Expansion of Group 2 Innate Lymphoid Cells in Patients with End-Stage Renal Disease and Their Clinical Significance

This information is current as Gao-Yu Liu, Xiao-Hui Deng, Xing Li, Ying-Jiao Cao, of September 24, 2021. Yan-Fang Xing, Pan Zhou, Ai-Hua Lei, Quan Yang, Kai Deng, Hui Zhang and Jie Zhou J Immunol published online 22 May 2020 http://www.jimmunol.org/content/early/2020/05/21/jimmun

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 22, 2020, doi:10.4049/jimmunol.1901095 The Journal of Immunology

Expansion of Group 2 Innate Lymphoid Cells in Patients with End-Stage Renal Disease and Their Clinical Signiﬁcance

Gao-Yu Liu,*,†,‡,1 Xiao-Hui Deng,*,†,‡,1 Xing Li,x,1 Ying-Jiao Cao,*,†,‡ Yan-Fang Xing,{ Pan Zhou,‡ Ai-Hua Lei,‡,‖ Quan Yang,# Kai Deng,‡ Hui Zhang,‡ and Jie Zhou*,†

Group 2 innate lymphoid cells (ILC2s) play an important role in the control of tissue inflammation and homeostasis. However, the role of ILC2s in patients with end-stage renal disease (ESRD) has never been illustrated. In this study, we investigated ILC2s in ESRD patients and their clinical significance. Results showed that the frequencies and absolute numbers of ILC2s, not group 1 innate lymphoid cells or innate lymphoid cell precursors, were significantly elevated in the peripheral blood of ESRD patients when compared with those from healthy donor controls. Moreover, ILC2s from ESRD patients displayed enhanced type 2 cytokine production and cell proliferation. Plasma from ESRD patients significantly increased ILC2 levels and enhanced their effector function

