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Doctoral thesis from the Department of Molecular Bioscience,
The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden
Role of alveolar epithelial cells in macrophage responses against
mycobacterial infections
Olga Daniela Chuquimia Flores
Stockholm, 2013
All previously published papers were reproduced with permissions from the publishers
Printed in Sweden by Universitetsservice AB, Stockholm 2013
Distributor: Stockholm University Library
© Olga Daniela Chuquimia Flores
ISBN 978-91-7447-631-6
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“Science can purify religion from error and superstition; religion can purify science from
idolatry and false absolutes”
Pope John Paul II (1920-2005)
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SUMMARY
This thesis aimed to investigate the role of alveolar epithelial cells (AEC) on immune
responses against mycobacterial infections, specifically, the role of AEC in modulating
macrophage functions through the secretion of broad variety of factors.
In paper I, we investigated the role of AEC in the defense against mycobacterial
infections. First, we compared murine AEC with interstitial macrophages, herein named PuM
in their ability to take up and control mycobacterial growth and their capacity as antigenpresenting cells. We found that AEC were able to internalize and control bacterial growth and
present antigens to T cells from immunized mice. In addition, both AEC and PuM exhibited
distinct patterns of secreted factors, and a more comprehensive profile of AEC responses
revealed that AEC were able to secrete different factors important to generate various effects
in other cells. The major finding of this study was that secreted AEC factors might modulate
and influence other immune cell types such as macrophages and T cells resident in the lungs.
Paper II: Since AEC secrete a broad variety of factors involved in activation and
differentiation of immune cells, we hypothesized that being in the interface; AEC may play
an important role in transmitting signals from the external to the internal compartment and in
modulating the activity of PuM. Thus, we prepared AEC-derived media and tested their
effect on bacteria and a number of macrophage functions a) migration, b) phagocytosis and
control of intracellular bacterial growth, and c) alteration in cell morphology and expression
of surface markers. We found that AEC-secreted factors had a dual effect, in one hand
controlling bacterial growth and on the other hand increasing macrophage activity. In paper
III, we first investigated the responsible mechanisms of intracellular bacterial growth control
mediated by AEC-derived media. We found that infected macrophages upon AEC-secreted
factors increased the control of intracellular bacterial growth by inducible nitric oxide
synthase-independent pathways. Compared with other macrophage types, PuM, did not
control the intracellular bacterial growth upon the well-known potent macrophage activator,
IFN-γ. We found that SOCS1 was involved in the un-responsiveness to IFN-γ by PuM to
control the intracellular bacterial growth. We suggested that PuM are restricted in their
inflammatory responses perhaps for avoiding tissue damage.
Overall, the current findings highlight the importance of AEC in the defense against
bacterial infection in the lungs by secreting factors involved in activation and differentiation
of immune cells such as macrophages.
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LIST OF PAPERS
This thesis is based on the following original papers (manuscripts), which will be referred to
by their roman numeral in the text.
I.
Olga D. Chuquimia, Dagbjort H. Petursdottir, Muhammad J. Rahman, Katharina
Hartl, Mahavir Singh and Carmen Fernández. The role of alveolar epithelial cells
in initiating and shaping pulmonary immune responses: communication between
innate and adaptive immune systems. PLoS One. 2012;7(2):e32125. Epub 2012
Feb 29.
II.
Olga D. Chuquimia*, Dagbjort H. Petursdottir*, Natalia Periolo and Carmen
Fernández. Alveolar epithelial cells are critical in protection of the respiratory
tract by secreting factors able to modulate the activity of pulmonary macrophages
and directly control bacterial growth. Infect Immun. 2013 Jan;81(1):381-9. doi:
10.1128/IAI.00950-12. Epub 2012 Nov 12.
III.
Olga D. Chuquimia*, Dagbjort H. Petursdottir* and Carmen Fernández. Soluble
factors from alveolar epithelial cells increase intracellular killing of BCG by
macrophages through nitric oxide independent mechanisms. Manuscript.
*These authors contributed equally to this work.
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LIST OF PAPERS (not included in this thesis)
The following original papers are relevant but not included in this thesis. The papers will be
cited by their roman numerals:
IV.
Muhammad J. Rahman*, Olga D. Chuquimia*, Dagbjort H. Petursdottir, Natalia
Periolo, Mahavir Singh and Carmen Fernández. Impact of toll-like receptor 2
deficiency on immune responses to mycobacterial antigens. Infect Immun. 2011
Nov;79(11):4649-56. Epub 2011 Aug 15.
V.
John Arko-Mensah*, Muhammad J. Rahman*, Irene R. Dégano, Olga D.
Chuquimia, Agathe L. Fotio, Irene Garcia, Carmen Fernández. Resistance to
mycobacterial infection: a pattern of early immune responses leads to a better
control of pulmonary infection in C57BL/6 compared with BALB/c mice.
Vaccine. 2009 Dec 9;27(52):7418-27. Epub 2009 Sep 5.
*These authors contributed equally to this work.
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TABLE OF CONTENTS
Page
SUMMARY
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LIST OF PAPERS
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LIST OF ABBREVIATIONS
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INTRODUCTION
Immune responses in the respiratory tract
Innate immunity
Pattern recognition receptors
Antimicrobial products in the respiratory tract
Cells
Alveolar epithelial cells
Macrophages
1. Macrophage polarization in the lung
Dendritic cells
Neutrophils
NK-cells
Phagocytosis
Cellular mechanisms of microbial killing in infected cells
Generation of ROS/RNS
Autophagy
Cytokines and chemokines
Mycobacterial infections in the respiratory tract
Tuberculosis
Pathogenesis of tuberculosis
Innate responses may prevent mycobacterial infections
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9
10
10
12
13
13
14
15
16
17
18
18
19
19
20
21
25
25
26
26
PRESENT STUDY
Aims
Materials and Methods
Results and Discussion
Paper I
Paper II
Paper III
28
28
29
31
31
34
37
CONCLUDING REMARKS AND FUTURE PERSPECTIVES
40
ACKNOWLEDGEMENTS
41
REFERENCES
43
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LIST OF ABBREVIATIONS
AEC I and II
AECsup
AECLPS
AM
AMP
AP
APC
ARG-I
Atg
BCG
BMM
DC
ELISA
GM-CSF
HK-BCG
IFN-α
IFN-γ
IL
iNOS
IP-10
kDa
KC
Lys-BCG
LPS
MCP-1
MHC
MIP-2
MMP-9
MMR
Mtb
NF-κB
NK
NOD
NOS
NO
NLR
PAMP
Pam3
PRR
PuM
RNI
ROI
RNS
ROS
s.c.
SOCS
SP
TB
TGF-β
TLR
TNF
Type I and II alveolar epithelial cells
Cell culture supernatant from AEC un-stimulated
Cell culture supernatant from AEC stimulated with LPS
Alveolar macrophages
Antimicrobial peptides
Antimicrobial products
Antigen-presenting cell
Arginase-I
Autophagy-related
Bacillus Calmette-Guérin
Bone marrow derived macrophages
Dendritic cells
Enzyme- linked immunosorbent assay
Granulocyte-macrophage colony-stimulating factor
Heat killed-BCG
Interferon alpha
Interferon gamma
Interleukin
Inducible nitric oxide synthase
Interferon gamma-induced protein 10 kDa
Kilo Dalton
Keratinocyte-derived chemokine
BCG lysate
Lipopolysaccharide
Monocyte-chemotactic protein-1
Major Histocompatibility complex
Macrophage-inflammatory protein-2
Matrix metallopeptidase-9
Mannose receptors
Mycobacterium tuberculosis
Nuclear factor kappa beta
Natural killer
Nucleotide-binding oligomerization domain
Nitric oxide synthase
Nitric oxide
NOD-like receptors
Pathogen-associated molecular patterns
Pam3Cys-Ser-(Lys)4 trihydrochloride
Pattern recognition receptors
Interstitial macrophages
Reactive nitrogen intermediates
Reactive oxygen intermediates
Reactive nitrogen species
Reactive oxygen species
Subcutaneously
Suppressor of cytokine signaling proteins
Surfactant proteins
Tuberculosis
Transforming growth factor beta-β
Toll-like receptors
Tumor-necrosis factor
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INTRODUCTION
Immune responses in the respiratory tract
The innate and adaptive immune systems are involved in the defense and protection
against invading microorganisms (control of pathogen presence and infection levels) in the
respiratory tract. An important function of the respiratory tract is the regulation of the local
immunological homeostasis (to minimize local damage at the cell surfaces) and therefore to
guarantee the integrity of gas exchange (1). The respiratory tract is divided in two parts: The
upper and the lower respiratory tract. The lower respiratory tract, considered to be sterile, is
also divided in two major compartments: conducting airways and the lung parenchyma. In
the conducting airway, the ciliated epithelium not only provides a physical barrier, but also
plays an important role as a first line of defense with the recognition of pathogens and the
secretion of effector molecules (2). Different immune cell populations are present within the
epithelium and mucosa in the conducting airways, such as dense networks of macrophages
and dendritic cells (DC) among other cell populations. Lymphocytes can be found either
singly or in clusters in the airway lamina propria and in the submucosa with effector and
memory CD4+ and CD8+ T cell phenotypes. Plasma cells and B cells are also present in the
airway mucosa (in the intraepithelial and within the underlying lamina propria) (3-5). Other
immune cells-types such as mast cells, basophils, eosinophils and neutrophils have also been
found in the lamina propria (6, 7). The lung parenchyma consists of alveoli that are
separated by fine vascularized interstitial tissue. 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 consists of 80-90% macrophages (as reflected by broncho-alveolar lavage
fluid composition) which are considered the first line of defense, the remainder being T cells
and DC. However, a large sequestered T-cell population with undefined role in the lung
parenchyma has been found. The lung parenchyma also contains neutrophils, B cells, mast
cells and other cell-types such as alveolar epithelial cells (AEC) (8-12). AEC in the lung
parenchyma have been found to play an important role in the local immune responses
secreting different antimicrobial molecules and factors important for activation, recruitment
and proliferation of immune cells, this will be discussed in detail later.
