Study On The Role And Mechanism Of Innate Lymphoid Cells In Kidney Disease

Recently,a new group of innate lymphoid cells(ILC s)have been discovered in the field of immunology,which lack specific receptors and can be activated independently of antigen presentation.According to the expression of transcription factors and the production of cytokines,ILCs can be divided into three subgroups:ILC1,ILC2 and ILC3.ILC1 expresses T-bet and produces IFN-? and TNF-?,ILC2 expresses GATA-3 and produces IL-4,IL-5 and IL-13,and ILC3 expresses RORyt and produces IL-17 and IL-22.ILCs are not only enriched in the mucosal barrier of the lung,intestine and skin,but also widely exist in peripheral parenchymal organs,such as the liver and kidney.It has been found to play an important role in a variety of inflammatory diseases.The incidence and mortality of chronic kidney disease(CKD)have been increasing year by year,which has become a threat to global health.Immune cells change significantly in kidney diseases,and play an important role in many processes of the disease,such as occurrence,progression,immune surveillance and tissue repair.Persistent inflammation is considered to be a key factor in CKD and significantly related to all-cause mortality in these patients.Inflammation in CKD is characterized by the changes in innate and adaptive immunity,especially activated monocytes and T cells in circulation.A clinical study has shown that ILC response changes significantly in multiple diseases(such as asthma,Crohn's disease and psoriasis),and our previous study has found the unique role of ILC subsets in animal models of kidney diseases and their therapeutic potential.However,ILC is rarely investigated in the clinical study on kidney diseases.Therefore,to clarify the changes of ILCs and their subsets in CKD patients,the relationships of ILCs and their subsets with various clinical indicators reflecting disease activities are a topic worthy of further exploration.In the study of animal models,it has been found that different subsets of ILC are vital to maintain homeostasis during physiological process,induce inflammation and damage,and promote tissue recovery in a pathological state.In-depth study has revealed that ILCs play a nonnegligible role in renal homeostasis and diseases.A previous study has found that ILC2s regulate inflammatory response and prevent renal dysfunction by activating regulatory immune cells and producing amphiregulin in animal models of acute and chronic kidney disease.In addition,it has been found that in the model of anti-glomerular basement membrane(anti-GBM)glomerulonephritis,renal ILC1s promote the development of glomerulonephritis.Recently,a new class of ILC subsets has been found in the intestine,and the number has increased significantly in the model of inflammatory bowel disease.They can inhibit the secretion of effector cytokines by ILC1s and ILC3s in the intestine and reduce intestinal inflammation by secreting IL-10.These ILC subsets with immunomodulatory effect are named innate regulatory lymphocytes(ILCregs).However,whether ILCregs are expressed in the kidney under physiological and pathological status,as well as their phenotype and biological functions have not been reported,which needs to be clarified.Therefore,to explore the expressions and functions of these cells in renal tissue is of great significance and value for finding new targets for the treatment of kidney diseases.Objectives1.By detecting the changes of total ILCs and ILC subsets in peripheral blood from patients with glomerular diseases of different types and healthy controls(HCs),to analyze the correlations of ILCs in peripheral blood with clinical indicators of the patients and clinical indicators of patients with CKD of various types,such as lupus nephritis(LN)and diabetic kidney disease(DKD),reflecting the degree of disease activity and the degree of kidney diseases,and preliminarily evaluate whether there are abnormalities of ILCs in patients with CKD and whether peripheral ILCs are correlated with CKD.2.By detecting the proportion and phenotype of ILCregs in human and mouse renal tissues,to preliminarily identify the distribution and phenotype of ILCregs in the kidney.3.By co-culturing in vitro induced and amplified ILCregs with ILC1s and M1 macrophages,to explore the regulatory mechanism of ILCregs in innate immunity.4.To induce the amplification of ILCregs in a mouse model of renal IRI using IL-2/IL-2 antibody complex,adoptively transfer ILCregs into model mice and antibody-specifically clear ILCregs,and explore the role of ILCregs in renal IRI by detecting renal function,injury,inflammatory level,inflammatory cell infiltration,apoptosis and proliferation.5.To adoptively transfer ILCregs amplified in vitro into the mouse model of renal IRI,and explore the mechanism of ILCregs in affecting the course of renal IRI by reducing inflammatory cell infiltration and inhibiting innate immunity by detecting the level of inflammatory cytokines,inflammatory cell infiltration and the phenotype of macrophages.Methods1.CKD patients were selected according to the inclusion and exclusion criteria.Peripheral venous blood samples were taken to detect total ILCs and ILC subsets in mononuclear cells in peripheral blood.Renal function,triglyceride and serum albumin were detected.The 24-hour