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. Author manuscript; available in PMC: 2014 Mar 15.
Published in final edited form as: J Immunol. 2013 Feb 8;190(6):2778–2790. doi: 10.4049/jimmunol.1202722

Interleukin-10 inhibits mature, fibrotic granuloma formation during Mycobacterium tuberculosis infection

Joshua C Cyktor *,‡, Bridget Carruthers ‡, Rachel A Kominsky *,‡, Gillian L Beamer *,†,‡,§, Paul Stromberg †, Joanne Turner *,‡
PMCID: PMC3594073  NIHMSID: NIHMS435210  PMID: 23396944

Abstract

Protective immunity and latent Mycobacterium tuberculosis (Mtb) infection in man are associated with the formation of mature protective granulomas within the lung. Unfortunately, understanding the importance of such structures has been hindered by the lack of small animal models that can develop mature granulomas. Here we describe for the first time the formation of mature, fibrotic Mtb-containing pulmonary granulomas in a mouse model of IL-10 deficiency (CBA/J IL-10−/−). Long term control of Mtb infection in the absence of IL-10 was also associated with an early and enhanced capacity for antigen-presentation and a significant increase in the generation of multifunctional T cells. While IL-10 deficiency is known to enhance TH1 immune responses in general, we demonstrate here using transient anti-IL-10R treatment that it is the presence of IL-10 in vivo during the first month of Mtb infection that plays a definitive role in the inhibition of optimum protective immunity that can establish the environment for mature granuloma formation. Although the importance of IL-10 during Mtb infection has been debated, our data demonstrate that in CBA/J mice, IL-10 plays a significant early inhibitory role in preventing the development of protective immunity associated with containment of Mtb infection.

Introduction

Mycobacterium tuberculosis (Mtb), the etiological agent responsible for the disease tuberculosis (TB), remains a severe threat to mankind, infecting nearly one third of the world’s population and causing over 1 million deaths each year (1). The majority of individuals that become infected with Mtb are capable of asymptomatic containment and control of the infection (2). During this asymptomatic phase, Mtb persists in balance with the immune system. In some individuals, this control can break down leading to reactivation and contagious TB disease. In man, protective immunity and the development of latent Mtb infection are associated with the formation of small, dense foci, or granulomas, within the lungs that serve to limit Mtb infection (3). Protective granulomas in humans are characterized by the containment of infection within a fibrotic capsule in which the central core can become necrotic (3, 4). Encapsulated, contained, necrosis is in contrast to the tissue necrosis associated with TB reactivation. In this environment of encapsulation, Mtb is thought to survive in a non-replicating persistent state until host immunity and granuloma structure break down (5, 6). Understanding the development and importance of granuloma structure has been challenging due to the paucity of animal models that develop structures resembling mature granulomas in humans. Indeed, the greatest limitation of the mouse model, the species most widely used for the study of Mtb disease progression, is its failure to generate granuloma structures that are fully representative of granulomas associated with protection in man (4, 7).

The factors that determine susceptibility to reactivation TB are not completely understood, but are complex, multifactorial and closely linked to immune competency. Robust TH1-driven immune responses with the generation of interferon-γ (IFN-γ)-secreting T cells are thought to be essential (8-10), yet it is unclear how other immune modulators influence the generation of protective immunity and long-term control of Mtb infection. The immunosuppressive cytokine interleukin-10 (IL-10) has been implicated in susceptibility to TB in both humans and animal models. Patients with active TB have increased levels of IL-10 in their serum (11, 12), pleural fluid (13), and/or bronchoalveolar lavage fluid (14), suggesting a link between elevated IL-10 and TB disease. It is unclear at this point whether the elevated levels of IL-10 detected in TB patients are produced to dampen the excessive inflammation associated with active disease, or if IL-10 directly causes TB reactivation in man. However, Mtb infection of IL-10-expressing transgenic mice indicates that IL-10 production can drive TB reactivation (15). Other murine studies have confirmed the relationship between IL-10 and TB disease progression by demonstrating that blocking or deleting the action of IL-10 can reduce the pulmonary bacterial load (16, 17) an event highly associated with increased IFN-γ production. The exact mechanism for how IL-10 influences the control of Mtb infection remains elusive, but its presence during reactivation TB implicates IL-10 as a significant mediator of TB disease progression.

To determine the precise role of IL-10 during Mtb infection and TB disease progression we developed IL-10 gene-disrupted mice on the CBA/J mouse strain background (CBA/J IL-10−/−). This Mtb-susceptible strain succumbs to aerosol Mtb infection within 150-200 days, which is associated with reduced TH1-mediated immunity, poorly organized granulomas, and abundant IL-10 production (16, 18). Deletion of IL-10 on the CBA/J mouse strain background provided a tractable system to elucidate the role of IL-10 in a model that naturally mimics the elevated levels of IL-10 seen in TB patients. Our findings demonstrate that CBA/J IL-10−/− mice were fully capable of controlling and containing Mtb infection which was, as expected, associated with an early and heightened TH1-mediated immune response. What was particularly striking, however, was that in the absence of IL-10 CBA/J mice formed mature protective granulomas with Mtb encapsulated within a fibrotic capsule that frequently surrounded a necrotic core. Furthermore, the protective phenotype of CBA/J IL-10−/− mice was recapitulated by removing the action of IL-10 throughout the first 21 days of Mtb infection in wild-type CBA/J mice. Our data demonstrate that the presence or absence of IL-10 during initial Mtb infection is a critical decision point for the generation of long-term protective immunity and the development of mature granulomas. We hypothesize that the early action of IL-10 in humans during an initial encounter with Mtb determines the long-term outcome of TB disease.

