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“Exponential growth of lung granulomas after Mycobacterium tuberculosis
infection in IRF8-deficient mice is associated to defective adaptive immunity”
PhD Coordinator
Prof. Marco Tripodi
Supervisor
Dott.ssa Lucia Gabriele
PhD Candidate
Laura Abalsamo
PhD in Pasteurian Sciences
XXIV Cycle
Contents
Abstract …………………………………………….……………...………………..4
1. INTRODUCTION ……………………………………..……...…………...…….6
1.1 Tuberculosis in the world …………………………………………………...…...6
1.2 Mycobacterium tuberculosis (Mtb) ………………………………....…………...9
1.3 Host-pathogen interaction ………………………………………….………......11
1.4 The immune response against Mtb ………………………………………..…...15
1.5 Interferon Regulatory Factor 8 (IRF8) …………………………………………19
1.6 The IRF8-/- mouse model ………………………………………………………21
2. PURPOSE OF THE THESIS …………………………………………………24
3. MATERIALS AND METHODS …………………………………………..….25
3.1 Bacteria …………………………………………………..………………….....25
3.2 Mice ………………………………………………………………....................25
3.3 Mice infection with Mtb ………………………………………………...….......25
3.4 Histopathologic analysis ……………………………………………….............26
3.5 Immunohistochemistry and Immunofluorescence analyses ……………….......27
3.6 Flow cytometry analyses ………………………………………………...…......29
2
4. RESULTS ………………………………………………………………………31
4.1 IRF8-deficiency causes high susceptibility to Mtb infection …………………..31
4.2 IRF8-/- mice exhibit extensive tissue damage following Mtb infection ………..34
4.3 Different recruitment of immune cells characterizes Mtb infection in IRF8-/- and
WTmice ………..…………………...……………………………………………....39
4.4 Impaired formation of pulmonary newly lymphoid structures in IRF8-/- mice
after Mtb-infection ……………………………………………………………...….44
4.5 IRF8 function is necessary for immune cell maintenance in lung granulomas
during Mtb-infection ……………………………………………………………….51
5. DISCUSSION …………………………………………………………………..57
6. REFERENCES …………………………………………………………….…...62
7. ACKNOWLEDGEMENTS …………………………………………………...67
3
Abstract
Pulmonary tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb)
infection, remains a global health problem of enormous proportions, causing 2
million deaths each year. It is estimated that one-third of the world population has
been exposed to or carry the pathogen, with 8 million new cases of active disease
per year. Both innate and adaptive immunity responses are known to play a pivotal
role in establishing a protective immunity during Mtb infection, and latest studies
suggested that additional genetic factors could affect the outcome of infection. The
interaction between the pathogen and the host immune system induces granulomas
development, structures characterized by bacterial components and immune cells
that represent key pathologic features of TB. Moreover, neogenesis of lymphoid
tissues in the lungs after Mtb infection contributes to the generation of local antigenspecific responses. To test the role of Interferon Regulatory Factor 8 (IRF8) in host
defenses against Mtb, we used mice deficient in IRF8 gene (IRF8-/- mice), and we
investigated the mycobacterial load and dissemination in the lesions, the functional
and structural properties of infiltrated tissues as well as the distribution and
interactions of host immune cells. This study shows that this transcription factor is
essential for the induction of an effective adaptive immunity associated to the
generation of competent pulmonary newly formed lymphoid structures and
granulomas characterized by a proper influx of immune cells able to restrain Mtb
4
infection. Together, these findings support the role of IRF8 as an important regulator
of host defenses against TB.
5
1. INTRODUCTION
1.1 Tuberculosis in the world
Tuberculosis (TB), an infectious disease caused by Mycobacterium
tuberculosis (Mtb), is more prevalent in the world today than at any other time in
human history, and it is a major cause of morbidity and mortality world-wide.
Approximately 1/3 of the human population is skin test positive for the infection
thus it thought to harbor the bacterium. The burden of disease is disproportionately
distributed across the planet with 22 countries bearing 80% of the total number of
cases of active disease. Not surprisingly, this distribution tracks predominantly with
socio-economic status, with sub-Saharan Africa being one of the most intensely
affected area followed by Asia (Figure 1). Smaller proportions of cases occur in
Eastern Mediterranean Region, European Region and Region of the Americas
(Russell 2011; Ahmad 2011).
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Figure 1 Estimated TB incidence rates in 2010, World Health Organization, Report 2011
There are no effective vaccines against Mtb infection and the only currently
approved vaccine, Mycobacterium bovis Bacillus Calmette Guérin (BCG), shows
slight protection against severe disease among some ethnic groups. In addition,
although drug therapy is effective, it requires treatment with multiple antibiotics for
many months, a procedure almost impossible to sustain in many parts of the world.
Importantly, such regimens may lead to the repeated selection of multi-drug resistant
7
strains, and their effectiveness depends on the existence of a well-operating health
care infrastructure that is beyond the resources and expertise of many of the most
seriously affected regions of the world. Nearly new 500.000 cases of multidrugresistant TB (MDRTB, defined as infection with Mtb strains resistant to the two
most important first-line drugs, rifampin and isoniazid) occurred in 2007. Moreover
by the end of 2008, extensively drug-resistant TB (XDR-TB, defined as MDRTB
strains additionally resistant to a fluoroquinolone and an injectable agent such as
kanamycin, amikacin, viomycin, or capreomycin) has been found in 55 countries
and territories of the world. While MDR-TB is difficult and expensive to treat,
XDR-TB is an untreatable disease in most of the developing countries (World
Health Organization, 2009).
The frequent co-infection of TB in HIV patients further complicates the
selection of an appropriate treatment regimen because increased pill burden
diminishes compliance, drug-drug interaction leads to sub-therapeutic concentration
of antiretrovirals, and overlapping toxic side effects increase safety concerns (Koul
et al., 2011).
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1.2 Mycobacterium tuberculosis
Mtb was first identified by the German scientist Robert Koch, who
announced the discovery on 1882.
Mtb is a member of the M. tuberculosis complex (MTBC) which includes six
other closely related species: M. bovis, M. africanum, M. microti, M. pinnipedii, M.
caprae and M. canetti. The MTBC members are genetically extremely related, in
fact the genome of M. tuberculosis shows <0.05% difference with M. bovis. All
MTBC members cause TB and exhibit distinct host range, even though they are not
tightly specie-restricted as, for example, M. caprae and M. canetti primarily infect
cattle but can also cause TB in other mammals including humans (Ahmad 2011).
Mtb is an obligate intracellular pathogen that can infect several animal
species, although human beings are the principal hosts. It is an aerobic, acid-fast,
non-motile, non-encapsulated and non-spore forming bacillus. It grows most
successfully in tissues with high oxygen content, such as the lungs (Lawn et al.,
2011). Mtb divides every 15–20 h being extremely slow as compared with other
bacteria. The slow replication rate and the ability to persist in a latent state result in
the need for long duration of both drug therapy of TB and preventive treatment of
Mtb-infected people.
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Compared with the cell walls of other bacteria, the lipid-rich cell wall of Mtb
is relatively impermeable to basic dyes unless combined with phenol. Thus Mtb is
neither gram positive nor gram negative but instead it is described as “acid-fast”
bacterium, since once stained it resists decolorization with acidified organic solvents
(Figure 2).
Figure 2 Photomicrograph revealing Mycobacterium tuberculosis bacteria using acid-fast ZiehlNeelsen staining.
