Modulating inflammation with interleukin 37 treatment ameliorates murine Autosomal Dominant Polycystic Kidney Disease

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a leading cause of kidney failure and is associated with substantial morbidity and mortality. Interstitial inflammation is attributed to the action of infiltrating macrophages and is a feature thought to aggravate disease progression. Here, we investigated the therapeutic potential of the anti-inflammatory IL37b cytokine as a treatment for ADPKD using genetic mouse models, demonstrating that transgenic expression of human IL37b reduced collecting duct cyst burden in both early and adult-onset ADPKD rodent models. Moreover, injection of recombinant human IL37b could also reduce cyst burden in early onset ADPKD mice, an observation not associated with increased macrophage number at early stages of cyst formation. Interestingly, transgenic IL37b expression also did not alter macrophage numbers in advanced disease. Whole kidney RNA-seq highlighted an IL37b-mediated upregulation of the interferon signaling pathway and single-cell RNA-seq established that these changes originate at least partly from kidney resident macrophages. We further found that blocking type I interferon signaling in mice expressing IL37b resulted in increased cyst number, confirming this as an important pathway by which IL37b exerts its beneficial effects. Thus, our studies show that IL37b promotes interferon signaling in kidney resident macrophages which suppresses cyst initiation, identifying this protein as a potential therapy for ADPKD. Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a leading cause of kidney failure and is associated with substantial morbidity and mortality. Interstitial inflammation is attributed to the action of infiltrating macrophages and is a feature thought to aggravate disease progression. Here, we investigated the therapeutic potential of the anti-inflammatory IL37b cytokine as a treatment for ADPKD using genetic mouse models, demonstrating that transgenic expression of human IL37b reduced collecting duct cyst burden in both early and adult-onset ADPKD rodent models. Moreover, injection of recombinant human IL37b could also reduce cyst burden in early onset ADPKD mice, an observation not associated with increased macrophage number at early stages of cyst formation. Interestingly, transgenic IL37b expression also did not alter macrophage numbers in advanced disease. Whole kidney RNA-seq highlighted an IL37b-mediated upregulation of the interferon signaling pathway and single-cell RNA-seq established that these changes originate at least partly from kidney resident macrophages. We further found that blocking type I interferon signaling in mice expressing IL37b resulted in increased cyst number, confirming this as an important pathway by which IL37b exerts its beneficial effects. Thus, our studies show that IL37b promotes interferon signaling in kidney resident macrophages which suppresses cyst initiation, identifying this protein as a potential therapy for ADPKD. Translational StatementInflammation is emerging as a critical driver of renal cyst formation in autosomal dominant polycystic kidney disease. Our mouse studies indicate that the modulation of inflammatory pathways using human interleukin-37 represents a potential approach for ameliorating disease severity. Inflammation is emerging as a critical driver of renal cyst formation in autosomal dominant polycystic kidney disease. Our mouse studies indicate that the modulation of inflammatory pathways using human interleukin-37 represents a potential approach for ameliorating disease severity. 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Kurtz E. et al.Mcp1 promotes macrophage-dependent cyst expansion in autosomal dominant polycystic kidney disease.J Am Soc Nephrol. 