Materials and Methods

Mice

CBA/J mice (Jackson laboratories, Bar Harbor, Maine) were crossed with C57BL/6 IL-10−/− mice (Jackson) for eight generations. At each cross progeny were ear-punched and DNA was screened for the presence of a neomycin cassette at the il10 gene locus and IL-10+/− mice were selected for further breeding. At the eighth generation, heterozygotes were crossed and IL-10-deficient homozygote CBA/J mice were selected. A homozygous breeder colony of CBA/J IL-10−/− mice was maintained thereafter.

4- to 8-week-old, specific pathogen-free, age/sex-matched CBA/J IL-10−/−, CBA/J wild-type (Jackson or National Cancer Institute, Frederick, MD), C57BL/6 wild-type (Jackson) and C57BL/6 IL-10−/− mice (Jackson) were maintained in ventilated cages inside a biosafety level 3 (BSL3) facility and provided with sterile food and water ad libitum. Experiments were unaffected by the recent CBA/J-C3H/HeN genetic contamination at NCI. All protocols were approved by The Ohio State University’s Institutional Laboratory Animal Care and Use Committee.

Mtb infection and Colony Forming Unit Enumeration

Mtb Erdman (ATCC no. 35801) was obtained from American Type Culture Collection. Stocks were grown in Proskauer-Beck liquid medium containing 0.05% Tween 80 to mid-log phase and frozen in 1 ml aliquots at −80°C. Mice were infected with Mtb Erdman using an inhalation exposure system (Glas-Col) calibrated to deliver 50–100 CFU to the lungs of each mouse, as previously described (19).

At specific time points post Mtb infection mice were sacrificed and lung, spleen and mediastinal lymph node (MLN) aseptically removed into sterile saline. Organs were homogenized and serial dilutions plated onto 7H11 agar supplemented with OADC as previously described (20). Plates were incubated at 37°C for 21 days in order to enumerate bacterial colonies and calculate the bacterial burden.

Lung cell isolation

Mice were euthanized by CO2 asphyxiation and lungs perfused with cold phosphate buffered saline containing 50 Units/mL of heparin through the right ventricle. Lungs from individual mice were mechanically disrupted using a GentleMACS dissociator (Miltenyi Biotec, Boston, MA) followed by collagenase A (type XI) (0.7 mg/mL, Sigma) and type IV bovine pancreatic DNAse (30μg/mL, Sigma) digestion at 37 °C for 30 minutes in GentleMACS C-tubes. Lung cell suspensions were passed through a 70 μm nylon cell screen and residual erythrocytes were lysed with Gey’s solution. Viable cells were determined by trypan blue exclusion.

Cell Purification

Single lung cell suspensions were adhered to sterile tissue culture dishes for 1hr at 37°C. Non-adherent cells were washed and removed from the plates. CD4+ and CD8+ T cells were obtained from the non-adherent cell fraction by magnetic cell separation (BD IMAG anti-CD4 particles GK1.5, anti-CD8 particles 53-6.7) and either placed directly into TRIzol reagent (Invitrogen, Grand Island, NY), homogenized, and frozen at −80°C or used for culture as described below. Purity of CD4+ and CD8+ T cell populations was determined to be greater than 90% for all experiments by flow cytometry using an LSRII flow cytometer (BD Biosciences, San Jose, CA). Adherent cells were scraped off the plates with a sterile razor blade, lysed in TRIzol, homogenized, and immediately frozen at −80°C.

Flow cytometry

Isolated lung cells or MLN were suspended in deficient RPMI (Mediatech, Manassas, VA) supplemented with 0.1% sodium azide (Sigma-Aldrich). Surface targets were detected as previously described (16). Specific antibodies were purchased from BD Biosciences: PerCP-Cy5.5 anti-CD3ε (145-2C11), allophycocyanin-Cy7 anti-CD4 (GK1.5), PE-Cy7 anti-CD8 (53-6.7), PE-Cy7 anti-IFN-γ (XMG1.2), FITC IA/IE (2G9), FITC CD44 (IM7), PE CD11c (HL3), PE CD69 (H1.2F3), APC CD11b (M1/70), APC CD62L (MEL-14). Cytokine levels were determined according to the manufacturer’s instructions for intracellular cytokine staining (Cytofix-Cytoperm fixation/permeabilization solution kit with BD GolgiStop, BD Biosciences), following a 4hr incubation with 10μg/mL anti-CD3 (145-2C11) and 1μg/mL anti-CD28 (37.51). Samples were read using a six color BD LSRII flow cytometer and analyzed with FACSDiva software (BD Biosciences).

Real-time PCR

RNA was purified using Qiagen RNeasy kit with RNeasy columns and quantified on a NanoDrop spectrophotometer (ThermoSCIENTIFIC, Waltham, MA). ifng, il17a, tnf, ciita, h2a, nos2, tgfb primer/probe sets were obtained from Applied Biosystems, Carlsbad, CA.

Histology

Caudal lung lobes were taken from infected CBA/J wild-type and IL-10−/− mice at various timepoints post-infection, inflated and stored in formalin as previously described (21). Tissue sections were prepared and stained with hemotoxylin and eosin, Ziehl-Neelson, or Masson’s trichrome, and were assessed by a board certified veterinary pathologist with no prior knowledge of experimental groups.

