IL-33–Mediated Expansion of Type 2 Innate Lymphoid Cells Protects from Progressive Glomerulosclerosis

Abstract

Innate lymphoid cells (ILCs) have an important role in the immune system's response to different forms of infectious and noninfectious pathologies. In particular, IL-5– and IL-13–producing type 2 ILCs (ILC2s) have been implicated in repair mechanisms that restore tissue integrity after injury. However, the presence of renal ILCs in humans has not been reported. In this study, we show that ILC populations are present in the healthy human kidney. A detailed characterization of kidney-residing ILC populations revealed that IL-33 receptor–positive ILC2s are a major ILC subtype in the kidney of humans and mice. Short-term IL-33 treatment in mice led to sustained expansion of IL-33 receptor–positive kidney ILC2s and ameliorated adriamycin-induced glomerulosclerosis. Furthermore, the expansion of ILC2s modulated the inflammatory response in the diseased kidney in favor of an anti-inflammatory milieu with a reduction of pathogenic myeloid cell infiltration and a marked accumulation of eosinophils that was required for tissue protection. In summary, kidney-residing ILC2s can be effectively expanded in the mouse kidney by IL-33 treatment and are central regulators of renal repair mechanisms. The presence of ILC2s in the human kidney tissue identifies these cells as attractive therapeutic targets for CKD in humans.

CKD affects around 10% of the Western population and is a major risk factor for cardiovascular mortality.¹ Regardless of the underlying cause, glomerular injury regularly results in proteinuria and progressive glomerulosclerosis with deteriorating kidney function.² In recent years, it has become evident that the immune system’s response to the initial injury can critically contribute to the progression of kidney damage. However, regulatory components of the immune system can also promote resolution of kidney injury and limit chronic inflammation.³ To develop novel therapeutic strategies for progressive CKD, it is therefore necessary to identify the immune cells and mediators that can shift the balance from chronic kidney inflammation toward resolution and restoration of tissue integrity.

Innate lymphoid cells (ILCs) are a newly described group of leukocytes that is defined by its lymphoid morphology and antigen-independent activation. According to a recently proposed nomenclature, ILCs can be subdivided into four groups that include conventional natural killer (NK) cells and three subsets of “helper-like” ILCs, differing in their tissue localization and functional characteristics.⁴ The helper-like ILC subsets are defined by their distinct profiles of cytokine production and transcription factor usage and referred to as T-bet⁺IFN-γ⁺ ILC1s, GATA3⁺IL-5⁺IL-13⁺ ILC2s, and ROR-γt⁺IL-17A⁺ and/or IL-22⁺ ILC3s. These ILC subtypes have mainly been studied in barrier organs, such as the gut, lung, and skin, where they promote tissue homeostasis and defense against different classes of pathogens, but also contribute to allergic and autoimmune diseases in mice and humans.^5,6 Recent evidence suggests that ILC2s especially are central regulators of repair mechanisms that are aimed at restoring homeostasis after acute injury in the lung,^7,8 intestine,⁹ and skin.¹⁰

The question of whether helper-like ILC subsets reside in the kidney of humans and mice and, if so, whether they might be therapeutically exploited in chronic kidney inflammation, has not been addressed so far.

In this study, we identify and characterize resident ILC populations in the human kidney for the first time. We further show that IL-33 receptor-positive (IL-33R⁺) ILC2s are a major ILC subtype in the healthy human and murine kidney. In line with their tissue regenerative capacity, expansion of ILC2s by short-term IL-33 treatment ameliorated progressive glomerulosclerosis and loss of kidney function induced by adriamycin injection in mice, a model of chronic kidney injury. ILC2s modulated the inflammatory response in the diseased kidney in favor of an anti-inflammatory milieu with a reduction of pathogenic neutrophils and mononuclear phagocytes and an increase of tissue-protective mediators, such as eosinophils and alternatively activated macrophages. We further show that the accumulation of eosinophils was required for the tissue-protective effect of ILC2s. Thus, we established that targeting ILC2s is a promising therapeutic strategy to promote tissue regeneration in progressive kidney disease.

Results

Distribution of Helper-Like ILC Subtypes Residing in the Naïve Human and Murine Kidney

Helper-like ILCs (hILCs) are a largely tissue-resident population of lymphocytes that varies in frequency between different organs and shows a tissue-specific distribution of the ILC1, ILC2, and ILC3 subtypes.¹¹ So far, there are no reports on resident ILC populations in the human kidney. Flow cytometric analysis of leukocytes isolated from healthy human kidney cortex showed that approximately 0.4% of renal lymphocytes displayed an hILC phenotype, defined as lineage marker–negative (Lin⁻), IL-7Rα–positive (CD127⁺). The vast majority of the Lin⁻CD127⁺ ILC population in the human kidney expressed the pan-hILC marker CD161 (Figure 1, A and C). Further subtyping revealed that the kidney-residing Lin⁻CD127⁺CD161⁺ population of hILCs contained CRTH2⁺ ILC2s, NKp44⁺, and NKp44⁻ CRTH2⁻CD117⁺ ILC3s, as well as a smaller population of CRTH2⁻CD117⁻NKp44⁻ ILC1s (Figure 1, A and D). Similar to the results obtained in human kidney tissue, flow cytometry of leukocytes isolated from the kidney of naïve C57BL/6 mice revealed a population of Lin⁻CD127⁺ hILCs that comprised around 0.8% of renal lymphocytes (Figure 1, B and C). Further characterization of hILCs residing in the naïve mouse kidney by transcription factor staining showed a distribution of ILC subtypes similar to the mouse lung,^8,12 with the majority of around 80% being GATA-3⁺ ILC2s and only minor populations of the (NK cell receptor–negative) ROR-γt⁺ ILC3s and NK1.1⁺ and NK1.1⁻T-bet⁺ ILC1 subsets (Figure 1, B and D). Of note, the T-bet⁺ ILC1 subset was negative for the NK cell transcription factor Eomes, excluding a contaminating NK cell population in the ILC1 gate (data not shown). Consistent with the ILC subtype distribution defined by transcription factor staining and surface marker expression in mice, kidney ILCs restimulated with phorbol 12-myristate 13-acetate and ionomycin mainly produced the type 2 cytokines IL-5 and IL-13, whereas IFN-γ and IL-17A were produced by smaller fractions (Supplemental Figure 1). Similar to ILC2 populations from other tissue locations, human and mouse ILC2s in the kidney expressed the IL-33 receptor T1/ST2 and the IL-2 receptor high-affinity chain CD25 (Figure 1E). Thus, despite a discrepancy in the abundance of the ILC3 subsets between the healthy mouse and human kidney, IL-33R⁺ ILC2s are a major kidney-residing hILC subset in both species.