2018; 29: 2471-2481Crossref PubMed Scopus (76) Google Scholar,29Gardner Jr., K.D. Evan A.P. Reed W.P. Accelerated renal cyst development in deconditioned germ-free rats.Kidney Int. 1986; 29: 1116-1123Abstract Full Text PDF PubMed Scopus (40) Google Scholar,30Gardner Jr., K.D. Reed W.P. Evan A.P. et al.Endotoxin provocation of experimental renal cystic disease.Kidney Int. 1987; 32: 329-334Abstract Full Text PDF PubMed Scopus (42) Google Scholar we speculated that repressing inflammation with IL-37b may reduce disease severity. In mouse models of ADPKD, we found that human IL-37b expression/treatment reduces cyst number. Interestingly, these effects appear to be mediated by kidney resident macrophages with heightened interferon (IFN) responses. This mechanism is distinct from macrophage recruitment, which is thought to drive proliferative cyst growth in later stage disease. Importantly, this animal study highlights the modulation of inflammation by IL-37b as a potential approach to reducing ADPKD severity. Pkd1tm2Ggg C57BL6J31Piontek K.B. Huso D.L. Grinberg A. et al.A functional floxed allele of Pkd1 that can be conditionally inactivated in vivo.J Am Soc Nephrol. 2004; 15: 3035-3043Crossref PubMed Scopus (128) Google Scholar mice were imported from the Jackson Laboratory (Stock number: 010671). IL37b-tg mice were generated as previously described32Nold M.F. Nold-Petry C.A. Zepp J.A. et al.IL-37 is a fundamental inhibitor of innate immunity.Nat Immunol. 2010; 11: 1014-1022Crossref PubMed Scopus (716) Google Scholar and were a gift from Prof. Marcel Nold and A/Prof. Claudia Nold (Hudson Institute of Medical Research, Australia). Hoxb7-cre mice33Yu J. Carroll T.J. McMahon A.P. Sonic hedgehog regulates proliferation and differentiation of mesenchymal cells in the mouse metanephric kidney.Development. 2002; 129: 5301-5312Crossref PubMed Google Scholar were provided by the McMahon Lab (University of Southern California, USA) and were predominantly C57BL6J with minor Swiss Weber contribution. The Pax8rtTA and TetO-Cre mouse lines have been previously described34Ma M. Tian X. Igarashi P. et al.Loss of cilia suppresses cyst growth in genetic models of autosomal dominant polycystic kidney disease.Nat Genet. 2013; 45: 1004-1012Crossref PubMed Scopus (251) Google Scholar, 35Perl A.K. Wert S.E. Nagy A. et al.Early restriction of peripheral and proximal cell lineages during formation of the lung.Proc Natl Acad Sci U S A. 2002; 99: 10482-10487Crossref PubMed Scopus (429) Google Scholar, 36Traykova-Brauch M. Schonig K. Greiner O. et al.An efficient and versatile system for acute and chronic modulation of renal tubular function in transgenic mice.Nat Med. 2008; 14: 979-984Crossref PubMed Scopus (222) Google Scholar and were a gift from Prof. Stefan Somlo (Yale School of Medicine, USA). Experiments were performed at the Monash University Animal Research Platform. Animals were housed and used for experiments under the approval of the Monash University Animal Ethics Committee. Time points for analysis are as indicated for postnatal day (P)0, P4, and P11 (neonatal model), whereas 12 weeks refers to P84 and 18 weeks to P124–P126. Genotyping for the Pkd1 wild type, floxed, and Δ alleles was performed as previously described.31Piontek K.B. Huso D.L. Grinberg A. et al.A functional floxed allele of Pkd1 that can be conditionally inactivated in vivo.J Am Soc Nephrol. 2004; 15: 3035-3043Crossref PubMed Scopus (128) Google Scholar Cre genotyping was performed using the Jackson Laboratory master generic Cre protocol with GoTaq green master mix (Promega). Pax8-rtTA and TetO-Cre genotyping was performed as previously described.34Ma M. Tian X. Igarashi P. et al.Loss of cilia suppresses cyst growth in genetic models of autosomal dominant polycystic kidney disease.Nat Genet. 