Immunohistochemistry

Paraffin embedded tissue sections were rehydrated and stained with monoclonal anti-human/mouse HIF1α IgG1 (R&D systems, Minneapolis, MN) or mouse IgG1 control (R&D systems). Antibodies were used at a concentration of 15μg/mL. Tissue treatment and staining was performed according to R&D systems cell & tissue staining kit (CTS002). Counterstaining was not performed on HIF1α/isotype sections. Paraffin embedded tissue sections were stained for CD3, B220, or F4/80 by The Ohio State Comparative Pathology and mouse phenotyping resource.

IL-10R Blockade

CBA/J mice were injected intraperitoneally with 1mg of anti-IL-10R (1B1.3A) or Rat IgG1 antibody (BioXCell, West Lebanon, NH) one day before aerosol infection with Mtb Erdman. On days 6, 13, 20 post-infection 0.2mg of antibody were injected to maintain the blockade. On day 21 post-infection lung and MLN were removed from a subset of mice for T cell purification (lung) or flow cytometry (MLN). Remaining groups of infected mice were sacrificed on day 120 post-infection without any further treatment past day 21. Right caudal lungs were fixed in formalin for sectioning while the remaining lobes were homogenized and plated for CFU enumeration.

Statistics

Statistical analysis performed using GraphPad Prism software for the Students t test per individual time point of each graph. * p<0.05, ** p<0.01, *** p<0.001

Results

IL-10 deficiency in CBA/J mice results in the development of mature granulomas and long-term control of Mtb infection

Increasing levels of IL-10 in the lung have been linked to TB disease progression in CBA/J mice (16, 22) and we therefore asked whether the complete absence of IL-10 would provide a significant survival advantage for CBA/J mice during Mtb infection. CBA/J IL-10−/− mice were created and infected with a low-dose aerosol of Mtb and the course of infection was compared to wild-type CBA/J mice. The development of pulmonary granulomas was initially similar between wild-type and IL-10−/− CBA/J mice but diverged significantly after day 60 (Fig 1a). By day 90 post-infection, CBA/J IL-10−/− mice had formed distinct organized structures with minimal involvement of surrounding lung tissue (Fig 1a). In contrast, wild-type CBA/J mice developed large, diffuse areas of unorganized pulmonary inflammation (Fig 1a), as previously reported (18). Closer examination of these organized structures in CBA/J IL-10−/− mice revealed the formation of mature granulomas defined as highly organized arrangements with foamy macrophages located in the center of the granuloma (23), surrounded by distinct layers of macrophages and tissue matrix encompassed by lymphocytes (Fig 1b). Mature granulomas were maintained at days 120 and 150 post infection, with increasing amounts of central degenerate macrophages and tissue matrix deposition (Fig 1b). Masson’s Trichrome staining revealed that mature granulomas in CBA/J IL-10−/− mice were encased in collagen (Fig 2a), indicating a substantial level of Mtb containment that has previously not been observed in the mouse model. Acid fast staining revealed that the Mtb bacilli inside granulomas of CBA/J IL-10−/− mice appeared faintly stained, singular, and fragmented in comparison to robust clumps of brightly stained bacilli normally found in wild-type mice (Fig 2a) (18). The histology data are summarized in Table I. Immuno-histochemical analyses for T and B cells revealed the presence of both lymphocyte subsets in and around the mature granulomas of CBA/J IL-10−/− mice (Fig 3b,d), which appeared more organized within the tissue than that observed in granulomas from WT mice (Fig 3a,c). Furthermore, staining for HIF1α as a surrogate marker of hypoxia revealed the presence of HIF1α positive cells within and around the mature granulomas of CBA/J IL-10−/− mice (Fig 3e,f). The abundant healthy lung tissue observed in CBA/J IL-10−/− mice stained negative for T and B cells, as well as HIF1α. Such extensive granuloma organization has not been reported in wild-type CBA/J (Fig 1, 2) or IL-10−/− C57BL/6 mice (24), indicating that the profound negative effect that IL-10 exerts on granuloma formation may be masked by host genetics in relatively resistant C57BL/6 mice and that CBA/J IL-10−/− mice provide an ideal model of human granuloma development and maturation.

Figure 1. CBA/J IL-10−/− pulmonary granuloma maturation.

Figure 1

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Wild-type and IL-10−/− CBA/J mice were infected with a low-dose aerosol of Mtb. At various timepoints post-infection lungs were removed, fixed in formalin, sectioned, and stained with hematoxylin and eosin. 10x H&E images of wild-type or IL-10−/− CBA/J lungs from day 21-150 post-infection (a). 40x H&E (b). Data representative of at least two independent experiments with 5 mice per group per timepoint. Scale bars reflect 1mm (a), 250μm (b).

Figure 2. CBA/J IL-10−/− pulmonary granuloma containment and Mtb burden.

Figure 2

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Wild-type and IL-10−/− CBA/J mice were infected with a low-dose aerosol of Mtb. At various timepoints post-infection lungs were removed, fixed in formalin, sectioned, and stained with Masson’s trichrome stain (a left, center) or Ziehl-Neelsen stain for acid-fast bacilli (a right). Day 120 post-infection at 20x (left) or 40x (center) magnification with Masson’s trichrome stain or 200x with Ziehl-Neelsen stain (right). Pulmonary and splenic Mtb CFU were enumerated on 7H11 plates (b-d). Data representative of at least two independent experiments with 5 mice per group per timepoint. * p<0.05, ** p<0.01, *** p<0.001 as obtained by Student’s t test.

Table 1.

