IL-17 produced by neutrophils regulates IFN-γ–mediated neutrophil migration in mouse kidney ischemia-reperfusion injury (original) (raw)
The IL-23/IL-17 axis is essential for the inflammatory response to kidney IRI. During the period of reperfusion following an ischemic event in kidney as well as numerous other organs, activation of the inflammatory system accompanies a complex immune cascade in response to ischemic injury. Increases in cytokines and chemokines mediate the influx of immune cells into kidney after injury. To test the involvement of the IL-23 cytokine pathway in injured kidneys, we initially investigated mRNA expression levels of the 2 subunits of IL-23, p40 and p19, in kidney by real-time PCR at different reperfusion times following kidney ischemia and sham operation (control). As shown in Figure 1A, both p40 and p19 mRNA expression increased significantly between 4 and 6 hours after IRI compared with sham-operated control mice. We then compared kidney function and pathology after IRI in p40–/–, p19–/–, and WT mice. Following 24 hours of reperfusion, there was a marked increase in plasma creatinine in WT mice, but p40–/– and p19–/– mice were protected from kidney injury as indicated by lower plasma creatinine levels (Figure 1B), less tubular necrosis (Figure 1C), and lower acute tubular necrosis (ATN) scores (Supplemental Table 1; supplemental material available online with this article; doi:10.1172/JCI38702DS1). The protective effect observed in p19–/– mice was also found when WT mice were pretreated with anti-p19 neutralization mAb (100 μg) 18 hours prior to IRI compared with isotype IgG1-treated mice (Figure 1B). IL-23 promotes the production of IL-17A, a member of a family of cytokines whose downstream mediators regulate neutrophil recruitment and function. We hypothesized that increased IL-23 expression drives the production of IL-17A to promote kidney injury through its receptor, IL-17R, following ischemia-reperfusion. The protective effects observed in kidney function and morphology in Il17a–/– and Il17r–/– mice after IRI compared with WT mice demonstrated that the IL-17 signaling pathway was involved in kidney IRI (Figure 1, D and E). Protection was also observed in WT mice pretreated with neutralization anti–IL-17A mAb (Figure 1D).
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.
Downstream pro-inflammatory cytokines and chemokines associated with the IL-23/IL-17 pathway mediate neutrophil recruitment in kidney IRI. Infiltration of neutrophils is one of the hallmarks of kidney IRI and may be mediated by production of downstream pro-inflammatory cytokines/chemokines of the IL-23/IL-17 pathway. Blocking neutrophil infiltration by using an anti-CXCR2 antibody prevented the increase in plasma creatinine levels and tubular necrosis in the kidney outer medulla following kidney IRI (Figure 2, A and B). Following kidney IRI, expression of the pro-inflammatory cytokines IL-6 and TNF-α was elevated in kidneys as early as 2 hours following reperfusion and increased at 4–6 hours (Figure 2C). Expression of CXCL1 and CXCL2, chemokines known to mediate neutrophil migration, increased compared with sham at 2 and 4 hours after reperfusion, respectively. CXCL1 remained elevated 24 hours following reperfusion (Figure 2C). No significant change in Cxcl5 mRNA expression was identified in the reperfused kidneys compared with sham (data not shown). The rise in neutrophil chemotactic factors preceded the known time course (22) for neutrophil infiltration into kidneys following IRI. To investigate the role of the IL-23/IL-17 axis in mediating cytokine and chemokine production following kidney IRI, Il6, Il1b, Tnfa, Cxcl1, and Cxcl2 mRNA levels were measured by real-time PCR in WT and KO mice after 6 hours of reperfusion. The increase in expression of IL-6, TNF-α, CXCL1, and CXCL2 observed in WT mouse kidneys after IRI was not found in p19–/–, Il17r–/–, or Il17a–/– mice (Figure 2D).
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.
The observed effects on cytokine and chemokine production prompted us to evaluate a role of the IL-23/IL-17 pathway in neutrophil infiltration after IRI. The marked increase in infiltration of CD11b+GR-1+ neutrophils in kidneys of WT mice after IRI was diminished in p40–/–, p19–/–, Il17r–/–, and Il17a–/– mouse kidneys after IRI, as revealed by FACS analysis (Supplemental Figure 1). Immunofluorescence showed CXCL1 production mainly in kidney tubule cells at 6 hours following reperfusion (data not shown), but intense labeling for CXCL1 was observed in infiltrating 7/4+ neutrophils in kidneys of WT mice 24 hours after IRI (Figure 3, A, E, and F), primarily in the outer medulla. A decrease in neutrophil recruitment in kidneys from p19–/–, Il17r–/–, and Il17a–/– mice that were protected from IRI was associated with low CXCL1 (Figure 3, B–D and G–J) and CXCL2 expression (data not shown). Collectively, these data strongly support the causal relationship between IL-23/IL-17 in neutrophil recruitment and the inflammatory immune response in kidneys after IRI.