In this thesis we will be focused in the innate branch of the respiratory tract, due to its
critical role for controlling infection in the early stages of exposure to invading
microorganisms and inhaled particles.
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Innate immunity in the lungs
The innate immune responses in the respiratory tract are composed of cellular,
antimicrobial and physical mechanisms. Once inhaled, particles or microorganisms arrive to
the mucosa surface in the upper respiratory tract, and then are trapped by mucus and removed
toward the pharynx and swallowed or expectorated. For those bacteria or viruses that arrived
into the alveoli, host cells of the innate immune branch are able to sense and recognize
conserved structures through biosensors “pattern-recognition receptors” (PRR), with a critical
role in the host defense. Also, epithelial cells, as well as local immune cells, produce and
secrete antimicrobial peptides to kill many microorganisms that have penetrated the mucous
layer by direct lysis, opsonisation and recruitment of inflammatory cells. Moreover, those
bacteria that are resistant to antimicrobial peptides are engulfed by phagocytes and killed by a
variety of reactive oxygen species produced by macrophages or neutrophils (13, 14). Innate
immunity also stimulates antigen-specific responses mediated by the adaptive immune
system.
Pattern-recognition receptors (PRR)
PRR can be broadly divided into five different classes: Toll like receptors (TLR),
Nucleotide-binding oligomerization domain (NOD)-like receptors (NLR), retinoic acidinducible gene-I (RIG-I)-like receptors (RLR), C-type lectins (CTL) and absent-in-melanoma
(AIM)-like receptors (ALR) (15). In the lungs, TLR members and some PRR are widely
expressed in macrophages, lung epithelial cells, intraepithelial DC, as well as, in endothelial
and stromal cells (1, 2, 15). These molecules are biosensors of microbial infection by
recognizing conserved microbial molecules, classically defined as pathogen-associated
molecular patterns (PAMP), and endogenous stress signals termed danger-associated
molecular patterns (DAMP). The engagement of PRR activates the production of cytokines,
interferons and chemokines on transcriptional and post-translational levels. Thus, PRR play a
key role in activating surrounding cells, in the regulation and recruitment of macrophages and
neutrophils and in the regulation of the expression of inducible antimicrobial peptides. PRR
also provide DC and macrophages with an obligatory signal for the induction and shaping of
subsequent T-cell responses (1, 15).
The well-known TLR comprise about 10 and 13 family members in humans and
mice, respectively. TLR are type I trans-membrane leucine-rich repeat proteins (between 19
and 25) where a single membrane proximal cysteine motive is involved in specific binding to
a wide variety of microbial- and endogenous-ligands (15, 16). TLR have a highly conserved
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intracellular signaling domain similar to the mammalian IL-1 receptor. After engagement of
this Toll/IL-1 receptor (TIR), the domain interacts with different adaptor molecules that
through activation of nuclear factor kappa beta (NF-κB) and/or IFN-regulatory factors (IRF)
leads to the transcription activation of a broad panel of genes (15, 16). In the respiratory tract
the lung is continuously exposed to a wide variety of airborne antigens and toxins, and
therefore it is essential to have an appropriate faster and selective immune response in this
organ. This response requires precise regulation of both pro-inflammatory and antiinflammatory responses. Thus, members of the TLR family are participating in initiating
innate as well as adaptive immune responses, following their binding to PAMP. For example,
the TLR2 binds to bacterial lipoproteins and lipoteichoic acid (LTA). TLR2 were also
described to mediate innate immunity to the induction and maintenance of adaptive immune
responses (Paper IV), TLR4 recognizes LPS from most gram-negative bacteria. TLR5
recognizes bacterial flagellin (monomer that makes up the filament of bacterial flagella),
TLR7 and TLR8 recognize single stranded RNA from viruses, and TLR9 mediates cellular
response to DNA containing un-methylated CpG motif present in bacterial DNA (17, 18).
How the conserved domains in Toll-like members are able to recognize different ligands
specifically is unclear, but hydrophobic interactions seem to be a prominent factor (17).
The NLR family sense PAMP in the cytosol (15, 16). NOD-like receptors are
involved in many processes, including autophagy induction, antiviral responses and initiation
of adaptive T cell responses (19-21). The NOD proteins NOD1 and NOD2 are the best
characterized members of this family and both recognize peptidoglycan fragments. NOD1
recognizes the peptide γ-D-glutamyl-meso-diaminopimelic acid (meso-DAP), which is found
on gram-negative bacteria while NOD2 is the receptor for muramyldipeptide (MDP), which
is a peptidoglycan constituent of both Gram-positive and Gram-negative bacteria (22, 23).
After recognition of the ligand, NOD not only can activate NF-κB and MAP kinase
pathways, but also can act in synergy with various TLR to enhance immune responses in
antigen presenting cells (24). The assembly of a number of proteins, including an NLR, procaspase-1 and the adapter apoptosis-associated speck-like protein (ASC) is denominated as
an inflammasome. The inflammasome activation is described as the production of caspase-1,
which cleaves pro-proteins of IL-1β and IL-18 to their biologically active forms. Pro-IL-1β
production is mediated by induction of the IL-1β gene through TLR and NOD stimulation,
which is then processed by caspase-induced after interaction with the NLR. The consequence
of inflammasome activation is a form of cell death termed pyroptosis, which results in
membrane disruption, leading to the release of IL-1β and other inflammatory cytokines. Two
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other NLR proteins are involved in inflammasome activation: IL1-β-converting enzyme
protease activating factor (IPAF), also known as NLRC4 (NLR CARD domain) which
recognizes cytosolic flagellin and NLR pyrin domain (NLRP3) which senses multiple PAMP
such as peptidoglycan and RNA (15, 25, 26).
Antimicrobial products in the respiratory tract
Several proteins and peptides with antimicrobial activity that act on invading
pathogens are secreted into the airway surface liquid (ASL) by the airway itself (27). The
antimicrobial products (AP) produced by the airway can be small cationic molecules, such as
the β-defensins, LL-37, and CCL20, or larger proteins, such as lysozymes, lactoferrin, and/or
mucins. Most of these products act cooperatively in their microbicidal activity and the degree
of their activity depends on the ionic strength of the solution (2, 27). Lysozyme and
lactoferrin are the most abundant proteins in ASL. Lysozyme is a 14-kDa enzyme produced
by neutrophils, monocytes, macrophages, and epithelial cells. This molecule enzymatically
cleaves glycosidic bonds of the bacterial membrane peptidoglycans or kills bacteria by a nonenzymatic mechanism (13, 14, 28). Lactoferrin is an 80-kDa cationic iron-binding protein,
also produced by neutrophils and epithelial cells. The function of lactoferrin is to inhibit
growth of iron-requiring bacteria and it can also be directly microbicidal through its Nterminal cationic fragment (13, 14, 28). On the other hand, antiproteinases, produced by
epithelial cells and macrophages, are molecules of low-molecular weight, positively charged,
containing numerous disulfide bonds all involved in the acute phase of inflammation which
protects against toxic effects of proteolytic enzymes released by phagocytic cells (27).
Antimicrobial peptides (AMP) are effector molecules of innate immunity with direct or
indirect antimicrobial effects against bacteria, fungi, protozoa and viruses. In humans and
other mammals, two main families of antimicrobial peptides are described: defensins (29)
and cathelicidins (30). AMP are secreted mainly by epithelial cells and neutrophils, but other
cells may contribute to their production. These molecules are involved in disruptive
interactions with the bacterial membrane (13, 14). AMP also have roles as mediators of
inflammation, with effects on epithelial and inflammatory cells, that can be derived into
diverse processes such as proliferation, immune induction, wound healing, cytokine release,
chemotaxis, protease-antiprotease balance, and redox homeostasis (13, 14). Other important
AMP involved in the defense of the respiratory tract, are the lung collectins: surfactant
protein (SP)-A and -D, which are produced by clara cells and type II AEC (27). These
molecules are able to bind, aggregate, and opsonize different microorganisms, including
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Gram-positive and Gram-negative bacteria and virus. SP-A and SP-D have also described to
enhance phagocytosis, killing, and clearance of microorganisms from the lung (31-33).
Cells
Alveolar epithelial cells (AEC)
Distal airway-epithelial cells and AEC are vital for maintenance of the pulmonary airblood barrier. Several studies have shown that airway epithelial cells express PRR, and
adhesion molecules on their surface and secrete various active molecules e.g. cytokines,
chemokines (Paper I, 34-36). 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, regulation and exacerbation of an inflammatory response
within the airways. The alveolar epithelium is composed of Type I AEC (AEC I) or
membranous pneumocytes and Type II AEC (AEC II) or granular pneumocytes.
