Figure 1.
Effect of human bronchial fibroblasts on CD4+ T cells transcription factor expression.
(A) Quantitative PCR analysis of RORc mRNA expression of CD4+ T cells. Fibroblasts and CD4+ T cells were cocultured during 14 hours, and then T cells were removed by PBS washing. RNA was then extracted from CD4+T cells. Data are presented as mean ± SEM of 8 asthmatics and 8 healthy controls. (B,C) RORγt and STAT3 expression in CD4+ T cells following coculture. Results are expressed as densitometric intensity of RORγt product over the densitometric intensity of the β-actin product or densitometric intensity of pSTAT3 over that of the total STAT3. Data are presented as mean ± SEM of 4 experiments.
Figure 2.
Effect of the co-culture on Th17 cells phenotype (IL17+/CCR6+).
(A, B) Bronchial fibroblasts and CD4+ T cells were co-cultured during 4 days and then T cells were retrieved by extensive PBS washing. CD4+ T cells were submitted to a flow cytometry analysis using both IL-17 and CCR6 monoclonal antibody. Results are showed as percentage of positive cells for both IL-17 and CCR6 (IL-17+/CCR6+). Data are representative of 8 asthmatics and 8 healthy controls.
Figure 3.
Effect of bronchial fibroblasts on Th17 cell lineage associated cytokines.
Quantitative PCR analysis of IL-17 and IL-22 gene expression by CD4+ T cells following co-culture with fibroblasts (A and B). C and D represent IL-17 and IL-22 protein levels in co-culture supernatants Data are presented as mean ± SEM of 8 experiments.
Figure 4.
Effect of CD4+ T cells on IL-6 expression by fibroblasts following co-culture.
Quantitative PCR analysis of IL-6 gene expression by bronchial fibroblasts following co-culture with CD4+ T cells (A). B- represent IL-6 protein levels in co-culture supernatants Data are presented as mean ± SEM of 8 experiments.
Figure 5.
IL-23, TGF-β and IL-1β expression by fibroblasts following co-culture with CD4+ T cells.
(A, B) IL-23 and TGF-β production were measured in culture supernatants after 24h of coculture. Data are presented as mean ± SEM of 6 experiments. (C) Quantitative PCR analysis of IL-1β mRNA expression by bronchial fibroblasts co-cultured with CD4+ T cells. Data are presented as mean ± SEM of 6 experiments.
Figure 6.
IL-23 stimulation of bronchial fibroblasts.
Quantitative PCR analysis of IL-6 (A) and IL-1β (B) mRNA expression by bronchial fibroblasts following stimulation with 100 ng/mL of recombinant IL-23. Cells were stimulated for 6 hours and total RNAs were extracted and qRT-PCRs were performed. Data are presented as mean ± SEM of 6 experiments.
Figure 7.
IL-1β stimulates bronchial fibroblasts expression of IL-23p19 mRNA.
Quantitative PCR analysis of IL-23p19 mRNA expression in bronchial fibroblasts stimulated with different doses of IL-1β. Data are presented as mean ± SEM of 8 experiments. * p< 0.001.
Figure 8.
Illustration of how bronchial fibroblasts could participate in Th17 cells phenotype maintains.
(1) CD4+ T cells stimulate fibroblasts to produce Th17 differentiating cytokines, thus creating a cytokine milieu prone to induce Th17 lineage specific transcription factor RORc and associated cytokines including IL-17 and IL-22 (2). Preservation of Th17 phenotype may involve an autoregulation loop and IL-23 by stimulation of IL-6, TGF-β and IL-1β expression (1).