after in vitro treatment. The expression of phosphorylation of STAT5 in ILC2s, as well as the amounts of IL-2 in plasma, were Downloaded from increased in ESRD patients when compared with those from healthy donors. Clinically, ESRD patients with higher ILC2 frequencies displayed lower incidence of infectious complications during a mean of 21 month follow-up study. The proportions of ILC2s were negatively correlated with the prognostic biomarkers of chronic kidney disease, including serum parathyroid hormone, creatinine, and phosphorus, whereas they were positively correlated with serum calcium. These observations indicate that ILC2s may play a protective role in ESRD. The Journal of Immunology, 2020, 205: 000–000. http://www.jimmunol.org/ he global prevalence of chronic kidney disease (CKD) is alterations in the immune system in ESRD patients contribute to their ∼13.4% (1). Around 120 million people in China suffer from enhanced susceptibility to infections (6, 8), the underlying mechanism T it (2). CKD therefore represents a huge health, economic, and of which remains to be fully understood. social burden worldwide. End-stage renal disease (ESRD) is the final Innate lymphoid cells (ILCs) are recently identified innate immune stage of CKD, which is characterized by permanent loss of kidney cells that do not express Ag-specific receptors. Distinct subsets of function and requires dialysis or kidney transplantation to sustain the ILCs use different transcription factors and secret different effector patient’s life (3, 4). ESRD patients displayed increased risk of infec- cytokines, which share high similarity with the corresponding helper tions, and this is the leading complication causing death (5–7). The T lymphocyte subsets (9, 10). However, the responses of ILCs are faster upon exposure to pathogens, tissue damage, or other envi- by guest on September 24, 2021 ronmental stimuli (11). Among the three groups of ILCs identified *Joint Program in Immunology, Department of Internal Medicine, Affiliated Guangzhou (9), group 2 ILCs (ILC2s) have been extensively studied. ILC2s Women and Children’s Medical Center, Zhongshan School of Medicine, Sun Yat-sen † reside in a variety of tissues, including mucosal barriers, fat tissue, University, Guangzhou 510623, China; Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Department of Immunology, School of Basic Med- liver, skin, kidney, and so on (12–16). It has been demonstrated that ical Sciences, Tianjin Medical University, Tianjin 300070, China; ‡Institute of Human ILC2s play important roles in a variety of physiological or patho- Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China; xDepartment of Medical Oncology, The Third Affiliated Hospital of Sun Yat-sen logical processes, including but not limited to airway hyperrespon- University, Guangzhou 510630, China; {Department of Nephrology, The Third Affiliated sive inflammation, defense against pathogens, tissue repair, and ‖ Hospital of Guangzhou Medical University, Guangzhou 510150, China; Institute of lipogenesis (10, 17–21). Interestingly, several reports revealed that Pathogenic Biology, Hengyang Medical College, University of South China, Hengyang 421008, China; and #Key Laboratory of Immunology, Sino-French Hoffmann Institute, ILC2s play a beneficial role in the protection against renal injury and School of Basic Medical Sciences, Guangzhou Medical University, Guangzhou 511436, the associated pathologies, including acute kidney injury, renal China ischemia-reperfusion injury, and progressive glomerulosclerosis in 1 G.-Y.L., X.-H.D., and X.L. contributed equally to this work. mice (16, 22–25). The therapeutic value of ILC2s is therefore sug- ORCIDs: 0000-0001-9145-4769 (X.L.); 0000-0002-2548-7252 (Q.Y.); 0000-0001- gested in the alleviation of renal diseases. However, the role of 5964-1599 (J.Z.). ILC2s in patients with ESRD has never been investigated. Received for publication September 10, 2019. Accepted for publication April 22, In this study, we evaluated ILC2s in patients with ESRD and further 2020. explored their clinical significance. Results showed that both the level This work was supported by the following grants (to J.Z.): the National Natural Science Foundation of China (81925018 and 81771665), the High-Level Talent and function of ILC2s were significantly elevated in ESRD patients Start-Up Funding of Tianjin Medical University, the Natural Science Foundation of when compared with healthy controls. Further studies indicated that Guangdong (2017B030311014), and the Science and Technology Program of the changes of ILC2s may be caused by certain soluble factors from Guangzhou (201605122045238). the plasma of ESRD patients. Importantly, ILC2s were negatively Address correspondence and reprint requests to Dr. Jie Zhou, Department of Immu- nology, School of Basic Medical Sciences, Tianjin Medical University, 22 Qixiangtai associated with the development of infections and the prognostic Road, Tianjin 300070, China. E-mail address: [email protected] biomarkers of CKD, indicating their potential protective role in ESRD. The online version of this article contains supplemental material. Abbreviations used in this article: CKD, chronic kidney disease; ESRD, end-stage renal disease; ILC, innate lymphoid cell; ILC1, group 1 ILC; ILC2, group 2 ILC; Materials and Methods ILCP, ILC precursor; Lin2, lineage-negative; MFI, mean fluorescence intensity; Study population PGD2, prostaglandin D2; PTH, parathyroid hormone. In this study, ESRD patients (n = 69) and healthy controls (n = 34) were Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 recruited from the Third Affiliated Hospital of Sun Yat-sen University and

