CD11c⁺ CD8⁺ T Cells Reduce Renal Fibrosis Following Ureteric Obstruction by Inducing Fibroblast Apoptosis

Abstract

Tubulointerstitial fibrosis is a common consequence of various kidney diseases that lead to end-stage renal failure, and lymphocyte infiltration plays an important role in renal fibrosis. We previously found that depletion of cluster of differentiation 8⁺ (CD8⁺) T cells increases renal fibrosis following ureteric obstruction, and interferon-γ (IFN-γ)-expressing CD8⁺ T cells contribute to this process. CD8⁺ T cells are cytotoxic T cells; however, whether their cytotoxic effect reduces fibrosis remains unknown. This study showed that CD8⁺ T cells isolated from obstructed kidney showed mRNA expression of the cytotoxicity-related genes perforin 1, granzyme A, granzyme B, and FAS ligand; additionally, CD8 knockout significantly reduced the expression levels of these genes in obstructed kidney. Infiltrated CD8⁺ T cells were distributed around fibroblasts, and they are associated with fibroblast apoptosis in obstructed kidney. Moreover, CD11c⁺ CD8⁺ T cells expressed higher levels of the cytotoxicity-related genes than CD11c^- CD8⁺ T cells, and infiltrated CD11c⁺ CD8⁺ T cells in obstructed kidney could induce fibroblast death in vitro. Results indicated that induction of fibroblast apoptosis partly contributed to the effect of CD8⁺ T cells on reduction of renal fibrosis. Given that inflammatory cells are involved in fibrosis, our results suggest that kidney fibrosis is a multifactorial process involving different arms of the immune system.

Keywords: T cells; fibroblasts; fibrosis; inflammation; kidney.

Figure 1

CD8 deficiency promotes renal fibrosis in unilateral ureteric obstruction (UUO) mice. (A) Masson’s Trichrome staining was performed to examine fibrosis at day 7; (B) Quantitative analysis of fibrosis area in UUO kidneys. CD8 knockout (KO) increases fibrosis in UUO kidney (* p < 0.05 vs. sham, # p < 0.05 vs. wild-type (WT) UUO; n = 5/group). Scale bars, 50 µm; (C) In fluorescence-activated cell sorting analysis, kidney CD8⁺ T cells were stained with anti-CD45-PerCP-Cy5.5, anti-CD3e-PE-Cy594, anti-CD8-APC-Cy7, and anti-CD4-PE to confirm the absence of CD8⁺ cells in CD8 KO mice and quantitate (D) CD8⁺ T cells or (E) CD4⁺ T cells. (* p < 0.05 vs. WT UUO 0 day, NS: no significant difference; n = 6/group).

Figure 1

CD8 deficiency promotes renal fibrosis in unilateral ureteric obstruction (UUO) mice. (A) Masson’s Trichrome staining was performed to examine fibrosis at day 7; (B) Quantitative analysis of fibrosis area in UUO kidneys. CD8 knockout (KO) increases fibrosis in UUO kidney (* p < 0.05 vs. sham, # p < 0.05 vs. wild-type (WT) UUO; n = 5/group). Scale bars, 50 µm; (C) In fluorescence-activated cell sorting analysis, kidney CD8⁺ T cells were stained with anti-CD45-PerCP-Cy5.5, anti-CD3e-PE-Cy594, anti-CD8-APC-Cy7, and anti-CD4-PE to confirm the absence of CD8⁺ cells in CD8 KO mice and quantitate (D) CD8⁺ T cells or (E) CD4⁺ T cells. (* p < 0.05 vs. WT UUO 0 day, NS: no significant difference; n = 6/group).

Figure 2

CD8⁺ T cell activation requires an inflammatory microenvironment. CD4⁺ T cells (CD45⁺ CD3⁺ CD4⁺ CD8⁻) and CD8⁺ T cells (CD45⁺ CD3⁺ CD4⁻ CD8⁺) were isolated from obstructed kidney or spleen. (A–D) mRNA levels of chemokines (CCL2, CCL3, CCL4, and CCL5) as determined in those CD4⁺ T cells or CD8⁺ T cells by quantitative RT-PCR (qRT-PCR; * p < 0.05 kidney vs. spleen; n = 5); (E–H) mRNA levels of cytotoxicity-related genes (PRE-1, GZMA, GZMB, and FASL) as determined in those CD4⁺ T cells or CD8⁺ T cells by qRT-PCR (* p < 0.05 kidney vs. spleen; n = 5).

Figure 3

CD8 KO reduces cytotoxicity and cell apoptosis in obstructed kidney. (A–D) mRNA expression levels of PRF-1, GZMA, GZMB, and FASL in whole kidney tissues were analyzed by quantitative RT-PCR (* p < 0.05, CD8 KO vs. WT; n = 5); (E) TUNEL staining is performed in kidney; TUNEL (green), nucleus (blue). The percentage of TUNEL-positive cells in total cells is calculated (n = 5, * p < 0.05).

Figure 4

CD8⁺ T cells are associated with apoptosis of fibroblast in obstructed kidney. (A) Double immunostaining in kidney was performed using anti-vimentin (red) and TUNEL (green) at UUO 5 or 7 days (bar = 50 µm); (B) Double immunostaining in kidney was performed using anti-vimentin (red) and anti-CD8 (green) at UUO 5 or 7 days (Bar = 50 µm); (C) Percentage of vimentin- and TUNEL-double positive cells among TUNEL-positive cells was calculated (* p < 0.05; n = 5).

Figure 5

CD11c⁺ CD8⁺ T cells mainly constitute the population of CD8 T cells expressing the mRNA of cytotoxin related factors in obstructed kidney. (A) CD11c⁻ CD8⁺ T cells and CD11c⁺ CD8⁺ T cells in kidney at UUO 5 days were separated through cell sorting after incubation of the total number of cells in antibody cocktail (anti-CD45, CD3e, CD8a, and CD11c). CD11c isotype control group: incubated in antibody cocktail (anti-CD45, CD3e, and CD8a and an isotype control of CD11c antibody); (B–E) mRNA levels of cytotoxin related factors (PRE-1, GZMA, GZMB, and FASL) as determined in those CD11c⁻ CD8⁺ T cells or CD11c⁺ CD8⁺ T cells by quantitative RT-PCR (* p < 0.05 vs. CD11c⁻ CD8⁺ T cells; n = 5).

Figure 6

CD11c⁺ CD8⁺ T cells isolated from obstructed kidney can induce fibroblast death in vitro. (A) Fibroblasts were co-cultured with CD11c⁻ CD8⁺ T cells or CD11c⁺ CD8⁺ T cells for 24 h; the cells were subsequently harvested to analyze the mortality of CD8⁻ CD3⁻ cells (fibroblast) through PI (Propidium Iodide) staining combined with flow cytometry; the CD8⁺ T cell-free set-up served as control; (B) Quantitative analysis of the percentage of PI staining-positive fibroblasts. (* p < 0.05 vs. fibroblasts plus CD11c⁻ CD8⁺ T cells; n = 5/group).