Acid fastness of Mtb is largely a result of the high content of mycolic acids,
lipids and polysaccharides of the cell wall. The complexity of the mycobacterial cell
10
wall constraints on protein secretion, but pathogenic mycobacteria have evolved
several systems to deal with secretion. These include the generic Sec-dependent
secretion pathway to transport proteins across the cytosolic membrane and a twinarginine transporter (Tat) system that is used to transport-fold molecules across the
membrane.
A more recently described secretion system is the “early secretory antigenic target of
6 kDa” (ESAT-6) system 1 (ESX-1), which is responsible for the secretion of
ESAT-6 and CFP-10 (culture filtrate protein of 10 kDa), both important T-cell
antigenic targets and essential for Mtb virulence (Pieters 2008).
1.3 Host-pathogen interaction
TB is a transmissible disease and patients with pulmonary TB are the most
important source of infection.
Infection is initiated by inhalation of droplet nuclei, which are particles of 1-5
μm diameter containing Mtb, expectorated by patients with active pulmonary TB
(open TB), typically when the patient coughs. The primary route of infection
involves the lungs, since inhaled droplets are engulfed by resident alveolar
phagocytic cells, namely macrophages and dendritic cells (DCs), that in turn invade
the subtending epithelial layer (Russell et al., 2010). This event induces a localized
11
inflammatory response that leads to the recruitment of mononuclear cells from
neighboring blood vessels, providing fresh host cells for the expanding bacterial
population. The activated T lymphocytes, macrophages, and other immune cells
form granulomas that wall off the growing necrotic tissue limiting further replication
and spread of the tubercle bacilli (Figure 3).
Figure 3 Mtb infection, course of the disease, and the immune mechanisms activated in TB. IFN,
interferon; IL, interleukin; LT, lymphotoxin; RN/OI, reactive nitrogen/oxygen intermediates; TNF,
tumour necrosis factor; TLR, toll-like receptor. Kaufmann SHE, 2004
12
Initially, the granuloma is an amorphous mass of macrophages, monocytes
and neutrophils. However, within this structure, macrophages differentiate into
several specialized cell types, including multinucleated giant cells, foamy and
epithelioid macrophages. With the development of an acquired immune response,
and the arrival of lymphocytes, the granuloma acquires a more organized structure:
the macrophage-rich center becomes surrounded by a mantle of lymphocytes that
may be enclosed within a fibrous cuff that marks the periphery of the structure.
In the vast majority of the infected individuals, an effective cell-mediated
immune response develops 2–8 weeks after infection stopping further multiplication
of the tubercle bacilli. However, in some individuals the pathogen is not completely
eradicated as Mtb has evolved effective strategies to evade the immune response
resulting in survival and persistence of some bacilli in the host.
Immunocompetent patients with active TB possess granulomas in all states of
development from Mtb containment to active bacterial replication, implying that the
fate of each granuloma is determined locally, not systemically. Active granuloma
exhibits extensive pathology and ultimately it ruptures and spills thousands of viable
bacilli into the airways, giving rise to a productive cough that facilitates aerosol
spread of infectious bacilli.
Recent molecular studies suggest that the recruitment of immune cells to the
granuloma is mycobacteria-dependent and part of a pathogen-directed virulence
13
programme. Therefore, on the one hand granuloma formation seems to function as a
host defence mechanism, and on the other hand this event offers apparent
advantages to Mtb. In fact, within granulomas Mtb may shield itself from immunebased killing mechanisms and escape therapeutic concentrations of anti-tuberculosis
drugs, thus promoting the emergence of drug-resistant strains (Paige et al., 2010).
Indeed, the capacity of Mtb to survive and cause disease is strongly correlated
to its ability to escape immune defense mechanisms. In this regard, the paradox is
that Mtb possess the remarkable capacity to survive within the hostile environment
of the macrophage. Despite the fact that these phagocytes are usually very effective
in internalizing and clearing most of the bacteria, Mtb has evolved several effective
evasion strategies, including the inhibition of phagosome-lysosome fusion and the
inhibition of phagosome acidification (Pieters 2008).
Mycobacterial cell wall lipids such as lipoarabinomannan (LAM) have been
demonstrated to modulate phagosome maturation. In fact, LAM incorporates into
the phagosomal membrane by preventing phosphatidylinositol 3-phosphate (PI3-P)
accumulation on phagosomal membranes and acquisition of Early Endosomal
Antigen 1 (EEA1), both events required for the loss of rab5 and its replacement with
rab7 in order to drive fusion with late endosomal and lysosome compartments. All
this causes the maturation arrest of phagosomes at the early endosomal stage (Russel
2011). Another well-established strategy elaborated by Mtb to subvert macrophage
14
activities is direct against superoxide and its downstream metabolites, namely
hydrogen peroxide and hypervalent iron, known to be highly toxic for many
microbes since some protein complexes, such as NADPH oxidase complex, are
recruited and activated into phagosomes to facilitate a rapid anti-microbial response.
In this regard, Mtb is known to possess several routes of avoidance of superoxide,
ranging from superoxide dismutase to the scavenging properties of its cell wall
lipidoglycans.
Finally Mtb can invade the cytosolic compartment of macrophage and inhibits
apoptosis by producing prostaglandins.
1.4 The immune response against Mtb
Mtb resides in cells of the myeloid lineage, notably macrophages. Detection
of Mtb by myeloid cells via pattern recognition receptors (PRRs) and processing of
mycobacterial antigens enable antigen-presenting cells (APCs) to activate T
lymphocytes as critical mediators of acquired immune control of infection.
Additional factors, namely cytokines and metabolic products released during
infection and cellular stress drive adaptive immunity. The tight crosstalk between
Mtb and host cells and the dynamic nature of the ensuing adaptive immune response
15
define whether pathology develops in the lungs or Mtb infection is controlled or
even eliminated.
The mycobacterial cell wall is complex and its interaction with APCs results
in simultaneous or consecutive engagement of an array of receptors, including Tolllike receptors (TLRs).
The role of members of this family during TB has been thoroughly
documented. TLR-2 recognizes much mycobacterial structures, including hsp65,
hsp70, 19 kDa lipoprotein, LAM, and phosphatidylinositol mannoside (PIM)
(Quesniaux et al., 2004), whereas Mtb nucleic acids containing CpG motifs, mostly
released after bacterial death, bind TLR-9 stimulating myeloid cell functions (Bafica
et al., 2005).
An important role for Mtb internalization has been attributed to mannose
receptor (MR), which is predominantly expressed in alveolar macrophages. MR
senses ManLam, but also higher order PIM, arabinomannan (AM), and
mannosylated proteins (Torrelles et al., 2006).
Finally, later studies suggest the importance of DC-SIGN, the major
phagocytic receptor on human DCs, in Mtb internalization and in controlling
immune responses during acute or chronic TB (Tanne et al., 2009; Schaefer et al.,
2008).
16
Signals transmitted by TLRs, NOD-like receptors, and C-lectin type receptors
are transduced by adapter molecules that activate nuclear factor-kB, activator
protein-1, mitogen-activated protein kinase, and IFN regulatory factors. The
activation of these molecules results in transcriptional responses culminating in
synthesis of cytokines and chemokines.