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).
Neutrophils are the major source of IL-17A in kidneys following IRI. We next performed a series of experiments to identify the cells in kidneys following IRI that produce IL-17A. After kidney ischemia and only 3 hours of reperfusion, by FACS analysis of intracellularly labeled cells, we found more IL-17A–producing CD45+ cells in IRI kidneys relative to sham (Figure 4A). Il17a–/– → WT and WT → WT bone marrow chimeras were created to examine the contribution of bone marrow–derived cells as the source of IL-17A in kidney IRI. Significant morphological (data not shown) and functional protection from kidney IRI was found in Il17a–/– → WT IRI mice compared with WT → WT mice. Plasma creatinine increased significantly in WT → WT mice after IRI, but not in Il17a–/– → WT IRI mice, which lacked IL-17A in bone marrow cells (Figure 4B). Similarly, fewer infiltrating neutrophils were found in kidneys of the protected chimeric mice by FACS analysis and immunostaining (data not shown). Therefore, we concluded that IL-17A–producing bone marrow–derived cells contribute to kidney injury. We then pretreated T cell– and B cell–deficient Rag1–/– mice with anti–IL-17A mAb before IRI and found that they had a reduction in injury similar to that of WT mice pretreated with anti–IL-17A mAb (Figure 4C), with less 7/4+ neutrophil infiltration and neutrophil CXCL1 expression (Figure 4D) following kidney IRI. These in vivo studies suggested that cells other than T, B, and NKT cells produce IL-17A and are involved in kidney IRI.
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.
To identify the leukocyte subset, we used an IL-17 secretion assay to simultaneously detect IL-17A–producing cells and their phenotype. Interestingly, IL-17A was secreted from the recruited neutrophils (Figure 5A) but not from CD4 or CD8 T cells or NK1.1+ cells (Supplemental Figure 2). Finally, to demonstrate functional significance of IL-17 production by neutrophils in kidney IRI, we adoptively transferred to recipient Il17a–/– mice neutrophils from the bone marrow of WT (WT → Il17a–/– mice) or Il17a–/– mice (Il17a–/– → Il17a–/– mice). Kidney injury was reconstituted in WT → Il17a–/– but not Il17a–/– → Il17a–/– mice (Figure 5B). Furthermore, in WT → Il17a–/– mice the reconstituted injury was blocked with 100 μg anti-mouse IL-17A mAb. These data demonstrate that neutrophils, rather than T, NKT, or B cells, are the major source of IL-17A production involved in kidney IRI.
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.
Impaired IFN-γ production by GR-1+ neutrophils in mice with deficient IL-23/IL-17A/IL-17R pathways. We have shown previously that GR-1+ neutrophils are the major immune source of IFN-γ following kidney IRI (20) and in the current study we showed that IL-23/IL-17 is important for neutrophil recruitment following IRI. Therefore, we investigated whether disruption of the IL-23/IL-17 pathway alters the IFN-γ–mediated immune response to kidney IRI. As expected, fewer IFN-γ–producing GR-1+ neutrophils infiltrated the kidney after IRI in IL-12–deficient (p35–/–) mice than in WT mice, but this was also found in p40–/–, p19–/–, Il17a–/–, and Il17r–/– mice (Table 1). Thus, blocking either the IL-23 or IL-17 pathway reduced not only neutrophil recruitment but also neutrophil IFN-γ production, imparting an important role of the IL-17 pathway in IFN-γ–mediated kidney IRI.
IFN-γ–producing GR-1+ neutrophils in WT, _p35_–/–, _p40_–/–, _p19_–/–, _Il17r_–/–, and _Il17a_–/– mouse kidneys
Both IL-12/IFN-γ and IL-23/IL-17 pathways are involved in activated NKT cell–mediated inflammation in kidney IRI. Previously we showed that CD1d-restricted NKT cell activation contributes critically to kidney IRI through IFN-γ production and regulation of neutrophil infiltration (20). Here we further demonstrate the involvement of the IL-12/IFN-γ pathway in IRI-induced kidney inflammation by using p35–/– and Ifng–/– mice. Compared with WT mice, ATN scores (Supplemental Table 1) and plasma creatinine levels (Figure 6A) were lower 24 hours after IRI in mice lacking p35 and IFN-γ signaling, and plasma creatinine was significantly lower following kidney IRI in mice treated with neutralizing anti–IFN-γ antibody (Figure 6A) compared with WT mice treated with IgG1 isotype control. In addition, following kidney IRI we observed an 81% reduction of CD11b+GR-1+ neutrophils infiltrating the kidney both in mice lacking IFN-γ (P < 0.05; n = 3–9) and in mice lacking p35 (P < 0.05; n = 5–9) compared with WT IRI mice, thus indicating that the IL-12/IFN-γ pathway is involved in neutrophil recruitment into kidney following IRI. Furthermore, IFN-γ deficiency abrogated the IRI-induced increase in pro-inflammatory cytokine and chemokine mRNA expression (Supplemental Table 2). Therefore, we concluded that both the IL-12/IFN-γ and IL-23/IL-17 pathways mediate neutrophil recruitment in kidney IRI.
