Department of Urology, Seoul National University College of Medicine, Seoul, Korea.
Abstract
Severe glomerular injury ultimately leads to tubulointerstitial fibrosis that determines patient outcome, but the immunological molecules connecting these processes remain undetermined. The present study addressed whether V-domain Ig suppressor of T cell activation (VISTA), constitutively expressed in kidney macrophages, plays a protective role in tubulointerstitial fibrotic transformation after acute antibody-mediated glomerulonephritis. After acute glomerular injury using nephrotoxic serum, tubules in the VISTA-deficient (Vsir-/-) kidney suffered more damage than those in WT kidneys. When interstitial immune cells were examined, the contact frequency of macrophages with infiltrated T cells increased and the immunometabolic features of T cells changed to showing high oxidative phosphorylation and fatty acid metabolism and overproduction of IFN-γ. The Vsir-/- parenchymal tissue cells responded to this altered milieu of interstitial immune cells as more IL-9 was produced, which augmented tubulointerstitial fibrosis. Blocking antibodies against IFN-γ and IL-9 protected the above pathological process in VISTA-depleted conditions. In human samples with acute glomerular injury (e.g., antineutrophil cytoplasmic autoantibody vasculitis), high VISTA expression in tubulointerstitial immune cells was associated with low tubulointerstitial fibrosis and good prognosis. Therefore, VISTA is a sentinel protein expressed in kidney macrophages that prevents tubulointerstitial fibrosis via the IFN-γ/IL-9 axis after acute antibody-mediated glomerular injury.
Keywords
FibrosisMacrophagesNephrologyT cells
Figures
Figure 1 VISTA expression in the NTN model. (A) Representative flow cytometry plot of R1 and R2 macrophages. Cells were gated from DAPI–CD45+Ly6G– subset. Proportions of R1 and R2 macrophages at day 8 after NTN induction (n = 7 per group). (B) UMAP plots of 86,254 cells pooled from 3 sources with C57BL/6 mice. PT, proximal tubule; DL-tAL, descending limb and thin ascending limb of loop of Henle; TAL, thick ascending limb of loop of Henle; DCT, distal convoluted tubule; PC, principal cell of collecting duct; IC, intercalated cell of collecting duct; ENDO, endothelial cell; PERI, pericyte; PODO, podocyte; Uro, urothelium; EC, erythrocyte. (C) Dot plots to identify clusters of parenchymal tissue cells (left) and immune cells (right). (D) Violin and dot plots for the expression levels of the Vsir gene in clusters. (E) Expression of VISTA protein in R1 and R2 macrophages at day 8 after NTN induction (n = 7 per group). A representative flow cytometry plot of VISTA is shown. (F) Representative image of kidney sections immunostained for DAPI, VISTA, and F4/80. Upper and lower images represent before and after NTN induction, respectively. Scale bar: 100 μm. (G) Expression of VISTA protein in other immune cells, such as CD4+ T (CD45+CD3+CD4+), CD8+ T (CD45+CD3+CD8+), double-negative (DN) T (CD45+CD3+CD4–CD8–), B (CD45+CD19+), and NK (CD45+CD3–NK1.1+) cells (n = 4 per group). A representative flow cytometry plot of VISTA after NTN induction is shown. Data are represented as mean ± SEM. P values were calculated using an unpaired Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001. Data represent at least 3 independent experiments.Figure 2 Deleterious parenchymal tissues in VISTA-depleted conditions. (A) Kidney damage markers in WT and Vsir–/– mice (n = 16 per group). uACR, random urine albumin-to-creatinine ratio; uPCR, random urine protein-to-creatinine ratio; BUN, blood urea nitrogen; Cr, creatinine. (B) Representative PAS staining images of NTN-induced kidneys. Scale bars: 100 μm (left); 50 μm (right). (C) Glomerular and tubular injury scores at day 8 after NTN induction (n = 13 per group). (D) Representative TUNEL images at day 8 after NTN induction (n = 13 per group). TUNEL+ cells (arrows) were calculated based on 10 randomly selected images. Original magnification, ×100. (E) Representative KIM-1 images at day 8 after NTN induction (n = 13 per group). KIM-1+ cells were calculated from 10 randomly selected images. Data are represented as mean ± SEM. P values were calculated using an unpaired Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001. Data represent at least 3 independent experiments.Figure 