IL-17 produced by neutrophils regulates IFN-gamma-mediated neutrophil migration in mouse kidney ischemia-reperfusion injury

Abstract

The IL-23/IL-17 and IL-12/IFN-gamma cytokine pathways have a role in chronic autoimmunity, which is considered mainly a dysfunction of adaptive immunity. The extent to which they contribute to innate immunity is, however, unknown. We used a mouse model of acute kidney ischemia-reperfusion injury (IRI) to test the hypothesis that early production of IL-23 and IL-12 following IRI activates downstream IL-17 and IFN-gamma signaling pathways and promotes kidney inflammation. Deficiency in IL-23, IL-17A, or IL-17 receptor (IL-17R) and mAb neutralization of CXCR2, the p19 subunit of IL-23, or IL-17A attenuated neutrophil infiltration in acute kidney IRI in mice. We further demonstrate that IL-17A produced by GR-1+ neutrophils was critical for kidney IRI in mice. Activation of the IL-12/IFN-gamma pathway and NKT cells by administering alpha-galactosylceramide-primed bone marrow-derived DCs increased IFN-gamma production following moderate IRI in WT mice but did not exacerbate injury or enhance IFN-gamma production in either Il17a-/- or Il17r-/- mice, which suggested that IL-17 signaling was proximal to IFN-gamma signaling. This was confirmed by the finding that IFN-gamma administration reversed the protection seen in Il17a-/- mice subjected to IRI, whereas IL-17A failed to reverse protection in Ifng-/- mice. These results demonstrate that the innate immune component of kidney IRI requires dual activation of the IL-12/IFN-gamma and IL-23/IL-17 signaling pathways and that neutrophil production of IL-17A is upstream of IL-12/IFN-gamma. These mechanisms might contribute to reperfusion injury in other organs.

Figure 1. The IL-23/IL-17 pathway contributes to kidney IRI.

(A) mRNA levels of p40 and p19 were measured by real-time PCR in kidneys after 28 minutes of ischemia and exposure to different times of reperfusion. Values are expressed as relative gene expression (compared with GAPDH) in sham-operated samples and IRI samples following different times of reperfusion. n = 3–5. *P < 0.05 compared with sham. (B) Plasma creatinine was measured as an indication of kidney function in mice exposed to sham operation or IRI (ischemia followed by 24 hours of reperfusion). IgG1, WT mice that received 100 μg IgG1 isotype control 18 hours prior to kidney IRI; anti-p19, WT mice that received anti-p19 mAb treatment 18 hours prior to kidney IRI. n = 4–18; *P < 0.05; **P < 0.01; ***P < 0.001. (C) Representative morphology (by H&E staining) of kidney outer medulla from WT, p40^–/–, and p19^–/– sham and IRI mice. (D) Plasma creatinine in WT, Il17r^–/–, and Il17a^–/–, IgG2a, and anti–IL-17A sham and IRI mice after 24 hours of reperfusion. n = 4–9; *P < 0.05; ***P < 0.001. (E) H&E staining of kidney outer medulla from WT, Il17r^–/–, and Il17a^–/– sham and IRI mice after 24 hours of reperfusion. In C and E, arrowheads indicate necrotic tubules. Scale bars: 100 μm. Values are mean ± SEM.

Figure 2. Activation of the IL-23/IL-17A/IL-17R pathway following kidney IRI increases kidney expression of proinflammatory cytokines and chemokines that mediate neutrophil recruitment.

(A and B) Blocking CXCR2 attenuated kidney IRI inflammation. (A) Plasma creatinine levels in WT mice that received anti-CXCR2 goat serum or goat serum control 18 hours and 1 hour prior to sham or IRI. n = 4–6; **P < 0.01. (B) Kidney morphology evaluated by H&E staining. Arrowheads indicate tubular injury. Scale bar: 10 μm. (C and D) Kidney mRNA expression level (C) of IL-23/IL-17 downstream cytokines (Tfna and Il6) and neutrophil chemoattractant chemokines (Cxcl1 and Cxcl2) was measured by real-time PCR at different time points following kidney reperfusion (2, 4, 6, and 24 hours; n = 2–5. *P < 0.05; ***P < 0.001 compared with sham) and (D) in WT, p40^–/–, p19^–/–, Il17a^–/–, and Il17r^–/– mice after 6 hours of kidney reperfusion (n = 2–8; *P < 0.05 compared with KO mice). Values are mean ± SEM.