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.
Our results showing that both the IL-12/IFN-γ and IL-23/IL-17 pathways mediate neutrophil recruitment in kidney IRI suggest that these pathways may also be important in NKT cell–mediated kidney injury. We posited that production of IL-12 and CD1d presentation of glycolipids by ischemia-reperfusion–activated DCs trigger and stimulate NKT cell–mediated kidney injury, and we further hypothesized that IL-23/IL-17 contributes to this process. To examine the interaction of NKT cells with IL-12/IFN-γ and IL-23/IL-17 pathways in promoting the kidney inflammatory response, we used bone marrow–derived DCs (BMDCs) primed with α-galactosylceramide (BMDC-αGalCer) to activate kidney NKT cells before a moderate (24-minute) subthreshold level of ischemic injury. This strategy has been used widely to modulate NKT cell activation in different mouse models and human disease, and substantial IFN-γ production, which contributes to tissue injury and inflammation, has been found after administration of BMDC-αGalCer (23–28). Compared with WT mice that received unprimed BMDCs and showed no significant change in kidney function after a moderate ischemic injury, a large increase in the level of plasma creatinine was found in WT mice treated with BMDC-αGalCer (Figure 6B). However, creatinine was not elevated by BMDC-αGalCer and IRI in mice deficient in NKT cells (Cd1d–/– and Ja18–/– mice) or Ifng–/– (Figure 6B), but it was increased in Tnfa–/– mice (1.55-fold ± 0.12-fold vs. 1.85-fold ± 0.66-fold compared with sham for Tnfa–/– and WT mice, respectively; P = NS). These results demonstrate that BMDC-αGalCer–mediated specific NKT cell activation exacerbates moderate kidney IRI inflammation through IFN-γ–dominant cytokine production.
To further investigate whether kidney injury by specific NKT cell activation is mediated by the IL-23/IL-17 pathway, we also adoptively transferred WT BMDC-αGalCer to p40–/–, p19–/–, Il17a–/–, and Il17r–/– mice (IL-23/IL-17A/R pathway) and, for comparison with the IL-12/IFN-γ pathway, to p35–/– and Ifng–/– mice. Interestingly, only WT mice that received BMDC-αGalCer before surgery showed significant functional (Figure 6C) and morphological injury (H&E staining; data not shown) and an anticipated infiltration of neutrophils (data not shown), including a population of GR-1+IFN-γ+ neutrophils in kidney (Figure 6D), following moderate kidney IRI. In addition, adoptive transfer of BMDC-αGalCer prior to surgery produced a dramatic increase in plasma IFN-γ levels in WT mice, which was consistent with higher plasma creatinine levels following kidney IRI, compared with mice deficient in NKT cells, IL-12, IL-23, IL-17, or IFN-γ (Figure 6E). These results suggest that both IL-12/IFN-γ and IL-23/IL-17 signal pathways are necessary for activated NKT cell–mediated kidney inflammation, neutrophil infiltration, and neutrophil IFN-γ production following IRI and that IL-23/IL-17 signaling regulates NKT cell IFN-γ production.
IL-17A is upstream of IFN-γ in the inflammatory process in kidney IRI. To further define in vivo the relationship between the IL-17 pathway and IFN-γ in kidney IRI, an optimal dose of exogenous recombinant mouse (rm) IL-17A (50 ng) was administered to Ifng–/– mice 1 hour prior to kidney surgery. The protection from injury observed in the control Ifng–/– mice (which received PBS) was not reconstituted by administration of rm IL-17A, as plasma creatinine did not increase in either group of mice after IRI relative to the sham groups (Figure 7A). Conversely, plasma creatinine increased significantly in Il17r–/– (data not shown) or Il17a–/– mice that received rm IFN-γ (30,000 U/mouse 1 hour before IRI) compared with vehicle-treated mice (Figure 7A). Similarly, reconstitution of the infiltration of CD11b+GR-1+ neutrophils after IRI was revealed by FACS analysis of kidneys from Il17a–/– mice given rm IFN-γ but not in Ifng–/– mice given rm IL-17A (Figure 7B). These results indicate that IL-17A is upstream of IFN-γ in the inflammatory response that occurs in kidney IRI.
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.
Taken together with our results described above (that mice deficient in not only p35/IFN-γ but also the IL-23/IL-17A/R pathway showed decreased neutrophil recruitment and IFN-γ production after IRI), these results suggest that cross-talk between the IL-12/IFN-γ and IL-23/IL-17 pathways promotes the cytokine cascade immune responses in kidney IRI.