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. Author manuscript; available in PMC: 2014 Jan 1.
Published in final edited form as: J Immunol. 2012 Nov 23;190(1):349–356. doi: 10.4049/jimmunol.1202225

TLR9-Dependent IL-23/IL-17 is Required for the Generation of Stachybotrys chartarum-induced Hypersensitivity Pneumonitis

Urvashi Bhan 1, Michael J Newstead 1, Xianying Zeng 1, Amy Podsaid 1, Moloy Goswami 1, Megan N Ballinger 1, Steven L Kunkel 1, Theodore J Standiford 1
PMCID: PMC3529778  NIHMSID: NIHMS417963  PMID: 23180821

Abstract

Hypersensitivity pneumonitis (HP) is an inflammatory lung disease that develops following repeated exposure to inhaled particulate antigen. Stachybotrys chartarum (SC) is a dimorphic fungus that has been implicated in a number of respiratory illnesses, including HP (1). In this study we have developed a murine model of SC- induced HP that reproduces pathology observed in human HP and hypothesized that TLR9–mediated IL-23/IL-17 responses are required for the generation of granulomatous inflammation induced by inhaled SC. Mice that undergo i.p. sensitization and i.t. challenge with 106 SC spores developed granulomatous inflammation with multinucleate giant cells, accompanied by increased accumulation of T cells. SC sensitization and challenge resulted in robust pulmonary expression of IL-17 and IL-23. SC-mediated granulomatous inflammation required intact IL-23/IL-17 responses and required TLR9, as TLR9−/− mice displayed reduced IL-17 and IL-23 expression in whole lung associated with decreased accumulation of IL-17 expressing CD4+ and γδ T cells. As compared to SC-sensitized dendritic cells (DC) isolated from WT mice, DC isolated from TLR9−/− mice had a reduced ability to produce IL-23 in responses to SC. Moreover, shRNA knockdown of IL-23 in DC abolished IL-17 production from splenocytes in response to antigen challenge. Finally, the i.t. reconstitution of IL-23 in TLR9−/− mice recapitulated the immunopathology observed in WT mice. In conclusion, our studies suggest that TLR9 is critical for development of Th17-mediated granulomatous inflammation in the lung in response to SC.

Keywords: Fungus, toll receptors, lung, cytokines

INTRODUCTION

Hypersensitivity pneumonitis (HP) is an inflammatory lung disease that develops following repeated exposure to inhaled particulate antigen (2). Exposure to these antigens occurs most frequently in certain occupations, hobbies, or within the home environment. Accumulating evidence suggests that molds may represent an underappreciated cause of HP. For instance, molds are increasingly being recognized as a cause of HP after exposure to contaminated humidifiers, heating ducts, and more recently flooded old buildings. Stachybotrys chartarum is a fungus that causes “black mold disease” and “sick building syndrome”, and has been speculated as a major offender in promoting HP(1, 3–5).

Histopathology in HP is characterized by dense accumulation of macrophages, lymphocytes, epithelioid cells and fibroblasts around indigestible particles or antigens (6). Hypersensitivity-type granulomas are believed to be induced by the interaction of activated T cells, macrophages and DC in an antigen-specific manner (7). DC are potent antigen presenting cells in the lungs and play a major regulatory role in T cell activation and differentiation. T cells are necessary for the development and progression of HP (8, 9). For instance, athymic nude mice that lack T cells are protected from the development of HP in response to inhaled antigenic challenge. In rodent models of HP, there is a shift in T cell population to CD8+ predominance by 24–48 hours post challenge with antigen (9–11). In humans with HP, there is an increase in the number of both CD4+ and D8+ T cells in bronchoalveolar lavage fluid (BALF) and/or lung interstitium (12–15). Adoptively transferred, sensitized CD4+ Th1 cells can cause HP in healthy animals (10). Mechanisms which promote T cell accumulation/activation have not been thoroughly defined.

Toll-like receptors (TLRs) are a family of type I transmembrane receptors that respond to pathogen-associated molecular patterns (PAMPs) expressed by a diverse group of infectious microorganisms, resulting in activation of the host’s immune system (16–19). The role of TLRs in the generation of HP is incompletely defined. Given the composition of fungal cell wall components (20, 21), the most relevant TLRs are TLR4, which binds to and is activated by LPS and fungal mannans, and TLR2, which recognize fungal β-glycans and zymosan (22, 23). TLR9 is of particular interest, as this TLR recognizes fungal DNA, and TLR9 has been shown to drive type 1 responses to both microbial and non-microbial antigens (24).

