Tertiary lymphoid organs are associated with the progression of kidney damage and regulated by interleukin-17A

Abstract

Background: Tertiary lymphoid organs (TLOs) occur after multiple chronic kidney injuries. interleukin-17A (IL-17A) has been reported to associate with the development of TLOs in inflammatory diseases. However, regulation of the renal TLOs and its clinical significance to the pathogenesis of chronic kidney injury are unknown. Methods: To evaluate the clinical significance and regulation of renal TLOs, we analyzed the progression of patients with kidney damage based on the existence and absence of TLOs in a larger multicenter cohort. We also blocked the recruitment of lymphocyte cells into the kidney by FTY720 (fingolimod) in vivo. Besides, we used aged IL-17A genetic knocked out mice and IL-17A-neutralizing antibody to explore the role of IL-17A in renal TLOs formation. Results: We demonstrated that renal TLOs of IgA nephropathy patients were associated with disease severity and were independent risk factors for renal progression after adjustment for age, sex, mean arterial pressure, proteinuria and, baseline eGFR and MEST-C score, especially in the early stage. Plasma levels of TLO-related chemokines CXCL13, CCL19, and CCL21 were higher in patients with renal TLOs. Inhibiting the formation of renal TLOs by FTY720 could reduce the intrarenal inflammation and fibrosis, and early intervention was found to be more effective. IL-17A was increased in renal TLOs models, and genetic depletion of IL-17A or treatment with anti-IL-17A antibody resulted in a marked reduction of the TLOs formation as well as alleviation of renal inflammation and fibrosis. Conclusion: These results indicate that TLOs are associated with the progression of kidney damage and regulated by IL-17A and may be effective targets for the treatment of kidney damage.

Keywords: IL-17A; Tertiary lymphoid organs; inflammation; kidney damage; progression.

Figure 1

Renal TLOs developed in patients with kidney damage and were associated with renal progression in IgAN patients. (A) Representative renal TLOs in kidneys of multiple pathological types were stained by PAS. CD4⁺ T cells, CD8⁺ T cells, and CD20⁺ B cells in TLOs were stained by immunohistochemistry. (B) The proportion of TLOs in renal biopsy specimens of IgAN (N = 1044), LN (N = 85), MN (N = 127), and MCD (N = 49) patients. (C-E) Comparison of interstitial inflammatory cells, including CD4⁺ T cells (C), CD8⁺ T cells (D), and CD20⁺ B cells (E) in renal biopsy specimens of IgAN patients with TLOs group (N = 43) and without TLOs group (N = 10). (F-H) Plasma levels of CXCL13 (F), CCL19 (G), and CCL21 (H) were increased in IgAN patients with renal TLOs (N = 32) compared with IgAN patients without renal TLOs (N = 41). (I-K) Kaplan-Meier curves of the renal combined event survival of all IgAN patients (I), IgAN patients with eGFR ≥ 60 mL/min/1.73 m² (J), IgAN patients with eGFR < 60 (mL/min/1.73 m²) (K) who were divided into three groups: without TLOs under 10 equivalent HPFs, with 1-2 TLOs under 10 equivalent HPFs, and ≥ 3 TLOs under 10 equivalent HPFs. PAS, Periodic acid-Schiff; IgAN, IgA nephropathy; LN, lupus nephritis; MN, membranous nephropathy; MCD, minimal change disease; eGFR, estimated glomerular filtration rate. *P < 0.05, **P < 0.01, ***P < 0.001. Data represent mean ± SEM, Scale bar = 20 µm. P-values were calculated using a two-tailed t test or Kaplan-Meier survival curves.

Figure 2

TLOs were induced in aged mouse kidneys after renal injury. (A) PAS and Masson staining. TLOs size of sham mice (N = 4), UUO mice (N = 5) (day 14), FA mice (N = 4) (day 21), and IRI mice (day 45 after 37-min IRI) (N = 6) and correlation between renal TLOs size and renal fibrosis score of mice at day 45 after 37-min IRI (N = 10). (B) TLOs size of mice after 30-min and 37-min IRI and sacrificed at day 30 and day 45 (N = 3 per group). (C) Correlation between TLOs size and mRNA level of fibronectin, collagen I, LTα, LTβ, CXCL13, and CCL19 in kidney of mice at day 45 after 37-min IRI (N = 10). (D) Immunofluorescence analysis of CD3 and B220, CD45 and CD21, podoplanin, LYVE-1 and αSMA, Ki67, and IL-17A. (E) Immunofluorescence analysis of CXCL13, CCL19 and CCL21 with podoplanin. (F) Renal fibrosis, inflammation, TLO-related chemokine, lymphotoxin and podoplanin, and Th17-related markers in kidneys of mice at day 45 after 37-min IRI analyzed by real-time PCR (N = 7 per group). UUO, unilateral ureteral obstruction; FA, folic acid; LYVE-1, lymphatic vessel endothelial hyaluronan receptor 1; αSMA, α-smooth muscle actin; IL-17A, Interleukin (IL)-17A. *P < 0.05, **P < 0.01, ***P < 0.001. Data represent mean ± SEM, Scale bar = 100 µm. P-values were calculated using a two-tailed t test.