2013; 45: 1004-1012Crossref PubMed Scopus (251) Google Scholar P0 and P4 kidneys were fixed whole in 4% paraformaldehyde for 16 hours at 4 °C, whereas P11 kidneys were cut in half and fixed in 10% neutral buffered formalin for 16 hours at room temperature. Adult kidneys were cut into halves, and 1 was fixed in 10% neutral buffered formalin for 24 to 48 hours at room temperature. One kidney was sent for histologic processing, and the other was snap-frozen in liquid nitrogen after dissection and stored at −80 °C for protein and gene expression analysis. RNA was extracted from snap-frozen kidney tissue using QiaShredders in conjunction with Qiagen RNeasy Mini Kits according to the manufacturer's protocol. Tissue was initially crushed using eppy pestles in RLT buffer. mRNA analysis was performed using the Nanostring nCounter GX Mouse PanCancer Immune Profiling panel, according to the manufacturer's protocol on an nCounter SPRINT Profiler. Analysis was performed with Rosalind (https://rosalind.bio/) using their default NanoString nCounter Advanced Analysis protocol, which divides counts within a lane by the geometric mean of the normalizer probes from the same lane. Housekeeping probes used for normalization were selected based on the geNorm algorithm as implemented in the NormqPCR R library1. P value adjustment was performed using the Benjamini-Hochberg method. Differential expression analysis was performed with a ±25% fold change cutoff with corrected P values of <0.05 to account for collecting duct mRNA dilution in whole-kidney RNA samples (Supplementary File S1). Pkd1Δ/Δ and Pkd1Δ/+ mice were injected subcutaneously with either saline or recombinant IL-37b (40 μg/kg of IL-37b Y85A variant37Ellisdon A.M. Nold-Petry C.A. D'Andrea L. et al.Homodimerization attenuates the anti-inflammatory activity of interleukin-37.Sci Immunol. 2017; 2eaaj1548Crossref PubMed Scopus (48) Google Scholar provided by A/Prof. Andrew Ellisdon [Monash Biomedicine Discovery Institute] in saline delivered at 10 μl/g) daily from P4 until P10. A cohort of Pkd1Δ/Δ mice were examined with daily handling but left untreated as a control. Mice were humanely culled at P11 for analysis, and kidneys were collected. To examine the IFN signaling pathway, Pkd1Δ/Δ;IL37b and Pkd1Δ/+;IL37b mice were subcutaneously injected with vehicle control (Dulbecco's phosphate-buffered saline), MAR1 (a gift of Prof. Paul Hertzog, Hudson Institute), or its isotype-matched IgG control at 3.3 mg/kg following established protocols.38Sheehan K.C. Lai K.S. Dunn G.P. et al.Blocking monoclonal antibodies specific for mouse IFN-alpha/beta receptor subunit 1 (IFNAR-1) from mice immunized by in vivo hydrodynamic transfection.J Interferon Cytokine Res. 2006; 26: 804-819Crossref PubMed Scopus (209) Google Scholar Fixed tissue was embedded in paraffin wax and sectioned at 4 μm for analysis. Antigen retrieval was performed in citrate buffer (pH 6) using a Tefal pressure cooker. Antibody staining was undertaken as previously described,39Cottle D.L. Kretzschmar K. Schweiger P.J. et al.c-MYC-induced sebaceous gland differentiation is controlled by an androgen receptor/p53 axis.Cell Rep. 2013; 3: 427-441Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar with 0.1% Triton X-100 included in blocking buffers. Cover slips were mounted using Prolong Gold (Invitrogen). Primary and secondary antibodies are listed in Tables 1 and 2. Imaging was performed using an Aperio fluorescent scanner, an Olympus VS200 confocal scanner, or a Leica DMi8.Table 1Primary antibodies/lectinsAntibodyDilutionSupplierCatalogBiotinylated Dolichos biflorus agglutinin1:250Vector LaboratoriesB-1035Biotinylated Lotus tertragonolobus lectin1:250Vector LaboratoriesB-1325Rabbit anti-UMOD1:500Abcamab207170Mouse anti-Uroplakin-III1:50Progen651108F4/80 Rabbit mAb1:250CSTD2S9RRabbit anti-CXCL11:100Thermo Fisher ScientificPA586508CXCL, CXC chemokine ligand; UMOD, uromodulin. Open table in a new tab Table 2Secondary antibodies and stainsAntibodyDilutionSupplierCatalogAlexa Fluor donkey anti-mouse IgG (H+L) 5551:600InvitrogenA31570Alexa Fluor donkey anti-rabbit IgG (H+L) 5551:600InvitrogenA31572Streptavidin, Alexa Fluor 4881:600InvitrogenS11223Dolichos biflorus agglutinin—FITC1:100Vector LaboratoriesFL-1031Alexa