Historically, granulomatous tissue responses that were once believed to be solely Th1-mediated have more recently been shown to require IL-17 (25–27). Accordingly, several groups have shown that the gene deletion or in vivo neutralization of IL-17 in an experimental model of HP driven by repeated Saccharopolyspora rectivirgula (SR)-Ag challenges results in protection from HP, indicating that IL-17 and Th17 cells are major driving factors (28, 29). IL-17 drives a pro-inflammatory immune response by inducing chemokine and chemoattractant production from resident immune and stromal cells (30, 31). Subsequently, neutrophils and other immune cells are recruited, thereby intensifying the inflammatory response (32). Th17 differentiation requires the presence of IL-6 and transforming growth factor-β (TGF-β), while expansion and growth of Th17 cells is regulated by IL-23 (33, 34); the exact signaling mechanisms that lead to Th17 differentiation during immune responses such as HP are unclear.

In our recent study, we found that TLR9 was necessary for the generation of HP in a murine model using sensitization and challenge in response to SC. In this study, we investigated the novel role of TLR9 in driving IL-17 responses in experimental HP, using a murine model, which involves repeated intraperitoneal sensitization and i.t challenge of mice with the S. chartarum. Our study indicates that IL-17 is an important cytokine mediator of SC-induced HP, and that the production of IL-17 in this model requires TLR9-dependent IL-23 expression from DC.

METHODS

Reagents

Murine recombinant cytokines were purchased from R&D Systems (Minneapolis, MN). Polyclonal antimurine cytokine antisera used in ELISA or neutralization experiments were produced by immunization of rabbits with either recombinant murine cytokines in multiple intradermal sites with complete Freund’s adjuvant. ELISA for quantization of murine IFN-γ was purchased from PBL Biomedical Laboratories.

Animals

SPF Balb/c mice were purchased from Jackson Labs. Breeding pairs of TLR9−/− mice generated by S. Akira (Osaka University, Osaka, Japan) were obtained from Coley Pharmaceutical Group and a colony was established at the University of Michigan (Ann Arbor, MI). These mice were generated on a BALB/c background (more than five backcrosses), are phenotypically normal in the uninfected state, and reproduce without difficulty. All mouse strains were housed in SPF conditions within the animal care facility (ULAM) until the day of sacrifice.

Intratracheal administration of SC conidia

Mice were injected with ketamine and xylazine by the i.p. route. After adequate anesthesia, mice were restrained in the supine position. The anterior aspect of the neck was exposed and an incision made to visualize the trachea. A 26 gauge needle was advanced endotracheally, and through this, 30 μl of normal saline containing SC conidia was injected. The animals were then allowed to recover, and the wound closed with surgical staples. No adverse effects were encountered with this procedure.

Intranasal administration of SC conidia

Animals were lightly anesthetized with ketamine and xylazine. After adequate anesthesia, 10–20 μl of a solution containing SC conidia was placed in the nares of mice until the solution was inhaled.

Intraperitoneal administration of SC conidia

Mice were injected i.p. with 50μl of a solution containing SC conidia.

IL-23 Reconstitution

Murine recombinant IL-23 was purchased from R&D Systems (Minneapolis, MN). Mice were anesthetized and 2μg rmIL-23 administered i.t. at the same time as i.t. challenge with SC spore.

Removal of various organs at the time of necropsy

Mice were euthanized at various intervals after SC sensitization and challenge by inhalation of carbon dioxide, exsanguinated, and organs (lungs, spleens) removed.

Splenocyte isolation

Splenic single-cell suspensions were prepared by homogenization through a 70-μm cell strainer (BD Biosciences). Splenic CD4+ and CD8+ T lymphocytes were positively selected using anti-mouse–conjugated MidiMACS beads, according to the manufacturer’s instructions (Miltenyi Biotec).

Total lung leukocyte preparation

Lungs were removed from euthanized animals and leukocytes prepared as previously described. Briefly, lungs were minced with scissors to a fine slurry in 15 ml/lung digestion buffer (RPMI/5% fetal calf serum/1 mg/ml collagenase (Boehringer Mannheim Biochemical)/30 μg/ml DNAse (Sigma, St. Louis, MO). Lung slurries were enzymatically digested for 30 minutes at 37°C. The total lung cell suspension was pelleted, re-suspended and spun through a 20% Percoll gradient to enrich for leukocytes prior to further analyses.