AEC I are squamous, large thin cells that cover 90-95% of the alveolar surface, and
are essentially involved in gaseous exchange. These cells have been reported to express
proteins involved in regulation of cell proliferation, ion transport and water flow, as well as
the, metabolism of peptides, among other functions (37, 38). The large and thin AEC I are not
only considered as a physical barrier able to facilitate the gas exchange in the lungs, but these
cells are also, believed to participle in the lung defense. Although very little is known with
respect to specific functions of AEC I in innate immunity, AEC I have been suggested to
contribute to the lung defense because of their expression of transferrin (an oxidant involved
in the catalysis of highly reactive hydroxyl radicals from superoxide and hydrogen peroxide)
(39). Also, AEC I cell lines stimulated with bacterial products were able to up-regulate TLR
and to induce the production of chemokines (40). Moreover, primary AEC I were found to
up-regulate TLR2 and the stimulator of interferon genes (STING) and induce CXCL5 during
pneumococcal pneumonia suggesting an antibacterial role of AEC I in the lungs (41).
AEC II are cuboidal cells that constitute 15% of total parenchymal lung cells and
cover about 7% of the total alveolar surface. Ultra structural criteria used to identify AEC II
are the presence of lamellar bodies, apical microvilli and specific junctional proteins (42-45).
These cells perform different functions, including the ion transport, alveolar repair in
response to injury and regulation of surfactant metabolism. AEC II is 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
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the clearance of a variety of lung pathogens by macrophages (33). AEC II are also considered
important immunologic modulators in the alveolar space due to their strategic location in the
interface between the outside and pulmonary vasculature. These cells secrete several
antimicrobial proteins, such as lysozymes, and complement components (e.g., C2, C3, C4
and C5) and a variety of cytokines, chemokines and factors, that may be involved in the
activation of pulmonary macrophages and other cell-types during lung inflammation (46-49) .
In addition, AEC II express PRR on their surfaces molecules such as TLR2 and TLR4 (50,
51), and they constitutively express MHC II, which might suggest a possible function of AEC
II as antigen-presenting cells in the lungs (52). A possible contribution of AEC II in T-cell
tolerance to exogenous or innocuous antigens in the lungs due to their lack of the expression
of co-stimulatory molecules needed for the activation of T cells has also been suggested (53).
Moreover, AEC II were proposed to contribute in balancing inflammatory and regulatory Tcell responses in the lung, by connecting innate and adaptive immune mechanisms, and to
establish peripheral T-cell tolerance to respiratory self-antigen (54).
Macrophages
In the lungs, macrophages are considered to be the first line of defense against inhaled
microorganisms. Macrophages play important roles in homeostasis, tissue remodeling and in
host defense through the phagocytosis and killing of microorganisms (55). Although,
macrophages are morphologically similar, it is possible that their function is regulated
according to their localization in the lungs (Paper IV, 56, 57) .In fact, different
subpopulations of macrophages in the human and mouse lung have been defined on the basis
of their anatomic location (56, 57). These are described as interstitial macrophages, located in
the narrow space between the alveolar epithelium and vascular endothelium, alveolar
macrophages (AM) residing in the alveolar spaces, and intravascular macrophages located in
the capillaries in the alveolar septa (56, 58, 59). AM and interstitial macrophages are
considered the major macrophage populations in the lungs. Both types of macrophages have
been described to differ in their functions. AM take up most of the particulate material that is
delivered intranasally to the alveolar space, but they do not migrate to regional lymphoid
nodes (55, 56, 58). In addition, AM are not considered to have a significant role in antigen
presentation (60). Interstitial macrophages are considered to be an intermediate stage between
monocytes and alveolar macrophages (61), and might play a role in preventing allergies
through DC modulation (62). Moreover, interstitial macrophages have been described to have
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roles in maintaining the homeostatic immunological balance in the lungs, such as limiting
local inflammatory responses and antigen presentation (58-60, 63).
Figure 1. Schematic diagram illustrating lung macrophage heterogeneity. Phenotypic and
functional characteristics of lung macrophages are related to their location within the
alveolus or interstitium. J Leukoc Biol. 2001 Aug;70(2):163-70 (58).
1. Macrophage polarization in the lung
A key functional characteristic of macrophages is their ability to differentiate
(polarize) in response to changes in their tissue micro-environment, and therefore they can
exhibit a marked functional and phenotypic heterogeneity (57, 64). Polarized macrophages
are classified into M1 phenotype (classically activated macrophages) or M2 phenotype
(alternatively activated macrophages) (65). M1 macrophages exhibit a phenotype
characterized by the production of high levels of IL-12 and IL-23, but low levels of IL-10,
high production of reactive oxygen intermediates, inflammatory cytokines and robust
bacterial killing. In addition, M1 macrophages can respond to Th1 inflammatory cytokines,
such as IFN-γ, GM-CSF, and TNF, and microbial products such as lipopolysaccharides
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(LPS), thereby mediating resistance against intracellular microorganisms (65-67). M2
macrophages are subdivided in M2a, M2b and M2c. M2a are induced by IL-4 and/or IL-13,
and are associated with Th2-immune responses, arginine metabolism and immune response to
helminths (68). The presence of immune complexes and agonists of TLR or IL-1 receptors
are found to induce M2b phenotype, which is involved in immune regulation and Th2
activation. M2c are induced by IL-10 or TGF-β or glucocorticoid hormones, and these cells
are characterized by their roles in suppressing immune responses and tissue remodeling and
healing, collagen production and fibrosis (70-73). Generally, the various subtypes of M2
macrophages share a phenotype characterized by high levels of scavenger, mannose and
galactose-type receptors and IL-10 but also low levels of IL-12 and IL-23 (66, 67, 70).
Interestingly, M2b have been found to produce (similarly to M1 cells) TNF, IL-1, and IL-6
(74, 75) and induce high levels of inducible nitric oxide synthase (iNOS) and nitric oxide
(NO) and related reactive nitrogen intermediates (RNI) (76).
Since lungs are exposed constantly to both harmless and pathogenic agents, the
immune response must be tightly controlled. In the steady stage, lung macrophages have been
described to exhibit a more M2 phenotype to avoid cellular damage in the alveoli. However,
resistance to intracellular pathogens such as mycobacteria needs a M1 polarization, but this
must be controlled, since excessive or prolonged M1 responses are harmful for the host (77,
78). Therefore, lung macrophages are believed to turn into a M1 phenotype in responses to
lung injury from pathogens or damaged tissues, and later to be replaced by M2 macrophages
that contribute to tissue fibrosis or repair (77). However, some intracellular bacteria have the
capacity to interfere with M1 polarization to survive and escape macrophage responses by
disrupting their microbicidal capacities or inducing macrophages to M2 phenotype (79, 80).
Additionally, to suppress the induction of adaptive immunity against harmless antigens,
macrophages have been found to down-modulate the antigen-presenting capacities of DC and
to suppress T-cell activation and antibody production by B cells in the airways (81, 82).
Dendritic cells
DC are antigen-presenting cells (APC) specialized in T-cell activation. In the
respiratory tract, DC form a tight network of cells within the epithelium and sub-mucosa of
the conducting airways, the lung parenchyma and the nasal mucosa (83, 84). DC have been
described to play important roles in the regulation of immune responses to inhaled particles
(allergens, pollutants and microbes). They are extremely efficient antigen-presenting cells,
but with weak phagocytic capacity. After engagement of TLR, DC recognize antigen, migrate
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to the regional lymph node and present the antigen to T cells to induce T-cell activation
relevant in T-cell mediated immunity and pulmonary humoral responses to infection (83, 84).
The different subsets of DC are: myeloid or conventional DC (mDC/cDC) that develops,
from bone marrow-derived monocytic precursors and plasmacytoid DC (pDC), which are
developmentally related to the lymphoid lineage (85). In the lung steady state, DC have been
found to exhibit continuous turnover, which is exacerbated upon inflammatory stimuli (86).
Human and mouse DC have been described to express different DC subsets in the lung. In
normal human lung parenchyma; the presence of CD11c-/BDCA-2+ (pDC) and two subsets
of mDCs CD11c+/BDCA[blood DC antigen]-1+ and CD11c+/BDCA-3+ have been described
(84, 86). 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. Also, DC can act as a potent APC in situ in some other
diseases (11, 87).
Two major DC subsets have been described in mouse lung, CD11b+CD11c+
myeloid/conventional DC (mDC/cDC) and CD11b-B220+ (pDC), where pDC are found
mainly in the lung interstitium, secreting large amounts of IFN-α in response to CpG motifs
or viral infections. Moreover, unlike mDC, pDC have poor APC activity, and there is no
evidence for pDC migration out from the lung (8, 12, 86).
Neutrophils
Neutrophils have a short life span. They are the first immune cells recruited from the
bloodstream to the site of inflammation (88). Neutrophils are able to phagocytose and kill
microbes through antimicrobial mechanisms (a combination of non-oxidative and oxidative
mechanisms) and the formation of neutrophil extracellular traps (NET) (88, 89). The
recruitment of neutrophils is an essential antibacterial defense mechanism in the lungs.
Neutrophils sense bacteria or bacterial products through the PRR, such as TLR and NLR
proteins. Bacterial recognition followed by activation of transcription factors, production of
chemokines, up regulation of cell adhesion molecules, and enhancement of cell-cell
interactions (88-90). KC (CXCL1) and MIP-2 (CXCL2) murine chemokines are the major
factors responsible for recruiting neutrophils. Both chemokines KC and MIP-2 are able to
bind the chemokine receptor, CXCR2. In humans, the homologs are IL-8 and GRO
(functionally similar to the IL-8 CXC chemokine family) (88, 89).
