LRH-1 agonism favours an immune-islet dialogue which protects against diabetes mellitus

ARTICLE LRH-1 agonism favours an immune-islet dialogue which protects against diabetes mellitus Nadia Cobo-Vuilleumier1, Petra I. Lorenzo1, Noelia García Rodríguez1, Irene de Gracia Herrera Gómez1, Esther Fuente-Martin1, Livia López-Noriega1, José Manuel Mellado-Gil1, Silvana-Yanina Romero-Zerbo2,3, Mathurin Baquié4, Christian Claude Lachaud1, Katja Stifter5, German Perdomo6, Marco Bugliani7, Vincenzo De Tata8, Domenico Bosco9, Geraldine Parnaud9, David Pozo 10, Abdelkrim Hmadcha 1,3, Javier P. Florido11, Miguel G. Toscano12, Peter de Haan12, Kristina Schoonjans13, Luis Sánchez Palazón14, Piero Marchetti7, Reinhold Schirmbeck5, Alejandro Martín-Montalvo1, Paolo Meda15, Bernat Soria1,3, Francisco-Javier Bermúdez-Silva 2,3, Luc St-Onge16 & Benoit R. Gauthier 1 Type 1 diabetes mellitus (T1DM) is due to the selective destruction of islet beta cells by immune cells. Current therapies focused on repressing the immune attack or stimulating beta cell regeneration still have limited clinical efficacy. Therefore, it is timely to identify innovative targets to dampen the immune process, while promoting beta cell survival and function. Liver receptor homologue-1 (LRH-1) is a nuclear receptor that represses inflammation in digestive organs, and protects pancreatic islets against apoptosis. Here, we show that BL001, a small LRH-1 agonist, impedes hyperglycemia progression and the immune-dependent inflammation of pancreas in murine models of T1DM, and beta cell apoptosis in islets of type 2 diabetic patients, while increasing beta cell mass and insulin secretion. Thus, we suggest that LRH-1 agonism favors a dialogue between immune and islet cells, which could be druggable to protect against diabetes mellitus. DOI: 10.1038/s41467-018-03943-0 OPEN 1Department of Cell Regeneration and Advanced Therapies, Andalusian Center for Molecular Biology and Regenerative Medicine-CABIMER, Junta de Andalucia-University of Pablo de Olavide-University of Seville-CSIC, Seville 41092, Spain. 2Unidad de Gestión Clínica Intercentros de Endocrinología y Nutrición, Instituto de Investigación Biomédica de Málaga (IBIMA) Hospital Regional Universitario de Málaga Universidad de Málaga, Málaga 29010, Spain. 3Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Madrid 28029, Spain. 4Neurix SA, Geneva 1228, Switzerland. 5Ulm University Hospital, Ulm 89081, Germany. 6Facultad de Ciencias de la Salud, Universidad de Burgos, Burgos 09001, Spain. 7Department Clinical and Experimental Medicine, University of Pisa—AOUP University Hospital, Pisa 56126, Italy. 8Department of Translational Research and of New Surgical and Medical Technologies, University of Pisa, Pisa 56126, Italy. 9Cell Isolation and Transplantation Centre, University Hospital, Geneva 1211, Switzerland. 10 Department of Cell Dynamics and Signalling, CABIMER-Andalusian Center for Molecular Biology and Regenerative Medicine, Seville 41092, Spain. 11 Clinical Bioinformatics Area, Fundación Progreso y Salud, Consejería de Salud, Seville 41013, Spain. 12 Amarna Therapeutics, Seville 41092, Spain. 13 Laboratory of Metabolic Signaling, EPFL, Lausanne 1015, Switzerland. 14 Biological Resources, CABIMER-Andalusian Center for Molecular Biology and Regenerative Medicine, Seville 41092, Spain. 15 Department of Cell Physiology and Metabolism, University of Geneva, Geneva 1211, Switzerland. 16 Neuried Munich 82061, Germany. Correspondence and requests for materials should be addressed to B.R.G. (email: [email protected]) NATURE COMMUNICATIONS | (2018) 9:1488 |DOI: 10.1038/s41467-018-03943-0 |www.nature.com/naturecommunications 1 1234567890():,; Type 1 diabetes mellitus (T1DM) is a CD4+and CD8+T- cell-dependent autoimmune disease that targets beta cell destruction, ultimately leading to hyperglycemia and insulin dependence. The collapse in tolerance to self-antigens, such as insulin, is precipitated by genetic and environmental factors1,2. To date, therapies aimed at inhibiting the immune system using anti-CD3 monoclonal antibodies or at neutralizing pro-inflammatory cytokines, have had limited success3,4. One of the reasons may be that inhibiting the immune and