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901095 2 ILC2 EXPANSION IN ESRD PATIENTS the Third Affiliated Hospital of Guangzhou Medical University (Guangzhou, Cell culture China) during the period between July 2017 and July 2019. All participants were screened for hepatitis B surface Ag, hepatitis C virus Ab, hepatitis D The ILC2 in vitro culture consisted of the following: equal numbers of virus Ag and Ab, and HIV Ab. Participants who had chronic hepatitis virus ILC2s (500 cells) from the peripheral blood of healthy controls and ESRD infection or other acute infections, were positive for HIV-1, or received patients were cultured with IL-2 (20 ng/ml; PeproTech), IL-7 (20 ng/ml; systemic corticosteroids or immunosuppressive agents were excluded PeproTech), and IL-33 (20 ng/ml; PeproTech) for 3 d, then the supernatants were collected for ELISA. from this study, and all patients met the criteria for ESRD as defined by 2 2 current international guidelines. A written informed consent was ob- The Lin cell culture consisted of the following: Lin cells (100,000 cells tained from all participants and/or their legal guardians at the time of per well) from healthy donors that were cultured with medium containing admission, and the study was approved by the Clinical Ethics Review 5% plasma from healthy controls or ESRD patients in the presence of IL-2 Board of the Third Affiliated Hospital of Sun Yat-sen University and the (20 ng/ml) plus IL-7 (20 ng/ml) in 96-well plates for 3 d. IL-33 (20 ng/ml) Third Affiliated Hospital of Guangzhou Medical University. The clini- administration was used as positive control. cal and demographic information of these participants was showed in ELISA Supplemental Tables I, II. The quantification of human IL-5 (Dakewe), IL-13 (Invitrogen), IL-2 PBMC isolation (BioLegend), IL-7 (Dakewe), IL-9 (BioLegend), IFN-a (Dakewe), The peripheral blood was collected and centrifuged (2500 rpm, 5 min, IFN-b (4A Biotech), IL-33 (R&D System), and IFN-g (R&D System) 4˚C), and the supernatants were frozen at 280˚C. PBMCs were isolated was measured by ELISA, following the manufacturers’ instructions. from fresh blood samples by Ficoll (Axis-Shield) centrifugation, as de- scribed previously (26). The isolated PBMCs were washed with ice-cold Patient treatment and follow-up PBS to remove the residual Ficoll-Hypaque solution and were analyzed A total of 60 patients were followed up for an average of 21 mo. Among immediately.