The control of infection critically depends on Mtb-specific CD4+ Th1 cell
response, which includes production of IFN- (Cooper 2009). IFN- is crucial for
activating macrophages and regulates tissue inflammation. Th1 cytokines imprint an
effector phenotype, which is characterized by nitric oxide radical synthesis
efficiently restricting Mtb growth. The initial innate response of macrophages, in
fact, shapes T-cell response, which in turn directs the ability of macrophages to
control infection in a classic feedback loop. The crucial role of IFN- in antibacterial
immune response is underlined by the fact that mice lacking IFN- develop large
necrotic pulmonary lesions associated with granulocytic infiltrates within weeks of
Mtb infection despite a similar bacterial burden as WT mice (Nandi et al., 2011).
CD4+ T cells carry out several functions that are important to control
infection within the granulomas. These include apoptosis of infected macrophages
through Fas/Fas ligand interaction, production of cytokines such as IL-2 and TNF-
17
and stimulation of macrophages or DCs to produce other immunoregulatory
cytokines such as IL-10, IL-12, and IL-15 (Ahmad 2011).
Hovewer, the magnitude of Th1 response is not associated with bacterial
clearance or increased resistance. Several potential mechanisms may account for the
failure of adaptive immune responses to eradicate Mtb: (i) generation of Mtbspecific CD4+ effector T-cells is delayed compared with responses to other
pathogens (Wolf et al., 2008); (ii) certain individuals, or strains of mice, may
develop inappropriate Th2 response (Wangoo et al., 2001) or imbalanced effector
Th1/Th17 (Chen et al., 2009) in response to infection; (iii) host regulatory
mechanisms that limit protective immunity as well as immune pathology, such as T
regulatory cells (T reg) (Scott-Browne et al., 2007), production of inhibitory
cytokines (Turner et al., 2002), and onset of T cell exhaustion (Reiley et al., 2010)
may inhibit the activity of effector T cells at the site of infection.
The wide range of immune components involved in protective immune
response against Mtb include CD8+ T-cells, that besides producing IFN-γ and other
cytokines, may exert cytotoxic effects toward Mtb-infected macrophages thus
playing an important role in providing immunity to TB. In fact, CD8+ T-cells can
directly kill Mtb via granulysin during both acute as well as chronic infection
(Grotzke et al., 2005).
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1.5 Interferon Regulatory Factor 8 (IRF8)
IRF8, also known as ICSBP (Interferon consensus sequence-binding protein),
is a component of IRF transcription factor family, acting as an important regulator of
IFN-inducible genes. IRF family members are composed of an N-terminal DNA
binding domain and a C-terminal IRF association domain (IAD), and act as
transcriptional regulators forming heterodimers to control expression of IFNresponsive genes via direct binding to IFN-stimulated response element (ISRE)
sequences. IRF8 interacts with IRF1, IRF2, or IRF4 and negatively regulates some
IFN-inducible genes carrying functional ISRE, such as ISG15. Several studies
indicate that IRF8 can act on a wide range of target elements even beyond the IRF
recognition sequence (IRS), such as the Ets site found in IL-12p40 promoter and the
IFN-γ activation site (GAS). In these cases, IRF8 binds to the elements indirectly
through protein-protein interactions and activates promoter activity. Clearly, IRF8
can act either as a repressor or activator, depending on the target DNA sequence,
presumably by interacting with different proteins (Tamura et al., 2002).
One of the proteins that directly interacts with IRF8 is the transcription factor PU.1,
which regulates the expression of numerous myeloid-specific genes. In fact, PU.1-/mice do not produce mature macrophages and have very few granulocytes (Simon
1998). At the same extend, microarray analysis identified a large number of genes
19
activated by IRF8 when myeloid progenitor cells differentiate into macrophages.
Among these, there are genes important for macrophage functionality, coding for
lysosomal/endosomal enzymes, including cystatin C, cathepsin C and lysozyme M
(Tamura et al., 2005).
IRF8 plays a critical role in the regulation of lineage commitment and
development of diverse myeloid cell populations such as DCs and granulocytes
(Tamura et al., 2002) thus resulting crucial in defense against intracellular
pathogens.
IRF8 expression is essentially undetectable in non-hematopoietic cells. It is
well expressed in bone marrow progenitor cells (where it controls the cell growth
and differentiation of myeloid cells at different developmental stages), in
macrophages, in DCs and B cells, whereas its expression is lower in T cells. It is
required for ontogeny and maturation of macrophages and DCs, for activation of
anti-microbial defenses, and for production of the Th1-polarizing cytokine IL-12 in
response to IFN- thus it is potentially important for protection against Mtb infection
(Marquis et al., 2011).
Recently, it has been reported that BXH-2 mice, harboring a recessive
mutation (R294C) in IRF8, fail to mount an effective T-cell mediated immune
response following M. bovis infection. Strikingly, continuous microbial vaccination
20
in BXH-2 is associated with the inability to form granulomas in infected organs as
compared to C57BL/6J mice (Turcotte et al., 2007).
Altogether these studies highlight the importance of IRF8 in orchestrating
innate and acquired immune responses during mycobacterial infection. They also
raise the possibility that mutations in these genes may be associated with severe
mycobacteriosis in humans.
Notably, it has been shown that genetic mutations in human IRF8 result in primary
immunodeficiency that affects the differentiation of mononuclear phagocytes thus
impairing antimycobacterial immunity (Hambleton 2011).
IRF8 represents a key modulator of the developmental maturation program of
plasmacytoid DCs (pDCs) since IRF8 deficient mice (IRF8-/-) lack completely this
subset of DCs and IRF8-/- bone marrow progenitor cells are defective in generating
pDCs in the Fms-like tyrosine kinase 3 ligand (Flt3-L)-based culture system
(Schiavoni et al., 2002).
1.6 The IRF8-/- mouse model
Several reports have provided evidence that IRF8 plays a critical role in
modulating the immune response by influencing the differentiation and maturation
of diverse immune cells and by affecting cytokine expression (Tamura et al., 2002).
21
IRF8-/- mice are immunodeficient and highly susceptible to infection with various
pathogens, including vaccinia and lymphocytic choriomeningitis viruses (Holtschke
et al., 1996), bacteria as Listeria monocytogenes and Yersinia enterocolitica (Fehr et
al., 1997; Hein et al., 2000), and parasites as Leishmania major and Toxoplasma
gondii (Giese et al., 1997; Scharton-Kersten et al., 1997).
These mice display several immune functional defects among which it is noteworthy
to mention the impaired macrophage function in producing IL-12p40, the deficiency
to produce IFN in response to activation stimuli such as LPS, the inability to mount
Th1-mediated response, as opposite to the capability to produce considerable levels
of IL-4 mRNA. IRF8-/- mice, besides lacking pDC development program, display a
marked impairment in the number and activation properties of CD8+ DCs in all
secondary lymphoid organs. Altogether these data unravel a pivotal role of IRF8 in
the regulation of the development and activation of DCs (Schiavoni et al., 2002).
IRF8-/- mice develop a chronic myelogenous leukemia (CML)-like syndrome,
where a systemic expansion of granulocytes, predominantly characterized by mature
neutrophils, and causing lymphadenopathy and hepatosplenomegaly, is followed by
a fatal blast crisis. In this regard, it has been shown that normal mice injected with
cells from mice in blast crisis develop acute leukemia within 6 weeks of transfer
(Holtschke et al., 1996). Therefore, it has been suggested that IRF8 may act as a
22
tumor suppressor regulating the proliferation and differentiation of hematopoietic
cells (Tamura et al., 2002).
