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. 2011 Jun 23;8(1):15.
doi: 10.1186/1476-9255-8-15.

T-bet controls severity of hypersensitivity pneumonitis

Hossam Aly Abdelsamed  1 , Meena Desai, Stephanie C Nance, Elizabeth A Fitzpatrick
Affiliations

T-bet controls severity of hypersensitivity pneumonitis

Hossam Aly Abdelsamed et al. J Inflamm (Lond). .

Abstract

Hypersensitivity Pneumonitis (HP) is an interstitial lung disease that develops following repeated exposure to inhaled environmental antigens. The disease is characterized by alveolitis, granuloma formation and in some patients' fibrosis. IFNγ plays a critical role in HP; in the absence of IFNγ granuloma formation does not occur. However, recent studies using animal models of HP have suggested that HP is a Th17 disease calling into question the role of IFNγ. In this study, we report that initially IFNγ production is dependent on IL-18 and the transcription factor T-bet, however as the disease continues IFNγ production is IL-18-independent and partially T-bet dependent. Although IFNγ production is required for granuloma formation its role is distinct from that of T-bet. Mice that are deficient in T-bet and exposed to S. rectivirgula develop more severe disease characterized by an exacerbated Th17 cell response, decreased Th1 cell response, and increased collagen production in the lung. T-bet-mediated protection does not appear to be due to the development of a protective Th1 response; shifting the balance from a Th17 predominant response to a Th1 response by inhibition of IL-6 also results in lung pathology. The results from this study suggest that both Th1 and Th17 cells can be pathogenic in this model and that IFNγ and T-bet play divergent roles in the disease process.

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Figures

Figure 1
Figure 1
Role of IL-18 and T-bet in IFNγ production in vitro. A.) Neutrophils (CD45+/CD11b+/Ly6G+/NK1.1-) were sorted from the lungs of WT mice exposed to S. rectivirgula one time. The cells were stimulated overnight with IL-12 (10 ng/ml), IL-15 (20 ng/ml) or IL-18 (20 ng/ml) alone or in combination. Culture supernatants were collected and IFNγ measured by ELISA. B.) Spleen cells were isolated from WT or T-bet-/- mice and stimulated overnight with S. rectivirgula (10 μg); IFNγ was measured in cell culture supernatants by ELISA.
Figure 2
Figure 2
IFNγ and IL-17 mRNA are decreased in T-bet-/- and IL-18R-/- mice following S. rectivirgula exposure. C57BL/6, IL-18-/-, or T-bet-/- mice (5/group) were intranasally exposed to S. rectivirgula (150 μg) or saline for 3 days. A.) RNA was isolated from lungs of individual mice, reverse transcribed, and real-time PCR performed using primers specific for MIP-2, IFNγ and IL-17. The results were normalized to the housekeeping gene HPRT and expressed as fold induction over unexposed mice. B.) IFNγ was measured in the BALF of individual mice by ELISA.
Figure 3
Figure 3
Increase in Th17 cells in T-bet KO mice following S. rectivirgula exposure. C57BL/6 or T-bet-/- mice (5/group) were intranasally exposed to S. rectivirgula (150 μg) or saline for 3 weeks. Lungs were removed and cells were isolated from individual mice as described in materials and methods. A) Lung cells were stimulated overnight with S. rectivirgula or media alone prior to intracellular cytokine staining. Lung cells from WT/saline exposed mice were pooled due to the low number of CD4+ T cells in the lungs of these mice. Cells were surface stained with antibodies to CD45, βTcR chain and CD4 followed by permeabilization and incubation with anti-IL-17 and IFNγ antibodies and run on a BD LSRII flow cytometer. The data was analyzed using DIVA software and the % of cells expressing IL-17 or IFNγ was obtained by gating on CD45+/βTcR+/CD4+ T cells. B) RNA was isolated from lungs of individual mice, reverse transcribed, and real-time PCR performed using primers specific for IFNγ and IL-17 the results were normalized to the housekeeping gene HPRT and expressed as fold induction over unexposed mice. * p < 0.05; T-bet-/- mice exposed to S. rectivirgula compared to WT mice exposed to S. rectivirgula.
Figure 4
Figure 4
Development of granulomas in lungs of WT and KO mice. Representative H & E stained lung sections from WT mice (A and C), IL-6KO mice (B), and T-Bet KO mice (D), exposed to S. rectivirgula for 3 weeks. IL-6 KO mice exposed to S. rectivirgula (B) form granulomas similar to WT mice exposed to S. rectivirgula (A). T-bet KO mice exposed to S. rectivirgula (D) demonstrated more severe granuloma formation compared to WT mice exposed to S. rectivirgula (C). (Original magnification × 63).
Figure 5
Figure 5
Increased collagen in lungs of T-bet mice exposed to S. rectivirgula . C57BL/6 or T-bet-/- mice (5/group) were intranasally exposed to S. rectivirgula (150 μg) or saline for 3 weeks. The right lung lobe was removed from mice and the collagen content was determined as described in methods. The results are expressed as amount of collagen/right lung lobe and represent the average+/- SD in each group of mice. *p < 0.05; T-bet KO mice exposed to S. rectivirgula compared to WT mice exposed to S. rectivirgula.
Figure 6
Figure 6
IL-6 is required for development of Th17 cells during HP. C57BL/6 or IL-6-/- mice (5/group) were intranasally exposed to S. rectivirgula (150 μg) or saline for 3 weeks. Lungs were removed and cells were isolated from individual mice as described in materials and methods. A). Lung cells were stimulated with media alone or PMA and ionomycin for 4 hrs prior to intracellular cytokine staining. Cells were surface stained with antibodies to CD45, βTcR chain and CD4 followed by permeabilization and incubation with anti-IL-17 and IFNγ antibodies and run on a BD LSRII flow cytometer. The data was analyzed using DIVA software and the % of cells expressing IL-17 or IFNγ was obtained by gating on CD45+/βTcR+/CD4+ T cells. B) RT-PCR was performed on RNA isolated from one lung lobe from individual WT or IL-6-/- mice (n = 4/group) exposed to saline or S. rectivirgula using primers specific for IL-17A and IL-17F. The housekeeping gene β-actin was used as an internal control.
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