Microscopic lung lesions from M. tuberculosis-infected mice

CBA/J CBA/J IL-10−/−
D21 Scattered small discrete foci of epithelioid Mφ; few
foamy Mφ, few PMN. Many acid fast bacilli in foci		 Scattered small discrete foci of epithelioid Mφ;
few foamy Mφ, few PMN. Mild multifocal PV
and PB lymphoid cuffs. Moderate numbers of acid
fast bacilli in lesions
D60 Slightly larger scattered discrete foci of epithelioid
Mφ; more foamy Mφ and scattered PMN in the
reaction. Multifocal PV and PB lymphoid cuffs;
some small airways have exudate. in the lumen.
Moderate numbers of acid fast bacilli in foci.		 Multifocal small discrete foci to confluent
extensive areas of granulomatous inflammation.
Epithelioid Mφ and more foamy Mφ mixed with
lymphocytes and PMN. PV and PB lymphoid
cuffs; occasional small airways with exudate. Few
acid fast bacilli with some foci devoid of bacteria.
D90 Multifocal small to larger discrete foci with some
confluence; epithelioid and many foamy Mφ.
Thick PV and PB lymphoid cuffs. Few PMN.
Many acid fast bacilli in all foci		 Multiple larger discrete mature granulomas with
many foamy Mφ in the center; some confluence.
Central PMN, necrosis with rim of epithelioid Mφ
and PV/PB lymphoid cuffs. Only scattered acid
fast bacilli with many attenuated and fragmented
D120 Multifocal small discrete foci with some confluent.
Epithelioid Mφ and many foamy Mφ; more PMN
and lymphocytes but no mature granulomas.
Multifocal PV and PB lymphoid cuffs.
Acid fast bacilli in all foci but some attenuated		 Large confluent foci with mature granulomas;
many foamy Mφ in the centers with necrosis,
pyknosis rimed by lymphocytes and PV/PB
cuffing. Two mature subpleural granulomas with
pleural rupture and fibrosis. Exudate in some small
airways. All foci with few acid fast bacilli but
pronounced in mature granulomas where they are
scarce, fragmented and attenuated.
D150 Large confluent foci of both epithelioid and foamy
Mφ. Thick PV and PB lymphoid cuffs. No mature
granulomas. Many PMN and pyknosis throughout foci.
Exudate in small airways. Many viable appearing
acid fast bacilli in the foci.		 Multifocal medium foci with occasional
confluence; Mature granulomas with many
epithelioid and foamy Mφ. Thick PV and PB
lymphoid cuffs. Focal fibrosis in granuloma with
necrosis, scattered PMN and lymphocytes.
Exudate in airways. Low numbers of acid fast
bacilli with few present in the mature granulomas
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Mφ = macrophage; PMN = polymorphonuclear cell; PV = Perivascular; PB = Peribronchiolar

Figure 3. CBA/J IL-10−/− pulmonary granuloma composition and hypoxia.

Figure 3

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Lung sections were processed from wild-type and IL-10−/− CBA/J mice infected with low (e, f) or high (a-d) dose aerosol of Mtb after 120 days of infection. Wild-type (a) or CBA/J IL-10−/− (b) lungs stained with CD3 and displayed at 20x. Wild-type (c) or CBA/J IL-10−/− (d) lungs stained with B220 and displayed at 20x. CBA/J IL-10−/− lung sections stained with HIF1α (e) or isotype control (f) and displayed at 10x.

Accordingly, the level of Mtb CFU in the lungs of CBA/J IL-10−/− mice was controlled throughout the course of infection, in contrast to escalating CFU observed in wild-type CBA/J mice (Fig 2b). Splenic analysis also displayed an enhanced control (Fig 2c). Challenge of CBA/J wild-type and CBA/J IL-10−/− mice with a higher dose of Mtb (approximately 500 CFU) showed similar findings for Mtb control in the lung as well as mature granuloma formation (not shown). Furthermore, CBA/J IL-10−/− mice survived significantly longer after low dose Mtb infection with a median survival time of 326 days, compared to 249.5 days for wild-type CBA/J mice (not shown). Interestingly, and despite a significant and sustained reduction in Mtb CFU in the lungs of CBA/J IL-10−/− mice by day 60 post infection, we observed no significant differences in lung or spleen CFU at earlier time points (Fig 2d), suggesting that immune responses were not accelerated in the absence of IL-10; a finding that was also verified using early anti-IL-10R treatment of CBA/J mice (not shown). Our studies therefore identify a model of IL-10 deficiency where low bacterial burden and extended survival is associated with the formation of mature, encapsulated granulomas.

Control of Mtb infection in CBA/J IL-10−/− mice is associated with an initial, robust TH1 immune response via increased expression of MHC class II on pulmonary antigen-presenting cells