Multiparameter flow cytometric analyses

Total lung leukocytes were isolated as described above. Lymphocyte subsets were analyzed by first gating on CD45 positive “lymphocyte sized” leukocytes, and then examined for lymphocyte-associated markers. Antibodies (PharMingen and Caltag) used for phenotyping include T cell markers (anti-CD4, anti-CD8, anti-CD69, B cells (anti-CD19), pan-NK cells (anti-DX5)γδ T cells (anti-γδ TCR). Cells were collected on a FACSCalibur cytometer (Becton Dickinson, San Jose, CA) using Cellquest software (Becton Dickinson). Analyses of data were performed using the Cellquest software package.

Cytokine ELISA

Lung homogenate, DC supernatants, or DC-splenocyte co-culture supernatants were collected and cytokine levels determined by a sandwich ELISA method as previously described. (35)

Generation of bone marrow-derived dendritic cells

Bone marrow was harvested from WT and mutant mice and seeded in tissue culture flasks in RPMI 1640 based complete media with 10 ng/ml murine rGM-CSF. Loosely adherent cells collected after 6–7 days and incubated with anti-CD11c antibody coupled to magnetic beads (Miltenyi Biotec, Auburn, CA). Cells were purified using positive selection for CD11c+ cells by running the cell suspension through a magnetic column. CD11c+ dendritic cells were plated overnight and re-suspended in fresh media the following day.

Histology

Lungs were harvested 3 days after stimulation and challenge with SC and were inflated, and fixed in 10% formalin. The fixed lung lobe was embedded in paraffin; 5 μm sections were then stained with hematoxylin-eosin. Images were captured using Olympus BX40 microscope and IP Lab Spectrum software (Signal Analytics Corp., Vienna, VA).

mRNA extraction and Real-Time (TaqMan) quantitative polymerase chain reaction

Total RNA from cells was isolated per manufacturer’s protocol for the RNAeasy Mini kit (Qiagen, Valencia, CA). RNA amounts were determined by spectrometric analysis at 260 nmλ. All primers were designed using Primer Express software (Applied Biosystems, Foster City, CA). Levels of mRNA were determined by real-time quantitative RT-PCR analysis using an ABI PRISM® 7000 Sequence Detection System (ABI/Perkin Elmer Co, Foster City, CA).

Retroviral transduction of DC

Expression vectors in pGFP-V-RS plasmid producing short-hairpin RNA (shRNA) against murine IL-23 mRNA were purchased from Origen Technologies. All shRNA constructs were prepared in the MSCV-based pLMP retroviral vectors. Supernatants from transfected cells were collected at 48 hr post transfection. Supernatants were added to DC for stable knockdown of IL-23 verified by real time PCR. The sequences of the shRNA that are generated from these vectors are as follows (only the sense strands are shown): sh-mIL-23, 5′-CAGAGCAGTAATAATGCTATGGCTGTTGC-3′. Transfected cells were seeded at 1 × 106 × 12-well plates. Cells were stimulated and lysed for mRNA extraction as described.

Statistical analyses

Statistical significance was determined using the unpaired, two-tailed Student t test and non-parametric Mann-Whitney test. Calculations were performed using InStat for Macintosh (GraphPad Software, San Diego, CA). In all cases, a p value of less than 0.05 will be considered significant.

RESULTS

Increased IL-17 mRNA and protein levels in whole lung

We have developed a murine model of HP utilizing the fungus SC, which has been associated with a variety of respiratory diseases, including sick building syndrome and black mold disease. This is a particularly attractive model, because SC is a clinically relevant cause of lung disease and is relatively understudied. In this model, Balb/c mice (WT) were sensitized with i.p. injections of 106 SCspores on day −14 and −7, and then administer 106 spores i.n. on day −5 and −3. At day 0, sensitized mice were administered 106 fungal spores i.t., then lungs removed at day 2 and day 5. As we have previously shown, WT mice had increased mononuclear cell infiltrates which occurred in a peribronchial distribution (Figure 1A, arrow), with the presence of giant cells and evidence of loose granuloma formation. By comparison, reduced peribronchial inflammation was observed in sensitized TLR9−/− mice challenged i.t. with SC, as well as a reduction in the total number of CD4+ (1B) and CD8+ T (1C) cells in mutant mice. Moreover, SC sensitization and challenge in WT mice resulted in an substantial increase in the percentage of CD4+ T cells expressing the activational marker CD69, whereas this increase in CD69 expression was blunted in CD4+ T cells from TLR9−/− mice (Figure 1D, 1E). There were no differences in number of CD69 expression in CD8+ T cells between the two groups (data not shown).