Figure 3

Transcriptome analysis of kidneys in aged IRI mice. (A) Volcano plot of gene expression changes. One thousand five hundred sixty-six upregulated genes and 152 downregulated genes with fold change ≥ 4 and adjusted P value ≤ 0.001 were found. (B) KEGG pathway functional enrichment analysis of all upregulated DEGs (FDR ≤ 0.01). (C) GO enrichment analysis of all the upregulated DEGs (FDR ≤ 0.01). (D) Heatmap of selected enriched terms (FDR ≤ 0.01) from KEGG pathway analysis of upregulated DEGs. GO, gene ontology; DEGs, differentially expressed genes; FDR, false discovery rate; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 4

Targeting TLOs formation has the potential to ameliorate renal fibrosis and inflammation. (A) Scheme. Aged mice were treated with 0.5 mg/kg body weight FTY720 or vehicle intraperitoneally 1 day before renal IRI followed by daily injection. (B) The percentage of B220⁺ B cells and CD3⁺ T cells analyzed by flow cytometry from blood (N = 6 per group). (C) Representative photomicrograph statistical graph for PAS and Masson pathology staining and representative immunofluorescence analysis of B220 (red) with CD3 (green) and podoplanin (green). (D) The percentage of B220⁺ B cells, CD3⁺ T cells, CD8⁺T cells and CD4⁺ T cells was analyzed by flow cytometry from the whole kidney in the vehicle group and FTY720 treated group (N = 6 per group). (E) Real-time PCR analysis of renal fibrosis, TLO-related markers, and inflammation in kidneys of the vehicle group and FTY720-treated group (N = 6 per group). (F) Scheme. Aged mice were treated with 0.5 mg/kg body weight FTY720 or vehicle intraperitoneally at day 15 or day 30 after renal IRI followed by daily injection. (G) Representative photomicrographs and statistical graph for PAS staining of the FTY720 treatment group starting from day -1, day 15, and day 30 (N = 3 per group). *P < 0.05, **P < 0.01, ***P < 0.001. Data represent mean ± SEM, Scale bar = 100 µm. P-values were calculated using a two-tailed t test.

Figure 5

The development of TLOs required IL-17A. (A) Representative photomicrograph statistical graph for PAS and Masson pathology staining and representative immunofluorescence analysis of B220 (red) with CD3 (green) and podoplanin (green) in the WT and Il17a^-/- groups. (B) The percentage of B220⁺ B cells, CD3⁺ T cells, CD8⁺ T cells, CD4⁺ T cells, and CD31^-GP38⁺ FRCs was analyzed by flow cytometry from the whole kidneys in the WT and Il17a^-/- groups (N = 6 per group). (C) Real-time PCR analysis of renal fibrosis, inflammation, and TLO-related markers in kidneys of the WT and Il17a^-/- groups (N = 6 per group). *P < 0.05, **P < 0.01, ***P < 0.001. Data represent mean ± SEM, Scale bar = 100 µm. P-values were calculated using a two-tailed t test.

Figure 6

Neutralization of IL-17A inhibited TLOs formation and alleviated renal fibrosis and inflammation of aged mice after IRI. (A) Scheme. Aged mice were injected intraperitoneally with either 50 µg of anti-IL-17A antibody or IgG at 1 day before renal IRI followed by daily injection (IgG group N = 5, IL-17A Ab N = 6). (B) Representative photomicrograph of pathology staining Masson pathology staining and representative immunofluorescence analysis of B220 (red) with CD3 (green) and podoplanin (green) in the IgG and IL-17A Ab groups. (C) The percentage of B220⁺ B cells, CD3⁺ T cells, CD8⁺ T cells, CD4⁺ T cells, and CD31^-GP38⁺ FRCs were analyzed by flow cytometry from whole kidneys in the IgG and IL-17A Ab groups (IgG group N = 5, IL-17A Ab N = 6). (D) Real-time PCR analysis of renal fibrosis, inflammation, and TLO-related markers in kidneys of the vehicle and IL-17A Ab groups (IgG group N = 5, IL-17A Ab N = 6). *P < 0.05, **P < 0.01, ***P < 0.001. Data represent mean ± SEM, Scale bar = 100 µm. P-values were calculated using a two-tailed t test.