Analysis of immune function and composition during Mtb infection showed that CBA/J IL-10−/− mice produced significantly more pulmonary IFN-γ (Fig 4a) and had increased numbers of IFN-γ+CD4+ T cells in the lung (Fig 4b) at day 21-30 post-infection only. Increased numbers of CD4+ T cells from CBA/J IL-10−/− mice also expressed the early activation marker CD69 at day 21 post-infection (Fig 4c) and secreted more IFN-γ in response to ex vivo T cell receptor (TcR) stimulation (Fig 4d) or Mtb antigen85 (Fig 4e) than wild-type CBA/J mice. Proportions of CD4+ T cells expressing CD69 or IFN-γ was moderately elevated at day 21 but did not reach significance (Fig S1). CD8+ T cells showed no significant differences in IFN-γ production or CD69 expression (not shown) at the protein level. CD4+ T cell activation in CBA/J IL-10−/− mice was also increased at early time points as determined by CD44hiCD62Llow expression (Fig S2a, b), as well as the expression of CD69 on naïve (CD44lowCD62Lhi), effector memory (CD44hiCD62Llow) or central memory (CD44hiCD62Lhi) T cell subsets in the lung (Fig S2c,d). Therefore T cells from CBA/J IL-10−/− mice showed an enhanced response during Mtb infection that peaked around 2-3 weeks of infection and then reduced to levels similar to wild-type mice. Despite this limited window of increased IFN-γ production in vivo we observed abundant capacity of CD4+ T cells to secrete Mtb-specific IFN-γ after in vitro culture with Ag85-pulsed bone marrow-derived dendritic cells at all timepoints tested (Fig 4e) indicating that CBA/J IL-10−/− mice generated and maintained a pool of Mtb-specific T cells within the lung, but these cells did not appear to be producing abundant IFN-γ in vivo during later timepoints of infection (Fig 4a). To determine the capacity of T cells to produce IFN-γ in the absence of ex vivo stimulation, we isolated CD4+ and CD8+ cells from Mtb-infected mice and analyzed purified subsets by real-time PCR for IFN-γ message, indicative of in vivo capacity. Both purified CD4+ and CD8+ cells from CBA/J IL-10−/− mice expressed significantly more IFN-γ mRNA than wild-type CBA/J mice at 21 days of infection with Mtb (Fig 4f) which subsequently reduced to levels similar to wild-type mice (not shown). This correlated with the amount of IFN-γ in lung homogenates (Fig 4a) and from T cells stimulated ex vivo (Fig 4d,e). IFN-γ mRNA from purified CD4+ and CD8+ cells was reduced at later timepoints (not shown). Together, these data demonstrate that IL-10 deficiency enables CBA/J mice to mount a relatively brief but potent TH1 response in vivo.

Figure 4. Pulmonary T cell responses in CBA/J IL-10−/− mice after Mtb infection.

Figure 4

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Wild-type and IL-10−/− CBA/J mice were infected with a low-dose aerosol of Mtb. At various timepoints post-infection lungs were removed and processed. IFN-γ ELISA of whole lung homogenate (a). Absolute number of CD4+ T cells (b) or CD69+CD4+ T cells (c). IFN-γ+CD4+ T cells as determined by flow cytometry after 4hr treatment with anti-CD3, anti-CD28 and GolgiSTOP (d). IFN-γ ELISpot of purified pulmonary CD4+ T cells cultured for 48hrs with Ag85-pulsed BMDCs. SFU=spot-forming units (e). Naïve mice did not respond to Ag85-pulsed BMDCs (no SFU; not shown). Relative units of IFN-γ mRNA in purified CD4+ or CD8+ T cells by real-time PCR (f). Naïve mice were used as a calibrator for the mRNA studies. Data representative of at least two independent experiments with 5 mice per group per timepoint. * p<0.05, ** p<0.01, *** p<0.001 as obtained by Student’s t test.

To understand the mechanism of this early robust TH1 response in CBA/J IL-10−/− mice, we analyzed MHC class II expression on the surface of pulmonary macrophages (CD11b+CD11c+ cells) isolated from Mtb-infected wild-type and CBA/J IL-10−/− mice. We again found that, only at early timepoints, CBA/J IL-10−/− mice had increased numbers (Fig 5a) and proportions (Fig 5b) of macrophages that expressed MHC class II on their surface as well as a significant increase in the proportion of cells that expressed MHC class II with higher mean fluorescence intensity (MFI) (Fig 5c). Increased MHC class II expression was verified by real-time PCR where adherent cells from the lungs of Mtb-infected CBA/J IL-10−/− mice had increased mRNA expression for MHC class II and the MHC class II transactivator, CIITA (Fig 5d). Similar to our findings for T cell responses in CBA/J IL-10−/− mice, significant increases in MHC class II expression and MFI were observed at early time points of Mtb infection only, indicating an increase in MHC class II-expressing cells in the lung, as well as an increased expression of MHC class II on individuals cells within the lung (Fig 5c). Compared to wild-type CBA/J mice, we observed no enhancement throughout infection in nos2 (iNOS) or TGFβ mRNA expression (Fig 5d) indicating that macrophages from Mtb-infected CBA/J IL-10−/− mice were not more bactericidal or compensating for the lack of IL-10 by secreting other immunosuppressive cytokines. We also failed to observe any differences in mRNA expression of arginase-1, which has been shown to mediate inflammation and affect alternative activation of macrophages during Mtb infection (25), between CBA/J IL-10−/− and WT mice (not shown). Since removal of IL-10 was linked to greater cell numbers, we investigated chemokine expression by antigen-presenting cells. As anticipated, cells from CBA/J IL-10−/− mice expressed significantly more CCL5 and more CXCL10 than wild-type mice at day 21 after Mtb infection (Fig 5d). Together, these data demonstrate that macrophages from CBA/J IL-10−/− mice have an enhanced capacity to present Mtb antigens and recruit inflammatory cells in vivo during early stages of infection.

Figure 5. Pulmonary APC responses in CBA/J IL-10−/− mice after Mtb infection.