The extensive studies on IRF8-/- mouse model have also allowed to unravel
the role of this transcription factor in dictating macrophagic versus granulocytic
development. In fact, IRF8-/- mice harbor increased numbers of progenitor cells in
adult bone marrow and fetal liver, which in vitro are hyperresponsive to both
granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte
colony-stimulating factor (G-CSF) differentiating preferentially in granulocytes.
In contrast, IRF8-/- colonies formed in presence of macrophage colonystimulating factor (M-CSF) were mostly granulocytes, indicating their reduced
response to M-CSF. Consequently, in bone marrow of IRF8-/- mice the cells of
macrophage lineage were significantly fewer to respect wilde-type counterparts
(Scheller et al., 1999).
This important role of IRF8 is also supported by the finding that its in vivo
expression begins early in hematopoiesis at higher levels in macrophages than in
granulocytes, suggesting that whereas IRF8 stimulates expression of genes
important for macrophage differentiation, it represses a series of genes required for
granulocyte differentiation.
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2. PURPOSE OF THE THESIS
The aim of this thesis is to investigate the role of IRF8 in determining host defense
against TB.
In particular, by using IRF8-/- mice, we show the importance of this transcription
factor in the development of immunological as well as pathological mechanisms
implicated in the establishment of a competent immune response during Mtb
infection. For this purpose, we infected IRF8-/- and the immunocompetent
counterpart WT-B6 mice with the pathogenic Mtb Erdman strain and we analyzed:
 the survival of mice and their ability to control the bacterial burden;
 the nature of the immune response elicited by Mtb and the recruitment
of different immune cells within inflammatory sites;
 the formation in the lungs of lymphoid tissues and granulomas, both
structures necessary for the priming and initiation of the adaptive immune
response;
 the tissue damage induced by the pronounced host inflammatory response
and by bacterial replication.
24
3. MATERIALS AND METHODS
3.1 Bacteria
Pathogenic Mtb Erdman strain was grown in Middlebrook 7H9 broth supplemented
with albumin, dextrose, and catalase. Mycobacteria were then harvested, suspended
in sterile phosphate buffered saline (PBS) pH 7.2, aliquoted and stored at -80 °C
until use. Before infection, aliquots of strain were grown on 7H10 plates to titer the
bacteria after thawing.
3.2 Mice
IRF8-/- and the congenic C57BL/6J wild-type (WT-B6) mice (5–7 wk old) were bred
and housed in a controlled pathogen-free environment in animal facilities at the
Istituto Superiore di Sanità. Genotypes of mice were confirmed by PCR testing of
tail genomic DNA. All procedures conducted on mice were in accordance with the
conditions specified by the local Ethical Committee guidelines.
3.3 Mice infection with Mtb
Eight-week-old IRF8-/- and WT-B6 mice (4 per group) were aerogenically infected
with Mtb Erdman strain using a Glas-Col chamber. At 8, 15 and 30 days post25
infection (p.i), mice were sacrificed for determination of mycobacterial burden in
organs. Their lungs and spleens were removed, homogenized in PBS supplemented
with 0,05% Tween 20, and serial dilutions were plated on Middlebrook 7H11-agar
plates containing 10% albumin-dextrose-catalase. Colony-forming units (CFUs)
were counted after incubation at 37°C for 28 days. All the infection studies were
performed in a biosafety level 3 facility.
3.4 Histopathologic analysis
The lung left lobes of Mtb-infected IRF8-/- and WT-B6 mice were perfused and fixed
with 10% paraformaldehyde in PBS and then embedded in paraffin for sectioning.
The tissue sections were stained with hematoxylin and eosin (H&E) reagent or with
Ziehl-Neelsen acid-fast stain and were evaluated the lesions morphology and
distribution by light microscopy. For each lung left lobe, at least three sections were
obtained, and for each section, the total surface area and the area with lesions were
measured and the averages calculated for each section and for each group (five lung
left lobes per group). Measurements were carried out using the Nikon Eclipse 80i
microscope, the Nikon DS-L2 camera control unit, and the dedicated software
3422.1001.1798.080117.
26
3.5 Immunohistochemistry and Immunofluorescence analyses
Immunohistochemistry and Immunofluorescence were performed using the labeled
streptavidin biotin (LSAB) method. Histologic sections (3 μm thick) from formalinfixed, paraffin-embedded tissue were mounted on positively charged Superfrost
slides (Fisher Scientific). Tissue sections were deparaffinized and rehydrated
through a series of graded alcohols. Antigens were retrieved by a high-temperature
heating method (slides were immersed in target retrieval solution at pH 6 (Dako
Cytomation), in a steamer (90-95 °C) with a 20-minute incubation for all antigens,
for which slides were kept in a water bath for 40 min at 97 °C. Tissues were then
blocked for endogenous peroxidase in 3% hydrogen peroxide in water, and for
nonspecific binding in PBS containing 2% BSA, stabilizing protein and 0.015 mol/L
sodium azide (Protein Block Serum-Free, DakoCytomation). Tissues were incubated
overnight at 4 °C with the antisera showed below. The sections were then incubated
at room temperature for 1h with the secondary antibody and subsequently with the
streptavidin–biotin–peroxidase complex (Dako Lab Kit peroxidase) at room
temperature for 45 min. The reaction was revealed using the chromogen 3,3'diaminobenzidine (DAB) (DakoCytomation) for Immunohistochemistry and Alexa
Fluor® 555 streptavidin (Invitrogen) for immunofluorescence (the section was
counterstained with blu Hoechst). The immunohistochemistry sections were
27
counterstained with Mayer's hematoxylin and then cover-slipped in 50:50
xylene/Permount (Fisher Scientific). Control slides, known to be positive for each
antibody, were incorporated into each run. The sections were analyzed under light
microscopy Nikon® 80i eclipse and confocal laser scanning microscopy Leica®
DMI4000 B. The Nikon® DS-L2 camera control unit was used for measurements,
equipped
with
software
Nikon®
image-analysis
program,
version
322.1001.1798.080117 and the dedicated software for the cellular automatic count
(Axiovision ver. 4.4, Zeiss).
Antibody
Clone
Costumers
Specificity
Concentration
CD3
PC3/188A
S.C.
Biotechnology
Lymphocyte T
1:100
CD4
H129.19
BD Pharmingen
Lymphocyte T
CD4+
1:30
CD8
JXYT8
S.C.
Biotechnology
Lymphocyte T
CD8+
1:100
Foxp3
eBio7979
eBioscience
Lymphocyte
Treg
1:120
DEC205
NLDC-145
Dendritics
Dendritic Cells
1:100
7/4
7/4
Caltag
Laboratories
Neutrophils
1:100
28
F4/80
CI:A3-1
Caltag
Laboratories
Macrophages
1:100
CD45R
RA3-6B2
BD Pharmingen
Lymphocyte B
1:50
Table 1 Antibodies list used in Immunohistochemistry analyses
3.6 Flow cytometry analyses
For phenotypic analysis of immune populations of Mtb-infected IRF8-/- and WT-B6
mice, the lungs from 4 mice were pooled and cut into small fragments. These latter
were digested in RPMI 1640 containing 10% FCS and 1 mg/ml type III collagenase
(Worthington Biochemicals), with periodic pipetting to break up fragments, for 25
min at room temperature. EDTA (0.1 M, pH 7.2) was added for an additional 5 min,
and then the omogenate was allowed to pass through a 100µm cell strainer. The
collected cells were washed and directly used for phenotypic analysis. The following
monoclonal antibodies (from BD PharMingen and eBiosciences) were used for
surface staining: anti-CD45R/B220 (RA3-6B2) PercP5.5; anti-CD11c-biotin (HL3);
anti-SiglecF PE; anti-CD11b APC; anti-Gr1 APC-eFluor 780 (RB6-8C5); anti-B220
PE-Cy7 (RA3-6B2); anti-CD8 FITC (53-6.7); anti-CD3 APC (145-2C11); anti-CD4
FITC (GK1.5). Biotinylated antibodies were detected with streptavidin-eFluor 450
29
(eBioscience). Stained cells were analyzed on a FACSsort® flow cytometer (Becton
Dickinson). Viable cells were selected for analysis based on forward- and sidescatter properties.