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Natural killer (NK) cells
NK cells are a small fraction of lymphocytes that are best known for their potent
cytotoxic activities against cancer cells and cells infected with virus and intracellular bacteria.
NK cells lack antigen specificity, and their activation occurs through target-cell recognition,
which is controlled by germ line encoded activating and inhibitory receptors (91, 92). These
receptors are NKR-P1 and Ly49. NKR-P1 binds to host cell carbohydrates, triggering the NK
cell to kill the host cell to which they are bound. The Ly49 molecule binds to the MHC-I
molecule, thus inhibiting the killing activity of the NK cells. If both signals are activated
simultaneously, the inhibitory one is dominant and the cell will not be killed. This recognition
allows NK cells to discriminate between normal cells from abnormal cells to finally kill the
altered target cells (91, 92). In addition, NK cells can secrete different inflammatory
cytokines and chemokines such as TNF, IFN-γ, IFN-α, MIP-1α and IL-22 (93-95). Studies in
animals have shown that after the spleen, the lung is the tissue containing the largest number
of NK cells (96, 97). The important role of NK cells in host defense in the lungs has been
demonstrated in several models including viral and intracellular bacterial infections e.g.
mycobacterial infections (98).
Phagocytosis
Phagocytosis is a mechanism by which phagocytes, such as macrophages and
neutrophils take up large particles into cells, which occurs by a receptor-mediated- and actindependent mechanism (99, 100). Phagocytosis is very complex due to the diversity of
receptors capable of stimulating phagocytosis, and because the capacity of a variety of
microbes to influence their fate once internalized (99, 101-103). In general, the diverse
phagocytic mechanisms start with microbial interactions with phagocytes, which stimulate
these cells to activate several complex signaling networks of phagocytosis. This activation is
generated either by direct recognition, for instance PRR and their ligands, or indirect
recognition, when the microbe has been opsonized by immunoglobulins or complement and
consequently is engaged by their receptors (FcγR and complement receptors respectively)
(101, 102). This interaction is followed by the polymerization of actin at the site of ingestion
and the internalization of the particle via an actin-based mechanism. Consequently, actin is
shed from the phagosome, and the phagosome matures by a series of fusion and fission
events with components of the endocytic pathway, culminating in the formation of the mature
phagolysosome. Since endosome-lysosome trafficking occurs primarily in association with
microtubules, phagosome maturation requires a coordinated interaction between actin and
18
tubulin based cytoskeletons (99, 101, 102, 104, 105). The process of phagocytosis normally
leads to kill and eliminate the pathogen depending on which receptors were engaged in
phagocytosis.
Cellular mechanisms of microbial killing in infected cells
In the lungs, there are different pathways that mediate the killing of microbes that
occasionally break the sterile status of the alveoli. After detection of inhaled pathogens by the
host cells (macrophages, neutrophils, AEC among other cells) there is an organized induction
of antimicrobial mechanisms to kill bacteria in infected cells as well as in the stimulation of
antigen-specific responses mediated by the adaptive-immune system.
Generation of reactive oxygen/nitrogen species (ROS/RNS)
Neutrophils and lung macrophages, especially AM, kill phagocytosed pathogens by
the generation of reactive oxygen/nitrogen species (ROS/RNS) such as the reactive oxygen
intermediates (ROI): superoxide anion (O2-) and hydrogen peroxide (H2O2), peroxyl (ROO-)
radical, the very reactive hydroxyl (OH-) radical and, the reactive nitrogen intermediates
(RNI): nitric oxide (NO), nitrogen dioxide (NO2-) and peroxynitrite (ONOO-) (106, 107).
ROI/RNI are involved in the activation of various signaling pathways to generate effector
functions and therefore in the initiation of different immune responses against microbes in the
lungs and other tissues (107-110). Various ROI, such as superoxide, are generated by the
assembly and activation of Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase
as a part of the respiratory burst in human and murine macrophages and neutrophils (107110). ROS also play roles as secondary messengers in many signaling pathways, such as NFκB, activating protein-1 (AP-1), mitogen-activating protein kinase (MAPK), and
phosphotidyl inositol-3 kinase (PI3K) pathways (107, 109, 110). Superoxide production by
AM upon LPS stimulation has been suggested to be important in the activation of NF-κB
pathways and the production of cytokines (107, 109, 110). The microbicidal activity of
neutrophils seems to be mostly through the formation of hypochlorous acid (HOCl) generated
from H2O2 by myeloperoxidase (MPO) in the presence of Cl- ions. Therefore, neutrophils are
mostly ROI dependent for the microbial killing (88) in the distal air spaces (111).
Activated macrophages seem to use RNI, such as NO, for their microbicidal capacity
against intracellular pathogens (112, 113). NO is generated by distinct isoforms of NO
synthase (NOS) enzymes with inducible NOS (iNOS) or NOS2 (114). NO, in conjunction
with ROI, is responsible for microbial DNA damage and alteration in microbial membrane
19
lipids and proteins (115, 116). The production of NO is limited by the competition of two
enzymes: NOS, specially the inducible isoform (iNOS) and the arginase (ARG) (I and II) for
their common substrate, L-arginine (117-119). The balance between the presences of both
enzymes depends on the cytokine environment in response to pathogens. Th2 cytokines, such
as IL-4, IL-10, IL-13, TGF-β and M-CSF, have been seen to induce the expression ARG-I
(118, 119), whereas IFN-γ, IL-1, TNF and GM-CSF (Th1 cytokines) have been shown to
induce iNOS (120, 121). The relative expression and regulation of NOS and ARG might be
dependent on the activation status of the macrophages due to other stimuli such as LPS (122,
123) (classic inducer of Th1 responses), while IFN-γ (124) has been described to induce both
ARG and NOS. Also, SP-D (produced by epithelial cells) may also influence the regulation
of both ARG and NOS (125, 126).
Autophagy
Autophagy (macroautophagy) is an important mechanism that provides a membranedependent mechanism for the sequestration, transport and lysosomal turnover of clearance of
intracellular components, including organelles, apoptotic bodies, and invading microbes (127,
128). The steps involved in the autophagy pathway are: a) formation of an isolation
membrane; b) autophagosome formation with encapsulated cargo; c) autophagosomelysosome fusion and digestion of lysosomal contents (degradative phase) (127). Autophagy
plays a critical role in innate immune processes upon microbial recognition (129-132) as well
as in adaptive immunity as the autophagosome may deliver ligands for its activation (129133).
There are more than thirty key components in the autophagic machinery, which are
encoded by autophagy-related (Atg) genes function at different steps in this process (134).
Two ubiquitin-like conjugation systems are essential for the autophagosome formation: the
Atg5-Atg12 conjugation system and the microtubule-associated protein-1 light chain (LC)
(Atg8) (135), as well as the Bcl-2-interacting protein, beclin 1 (134). TOR (the target of
rapamycin) is a key regulator of autophagy where the mammalian TOR (mTOR) has been
described as a central negative regulator of autophagy that can directly phosphorylate ULK1
and mAtg13 and inhibits ULK1 kinase activity, which is essential for autophagy induction
(136). Thus, autophagy is regulated through mTOR by the presence of microbes, processes
downstream of PRR and immune cytokine activation and the TAB2-TAB3-TAK1-IKK
signaling axis (129, 131, 137).
20
Autophagy contributes to control microbial infections through various mechanisms,
including regulation and activation of PRR pathways, such as TLR, RIG-I and NLR (138142), regulation of microbial killing, IL-1β production (143, 144) and T-cell selection
through the MHC II molecules (145-147). In the lungs, autophagy has been reported to be
involved in the elimination of intracellular microbes, including Mycobacterium tuberculosis
(Mtb) (148). Autophagy can also be activated through the beclin-1-Atg7-Atg5 canonical
pathways in AM cell line infected with P. aeruginosa (149).
Cytokines and chemokines
The important functions of some cytokines and chemokines involved in the
macrophage and other innate cell activation in the respiratory tract will be described in this
segment due to their critical involvement in innate defense against mycobacterial and other
respiratory infection and in determining the subsequent adaptive T-cell response.
Figure 2. Role of innate cytokines in mycobacterial infection. Mucosal Immunology (2011) 4,
252–260; doi:10.1038/mi.2011.13; published online 23 March 2011 (160)
21
Tumor necrosis factor (TNF)
TNF is a pro-inflammatory cytokine produced by monocytes, macrophages, DC and T
cells in response to infection, bacterial products and other stimuli (150, 151). In the immune
system, TNF is crucial in mediating inflammation, promoting cell growth, apoptosis, and
enhancing the cellular immune response (152, 153). TNF mediates its activity by binding two
types of receptors, TNFR1 and TNFR2, expressed on diverse cell membranes to initiate
cytoplasmic signaling pathways following receptor-ligand interaction (152-154). Together
with IFN-γ, TNF activates macrophages to prevent the growth or to kill intracellular
microorganisms through the generation of ROI and RNI (155-157). It has been found that
TNF plays a key role in granuloma formation, inducing macrophage activation, and in the
resistance to Mtb infection. (158-160).