them, 58 patients were treated with hemodialysis, two patients were Downloaded from Flow cytometric analysis treated with peritoneal dialysis. During the follow-up, six patients died of renal failure–related complications, 33 patients developed infectious compli- PBMCsuspensionswerepreparedandstainedwithfluorochrome- cations, and 12 patients had cardiovascular events. In addition, the information conjugated Abs. Cells were acquired on the LSRFortessa flow cytom- for about six patients’ infectious status and one patient’s cardiovascular disease eter (BD Biosciences), and data were analyzed with FlowJo V10.5.3 status was missing and therefore was not included. (FlowJo). The gating strategies for ILCs and ILC precursors (ILCPs) were as follows: CD45+Lin2CD127+CD1172CRTH22CD161+ for Statistics group 1 ILCs (ILC1s), CD45+Lin2CD127+CRTH2+CD161+ for ILC2s, http://www.jimmunol.org/ + 2 + + 2 Clinical and immunological parameters were compared by nonparametric and CD45 Lin CD127 CD117 CRTH2 for ILCPs (Fig. 1A). For Mann–Whitney U tests or x2 test. For other data, statistical analyses were intracellular cytokine staining, PBMCs were stimulated in complete done using unpaired t test. Correlations between different parameters were RPMI 1640 (Life Technologies) with 50 ng/ml PMA (Sigma-Aldrich), analyzed using a Spearman rank test and linear regression. Statistical tests 1 mg/ml ionomycin (Sigma-Aldrich), and 1 mg/ml brefeldin A (Invitrogen) were performed using GraphPad version 6.0. for 4 h. Cells were then stained with the Abs against surface markers, fixed, and permeabilized using an Intracellular Fixation and Permeabilization Buffer Set (eBioscience). After that, cells were stained with the Abs against Results cytokines. For the staining of transcription factor, PBMCs were stained ILC2s are expanded in patients with ESRD with the Abs against surface markers, fixed, and permeabilized according To investigate the potential role of ILCs in ESRD, we first evaluated to the manufacturer’s instructions (Foxp3 Transcription Factor Staining by guest on September 24, 2021 Buffer Set; eBioscience). For the detection of PMA-induced phosphory- the levels of ILCs in the peripheral blood of ESRD patients by flow lation, PBMCs were stained with the Abs against surface markers, then cytometric analysis. The gating strategies of ILCs and ILCPs treated with DMSO (Sigma-Aldrich) or PMA (50 ng/ml) for 10 min. After were as follows: ILC2s (CD45+Lin2CD127+CRTH2+CD161+), that, cells were fixed, permeabilized, and stained with anti–p-STAT5 or ILC1s (CD45+Lin2CD127+CD1172CRTH22CD161+), and ILCPs anti–p-ERK1/2. For ILC2 sorting, a BD FACSAria III cell sorter (BD + 2 + + 2 Bioscience) was used. (CD45 Lin CD127 CD117 CRTH2 ) (Fig. 1A) (27–31). Results Human Abs used in this study were as follows: human hematopoietic showed that both the frequencies and absolute cell counts of ILC2s lineage–allophycocyanin (Invitrogen), MagniSort Human Hematopoi- were significantly increased in patients with ESRD when com- etic Lineage Depletion Kit (Invitrogen), streptavidin–allophycocyanin pared with those from healthy donor controls (Fig. 1B, 1C) (32). (eBioscience), streptavidin-PE-Cyanine 7 (Invitrogen), streptavidin–FITC In contrast with the elevation in ILC2s, no noticeable changes (Invitrogen), anti-CD45-allophycocyanin/Cy 7 (HI30; BioLegend), anti-CD45-PE-Cyanine 7 (HI30; eBioscience), anti-CD127-eFlour 450 were observed in the frequencies and absolute numbers of ILC1s (eBioRDR5; Invitrogen), anti-CD294 (CRTH2)-PE (BM16; Invitrogen), anti- or ILCPs in patients with ESRD (Fig. 1D, 1E), indicating that the CD117 (c-kit)-FITC (104D2; BioLegend), anti-CD161-PE/Cy 7 (HP- changes were specifically observed in ILC2s (32). 3G10; BioLegend), anti-mouse/human IL-5–allophycocyanin (TRFK5; Human ILC2s have two distinct subsets: CD117+ and CD1172 BioLegend), anti–IL-13-PerCP/Cy 5.5 (JES10-5A2; BioLegend), anti-CD3- 2 PE-Cyanine 7 (UCHT1; eBioscience), anti-CD4-PE (OKT4; eBioscience), ILC2s. They have different functional plasticity. CD117 ILC2s are anti-CD8-FITC (3B5; eBioscience), anti-CD19-allophycocyanin-eFluor 780 more mature and lineage-determined and they secret higher amounts (HIB19; eBioscience), anti-CD25-PE-Cyanine 7 (BC96; eBioscience), anti- of effector cytokines than CD117+ ILC2s (28, 33). CD117+ ILC2s Ki-67-FITC (20Raj1; Invitrogen), anti-GATA3-PerCP-eFluor 710 (TWAJ; share some signatures with ILC3s and may contribute to IL-17– eBioscience), anti–p-ERK1/2-allophycocyanin (MILAN8R; Invitrogen), mediated pathology (33, 34). We next evaluated the changes of and anti–p-STAT5-allophycocyanin (SRBCZX; eBioscience). For the lineage mixtures in Figs. 1A–E, 2F, 3E, and 4A–G and in Supplemental these two ILC2 subsets in ESRD patients. Results showed that the + Fig. 1A, 1B, 1F, and 1G, the lineage mixtures included anti-CD2, anti- elevation of ILC2s in ESRD patients were observed in both CD117 CD3, anti-CD4, anti-CD16, anti-CD19, anti-CD56, and anti-CD235a. In and CD1172 ILC2s, although the frequencies of CD1172 ILC2s Figs. 2A–D, 2G, 3A, 3B, 3D, 3F, and 3G and in Supplemental Fig. 1C–E, were significantly higher than CD117+ ILC2s (Supplemental Fig. 1A, the lineage mixtures included anti-CD2, anti-CD3, anti-CD10, anti- 2 1B) (32). Consistent with previous reports, CD117 ILC2s produced CD11b, anti-CD14, anti-CD16, anti-CD19, anti-CD56, anti-CD123, + and anti-CD235a. higher levels of IL-5 and IL-13 when compared with CD117 ILC2s, whichwereobservedinbothhealthycontrolsandESRDpatients Lineage-negative cell isolation (Supplemental Fig. 1C, 1D) (28, 33). And the expression of GATA3 2 For human lineage-negative (Lin ) cell isolation, PBMCs were depleted in CD1172 ILC2s was relatively higher than CD117+ ILC2s, as of T cells, B cells, NK cells, myeloid cells, granulocytes, and RBCs by expected (Supplemental Fig. 1E) (33). Expression of CD161 was labeling with biotin-conjugated anti-CD2, anti-CD3, anti-CD10, anti- CD11b, anti-CD14, anti-CD16, anti-CD19, anti-CD56, anti-CD123, and comparable in these two subsets (Supplemental Fig. 1F). anti-CD235a from MagniSort Human Hematopoietic Lineage Depletion Consistent with previous reports (35–38), the percentages of dis- Kit, following the manufacturer’s instructions (Invitrogen). tinct T cell subsets, including CD3+ T cells, CD3+CD4+ T cells, The Journal of Immunology 3 Downloaded from