Figure 1. Lung IL-17 levels post SC challenge.

Figure 1

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Shown are 10x magnification micrographs of H&E staining of lung sections obtained from sensitized WT and TLR9−/− mice challenged with Stachybotrys chartarum at Day 2 (Figure 1A). Accumulation of neutrophils and mononuclear cells within the interstitial compartments, and a predominantly lymphocytic infiltration within the peribronchial regions, as well as multinucleated giant cells (arrows) and loose epitheloid granulomas within both the interstitial and peribronchial areas in SC challenged mice. WT and TLR9−/− mice were sensitized and challenged with SC as described and lungs harvested at Day 3, lung collagenase digests were performed and lymphocytes purified. Flow cytometric analysis was performed and number of CD4+ T cells (Figure 1B), CD8+ T cells (Figure 1C), and CD4+, CD69+ cells (Figure 1D, 1E) is shown. n=5 in each experiment, mean ± SEM of two experiments. *p<0.01as compared to sensitized WT mice.

Interleukin 17 has previously been causally linked to hypersensitivity responses in a murine SR-Ag induced HP model (29). To determine if protection against the development of HP in TLR9−/− mice was attributable to differences in the expression of IL-17, WT and TLR9−/− mice were sensitized and i.t. challenged with SC, then IL-17 mRNA and protein levels measured in lung homogenates. In WT animals, sensitization and subsequent challenge with SC induced a vigorous time-dependent expression of IL-17 mRNA (Figure 2A) and protein (Figure 2B) in whole lung. By comparison, mRNA IL-17 mRNA and protein levels were substantially diminished in the lungs of TLR9 deficient mice after sensitization and challenge with SC (37% and 48% decrease, respectively).

Figure 2. IL-17 expression in WT and TLR9−/− mice post SC challenge.

Figure 2

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WT and TLR9−/− mice were sensitized and i.t. challenged with SC, then IL-17 mRNA (Figure 2A) and protein levels by ELISA (Figure 2B) measured in lung homogenates. WT mice were administered purified polyclonal rabbit anti-mouse anti–IL-17 antibody or control IgG 3 days prior to and the day of i.t. challenge with SC. Histological examination of lungs was performed 3 days after sensitization and SC challenge (Figure 2C). WT and TLR9−/− mice were sensitized and challenged with SC as well as treated with either purified polyclonal rabbit anti-mouse anti–IL-17 antibody or control IgG 3 days prior to and the day of i.t. challenge with SC. Flow cytometric analysis was performed and number of CD4+ T cells (Figure 2D) and CD8+ T cells (Figure 2E) is shown as well as CD69+ CD4+ T cells (Figure 2F, 2G). n=4–5 for each experiment, mean ± SEM of two experiments. * p<0.05 as compared to sensitized WT mice.

Immunoneutralization of IL-17 has effectively attenuated disease in several experimental models of disease, including allergen-induced contact hypersensitivity responses and SR-Ag–induced HP (31). To establish that IL-17 contributed meaningfully to SC-induced HP, WT mice were administered purified polyclonal rabbit anti-mouse anti–IL-17 antibody 100μg or control IgG 3 days prior to and the day of i.t. challenge with SC. Histological examination of lungs 3 days after sensitization and SC challenge revealed a decreased inflammatory response in mice that received anti–IL-17 antibody (Figure 2C) and reduced CD4+ T but not CD8+ T cell accumulation, as determined by flow cytometry (Figure 2D, 2E, respectively). Moreover, there was a substantial decrease in the number of activated CD4+ T cells, as measured by expression of CD69 in mice treated with the IL-17 antibody as compared to control antibody treated mice (Figure 2F, 2G).