Interleukin (IL)-12
IL-12 is a bioactive IL-12 p70 heterodimer protein produced by different cells, such
as monocytes/macrophages, neutrophils and DC, upon different stimuli such as cytokines and
pathogens (161-163). IL-12 is composed of two covalently linked subunits: p35 and p40
(shared with IL-23). Together with IL-23 and IL-27, IL-12 has been described as important
Th1 mediator for inducing and maintaining Th1 immunity (164-166). Therefore, IL-12 plays
a crucial role in bactericidal activity and antigen presentation by inducing the production of
IFN-γ on NK- and T cells (162, 166-168). IL-12 is a key player in host defense against
intracellular bacteria such as mycobacteria (160). The protective role of IL-12 can be inferred
from the observation that IL-12 deficient mice are highly susceptible to mycobacterial
infections (169). Similarly to other cytokines, IL-12 is a regulatory cytokine that connects the
innate and adaptive host response to mycobacteria and probably exerts its protective effects
mainly through the induction of IFN-γ.
Interferon (IFN)-γ
IFN-γ is a homodimeric glycoprotein type II interferon cytokine secreted by activated
immune cells, primarily T (CD8+ T cells and CD4+ Th1 cells), NK and NKT cells (170). IFNγ plays a critical role in both innate and adaptive responses against viruses and intracellular
microorganisms. A wide range of cellular responses are regulated by IFN-γ, such as
promoting cell-mediated immune responses, increased antigen presentation and production of
pro-inflammatory cytokines (170, 171). The Janus kinase (JAK)/Signal Transducer and
Activator of Transcription (STAT) signaling pathway are the primarily mechanism through
22
which IFN-γ gene expression is primary induced (171, 172). IFN-γ together with TLRbinding pathogens (virus or bacteria), induces the classical activation of macrophages
increasing intracellular killing of phagocytosed microorganisms and promotes recruitment of
additional antimicrobial cells (170). Thus, protective anti-mycobacterial or viral immune
responses involve mainly IFN-γ secreted by T cells to activate macrophages and induce their
microbicidal functions (170). It has been well established that IFN-γ provides protective
function against Mtb and other mycobacteria in both mice and humans (160, 173). However,
even if IFN-γ is important to generate immune responses and protection against Mtb, it is not
enough for eliminating these mycobacteria (174).
IL-6
IL-6 is a pleiotropic cytokine, which plays a major role in hematopoiesis, T- and Bcell differentiation, and inflammation. IL-6 is secreted by T cells and macrophages as part of
the inflammatory response to trauma, such as burns or other tissue damage. Bacterial and
viral infection and microbial products such as LPS also induce IL-6 (175, 176). IL-6, which
has both pro and anti-inflammatory properties, is produced early during mycobacterial
infection and at the site of infection (160). IL-6-deficient mice display increased
susceptibility to Mtb infection, which suggests a protective role of IL-6 (177). However, IL-6
secretion by macrophages infected with Mtb may contribute to the inability of IFN-γ to
eradicate Mtb infection (178).
IL-1β
IL-1β is a member of the IL-1 family of cytokines mainly produced by monocytes,
neutrophils, macrophages, keratinocytes, epithelial cells and DC. This pro-inflammatory
cytokine is secreted at the sites of infection or injury, and regulates a broad variety of
physiological responses. This cytokine is an important mediator of the inflammatory
response, and is involved in a variety of cellular activities, including cellular proliferation,
differentiation, and apoptosis (179). Pro-IL-1β is produced in response to inflammatory
stimuli such as TLR ligands and converted into mature IL-1β (17 kDa) by caspase-1
(activated by inflammasomes for instance: NLRP3) to be able to bind specific receptors (IL1RI and IL-1RII) and therefore activate cells (179, 180). In vitro studies have shown that IL1β secretion depends in part on macrophage autophagy through the regulation of NLRP3
inflammasome and inhibition of pro-IL-1β upon LPS (144). Also, IL-1β has been found to
cause lung inflammatory diseases and enhance the production of KC, MIP-2 and matrix
23
metalloproteases (MMP)-9 and -12 in the lungs of transgenic mice (181). IL-1β deficient
mice have been found to be acutely susceptible to Mtb infections, suggesting the importance
of this cytokine in the host’s resistance to Mtb (182).
Granulocyte-macrophage colony-stimulating factor (GM-CSF)
GM-CSF is a 22 kDa glycoprotein secreted by a wide variety of cell types, including
fibroblasts, endothelial cells, T cells, macrophages, mesothelial, epithelial cells and many
types of tumor cells (183-185). GM-CSF promotes the growth of myeloid progenitor cells
and the activation of mature neutrophils, eosinophils, and monocytes (185). Bacterial
endotoxins and inflammatory cytokines, such as IL-1, IL-6, and TNF, are found to induce
GM-CSF secretion (184-186). GM-CSF is a cytokine not only with the capacity to increase
antigen-induced immune responses, but also to alter the Th1/Th2 cytokine balance (184). AM
from GM-CSF-deficient mice have shown to reduce the phagocytic capacity of macrophages
capacity and their ability to kill bacteria, suggesting an arrest at an early stage of
differentiation (187). GM-CSF deficient mice have been found to succumb more rapidly from
severe necrosis when exposed to aerosol infection of Mtb, because of their inability to mount
a Th1 response (188).
Monocyte chemotactic protein (MCP)-1/CCL2
MCP-1 is an 8.7 kDa CC chemokine produced by monocytes, macrophages, and
epithelial cells in the lungs (189, 190). The principal role of MCP-1 is the induction of
chemotaxis, proliferation and cytokine secretion in monocytes/macrophages, mast cells, and a
certain subset of human T lymphocytes (191). Mycobacteria such as Mtb preferentially
induce production of MCP-1 by monocytes, and to direct T cells to the site of infection in the
lung perhaps to help in the granuloma formation (192).
Murine keratinocyte-derived (KC) chemokine/CXCL1 and macrophage-inflammatory
protein (MIP)-2/CXCL2
KC and MIP-2 are also considered homologs to the human GRO chemokines that are
functionally similar to the human IL-8 CXC chemokine family (193, 194). Both are closely
related (195) and are the major chemoattractants responsible for recruiting neutrophils in
mice at the site of infection from blood stream. Both chemokines bind to the chemokine
receptor, CXCR2 (196). Macrophages are the main sources for MIP-2 and KC, but
24
endothelial, mesothelial and alveolar epithelial cells can produce both chemokines (21, 36,
38, 46). The MIP-2 mRNA expression has been shown to be induced in mice infected with
different Mtb strains (194). Also, lipoarabinomannan (LAM), a cell wall component of Mtb
has been found to induce MIP-2 and KC in the lungs (197).
Interleukin-8/CXCL8
IL-8 is a human CXC chemokine structurally related to the growth related oncogene-α
(GROα). IL-8 is produced by macrophages, fibroblasts, endothelial- and epithelial cells upon
different stimuli including inflammatory signals, chemicals and environmental stresses,
(198). The principal function of IL-8 is to attract neutrophils, T lymphocytes, and possibly
monocytes to the site of infection (198). During lung inflammation, IL-8 is secreted at high
levels to attract and activate neutrophils to the injured tissue (199, 200). Human monocytes
infected with Mtb have been found to affect the IL-8 production (201) and apparently TNF
and IL-1β are the responsible to control IL-8 production (202).
Mycobacterial infection in the respiratory tract
Tuberculosis
Tuberculosis (TB) is an infectious disease caused by 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 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 possible reduction in HIV
prevalence. Of these HIV-positive TB cases, approximately 80% are in the African region
(203).
Bacillus Calmette-Guérin (BCG) is the only available vaccine against TB, which is
prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus,
Mycobacterium bovis (204). BCG vaccination does not confer protection against adult
pulmonary TB but it is effective against childhood tuberculous meningitis and miliary
disease. Until now, there is no new vaccine able to achieve a level of protection better than
BCG.
25
Pathogenesis of Tuberculosis
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 lung macrophages
(205, 206). 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 (207). Also, Mtb is reported to
invade both alveolar epithelial cells, as well as other surrounding cells in the respiratory tract
(208-210). 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
(211-213). 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 (205, 206). The granuloma formation prevents the dissemination of Mtb
and provides a local environment for communication between immune cells. However, the
granuloma formation does not always eliminate the mycobacteria. 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 mechanisms have 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 (205, 206). Around
15% of TB patients develop extra pulmonary TB in the pleura, lymphatics, bones, genitourinary system, meninges, peritoneum, or skin.
Innate responses may prevent mycobacterial infections
It is believed that only a small fraction of individuals (without previous vaccination or
exposure to (Mtb) in close contact with TB patients develops active TB (203). These findings
support the hypothesis that immune surveillance and early protection against Mtb, depends
on the generation of an effective early innate response. For that reason, an early activation
and recruitment of cells such as macrophages and neutrophils to the site of the infection may
contribute to the early control of Mtb infection through the production of ROS/RNI and other
pro-inflammatory factors (214-216). Recently, other resident cells in the lungs such as
26
alveolar epithelial cells have also been suggested to play different roles during Mtb infection
(217). During early phase of mycobacteria infection epithelial cells, together with
macrophages, were found to secrete Lipocain 2, which is important in the host’s defense
against Mtb (218). Moreover, epithelial cells may modulate resident phagocytes to kill
mycobacteria through the secretion of a broad variety of factors as we described in this thesis.
Furthermore, the production of antimicrobial peptides by local cells including epithelial cells
may also mediate the direct killing of mycobacteria.