and morning urine were collected for examination,the 24-hour urine volume was recorded,the 24-hour total urine protein excretion rate(UPER)and albumin-creatinine ratio(ACR)were detected,and eGFR was calculated using the CKD-EPI formula.In addition,the height and body weight of DKD patients were recorded,and fasting blood glucose and glycated haemoglobin(HbA1C)were detected.For LN patients,blood routine and component 3(C3)were detected.2.The mouse model of renal ischemia-reperfusion injury(IRI)was established using adult male C57BL/6,Rag-/-and IL-10-GFP mice,weighing 20-25 g.3.The proportion and phenotype of renal ILCregs were identified by flow cytometry.Renal ILCregs were selected by flow cytometry,and the in vitro amplification scheme for ILCregs was established.4.ILCregs were co-cultured with ILC1s and M1 macrophages in vitro.The levels of effector cytokines secreted by ILC1s and M1 macrophages such as IFN-?,IL-1? and TNF-? were detected by ELISA.5.The induced ILCregs in renal IRI model were specifically cleared by IL-2/IL-2 antibody complex or anti-CD25 antibody pretreatment,and adoptive transfer of ILCregs.Renal tubular injury was observed by PAS staining,apoptosis of renal tubular cells was detected by TUNEL,proliferation of tubular cells was detected by Ki67 staining,and the proportions of ILCregs,ILC1s,ILC2s and ILC3s in renal tissue was determined using flow cytometry.6.ILCregs amplified in vitro were adoptively transferred into renal IRI model mice.Renal tubular injury was observed by PAS staining,apoptosis of renal tubular cells was detected by TUNEL,proliferation of tubular cells was detected by Ki67 staining,and the proportion of ILCregs in renal tissue was determined using flow cytometry.Macrophages were sorted by flow cytometry,and the expressions of inducible nitric oxide synthase(iNOS),tumor necrosis factor-?(TNF-?),interleukin(IL)-1?,CC chemokine ligand 2(CCL2),mannose receptor(MR),arginase,heme oxygenase-1(HO-1)and IL-10 mRNA were detected by real-time PCR.7.Descriptive statistics of numerical variables,such as variables subject to normal distribution,were expressed as mean ± standard deviation(SD),otherwise,they were expressed as median and full range.Renal function indicators were continuous variables,expressed as mean ± SD,and subjected to logarithmic transformation to stabilize the variance.Two-tailed t-test was used for independent samples in the two groups.If the variance is uneven,Welch correction is used.Samples in multiple groups were compared using the one-way analysis of variance,and multiple pairwise comparisons were performed by the Tukey's method.In correlation analysis,Pearson correlation analysis was used for parametric test,and Spearman correlation analysis for nonparametric test.Statistical analysis was carried out using Prism version 8(GraphPad Software,San Diego,CA),with ?=0.05 as statistically significant.Results1.According to the inclusion and exclusion criteria,82 patients with CKD and 16 HCs were included in this study,including 21 patients with IgA nephropathy(IgAN),11 patients with membranous nephropathy(MN),28 patients with LN and 22 patients with DKD,of which 41 were male.Compared with the HC group,the total ILCs in the DKD group were significantly higher(P<0.001),but no statistical significance was found in the proportion of total ILCs between MN and IgAN patients.The proportion of ILC1s in CD45+lymphocytes in the DKD and LN groups was significantly higher than that in the HC group,with statistical differences.The proportion of ILC2s in CD45+lymphocytes increased significantly in patients with DKD(P<0.05),but showed no significant changes in other groups.2.In LN patients,the proportions of total ILCs and ILC1s in CD45+lymphocytes increased significantly during the active period,with statistically significant differences.ILC2s and ILC3s had no significant differences between the two groups.The proportions of total ILCs and ILC1s in CD45+ lymphocytes were negatively correlated with C3 level and WBC count,and the proportion of ILCs was positively correlated with proteinuria quantification.In DKD patients,the proportions of total ILCs and ILC1s in CD45+lymphocytes were positively correlated with BMI and HbA1c,the proportions of total ILCs,ILC1s and ILC2s in CD45+cells were positively correlated with eGFR,the proportion of total ILCs was also positively correlated with ACR,and the proportion of ILC3s in CD45+lymphocytes was positively correlated with BMI and ACR.In all CKD patients included in the study,the proportions of total ILCs and ILC1s in CD45+lymphocytes were negatively correlated with eGFR while positively correlated with ACR level.On the contrary,there were no significant correlations between the proportions of ILC2s and ILC3s in CD45+lymphocytes and clinical indicators of renal function.3.Lin-CD127+CD161+IL-10+ cells were defined as human ILCregs,accounting for about 4.4%of the total renal ILCs.ILCregs in human renal tissue expressed high-affinity IL-2 receptor CD25 and T cell-induced costimulatory molecules,but lacked ILC2 markers CRTH2 and KLRG1.Lin-CD127+CD90+IL-10+cells were defined as mouse renal ILCregs,accounting for about 2.7%of the total renal ILCs.Mouse renal ILCregs expressed CD25 and ICOS,but lacked ILC2-characteristic markers ST2 and KLRG1.4.ILCregs were isolated and purified from