Negative Regulation of Pulmonary Th17 Responses by C3a Anaphylatoxin during Allergic Inflammation in Mice

<div><p>Activation of complement is one of the earliest immune responses to exogenous threats, resulting in various cleavage products including anaphylatoxin C3a. In addition to its contribution to host defense, C3a has been shown to mediate Th2 responses in animal models of asthma. However, the role of C3a on pulmonary Th17 responses during allergic inflammation remains unclear. Here, we show that mice deficient in C3a receptor (C3aR) exhibited (i) higher percentages of endogenous IL-17-producing CD4⁺ T cells in the lungs, (ii) higher amounts of IL-17 in the bronchoalveolar lavage fluid, and (iii) more neutrophils in the lungs than wild-type mice when challenged with intranasal allergens. Moreover, adoptive transfer experiments showed that the frequencies of antigen-specific IL-17-producing CD4⁺ T cells were significantly higher in the lungs and bronchial lymph nodes of C3aR-deficient recipients than those of wild-types recipients. Bone-marrow reconstitution study indicated that C3aR-deficiency on hematopoietic cells was required for the increased Th17 responses. Furthermore, C3aR-deficient mice exhibited increased percentages of Foxp3⁺ regulatory T cells; however, depletion of these cells minimally affected the induction of antigen-specific Th17 cell population in the lungs. Neutralization of IL-17 significantly reduced the number of neutrophils in bronchoalveolar lavage fluid of C3aR-deficient mice. Our findings demonstrate that C3a signals negatively regulate antigen-specific Th17 responses during allergic lung inflammation and the size of Foxp3⁺ regulatory T cell population in the periphery.</p> </div

Foxp3⁺ regulatory T cells in C3aR^−/− mice.

<p><i>A</i> and <i>B</i>, Lymphoid cells from the indicated organs were obtained from wild-type and C3aR^−/− mice (<i>A</i>), or the spleens from bone-marrow reconstituted mice (<i>B</i>) generated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052666#pone-0052666-g005" target="_blank"><i>Figure 5</i></a>. The percentage of Foxp3⁺ cells among CD4⁺ (spleen, lung, inguinal lymph node (iLN)) or CD4⁺CD8⁻ (thymus) cells was analyzed by flow cytometry. <i>C</i>, Groups of C3aR^−/− mice were intraperitoneally injected with control IgG or anti-CD25 (PC-61) (n = 3–4) on day −2, day 0, and day 2. The mice were intravenously injected with CD45.1⁺ OT-II T cells on day -1, followed by subsequent intranasal injection with Asp/OVA on day 0, 2, 4, and 6. On day 7, lymphoid cells from the lung or draining lymph nodes were obtained, and the expression of IL-17 by CD4⁺ T cells was analyzed by intracellular staining. Data shown represent three (<i>A</i>) or two (<i>B</i> & <i>C</i>) independent experiments. *, p<0.05 or **, p<0.01 in comparison with WT (<i>A</i>), or isotype control IgG treatment (<i>C</i>).</p

C3aR-deficiency on hematopoietic cells induces the increased pulmonary Th17 responses.

<p>Bone marrow cells from wild-type or C3aR^−/− mice were adoptively transferred into wild-type or C3aR^−/− mice (n = 3–4 per group; 5–10×10⁶ cells/transfer). Six to eight weeks later, the reconstituted mice were injected i.v. with CD45.1⁺OT-II T cells, and were further challenged with intranasal allergen every other day for a total of four times. One day after the final challenge, the expression of IL-17 and IFN-γ by CD45.1⁺ donor CD4⁺ T cells in the lungs and draining lymph nodes was analyzed by flow cytometry. Bars in <i>B</i> are mean values. Data shown represent two independent experiments. *, p<0.05 in comparison with WT bone marrow reconstituted mice.</p

Antigen-specific pulmonary Th17 responses in C3aR-deficient mice.

<p><i>A–C</i>, Groups of C3aR^−/− and wild-type mice (n = 7–9) were i.v. injected with CD45.1⁺ OT-II T cells (5×10⁶ cells/transfer; day -1), and were intranasally injected with the mixture of <i>Aspergillus</i> proteinase plus OVA on day 0, 2, 4, 6. On day 7, lymphoid cells from the lung (<i>B</i>) and draining LNs (<i>C</i>) were restimulated with PMA and ionomycin in the presence of brefeldin A and monensin, stained with anti-CD45.1 and CD4, and the expression of IL-17, IFN-γ, or IL-4 + IL-5 by CD45.1⁺CD4⁺ donor T cells was analyzed by intracellular staining. Left panels in <i>A</i> illustrate gating strategy. Bars in <i>B</i> and <i>C</i> show the mean values. Data shown represent at least three independent experiments. *, p<0.05 or ***, p<0.001 in comparison with wild-type recipients.</p

Expression of C3, C3a receptor and C3a in the lung upon allergenic challenge.

<p>C57BL/6 mice were intranasally challenged with the mixture of <i>Aspergillus</i> proteinase and OVA (<i>A-C</i>). The lungs and BAL fluid were collected from the treated mice (n = 3) at the indicated time points. C57BL/6 mice (n = 3) were intranasally injected with intact or boiled <i>Aspergillus</i> proteinase, and the lungs and BAL fluid were collected 24 hours after the treatment (<i>D</i>). The mRNA transcript levels of <i>C3</i> (<i>A</i>) and <i>C3ar1</i> (<i>B</i>) in the lungs were measured by quantitative RT-PCR. The concentration of C3a in the lung homogenate and BAL fluid was measured by ELISA (<i>C & D</i>). *, p<0.05 or **, p<0.01 in comparison with 0 hr time point.</p

Increased IL-17 and neutrophils in the BAL fluid of C3aR-deficient mice after allergenic challenges.

<p>Groups of C3aR^−/− and wild-type mice (n = 4) were intranasally administered with allergen every other days for four times. One day after the last challenge, the lungs and BAL fluid were obtained. The concentration of IL-17 (<i>A</i>) and the numbers of macrophages, lymphocytes, eosinophils and neutrophils were evaluated (<i>B</i>). The levels of mRNA transcript of the indicated genes were determined by quantitative RT-PCR and were normalized with expression levels of <i>Actb</i> (<i>C</i>). Histology of the lungs were examined by H&E and periodic acid-Schiff (PAS) staining (×20 magnification) and visualized by light microscope (<i>D</i>). Data shown are mean ± SE, and represent two independent experiments. *, p<0.05 in comparison with WT control.</p

Effect of IL-17 neutralization on the lung inflammation of C3aR^−/− mice.

<p>Groups of C3aR^−/− mice were (n = 3–4) intranasally challenge with Asp/OVA every other days four times (day 0, 2, 4, 6), and i.p. injected with isotype control antibody or with IL-17 neutralizing antibody on day 0, 2, 4. Twenty-four hours after the last challenge, mice were anesthetized, mechanically ventilated, and airway responses to increasing doses of intravenous acetylcholine were measured. AHR is expressed as the percentage of changes from baseline (<i>B</i>). The BAL fluid and lungs were collected, and the amounts of IL-17 (<i>A</i>) and number of the indicated cell population (<i>C</i>) in the BAL fluid were measured. Histology of the lungs was examined by H&E and PAS staining (×20 magnification) and visualized by light microscope (<i>D</i>). Data shown are mean ± SE. *, p<0.05 in comparison with control antibody treated C3aR^−/− mice.</p