27
PRESENT STUDY
Aims
General aim
The overall aim of this study was to determine the role of AEC in the immune response
against mycobacterial infection, with particularly focus on the influence of AEC-secreted
factors on macrophages, to maintain the local responses in the respiratory system using
murine models.
Specific aims
-
To evaluate the role of AEC in pulmonary immune responses to mycobacteria and
different stimuli including mycobacterial products.
-
To evaluate the effects of factors secreted by AEC on bacteria and a number of
macrophage functions.
-
To investigate the responsible mechanisms of bacterial killing by macrophages
mediated by AEC-secreted factors.
28
Materials 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,
-
Cell separation by magnetic beads
-
ELISA
-
Cytokine array
-
Flow cytometry
-
Fluorescence microscopy
-
Luminescence assay
-
Trans-membrane migration assay
-
Wound healing assay
-
Quantitative RT-PCR
Here, there will be a description of GFP-BCG quantification as well as the isolation and
phenotype determinations of AEC and PuM, because these methods were used in all the
different studies in the present thesis.
Quantification of GFP-BCG
For a rapid quantification of BCG in our cultures, we used GFP-BCG. To construct
the GFP-BCG strain, M. bovis BCG was transformed with the dual reporter plasmid
containing the human codon-optimized and fluorescence-enhanced EGFP and the luxAB
genes from Vibrio harveyi (219). This is very convenient since bacteria contents can be
quantified immediately by luminescence, while the classical evaluation of BCG growth in
agar plates takes between 2-3 weeks. Luminescence is expressed as relative luminescence
unit (RLU). To determine the RLU, Decanal (Sigma-Aldrich) was used as a specific substrate
for the bacterial enzyme luxAB. Decanal was dissolved in 70% ethanol and added to the
lysates at a final concentration of 0.01%. The samples were mixed immediately and the RLU
was measured after 15 seconds in a Modulus, Turner Bio Systems luminometer.
Isolation and phenotype determination of AEC and PuM
Total pulmonary cells were prepared using Corti’s protocol (220) with previously
described modifications (Paper I, Paper IV). In short, CD45+ cells were obtained from total
lung cells using MACS (Miltenyi, Germany) and subsequently cultured for 48h in RPMI
29
(Gibco-Invitrogen, Paisley, UK) supplemented with 10% FCS, 2mM L-glutamine, 100 U/ml
penicillin, 100 µg/ml streptomycin, 0.02 M Hepes, and 0.05 M 2-mercaptoethanol (Sigma) at
37ºC and 5% CO2. PuM were isolated by adhesion to get rid of cellular debris and nonadherent cells such as DC. Cells isolated from lung parenchyma, and not from
bronchoalveolar lavage, are enriched in interstitial macrophages (221-223). In average, 98%
of the adherent cells using this methodology were positive for the macrophage marker F4/80.
Isolated PuM were in average 80% of F4/80+CD11c+ and 30% of F4/80+CD11b+ (Table 2;
Paper II). AEC were obtained by depleting CD45+ and CD146+ cells from lung preparations
using MACS. ~92-95% of these CD45-CD146- cells exhibited an AEC phenotype, where
approximately 22% expressed podoplanin (AEC I) and approximately 72% expressed CD74
(AEC II) as determined by flow cytometry.
All mouse experiments were approved and performed in accordance with the guidelines of
the Animal Research Ethics Board at Stockholm University.
30
Results and Discussion
Paper I
The role of alveolar epithelial cell in initiating and shaping pulmonary immune responses.
Communication between the innate and adaptive immune systems
Macrophages and DC are considered to be key players in the defense against
mycobacterial infections in the lungs. However, other cell populations in the lower
respiratory tract, such as epithelial cells, have been suggested to play important roles in the
pathogenesis and defense against mycobacterial and other infections. In the lungs, the
alveolar compartment is lined with membranous pneumocytes, known as Type I alveolar
epithelial cells (AEC I), and granular pneumocytes, type II alveolar epithelial cells (AEC II).
Both types of cells play important roles not only in regulating the gas exchange in the body
but also immune responses in the lungs (224). AEC have been considered to play an
important role during mycobacterial infections due to their strategic localization in the
alveoli, expression of immune markers such as TLR (2) and MHC II (225) and close
interaction with other cells, especially with macrophages. In addition, AEC secrete a broad
variety of antimicrobial products, cytokines, chemokines and other factors.
In Paper I, we first analyzed the phenotypic characterization of freshly AEC isolated
from mouse lung. FACS analysis showed that in average 92-95% of the cells displayed AEC
phenotype, and from them approximately 70% corresponded to AEC II phenotype, while
22% corresponded to AEC I. We next, compared AEC with interstitial macrophages, herein
named PuM, in their ability to generate immune responses against mycobacterial products
and BCG. We first analyzed the ability to take up bacteria and the capacity to control the
intracellular bacterial growth by AEC in comparison with PuM infected with BCG. Our data
showed that even if PuM were more efficient in both capacities, AEC 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 (226, 227). We also
performed in vitro experiments to compare the capacity of primary AEC to act as APC
compared to professional APC, for instance PuM. Previous studies have shown that AEC II
express constitutively MHC II (225), and that murine AEC II can present mycobacterial
antigens to T cells (228). Our findings showed that AEC pulsed in vitro 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 lower
31
compared to that seen with pulsed PuM. These results confirmed the capacity of AEC, most
probably by AEC II, to take up, process and present antigens. Therefore, we support previous
data suggesting a possible role as APC of AEC in the adaptive responses 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 distinct compartments of
AEC and T cells. Consequently, AEC have to promote the migration of T cells from the
peripheral blood and other compartments to the lung to generate a successful antigen
presentation. Also, another crucial factor is the expression of co-stimulatory molecules on
AEC. In humans and mice, AEC II express a low grade or lack of expression of classical costimulatory molecules (53, 229). The lack of co-stimulatory molecules in AEC II may induce
T-cell tolerance to suppress inflammatory responses in the lungs against harmless antigens
(53). Moreover, another study showed that AEC II are able to induce regulatory peripheral T
cells inducing tolerance against self-antigens in the lungs through the secretion of factors
such as TGF-β (230). Thus, AEC participation in the lung immune defense is likely to happen
through the secretion of factors that can modulate the activation and function of different cell
types present in the lungs.
In the lungs, the production of cytokines, chemokines and other factors by resident
cells decides the outcome of inflammatory responses in this tissue. Although, immune cells
such as macrophages and DC secrete many of these factors; AEC and other cells in the lungs
are able to produce many factors constitutively or upon stimulation (231, 232). To gain a
better understanding of the role of AEC in the production of factors against mycobacteria, we
first compared the production of MCP-1, MIP-2, KC, TNF, MMP-9 and IL-12 in primary
AEC with that of PuM upon stimulation. 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 was mostly secreted by primary AEC, while PuM were the main producers of MIP-2
(homologue in mouse of human IL-8). However, even if the levels of MIP-2 secreted by AEC
were lower than PuM, these levels were comparable with IL-8 levels secreted by human
epithelial cells (233).
Since macrophages secrete TNF, and MCP-1 can activate macrophages, the possible
influence of PuM on AEC and vice versa was suggested when primary AEC secreted high
amounts of MCP-1 upon TNF. The major ligands may be present in the BCG cell wall due to
32
the fact that Lys-BCG was not as good stimulator compared with HK-BCG. TNF and IL-12
were only produced by PuM upon LPS stimulation (data not shown). The role of MMP-9, a
molecule involved in the granuloma formation (234, 235) was also investigated. AEC but not
PuM were good secretors of MMP-9 upon TNF stimulation.
Since AEC are able to produce factors such as MCP-1 and MIP-2, we aimed to
determine a more complete profile of different factors produced by AEC. Cells were
stimulated with TLR ligands [Flagellin, Pam3Cys-Ser-(Lys)4 trihydrochloride (Pam3) and
LPS], IFNs (IFN-γ and IFN-α) and mycobacterial products, such as virulent HK-Mtb, HKMtb attenuated phoP mutant
strain
SO2 (SO2), HK-BCG and Lys-BCG. The cell
supernatants were collected and analyzed with a proteome profile array. We found that a
broad array of different factors was produced by AEC namely: G-CSF, GM-CSF, M-CSF,
KC, MCP-1, MIP-1, MIP-2, TIMP-1, IL-6, and IP-10. The analysis of un-stimulated AEC
showed that these cells were able to constitutively produce some of the factors (GM-CSF, MCSF, 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.
We also evaluated the levels of some factors because higher levels of MCP-1 were
secreted by AEC upon LPS and TNF. We found that GM-CSF is probably the main growth
factor produced by AEC as a result of undetectable levels of M-CSF even after stimulation of
AEC (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 induction
via TLR whereas IP-10 and RANTES were mostly induced by IFNs. Therefore, the
interaction between AEC and lymphocytes is also possible since IFNs (mainly produced by
lymphocytes) were able to induce the secretion by AEC of IP-10 and RANTES. Thus, AEC
are perhaps assisting in the recruitment of circulating lymphocytes to areas of injury,
inflammation, or viral infection in the lungs. Flagellin was the weakest inducer of the three
different TLR ligands.
Together, our data have shown a role for AEC in internalization of mycobacteria and
a role as APC. However, even if AEC were able to process and present mycobacterial antigen
to T cells, their role as APC appears to be secondary, since AEC and T cells belong to
different compartments. We have also extended the knowledge about AEC as a
multifunctional cell type by determining the secretion of a broad variety of factors in
response to several stimuli. Our findings suggested a possible cell-cell interaction between
AEC and other resident cell populations in the lungs as evidenced by the production of
33
cytokines, chemokines and other factors by AEC. We concluded that AEC are involved
actively in inducing and modulating different effects on other cell types resident in the lungs
such as monocytes, macrophages, DC, and T cells important in the of the respiratory tract.