Figure 1.

IL-33R⁺ ILC2s are a major ILC population in the human and murine kidney. Flow cytometric analyses of leukocytes isolated from healthy human and mouse (C57BL/6) kidney tissue. (A and B) Representative plots of human kidney cells (A) stained for CD45, CD127 (IL-7Rα), CD161, CRTH2, CD117 (c-kit), NKp44, and lineage markers (Lin = CD1a, CD3, CD4, CD11c, CD14, CD16, CD19, CD34, CD56, CD94, CD123, γδ-TCR, αβ-TCR, FcεR1α, and BDCA1); and mouse kidney cells (B) stained for CD45, CD127, CD90.2, GATA-3, T-bet, ROR-γt, Eomes, NK1.1, and lineage markers (Lin = CD3, CD4, CD8, β-TCR, γδ-TCR, CD19, CD11b, CD11c, GR-1, CD49b, Ter119). T-bet⁺ ILC1s were Eomes⁻, excluding a contaminating NK cell population in the ILC1 gate. Numbers indicate the percentage of cells in each gate. (C) Frequency of human and mouse ILCs in percentage of total CD45⁺ lymphocytes. (D) Frequency of the ILC1, ILC2, and ILC3 subtypes within the total ILC population (human total ILC: CD127⁺Lin⁻CD161⁺; mouse total ILC: CD127⁺Lin⁻). (E) Representative flow cytometry analysis of IL-33R (T1/ST2) and CD25 expression on kidney-residing ILC2s in humans and mice. The mouse data represent at least three independent experiments with similar results. Symbols represent individual data points and the horizontal lines indicate the median.

Effective Expansion of Kidney-Residing ILC2s by Short-Term IL-33 Treatment

In order to investigate the function of ILC2s in a kidney disease model, we aimed at establishing a strategy to manipulate their abundance in the kidney. Thus, we addressed the question of how kidney IL-33R⁺ ILC2s respond to short-term IL-33 treatment, a treatment that has been used to expand ILC2s residing in other tissue locations.^13–15 Analysis of leukocytes isolated from the kidneys of C57BL/6 mice 7 days after treatment with four daily doses of recombinant IL-33 (400 ng intraperitoneally each) showed a massive increase in GATA-3⁺Lin⁻ ILC2 frequencies and numbers as compared with PBS-treated controls (Figure 2, A and B). IL-33–induced ILC2 accumulation in the kidney was maintained at a high level for up to 8 weeks after a single course of four IL-33 injections, whereas the ILC2 increase was more transient in the spleen (Figure 2C). In the kidney, ILC2 accumulation was accompanied by a sustained increase of Il5 and Il13 mRNA expression (Figure 2D). To assess a potential effect of IL-33 treatment on other kidney-residing leukocyte subsets, we analyzed IL-33R expression on various lymphocytic and myeloid cell populations in the naïve C57BL/6 kidney. These analyses showed that, among leukocyte populations in the murine kidney, high-level IL-33 receptor expression is unique to ILC2s (see Figure 1E, Supplemental Figure 2). However, although eosinophils lacked expression of the IL-33R, they were strongly expanded after IL-33 treatment (Figure 2E), suggesting an indirect effect mediated by ILC2-derived IL-5.¹⁶ As compared with ILC2s and eosinophils, leukocyte populations in the kidney, spleen, and peripheral blood were only modestly altered in response to IL-33 treatment (Figure 2E, Supplemental Figures 2B and 3, A and B). Consistent with previous reports about IL-33 responsiveness of a subset of regulatory T cells,¹⁷ we also observed a moderate increase of Treg numbers in the kidney, spleen, and peripheral blood after IL-33 treatment (Figure 2E, Supplemental Figure 3, A and B). However, Tregs isolated from the spleens of IL-33–treated mice showed a similar suppressive capacity, as compared with Tregs from PBS-treated controls (Supplemental Figure 3C). Taken together, we were able to establish a 4-day IL-33 treatment course as an efficient tool to induce a sustained increase in ILC2 abundance in the murine kidney.

Figure 2.