30
4. RESULTS
4.1 IRF8-deficiency causes high susceptibility to Mtb infection
To investigate the role that IRF8 plays in the control of TB, we infected IRF8-/- and
WT-B6 mice with ~ 100 colony-forming units (CFUs) dose of the highly virulent
strain Mtb Erdman via the aerosol route, and at different time points we evaluated
the survival of mice and the bacterial load (Figure 4).
Aerogenic
Mtb infection
0
-
1
CFU count
Histopathology
2
4
weeks
Figure 4 Experimental model of in vivo Mtb infection. IRF8-/- and WT-B6 mice were aerogenically
infected with Mtb Erdman strain at t=0. At 1, 2 and 4 week postinfection the mice were sacrificed to
assess mycobacterial colonization and histopathology.
In WT-B6 immunocompetent mice, infection with Mtb results in a chronic disease
characterized by two distinct phases: 1) during the first 15 days of infection Mtb
31
highly replicates in the lungs and spleen of mice growing exponentially and bacterial
burden peaks between 2 and 4 weeks post aerosolization; 2) by day 30, as the
infection progresses from the acute phase into the chronic persistent stage, bacterial
load is hold stationary with proficient granuloma formation and limited pathology
keeping the mice clinically well (Figure 5).
8
acute infection
chronic infection
7
Log CFU/organ
Lung
6
5
Spleen
4
3
2
7 14
28
70
150
Days post-infection
Figure 5 Typical pattern of lung and spleen colonization (CFU) by virulent Mtb Erdman TMC 107 in
C57Bl/6 mice following aerogenic infection.
32
Accordingly, in our experiments WT-B6 mice were chronically infected exhibiting
no apparent distress and surviving up to 150 days whereas the survival time of IRF8/-
mice was significantly shortened with all animals succumbing from infection by 40
days, indicating that IRF8 is responsible for a significant portion of anti-TB
immunity (Figure 6, panel A). Of interest, up to 15 days active Mtb replication was
observed in both mouse strains and no significant differences in the bacterial
burdens were found in the lungs of IRF8-/- and WT-B6 mice (Figure 6, panel B).
However, at time points later than 15 days p.i., the bacterial growth remained
exponential only in IRF8-/- mice resulting in a massive bacterial load in the lungs and
spleen of these animals by 30 days p.i. (Figure 6, panel B). Taken together, these
results indicate that IRF8 deficiency affects the resistance to Mtb infection and
define IRF8-/- mice as a potential mouse model of acute Mtb infection as compared
to chronically infected WT-B6 mice.
33
B
Percent survival
A
Days
Days
Figure 6 Survival of IRF8-/- and WT mice and bacterial load in target organs following aerogenous
infection with the virulent Mtb Erdman strain. (A) Surviving mice (n=5 per group). (B) Bacterial
loads, determined as CFU, in spleens and lungs at 8, 15 and 30 days p.i. Data are expressed as mean
± SEM of each individual mouse organ out of at least 3. One representative experiment out of four
independent is shown.
4.2 IRF8-/- mice exhibit extensive tissue damage following Mtb infection
To investigate the extent of lung pathology during acute and chronic Mtb
infection in IRF8-/- and WT-B6 mice, we performed macroscopic and
histopathological analysis of granuloma structures. At early p.i. time (15 days), the
lungs of both mouse strains contained few limited granuloma foci (data not shown),
34
while at 30 days p.i. IRF8-/- mice had enlarged lungs with gigantic white nodules as
compared with WT-B6 counterparts exhibiting normal lung size with discrete
inflammatory lesions (Figure 7).
WT
IRF8-/-
Figure 7 Uncontrolled growth of pulmonary granulomas at late fase of Mtb infection in IRF8-/- mice.
Granuloma formation in the lungs of IRF8-/- and WT-B6 mice infected with 100 CFU of Mtb Erdman
via the aerosol route at day 30 p.i. One representative experiment of three is shown.
Afterward, we performed a quantitative microscopic analysis to objectively assess
the extent of tissue damage. To this end, we evaluated in each lung left lobe the
median granuloma surface area, the median total tissue surface area with lesions and
the ratio of the tissue surface area with lesions to the total lung surface. Of interest,
we found that while at 15 days p.i. these parameters where almost undetectable in
both mouse strains, at 30 days p.i. all three were strikingly up-modulated in IRF8-/35
mice with respect to WT-B6 counterparts, confirming the impressive mycobacterial
colonization occurring in deficient mice in the time lapse between 15 and 30 days
p.i. (Figure 8, panels A, B and C).
A
B
C
Lesions area
Day 15
Day 30
Ratio
% lesions
Area (mm2x10-6)
Area (mm2x10-6)
Granulomas
Day 15
Day 30
Day 15
Day 30
Figure 8. Quantitative analyses of tissue damage of the lungs of WT-B6 and IRF8-/- mice infected
with Mtb. (A) The median granuloma surface area; (B) The median total tissue surface area with
lesions; (C) The ratio between the tissue surface area with lesions with respect to the total area of
lung expressed as percentage. At least three sections per lung and five lungs per group were analyzed
and representative slides are shown.
Of interest, when lung sections were examined microscopically by hematoxylin and
eosin staining (Figure 9), at 15 days p.i. we found the occurrence of neogenesis of
lymphoid structures characterized by patches of immune infiltrations surrounding
36
some blood vessels as well as moderate infiltration of mononuclear cells
contiguously to the bronchial tree predominantly in WT-B6 mice with respect to
IRF8-/- mice showing very limited numbers of immune infiltrates.
IRF8-/A1
A2
WT
B1
A3
C1
C2
B2
B3
D1
C3
15 days
D2
D3
30 days
Figure 9 Histopathological analysis of lung left lobes of IRF8-/- and WT mice infected with Mtb. At
15 and 30 days p.i. the lung tissues were isolated from mice, formalin-fixed, paraffin-embedded, cut
to microtome and coloured with Haematoxylin-Eosin and Ziehl-Neelsen acid-fast stain. At least three
sections per lung and five lungs per group were analyzed. Representative slides are shown.
Magnification are 10 (left panels), 63 (upper right panels) and 40 (bottom right panels).
37
Moreover, discrete granulomas were observed at the same extent in the lung
parenchyma of both stains, although with different structure being granulomas of
deficient mice less organized (Figure 9, panels A and B). At the latter time point of
infection a diffuse destruction of lung parenchyma with an exceptional high
frequency of acid-fast bacilli was observed in the lesions of Mtb-infected IRF8-/mice (Figure 9, panels C and D). In particular, the exuberant damage of the lungs of
deficient mice at 30 days p.i. resulted in the appearance of cavitary granulomatous
lesions with extensive areas of necrosis whereas in WT animals well-organized nonnecrotizing granulomas were observed (Figure 9, panels C and D). Moreover, while
in WT-B6 mice the granulomatous foci were characterized by the accumulation of
macrophages, holding intracellular Mtb bacilli, and lymphocytes scattered in defined
areas, the granulomas of IRF8-/- mice exhibited multifocal foamy macrophages, of
which many appeared lysed and harboring an enormous amount of bacilli (Figure 9,
panels C2 and D2). Together, these data clearly demonstrate that IRF8 loss-offunction is detrimental for restricting Mtb replication in chronically infected
immunocompetent B6 mice and suggest its causative role in lung pathogenesis
occurring at late times after Mtb entry.