3 Immunometabolic changes in T cells. (A) UMAP plots of 7396 sorted immune cells. (B) Dot plot to identify clusters of immune cells. pDC, plasmacytoid dendritic cell; NKT, NK T cell; ILC2, type 2 innate lymphoid cell. (C) Proportions of all cell types in WT and Vsir–/– kidneys (upper). Proportions of EM-like T cells within each T cell lineage (lower). (D) Heatmap of logarithmic fold changes of cytokine genes in T cells from Vsir–/– mice compared with WT mice. (E) Hallmark gene set enrichment analysis in order of normalized enrichment score (ES). (F) Correlation between metabolic features and Ifng level in EM-like T cells. Line and area represent nonlinear relationship and 95% CIs, respectively. Blue and red dots represent T cells obtained from the kidneys of WT and Vsir–/– mice, respectively. (G) Proportions of T cell subsets evaluated by flow cytometry (n = 7 per group). TN, naive T cells; TEM, effector memory T cells; TE, effector T cells; TCM, central memory T cells. (H) Proportions of cytokine-producing T cells evaluated by flow cytometry (n = 11 per group). (I) Proportions of IFN-γ+ cells in CD4+, CD8+, and other immune cells (n = 11 per group). P values were calculated using an unpaired Student’s t test for 2 groups or ANOVA with Tukey’s test for multiple comparison. *P < 0.05; **P < 0.01; ***P < 0.001. Data represent 2 or 3 independent experiments.Figure 4 Contact between macrophages and T cells. (A) Representative flow cytometry plot of CD72 in R1 and R2 macrophages. (B) Representative plot of kidney sections immunostained for F4/80, CD72, and CD3 and analysis process to identify the contact frequency between macrophages and T cells. Scale bars: 50 μm. (C) Density of macrophages around T cells. (D) Density of macrophage subsets in WT (left) and Vsir–/– (right) mice. (E) Numbers of ligand-receptor pairs between macrophage and T cell clusters. (F) Chord diagrams of the top 30 interactions between macrophage and T cell clusters. The ligand-receptor interaction scores (LR scores) were bounded by 0 and 1, and a high score represented a strong interaction. (G) Chord diagrams of all ligand-receptor pairs between EM-like CD8+ T cells and each macrophage cluster (see the raw data in Supplemental Data 1). Data represent 2 or 3 independent experiments.Figure 5 Fibrotic parenchyma in VISTA-depleted kidneys. (A) Masson trichrome–positive area in WT and Vsir–/– kidneys and their representative plots. Scale bars: 100 μm. (B) Sirius red–positive area WT and Vsir–/– kidneys and their representative plots. Scale bars: 100 μm. (C) αSMA-positive area in WT and Vsir–/– kidneys, and their representative plots. Scale bars: 100 μm. (D) Cytokine-gene expression of kidney parenchyma. (E) Representative plots of kidney sections immunostained for IL-9 and comparison of the IL-9+ area between WT and Vsir–/– kidneys. Scale bars: 100 μm. Data are represented as mean ± SEM (n = 11–13 per group). P values were calculated using an unpaired Student’s t test. *P < 0.05; ***P < 0.001. Data represent at least 3 independent experiments.Figure 6 Overwhelming of the IFN-γ/IL-9 axis in the late phase of VISTA-depleted kidneys. (A) Survival rates of NTN-induced WT and Vsir–/– mice (n = 12 per group). Statistical significance for survival was calculated using the Kaplan-Meier method with the log-rank test. (B) Kidney damage markers in WT and Vsir–/– mice that had survived to day 36 after NTN induction. (C) Representative PAS-staining images of kidneys at day 36 after NTN induction. Scale bars: 100 μm (left); 50 μm (right). (D) Glomerular and tubular injury scores at day 36 after NTN induction. (E) Proportions of cytokine-producing T cells evaluated by flow cytometry. (F) Proportions of IFN-γ+ cells in CD4+, CD8+, and other immune cells. (G) Representative plots of kidney sections immunostained for IL-9 and comparison of the IL-9+ area between WT and Vsir–/– kidneys. (H) Representative Masson trichrome plots of kidney sections and comparison between WT and Vsir–/– kidneys. (I) Representative Sirius red plots of kidney sections and comparison between WT and Vsir–/– kidneys. (J) Representative Sirius red plots of kidney sections immunostained for αSMA, and comparison between WT and Vsir–/– kidneys. Data are represented as mean ± SEM (n = 7–12 per group). Scale bars: 100 μm. P values were calculated using an