Sustained expansion of kidney-residing ILC2s after short-term IL-33 treatment. C57BL/6 mice were treated with IL-33 (400 ng intraperitoneally on four consecutive days) or PBS. (A) Representative flow cytometry of leukocytes isolated from the kidney at day 7 after start of treatment. Plots are gated for CD45⁺ lymphoid cells and numbers indicate the percentage of events in the gate. (B) Frequency and absolute number of ILC2s (Lin⁻GATA-3⁺) in the kidneys at day 7 (n=8 per group). (C) Absolute numbers of ILC2s in the kidney and spleen in PBS-injected controls and at weeks 1–13 after start of IL-33 treatment (n=3–7 per IL-33–treated group, n=12 for controls). (D) Quantitative RT-PCR analysis of Il5 and Il13 mRNA transcripts in the kidneys of IL-33–treated mice relative to PBS-injected controls (numbers as in [C]). (E) Increase in absolute cell numbers of the indicated leukocyte subsets in kidneys of IL-33–treated mice relative to PBS-injected controls at day 7 (n=4–10 per IL-33–treated group, n=5 for controls). Data in (B–E) are pooled from two independent experiments with similar results. Symbols in (B, C, and E) represent individual data points and the horizontal lines indicate the median. Symbols in (D) represent mean±SEM (*P<0.05, **P<0.01, ***P<0.001).

Localization of ILC2s in the Kidney

In the next step, we wanted to analyze the localization of ILC2s in the kidney. In flow cytometric analyses, we established that a combination of CD127 and GATA-3 reliably identifies ILC2s in the kidney, because other GATA-3⁺CD127⁺ cells (mainly CD4⁺) were <5% of the total GATA-3⁺CD127⁺ cells in controls and IL-33–treated mice (Figure 3, A and B). Immunohistochemical costaining for GATA-3 and CD127 clearly identified GATA-3⁺CD127⁺ ILC2s in the tubulointerstitial compartment of the kidney of control and IL-33–treated wildtype mice and in IL-33–treated Rag1^−/− mice (Figure 3C). More detailed analyses of the spatial distribution of ILC2s in IL-33–treated Rag1^−/− mice by including lectin staining as an endothelial marker revealed that they can be found in the tubulointerstitial location (both distant from and close to peritubular capillaries), as well as in the glomerular tuft (Figure 3D).

Figure 3.

ILC2s are localized in the glomerular and tubulointerstitial comparment. (A) Representative flow cytometric analyses of kidney leukocytes isolated from wildtype C57BL/6 mice at day 7 after IL-33 (400 ng intraperitoneally on four consecutive days) or PBS treatment. Left panel is gated for CD45⁺ lymphoid cells. Numbers indicate the percentage of cells in the gate or quadrants. (B) Percentage of ILC2s (Lin⁻CD4⁻) and CD4⁺ Th2 cells (Lin⁺CD4⁺) in the CD127⁺GATA-3⁺ gate in PBS- (n=3) and IL-33–treated (n=5) mice. (C) Representative confocal microscopy images of kidney sections from PBS- and IL-33–treated wildtype mice, as well as from IL-33–treated Rag1^−/− mice costained for CD127 and GATA-3. ILC2s (arrows) are found in wildtype and Rag1^−/− mice, whereas CD127-single–positive T cells (arrowheads) are absent in Rag1^−/− mice. (D) Representative confocal microscopy images of kidney sections of IL-33–treated Rag1^−/− mice costained for CD127, GATA-3, and endothelial lectin binding. ILC2s (arrows) are found in the tubulointerstitial compartment and within the glomerular tuft (dotted line). Symbols in (B) represent individual data points and the horizontal lines indicate the median. Data in (A and B) are representative of at least three independent experiments with similar results.

IL-33–Mediated ILC2 Expansion Ameliorates Progressive Glomerulosclerosis

ILC2s have been described to maintain tissue integrity and promote repair functions in barrier organs, such as the lung, skin, and intestine.^7–10 In order to investigate if the IL-33–mediated expansion of ILC2s could be used as a therapeutic strategy to alter the course of CKD, we induced adriamycin-induced nephropathy (AN) in BALB/c mice, a model of progressive glomerulosclerosis with proteinuria.¹⁸ IL-33 treatment was started at day 5 after disease induction (Figure 4A), a time point at which the initial glomerular damage is established. Flow cytometric analyses 2 weeks after disease induction confirmed the strong and persistent expansion of GATA-3⁺ ILC2s in kidneys of IL-33–treated animals, whereas the diseased PBS-treated mice showed ILC2 frequencies and numbers comparable to naïve controls (Figure 4, B and C). To investigate if the IL-33–induced ILC2 accumulation affects the course of CKD, we assessed parameters of kidney damage at day 14 after disease induction. Periodic acid–Schiff (PAS)–stained kidney sections showed a severe glomerular and tubular pathology in PBS-treated mice that was significantly attenuated in the IL-33–treated group (Figure 4D), as shown by reduced glomerular and tubulointerstitial injury scores (Figure 4E). In line, analyses of kidney function parameters showed significantly reduced albuminuria and cholesterol levels, as laboratory markers of nephrotic syndrome, as well as significantly reduced blood urea nitrogen levels, as a marker of renal failure, in IL-33–treated mice (Figure 4, F and G). Thus, IL-33 administration leads to a sustained expansion of ILC2s and, even if initiated 5 days after induction of glomerular damage, significantly attenuates the course of progressive glomerulosclerosis.

Figure 4.

