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. 2010 May;130(1):125-36.
doi: 10.1111/j.1365-2567.2009.03219.x. Epub 2010 Jan 11.

Toll-like receptor 6 drives interleukin-17A expression during experimental hypersensitivity pneumonitis

Daniel J Fong  1 , Cory M Hogaboam, Yosuke Matsuno, Shizuo Akira, Satoshi Uematsu, Amrita D Joshi
Affiliations

Toll-like receptor 6 drives interleukin-17A expression during experimental hypersensitivity pneumonitis

Daniel J Fong et al. Immunology. 2010 May.

Abstract

Hypersensitivity pneumonitis (HP) is a T-cell-driven disease that is histologically characterized by diffuse mononuclear cell infiltrates and loosely formed granulomas in the lungs. We have previously reported that interleukin-17A (IL-17A) contributes to the development of experimental HP, and that the pattern recognition receptor Toll-like receptor 6 (TLR6) might be a factor in the initiation of this response. Using a well-established murine model of Saccharopolyspora rectivirgula-induced HP, we investigated the role of TLR6 in the immunopathogenesis of this disease. In the absence of TLR6 signalling, mice that received multiple challenges with S. rectivirgula-antigen (SR-Ag) had significantly less lung inflammation compared with C57BL/6 mice (wild-type; WT) similarly challenged with SR-Ag. Flow cytometric analysis of whole lung samples from SR-Ag-challenged mice showed that TLR6(-/-) mice had a decreased CD4(+) : CD8(+) T-cell ratio compared with WT mice. Cytokine analysis at various days after the final SR-Ag challenge revealed that whole lungs from TLR6(-/-) mice contained significantly less IL-17A than lungs from WT mice with HP. The IL-17A-driving cytokines IL-21 and IL-23 were also expressed at lower levels in SR-Ag-challenged TLR6(-/-) mice, when compared with SR-Ag-challenged WT mice. Other pro-inflammatory cytokines, namely interferon-gamma and RANTES, were also found to be regulated by TLR6 signalling. Anti-TLR6 neutralizing antibody treatment of dispersed lung cells significantly impaired SR-Ag-induced IL-17A and IL-6 generation. Together, these results indicate that TLR6 plays a pivotal role in the development and severity of HP via its role in IL-17A production.