Figure 3. Immunofluorescent labeling demonstrates recruitment of 7/4+ neutrophils and CXCL1 expression in kidneys following IRI.

(A–D) Panoramic views of kidney through the entire extent of cortex (lateral, left) and medulla (medial, right) were generated by stitching together 4 overlapping images (Adobe Photoshop). Labels at the top delineating approximate boundaries of cortex and inner and outer medulla apply to A and B (C and D are at a slightly higher magnification). (A) A large influx of 7/4⁺ neutrophils (FITC epifluorescence; green) was seen in the kidney outer medulla 24 hours after IRI in WT mice. Some CXCL1 immunoreactivity (Cy3 epifluorescence; red) was evident in medullary tubule cells, but most was associated with infiltrating neutrophils. (B–D) Significantly less neutrophil recruitment and CXCL1 expression were observed in p19^–/–, Il17r^–/–, and Il17a^–/– IRI mouse kidneys. (E–J) Higher magnification of images shows substantial neutrophil infiltration and colocalization (yellow) of CXCL1 and 7/4 immunoreactivity in neutrophils in medulla of WT IRI kidney (E and F) but not in kidneys from the KO mice that were protected from injury (G–J). Nuclei were labeled with DAPI (blue). Scale bars: 100 μm (A–D); 10 μm (F); 40 μm (E, G–J).

Figure 4. IL-17A produced from non-T and non-B bone marrow–derived cells contributes to kidney IRI.

(A) Kidney CD45⁺ cells were isolated from sham and IRI mouse kidneys after 3 hours of reperfusion and restimulated in vitro as described in Methods. Intracellular IL-17A was measured by FACS, and the number of IL-17A–producing cells was evaluated with Caltag Counting Beads. n = 3; ***P < 0.001. (B) Plasma creatinine in WT → WT and Il17a^–/– → WT chimeric mice 24 hours after kidney IRI. n = 4–7; ***P < 0.001. (C) Plasma creatinine after kidney IRI in WT and Rag1^–/– mice that received anti–IL-17A mAb or IgG2a isotype control. n = 3–6; ***P < 0.001. (D) Recruitment of 7/4⁺ cells (green) co-expressing CXCL1 (red) in kidney sections from WT and Rag1^–/– IRI mice treated with isotype control (IgG2a) or anti–IL-17A neutralizing antibody. Nuclei were labeled with DAPI (blue). Scale bar: 50 μm. Values are mean ± SEM.

Figure 5. IL-17A produced by PMNs contributes to kidney IRI inflammation.

(A) Identification of IL-17A–secreting cells in kidneys 3 hours after sham or IRI. Mouse kidney leukocytes were restimulated, and the secreted form of IL-17A was measured as described in Methods. IL-17A–secreting CD45⁺GR-1⁺7-AAD^– cells were measured by FACS. The numbers within each box in the contour diagrams indicate the percentage of CD45⁺7-AAD^– cells gated. (B) WT or Il17a^–/– PMNs were isolated from bone marrow and were adoptively transferred to Il17a^–/– mice at the onset of kidney IRI, and in 1 group (WT → Il17a^–/– + anti–IL-17A) 100 μg anti–IL-17A mAb was administered at the onset of IRI. Plasma creatinine was measured following 24 hours of kidney reperfusion. Values are mean ± SEM. n = 4–7; **P < 0.01; ***P < 0.001.

Figure 6. Both IL-12/IFN-γ and IL-23/IL-17 pathways are necessary in activated NKT cell–promoted kidney IRI inflammation.

