Figure 5. C3aR is critical in cigarette smoke induced emphysema.

WT and C3ar^−/− mice were exposed to cigarette smoke or air for 6 month. (a) Micro-CT quantification of lung volume in indicated groups of mice, and (b) Mean Linear Intercept (MLI) was measured using unbiased morphometry in the same groups of mice (n=10). **P<0.01 as determined by the student t test and one-way ANOVA with Bonferroni's multiple comparison. (c) Bronchoalveolar Lavage (BAL) fluid analyses from the same group of mice (n=4 or 5 per group) showing macrophages (Mac), lymphocytes (Lymph) and neutrophils (Neu). *P<0.05 as determined by the student t test. (d) Expression of Mmp9 and (e) Mmp12 mRNA in BAL cells was measured by quantitative reverse transcription PCR (qPCR). **P<0.01, as determined by one-way ANOVA with Bonferroni's multiple comparison. (f) Representatives and (g) cumulative of flow cytometry analysis of B220⁻ CD11b⁺CD11c⁺ mDCs (enclosed population) in the lung of the same groups of mice (n=4 or 5 mice in each group). **P<0.01, as determined by one-way ANOVA with Bonferroni's multiple comparison. Cumulative intracellular cytokine staining of IL-17A in αβ (h), and γδ CD3⁺/CD4⁺ T cells (i) (n=4 or 5 in each group). Numbers in each quadrant indicate % positive cells for the indicated cytokines. *P<0.05, as determined by one-way ANOVA with Bonferroni's multiple comparison. (j) Expression of Il6 and (k) Il1beta mRNA in BAL cells were measured by quantitative reverse transcription PCR (qPCR). ***p<0.001 as determined by one-way ANOVA test with Bonferroni's multiple comparison. (l) Expression of Cd86 mRNA in BAL cells was measured by quantitative reverse transcription PCR (qPCR). **P<0.01, as determined by one-way ANOVA with Bonferroni's multiple comparison. (m) CD11c⁺ mDCs (2×10⁴) isolated from whole lung homogenates isolated from WT, C3ar^−/− and C3^−/− mice exposed to 6 month of air or smoke, were cultured in complete media for 3 days, and the concentration of IL6 in the supernatant was measured by multiplex assay. ***p<0.001 as determined by one-way ANOVA test with Bonferroni's multiple comparison. (n) CD4⁺ splenic T cells isolated from naïve WT mice were co-cultured with CD11c⁺ mDCs shown in (m) in 10:1 ratio (CD4⁺ T cells and CD11c⁺ mDCs) for 3 days, and the concentration of IL17 in the supernatant was measured by multiplex assay. CD4⁺ T cells (2×10⁵) without CD11c⁺ mDCs was cultured for 3 days as control. ***p<0.001 as determined by one-way ANOVA test with Bonferroni's multiple comparison. All mRNA gene expression was normalized to 18S ribosomal RNA expression and analyzed by ΔΔCt method. Results are expressed as mean±s.e.m, from 3 independent experiments with 4-5 mice in each group.