the renal tissue of IL-10-GFP mice,and incubated with IL-2,IL-7 and TGF-?,respectively,for 3,6 and 12 d.ILCregs in different groups were counted at each time point.After stimulation with different cytokines,the number of ILCregs increased with time.IL-2+IL-7+TGF-?Compared with other groups,ILCregs in the IL-2+IL-7+TGF-? group increased significantly on d 3,6 and 12,with statistically significant differences.The number of ILCregs on d 12 after cell culture was>40 times larger than that at the beginning of amplification.The amplified ILCregs still retained the key markers of characteristic membrane expression and transcription factors,including CD 127,CD90,CD25,ICOS and Id3.ILCregs amplified in vitro were co-cultured according to different cell density ratios,and compared with the negative control group with only ILC1s and ILCregs.The culture system was maintained for 3 d,and the level of IFN-? in the culture supernatant of different groups was detected.With the increase in the proportion of ILCregs,the level of IFN-? secreted by activated ILC1s decreased.After adding anti-IL-10 and TGF-? neutralizing antibody,the level of IFN-y secreted by ILC1s in the culture supernatant was significantly higher than that without antibodies,with a statistically significant difference.The amplified ILCregs were incubated with M1 macrophages,with or without anti-IL-10 and TGF-? neutralizing antibody to block the inhibitory function of ILCregs.The results showed that after adding neutralizing antibody,the levels of TNF-? and IL-1? secreted by M1 macrophages in the culture supernatant were significantly higher than those in the non-antibody group,with statistically significant differences.5.After pretreatment of Rag-/-mice with IL-2/IL-2 antibody complex(IL-2C),it was found that ILCregs in the kidney increased significantly.IL-2C pretreatment(IRI+IL-2C group)and simple disease model(IRI+Vehicle group)could improve renal function and alleviate renal injury.The apoptosis and proliferation of renal tubular epithelial cells in the IRI+IL-2C group were detected using terminal deoxyribonucleotide transferase-mediated dUTP nick-end labeling and Ki67 staining,revealing that compared with the IRI+Vehicle group,apoptosis was alleviated and cell proliferation was improved.The proportion of renal IL-10+ILCregs in total ILCs increased significantly in the IRI+IL-2C group.6.After pretreatment with IL-2C,ILCregs in mice were cleared with anti-CD25 antibody(PC61)(IRI+IL-2C/PC61 group).The results demonstrated that in the IRI+IL-2C/PC61 group,the protective effects of IL-2C intervention in improving renal function,reducing tubular cell injury and apoptosis and promoting cell proliferation were offset.Compared with the disease model group only intervened with IL-2C(IRI+IL-2C group),there were statistically significant differences in creatinine level,tubular injury score,apoptotic cells and Ki67-expressing cells.Additionally,after PC61 intervention,flow cytometry analysis presented that renal ILCregs were significantly less in the IRI+PC61 group compared with the IRI+Vehicle group and in the IRI+IL-2C/PC61 group compared with the IRI+IL-2C group.7.ILCregs amplified in vitro were adoptively transferred into C57BL/6 mice 24 h before the establishment of IRI model(pre-ILCreg group)or 4 h after the establishment(post-ILCreg group).Both pre-ILCreg and post-ILCreg groups showed that compared with the IRI group,renal tubular injury,serum creatinine level and renal tubular epithelial cell apoptosis were significantly reduced,and renal tubular epithelial cell proliferation was significant.Compared with the IRI group,the inflammatory cytokines TNF-?,IL-1? and IL-6,CXC chemokine ligand 1(CXCL1)and CXC chemokine ligand 2(CXCL2)in renal tissue of the pre-ILCreg and post-ILCreg groups decreased significantly,and the neutrophil infiltration of Gr-1+reduced significantly.In the pre-ILCreg and post-ILCreg groups,M1 markers in macrophages including inducible nitric oxide synthase,TNF-?,IL-1? and CCL2 decreased significantly,while M2 markers including mannose receptor,arginase,heme oxygenase-1 and IL-10 increased significantly.Conclusions1.In DKD patients,the incidences of total ILCs and ILC subsets in peripheral blood change significantly,and show good correlations with some clinical indicators.The proportions of total ILCs and ILC Is are positively correlated with BMI and HBA1c,and the proportion of ILC3s is positively correlated with BMI and ACR concentration.2.In LN patients,the proportion of ILC1s in peripheral blood increases while the proportion of ILC2s decreases.The proportions of total ILCs and ILC1s are correlated with clinical indicators(WBC and C3)reflecting the degree of disease activities.3.In CKD patients,total ILCs and ILC1s in peripheral blood are significantly correlated with the clinical indicators of renal function(ACR and eGFR).4.ILCregs are identified in human and mouse renal tissues,human renal ILCregs(Lin-CD127+CD161+IL-10)and mouse renal ILCregs(Lin-CD127+CD90+IL-10+).5.Renal ILCregs can inhibit innate immune response,M1 macrophages and ILC Is in vitro.6.In vivo amplification or adoptive transfer of ILCregs can reduce renal injury in renal IRI model.7.ILCregs reduce renal injury by infiltrating inflammatory cells in renal tissue with IRI,and promoting macrophage M1/M2 polarization.