (167,168). We found that AEC II but not PM were good

secretors of MMP-9 upon TNF stimulation.

In this study, we found some different results compared with previous reports in

human studies. However, even if the levels of MIP-2 secreted by murine primary AEC II

were lower than PM our results were comparable with previous reports of IL-8 levels

secreted by human epithelial cells (187). We also considered that the health status of mouse

33

lung cells can be secured compared with the samples of human lung cells, which come from

unhealthy lung tissues. Thus, there is not the guarantee that these lung cells are not activated

or anergized. In addition, we found some contradictory results when we used a commercial

AEC II cell line. We concluded that there is not a cell line that exhibit the full range of known

primary AEC II functions.

As we and others demonstrated that AEC II are able to produce factors such as MCP-

1 and MIP-2 chemokines, we aimed to determine a more complete profile of different factors

produced by AEC II in response to stimuli such as mycobacterial products, TLR ligands and

IFNs. AEC II were stimulated with HK-BCG, Lys-BCG, LPS, Flagellin, Pam3Cys-Ser-(Lys)4

trihydrochloride (Pam3), IFN-γ and IFN-α in vitro. Supernatants were collected and were

analysed with the R&D mouse proteome profile array. This protein array allowed us to

determine of 40 different factors including growth factors. We found that a broad array of

different factors was produced by AEC II namely: G-CSF, GM-CSF, M-CSF, KC, MCP-1,

MIP-1, MIP-2, TIMP-1, IL-6, and IP-10. The analysis of unstimulated AEC II showed that

these cells were able to produce constitutively some of the factors (GM-CSF, M-CSF, MCP-

1, TIMP-1, IL-6, and IP-10) while other factors (G-CSF, MIP-1, and MIP-2) were secreted

only by stimulated AEC II.

We also considered the importance of evaluating levels of some factors because

higher levels of MCP-1 secreted by AECII upon LPS and TNF. We found that GM-CSF is

probably the main growth factor produced by AEC II as a result of undetectable levels of M-

CSF even after the stimulation of AEC II (data not shown). Moreover, increased levels of

GM-CSF were found after LPS and Pam3 stimulation. In addition, MCP-1, KC and IL-6

were strongly produced after the induction via TLRs whereas IP-10 and RANTES were

mostly induced by IFNs. Therefore, the interaction between AEC II and lymphocytes is also

possible due to the IFNs (produced by lymphocytes) which were able to induce the

expression chemokines (IP-10 and RANTES) assisting in the recruitment of circulating

lymphocytes to areas of injury, inflammation, or viral infection. Flagellin was the weakest

inducer of the three different TLRs ligands. These results suggested that AEC II are involved

actively to induce different effects on other cell types in the lungs such as monocytes,

macrophages, DC, and T cells.

In this study, we confirmed and provided more evidence of a novel role for AEC II in

the lung to generate local responses against mycobacterial infection. Our findings suggested a

possible interaction of AEC II with other local cell populations in the lungs (for instance PM)

34

which is considered crucial in the responses against Mtb. Although PM were more efficient,

murine AEC II were also able to internalization and the control growth of BCG. In addition,

AEC II were able to process and present mycobacterial antigen to T cells. However, their role

as APC appears to be secondary since AEC II and T cells belong to different compartments.

The main findings in our study were the communication between AEC II and other cell

populations in the lungs (PM and lymphocytes) as evidenced by the production of cytokines,

chemokines and other factors by AEC II upon TLRs and IFNs stimuli.

35

Concluding Remarks

Even though, the roles of components of innate immune responses against

mycobacterial infection have been extensively studied, the results of this thesis provide more

evidence that TLR signalling pathway, for instance TLR2, links both innate and adaptive

immune responses. We have demonstrated that the antigen presentation is affected when

both, APC and T cells are TLR2 deficient. We have also suggested that the functional activity

and phenotype expression of the two types of macrophages used in this study (BMM and

PM) are influenced by the local tissue environment. Furthermore, we provided more evidence

of a novel role for AEC II in response to mycobacterial infection and TLRs and IFNs

stimulation. In addition, the communication between AEC II and other cell populations in the

lungs (PM and lymphocytes) was evidenced in this study through the production of

cytokines, chemokines and other factors.

Future Plans

The results from the paper I have suggested that immune responses in local resident

macrophages in the lungs might be restricted by the local airway environment. In the paper II

we suggested that AEC II are playing an important role in the lung immune responses

through the secretion of different factors. Indeed, these factors produced by AEC II are

important to activate resident cells such as macrophages or to induce cell migration from

other compartments, for instance T cells. In order to increase the understanding of the role of

AEC II and their influence in the local responses against mycobacterial infection further

evaluation of the functional capacities of AEC II might be important to elucidate potential

function by AEC II in the airway surfaces. There is some evidence that activated AEC II

promote the migration of macrophages (188) Also, we and other have demonstrated a strong

production of MCP-1 by AEC II. Thus, it is important to evaluate the influence of AEC II

stimulated with mycobacterial products, TLRs and IFNs in the migration of different resident

cell in the lungs. Furthermore, we will also investigate the influence of AEC II in other

functional capacities such as uptake and growth control in macrophages. Moreover, it will be

also important to investigate the influence of others cell types on AEC II.

36

ACKNOWLEDGEMENTS

I would like to thank my supervisor Professor Carmen Fernández for giving me the trust and

the opportunity to work in her group. I sincerely grateful to you Carmen, for your large

support since the time I arrived Sweden. Muchas gracias Carmen!

My special thanks to my co-authors and collaborators Katharina Hartl, Dagbjört Petursdottir,

Natalia Periolo and in particular to Jubayer Rahman for being my friends and always helping

me when I asked for.

To all the seniors at Immunology, Marita Troye-Blomberg, Eva Sverremark-Ekström, Klavs

Berzins and Eva Severinson for all the nice discussions and advices.

To Maggan, Gelana Yadeta and Anna-Leena Jarva for all your help.

To all the staff at the animal house for being friendly and helpful.

To all the past and present students at Immunology and other Departments, in particular to

Jacqueline Calla, Irene Roman, Andrea Sommer, Katarina Tiklova, Natalija Gerasimcik, and

Olivia Simone for giving me your friendship and help.

To all my friends in and out of Sweden.

Finally, I would like to thank my family for being my strength and inspiration to continue my

PhD studies. Papí, Mamí, Carmen, Enrique and Gustavo; your love and support are the most

important pieces of my life. Todo lo que hice, hago y haré siempre será para y por ustedes!

37

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