Decreased expression of IL-17 by TLR9−/− lung T cells in-vivo

To determine if T cells were the cellular source of IL-17 production in response to SC, WT and TLR9−/− mice were sensitized and challenged as described, lungs harvested at day 3 post challenge, and collagenase digests performed. The numbers of IL-17 expressing T cell populations present in the lungs of WT and TLR9−/− mice were quantitated at baseline and post-SC administration by flow cytometry. As shown in Figure 3A and C, SC sensitization and challenge resulted in an increase in the percentage and total number of CD4+ T cells expressing IL-17, as well as percentage and total number of γδ T cells (Figure 3B, 3D) expressing IL-17 (p <0.05) in WT mice, as compared to untreated controls. By comparison, the number of CD4+ T cells and γδ T cells expressing cell-associated IL-17 was significantly diminished in TLR9 deficient mice post SC sensitization.

Figure 3. Recruitment of IL-17 expressing T cells in WT and TLR9−/− mice post SC sensitization and challenge.

Figure 3

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WT and TLR9−/− mice were sensitized and challenged with SC as described and lungs harvested at Day 3, lung collagenase digests were performed and lymphocytes purified. Flow cytometric analysis was performed and percentage and number of γδ (Figure 3B, 3D) and CD4+ T cells (Figure 3A, 3C) with intracellular IL-17 is shown. n=5 in each experiment, mean ± SEM of two experiments. * p<0.05 as compared to untreated controls. #p<0.05 as compared to SC challenged WT mice.

Reduced IL-23 production by DC isolated from sensitized TLR9−/− mice

Dendritic cells (DC) are required for the development of antigen-specific type 1 cytokine responses, particularly IFN-γ production by T cells (36). To determine if the reduced granulomatous inflammation and T cell-derived IL-17 production observed in TLR9−/− mice was secondary to altered DC IL-23 production, WT and TLR9−/− mice were sensitized (i.p. and i.n.) as described previously and bone marrow harvested on day 0. Bone marrow cells were cultured x 6 days in GM-CSF, DC purified by CD11c+ magnetic beads separation on Day 6, stimulated with SC spores in a ratio of 1:10 (DC: SC spore), and spontaneous ex-vivo production of IL-23 measured by ELISA (Figure 4B) and IL-23 p19 mRNA expression determined by real time PCR (Figure 4A). As shown in Figure 4A, incubation of sensitized WT DC with SC resulted in considerable induction of IL-23 p19 mRNA and IL-23 protein. The expression of IL-23 p19 mRNA and IL-23 protein was substantially attenuated in BMDC isolated from TLR9−/− mice (p<0.05).

Figure 4. DC IL-23 production in-vitro post SC challenge.

Figure 4

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WT and TLR9−/− mice were sensitized and challenged with SC and bone marrow harvested at Day 2. Bone marrow cells were cultured with GM-CSF for 6 days and DC purified using magnetic bead separation and stimulated with SC spores in a ratio of 1:10. Spontaneous in-vitro IL-23 production was measured in the supernatants with ELISA (Figure 4B) and IL-23 mRNA measured by real time (Figure 4A) n=4–5 in each group, experiments performed twice. *p<0.05 as compared to untreated controls. #p<0.05 as compared to SC challenged WT mice.

IL-23p19 shRNA silences IL-23 expression and blocks DC-stimulated IL-17 production by T cells

To assess the contribution of IL-23 production from SC-sensitized DC to expression of IL-17 from T cells, bone marrow was isolated from sensitized WT mice on day 0, then DC maturated x 6 days in GM-CSF. DC were then incubated with either control or IL-23 p19 shRNA to extinguish DC-derived IL-23 expression. Transfection efficiency of primary DC was high, with greater than 90% of cells expressing GFP (Figure 5A). Stimulation with SC resulted in strong expression of IL-23 p19 mRNA expression from sensitized WT DC incubated with control shRNA (Figure 5B). By comparison, treatment with IL-23 p19 shRNA nearly completely silenced IL-23 p19 expression from SC challenged DC. Knockdown was specific for IL-23p19, as this treatment had no effect on DC-derived IL-12 p40 mRNA expression (Figure 5C). To assess if DC-derived IL-23 was required for SC-induced IL-17 production from T cells, WT DC incubated with control or IL-23 p19 shRNA were co-cultured with splenic T cells harvested from naïve WT mice for 18 hours. T cells co-cultured with WT DC treated with control siRNA produced large quantities of IL-17 (22-fold increase over unstimulated cells). The induction of IL-17 was dependent on DC-derived IL-23, as IL-17 expression was nearly completely abolished when co-cultured with IL-23 shRNA treated DC (Figure 5D). Collectively, these findings suggest that the antigen-specific induction of IL-17 by T cells in response to SC is dependent on DC-derived IL-23.