Paper II
Alveolar epithelial cells are critical in protection of the respiratory tract by secreting factors
able to modulate the activity of pulmonary macrophages and directly control bacterial
growth
We (Paper I) and others have previously described that AEC (type I and II) are not
only considered part of the physical and functional barrier in the lungs, but these cells are
also actively involved in the clearance of inhaled pathogens. Although, both type I and II
AEC are constantly contributing to the lung defense, many studies have focused on AEC II,
because these cells were found to be more immunologically active. Even though AEC can
present antigen to specific T cells (44, 46, 49, 52, 236, 237), it has been suggested that AEC
contribution in the lung defense is mainly through their production of a broad variety of
antimicrobial products and their capacity to promote inflammatory responses by secreting
factors such as cytokines and chemokines involved in activation and differentiation of
immune cells (44, 46, 49, 52, 236, 237). Macrophages are also considered important in the
defense of the respiratory tract. The lower respiratory tract has two macrophage populations:
alveolar macrophages (AM) in the alveoli and interstitial macrophages located in the
insterstitium (56) herein named PuM. It has been described that the macrophage function is
regulated
according
to
their
anatomic
localization
and
exposure
to
different
microenvironments in the lungs (56, 238).
In the present study, we hypothesized that being in the interface; AEC may play an
important role in transmitting signals from the external to the internal compartment and in
modulating the activity of PuM. Thus, we evaluated the effect of secreted factors by AEC in a
number of macrophage functions. In Paper I, we described that AEC isolated from mouse
lungs are able to secrete different factors with or without stimulation. For this reason, we
chose to prepare two types of AEC-derived media: Cell supernatants were prepared and
collected from AEC un-stimulated and stimulated in vitro with lipopolysaccharide (LPS) and
named as AECsup and AECLPS, respectively. We also preferentially worked with PuM,
because these cells are in close contact with AEC and with other immune cells in the lung
34
interstitium. PuM were isolated and purified using a previously described system to produce
high purity of interstitial macrophages (221-223).
Previous studies have shown that AEC-derived media attract human monocytes, Tcells and neutrophils isolated from peripheral blood (49, 239), but there are no reports related
to the migration of resident lung cells. Therefore, we first evaluated whether AEC could
promote migration of local pulmonary cells, and whether a specific cell type is preferentially
influenced by AEC-derived factors. Using a transwell-migration assay we found that higher
numbers of lung cells migrated towards AEC-derived media than towards medium alone. The
phenotypic analysis of the migratory cells showed that most of them were F4/80+/CD11c-.
Since AEC-derived media induced mostly the migration of lung cells with the F4/80+
phenotype, we used the scratch assay (wound healing assay) to specifically assess
macrophage migration. For this, we evaluated the kinetics of the cell line J774 and PuM upon
treatment with AEC-derived media, and found that J774 cells started migrating at an earlier
time-point (after 4 h) than primary PuM (after 24 h) and for both cell-types, AEC-derived
media increased migration compared with the control. Studies of neutralization of MCP-1,
IL-8 and GRO- from culture medium of human AEC II have shown a reduced migration of
blood monocytes toward these human AEC II derived media (49, 239, 240). Therefore, it is
likely that in our system these factors play a major role in inducing the macrophage
migration, since we have previously shown that AEC secrete the murine IL-8 homologs KC
and MIP-2 and high levels of MCP-1 upon stimulation with LPS (Paper I).
Next, we evaluated the effect of AEC-derived media on other macrophage functions,
such as phagocytosis and control of intracellular bacterial growth. Both macrophage
functions are crucial for preventing infections (clearance and destroying of invading
pathogens) and therefore to maintain the homeostatic immunological balance in the lungs.
We found that PuM and bone marrow derived macrophages (BMM) increased bacterial
uptake and also reduced the bacterial growth upon AEC-derived media treatment. Moreover,
we also assessed the need for a constant presence of AEC-derived factors in the process of
intracellular growth control and found that after infection, in the absence of AEC-derived
media the reduction of intracellular bacterial growth was lower than treated cells. The fact
that AEC-derived media increased both macrophage functions is in line with the crosstalk
between AEC and macrophage to promote host defense in the lungs, similarly to what others
have previously described using AM (241). Several factors produced by AEC, for instance,
GM-CSF (242) and surfactant proteins (33), may increase phagocytosis as well as
35
intracellular growth control. However, further evaluation of the mechanisms by which these
AEC secreted factors increased both macrophage functions must be evaluated.
The effects of AEC-derived media on macrophage activation suggested that this
activation could be correlated with the up-regulation of surface marker expression. Thus, we
also evaluated the expression of some co-stimulatory molecules, MHC II, MMR (mannose
receptors), CD11b and CD11c on PuM upon AECsup. We found that MMR and CD11b were
up-regulated in PuM upon AECsup. There was not a major effect on the expression of MHC II
or any of the tested co-stimulatory molecules. Since we observed morphological changes in
cells treated with AEC-derived media, we also investigated changes in cell morphology and
actin cytoskeleton using microscopy. We found that upon AECsup treatment of BCG-infected
PuM, an increased appearance of elongated cells, resembling the M2-type phenotype
according to a model described by Vereykel et al, was seen (243). Thus, these morphological
changes together with the increased expression of MMR, a receptor known to be expressed
preferentially by M2 macrophages (55, 58, 238), suggest that AEC-derived media can induce
a M2 polarization which is considered to be less inflammatory and less active in bacterial
killing, perhaps by keeping the integrity of the tissue (70, 244).
Finally, we evaluated the direct effect of the AEC-derived media on bacteria, We
determined whether AEC-derived media had opsonizing effects since AEC have been
reported to secrete factors (opsonins), which enhance the engulfing of bacteria by phagocytes
(32, 245). However, AECsup did not increase the mycobacterial uptake by macrophages,
indicating that the effects of AEC-derived media were on cells. Since AEC have also been
found to secrete a number of antimicrobial products e.g. complement, lysozymes,
cathelicidin, β-defensins and SP (224, 246), we evaluated the direct bacterial killing by AECderived media and compared it with gentamicin. The results show that, BCG treated with
AECsup did not inhibit the growth after 4 h, whereas after 24 h AECsup was as effective as
gentamicin in killing the mycobacteria. The possible mechanism(s) behind this effective BCG
killing in our system is comparable with those of other antimicrobial peptides like
cathelicidins (30).
Thus, factors secreted by AEC have a clear effect on both macrophages and bacteria.
We show here that AEC-derived media induced the migration of lung cells, especially cells
with F4/80 phenotype. Moreover, AEC-derived factors increased phagocytosis and promoted
effective control of intracellular mycobacterial growth, which is in line with the prevention of
bacterial infections in the lungs and dissemination to other places in the body. In addition, our
data suggest that macrophages might polarize towards a more M2-like phenotype upon AEC36
derived media treatment. However, it seems to be controversial that the M2 phenotype
(ascribed as more regulatory than inflammatory cells) is able to control the intracellular BCG
growth. Therefore a further evaluation must be performed. Finally, we found and effective
bacterial killing of BCG, but it is difficult to speculate by which mechanism the bacterial
killing was achieved in our system. Together, we observed that AEC-secreted factors had a
dual effect, in both the control of bacterial growth and in increasing macrophage activity.
Moreover, these effects might be the result of not only the activity of individual factors but
rather to the combination of different factors possibly acting in an additive or synergistic
manner.
Paper III
Soluble factors from alveolar epithelial cells increase intracellular killing of BCG by
macrophages through nitric oxide independent mechanisms
Activated macrophages have been broadly classified into two groups: M1 and M2
cells (65). M1 cells are described to prevent bacterial phagosome escape and to stimulate
intracellular killing of bacteria. Th1 cytokines, such as IFN-γ and microbial products, have
been described to induce iNOS and produce ROS on macrophages with a M1 phenotype
(120, 121). On the other hand, Th2 cytokines have been found to induce ARG-I on
macrophages with a M2 phenotype depicting immunoregulatory properties with poor
microbicidal capacities (118, 119). Lung macrophages have been described to exhibit a more
M2-like phenotype in the steady stage to avoid damage in the tissue. However, it is believed
that these cells turn to a more M1-like phenotype, to accomplish microbicidal competences in
responses to lung injury from pathogens or damage tissues to be later replaced by M2 cells
(77, 78). For controlling intracellular pathogens in the mature phagosome, macrophage use
different antimicrobial mechanisms such as phagosome acidification, activation of iNOS to
produce RNI (112, 113) and activation of the NADPH oxidase (107-110), among other
molecules. Autophagy is another antimicrobial mechanism for killing of intracellular
pathogens through various pathways (127, 128), including the regulation of IL-1β production
(143, 144).