IL-33 treatment ameliorates the clinical course of AN. (A) PBS or IL-33 (400 ng) was injected intraperitoneally on days 5–8 after induction of AN in BALB/c mice. Mice were analyzed at day 14. (B) Representative flow cytometry plots and (C) absolute numbers of Lin⁻GATA-3⁺ ILC2s in the kidney of naïve control mice (n=4) and both AN groups (n=13–16). Numbers in B indicate the percentage of cells in the gate. (D) Representative photographs (original magnification, ×200) of PAS-stained kidney sections, (E) histopathologic quantification of glomerular and tubulointerstitial damage, and (F) analyses of renal function parameters in naïve control mice (n=6), PBS-treated mice with AN (n=20), and IL-33–treated mice with AN (n=17). Data are pooled from at least three independent experiments with similar results. Symbols represent individual data points and the horizontal lines indicate the median. (*P<0.05, **P<0.01, ***P<0.001).

IL-33 Treatment Induces a Tissue-Protective Type 2 Response in the Kidney

In order to investigate the mechanisms underlying IL-33–mediated tissue protection, we performed a detailed analysis of the kidney inflammatory milieu in IL-33– and PBS-treated mice with AN. In IL-33–treated animals, positivity of ILCs for IL-5 and IL-13 was >50% (Figure 5A) and the absolute number of IL-5⁺IL-13⁺ ILCs was significantly increased (Figure 5B). In line, levels of renal Il5 and Il13 mRNA transcripts were significantly elevated in IL-33–treated mice (Figure 5C), whereas IFN-γ, TNF-α, and GM-CSF expression was downregulated. We did not observe upregulation of the growth factor amphiregulin, which is secreted by ILC2 in other tissue locations, in the IL-33–treated group and, regardless of the treatment group, IL-4 (and also IL-17A) was undetectable in most samples. Consistent with the shift of the cytokine milieu toward a type 2 response, mRNA expression of macrophage activation markers in the kidney parenchyma was skewed toward an alternatively activated phenotype, with increased expression of arginase-1 and downregulation of the inducible nitric oxide synthase (Figure 5D). It is, however, noteworthy that arginase-1 can also be produced by ILC2s themselves,¹⁹ which most likely contributes to the increased Arg1 expression in IL-33–treated mice. mRNA expression analysis of a panel of chemokines showed a significant reduction of the neutrophil attractants CXCL1 and CXCL2, as well as of the T cell and monocyte attractant CCL5. Other chemokines involved in CD4⁺ T cell (CCL20, CXCL10) and eosinophil (CCL11) migration remained unchanged (Figure 5E). The most striking change in the cellular infiltrate, beside the massive accumulation of ILC2s, was the significant increase in eosinophil abundance (Figure 5F). In accordance with the reduced levels of CXCL1, CXCL2, and CCL5, we observed reduced infiltration of neutrophils and F4/80^intCD11b^hi mononuclear phagocytes (Figure 5, G and H), which have both been implicated in the progression of renal tissue damage in models of inflammatory kidney diseases.^20,21 Quantification of neutrophils in kidney sections stained for GR-1 (Ly6G/C) confirmed their reduced accumulation in the IL-33–treated group (Figure 5I).

Figure 5.

IL-33 treatment induces a protective type 2 response in the kidney. PBS or IL-33 (400 ng) was injected intraperitoneally on days 5–8 after induction of AN in BALB/c mice (see Figure 4A). Mice were analyzed at day 14. (A) Representative flow cytometric analyses of leukocytes isolated from the kidney, stimulated with phorbol 12-myristate 13-acetate and ionomycin, and stained intracellularly for IL-5 and IL-13. Plots are gated for CD45⁺CD90.2⁺Lin⁻ total ILCs. Numbers indicate the percentage of cells in the gate. (B) Absolute numbers of IL-5⁺IL-13⁺ ILCs in the kidney of naïve controls (n=4) and both AN groups (n=5 per group). (C–E) Quantitative RT-PCR analyses of the indicated mRNA transcripts in the kidney of naïve control mice (n=3), PBS-treated mice with AN (n=10), and IL-33–treated mice with AN (n=8). (F–H) Representative flow cytometry plots and absolute numbers of (F) eosinophils, (G) neutrophils, and (H) mononuclear phagocytes (MNP) in the kidney of naïve control mice (n=4), PBS-treated mice with AN (n=11), and IL-33–treated mice with AN (n=10). (I) Representative photographs of kidney sections stained for the neutrophil marker GR-1 (original magnification, ×200) and histologic quantification of GR-1–positive neutrophils in the three groups (naïve controls: n=4; AN + PBS: n=16; AN + IL-33: n=13); lpf, low-power field. Data are pooled from two to three independent experiments with similar results. Symbols in (B and F–I) represent individual data points and the horizontal lines indicate the median. Symbols in (C–E) represent mean±SEM (*P<0.05, **P<0.01, ***P<0.001).

The abundance of total CD4⁺ and CD8⁺ T cells was unaffected by IL-33 treatment, but we observed a significant reduction in γδ T cells, which have also been shown to be pathogenic in models of renal inflammation²² (Supplemental Figure 4A). As described before for C57BL/6 mice (see Figure 2), we again observed a moderate accumulation of Tregs in the kidneys of nephritic mice treated with IL-33 (Supplemental Figure 4A). In addition, GATA-3⁺ CD4⁺ Th2 cells were significantly increased after IL-33 treatment. However, this subset was unlikely to contribute significantly to the overall amount of IL-5 and IL-13, because it was two orders of magnitude less abundant than ILC2s (Figure 4C, Supplemental Figure 4A). Further analyses of ILC and T cell subsets in the kidney showed no significant difference in the abundance of Th17 cells and ILC3s (Supplemental Figure 4A). Finally, except for the expected increase in ILC2s and eosinophils, lymphocyte subsets in the spleen remained largely unchanged after IL-33 treatment of mice with AN (Supplemental Figure 4, B and C). In summary, IL-33 treatment modulated the inflammatory response in the diseased kidney in favor of an anti-inflammatory milieu with a reduction of pathogenic cell types and an increase of tissue-protective mediators.