unpaired Student’s t test for 2 groups or ANOVA with Tukey’s test for multiple comparison. *P < 0.05; **P < 0.01; ***P < 0.001. Data represent 2 or 3 independent experiments.Figure 7 Confirmation of the IFN-γ/IL-9 fibrosis axis. (A) Kidney damage markers in vehicle- and rmIFN-γ-treated WT kidneys. (B) Representative plots of kidney sections immunostained for IL-9 and comparison of the IL-9+ area between vehicle- and rmIFN-γ–treated kidneys. (C) Kidney damage markers in control antibody- and anti–IFN-γ antibody–treated Vsir–/– kidneys. (D) Representative plots of kidney sections immunostained for IL-9 and comparison of the IL-9+ area between control antibody- and anti–IFN-γ antibody–treated kidneys. (E) Kidney damage markers in NTN-induced Rag1–/– mice with adoptive transfer of naive CD8+ T cells from WT and Ifng–/– mice. (F) Representative plots of kidney sections immunostained for IL-9 and comparison of the IL-9+ area in NTN-induced Rag1–/– kidneys between WT mouse-derived and Ifng–/– mouse–derived naive CD8+ T cell transfer. (G) Kidney damage markers in vehicle- and rmIL-9–treated WT kidneys. (H) Representative plots of kidney sections immunostained for Sirius red and comparison of the Sirius red+ area between vehicle- and rmIL-9-treated WT kidneys. (I) Kidney damage markers in control antibody- and anti–IL-9 antibody–treated Vsir–/– kidneys. (J) Representative plots of kidney sections immunostained for Sirius red and comparison of the Sirius red+ area between control antibody– and anti–IL-9 antibody–treated Vsir–/– kidneys. Scale bars: 100 μm. (K) Representative PAS staining images of control antibody- and anti–IL-9 antibody–treated Vsir–/– kidneys. Scale bars: 100 μm. (L) Glomerular and tubular injury scores in control antibody– and anti–IL-9 antibody–treated Vsir–/– kidneys. (M) Proportions of T cell subsets in control antibody– and anti–IL-9 antibody–treated Vsir–/– kidneys. Data are represented as mean ± SEM (n = 4–7 per group). P values were calculated using an unpaired Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001. Data represent 2 or 3 independent experiments.Figure 8 Minimal effects of VISTA-depleted T cells in the NTN model. (A) Schematic of adoptive transfer experiments. Infiltrated cell numbers in the spleen and kidney after adoptive transfer of naive CD4+ T cells to NTN-induced Rag1–/– mice. (B) Infiltrated cell numbers in the spleen and kidney after adoptive transfer of naive CD8+ T cells to NTN-induced Rag1–/– mice. The adoptive transfer scheme was the same as in A. (C) Proportion of CD44+ cells among each T cell lineage in the spleen. (D) Proportion of CD44+ cells among each T cell lineage in the kidney. (E) Infiltrated cell numbers of WT mouse– and Vsir–/– mouse–derived CD3+ T cells after NTN induction in a competitive assay. The adoptive transfer scheme was the same as in A. (F) Proportion of CD44+ cells among each T cell lineage in the spleen. (G) Proportion of CD44+ cells among each T cell lineage in the kidney. Data are represented as mean ± SEM (n = 3–4 per group). P values were calculated using an unpaired Student’s t test. All the differences between groups were not significant.Figure 9 Translation of results to human glomerulonephritis. (A) UMAP plots of 55,107 cells from pooled data. (B) Dot plots to identify clusters of parenchymal tissue cells (left) and immune cells (right). ENDO, endocyte; MES, mesangial cell. (C) Expression of the C10orf54 gene in the UMAP plot (upper) and clusters (lower). (D) Dot plot for the gene expression of PSGL-1 and other immune checkpoints. (E) Representative image of kidney sections with ANCA vasculitis immunostained for VISTA. Scale bars: 200 μm (left); 100 μm (right). (F) Correlations of VISTA expression with the IL-9+ (left) and Sirius red+ (right) areas in ANCA vasculitis. The correlation coefficients were measured using Pearson’s correlation test. (G) Kaplan-Meier curve of composite renal risk according to VISTA expression. Composite renal risk was defined as a doubling of serum creatinine, development of end-stage renal disease, or death. Statistical significance for survival was calculated using the Kaplan-Meier method with the log-rank test.
그림 출처: PubMed Central 공개 논문 (PMC8718152)
그 밖의 정보
출판 형태
Journal Article, Research Support, Non-U.S. Gov't
저널
The Journal of clinical investigation · eISSN 1558-8238