inflammatory reactions in the pancreas impairs the repairing and regeneration capabilities of a functional beta cells mass5,6, as observed during wound healing7. Novel agents that could guide a proinflammatory autoimmune destructive environment toward an anti-inflammatory milieu facilitating islet regeneration, would define a novel class of antidiabetic therapies. The liver receptor homolog-1 (LRH-1, or NR5A2) is a member of the NR5A family of nuclear receptors, which plays a pivotal role in early embryonic development, and specifies the endodermal lineage8. In the liver, LRH-1 modulates the expression of genes involved in cholesterol and bile acid metabolism, as well as in glucose homeostasis9, attenuates the hepatic acute phase response, which is triggered upon increases of pro-inflammatory cytokines, and protects against endoplasmic reticulum stress10,11. In the intestine, LRH-1, modulates the enterocyte renewal and regulates the local immune system via production of glucocorticoids12. In the pancreas, LRH-1 regulates the expression of genes involved in digestive functions, and protects the endocrine islets against cytokine- and streptozotocin-induced apoptosis13,14, while stimulating the production of enzymes involved in glucocorticoids biosynthesis14. In view of the above, specifically of the possibility that LRH-1 could elicit an islet-driven anti-inflammatory microenvironment, we posited that upregulating LRH-1 activity could have beneficial therapeutic effects in diabetes mellitus (DM). Natural phospholipids physiologically stimulates LRH-1 activity15,16, decreasing hepatic steatosis and improving glucose homeostasis in animal models of insulin resistance17. Given that LRH-1 can also be activated by smaller, non-polar bicyclic compounds18, we have synthesized a compound termed BL001, which we have tested in mouse models of T1DM, as well as in pancreatic islets from patients affected by Type 2 DM (T2DM). Here we report that the long-term in vivo administration of BL001 prevents the development of diabetes in mice, through the combined maintenance of a functional islet beta cell mass and the release of anti-inflammatory factors, which contribute to the islet regeneration effect. We further report that BL001 also protects human islet cells from apoptosis and improves impaired insulin secretion as well as beta cell survival in the pancreatic islets of T2DM patients. The data define LRH-1 as a novel therapeutic target for the treatment of T1DM. Results BL001 activates LHR-1 without cytotoxic or metabolic effects. The chemical structure of BL001, which specifically binds to and activates LRH-118, is depicted in Supplementary Fig. 1a. The effects of the drug on LRH-1 activity, cell viability, and toxicity are described in Supplementary Fig. 1b–e. Pharmacokinetic and safety profiling of BL001 were studied in C57BL/6 and RIP-B7.1 mice, respectively. An i.p. injection of 10 mg/kg b.w. BL001 led to peak plasma concentrations of 3.6 μg/ml (≈8μM) after 0.2 h, and a half-life of 9.4 h. Daily injections during 24 weeks did not reveal macroscopic organ alterations in BL001-treated RIP-B7.1 mice (Supplementary Fig. 2a, b), which also featured normal plasma levels of total cholesterol and triglycerides up to 8 weeks of treatment (Supplementary Fig. 3a, b). Insulin sensitivity was not altered by this BL001 treatment (Supplementary Fig. 3c). BL001 blunts apoptosis and attenuates diabetes in mice.To assess the anti-apoptotic effect of BL001, mouse islets were exposed to 10 μM BL001 in the presence of 2 ng/ml IL1beta, 28 ng/ml TNFalpha and 833 ng/ml IFNgamma. The drug prevented the cytokine-induced islet cell death (Fig. 1a). A substantial loss of LRH-1 transcript and protein by RNAi, sensitized BL001-treated islets to the cytokine-induced apoptosis (Fig. 1b–d). The antidiabetic role of BL001 was next evaluated in animal models of T1DM. C57BL/6 male mice that received 150 mg/kg b.w. streptozotocin (STZ) developed diabetes within 4 weeks (Fig. 1e, Supplementary Fig. 4a). The incidence of diabetes was decreased after a 5 day pre-treatment with 10 mg/kg b.w. BL001 (Fig. 1e, Supplementary Fig. 4a), which decreased the loss of insulincontaining beta cells (Fig. 1g), and increased