IL-33–Mediated Tissue Protection Is ILC Dependent

In the next set of experiments, we addressed the question of whether ILCs are necessary and sufficient for the IL-33 effect on inflammatory mediators and cells of the myeloid lineage (eosinophils, neutrophils, mononuclear phagocytes), by subjecting BALB/c Rag2^−/− mice (lacking T and B cells) and BALB/c Rag2^−/−Il2rcg^−/− (lacking also all ILC populations) with AN to PBS or IL-33 treatment.

Flow cytometric analyses confirmed a marked expansion of ILC2s in the kidneys of IL-33–treated Rag2^−/− mice, whereas ILCs were absent in Rag2^−/−Il2rcg^−/− mice (Figure 6A). The IL-33–induced upregulation of Il5 and Il13 transcripts, the increased expression of arginase-1, and the accumulation of eosinophils were T cell independent and completely ILC dependent (Figure 6, C and D). Most importantly, the IL-33–induced reduction of neutrophil and mononuclear phagocyte infiltration into the kidney required the presence of ILCs, because it was maintained in ILC-sufficient Rag2^−/− mice, but absent in ILC-deficient Rag2^−/−Il2rcg^−/− mice (Figure 6, D and E). In line, IL-33 was effective in reducing glomerular histopathology in ILC-sufficient mice, whereas it failed to do so in the ILC-deficient setting (Figure 6, F and G). However, as described before,²³ AN was exacerbated in these immunodeficient mice, leading to aggravation of renal function impairment in Rag2^−/− animals that could only partially be rescued by ILC2 expansion (Figure 6H). Taken together, these data show that the beneficial effects of IL-33 treatment can be attributed to ILC2 expansion.

Figure 6.

IL-33–mediated tissue protection is ILC dependent. PBS or IL-33 (400 ng) was injected intraperitoneally on days 5–8 after induction of AN in BALB/c Rag2^−/− mice (PBS: n=8; IL-33: n=7) and BALB/c Rag2^−/−Il2rcg^−/− (PBS: n=8; IL-33: n=7). Mice were analyzed at day 14. (A) Representative flow cytometric analyses of leukocytes isolated from the kidney of BALB/c Rag2^−/− mice and BALB/c Rag2^−/−Il2rcg^−/− with IL-33 or PBS treatment. Plots are gated for CD45⁺ lymphocytes. Numbers indicate the percentage of cells in the gate. (B) Absolute numbers of ILC2s in the kidney of the respective groups. (C) Quantitative RT-PCR analyses of the indicated mRNA transcripts in the kidney of BALB/c Rag2^−/− mice and BALB/c Rag2^−/−Il2rcg^−/− with IL-33 or PBS treatment. (D and E) Absolute numbers of eosinophils, neutrophils, and CD11b^hiF4/80^int mononuclear phagocytes (MNP) (D), as well as histologic quantification of GR-1–positive neutrophils (E), in the respective groups. (F and G) Histopathologic quantification of glomerular damage (F) and representative photographs (G) (original magnification, ×200) of PAS-stained kidney sections. (H) Analyses of renal function parameters in the four groups. Data are representative for three independent experiments with similar results. Symbols in (B, D–F, and H) represent individual data points and the horizontal lines indicate the median. Symbols in (C) represent mean±SEM (*P<0.05, **P<0.01, ***P<0.001).

IL-33–Mediated Tissue Protection Requires Eosinophils

Because the accumulation of eosinophils in the kidneys of IL-33–treated mice was the most striking alteration of the inflammatory infiltrate, beside the expansion of ILC2s, we decided to address the role of eosinophils in IL-33–mediated protection from kidney injury in the AN model. To this end, we subjected eosinophil-deficient ΔdblGATA mice to AN with or without IL-33 treatment. Consistent with an important role for eosinophils in ILC2-mediated tissue protection, IL-33 treatment in the absence of eosinophils failed to protect mice from glomerulosclerosis, proteinuria, and renal function impairment, despite an unaltered expansion of ILC2s (Figure 7, A–D). It is therefore likely that the ILC2 effect is, at least in large parts, due to their role in regulating the abundance and function of other immune cells, such as eosinophils.

Figure 7.

Eosinophils are required for IL-33–mediated tissue protection. PBS or IL-33 (400 ng) was injected intraperitoneally on days 5–8 after induction of AN in ΔdblGATA mice. Mice were analyzed at day 14. (A) Representative flow cytometry plots and (B) absolute numbers of Lin⁻GATA-3⁺ ILC2s and CD11b⁺SiglecF⁺ eosinophils in the kidney of ΔdblGATA mice treated with PBS or IL-33 (n=7–8). Numbers in (A) indicate the percentage of cells in the gate. (C) Histopathologic quantification of glomerular damage and (D) analyses of renal function parameters in the two groups. Data represent one of two independent experiments with similar results. Symbols represent individual data points and the horizontal lines indicate the median. (***P<0.001).