Figure 5. DC function post treatment with IL-23 shRNA.

Figure 5

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Bone marrow cells were harvested from WT mice, cells were cultured with GM-CSF and DC purified by CD11c+ magnetic beads at Day 6, DC were treated with either control shRNA or shRNA against IL-23. Florescent microscopy was performed to access transfection efficiency (Figure 5A) WT DC treated with control or shRNA for IL-23 stimulated with SC spores (1:10) for 1 hour and then co-cultured with splenic T cells harvested from naïve WT mice for 18 hours. DC expression of IL-23p19 (figure 5B), IL-12p40 (Figure 5C), and T cell expression of IL-17 (Figure 5D) was measured by real time PCR. n=4–5 in each group, each experiment repeated twice and results represent mean ± SEM. *p<0.05 as compared to controls shRNA treated cells.

Administration of IL-23 to TLR9−/− mice recapitulates HP phenotype

Having observed diminished IL-23 production by DC and in whole lung of TLR9−/− mice post SC sensitization and challenge, we next evaluated if reconstitution of IL-23 would restore a granulomatous hypersensitivity response in TLR9−/− mice. WT and TLR9−/− mice were sensitized and challenged as previously described, with some animals receiving rmIL-23 (2μg) i.t. at the time of i.t. SC challenge (day 0). Lungs were harvested 3 days post challenge. As shown in Figure 6, WT mice displayed evidence of granulomatous inflammation, as manifest by accumulation of CD4+ T cells (Figure 6B), which was considerably diminished in TLR9−/− mice. Interestingly, administration of mrIL-23 to TLR9−/− mice resulted in histopathology (Figure 6A) and CD4+ accumulation (Figure 6B) indistinguishable from SC-challenged WT mice. Moreover, treatment of mutant mice with IL-23 significantly increased the percentage of CD4+ T cells expressing CD69 to levels observed in WT animals (Figure 6C, 6D). This suggests that the defect in granulomatous responses to SC observed in TLR9−/− mice is largely attributable to defects in the production of IL-23.

Figure 6. Histopathology and Flow-cytometry post reconstitution of IL-23 in WT and TLR9−/− mice post SC sensitization and challenge.

Figure 6

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TLR9−/− mice were sensitized and challenged with SC and with or without reconstitution of murine recombinant IL-23 at the time of challenge. Lungs harvested at Day 3 post challenge and representative histology is shown in Figure 6A. WT and TLR9−/− mice were sensitized and challenged with SC as well as treated with either saline or rmIL-23 the day of i.t. challenge with SC. Flow cytometric analysis was performed and number of CD4+ T cells (Figure 6B) and CD4+, CD69+ T cells (Figure 6C,6D) is shown. n=4 in each group, *p<0.05 as compared to WT mice, #p<0.05 as compared to TLR9−/− mice post SC challenge without IL-23 reconstitution.

DISCUSSION

In this study, we show for the first time the critical role of TLR9 in the generation of the Th17 response in a mouse model of SC-induced HP. Several PRR have previously been implicated in the immunopathogenesis of HP, including TLR2 and TLR6, as well as the downstream adaptor protein MyD88 in a SR mouse model of farmer’s lung disease (37, 38). We and others have identified the importance of TLR9 in the generation of a type 1 granulomatous response to antigen sensitization and challenge. Moreover, this TLR is required for granuloma formation and IFN-γ production in certain Gram-positive infection (Propioniacterium acnes) (39) and in response to mycobacterial antigen (40). Interestingly, many diseases thought to be entirely Th1 dependent have more recently been shown to require activation of the Th17 pathway. In this study, we found a reduced pulmonary granulomatous inflammatory response to sensitization and challenge with SC associated with decreased IL-17 production in TLR9−/− mice as compared to their WT counterpart. Reduced IL-17 production in mutant mice was attributable, at least in part, to defective IL-23 production from DC. Taken together these findings implicate TLR9 in mediating IL-17 driven hypersensitivity pneumonitis in response to SC.

