Fig 2: CD4+ T cell proliferation and IL-17A levels are increased by RSV and C5a but reduced by C5aRA via human mesangial cell antigen presentation. (A) Effect of RSV infection on HMCs antigen presentation function. CD80 and CD86 expression in RSV-infected HMCs assessed by real-time PCR. (B) Effect of RSV infection and C5a stimulation on CD4+ T cell proliferation. (C) C5aRA and costimulatory antibodies decrease Ki67+CD4+ T cell proportions in the coculture system of HMCs and CD4+ T cells in response to RSV infection. Ki67+CD4+ T cell percentages were detected by flow cytometry (B,C). *P < 0.05, **P < 0.01, ***P < 0.001 vs. RSV+HMCs+CD4+T group. ?P < 0.05, ??P < 0.01 vs. RSV+HMCs+CD4+T+anti-CD80+anti-CD86 group. (D) Effect of RSV and C5a stimulation on IL-17A expression. (E) IL-17A levels are downregulated by C5aRA and co-stimulatory inhibitor treatment in the coculture system of HMCs and CD4+ T cells in response to RSV infection. Total RNA extracted from CD4+T cells were collected and then IL-17A levels were assessed by real-time PCR (D,E). **P < 0.01, ***P < 0.001 vs. RSV+HMCs+CD4+T group. ?P < 0.05, ??P < 0.01 vs. RSV+HMCs+CD4+T+anti-CD80+anti-CD86 group. Data are expressed as mean ± sem and each experiment was performed in triplicate repeated in cells, n = 3, t-test.

Fig 3: The effect of C5a on RPE cell pro-fibrotic mediator production. Primary mouse RPE cells were treated with C5a (50 ng/mL) for different times. The supernatants were collected for ELISA analysis of VEGF-a, IL-6, TGF-ß1, and TGF-ß2. A VEGF-A production in control and C5a-treated RPE cells at different times. (B) IL-6 production in control and C5a-treated RPE cells at different times. C, D The production of TGF-ß1 (C) and TGF- ß2 (D) in control and C5a-treated RPE cells at different times. Mean ± SEM, N = 4–6, *p < 0.05, Student t-test

Fig 4: Epithelial/Mesenchymal marker expression in human eyes. A The expression of CDH1, FN1, ACTA2, C5AR1 and C3AR1 in different types of choroidal cells from macular and peripheral area identified by the Spectacle platform [28] using the dataset of dataset of Voigt et al. [29]. B–E Immunohistochemistry of human paraffin sections from the non-lesion area (B) and macular fibrosis lesion area (C–E) of a nAMD donor eye stained for C5aR (green) and a-SMA (red) and DAPI. Thin arrows in B indicate C5aR expression in RPE cells. Small arrows in B and D indicate choroidal C5aR+aSMA- cells. Asterisks in C indicating clumps of pigmented cells that were C5aR+a-SMA+. D High-magnification view of the rectangle area in C. E C5aR and a-SMA expression inside the fibrotic lesion. Big arrows in D and E indicate C5aR+ a-SMA+ cells. Scale bar (D, E) = 50 µm. BrM: Bruch’s membrane; Fibr: fibrosis; Ch: choroid; RPE: RPE; Re: retina; Sc: sclera

Fig 5: Expression of C5a and C5aR in primary mouse RPE cells. Mouse RPE cells were treated with 10 ng/mL TGF-ß2 for different times. A C5aR representative immunofluorescence image of RPE cells under normal conditions and after the exposure to TGF-ß2 for 48 h. Scale bar = 50 µm. Arrows indicate C5aR expression. Arrowhead, spindle-shaped RPE cells. B Western blot analysis of C5aR expression in RPE cells treated with TGF-ß2 for different times. Data were representative of 2–3 repeated studies and expressed as fold change of control of each time point. C, D ELISA quantification of protein concentration of C5 (C) or C5a (D) in the supernatants of control and TGF-ß2-treated RPE cells. Data were normalized to supernatant total protein. Mean ± SEM. n = 3–6. *p < 0.05; ****p < 0.001, Student t-test