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. 2013 Aug 30;8(8):e73143.
doi: 10.1371/journal.pone.0073143. eCollection 2013.

TLR2 regulates neutrophil recruitment and cytokine production with minor contributions from TLR9 during hypersensitivity pneumonitis

Kelly Andrews  1 , Hossam Abdelsamed, Ae-Kyung Yi, Mark A Miller, Elizabeth A Fitzpatrick
Affiliations

TLR2 regulates neutrophil recruitment and cytokine production with minor contributions from TLR9 during hypersensitivity pneumonitis

Kelly Andrews et al. PLoS One. .

Abstract

Hypersensitivity pneumonitis (HP) is an interstitial lung disease that develops following repeated exposure to environmental antigens. The disease results in alveolitis, granuloma formation and may progress to a fibrotic chronic form, which is associated with significant morbidity and mortality. The severity of the disease correlates with a neutrophil rich influx and an IL-17 response. We used the Saccharopolysporarectivirgula (SR) model of HP to determine whether Toll-like receptors (TLR) 2 and 9 cooperate in neutrophil recruitment and IL-17-associated cytokine production during the development of HP. Stimulation of bone marrow derived macrophages (BMDMs) from C57BL/6, MyD88(-/-) and TLR2/9(-/-) mice with SR demonstrate that SR is a strong inducer of neutrophil chemokines and growth factors. The cytokines induced by SR were MyD88-dependent and, of those, most were partially or completely dependent on TLRs 2 and 9. Following in vivo exposure to SR, CXCL2 production and neutrophil recruitment were reduced in TLR2(-/-) and TLR2/9(-/-) mice suggesting that the response was largely dependent on TLR2; however the reduction was greatest in the TLR2/9(-/-) double knockout mice indicating TLR9 may also contribute to the response. There was a reduction in the levels of pro-inflammatory cytokines TNFα and IL-6 as well as CCL3 and CCL4 in the BALF from TLR2/9(-/-) mice compared to WT and single knockout (SKO) mice exposed one time to SR. The decrease in neutrophil recruitment and TNFα production in the TLR2/9(-/-) mice was maintained throughout 3 weeks of SR exposures in comparison to WT and SKO mice. Both TLRs 2 and 9 contributed to the Th17 response; there was a decrease in Th17 cells and IL-17 mRNA in the TLR2/9(-/-) mice in comparison to the WT and SKO mice. Despite the effects on neutrophil recruitment and the IL-17 response, TLR2/9(-/-) mice developed granuloma formation similarly to WT and SKO mice suggesting that there are additional mediators and pattern recognition receptors involved in the disease.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Cytokine production by WT and mutant BMDMs following SR stimulation.
WT, TLR2/9-/- and MyD88-/- BMDMs were cultured with or without SR (0.05 mg/ml) for 24 h and cell culture supernatants analyzed for cytokine production by a 32-plex bead based ELISA. The results are expressed as the mean ± SD (n = 4 per group). *p < 0.05.
Figure 2
Figure 2. Contribution of TLRs 2 and 9 to inflammation following a single SR exposure.
WT, TLR2-/-, TLR9-/-, and TLR2/9-/- mice were exposed to SR and analyzed 6 h after one exposure. (A) BAL was performed and alveolitis determined using trypan blue dye exclusion. (B) Cells recovered from the BAL fluid were incubated with antibody to CD11b and Gr1 and analyzed by flow cytometry to determine the number of neutrophils (CD11b+/Gr1high) in each group. (C) Expression of mRNA for cytokines was determined by qRT-PCR on RNA isolated from individual lung lobes (n = 3-5 mice per group) and is expressed as fold induction over WT unexposed mice. The data represent mean ± SD of duplicate samples and significance was determined using one-way ANOVA with Tukey’ post-hoc test (*p < 0.05 compared to WT / SR exposed mice). (D) The cell-free BAL fluid was analyzed for CXCL2, TNFα and IL-6 by ELISA and CCL3 and CCL4 by Milliplex bead based ELISA. Data represent the mean ± SD (n = 5 per group) significance was determined using one-way ANOVA with Tukey’ post-hoc test (*p < 0.05 compared to WT SR exposed mice).
Figure 3
Figure 3. Neutrophil recruitment following repeated SR exposures is primarily dependent on TLR2.
WT, TLR2-/-, TLR9-/-, and TLR2/9-/- mice were exposed to SR 3 times / week for 3 weeks and analyzed 1 day after the last exposure. (A) BAL was performed and alveolitis determined using trypan blue dye exclusion. (B) Cells recovered from the BAL fluid were incubated with antibody to CD11b and Gr1 and analyzed by flow cytometry to determine the number of neutrophils (CD11b+/Gr1high) in each group. (C) Expression of mRNA for cytokines was determined by qRT-PCR on RNA isolated from individual lung lobes (n = 3-5 mice per group) and is expressed as fold induction over WT unexposed mice. The data represent mean ± SD of duplicate samples and significance was determined using one-way ANOVA with Tukey’ post-hoc test (*p < 0.05 compared to WT SR exposed mice).
Figure 4
Figure 4. TLRs 2 and 9 contribute to Th1- and Th17-associated cytokine production following repeated SR exposures.
WT, TLR2-/-, TLR9-/- and TLR2/9-/- mice were exposed to SR 3 times / week for 3 weeks and analyzed 1 day after the last exposure. Lungs were removed and qRT-PCR of RNA isolated from individual lung lobes was performed to measure the expression of individual cytokines using specific primers. The results were normalized to the housekeeping gene HPRT and expressed as fold induction over unexposed mice. Significance was determined using one-way ANOVA with Tukey’ post-hoc test (*p < 0.05 compared to WT SR exposed mice).
Figure 5
Figure 5. TLRs 2 and 9 cooperate in generation of the Th17 response following repeated SR exposures.
WT, TLR2-/-, TLR9-/-, and TLR2/9-/- mice were exposed to SR 3 times / week for 3 weeks and analyzed 4 days after the last exposure. Lung cells were stimulated with SR lysate or media alone for 6 hrs and intracellular cytokine staining performed to measure expression of IL-17A and IL-10 by CD4+/βTcR+ cells. Representative dot plots are shown (n = 3; repeated 2 times) Mean ± SD of CD4+/IL-17A+ WT unexposed = 0 ± 0%; CD4+/IL-17A+ WT exposed = 6.7 ± 2.2%; CD4+/IL-17A+ TLR2-/- exposed = 4.8 ± 0.8%; CD4+/IL-17A+ TLR9-/- exposed = 5.4 ± 1.0%; CD4+/IL-17A+ TLR2/9-/- exposed = *2.5 ± 1.4%. Significance was determined using one-way ANOVA (*p < 0.05 compared to WT SR exposed mice).
Figure 6
Figure 6. Granuloma development is not dependent on TLRs 2 and 9.
WT, TLR2-/-, TLR9-/-, and TLR2/9-/- mice were exposed to SR 3 times / week for 3 weeks and analyzed 3 days after the last exposure. Representative H&E stained lung sections from unexposed WT mice (A), WT mice (B), TLR2-/- mice (C), TLR9-/- mice (D) and TLR2/9-/- mice (E) exposed to SR (Original magnification x4).
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