Several recent reports have identified the contribution of Th17 pathway to the generation of HP. In a murine model of farmers lung disease, IL-17 is produced upon repeated challenges with SR, which is associated with higher percentage of Th17 cells inthe lungs of mice exposed to SR-Ag compared with saline-challengedmice. Genetic deletion of IL-17 or antibody-mediated immunodepletionresulted in protection against the disease, as manifest by decreased cell infiltration and lower production ofchemokines and cytokines in the lungs of SR-Ag challenged mice. Similarly, IL-17 neutralization studies indicated that this cytokine was necessary for SC-induced lung pathology. A recent study by Fong and associates has implicated TLR6 as a pivotal TLR in the production of IL-17 and the development of HP (37). However, IL-17 production in response to SR-Ag can occur in a fashion independent of TLR6, suggesting that other TLRs might contribute to the development of IL-17-mediated HP. Interestingly, mice deficient in TLR9 had a significantly blunted IL-17 response to sensitization and challenge with SC, in association with reduced accumulation of IL-17 expressing CD4+ and γδ T cells. Impaired production of IL-17 in TLR9−/− mice was not limited to lung T cells, as splenocytes isolated from TLR9-deficient mice post Ag sensitization displayed reduced IL-17 production ex-vivo (data not shown). Collectively, this and other studies implicate IL-17 as a major driver of antigen-specific granulomatous inflammation. Consistent with this notion, high levels of IL-17 have recently been described in the bronchoalveolar lavage fluid recovered from patients with HP (41). IL-17 has been previously implicated in other inflammatory processes such as EAE, collagen induced arthritis, and autoimmune diseases, in part by up-regulating the expression of proinflammatory cytokines and chemokines such as IL-6, IL-8, TNF-α, CXCL9, and CXCL10.(30, 42, 43) This cytokine has also been shown to be involved in activation and expansion of neutrophils. Furthermore, IL-17 induces the expression of acute phase response genes and antimicrobial genes which can all contribute to deleterious inflammation at the site if injury or antigen exposure. (44, 45)

Dendritic cells are the major antigen presenting cell in the lung that recognize and present antigen to T cells, leading to initiation of antigen-specific immune responses. TLR9 is a key pathogen recognition receptor mediating DC recruitment and effector responses during microbial challenge. For example, we have shown previously that TLR9 is required for recruitment and activation of DC in murine models of Gram-negative bacterial pneumonia and SC-induced HP (46–48). Similarly, Daito and colleagues have recently identified the importance of TLR9-mediated DC responses in Mycobacterium avium-induced granulomatous inflammation (49). In the aforementioned study, TLR9−/− mice failed to mount a Th1-skewed cytokine response and lung peribronchial, perivascular and parenchymal inflammation after exposure to a killed M. avium strain. Moreover, lung CD11c+ cells from wild-type mice transferred intravenously into TLR9−/− recipients reconstituted M. avium-induced HP-like reactions in mutant mice, indicating an important role for TLR9-mediated DC responses though a yet to be defined mechanism. To our knowledge, our study is the first demonstration of the role of TLR9 in driving antigen-specific production of IL-23 by DC, which promotes Th17-mediated granulomatous inflammation.

IL-23 is an important cytokine produced by DC that leads to skewing towards a Th17 response by T cells. IL-23 is a heterodimic protein consisting of a 40-kD protein (p40) and the 19 kD protein p19. (50) The IL-23 p19 component is produced in large amounts by activated DC, macrophages, DC, T cells, and endothelial cells, whereas only activated DC and macrophages concomitantly expressing both p19 and the p40 subunit (51). Activation of Th17 pathway and production of IL-17 from T cells is primarily mediated by IL-23.(52) In this study, we found impaired IL-23 production by TLR9−/− DC in response to SC antigen, as well as blunted expression of IL-23 in the lungs of TLR9−/− mice post SC sensitization and challenge. Importantly, intrapulmonary reconstitution of IL-23 in TLR9−/− mice recapitulated the in-vivo phenotype observed in the lungs of SC-challenged WT mice. These findings are consistent with the hypothesis that impairment in IL-23 production by DC is the primary defect contributing to reductions in T cell derived IL-17 expression in TLR9 deficient mice.

In summary, this and our previous studies implicate both Th1 and Th17 pathways in generation of HP in response to SC. Moreover, TLR9-mediated DC responses are critical for antigen specific T cell responses in this model. TLR9 signaling may serve as a therapeutic target in the treatment of patients with this and other forms of granulomatous lung disease.

Acknowledgments

This grant is supported by NIH/NHLBI grants K08HL094762, HL25243, and HL097564.

Special thanks to Pam Lincoln for IL-17 production and purification.

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