Unexpected kidney-restricted role for IL-17 receptor signaling in defense against systemic Candida albicans infection

Abstract

Kidney injury is a frequent outcome in patients with disseminated Candida albicans fungal infections. IL-17 receptor (IL-17R) signaling is critical for renal protection against disseminated candidiasis, but the identity and function of IL-17-responsive cells in mediating renal defense remains an active area of debate. Using BM chimeras, we found that IL-17R signaling is required only in nonhematopoietic cells for immunity to systemic C. albicans infection. Since renal tubular epithelial cells (RTEC) are highly responsive to IL-17 in vitro, we hypothesized that RTEC might be the dominant target of IL-17 activity in the infected kidney. We generated mice with a conditional deletion of IL-17 receptor A (Il17ra) in RTEC (Il17raΔRTEC). Strikingly, Il17raΔRTEC mice showed enhanced kidney damage and early mortality following systemic infection, very similar to Il17ra-/- animals. Increased susceptibility to candidiasis in Il17raΔRTEC mice was associated with diminished activation of the renal protective Kallikrein-kinin system (KKS), resulting in reduced apoptosis of kidney-resident cells during hyphal invasion. Moreover, protection was restored by treatment with bradykinin, the major end-product of KKS activation, which was mediated dominantly via bradykinin receptor b1. These data show that IL-17R signaling in RTEC is necessary and likely sufficient for IL-17-mediated renal defense against fatal systemic C. albicans infection.

Keywords: Cytokines; Fungal infections; Immunology; Infectious disease; Innate immunity.

Figure 1. Innate TCRγδ⁺ T cells produce IL-17 in C.

albicans–infected kidney. (A) WT mice were either subjected to disseminated C. albicans infection or left uninfected (sham) (n = 8). At day 2 p.i., renal mRNA levels of Il17a, Il17f, Il17c, and Il17e were quantified by qPCR. (B) Single cell suspensions from the perfused kidneys of C. albicans infected or sham IL-17^eYFP mice (n = 6) were used to determine the number of eYFP⁺ cells (gated on live CD45⁺) at day 2 p.i. Absolute numbers of innate TCRγδ⁺ and TCRαβ⁺ T cells producing IL-17 were evaluated by gating on live CD45⁺eYFP⁺ cells. (C) Percentages of renal Ki-67⁺TCRγδ⁺ and Ki-67⁺TCRαβ⁺ cells from C. albicans–infected (n = 9) or sham mice (n = 5) (gated on live CD45⁺ cells) at day 2 p.i. Numbers in the zebra (B) and contour (C) plots reflect percentages of cells. In the dot plots, each dot represents an individual mouse, and data are represented as mean ± SD and compared by 2-tailed Student’s t test for A and B and by 1-way ANOVA for C. Data are pooled from 2 independent experiments for A–C.*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 2. RTEC-specific expression of IL-17RA is required for antifungal immunity in the kidney.

(A) BM cells from Il17ra^−/− (CD45.2⁺) and WT (CD45.1⁺) mice were adoptively transferred into irradiated (9 Gy) Il17ra^−/− or WT recipients (n = 14). Six weeks later, mice were systemically infected with C. albicans, and survival was evaluated over 14 days. Don, donor; rec, recipients. (B) Total kidney cells from WT, Il17ra^–/–, control, and Il17ra^ΔRTEC mice (n = 3) were evaluated for IL-17RA expression on RTEC (live CD45^–CD133⁺). The number in the contour plot reflects percentage of cells. Histogram is representative of 1 of 3 independent experiments. (C) Control, Il17ra^ΔRTEC, and Il17ra^–/– mice were systemically infected with C. albicans (CAF2-1 strain; 1 × 10⁵ CFU) (n = 15–16) or left uninfected (n = 3–5). Mice were evaluated for survival over 14 days. (D) Control, Il17ra^ΔRTEC, and Il17ra^–/– mice were systemically infected with C. albicans (HUN96 strain; 5 × 10⁵ CFU) (n = 8–11) or left uninfected (n = 2). Mice were evaluated for survival over 14 days. (E) Il17ra^fl/flNpsh2^Cre- (control) and Il17ra^fl/flNpsh2^Cre+ (Il17ra^ΔPOD) mice were either subjected to infection (n = 8–10) or left uninfected (n = 3) and were evaluated for survival over 14 days. Data are pooled from 3 independent experiments for A, C, and E and 2 independent experiments for D. The comparison of survival curves were performed by Log-rank (Mantel-Cox) test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 3. NK cells are functional in Il17ra^ΔRTEC mice.

(A) BM cells from WT, Il17ra^–/–, control, and Il17ra^ΔRTEC mice (n = 4) were evaluated for IL-17RA expression on mature NK (mNK) (liveCD45⁺CD3^–NKG2d⁺CD49b⁺CD94⁺), immature NK (iNK) (liveCD45⁺CD3^–NKG2d⁺CD49b^–CD94⁺), and precursor NK (NKP) (liveCD45⁺CD3^–NKG2d⁺CD49b^–CD94^–) cells by flow cytometry. (B) WT, Il17ra^–/–, control, and Il17ra^ΔRTEC mice were either injected i.p. with LPS (1.5 ng/ml) (n = 4) or left untreated (n = 2). Five hours later, splenic NK cells (gated on liveCD45⁺CD3^–NKp46⁺CD49b⁺CD69⁺) were evaluated for IFNγ production by intracellular cytokine staining. Numbers in the dot plot indicate percentages of cells. In the dot plots, each dot represents individual an mouse, and data are represented as mean ± SD. Data were pooled from 3 and 2 independent experiments for A and B, respectively, and analyzed by 1-way ANOVA. ***P < 0.001.