FIGURE 1. Expansion of ILC2s in patients with ESRD. (A) Gating strategies of ILCs and ILCPs in the peripheral blood by flow cytometric analysis. (B) Representative flow cytometric plots of ILC2s from patients with ESRD and healthy controls. (C) The percentages and http://www.jimmunol.org/ absolute numbers of ILC2s from (B)(ESRD, n = 60; control, n =29).(D and E) The percentages and absolute numbers of ILC1s (D) and ILCPs (E) from patients with ESRD (n = 60) and healthy controls (n = 29). Both representative plots (left) and statistical results (right) were shown. In all plots, mean 6 SD are shown. Re- sults are representative of at least three independent experiments. **p , 0.01, ***p , 0.001 by by guest on September 24, 2021 unpaired t-test. ns, not significant.

CD3+CD8+ T cells, and CD4+CD25+ regulatory T cells, displayed To determine the function of ILC2s from ESRD patients, their no statistical differences between ESRD patients and healthy con- intracellular cytokine production was measured by flow cytom- trols (Supplemental Fig. 2A, 2B). The percentages of CD19+ B cells etry. Results showed that the proportions and absolute cell num- were decreased in ESRD patients (Supplemental Fig. 2C). bers of IL-5+IL-13+ ILC2s were significantly higher in ESRD patients than healthy controls (Fig. 2A, 2B), which led to higher The function and proliferation of ILC2s are enhanced in proteinlevelsofIL-5andIL-13onESRD-derivedILC2s, ESRD patients as indicated by mean fluorescence intensity (MFI) (Fig. 2C, ILC2s are characterized by their capability to produce type 2 Supplemental Fig. 1C, 1D). Meanwhile, flow cytometric analysis effector cytokines, predominantly IL-5 and IL-13 (10, 27). for Ki-67 showed that the proliferation of ILC2s from ESRD 4 ILC2 EXPANSION IN ESRD PATIENTS Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 2. ILC2s from patients with ESRD display enhanced function and proliferation. (A) Representative ﬂow cytometric plots of IL-5+IL-13+ ILC2s from ESRD patients and healthy controls and the isotype of IL-5 and IL-13 were shown. ILC2s were gated on CD45+Lin2CD127+CRTH2+.(B) The percentages and absolute numbers of IL-5+IL-13+ ILC2s in (A) (ESRD, n = 12; control, n = 7). (C) The MFI of IL-5 and IL-13 from (A) (ESRD, n =6; control, n = 4). (D) The proliferation of ILC2s (CD45+Lin2CD127+CRTH2+CD161+) was measured by Ki-67 staining. The percentages (n = 9) and absolute numbers (n = 6) of ILC2s were shown. (E) The levels of IL-5 (ESRD, n = 9; control, n = 8) and IL-13 (ESRD, n = 11; control, n = 10) in the plasma from ESRD patients and controls were measured by ELISA. (F) The correlation between ILC2 cell counts with plasma IL-5 and IL-13 levels in ESRD patients and healthy controls was analyzed by linear regression (n = 16). (G) Equal numbers of ILC2s from healthy controls and ESRD patients were cultured with IL-2 (20 ng/ml), IL-7 (20 ng/ml), and IL-33 (20 ng/ml) for 3 d. The concentrations of IL-5 (n = 8) and IL-13 (n = 7) in the supernatants were measured by ELISA. In all plots, data represent mean 6 SD. Results are representative of three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001 by unpaired t test. The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 3. Plasma from ESRD patients promotes the expansion and function of ILC2s in vitro. (A and B)Lin2 cells were cultured with 5% plasma from ESRD patients or healthy controls in the presence of IL-2 (20 ng/ml) and IL-7 (20 ng/ml) for 3 d. The frequencies and absolute cell numbers of ILC2s (A) and IL-5+IL-13+ ILC2s (B) were determined by flow cytometry (IL-33, n =4;control,n =4;ESRD,n =6).(C) The concentrations of IL-33 in the plasma from ESRD patients (n =5) and healthy controls (n =4)weremeasuredbyELISA.