Fluor donkey anti-rabbit IgG (H+L) 4881:600InvitrogenA212064′,6-Diamidino-2-phenylindole1:1000DakoD1306FITC, fluorescein isothiocyanate. Open table in a new tab CXCL, CXC chemokine ligand; UMOD, uromodulin. FITC, fluorescein isothiocyanate. Kidneys were dissociated by digesting tissue using collagenase IV (Sigma C51380; 4 mg/ml), DNAseI (Sigma DN25; 1 mg/ml), Dispase (Gibco 17105-041; 2 mg/ml), and hyaluronidase (Sigma H3506-1G; 2 mg/ml) in Dulbecco's phosphate-buffered saline (Thermo Fisher) for 20 minutes at 37 °C with agitation. Cell suspensions were filtered through a 70 μm membrane before resuspension. For single-cell RNA sequencing (RNA-seq) at P4, directly conjugated antibodies (CD11b and F480) were used to sort for macrophages. Cells were gated by live cells, and at least 20,000 double-positive cells were collected for single-cell RNA-seq. For the analyses at P11, a cocktail of directly conjugated antibodies was used, including anti-CD45, F480, CD11b, and Ly6C, and added to the suspended cells. Cells were Fc-blocked at room temperature before antibody incubation. Fc blocker comprised rat anti-mouse CD16/CD32 (Cat#12-0161-82) and normal rat serum (Cat#19851) in fluorescence-activated cell sorting buffer (2% fetal calf serum, 1 mM ethylenediamine tetraacetic acid, and 0.05% NaN3 in 1×phosphate-buffered saline). Analysis was undertaken using a Fortessa X20 (BD Sciences) and FlowJo Software (Tree Star Inc.). Cells were gated by live cells and CD45 positive cells, and then separated into positive cell populations. Directly conjugated antibodies are listed in Table 3.Table 3Flow cytometry antibodiesAntibodyDilutionSupplierCatalogPE anti-mouse/human CD11b antibody1:500BioLegend101208Pacific Blue anti-mouse F4/80 Antibody1:200BioLegend123124Fixable viability dye V5061:1000Thermo Fisher Scientific65-0866-14CD45 eVolve 6451:200Thermo Fisher Scientific86-0451-42F4/80 BUV4961:200BD Biosciences750644Ly6C BV7111:200BioLegend128037CD11b APC-Cy71:200TONBO Biosciences25-0112-U100PE, phycoerythrin. Open table in a new tab PE, phycoerythrin. Kidney protein lysates were extracted by homogenization in 0.25% NP-40 Tris-buffered saline (pH 7.2–7.5), and concentrations were measured using a BioRad DC reagent kit. Inflammatory markers were analyzed using a 40 factor RayBiotech Quantibody Mouse inflammation Array Q1 kit (QAM-INF-1-1). Fluorescence was detected with a Genepix 4000B slide scanner. The RayBiotech Quantibody Mouse Inflammation Array Q1 excel tool was used to perform analysis. Snap-frozen P0 kidneys were crushed using micropestles in RLT buffer, and RNA was extracted using QiaShredders followed by Qiagen RNeasy Mini Kits. RNA was sent to GeneWiz for quality assessment, library preparation, and sequencing using the Illumina HiSeqX10 sequencer. Sequence data were uploaded onto Galaxy servers and processed with an automatic adapter trimmer and mapped with HiSat2 to the Mus musculus GRCm38_v100 genome assembly. The resulting Bam files were exported and analyzed in the Seqmonk version 1.47.1 software. A minimum of 23 million reads were obtained per sample. Differentially expressed genes between the groups were determined using the count-based DeSeq2 method, with a multiple testing–corrected P value cutoff of < 0.05. Significant pathway changes were analyzed using Metascape.40Zhou Y. Zhou B. Pache L. et al.Metascape provides a biologist-oriented resource for the analysis of systems-level datasets.Nat Commun. 