Discussion

Accumulating evidence has led to the hypothesis that there is a link between type 2 immunity and the repair mechanisms that are initiated in response to tissue injury. Indeed, the type 2 immune response that is typically triggered by parasitic infections shares key mediators with the wound healing response aimed at restoring tissue integrity. From an evolutionary perspective this relationship seems advantageous, because the physical disruption of tissues often caused by migrating multicellular pathogens, such as helminthes, must be repaired rapidly in order to prevent microbial invasion at the sites of injury.²⁴

Type 2 immunity is a highly complex system that involves multiple cell types and mediators, including the type 2 cytokines IL-4, IL-5, IL-9, and IL-13; Th2 cells; eosinophils; mast cells; and basophils, as well as alternatively activated macrophages (AAMΦ) and IgE-production by B cells.^24,25 In recent years, it has become evident that ILC2s are among the central regulators of the type 2 immune response in mice and men.²⁶ In the context of tissue damage, the type 2 response is thought to facilitate the restoration of tissue homeostasis by inhibiting tissue destructive type 1 inflammation and promoting matrix deposition and remodeling.^25,27 Only in the last few years has it been appreciated that therapeutic targeting of the type 2 response, and especially of ILC2s,²⁸ might not only be of relevance in the setting of helminth infections and allergic disease, but could also be exploited to enhance endogenous mechanisms of regeneration after different forms of injury.^{7–10,25–27,29}

In this study, we apply this emerging concept to develop novel therapeutic strategies for CKD, a global health burden that affects around 10% of the Western population and is a major risk factor for cardiovascular mortality.¹ For the first time, we identify IL-33R⁺ ILC2s as a major ILC population in the human and mouse kidney and show that therapeutic expansion of ILC2s by IL-33 treatment shifts the inflammatory milieu after kidney injury toward a type 2 response that ameliorates the course of progressive, proteinuric CKD induced by injection of adriamycin in BALB/c mice.

In the last years, it has been recognized that the cytokines IL-25, IL-33, and thymic stromal lymphopoietin that are released after epithelial cell damage are potent inducers of ILC2-mediated type 2 responses and tissue repair mechanisms in the intestine, lung, and skin.³⁰ With respect to a potential role of ILCs in kidney disease, there is only one study that describes an expansion of ILC2s in the mouse kidney after IL-25 application. In a pre-emptive treatment approach, these IL-25–elicited ILCs were shown to ameliorate the course of ischemia/reperfusion-induced AKI by promoting alternative activation of macrophages that downregulate the inflammatory response and provide survival and proliferation signals to damaged tubular epithelial cells in the kidney.³¹ According to another report, a high dose of IL-33 (1 µg twice a day for 3 days) resulted in the aggravation of AKI in a model of cisplatin-induced tubular epithelial cell toxicity at a short time point (day 3).³² However, in this study, ILCs were not addressed and the IL-33 effect was attributed to CD4⁺ T cells. Thus, the evidence for a potential ILC-mediated protection from AKI is still controversial. The effect of IL-25 treatment was also investigated in AN, but again ILCs were not addressed in this study and the beneficial IL-25 effect depended on the presence of T cells.³³

In summary, there are no published reports that provide a detailed characterization of kidney ILC populations in mice and the question of whether ILC populations can be found in the human kidney has not been addressed so far. Here, we show that helper-like ILCs constitute 0.5%–3% of leukocytes in the murine and human kidney and that ILC2s are a major subtype of kidney ILCs in both species. The high expression of the IL-33R on kidney-residing ILC2s prompted us to explore IL-33 therapy as a way to enhance the type 2 response in the kidney of mice by modulating ILC2 abundance. We found a remarkable responsiveness of kidney-residing IL-33R⁺ ILC2s to a 4-day IL-33 treatment course (400 ng/d), resulting in a rapid accumulation of ILC2s in the kidney tissue that was accompanied by increased type 2 cytokine expression and was sustained for at least 8 weeks. Most importantly, the short-term IL-33 treatment, which was applied at a time point at which the glomerular damage is established, ameliorated parameters of histopathologic injury and renal function at later stages of adriamycin-induced proteinuric CKD in mice.

In the literature, there is robust evidence that IL-33 treatment can affect the function of a variety of different cell types,³⁴ and IL-33 treatment has been used in different experimental settings to modulate the inflammatory response in infection,^35,36 autoimmune disease,³⁷ and sterile inflammation.^9,10,38 Many of these studies were done before the emergence of ILCs as a separate cell subset and the unique responsiveness of ILC2s to IL-33 that we now know of makes it tempting to speculate that some of the IL-33 effects on other cell types described in in vivo models may indeed be attributed to indirect effects mediated by IL-33–elicited ILC2s. With respect to the kidney-residing leukocyte populations, we found no evidence for a significant stimulatory effect of IL-33 on cell types other than ILC2s, except for a mild increase of Tregs and Th2 cells. Moreover, we did not find evidence for an improved function of Tregs isolated from IL-33–treated mice, as described in other model systems.³⁹ The potential IL-33 effects on T cells, however, were not essential for the IL-33–induced protective type 2 response in the kidney, as illustrated by the fact that IL-33 treatment was able to reduce glomeruloclerosis in T cell–deficient Rag2^−/− mice, but not in Rag2^−/−Il2rg^−/− mice that also lack ILCs. Of note, the IL-33–mediated protection in Rag2^−/− mice was incomplete and we cannot fully exclude that a direct IL-33 effect on Tregs (or a potential ILC2-Treg interaction) may contribute to the amelioration of kidney damage observed in WT mice treated with IL-33. Moreover, the preferential cellular target of IL-33 action most likely depends on the inflammatory context in which IL-33 is used³⁴ and may therefore greatly vary between e.g., viral infection,³⁶ sepsis,³⁵ AKI,³² and a setting of chronic sterile inflammation, such as CKD. These considerations are also critical for assessing the risk for potential side effects of chronic IL-33 exposure, such as the development of allergic disease and the excessive activation of wound repair mechanisms resulting in tissue fibrosis,⁴⁰ which are likely to be determined by the dose, duration, and context of the cytokine application.³⁴