(D) The MFI of GATA3 in ILC2s of ESRD patients and healthy controls was shown by flow cytometry (n =4). (E) The MFI of CRTH2 on ILC2s of ESRD patients (n = 17) and healthy controls (n = 7). (F) The levels of p-ERK1/2 in ILC2s were measured by flow cytometry (n = 4). (G) The levels of p-STAT5 in ILC2s were measured by flow cytometry (n = 3–4). (H) The amounts of IL-2, IL-7, IL-9, IFN-a, IFN-b,and IFN-g in plasma from ESRD patients and healthy controls were measured by ELISA (n = 5–16). ILC2s were gated on CD45+Lin2CD127+CRTH2+. In all plots, mean 6 SD are shown. Results are representative of two to three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001 by unpaired t test. ns, not significant. 6 ILC2 EXPANSION IN ESRD PATIENTS Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 4. ILC2s are negatively associated with infectious events in patients with ESRD. (A) The percentages of ILC2s in the peripheral blood from ESRD patients with (n = 33) or without (n = 15) infectious events were shown. (B) The correlation between ILC2 proportions in the peripheral blood and the infectious events per month of ESRD patients during follow-up was illustrated by linear regression (n = 48). (C) The percentages of ILC2s in the peripheral blood from ESRD patients with (n = 12) or without (n = 41) cardiovascular diseases (CVD) were shown. (D) The correlation between serum PTH with the proportions of ILCs and ILCPs in the peripheral blood from ESRD patients was illustrated by linear regression (n = 50). (E) The correlation between serum creatinine with ILCs and ILCPs in the peripheral blood from ESRD patients was illustrated by linear regression (n = 50). (F and G) The correlation between the proportions of ILC2s in the peripheral blood with serum phosphorus (n = 48) (F) and calcium (Figure legend continues) The Journal of Immunology 7 patients was increased more than 3-fold when compared with developed infectious complications. To investigate whether the those from healthy controls (Fig. 2D). The amounts of IL-5 and elevation of ILC2s was associated with the increased susceptibility IL-13 in the plasma from ESRD patients were significantly to infectious events, ESRD patients were divided into two groups higher than healthy controls (Fig. 2E), which was consistent with a based on their development of infections during the follow-up. It previous report (39). Importantly, linear regression analysis showed was found that ESRD patients who eventually developed infectious that the absolute cell numbers of ILC2s were positively correlated complications displayed lower frequencies of ILC2s when com- with the plasma IL-5 and IL-13 amounts (Fig. 2F), indicating that pared with those who did not (Fig. 4A). Notably, the percentages of ILC2s may represent one of the major sources of type 2 cytokines in ILC2s were negatively correlated with the development of infec- circulation. For further confirmation, equal numbers of ILC2s from tious events (Fig. 4B). Cardiovascular disease is another leading ESRD patients and healthy controls were sorted and cultured in the cause of death among ESRD patients (7). There were no differ- presence of IL-2, IL-7, and IL-33 for 3 d. Results, as revealed by ences in ILC2 levels between patients who developed cardiovas- ELISA, showed that ESRD-derived ILC2s secreted much higher cular complications or not (Fig. 4C). amounts of IL-5 and IL-13 than controls (Fig. 2G). These results Parathyroid hormone (PTH) and creatinine are well-recognized indicate that both the effector function and cell proliferation of biomarkers reflecting kidney function (3, 50). It was reported that ILC2s are remarkably enhanced in patients with ESRD. moderate-to-large elevations in serum PTH, creatinine, or both are associated with increased morbidity and mortality in ESRD pa- Plasma from ESRD patients promotes the expansion and tients (51–53). We therefore analyzed the association between activation of ILC2s ILC2s with PTH and creatinine. Results showed that the per-