Figure 5

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Wild-type and IL-10−/− CBA/J mice were infected with a low-dose aerosol of Mtb. At various timepoints post-infection lungs were removed and processed. Absolute number (a), proportions (b), or mean fluorescence intensity (c) of lung cells positive for CD11c+CD11b+IA/IE+ as determined by flow cytometry. Histograms represent day 30 post-infection. Black lines = infected samples; grey lines = isotype control. Relative units of MHC class II (h2a), CIITA (ciita), CCL5 (ccl5), TGFβ (tgfb), iNOS (nos2), CXCL10 (cxcl10) mRNA in adherent pulmonary cells by real-time PCR at day 21 post-infection (d). Data representative of at least two independent experiments with 5 mice per group per timepoint. * p<0.05, ** p<0.01, *** p<0.001 as obtained by Student’s t test.

TH1 responses in the draining lymph nodes of CBA/J IL-10−/− mice are not increased before day 21 of Mtb infection

To determine the kinetics for the generation of protective TH1 responses in CBA/J IL-10−/− mice, we analyzed the composition and phenotype of MLN cells from day 0 to day 21 of Mtb infection. We observed no difference in the number of total MLN cells (Fig 6a), CD4+ T cells (Fig 6b), CD8+ T cells (not shown), or activated CD4+ T cells (Fig 6c) prior to day 21 of Mtb infection after which CBA/J IL-10−/− mice had significantly more (Fig 6a, b, c). Ex vivo TcR stimulation of MLN CD4+ T cells demonstrated no significant increase in IFN-γ+CD4+ T cells (Fig 6d) and TNF+CD4+ T cells (Fig 6e) until day 21 of Mtb infection, showing that the absence of IL-10 did not accelerate priming or activation of T cells. At day 21 of Mtb infection CBA/J IL-10−/− mice had significantly more activated CD4+ and CD8+ T cells that could secrete IFN-γ or TNF indicating that the absence of IL-10 led to enhanced functional activation of T cells in the MLN. As a measure of protective TH1 effector cells (26) we analyzed the number of polyfunctional TNF+IFN-γ+ CD4+ T cells in the MLN and found those to be significantly increased at day 21 post-infection in CBA/J IL-10−/− mice (Fig 6f). We also examined the expression of IFN-γ mRNA in pulmonary T cells from CBA/J IL-10−/− mice at these same early timepoints during Mtb infection. Similar to MLN, we observed no alteration in the amount of IFN-γ mRNA detected in CD4+ or CD8+ T cells in the lungs of CBA/J IL-10−/− mice until day 21 of Mtb infection (not shown). Therefore our data indicate that IL-10 can influence the functional properties of CD4+ T cells throughout the first three weeks of infection with Mtb but appear to play no role in accelerating the initial immune response within the MLN or lung.

Figure 6. Early lymph node and lung responses to Mtb infection.

Figure 6

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Wild-type and IL-10−/− CBA/J mice were infected with a low-dose aerosol of Mtb. At various timepoints post-infection, MLN were obtained and processed for analysis. Total number of MLN cells determined by trypan blue exclusion (a), absolute number of CD4+ T cells (b) or CD69+CD4+ T cells (c) as determined by flow cytometry. Absolute number of IFN-γ+CD4+ T cells (d), TNF+CD4+ T cells (e), or IFN-γ+TNF+CD4+ T cells (f) as determined by flow cytometry after 4hr treatment with anti-CD3, anti-CD28 and GolgiSTOP. All data representative of two independent experiments with 5 mice per group per timepoint. * p<0.05, ** p<0.01, *** p<0.001 as obtained by Student’s t test.

Early IL-10R blockade of CBA/J mice recapitulates the CBA/J IL-10−/− mouse phenotype during Mtb infection

The concept that IL-10 mediated enhancement of early TH1 immune responses observed during the first 21 days of Mtb infection could be responsible for the long term control of Mtb infection, as well as late generation of mature granulomas, was tested. Wild-type CBA/J mice were treated with anti-IL-10R for the first 21 days of infection and T cell function, control of Mtb infection, and granuloma formation was assessed. Early anti-IL-10R treatment of CBA/J mice significantly increased the total number of cells within the MLN at day 21 post Mtb infection, with an associated increase in the total number CD4+ and CD8+ T cells (Fig 7a). The total number of CD69+ CD4+ and CD8+ T cells was significantly increased (Fig 7b) at day 21 post Mtb infection. After anti-IL-10R treatment, purified pulmonary CD4+ T cells expressed more IFN-γ mRNA (Fig 8a) whereas IL-17 and TNF mRNA was unaltered. Purified pulmonary antigen-presenting cells expressed significantly more MHC class II and CCL5 mRNA (Fig 8b), confirming and further highlighting the significant impact of IL-10 on CD4+ T cell and antigen presenting cell interactions.

Figure 7. IL-10R Blockade of wild-type CBA/J mice.

Figure 7

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Wild-type CBA/J mice were treated with anti-IL-10R or Rat IgG1 isotype control antibody from day (−1) to day 20 of Mtb infection. At day 21 a subset of mice were euthanized and MLN were harvested for analysis. Total MLN cells determined by trypan blue exclusion, absolute number of CD4+ T cells, CD8+ T cells (a) or CD69+CD4+ T cells (b) as determined by flow cytometry. At day 120 remaining mice were sacrificed and pulmonary CFU enumerated on 7H11 plates (c). IL-10R-treated lung at day 120 post-infection with H&E stain at 10x (d box 1), Masson’s trichrome stain at 20x or (d box 2) 100x. (d box 3). All data sets representative of two independent experiments with 5 mice per group per experiment. Control group = No treatment and Rat IgG1 groups combined for analysis. * p<0.05, ** p<0.01, *** p<0.001 as obtained by Student’s t test.