38
4.3 Different recruitment of immune cells characterizes Mtb infection in IRF8-/and WT mice
Previous studies revealed that TB control depends by immune cell
recruitment and antigen-specific T cells generation at the site of mycobacterial
replication. In addition, coordinated activation of immune cells is crucial for a
productive granuloma, and, given that 90% of all Mtb infections do not transform
into active disease, containment appears highly effective (Ulrichs et al., 2004).
Paradoxically, a pronounced immune response in the infected lungs is responsible of
the strong tissue damage that is detrimental for the host (Nandi et al., 2011). Thus,
we assess the infiltrated tissue composition of IRF8-/- and WT-B6 mice in order to
establish whether the extremely different pulmonary bacterial burden between the
two mouse strains was associated with the occurrence of different inflammatory
infiltrates. In agreement with the already described altered distribution of immune
cells in the blood and peripheral lymphoid organs of IRF8-/-, we found a profound
alteration of immune populations also in the lungs of these mice. As shown in
Figure 10A, a significantly increased number of Gr-1+CD11b+CD11c- granulocytes
were found in the lungs of the deficient mice with respect to the WT counterpart
(26,9%
vs
11,5%
of
total
CD45+
immune
cells).
Conversely,
SiglecF+CD11blowCD11c+ macrophages were drastically reduced in IRF8-/- mice as
compared to WT-B6 animals (5,2% vs 42,6% of total CD45+ immune cells).
39
A
B
C
Figure 10 Immune cell populations in lungs of IRF8-/- and WT mice. Lungs from naïve IRF8-/- and
WT mice were labelled with a panel of fluorescent antibodies against the indicated surface markers.
A) Percent values of each indicated cell population in gated CD45 + leukocytes. B) Mean percentages
of lung granulocytes, macrophages and DCs ± SD (n=6 mice per group). C) Ratio values between
granulocytes and macrophages ± SEM (n=6 mice per group). Data are representative of one
experiment out of four.
40
Moreover, SiglecF+Gr1-CD11c+ DCs as well as CD3+CD4+ and CD3+CD8+ T
lymphocytes proved to be notably reduced in the lungs of the former as compared to
the latter animals. Of interest, these results revealed that granulocytes represent the
predominant immune population in the lungs of IRF8-/- mice as opposite to the
situation of WT animals where macrophages predominate (Figure 10, panel B). This
peculiar immune setting is distinguished by a granulocyte/macrophage ratio 4 fold
higher in deficient animals with respect to the WT counterpart (Figure 10, panel C).
Next, we assessed the kinetics of influx of specific immune cells into the
lungs of acute Mtb-infected IRF8-/- vs chronic Mtb-infected WT-B6 mice. Thus, we
performed confocal laser-scanning microscopy (CLSM) analyses to depict lung
immune infiltrates during the specific phases of infection. We found that CD3+ T
lymphocytes, including both CD4+ and CD8+ T cells did not differ significantly
between the two groups of mice for up 15 days post Mtb infection when these cells
decreased sharply only in the lungs of IRF8-/- mice (Figure 11).
41
Figure 11 Altered lymphocytic infiltrates in lungs of IRF8-/- and WT mice infected with Mtb. Lung
tissue sections from Mtb-infected IRF8-/- and WT mice (day 15 and 30 p.i.) were stained with a panel
of antibodies against the indicated surface markers. Absolute number of cells expressing the indicated
markers in each individual lung section is reported. Data represents the mean cell counts of five fields
(1 field was 0,16 mm2 at 400x magnification) of each slide (4 slides of each group) ± SD. Three mice
per group were considered.
Moreover, by 15 days p.i. Foxp3+ Treg were significantly higher in the lungs
of WT-B6 mice with respect to the IRF8-/- counterparts in which they further
declined rapidly during the late phase of infection (Figure 11). The succeeding
evaluation of the frequency of myeloid cells in the lungs of Mtb-infected mice
revealed a sharp accumulation of 7/4+ neutrophils in the lungs of IRF8-/- mice,
42
resulting significantly higher with respect to WT-B6 counterparts (Figure 12).
Conversely, both DEC205+ DCs and F4/80+ macrophages were present at the same
levels in both IRF8-/- and WT mice by 15 days p.i.
Figure 12 Altered myeloid infiltrates in the lungs of IRF8-/- and WT mice infected with Mtb. Lung
tissue sections from Mtb-infected IRF8-/- and WT mice (day 15 and 30 p.i.) were stained with a panel
of antibodies against the indicated surface markers. Absolute number of cells expressing the indicated
markers in each individual lung section is reported. Data represents the mean cell counts of five fields
(1 field was 0,16 mm2 at 400x magnification) of each slide (4 slides of each group) ± SD. Three mice
per group were considered.
43
However, by 30 days p.i. DEC205+ DCs remained substantially unchanged in the
lungs of the two groups whereas F4/80+ macrophages increased significantly only in
WT-B6 mice (Figure 12). Collectively, these results demonstrate that the
uncontrolled growth of Mtb in the lungs of IRF8-/- was associated with a sudden loss
of infiltrating CD3+CD4+ and CD3+CD8+ T lymphocytes as well as Foxp3+ Treg
cells. On the contrary, at the same time points elevated levels of neutrophils were
present in the lungs of deficient mice confirming a role of these cells in the
pathology of TB. Taken together, these results suggest that lung immune
populations of IRF8-/- mice cannot contain Mtb replication and provide its clearance.
4.4 Impaired formation of pulmonary newly lymphoid structures in IRF8-/mice after Mtb infection
In previous studies the development of pulmonary newly lymphoid structures
has been described during TB in mice. These so-called “tertiary lymphoid organs”
are defined as ectopic lymphoid-cell accumulations that arise in non-lymphoid
tissues and develop under conditions of chronic inflammation (Kahnert et al., 2007).
They are functional sites where protective lymphocytic responses are mounted.
Thus, we investigated whether the occurrence of lymphoid neogenesis in the lungs
following Mtb infection could reflect the divergent course of the disease in IRF8-/mice as compared to WT animals. Therefore, we examined the areas of
44
inflammatory infiltration within the lungs of infected mice throughout the course of
disease by performing IHC examination of these tissues from both mouse strains at
day 8, as well as at days 15 and 30 p.i. As shown in Figure 13, at day 8 p.i., sections
from lungs revealed the presence of marked infiltration of CD3+, CD4+ and CD8+ T
lymphocytes surrounding the vessels and bronchial tree of lungs of WT-B6 mice
whereas in IRF8-/- mice T-cell infiltrates were significantly less and more diffusely
dispersed across the pulmonary tissue (Figure 13, panel A).