We next explored the mechanism of ILC2 elevation in ESRD pa- centages of ILC2s but not ILC1s or ILCPs in the peripheral blood Downloaded from 2 tients. Lin cells from the peripheral blood of healthy donors was were negatively correlated with serum PTH and creatinine in cultured with IL-2 plus IL-7 for 3 d in the presence of 5% plasma ESRD patients (Fig. 4D, 4E). Abnormalities in serum phosphorus from healthy controls or ESRD patients. IL-33 was used as positive and calcium are commonly observed in patients with CKD (52). It control. Results showed that the absolute cell counts of ILC2s was found that ILC2s were negatively correlated with serum (Fig. 3A), as well as their secretion of IL-5 and IL-13 (Fig. 3B), phosphorus (Fig. 4F), although they were positively correlated

were dramatically enhanced after treatment with plasma from ESRD with serum calcium (Fig. 4G) in ESRD patients. Interestingly, it http://www.jimmunol.org/ patients when compared with those from healthy controls. IL-33 en- was found that patients who displayed higher levels of PTH were hanced the expansion and function of ILC2s as expected (Fig. 3A, more prone to develop infections (Fig. 4H). The levels of creati- 3B). These observations indicate that certain soluble factors from nine, phosphorus, or calcium did not influence the development of ESRD plasma may drive the expansion and activation of ILC2s. infections in ESRD patients (Fig. 4I–K). Collectively, these ob- We next investigated the potential signaling events underlying servations indicate that ILC2s are negatively correlated with the the elevation of ILC2s observed in ESRD patients. IL-33 and its development of infectious events and the prognostic biomarkers in downstream transcription factor GATA3 play a critical role in the ESRD patients. dictation of ILC2 homeostasis and function (40–42). However, no noticeable differences were observed in either the amounts of Discussion by guest on September 24, 2021 plasma IL-33 or GATA3 expression in ILC2s between ESRD Both innate immunity and adaptive immunity participate in the patients and healthy controls (Fig. 3C, 3D, Supplemental Fig. 1E). progression of ESRD (6, 8). However, the role of ILCs, recently Prostaglandin D2 (PGD2) was known to be an activator of ILC2s defined important innate immune cells, in ESRD remains to be (43). The expression of CRTH2, a receptor for PGD2, was sig- explored. In this study, the levels and the function of ILC2s in the nificantly increased in ESRD-derived ILC2s when compared with peripheral blood of ESRD patients, as well as their clinical ILC2s from healthy controls (Fig. 3E), especially for CD1172 significance, were investigated. ILC2s (Supplemental Fig. 1G). The phosphorylation of ERK1/2 ILC2s have been demonstrated to play important roles in tissue and phosphorylation of STAT5 are important intracellular signal- homeostasis, inflammation, defense against pathogens, and lipid ing to induce ILC2 activation (41, 44, 45). It was found that the metabolism (9, 10, 54). The potential role of ILC2s in kidney levels of p-ERK1/2 in ILC2s did not display noticeable differ- function has been suggested in some pathologies related to kidney ences between ESRD patients and healthy controls (Fig. 3F), injury (16, 22–25). In this study, it was found that the frequencies whereas p-STAT5 levels were significantly elevated in ESRD- of ILC2s in the peripheral blood of ESRD patients were negatively derived ILC2s, especially after PMA stimulation (Fig. 3G). In correlated with the development of infectious complications as support of the elevation of p-STAT5, the amounts of plasma IL-2 well as prognostic biomarkers of CKD (PTH, creatinine, phos- were increased in ESRD patients compared with healthy controls phorus). ILC2s from ESRD patients displayed a stronger capa- (Fig. 3H) (39, 46), whereas other cytokines known to be upstream bility to produce effector cytokines and a higher proliferation rate of p-STAT5, including IL-7, IL-9, IFN-a,IFN-b,andIFN-g when compared with ILC2s from the healthy donors. ESRD pa- (47–49), did not show noticeable differences (Fig. 3H). These tients with higher levels of PTH displayed increased susceptibility observations indicate that IL-2–triggered STAT5 signaling may of infectious events. These observations further support the role of explain the elevation of ILC2s observed in ESRD patients. ILC2s in the protection against renal dysfunction. Modulation of ILC2s may have therapeutic value in ESRD. ILC2s are negatively correlated with the incidence of infectious Human ILC2s include two subsets: CD117+ and CD1172 ILC2s complications in ESRD patients (28, 33). Both ILC2 subsets were increased in ESRD patients We next investigated the clinical significance of ILC2s in ESRD. when compared with those from healthy controls, although the During a mean of the 21-mo follow-up, a total of 33 patients frequencies of CD1172 ILC2s were significantly higher than