In the present study we investigated the mechanisms responsible for the control of
intracellular bacterial growth in interstitial macrophages (PuM) mediated by AEC-derived
37
media and the well-known potent macrophage activator, IFN-γ. It is extensively described
that IFN-γ induces mainly killing of intracellular pathogens through the up-regulation of
iNOS and NO production (247). Moreover, we have also previously reported that AECderived media from un-stimulated cells (AECsup) enhance the control of intracellular bacterial
growth in PuM (Paper II). We first chose to evaluate and compare the microbicidal
activation by IFN-γ on PuM and BMM infected with BCG. We found that, in contrast to
BMM, previously described to control the BCG growth upon IFN-γ treatment (Paper V),
PuM treated with IFN-γ did not reduce the intracellular BCG growth. However both types of
cells produce NO. We therefore investigated if this unresponsiveness to IFN-γ by PuM was a
characteristic of these cells that reside in the interstitial lung compartment. For this reason,
we evaluated and compared PuM with other primary macrophages, such as AM (described to
take up and kill most of the particulate delivered into the alveolar space) (55, 56, 58) and
peritoneal exudate macrophages (PEM) (described to respond to IFN-γ for activating their
microbicidal capacities) (248), in their capacity to control intracellular bacterial growth
mediated by IFN-γ and AEC-derived media. We found that compared with PuM, AM and
PEM were able to reduce the intracellular bacterial growth upon IFN-γ treatment. As
expected, AECsup treatment mediated the reduction of BCG growth on PuM, AM and PEM,
but without NO production. This suggests that AEC-derived factors may mediate
antimicrobial mechanism by NOS-independent pathways. Also, PuM have been described to
play more regulatory roles, such as limiting local inflammatory responses (58-60, 63),
compared with AM and PEM. We also investigated the role of SOCS1 in PuM and BMM.
SOCS1 is a molecule to be considered as a critical inhibitor of IFN- signaling (249, 250).
We first evaluated the control of intracellular bacterial growth by PuM isolated from IFN--/SOCS1-/- mice upon IFN-γ and AECsup treatments. We found that PuM from SOCS1deficient mice were able to control the intracellular BCG growth after IFN-γ treatment to a
similar level as the AECsup treatment. This finding was confirmed by evaluating gene
expression of SOCS1 in PuM and BMM upon treatment with IFN-γ and AECsup. PuM
significantly induced SOCS1 upon IFN-γ treatment while AECsup did not induce SOCS1.
Thus, the finding that SOCS1 is involved in the un-responsiveness to IFN-γ by PuM to
control the bacterial growth demonstrate that these cells are by some means restricted in their
responses to IFN-γ perhaps for preventing tissue damage in the lungs.
The secretion of IL-12, IP-10 and IL-6 from BCG infected-PuM and -BMM upon
AECsup and IFN-γ treatments was also evaluated. Even if PuM secreted lower levels of these
38
factors than BMM, upon IFN-γ treatment, IP-10 was secreted earlier by BMM but not by
PuM, while increased IL-6 secretion was found in PuM stimulated with 24 h AECsup. It has
been shown that activated M1 macrophages produce IP-10 in order to attract T cells (79, 251)
while the role of IL-6 in macrophage polarization is classically involved in the induction of
M1 phenotype (79).
We next evaluated the expression of molecules such as iNOS, ARG-I and IL-1β,
involved in the control of intracellular bacterial growth by macrophages mediated by both
IFN-γ and AECsup. iNOS expression was induced in both BMM and PuM upon IFN-γ
treatment but to a lower extent in PuM in early induction. As expected, IFN-γ did not affect
the induction of ARG-I in both cell-types. AECsup treatment did not affect the expression of
iNOS in neither PuM nor BMM, while ARG-I expression was increased significantly in both
cell-types. We also confirmed this finding by performing iNOS inhibition and measuring NO
production. Together, our data confirmed that AEC-derived media mediated the microbial
killing by iNOS-independent pathways. ARG-I induction by AEC-derived media correlated
with our previously report that AEC-derived factors induce MMR and changes in the shape
compatible to M2-type phenotype (Paper II). However, it is difficult to define which
mechanisms were activated by AEC-derived factors to enhance the intracellular bacterial
control by inducing ARG-I in our system.
To better understand how AEC-derived media increased intracellular bacterial
growth control of BCG in macrophages by iNOS-independent pathways we also analyzed the
expression of IL-1β. We found that both IFN-γ and AECsup treatments induced IL-1β in both
BMM and PuM. IL-1β is a cytokine described to be important in the host resistance to Mtb
(182).
Recently, IL-1β has also been described to be involved in autophagy another
antimicrobial mechanism used to kill intracellular pathogens (144, 252). However, further
experiments must be performed to determine the role of IL-1β in our system.
Together, our data suggest that PuM are restricted in their inflammatory responses in
the lung interstitium and that AEC-secreted factors enhance the microbicidal capacities of
macrophages by iNOS-independent mechanisms.
39
CONCLUDING REMARKS AND FUTURE PERSPECTIVES
Our studies have shown that non-immune cells resident in the lungs such as AEC are
constantly participating in the defense in the lower respiratory tract against intracellular
pathogens. Here, we extended the knowledge about AEC regarding internalization and
control of intracellular growth of mycobacteria and their role as APC. We also suggested that
AEC are mostly involved in inducing and modulating different effects on other cell-types
resident in the lungs as evidenced by the secretion of a broad variety of factors in response to
several stimuli. Moreover, our data shown that factors secreted by AEC have a clear effect on
both the control of bacteria growth and in inducing macrophage activity as demonstrated by
the their effects in a) migration, b) phagocytosis and control of intracellular bacterial growth
and c) up-regulation of MMR and alterations of cell morphology with characters similar to
that ascribed to M2 cells. In addition, we also have shown that AEC-secreted factors from unstimulated cells induced intracellular bacterial growth in macrophages through iNOSindependent pathways because ARG-I but not iNOS was induced. Here, we suggested that
the mechanisms behind the intracellular growth control mediated by AEC-secreted factors
may be linked to IL-1β production because AEC-derived factors induced IL-1β on
macrophages infected with mycobacteria. Further studies should be investigated such as the
role of IL-1β in the microbicidal capacity of macrophages induced by AEC-derived media. In
addition, it would be of great interest to evaluate other microbicidal mechanisms such as
autophagy mediated upon treatment with AEC-derived media. A remarkable finding was that
compared with other primary macrophages, PuM were not able to control bacterial growth
upon IFN-γ treatment. We have found that SOCS1 are involved in the un-responsiveness to
INF-γ by PuM to control intracellular bacterial growth since a significantly more pronounced
induction of SOCS1 in these cells was found and PuM from SOCS1-deficient mice were able
to control the intracellular bacterial growth. These results suggested that PuM are restricted in
their inflammatory responses perhaps for avoiding tissue damage because strong
inflammatory responses.
We suggested that the effect(s) of secreted AEC factors might be the result not only
by the activity of individual factors but rather to the combination of different factors possibly
acting in additive or synergistic manners. Therefore, despite we have evaluated extensively
the presence of some factors secreted by AEC, it would be of great interest to identify the
factors secreted by AEC involved in mediating the intracellular bacterial growth control by
macrophages in our system.
40
ACKNOWLEDGEMENTS
This study was carried at the Department of Immunology, Stockholm University. I would like
to thank to all who have contributed in any way in making this work possible, in particular,
my supervisor Prof. Carmen Fernández for accepting me as a PhD student in her group. I
sincerely grateful to you Carmen, for all the support, guidance and advices you gave me
during these years. Muchas gracias Carmen!
My special thanks to my co-authors and collaborators Katharina Hartl, Natalia Periolo,
Dagbjört Petursdottir and especially to my friend Jubayer Rahman for helping me when I
asked for and for all the moments shared in the lab.
The seniors at Immunology: Marita Troye-Blomberg, Eva Sverremark-Ekström, Klavs
Berzins and Eva Severinson. Thank you for all the nice discussions and advices.
Thanks to Margaretha Hagstedt “Maggan”, Gelana Yadeta and Anna-Leena Jarva for all your
invaluable help and assistance.
Many thanks to all the staff at the animal facilities for being friendly and helpful.
Thanks to all the past and present colleagues at the Immunology and other Departments,
specially, Irene Roman, for introducing me in the lab work and for your friendship and help.
Andrea Sommer, for helping me with the FACS and for the unforgettable 2010. Natalija
Gerasimcik, thanks for your big heart my friend and all the help with the lab work and
personal issues. “Naty”, thanks for being my “Argentinian” friend and for your help in the
lab! Katarina Tiklova, thanks for all your help, advices and support in the good and bad times
here at SU, and outside and for introducing me with your lovely family and group of friends.
I will never forget you all!
To all my friends in and out of Sweden, particularly, my dear friends Ying and Irina because
you both help me since the beginning even though you did not know me well! I will always
appreciate your help and friendship. My Brazilian friends, Thais and André because of you I
could enjoy Stockholm and Sweden! Annalisa and Sergio, thank you guys for being such
amazing friends!
41
To my Bolivian friends, Edda, Leslie, Jenny and Brenda because despite the distance you
were always there to cheer me up. Las quiero mucho amigas!
Finally, I would like to thank to my beloved family “my treasure” for being my strength and
inspiration to continue my PhD studies during these years. This thesis would not have been
possible without your unconditional love, support and encouragement. Papí, Mamí, Carmen,
Enrique and Gustavo muchas gracias por todo!! Por alentarme y darme las fuerzas necesarias
para continuar en los buenos y malos tiempos, por estar conmigo siempre que lo necesité a
pesar de la distancia. Sin ustedes nunca lo habría logrado! Muy pronto estaremos juntos otra
vez!!!
This work was supported by grant FP7 (HEALTH-F3-2008-200732) from the European
Commission.
42
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