(A–C) Plasma creatinine levels in mice 24 hours after kidney IRI. (A) Plasma creatinine levels in WT, p35^–/–, and Ifng^–/– mice or in WT mice after administration of anti–IFN-γ mAb or isotype control IgG1. n = 2–11; ***P < 0.001. (B) Plasma creatinine levels after adoptive transfer of 6 × 10⁵ unprimed DCs or BMDC-αGalCer (DC-αGC) to NKT cell–deficient WT mice (Cd1d^–/– and Ja18^–/–) or Ifng^–/– mice. BMDC-aGalCer was used to specifically activate NKT cells, which promoted moderate kidney IRI (24 minutes of ischemia) in WT mice. n = 4–12; ***P < 0.001 compared with WT BMDC-αGalCer IRI. (C and D) Adoptive transfer of WT BMDC-αGalCer to WT or KO mice prior to sham operation or moderate (24 minutes ischemia) IRI. (C) Plasma creatinine in WT mice or mice with disrupted IL-12/IFN-γ (p40^–/–, p35^–/–, Ifng^–/–) or IL-23/IL-17 pathway (p40^–/–, p19^–/–, Il17a^–/–, Il17r^–/–). n = 4–8; **P < 0.01; ***P < 0.001 compared with WT IRI. (D) The number of IFN-γ–producing GR-1⁺ neutrophils in the kidney was determined by FACS intracellular staining analysis in WT and IL-12/IFN-γ KO and IL-23/IL-17A/IL-17R KO mice. n = 4–6; ***P < 0.001 compared with KO IRI mice. (E) Plasma IFN-γ levels (measured by ELISA) in mice after adoptive transfer of DCs or BMDC-αGalCer. n = 2–7; **P < 0.01, compared with BMDC-αGalCer–treated IRI KO mice. All values are mean ± SEM.

Figure 7. IL-17A is upstream of IFN-γ production in kidney IRI.

Plasma creatinine (A) and neutrophil infiltration (B) in Ifng^–/– mice that received PBS or 50 ng rm IL-17A 1 hour before kidney IRI or Il17a^–/– mice treated with PBS (vehicle) or 30,000 U rm IFN-γ 1 hour prior to kidney IRI or sham surgery. (A) n = 4–6; ***P < 0.001. (B) Kidney CD11b⁺GR-1⁺ neutrophils were measured by FACS. n = 2–4; *P < 0.05. Values are mean ± SEM.

Figure 8. Model summarizing the role of the IL-12/IFN-γ and IL-23/IL-17 axes of the innate immune response in kidney IRI.

(A) Injury to kidney epithelial cells early (6 hours) following IRI promotes inflammation by increasing pro-inflammatory cytokine/chemokine production, including neutrophil chemoattractant chemokines (CXCL1 and CXCL2). CXCL1 and CXCL2 mediate PMN recruitment and kidney injury through the chemokine receptor CXCR2 (expressed mainly on neutrophils). Autocrine CXCL1/2 production by infiltrating PMNs likely stimulates additional infiltration. (B) Both IL-12/IFN-γ and IL-23/IL-17 pathways are activated in kidney IRI. Kidney resident DCs initiate the immune response following kidney IRI by secreting IL-12 and IL-23. DCs activate NKT cells by CD1d-mediated glycolipid presentation and CD40/CD40L costimulation. IL-12 mediates NKT cell activation and IFN-γ production, which further stimulates neutrophil IFN-γ production and infiltration of PMNs and other immune cells (CD4⁺ and NKT cells and macrophages; not shown) that contribute to kidney injury. IL-23 is also involved in NKT cell activation (our unpublished observations). IL-23 (with TGF-β and IL-6; not shown) is known to induce Th17 cell differentiation, but neutrophils, not Th17 cells, are the predominant source of IL-17A in the innate immune response in kidney IRI. IL-23 and its downstream cytokine IL-17A induce pro-inflammatory cytokine (IL-6, IL-1β, TNF-α) and chemokine (CXCL1/2) (not shown) production to promote kidney inflammation. (C) The IL-23/IL-17 axis is proximal to and necessary for IL-12/IFN-γ–mediated tissue injury through NKT cell activation. IL-17A produced from PMNs regulates NKT cell activation, IFN-γ production, PMN infiltration and tissue injury in kidney IRI. Therefore, in addition to IL-12/IFN-γ, IL-23 and IL-17 signal pathways also participate in the innate immune response to IRI by stimulating activated NKT cell–mediated kidney inflammation and PMN infiltration.