Figure 8. TH1 mRNA expression after IL-10R Blockade of wild-type CBA/J mice.

Figure 8

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Wild-type CBA/J mice were treated with anti-IL-10R or Rat IgG1 isotype control antibody from day (−1) to day 20 of Mtb infection. At day 21 a subset of mice were euthanized and lungs were harvested for analysis. Relative units of IFN-γ (ifng), TNF (tnf), IL-17 (il17a) mRNA in purified CD4+ or CD8+ T cells by real-time PCR (a). Relative units of MHC class II (h2a), CIITA (ciita), CCL5 (ccl5), TGFβ (tgfb), iNOS (nos2), CXCL10 (cxcl10) mRNA in adherent pulmonary cells by real-time PCR (b). Control group = No treatment and Rat IgG1 groups combined for analysis. * p<0.05, ** p<0.01, *** p<0.001 as obtained by Student’s t test.

Anti-IL-10R treatment was halted at day 21 and a group of mice were maintained for an additional 100 days to determine the long-term phenotype. CBA/J mice treated with anti-IL-10R for only the first 21 days of Mtb infection exhibited a significantly reduced pulmonary Mtb burden (Fig 7c), similar to that observed in CBA/J IL-10−/− mice. Most striking, however, was that at day 120 post-Mtb infection anti-IL-10R treated mice had developed mature granulomas (Fig 7d), some with a prominent fibrotic capsule (Fig 7d box 2,3) and minimal Mtb within the granulomas (not shown), recapitulating the IL-10−/− phenotype. Granulomas in isotype treated CBA/J mice (not shown) resembled those seen in wild type CBA/J mice (Fig 1, 2). Our data define the first few weeks of Mtb infection as the most critical time to establish long term protective immunity, including the establishment of mature granulomas, and demonstrate that IL-10 plays a pivotal role in this event.

Discussion

IL-10 is highly associated with TB disease in humans but its exact role has been elusive to define because most IL-10−/− mice have been generated on relatively resistant mouse strain backgrounds, which show only limited changes after Mtb infection in the absence of IL-10 (27-29). Here we created IL-10−/− mice on a CBA/J Mtb-susceptible mouse strain background, which normally produces abundant IL-10 during Mtb infection (16). In this regard, the CBA/J mouse strain is thought to better reflect humans that are susceptible to TB disease progression (30). Our data demonstrate that in a Mtb-susceptible mouse strain, IL-10 plays a critical role in preventing the optimum generation of protective T cells, including polyfunctional T cells, and perhaps most significant was our demonstration that IL-10 inhibits the control of Mtb infection and the formation of mature, protective granulomas. Our data also clearly demonstrate that the action of IL-10 during the first three weeks of Mtb infection only was sufficient to alter the phenotype of CBA/J mice to one of relative resistance. Therefore, deletion of IL-10 on an Mtb-susceptible mouse strain leads to a highly significant reduction in CFU.

The most significant phenotypic change that was observed in CBA/J IL-10−/− mice was the generation of small, mature, protective granulomas, with Mtb localized in fibrotic capsules containing central necrotic cores. These mature granulomas were surrounded by T and B cells, indicating a level of immune control in and around the granuloma. We also observed the presence of HIF1α within cells that were circumventing mature granulomas. The presence of HIF1α is a surrogate marker of hypoxia (31, 32) indicating that the fibrotic capsule served to restrict oxygen to the central core. Indeed, Mtb within these mature granulomas appeared fragmented, likely a reflection of modified mycolic acid expression, and perhaps reflecting Mtb adaptation to a hostile granuloma environment. Such mature granulomas have not been observed in mice in the context of a protective response (4). Previous studies have demonstrated that altered serine protease activity in highly susceptible nos2−/− mice can lead to human-like Mtb granuloma formation early after infection (33) and although these lesions exhibited more organization than typical murine Mtb granulomas, nos2−/− granulomas did not develop a fibrotic capsule and were generated via an ear dermis Mtb infection route (33). Similarly, other studies have demonstrated necrotic, encapsulated granulomas in mice possessing the sst1 genetic susceptibility locus (34), although encapsulated granulomas were associated with increasing bacterial loads during primary Mtb infection and only associated with low CFU following a course of isoniazid treatment. Similar to other susceptible mouse strains (35-39), the CBA/J mouse can form large coalescing nodular structures during Mtb infection however these are highly associated with end stages of disease, abundant CFU, and substantial lung involvement (not shown). This does however indicate a predisposition to develop fibrotic structures in CBA/J and other susceptible mouse strains that, as we show here, can lead to mature granulomas that are associated with protection in the absence of IL-10. The presence of small mature granulomas that are associated with reduced bacterial loads are more frequently associated with latent Mtb infection in humans (40, 41). The CBA/J IL-10−/− mouse therefore provides us with a small animal model to study the development of protective granuloma formation during Mtb infection and also strongly implicates a role for IL-10 in inhibiting this process. We, and others, have previously shown that anti-IL-10R treatment of CBA/J mice during chronic Mtb infection (days 90-120 of infection) can similarly reduce the Mtb lung CFU (16, 17, 22). Where these studies differ from our current work, however, is that treatment of mice with anti-IL-10R during late stages of Mtb infection failed to modify granuloma structure. Therefore, although IL-10 can have an immuno-modulatory role throughout the course of Mtb infection, it is clear that the influence of IL-10 during T cell priming and activation has the most significant impact on long-term containment of Mtb infection.