(n = 48) (G) in ESRD patients was illustrated by linear regression. (H–K) The levels of PTH (H), creatinine (I), phosphorus (J), and calcium (K) in ESRD patients with (n = 32 or 33) or without (n = 15) infectious events were shown. In all plots, data represent mean 6 SD. *p , 0.05 by unpaired t test. ns, not signiﬁcant. 8 ILC2 EXPANSION IN ESRD PATIENTS

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Supplemental Figure 2. The levels of distinct immune cell types in the peripheral blood of patients with ESRD and healthy controls. (A) The frequencies of T cell subsets (CD3+ T cells, CD3+CD4+ T cells, and CD3+CD8+ T cells) from ESRD patients and healthy controls (n = 5). (B) The frequencies of CD4+CD25+ regulatory T cells (Tregs) from ESRD patients and healthy controls (n = 5). (C) The frequencies of CD19+ B cells from ESRD patients (n = 6) and healthy controls (n = 7). In all plots, data represent mean ± SD. Results are representative of two independent experiments. *P < 0.05; ns, no significant. Statistical analyses were done using unpaired t-test. Supplementary Table I. Clinical and demographic characteristic of healthy controls and patients with ESRD.

Healthy controls Patients with ESRD

Age (<45/>45) 34/0 28/41

Gender (Male/Female ) 18/16 50/19

Diabetes mellitus — 12

Hypertension — 6

Lupus nephritis — 4

Obstruction — 4

Ig A nephropathy — 2

Cardiovascular disease — 2

Proteinuria — 1

Liver transplantation — 1

Other causes — 37

Above shows the causes of ESRD in our current investigation. Supplementary Table II. Baseline characteristic of healthy controls and patients with ESRD.

Healthy controls Patients with ESRD Characteristic P value n Mean ± SD n Mean ± SD

PTH (pg/mL) — — 65 511.92±554.74 — — 2.2±0.21 Calcium (mmol/L) — — 63 Phosphorus — — 63 1.72±0.54 — (mmol/mL)

BUN (mmol/L) 31 4.66±1.17 37 25.01±5.7 < 0.0001

Serum creatinine 32 73.88±13.72 64 1168.28±310.7 < 0.0001 (umol/L)

eGFR 19 112.34±9.95 28 4.04±1.53 < 0.0001

WBC (*109/L) 32 6.68±1.27 62 6.08±1.7 0.0836

RBC (*1012/L) 32 5.1±0.58 61 3.55±0.64 < 0.0001

HGB (g/L) 32 144.5±14.08 65 105.4±15.21 < 0.0001

PLT (*109/L) 31 256±44.47 62 195.52±75.39 < 0.0001

NEUT (*108/L) 31 3.69±0.95 62 3.83±1.41 0.6134

PTH, parathyroid hormone; BUN, blood urea nitrogen; eGFR, estimated glomerular filtration rate; WBC, white blood cell; RBC, red blood cell; HGB, hemoglobin; PLT, platelet count; NEUT, neutrophil count. The reference range of clinical parameters of healthy controls: PTH, 12-65 pg/mL; calcium, 2.03-2.65 mmol/L; phosphorus, 0.74-1.52 mmol/L.