The downstream mechanisms that ILC2s might use to protect from progressive kidney damage include IL-5–induced accumulation of eosinophils, a cell type that has been shown to promote tissue regeneration,⁴¹ and IL-13–induced alternative activation of macrophages^31,42,43 that have been shown to promote kidney regeneration.⁴⁴ By subjecting eosinophil-deficient ΔdblGATA mice to AN with or without IL-33 treatment, we could show that IL-33 treatment in the absence of eosinophils failed to protect mice from AN, suggesting an important role for this cell type in ILC2-mediated tissue protection. It is therefore likely that the ILC2 effect is, at least in large parts, due to their role in regulating the abundance and function of other immune cells in the kidney tissue.

Taken together, we show here that ILC2s reside in the healthy kidney, proliferate vigorously in response to a short course of in vivo IL-33 therapy, have a long half-life in the kidney tissue, and are potent mediators of a type 2 response that promotes damage control in experimental CKD. Thus, we identify ILC2-directed cytokine therapy as a potential therapeutic strategy for chronic kidney inflammation. Further investigations of the different ILC subsets residing in the human kidney, their response to ex vivo cytokine stimulation, and a correlation of ILC subtype abundance with specific disease entities are needed to advance our understanding of this complex cell population in the human kidney.

Concise Methods

Animals, Progressive Glomerulosclerosis, and IL-33 Treatment

C57BL/6 wildtype mice, C57BL/6 Rag1^−/− mice, BALB/c wildtype mice, BALB/c Rag2^−/− mice, and BALB/c Rag2^−/−Il2rcg^−/− mice were bred in the animal facility of the University Medical Centre Hamburg-Eppendorf under specific pathogen-free conditions. ΔdblGATA mice were provided by S. Rausch (Berlin, Germany) or ordered from the Jackson Laboratory. Adult male and female mice with the appropriate sex- and age-matched controls were used in all experiments. For induction of progressive glomerulosclerosis, BALB/c mice were injected intravenously with 12 µg adriamycin (cell pharm GmBH, Germany) per gram body wt. For ILC expansion, mice were injected intraperitoneally with 400 ng rmIL-33 (BioLegend) in 200 µl PBS on 4 consecutive days. Controls received 200 µl PBS. For urine sample collection, mice were housed in metabolic cages for 6 hours. Urinary albumin excretion was determined by standard ELISA analysis (Mice-Albumin Kit; Bethyl Laboratories, Montgomery, TX). Urinary creatinine, blood urea nitrogen, and serum cholesterol were measured using standard laboratory methods.

Human Material

Human kidney cortex specimens were obtained from patients that underwent partial nephrectomy because of suspected renal carcinoma. After evaluation by the pathologist, samples of macroscopic healthy, tumor-free kidney cortex were excised from the residual tissue that was not needed for diagnostic purposes and processed as described below.

Cell Isolation

For isolation of leukocytes from the mouse kidney, the tissue was cut into small pieces, digested in complete medium (RPMI 1640, 10% FCS, 1% HEPES, 1% penicillin/streptomycin; all Gibco) with collagenase D (0.4 mg/ml; Roche) and DNase I (100 µg/ml; Roche) for 45 minutes at 37°C while rotating on a MACSmix tube rotator (Miltenyi), and then further dispersed by using a gentleMACS dissociator (Miltenyi). For human kidney samples, collagenase VIII (0.5 mg/ml; Sigma-Aldrich) was used instead of collagenase D. Further leukocyte purification was achieved by Percoll gradient centrifugation (37.5%). Murine splenocytes were obtained by mashing spleens through a 70 µm strainer with PBS containing 1% FCS. After subsequent erythrocyte lysis with ammonium chloride, cell suspensions of kidneys and spleens were filtered through a 50 µm strainer and used for further analyses.

Flow Cytometry

To characterize ILC subsets, cell suspensions of mouse kidneys were stained with fluorochrome-coupled antibodies against CD45 (30-F11), IL-7Rα (CD127; A7R34), Thy1.2 (CD90.2; 30-H12), and a combination of lineage markers (Lin) including CD3 (145–2C11), CD4 (RM4–5), CD8 (53–6.7), CD11b (M1/70), CD11c (HL-3), CD19 (6D5), CD49b (DX5), TCR-β (H57–597), TCR-γδ (GL3), GR-1 (RB6–8C5), and Ter119 (Ter119). Intranuclear staining, using antibodies against GATA-3 (L50–823), T-bet (4B10), ROR-γt (B2D), FoxP3 (FJK-16s), and Eomes (Dan11mag), was done with the Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer’s instructions. For further characterization of ILC surface marker expression, antibodies against CD25 (PC61.5) and IL-33R (DJ8; MD Bioproducts) were used. For characterization of other leukocyte subtypes, antibodies against CD11b, Ly6G (1A8), SiglecF (E50–2440), CD11c, F4/80 (BM8), CD4, CD8, TCR-γδ, ckit (CD117; 2B8), CD49b (HMα2), and FcεRIα (MAR-1) were used. For intracellular cytokine staining, isolated leukocytes were restimulated with phorbol 12-myristate 13-acetate (1 µg/ml; Sigma-Aldrich) and ionomycin (1 µg/ml; Calbiochem) in the presence of brefeldin A (10 µg/ml; Sigma-Aldrich) for 2.5 hours, stained for surface markers as described above, fixed with formalin (3.8%; Sigma-Aldrich), permeabilized with IGEPAL CA-630 (0.1%; Sigma-Aldrich), and stained with a combination of fluorochrome-coupled antibodies against IL-5 (TRFK5), IL-13 (eBio13A), IL-17A (TC11–18H10), and IFN-γ (XMG1.2). To identify ILC subsets in the human kidney, a combination of the following antibodies was used: CD45 (2D1), IL-7Rα (CD127; A019D7), CD161 (HP-3G10), CRTH2 (CD294; BM16), NKp44 (P44–8), cKit (CD117; 104D2), CD25 (BC96), IL-33R (B4E6; MD Bioproducts), and lineage (Lin = CD1a [HI149], CD34 [581], CD123 [6H6], CD8 [RPA-T8], CD4 [RPA-T4], CD94 [DX22], TCR-γδ [B1], CD3 [OKT3], TCRαβ [IP26], CD19 [HIB19], CD14 [HCD14], FcER1α [AER-37], BDCA2 [AC144], CD56 [HCD56], CD11c [3.9], CD16 [3G8]). All antibodies were obtained from BioLegend, BD Biosciences, or eBioscience unless otherwise indicated. Absolute numbers of CD45⁺ cells in kidney cell suspensions were determined by staining with fluorochrome-coupled anti-CD45 combined with the use of cell counting beads (Countbright; Invitrogen). All samples were acquired on an LSRII flow cytometer (BD Biosciences) and analyzed with the FlowJo software (Treestar Inc.).