The mechanism that leads to the formation of mature granulomas, although dependent on IL-10, is currently unclear. Measurement of tissue remodeling molecules (TGFβ, VEGF) or immune modulators known to be specifically influenced by IL-10 (25) (iNOS, arginase) showed no significant changes. CBA/J IL-10−/− mice did have a significant but transient increase in Ipr1 mRNA expression (p<0.05 at day 60 only; not shown), indicating a potential role for sst1 that may warrant further investigation. We have primarily observed significant changes in early immune events such as MHC class II expression and T cell function, as well as chemokine expression, but how these early events lead to long term and late changes within the lung remains elusive. Given that several of the known immuno-modulators of Mtb infection did not change in our CBA/J IL-10−/− mice before or during mature granuloma formation, an alternative unbiased approach to deciphering this process should be considered. Such an approach may reveal new participants in granuloma formation that have not been previously identified.

Early immune events during Mtb infection that can be influenced by IL-10 include the interaction of Mtb with host alveolar macrophages and DCs within the lung and activation of the adaptive immune system (16, 42-44). IL-10 is known to alter macrophage function, including the trafficking of Mtb to phago-lysomal compartments (43) and the capacity to express MHC class II on the cell surface as we and others (45) have shown. In the absence of IL-10 it is likely that pulmonary macrophages are more potent local activators of T cells, an event that is supported by the robust and early T cell responses we observed in the lungs of Mtb-infected CBA/J IL-10−/− mice, and our previous findings blocking the action of IL-10 during chronic Mtb infection in CBA/J mice (16). Furthermore, we observed altered T cell function within the MLN, suggesting that DC-T cell interactions may also be enhanced in the absence of IL-10. In contrast to previous studies (17), we found no evidence for an accelerated immune response in the absence of IL-10. The activation and expansion of T cell subsets in the MLN were unaltered throughout the first 18 days of infection, a period of time when Mtb is known to be trafficked to the MLN for presentation to T cells (46-49). We did observe enhanced immune responses at day 21 of Mtb infection in both MLN and lung and, more specifically, we observed a significant increase in the number of polyfunctional T cells within the MLN in the absence of IL-10. Protective polyfunctional T cells have been identified in several infection models (50-52) and more recently have been described in mouse models and human studies of TB (53-55). Although the current literature is somewhat contradictory regarding the protective or detrimental role of polyfunctional T cells during Mtb infection, our data clearly associate the presence of polyfunctional T cells with enhanced protective immunity and improved control of Mtb infection.

An important consideration is why such a dramatic role for IL-10 is only evident in an Mtb-susceptible mouse strain. IL-10 deficiency on the relatively Mtb resistant BALB/c and C57BL/6 mouse strain backgrounds leads to moderate reductions in organ CFU (17, 27, 45), and while TH1 immune responses are also enhanced, no mature granuloma formation has been observed at least throughout the first 200 days of infection (unpublished observations). Furthermore, Higgins et al showed that C57BL/6 IL-10−/− mice generated excessive inflammation as Mtb infection progressed resulting in sudden mortality at late stages of infection (45). This is in stark contrast to our observations with CBA/J mice where the absence of IL-10 provides a distinct survival advantage. It is likely that the complex and currently undefined genetic variations between Mtb resistant and susceptible mouse strains, such as those influencing the formation of fibrotic structures, provide a unique environment in which IL-10 can act, that in some genetic backgrounds has a profound impact on disease outcome. Such dramatic shifts in outcome highlight the benefits of studying Mtb infection in multiple different mouse strains capable of more accurately modeling the broad disease spectrum observed in man.

In summary, our studies using the CBA/J IL-10−/− mouse model identify a significant negative influence for IL-10 on the control of Mtb infection, providing conclusive evidence that IL-10 is a correlate of TB disease progression. Furthermore, we identify a previously unappreciated role for IL-10 in the formation of mature granulomas that resemble those previously described in humans (23, 41). In this regard, the CBA/J IL-10−/− mouse provides a unique and valuable small animal model system to understand granuloma development during Mtb infection. We also demonstrate that the influence of IL-10 is not limited to chronic Mtb infection as we (15, 16) and others (45, 56) have previously described. Early blockade of the IL-10R during Mtb infection afforded CBA/J mice with a similar protective advantage as CBA/J IL10−/− mice. This last finding may have significant application for the design of Mtb vaccine regimens in humans. While our study does not directly address the effect of IL-10 deficiency on vaccination, the prospect of using anti-IL-10R treatment during Mycobacterium bovis BCG vaccination has recently been shown to boost efficacy (57). Inclusion of anti-IL-10R with current or newly developed vaccines could provide susceptible individuals with the capacity to establish long-term protective granulomatous containment of Mtb.

Supplementary Material

1
2

Acknowledgements

Histology services were performed by Alan Fletchner and Anne Saulsbery in The Ohio State University’s College of Veterinary Medicine, Department of Veterinary Biosciences. Aperio slide scanning was performed by Shelly Haramia in The Ohio State University’s College of Veterinary Medicine, Department of Veterinary Biosciences. We would like to thank Drs. Andrea Cooper, Jordi Torrelles, and Amy Lovett-Racke for their insightful and valuable comments. The following reagent was obtained through BEI Resources, NIAID, NIH:Mycobacterium tuberculosis Ag85 Complex, Purified Native Protein from Strain H37Rv, NR-14855.

Support was provided by the NIH R01 (AI-064522; JT), NIH K08 (AI-071111; GB).

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Supplementary Materials

1
NIHMS435210-supplement-1.docx (127.8KB, docx)
2
NIHMS435210-supplement-2.docx (278.1KB, docx)

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