In addition, the areas surrounding the bronchial tree of WT-B16 mice had a
significant accumulation of Foxp3+ cells suggesting a consistent recruitment of Treg
whereas this lymphocytic population was barely detectable in the IRF8-/- counterpart
(Figure 13, panel A). On the contrary, at this early time point an accumulation of
lymphoid follicles characterized by the presence of B220+ cells was clearly visible at
the same degree in lymphoid structure of both IRF8-/- and WT-B6 mice (Figure 13,
panel B). The succeeding analysis of myeloid cells in newly formed lymphoid
structures revealed that at day 8 post Mtb infection, F4/80+ macrophages were slight
inferior in lung inflammatory areas of IRF8-/- mice with respect to the WT-B6
counterpart (Figure 13, panel B). Similarly, DEC205+ DCs were markedly present
surrounding vessels of WT-B6 mice whereas a reduced focal infiltration of these
cells characterized IRF8-/- lung sections (Figure 13, panel C). On the contrary, we
45
found a more significant recruitment of 7/4+ neutrophils into lung lymphoid
structures of IRF8-/- mice with respect to the WT counterpart (Figure 13, panel C).
Figure 13 Immunohystochemical analysis of pulmonary lymphoid structures in Mtb-infected IRF8-/and WT mice at early time after Mtb infection. Three mice per group were aerogenically infected,
lung tissues were isolated from mice at day 8 p.i. and stained with the reported antibodies.
Representative slides are shown and magnification is 40.
46
As the disease progressed at day 15 p.i. CD3+, CD4+ and CD8+ T cells as well as
Foxp3+ T cells were present in even more significant minor amounts in the lymphoid
areas of lungs of IRF8-/- mice with respect to WT counterparts (Figure 14, panel A).
Of interest, B220+ cells were present at the same degree in lymphoid structures of
both IRF8-/- and WT-B6 mice whereas the numbers of DEC205+ DCs and F4/80+
macrophages were slightly lower in the former group (Figure14, panel B and C).
Conversely, 7/4+ neutrophils infiltrating the tissues surrounding the vessels and
airways were present in substantially higher amounts in IRF8-/- lungs with respect to
the WT counterpart (Figure 14, panels C).
47
Figure 14 Immunohystochemical analysis of immune cells infiltrating pulmonary lymphoid
structures in Mtb-infected IRF8-/- and WT mice at day 15 after Mtb infection. Three mice per group
were aerogenically infected, lung tissues were isolated from mice at day 15 p.i. and stained with the
reported antibodies. Representative slides are shown and magnification is 40.
At day 30 p.i., a significant increase of both lymphocytic populations, namely CD3+,
CD4+, CD8+, B220+ cells and Foxp3+ Treg, and myeloid cells, such as DEC205+
48
DCs and F40/80+ macrophages, were observed in well defined pulmonary
inflammatory lymphoid structures of WT-B6 lungs whereas in IRF8-/- mice these
cells were barely revealed by a diffusely dispersed signal across the pulmonary
parenchyma due to the advanced disorganization of lung tissues characterized by a
prominent inflammation (Figure 15, panels A, B and C).
Lastly, staining of 7/4+ neutrophils at the latest stage of infection provided
confirmation of the persistence of these cells in lungs of IRF8-/- mice throughout the
course of the disease. Together, these results revealed a defective formation of
pulmonary newly lymphoid structures in IRF8-/- mice as compared with the prompt
early occurrence of lymphoid neogenesis in the lungs of WT-B6 mice. Moreover,
the persistence of high numbers of neutrophils in the newly formed lymphoid
structures throughout the course of the disease characterized the deficient adoptive
immunity occurring in IRF8-/- mice.
49
Figure 15 Immunohystochemical analysis of immune cells infiltrating pulmonary lymphoid
structures in Mtb-infected IRF8-/- and WT mice at late stage of Mtb infection. Three mice per group
were aerogenically infected, lung tissues were isolated from mice at day 30 p.i. and stained with the
reported antibodies. Representative slides are shown and magnification is 40.
50
4.5 IRF8 function is necessary for immune cell maintenance in lung granulomas
during Mtb-infection
Since the early times of pathology, the formation of lung granulomas has
been considered the hallmark of pulmonary TB. Containment of Mtb in these
structures prevents the pathogen from dissemination throughout the host organism
and focuses the immune response to the site of mycobacterial persistence (Ulrichs et
al., 2004). Thus, to analyze the role of immune components of the granulomas in the
defective induction of acquired T cell response against Mtb, we examined the influx
of lymphoid and myeloid cells into granulomatous lesions of IRF8-/- mice with
respect to WT counterparts. Immunohistological analysis of immune infiltrates
within lung tissues revealed a spatio-temporal sequence of cellular infiltration to site
of Mtb infection. In particular, the examination of CD3+CD4+ and CD3+CD8+ T
lymphocytes revealed that at 8 days p.i. significant higher numbers of these cells
were present at the surrounding T cell-rich outer layers of granulomatous structures
of IRF8-/- lungs as compared to those of WT animals. Conversely, at this time point
reduced Foxp3+ Treg infiltrated the granulomas of deficient mice with respect to
WT animals (Figure 16, panel A). Of interest, B220+ B lymphocytes as well as
F4/80+ macrophages were recruited at the same extent in the granulomas of both
mouse strains (Figure 16, panel B). The further analysis of myeloid immune
populations revealed that DEC205+ DCs as well as 7/4+ neutrophils were slightly
51
higher detected in a diffuse pattern in the inner layers of IRF8-/- granulomas as
compared to the WT counterpart (Figure 16, panel C).
Figure 16 Immunohystochemical analysis of immune cells infiltrating lung granulomas in Mtbinfected IRF8-/- and WT mice at early time following Mtb entry. Three mice per group were
aerogenically infected, lung tissues were isolated from mice at day 8 p.i. and stained with the reported
antibodies. Representative slides are shown and magnification is 20.
52
Altogether these data clearly show that, during the early phase of infection,
along with an advanced lost of structural integrity and an apparent disorganization of
the pulmonary tissues and despite the immunodeficiency of IRF8 -/- mice, evident
also in the inefficacy to promptly generate pulmonary newly formed lymphoid
structures, T lymphocytes as well as DCs and neutrophils were more efficiently
recruited in the granulomatous lesions of deficient mice with respect to
immunocompetent animals.
The further immune characterization of the lungs during disease progression
revealed a profound defective recruitment of T cells in IRF8-/- granulomas when
multifocal nodular lesions characterized by foamy alveolar macrophages became
predominantly evident only in these mice (Figure 17, panels B and D). In fact, we
found that at 15 days p.i. there was a significant deficiency of recruitment of
CD3+CD4+ and CD3+CD8+ T lymphocytes as well as Foxp3+ Treg in the
granulomatous lesions of deficient mice as compared to the clear-cut homing of
these cells to WT-B6 granulomas (Figure 17, panel A). On the contrary, at this time
point DEC205+ DCs as well 7/4+ neutrophils accumulated in a diffuse pattern in
granulomas of IRF8-/- mice at levels comparable to those observed in the
immunocompetent counterpart (Figure 17, panel C).
53
Figure 17 Immunohystochemical analysis of immune cells infiltrating lung granulomas in Mtbinfected IRF8-/- and WT mice at day 15 after Mtb infection. Three mice per group were aerogenically
infected, lung tissues were isolated from mice at day 15 p.i. and stained with the reported antibodies.
Representative slides are shown and magnification is 20.
