IL-25 induces M2 macrophages and reduces renal injury in proteinuric kidney disease

Abstract

The kidney contains receptors for the cytokine IL-25, but the effects of IL-25 in CKD are unknown. Here, we induced adriamycin nephropathy in both BALB/c mice and severe combined immunodeficient (SCID) mice, and we injected IL-25 for 7 consecutive days starting at day 5 after adriamycin administration. BALB/c mice treated with IL-25 had less glomerulosclerosis, tubular atrophy, interstitial expansion, and proteinuria than control mice at day 28. IL-25 increased the levels of IL-4 and IL-13 in serum, kidney, renal draining lymph nodes, and CD4+ lymphocytes. IL-25 also directly suppressed effector macrophages in vitro and in vivo and induced alternatively activated (M2) macrophages in vivo. However, in SCID mice and in BALB/c mice treated with IL-4/13-neutralizing antibody, IL-25 failed to protect against renal injury and did not induce M2. In conclusion, IL-25 protects against renal injury in adriamycin nephropathy in mice by, at least in part, inducing Th2 immune responses.

Figure 1.

IL-25 protected against renal injury in AN BALB/c mice. (A) BALB/c mice were injected daily with IL-25 from day 5 to day 12 after ADR injection. Mice were killed on day 14 and day 28. (B) Body weight in normal, AN + vehicle, and AN + IL-25 groups on days 0 to 28. (C) Proteinuria at weeks 2 and 4. (D) PAS-stained sections of renal cortices at week 4 (×200). (E) Quantitative assessment of glomerular sclerosis, tubular damage, and interstitial volume. The values represent the mean ± SEM of evaluations from each group (n = 8 per group). *P < 0.05, and **P < 0.01 versus AN + vehicle.

Figure 2.

IL-25 induced peripheral Th2 responses. (A) IL-4, IL-5, and IL-13 levels in serum were assessed in normal, AN + vehicle, and AN + IL-25 groups at weeks 2 and 4. (B) The levels of cytokine expression were measured by ELISA in splenic CD4+ T cells stimulated for 3 days with anti-CD3/CD28 (1 μg/ml) and then restimulated for 24 hours with 1 μg/ml of anti-CD3. (C and D) Intracellular IL-4 and IL-13 expression was analyzed by flow cytometry in CD4+ T cells stimulated with ionomycin and PMA in the presence of GolgiStop for 5 hours. Numbers above the gates indicate the percentage of cells stained positive for each respective cytokine. (E and F) The mRNA expression of IL-4 and IL-13 in kidney (E) and RDLN (F) was examined by real-time PCR and expressed relative to the control of each experiment. The values represent the mean ± SEM of evaluations from each group (n = 8 per group). *P < 0.05, **P < 0.01, and ***P < 0.001 versus AN + vehicle.

Figure 3.

IL-25 suppressed endogenous renal macrophages in vivo and M1 macrophages in vitro. After administration of IL-25, CD11b+ endogenous renal macrophages at week 2 and week 4 were purified by flow cytometry. The expression of TNFα and IL-12 by endogenous renal macrophages was analyzed by flow cytometry (A and B) and the mRNA expression of iNOS, CCL2, IL-1β, and IL-6 was examined by real-time PCR (C). The values represent the mean ± SEM of evaluations from each group (n = 8 per group). *P < 0.05, and **P < 0.01 versus AN + vehicle. (D) The mRNA expression of iNOS, TNFα, CCL2, IL-1β, IL-6, and IL-12 by bone marrow macrophages preincubated with medium (M) or IL-25 (100 ng/ml) for 1 hour and stimulated or not with LPS or IFNγ for a further 6 h in vitro. The values represent the mean ± SEM of evaluations from each group (n = 4 per group). #P < 0.05 and ##P < 0.01 versus No IL-25. (E and F) Resting macrophages (M0) were cultured with LPS (to become M1 macrophages) in the presence of various concentrations (ng/ml) of mouse recombinant IL-25 for 24 hours. Cells were co-cultured with FITC-labeled dextran for 45 minutes. The uptake of fluorescence beads (mean fluorescence intensity) was determined by flow cytometry. In parallel, macrophages were cultured with LPS in the presence of various concentrations (ng/ml) of mouse recombinant IL-25 for 48 hours, and NO was measured in the culture supernatant. The values represent the mean ± SEM of evaluations from each group (n = 4 per group). @P < 0.05 and @@P < 0.01 versus M1.

Figure 4.

IL-25 induced alternatively activated macrophages. CD11b+ endogenous renal macrophages were sorted by flow cytometry at weeks 2 and 4. The expression of MR was assessed by flow cytometry (A), and the mRNA expression of arginase, FIZZ1, YM1, and IL-10 was quantified by real-time PCR (B). The values represent the mean ± SEM of evaluations from each group (n = 8 per group). **P < 0.01 and ***P < 0.001 versus AN + vehicle and normal. (C) The mRNA expression of MR, arginase, FIZZ1, YM1, IL-10, and CCL-17 was examined by real-time PCR in bone marrow macrophages preincubated for 1 hour with medium (M) or IL-25 (100 ng/ml) combined or not with IL-4 (10 ng/ml) or IL-13 (10 ng/ml) for a further 6 hours in vitro. The values represent the mean ± SEM of evaluations from each group (n = 4 per group). #P < 0.05 versus No IL-25. (D) Naïve CD4+ T cells preincubated or not with mouse recombinant IL-25 were co-cultured with bone marrow–derived macrophages (M0) in the presence of IL-4/13 neutralizing antibodies or control rat IgG1 for 24 hours. The mRNA expression of MR, arginase, FIZZ1, YM1, IL-10, and CCL-17 in bone marrow macrophages was examined by real-time PCR. The values represent the mean ± SEM of evaluations from each group (n = 4 per group). &&P < 0.01, versus the other three groups.

Figure 5.

IL-25 protected against injury in AN in an IL-4/13–dependent manner. (A) Serum creatinine, creatinine clearance, and proteinuria were assessed at weeks 2 and 4. (B) PAS-stained sections of renal cortices at week 4 (×200). (C) Quantitative assessment of glomerular sclerosis, tubular damage, and interstitial volume expansion. The values represent the mean ± SEM of evaluations from each group (n = 8 per group). $P < 0.05 and $$P < 0.01 versus the other three groups.

Figure 6.

IL-25 induced M2 macrophages and suppressed endogenous renal macrophages in an IL-4/13–dependent manner. The percentage of endogenous renal macrophages expressing MR, TNFα, and IL-12 was measured by flow cytometry (A and C) and the mRNA expression of arginase, FIZZ1, YM1, IL-10, iNOS, CCL2, IL-1β, and IL-6 was quantified by real-time PCR (B and D). The values represent the mean ± SEM of evaluations from each group (n = 8 per group). $P < 0.05 and $$P < 0.01 versus the other three groups.

Figure 7.

IL-25 reduced inflammatory infiltrates in a IL4/13-dependent manner. Numbers of F4/80+ macrophages (A) and CD4+ and CD8+ T cells (B and C) were assessed by immunofluorescence staining in renal cortex of mice at week 2 and week 4. The values represent the mean ± SEM of evaluations from each group (n = 8 per group). $P < 0.05, and $$P < 0.01 versus the other three groups.

Figure 8.

IL-25 failed to protect against renal injury in AN SCID mice. (A) Proteinuria at weeks 2 and 4. (B) PAS-stained sections of renal cortices at week 4 (×200). (C) Quantitative assessment of glomerular sclerosis, tubular damage, and interstitial volume. The values represent the mean ± SEM of evaluations from each group (n = 8 per group).