Figure 4. Early renal innate response in Il17ra^ΔRTEC mice after disseminated candidiasis.

Control, Il17ra^ΔRTEC (n = 8–14) and Il17ra^–/– mice (n = 6) were subjected to systemic C. albicans infection. (A) Fungal burden was measured in the kidneys at days 2 and 5 p.i. Data for surviving Il17ra^–/– mice (n = 4) are shown for day 5 p.i. (B) The kidneys of Il17ra^–/–, control, and Il17ra^ΔRTEC mice (n = 5–6) were evaluated for kidney infiltrating neutrophils (live CD45⁺Ly6G⁺CD11b⁺), macrophages (live CD45⁺F4/80⁺CD11b⁺), and NK cells (live CD45⁺CD3^–NK1.1⁺) at day 2 p.i. (C) Renal transcript expression of Cxcl5, S100a8, and Defb1 was measured by qPCR in control and Il17ra^ΔRTEC mice (n = 7–10) at day 2 p.i. Each dot represents an individual mouse, and data are represented as mean ± SD. Data are pooled from 2 independent experiments for A and B and 3 independent experiments for C and were analyzed by 1-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5. Il17ra^ΔRTEC mice demonstrated aggravated renal damage following systemic C.

albicansinfection. Control and Il17ra^ΔRTEC mice were either subjected to systemic C. albicans infection (n = 6–8) or left uninfected (n = 4). At day 7 p.i., mice were evaluated for (A) histopathology and inflammatory cell influx by H&E staining (original magnification, 100×); (B) serum BUN level at day 7 p.i.; and (C) NGAL and KIM1 expression by IHC. Representative photomicrographs from 2 independent experiments for A and C. Original magnification, 100×. Pooled results from 2 independent experiments for B. Each dot represents an individual mouse, and data are represented as mean ± SD and analyzed by 1-way ANOVA. ***P < 0.001. Scale bars: 400 µm.

Figure 6. Increased apoptosis of RTEC in the absence of IL-17RA signaling following disseminated candidiasis.

Control and Il17ra^ΔRTEC mice were subjected to systemic C. albicans infection (n = 6) or left uninfected (n = 4). The percentages of (A) apoptotic kidney-resident tubular epithelial cells (gated on CD45^–CD133⁺cells) were detected by 7-AAD staining, and (B) percentages of active Caspase-3⁺ kidney-resident tubular epithelial cells (gated on CD45^–CD133⁺ cells) were quantified by DEVD-FMK staining. Numbers in the contour plot reflect percentages of cells. In the dot plots, each dot represents an individual mouse, and data are represented as mean ± SD. Pooled data are from 2 independent experiments and analyzed by 1-way ANOVA. ***P < 0.001; ****P < 0.0001.

Figure 7. Diminished activation of KKS in the C.

albicans–infected kidney ofIl17ra^ΔRTECmice. Control and Il17ra^ΔRTEC mice (n = 8–9) were subjected to systemic C. albicans infection. (A) At day 2 p.i., renal transcript expression of Klk1 and Klk1b26 was measured by qPCR. Each dot represents an individual mouse, and the data are represented as mean ± SD. Data were pooled from 2 independent experiments and analyzed by 2-tailed Student’s t test. (B) Serial kidney sections (n = 6) were stained for Klk1. Original magnification, 100×; scale bars: 400 µm. Representative photomicrographs from 2 independent experiments. (C) Il17ra^ΔRTEC mice (n = 12–15) were treated ± bradykinin (300 nmol/kg/day) starting day –1 (relative to infection) and then daily over 14 days. Control mice were left untreated. Both Il17ra^ΔRTEC and control mice were infected systemically with C. albicans. Sham mice were treated ± bradykinin (n = 3–5). Survival was assessed over 14 days. Data are pooled from 4 independent experiments and analyzed by Log-rank (Mantel Cox) test. (D) WT mice (n = 3–8) were subjected to systemic C. albicans infection. At day 2 p.i., renal transcript expression of Bdkrb1 and Bdkrb2 was quantified by qPCR. Each dot represents individual mouse, and data are represented as mean ± SD. Data are pooled from 2 independent experiments and analyzed by 2-tailed Student’s t test (E) WT mice (n = 10–11) were treated with either Bdkrb1 (R-715; 1 mg/kg/day) or Bdkrb2 (HOE-140; 1 mg/kg/day) antagonists or PBS starting day –1 day relative to infection and then daily over 14 days. Sham mice were treated with the antagonists only (n = 3). Mice were evaluated for survival over 14 days. The data are pooled from 2 independent experiments and analyzed by Log-rank (Mantel Cox) test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.