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. 2016 Mar 1;310(5):L393-402.
doi: 10.1152/ajplung.00305.2015. Epub 2015 Dec 30.

Chronic hypersensitivity pneumonitis caused by Saccharopolyspora rectivirgula is not associated with a switch to a Th2 response

Kelly Andrews  1 , Manik C Ghosh  2 , Andreas Schwingshackl  3 , Gabriel Rapalo  2 , Charlean Luellen  2 , Christopher M Waters  2 , Elizabeth A Fitzpatrick  4
Affiliations

Chronic hypersensitivity pneumonitis caused by Saccharopolyspora rectivirgula is not associated with a switch to a Th2 response

Kelly Andrews et al. Am J Physiol Lung Cell Mol Physiol. .

Abstract

Hypersensitivity pneumonitis (HP) is an immune-mediated interstitial lung disease that develops following repeated exposure to inhaled environmental antigens. The disease results in alveolitis and granuloma formation and may progress to a chronic form associated with fibrosis; a greater understanding of the immunopathogenic mechanisms leading to chronic HP is needed. We used the Saccharopolyspora rectivirgula (SR) mouse model of HP to determine the extent to which a switch to a Th2-type immune response is associated with chronic HP. Exposure of wild-type (WT) and tlr2/9(-/-) mice to SR for 14 wk resulted in neutrophilic and lymphocytic alveolitis that was not dependent on Toll-like receptors (TLRs) 2 and 9. Long-term exposure of WT mice to SR resulted in a significant increase in collagen deposition, protein leakage, and IL-1α accompanied by a decrease in quasistatic compliance and total lung capacity compared with unexposed mice. This was associated with an increase in IL-17 but not IL-4 production or recruitment of Th2 cells. tlr2/9(-/-) mice exhibited an increase in protein leakage but less IL-1α and collagen deposition in the lungs compared with WT mice, yet they still displayed a decrease in quasistatic compliance, although total lung capacity was not affected. These mice exhibited an increase in both IL-13 and IL-17, which suggests that IL-13 may ameliorate some of the lung damage caused by long-term SR exposure. Our results suggest that lung pathology following long-term SR exposure in WT mice is associated with the IL-17 response and that TLRs 2 and 9 may inhibit the development of the IL-13/Th2 response.

Keywords: Toll-like receptors 2 and 9; fibrosis; hypersensitivity pneumonitis.

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Figures

Fig. 1.
Fig. 1.
Neutrophil chemokine production in wild-type (WT) and tlr2/9−/− mice following long-term exposure to Saccharopolyspora rectivirgula (SR). WT and tlr2/9−/− mice were exposed to SR 3 times per week for 14 wk and analyzed 1 day after the last exposure. The cell-free bronchoalveolar lavage (BAL) fluid (BALF) was analyzed for CXCL1 and CXCL2 by a bead-based ELISA. Values are given as means ± SD (n = 4 per group). Significance was determined by 1-way ANOVA with a Tukey's post hoc test (*P < 0.05 compared with WT SR-exposed mice).
Fig. 2.
Fig. 2.
Long-term SR exposure results in decreased barrier integrity and increased inflammation in WT and tlr2/9−/− mice. WT and tlr2/9−/− mice were exposed to SR 3 times per week for 4 or 14 wk and analyzed 2 days after the last exposure. A: BAL was performed on mice and the concentration of total protein in the BALF was determined by the Bio-Rad protein assay. Significance was determined by 1-way ANOVA with a Tukey's post hoc test. *P < 0.05. B: lung homogenates were prepared and IL-1α expression was measured by ELISA. C: representative hematoxylin and eosin-stained lung sections from unexposed WT mice or 14-wk SR-exposed WT and tlr2/9−/− mice (original magnification ×10).
Fig. 3.
Fig. 3.
Long-term SR exposure results in an increase in collagen deposition in lungs of WT and tlr2/9−/− mice. Lung tissue from mice exposed to SR for 14 wk was harvested for histological examination. Lung slides were stained with Masson's trichrome, then digitized for image analysis. Blue-stained collagen pixels were quantified using the positive pixel algorithm. Representative images and the corresponding positive pixel markup image were captured at ×2.2 (A, C, and E) or ×18 (B, D, and F). Values are given as means ± SD of n = 5/group. Significance was determined by 1-way ANOVA with a Tukey's post hoc test. *P < 0.05.
Fig. 4.
Fig. 4.
WT and tlr2/9−/− mice exposed long term to SR develop reduced compliance and total lung capacity. Mice were exposed to SR for 15 wk, and lung function measured by flexiVent 3 days postexposure. Quasistatic compliance and total lung capacity were determined by a pressure-volume curve. Each point represents the average value of 2 measurements for each mouse. Values are given as means ± SD of n = 4–6/group. Significance was determined by 1-way ANOVA with Tukey's post hoc test. *P < 0.05.
Fig. 5.
Fig. 5.
Long-term SR exposure correlates with an IL-17 response and not IL-4. WT and tlr2/9−/− mice were exposed to SR for 14 wk and analyzed 3 days after exposure. A: expression of mRNA for cytokines was determined by quantitative RT-PCR on RNA isolated from individual lung lobes (n = 5–7 mice per group) and expressed as fold induction over WT unexposed mice. Values are given as means ± SD of duplicate samples; significance was determined by 1-way ANOVA with a Tukey's post hoc test (*P < 0.05 compared with WT SR-exposed mice). B: PCR products for IL-4 were electrophoresed on an agarose gel and stained with ethidium bromide to visualize the bands. C: cell-free BALF was analyzed for IL-17A by a bead-based ELISA. Values are given as means ± SD (n = 5 per group); significance was determined by 1-way ANOVA with Tukey's post hoc test (*P < 0.05 compared with WT SR-exposed mice). D and E: lungs were harvested from mice exposed to SR for 14 wk and digested to obtain a single cell suspension. The cells were stimulated with media or PMA/ionomycin to induce cytokine production. D: representative gating strategy for intracellular cytokine staining: a lymphocyte gate was set based on forward and side scatter characteristics, followed by expression of CD45 to gate out nonhematopoietic lung cells. The CD45+ T cells were gated on the basis of expression of the β chain of the TcR and lack of expression of F4/80. Isotype controls were used to set the gates to determine the contribution of CD4+ and CD4− T cells to IL-4 or IL-17 production. E: the percentage of CD45+/CD4+/βTcR+ T cells expressing IL-17 (Th17) or IL-4 (Th2) was determined by intracellular flow cytometry. Values are given as means ± SD of n = 5/group. Significance was determined by 2-way ANOVA with a Bonferroni post hoc test. *P < 0.05.
Fig. 6.
Fig. 6.
Expression of inflammasome components in the lung following long-term exposure to SR. WT and tlr2/9−/− mice were exposed to SR 3 times per week for 14 wk and analyzed 2 days after the last exposure. A: lung homogenates were prepared and IL-1β expression was measured by ELISA. B: expression of mRNA for NLRP3 and caspase-1 was determined by qRT-PCR on RNA isolated from individual lung lobes (n = 5 mice per group) and is expressed as fold induction over WT unexposed mice. C: bone marrow-derived macrophages were prepared from WT and tlr2/9−/− mice and stimulated with LPS or SR for 3 h followed by ATP for 45 min. Culture supernatants were collected and IL-1β was measured by ELISA. Significance was determined by 2-way ANOVA with a Tukey's post hoc test. *P < 0.05.
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