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. 2011 Dec;179(6):2779-87.
doi: 10.1016/j.ajpath.2011.08.019. Epub 2011 Oct 5.

Stachybotrys chartarum-induced hypersensitivity pneumonitis is TLR9 dependent

Urvashi Bhan  1 , Michael J Newstead, Xianying Zeng, Megan N Ballinger, Louis R Standiford, Theodore J Standiford
Affiliations

Stachybotrys chartarum-induced hypersensitivity pneumonitis is TLR9 dependent

Urvashi Bhan et al. Am J Pathol. 2011 Dec.

Abstract

Hypersensitivity pneumonitis (HP), an inflammatory lung disease, develops after repeated exposure to inhaled particulate antigen and is characterized by a vigorous T helper type 1-mediated immune response, resulting in the release of IL-12 and interferon (IFN)-γ. These T helper type 1 cytokines may participate in the pathogenesis of HP. Stachybotrys chartarum (SC) is a dimorphic fungus implicated in a number of respiratory illnesses, including HP. Here, we have developed a murine model of SC-induced HP that reproduces pathology observed in human HP and hypothesized that toll receptor-like 9 (TLR9)-mediated dendritic cell responses are required for the generation of granulomatous inflammation induced by inhaled SC. Mice sensitized and challenged with 10(6) SC spores develop granulomatous inflammation with multinucleate giant cells, accompanied by increased accumulation of neutrophils and CD4(+) and CD8(+) T cells. SC sensitization and challenge resulted in robust pulmonary expression of tumor necrosis factor-α, IL-12, and IFN-γ. SC-mediated granulomatous inflammation required IFN-γ and was TLR9 dependent, because TLR9(-/-) mice displayed reduced peribronchial inflammation, decreased accumulation and/or activation of polymorphonuclear (PMN) and CD4(+) and CD8(+) T cells, and reduced lung expression of type 1 cytokines and chemokines. T-cell production of IFN-γ was IL-12 dependent. Our studies suggest that TLR9 is critical for dendritic cell-mediated development of a type 1 granulomatous inflammation in the lung in response to SC.

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Figures

Figure 1
Figure 1
Lung histopathology after SC challenge. H&E staining of lung sections obtained from mice after saline (A) or nonsensitized (B) or sensitized mice challenged with SC at day 2 (C) or day 5 (D). Accumulation of neutrophils and mononuclear cells is shown within the interstitial compartments, and a predominantly lymphocytic infiltration within the peribronchial regions as indicated by thin arrows, as well as multinucleated giant cells (thicker arrow) and loose epitheloid granulomas within both the interstitial and peribronchial areas in SC-challenged mice. Four to five mice were used per group, and the experiments were repeated twice. Original magnification, ×40. N = 4 to 5; mean (SEM of two experiments).
Figure 2
Figure 2
A: Accumulation of inflammatory cells after SC challenge. WT mice were sensitized and challenged with SC as described, lungs were harvested at day 2 and day 5 after challenge, lung collagenase digests were performed, total number of leukocytes were counted with a hemocytometer, cytospins were performed, and total PMN cell were quantitated. B: Flow cytometry was performed, and total CD4+ and CD8+ T cells were quantitated. N = 4 to 5; mean ± SEM of two experiments. *P < 0.05 compared with untreated controls.
Figure 3
Figure 3
A: Type 1 cytokine expression in lung and lung histopathology in IFNγ−/− mice after SC sensitization and challenge. WT mice were sensitized and challenged with SC as described, and lungs were harvested at day 2 and day 5. Relative expression of IFNγ, TNF-α, and IL-12 p40 was measured by real-time PCR. Error bars indicate mean (SEM of two experiments). B: H&E staining was performed on lung sections harvested from WT and IFN-γ−/− mice at day 2 after SC challenge. N = 5 in each experiment, repeated twice.
Figure 4
Figure 4
Lung histopathology and inflammatory cell accumulation in WT and TLR9−/− mice after SC challenge. WT and TLR9−/− mice were sensitized and challenged with SC, and lungs were harvested at day 2. H&E staining was performed on lung sections as shown in A and B. C: Lung collagenase digests were performed, and total leukocytes were quantitated in different groups with a hemocytometer. D: Total PMN cells were quantified on cytospins. N = 4 to 5 in each group; experiments were performed twice. *P < 0.05 compared with untreated controls; **P < 0.05 compared with SC challenged WT mice.
Figure 5
Figure 5
Accumulation and activation of T cells and DCs in WT and TLR9−/− mice after SC challenge. WT and TLR9−/− mice were sensitized and challenged with SC, and lungs were harvested at day 2. Lung collagenase digests were performed, and total number of CD4+ T cells (A), activated CD4+ T cells (B), total number of CD8+ T cells (C), activated CD8+ T cells (D), total number of DCs [both myeoloid dendritic cell (mDC) (E) and plasmacytoid dendritic cell (pDC) (F)], as well as activated mDCs indicated by expression of costimulatory marker CD80 (G) and CD40 (H) were measured by flow cytometry. N = 6 to 8 in each group; experiments were performed twice. Results are expressed as mean ± SEM. *P < 0.05 compared with untreated controls; **P < 0.05 compared with WT mice after SC.
Figure 6
Figure 6
Cytokine and chemokine levels in lungs of WT and TLR9−/− after SC sensitization and challenge. WT and TLR9−/− mice were sensitized and challenged with either saline or SC, and lungs were harvested at day 2 and day 5. Cytokine levels of IFN-γ, TNF-α, IL-12 (A) and chemokine levels of keratinocyte-derived chemokine (KC), MIP-2, and IFN-inducible protein 10 (IP-10) (B) were measured by ELISA. N = 6 to 8 in each group; experiments were performed twice. Results are expressed as mean ± SEM. *P < 0.05 compared with untreated controls; **P < 0.05 compared with WT mice after SC sensitization and challenge.
Figure 7
Figure 7
Production of IL-12 by sensitized bone marrow DCs and lung DCs harvested from WT and TLR9−/− and antigen presentation by WT and TLR9−/− DCs. WT and TLR9−/− mice were sensitized by SC, and bone marrow cells were harvested at day 0, cells were cultured with GM-CSF, and DCs were purified by CD11c+ magnetic beads at day 6 and stimulated with SC spores (1:10). IL-12 protein levels were measured by ELISA in supernatant fluids collected at 18 hours after stimulation (B) and IL-12 p40 mRNA expression was measured by real-time PCR (A). In a separate experiment, sensitized bone marrow DCs from WT and TLR9−/− mice were cocultured with naive WT splenic T cells for 18 hours, and their antigen-presenting function was measured by T-cell IFN-γ production in the presence or absence of IL-12 antibody by ELISA (C). Lung DCs were harvested from sensitized WT and TLR9−/− mice as described previously and stimulated with SC, and cell supernatant levels of IL-12 were measured by ELISA (D). N = 4 to 5 in each group; each experiment was repeated twice. Results represent mean ± SEM. *P < 0.05 compared with untreated controls; **P < 0.05 compared with WT mice after SC or *P <0.01 compared with IL-12 antibody (Ab) treatment.
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