In Vitro Suppression Assay

CD4⁺ T cells from splenocytes of PBS- or IL-33–treated C57BL/6 mice were enriched via MACS (Miltenyi) according to the manufacturer’s instructions using biotinylated antibodies and anti-biotin microbeads depleting all other cell types. Enriched CD4⁺ T cells were stained with antibodies against CD3, CD4, CD25, and CD127. CD4⁺CD25⁻ T_eff were sorted via FACS from the PBS group and CD4⁺CD25⁺CD127⁻ T_reg were sorted from PBS- and IL-33–treated animals using a FACSFusion (BD Biosciences). Five ×10⁴ T_eff were cocultured with titrated numbers of T_reg in complete medium (RPMI 1640, 10% FCS, 1% HEPES, 1% penicillin/streptomycin, 50 µM β-ME; all Gibco) in 96-well plates precoated with anti-CD3 mAb (5 µg/ml, clone 145–2C11; BD Biosciences) for 4 days. Cytokine concentrations in the supernatant were measured by using a bead-based cytokine array (LEGENDplex; BioLegend) according to the manufacturer’s instructions.

Histopathology and Immunohistochemistry

Formalin-fixed, paraffin-embedded kidney sections were stained with PAS according to standard laboratory procedures and assessed for histopathology in a blinded fashion. Glomerular sclerosis (deposition of PAS-positive material) was scored from zero to four in 50 glomeruli per mouse. Tubular damage was assessed on the basis of formerly described methods.⁴⁵ In brief, photographs of nonoverlapping cortical areas from PAS-stained kidney sections were assessed for percentage of tubulointerstitial injury by superimposition of a grid and subsequent counting of the area displaying dilated, atrophic, or cast-filled tubules. For neutrophil quantification, paraffin-embedded sections were stained with an antibody directed against the neutrophil marker GR-1 (Ly6 G/C) (NIMP-R14; Hycult Biotech, The Netherlands) and developed with a polymer-based secondary antibody alkaline phosphatase kit (POLAP; Zytomed, Berlin, Germany). GR-1⁺ cells were counted in at least ten low-power fields (original magnification, ×200) per section. Slides were evaluated with an Axioskop light microscope (Zeiss, Jena, Germany) and photographed with an Axiocam HRc camera (Zeiss). For immunofluorescence staining, 2 μm paraffin sections were deparaffinized and rehydrated to water. Antigen retrieval was performed by constant boiling in DAKO antigen retrieval buffer pH 6. Unspecific binding was blocked with 5% horse serum (vector) and 0.05% triton-X100 in PBS. Primary antibodies (rabbit-CD127; LSBio; goat-GATA-3; R&D Systems) were incubated in 5% horse serum overnight at 4°C. GATA-3 antibody binding was visualized by using a Cy3- or AF647-coupled anti-goat and CD127 antibody binding by using an AF488-coupled anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories). DNA was counterstained with Draq5 (Molecular Probes). Staining of the endothelium was achieved by perfusion with lectin (40 µg/ml, DyLight 594 lycopersicon esculentum; Vector laboratories, Burlingame, CA), as previously described.⁴⁶ Stainings were evaluated with an LSM 510 Meta confocal microscope using the LSM software (Zeiss).

Quantitative Real-Time RT-PCR Analyses

Total RNA of the renal cortex was prepared according to standard laboratory methods. After reverse transcription, TaqMan Gene Expression Assays and a StepOnePlus Real-Time PCR system (both Thermo Fisher Scientific) were used for quantification of the housekeeping gene (Hprt1) and the genes of interest.

Statistical Analyses

The paired t test was used for comparison between two groups. In the case of three or more groups, one-way ANOVA was used followed by a post hoc analysis with Newman–Keuls test for multiple comparisons. A P value of <0.05 was considered to be statistically significant.

Study Approval

All animal experiments were performed according to national and institutional animal care and ethical guidelines and were approved by the local authorities (approval numbers G46/14 and G122/15; Behörde für Verbraucherschutz). For human kidney samples, written consent for the use of residual materials was obtained from all patients.

Disclosures

None.