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Figures

Figure 1
Figure 1
Pathogen recognition receptor expression in experimental hypersensitivity pneumonitis (HP). Whole lung samples were analysed by Taqman for gene expression. (a) Transcript expression of Toll-like receptor 6 (TLR6) in whole lungs of wild-type (WT) mice at 1, 4 and 8 days after their last exposure to Saccharopolyspora rectivirgula antigen (SR-Ag) or saline. Transcript expression for TLR6 (b), TLR2 (c), and Dectin-1 (d), in whole lungs from WT and TLR6−/− mice at 1, 4, 8 and 16 days after final SR-Ag challenge. Results are expressed as fold change over transcript expression in WT samples collected at day 1 after the last saline (a), or SR-Ag (b–d) challenge. Data represent the mean ± SEM, with n = 4 or n = 5 for each group. One-way analysis of variance and Newman–Keuls multiple comparison test were used to analyse significance between groups. *P ≤ 0·05, ***P ≤ 0·001.
Figure 2
Figure 2
Histological examination of whole lung samples from wild-type (WT) and Toll-like receptor-6-deficient (TLR6−/−) mice 1 day after the last Saccharopolyspora rectivirgula antigen (SR-Ag) challenge: magnification, 100 × (a–c), and 200 × (d–f). SR-Ag-challenged lungs from TLR6−/− mice (c,f) and saline-challenged lungs from WT mice (a,d), show reduced inflammation when compared with SR-Ag-challenged WT lungs (b,e). Histological scoring of whole lung sections revealed a higher number of fields devoid of inflammatory infiltrate (g) and a lower percentage of abnormal fields (h) in SR-Ag-challenged TLR6−/− mice when compared with SR-Ag-challenged WT mice. Data shown are mean ± SEM, with n = 4 or n = 5 for each group. One-way analysis of variance and Newman–Keuls multiple comparison test were used to analyse histological score significance between groups, and Student’s t-test was used to determine significance between WT and TLR6−/− lungs. *P ≤ 0·05, ***P ≤ 0·001.
Figure 3
Figure 3
Role of Toll-like receptor 6 (TLR6) in whole lung cellularity during experimental hypersensitivity pneumonitis (HP). (a) Total cell count of dispersed whole lung cells from mice at 1 day after the last Saccharopolyspora rectivirgula antigen (SR-Ag) challenge showed decreased number of cells from TLR6−/− mice compared with SR-Ag-challenged wild-type (WT) mice. (b) Flow cytometric analysis of dispersed lung cells showed a decreased CD4+ : CD8+ ratio in the lungs of TLR6−/− mice at 4 days after the last SR-Ag antigen challenge, when compared with SR-Ag-challenged WT mice. Data shown are mean ± SEM, with n = 4 or n = 5 for each group. Student’s t-test was used to determine significance between groups *P ≤ 0·05, ***P ≤ 0·001.
Figure 4
Figure 4
Interleukin-17A (IL-17A) and IL-17A-associated cytokine transcripts were decreased in Toll-like receptor-6-deficient (TLR6−/−) mice with experimental hypersensitivity pneumonitis (HP). Cyokine transcript analysis of whole lung samples was performed by Taqman. Transcript expression of IL-17A (a), IL-23 (b), IL-21 (c) and IL-6 (d), in whole lungs of wild-type (WT) and TLR6−/− mice at 1, 4, 8 and 16 days after the last Saccharopolyspora rectivirgula antigen (SR-Ag) challenge was determined. Results are expressed as fold change over transcript expression in WT samples collected at day 1 after the last SR-Ag challenge. Data represent the mean ± SEM, with n = 4 or n = 5 for each group. One-way analysis of variance and Newman–Keuls multiple comparison test were used to analyse significance between groups. **P ≤ 0·01, ***P ≤ 0·001.
Figure 5
Figure 5
Protein levels in lungs of mice with experimental hypersensitivity pneumonitis (HP). Lungs were excised from Toll-like receptor-6-deficient (TLR6−/−)and wild-type (WT) mice at 1, 4, 8 and 16 days after the last Saccharopolyspora rectivirgula antigen challenge and analysed using a multiplex bead-based assay or enzyme-linked immunosorbent assay. Interleukin-17A (IL-17A) (a), IL-6 (b), interferon-γ (IFN-γ) (c) and IL-13 (d) protein levels were measured. Data represent the mean ± SEM, with n = 4 or n = 5 for each group. One-way analysis of variance and Newman–Keuls multiple comparison test were used to analyse significance between groups. *P ≤ 0·05.
Figure 6
Figure 6
Toll-like receptor 6 (TLR6) regulation of cytokine generation in Saccharopolyspora rectivirgula antigen (SR-Ag) -driven recall responses by dispersed lung cells. At days 1, 4, 8 and 16 days after the final SR-Ag challenge, dispersed lung cells were cultured for 24 hr with and without SR-Ag. Cell-free supernatants were collected for protein analysis and cells were processed for Taqman analysis. interelukin-17A (IL-17A) (a), IL-6 (b), interferon-γ (IFN-γ) (c), and regulated on activation normal T-cell expressed and secreted (RANTES) (d) transcript analyses of cell cultures were carried out by Taqman. Results are expressed as fold change over transcript expression in wild-type (WT) samples collected at day 1 after the last SR-Ag challenge. Fifty microlitres of cell-free supernatant was analysed using a multiplex bead-based assay or enzyme-linked immunosorbent assay, to determine protein levels of IL-17A (e), IL-6 (f), IFN-γ (g) and RANTES (h). Data represent the mean ± SEM, with n = 3 for each group. One-way analysis of variance and Newman–Keuls multiple comparison test were used to analyse significance between the WT and TLR6−/− groups. *P ≤ 0·05, **P ≤ 0·01, ***P ≤ 0·001.
Figure 7
Figure 7
Anti-Toll-like receptor 6 (TLR6) neutralizing antibody reduced interleukin-17A (IL-17A) and IL-17A-associated transcript levels. At day 1 after the final Saccharopolyspora rectivirgula antigen (SR-Ag) challenge, dispersed lung cells were cultured with and without SR-Ag restimulation. The role of TLR6 and dectin-1 were analysed using anti-TLR6 or anti-dectin-1 neutralizing antibody. Immunoglobulin G was used as a control for these experiments. Results are expressed as fold change over transcript expression in samples cultured in medium alone. Cytokine transcript analysis of IL-17A (a), IL-21 (b), IL-23 (c), and IL-6 (d) were determined by Taqman. Data represent the mean ± SEM, with n = 3 for each group.
Figure 8
Figure 8
Anti-Toll-like receptor 6 (TLR6) neutralizing antibody reduced interleukin-17A (IL-17A) and IL-17A-associated cytokine protein levels. At day 1 after the final Saccharopolyspora rectivirgula antigen (SR-Ag) challenge, dispersed lung cells were cultured with and without SR-Ag restimulation. The roles of TLR6 and dectin-1 were analysed by using anti-TLR6 or anti-dectin-1 neutralizing antibody. Immunoglobulin G was used as a control for these experiments. IL-17A (a), IL-6 (b), and interferion-γ (IFN-γ) (c) protein levels were measured using a multiplex bead-based assay or enzyme-linked immunosorbent assay. Data represent the mean ± SEM, with n = 3 for each group. One-way analysis of variance and Newman–Keuls multiple comparison test were used to analyse significance between groups. *P ≤ 0·05.
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