2019; 10: 1523Crossref PubMed Scopus (6754) Google Scholar Venn diagrams were created using Venny 2.141Oliveros J.C. (2007–2015) An interactive tool for comparing lists with Venn's diagrams.http://bioinfogp.cnb.csic.es/tools/venny/index.htmlDate accessed: January 21, 2024Google Scholar (Supplementary File S2). Single-cell RNA-seq was undertaken by Micromon Genomics (Monash University) using 10x Genomics kits and an Illumina HiSeq. Raw files were processed using Cell Ranger in file aggregation mode and default settings for individual files before analysis by 10x Genomics Loupe Browser software. t-distributed stochastic neighbor embedding plots and differentially expressed gene lists were generated in Loupe Browser. Macrophages were classified as M1 (Il1r1, Tlr2, Tlr4, Nos2, Socs3, Cd80, Il12a, Il12b, Myd88 expressing), M2 (Cd163, Il10, Tgm2, Il1r2, Arg1, Retnl1, Chil3, Chil4, Cd200r1, Mrc1, Msr1, Tlr1, Tlr8, Vegfb expressing), infiltrating (Ccr2, Plac8, Lyz2, Cebpb, Itgam expressing), or resident (Cd81, Cd74, Fcgr1, C1qc, Apoe, Adgre1 expressing). Significant pathway changes were analyzed using Metascape40Zhou Y. Zhou B. Pache L. et al.Metascape provides a biologist-oriented resource for the analysis of systems-level datasets.Nat Commun. 2019; 10: 1523Crossref PubMed Scopus (6754) Google Scholar and String version 11.5 (Supplementary File S3). Cyst phenotypes (cystic index, number, and size) were profiled using a cross-sectional kidney taken medially, along the longest axis of the organ. Cystic index was calculated as the proportion of cystic space in the total kidney area; number reflects the average number of cysts within each section, whereas size is the average cross-sectional cyst size. Collecting duct cysts were identified based on the expression of Dolichos biflorus agglutinin. All analysis was performed in a semiautomated process using Fiji.42Schindelin J. Arganda-Carreras I. Frise E. et al.Fiji: an open-source platform for biological-image analysis.Nat Methods. 2012; 9: 676-682Crossref PubMed Scopus (37833) Google Scholar Samples were annotated by mouse ID number. Unless otherwise stated, datasets were analyzed in Graphpad Prism version 9.0 using either a 1-way analysis of variance or an unpaired Student's t test, assuming equal variance, where P < 0.05 is considered significant. One- or two-tailed tests were used as appropriate for the hypothesis. Data are presented as mean ± SEM. The examination of public expression datasets was performed using list importing, intersection, and graphic tools in R-studio. To explore the immune mechanisms contributing to cystogenesis, Pkd1-floxed conditional mice (Pkd1fl/fl)31Piontek K.B. Huso D.L. Grinberg A. et al.A functional floxed allele of Pkd1 that can be conditionally inactivated in vivo.J Am Soc Nephrol. 2004; 15: 3035-3043Crossref PubMed Scopus (128) Google Scholar were crossed with Hoxb7-cre mice, and whole-kidney mRNA was extracted at P4 for analysis using a NanoString mouse PanCancer Immune Profiling panel (Supplementary File S1). The NanoString results demonstrated that IL-18R1 and IL-1R8, the receptors of anti-inflammatory IL-37, were dysregulated (Figure 1a–c). However, as mice lack an IL-37 homolog, we wondered how restoration of IL-37 signaling may influence disease. To determine whether transgenic expression of human IL37b could prevent or curtail cyst formation, ADPKD mice were crossed with IL37-tg mice to examine cyst formation in collecting ducts. This resulted in mice with either 0, 1, or 2 alleles of the IL37 transgene, and the cystic phenotype was subsequently examined over the course of disease progression (Figure 1d). IL-37b mice have metabolic changes that may impact body weight43Kuipers E.N. van Dam A.D. Ballak D.B. et al.IL-37 expression reduces lean body mass in mice by reducing food intake.Int J Mol Sci. 2018; 19: 2264Crossref PubMed Scopus (5) Google Scholar and confound analysis of kidney to body weight ratios. Consequently, cystic index, number, and size were used as a measure of disease severity. All Pkd1Δ/Δ mice had extensive collecting duct–derived cysts and enlarged kidneys, a finding consistent with previous studies.44Paul B.M. Vassmer D. Taylor A. et al.Ectopic expression of Cux1 is associated with reduced p27 expression and increased apoptosis during late stage cyst progression upon inactivation of Pkd1 in collecting ducts.Dev Dyn. 2011; 240: 1493-1501Crossref PubMed Scopus (13) Google Scholar Cystic index was reduced in