IHC analysis of lung tissues at late stage of infection confirmed the profound defect
of T lymphocyte recruitment into granulomatous lesions of IRF8-/- mice at this time
point. As expected, in WT-B6 animals the structural integrity of the granulomas
correlated with a significant presence of CD4+ and CD8+ T lymphocytes as well as
54
Foxp3+ Treg whereas in IRF8-/- granulomas, characterized by a disorganized caseous
structure with extensive necrotic areas, few CD3+ as well as CD4+, CD8+ and
Foxp3+ cells were observed (Figure 18, panel A). Moreover, few aggregates of
DEC205+ DCs were found in the granulomatous lesions of IRF8-/- mice with respect
to WT animals whereas no differences were observed in B220+ B lymphocytes as
well as F4/80+ macrophages between the two strains of mice (Figure 18, panel B).
Of interest, at this late time point elevated numbers of neutrophils were located in
the granulomatous tissues of IRF8-/- mice with respect to WT animals (Figure 18,
panel C). Together, these results suggest that the recruitment of T cells as well as
DCs into tuberculous granulomas of IRF8-/- mice was not impaired per se, as it was
considerable in the early phase of infection, but the homing of immune infiltrates to
these sites gradually declined as the disease progressed along with a significant and
persistent maintenance of neutrophils.
55
Figure 18 Immunohystochemical analysis of immune infiltrates of lung granulomas of Mtb-infected
IRF8-/- and WT mice at late stage of Mtb infection. Three mice per group were aerogenically infected,
lung tissues were isolated from mice at day 30 p.i. and stained with the reported antibodies.
Representative slides are shown and magnification is 20.
56
5 DISCUSSION
Primary Mtb infection is, in the 90% of cases, associated with containment of
mycobacterial growth in small granulomas where the bacillus is controlled and
persists as latent infection (Young et al., 2009). Despite this, TB remains a major
global health problem also for immunocompetent subjects due to: 1) the emergence
of Mtb strains resistant to antibiotics; 2) the co-infection with HIV; 3) the increase
opportunity of infection for migratory flow of subjects from endemic areas.
Susceptibility to clinical TB is known to be influenced by diverse
components such as genetic variation within host populations and the nature of host
immune response (Behr et al., 2010). The evidence of a human genetic contribution
to TB susceptibility is now growing, in fact genetic variants of the natural
resistance-associated macrophage protein (NRAMP), of the vitamin D receptor
(VDR) and of IFN- pathways are known to affect susceptibility (Lawn et al., 2011).
In the past years, extensive genetic analyses of individuals who are highly
susceptible to non-TB mycobacterial infections, together with studies of human
populations in areas endemic for TB, has provided valuable information about key
resistance pathways required to protect the host against disease progression (Fortin
et al., 2007).
57
Very recently, this approach has revealed that genetic mutations in IRF8 gene
determine human primary immunodeficiency, which affects the functional activity
of DCs thus resulting detrimental in anti-mycobacterial immune response
(Hambleton et al., 2011). Indeed, the importance of IRF8 in immunity against
mycobacteria was already revealed by previous studies that identified this factor as a
critical regulator of host defenses against BCG and in some settings against Mtb. In
fact, Gros and collaborators reported that BXH-2 mice, bearing the defective IRF8
R394C allele, are extremely susceptible to systemic Mtb infection, showing
uncontrolled replication, quick dissemination and rapidly fatal disease (Marquis et
al., 2009). In this regard, it is worthwhile to underlie that IRF8 is a pivotal lineagespecific transcription factor of myeloid cells and plays a critical role in IFN-I
signaling pathways governing both innate and acquired immunity (Wang et al.,
2009).
A growing body of evidences suggests that Mtb infection induces the
formation of “ectopic lymphoid structures” in the infected lungs. These structures
arise from lymphoid neo-organogenesis under conditions of chronic inflammation
and are implicated in the initiation of local immune response (Kahnert et al., 2007).
This latter event occurs also into granulomas, a dynamic structure that is the
hallmark of Mtb infection. Importantly, besides physically restricting bacterial
growth by environmental stresses such as reactive oxygen and nitrogen
58
intermediates, granulomas are sites of immunological priming and events occurring
herein as well as in pulmonary newly formed lymphoid structures reflect systemic
immune responses. The proximity of lymphoid structures to granulomas suggests
that both are functional site where protective lymphocyte responses were mounted
for the immune control of TB (Kahnert et al., 2007). In this light, both granuloma
formation and the generation of newly formed lymphoid structures can be
considered as the contribution that lung tissue makes to the generation of antigenspecific immune responses in situ during Mtb infection.
In this thesis we deepened the role of IRF8 during Mtb infection through
aerosol route, the natural entry of this bacterium in humans. To this end, we focused
on the dissection of lung immune response throughout the course of TB in IRF8-/mice with respect to the WT-B6 counterpart. In particular, we addressed the control
of this transcription factor on the development of immunological and pathological
features that dictate the outcome of the disease.
Hence, we demonstrate that respect to immunocompetent WT-B6 animals:
 IRF8-/- mice are extremely susceptible to Mtb infection, being completely
unable to limit bacterial replication in spleen and lungs, and develop an
acute TB disease;
 Although during the early phase of Mtb infection they are able to recruit into
the lungs T and B lymphocytes as well as macrophages and DCs, at later
59
stages of the disease IRF8-/- mice show a clear-cut defective pulmonary
recruitment of T cells, essential to generate a protective antimycobacterial
immunity;
 Although they are characterized by a substantial influx of both T and
myeloid cells into the granulomas at early stage of the disease, IRF8-/- mice
are unable to maintain a competent local adaptive immunity response in the
lungs at late phase of the disease due to the impaired formation of
pulmonary newly formed lymphoid structures and the defective access of
CD4+ and CD8+ T cells, Treg and DCs to granulomas;
 IRF8-/- mice are characterized by advanced lung tissue damage due to a wide
inflammatory process caused by a persistent maintenance of neutrophils
throughout the course of TB.
Taken as a whole, the results of the present study show that the competence
of antimycobacterial immune response is impaired by IRF8 deficiency in mouse TB,
and that the defective recruitment/maintenance of immune cells deputed to bacterial
eradication in lymphoid structures and granulomas concur to the extent of lung
tissue damage and bacterial dissemination. Moreover, these data confirm a
prominent role of neutrophils in the pathology of TB since their persistence in all
phases of the disease is a hallmark of Mtb infection of IRF8-/- mice.
60
Importantly, this study establishes a crucial role of IRF8 in late time of Mtb
infection, when the recruitment of some immune populations rather than others to
the lungs causes immunopathology, compounding the loss of bacterial control and
worsening the outcome of infection. Therefore, we uncovered a new important
function of IRF8 as a pivotal regulator of key events, such as the generation of
competent pulmonary newly formed lymphoid structures and granulomas, essential
to elicit protective immunity to control Mtb infection. Importantly, this study also
defines IRF8-/- mice as an acute mouse model of Mtb to be compared to chronically
Mtb-infected immunocompetent WT-B6 mice avoiding the bias of different mouse
strain genetic backgrounds possessed by all available mouse models in TB research.
61
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7. ACKNOWLEDGEMENTS
I would like to take the opportunity of thanking the Unit of Experimental
Immunotherapy at Istituto Superiore di Sanità (Roma), especially Dr. Lucia
Gabriele, Dr. Giovanna Schiavoni and Dr. Fabrizio Mattei, who sustained this
work.
I gratefully acknowledge Prof. Giovanni Delogu, Università Cattolica del Sacro
Cuore (Roma), and Dr. Stefano Rocca, Università degli studi di Sassari (Sassari).
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