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.

Keywords: IL-33; glomerulosclerosis; innate lymphoid cells.

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.

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).

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.

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).

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).

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).

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).