the proportion of cells staining for both insulin and glucagon (Fig. 1h, i). Moreover, 30% of the mice that developed diabetes returned to normoglycemia 4 weeks after a daily injection of 10 mg/kg b.w. BL001, starting 1 week after the STZ injection (Fig. 1f, Supplementary Fig. 4b). To evaluate the effect of BL001 against an autoimmune attack, we studied RIP-B7.1 mice, a model mimicking the etiology of T1DM19, without gender influence20. Eighty percent of RIP-B7.1 mice immunized against insulin developed diabetes within 8 weeks (Fig. 1j, Supplementary Fig. 4c), when receiving only a vehicle solution. This proportion dropped to 43% in mice treated with 10 mg/kg b.w. BL001 for 5 days prior to immunization (Fig. 1j, Supplementary Fig. 4c). Still, a similar increase in the proliferation of T cells in response to insulin was also detected in the splenocytes of mice treated or not with BL001, both 4 and 8 weeks after the insulin immunization (Fig. 1k), confirming that all mice had mounted an in vivo autoimmune attack. At these time points, immunostaining showed a near-complete destruction of beta cells in the islets of the control immunized mice, and the persistence of sizable numbers of these cells in the immunized mice treated with BL001 (Fig. 1l). In the presence of hyperglycemia, the latter animals also featured islets with a significant increase in the number of PDX1+cells as compared to immunized mice (Fig. 1m, n). Mice in which the BL001 treatment was initiated 5 days after the immunization, featured an incidence of diabetes similar to that of controls for the first 5 weeks (Fig. 1o, Supplementary Fig. 4d). Thereafter, however, the incidence of diabetes was lower (~30%) in the BL001-treated mice than in immunized controls (~80%; Fig. 1o, Supplementary Fig. 4d). A similar decrease in diabetes incidence was observed in female NOD mice treated with BL001 from week 12 (Fig. 1p), an age at which these animals feature an ongoing autoimmune attack, and reduced insulin content. To establish whether the in vivo effects of BL001 were mediated via LRH-1, we generated mice in which the Lrh1 gene was selectively disrupted in beta cells. To this end, Lrh1 lox/lox mice were crossed with RIP-Cre mice. We found that all mice carrying two deleted alleles (βLRH-1−/−), and 75% of the progenies carrying one normal allele (βLRH-1−/+) died within 3 weeks of life (Supplementary Fig. 5a). Immunofluorescence staining of islets from day 1 neonatal pups, showed an atypical islet morphology, with a normal proportion of insulin-containing beta cells surrounded by an increased proportion of glucagoncontaining alpha cells in homozygous mice (Supplementary Fig. 5b–e). BL001 inhibits insulitis and alters serum cytokine profile.To unravel the mechanism whereby BL001 impedes the progression of diabetes, we evaluated the lymphocytic infiltration of islets (insulitis) in mice pre-treated or not with BL001. Four weeks after immunization, a sizable insulitis was detected in the pancreas of ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03943-0 2NATURE COMMUNICATIONS | (2018) 9:1488 |DOI: 10.1038/s41467-018-03943-0 |www.nature.com/naturecommunications both BL001-treated and control mice (Fig. 2a, b). However, 4 weeks later, the normoglycemic BL001-treated mice were nearly free of insulitis, in contrast to controls which displayed strong infiltration (Fig. 2c, d). Accordingly, no CD4+or CD8+T cells were found in the islets of the former animals (Supplementary Fig. 6), which also showed significantly higher levels of circulating IL10, IL5, IL6 (Fig. 2e, f), CCL2, CCL4 (Fig. 2g, h), and TGFbeta (Fig. 2i, j) than immunized controls at 8 but not 4 weeks. In contrast, no significant differences were observed in levels of INFgamma (0.6965 ± 0.0931 versus 0.4989 ± 0.0733 p=0.1072, unpaired Student’sttest), TNFalpha (5.925 ± 0.815 versus 6.648 ± 0.659 p=0.4982, unpaired Student’sttest), IL1beta (0.5267 ± DAPI LRH1 siSC siLRH1 CTRL STZ BL Pre-treatment + STZ STZ + BL Post treatment DAPI GLUC INS DAPI GLUC INS STZ + BL Post-treatment DAPI PDX1 INS DAPI GLUC INS 4 Weeks STZ 8 Weeks a 1.5 ** ** ** n = 6 n = 6 1.0 Cell death (A405-A490) 0.5 C CK CK + BL BL 0.0 120 STZ (n = 5) BL STZ (n = 12) STZ (n = 5) BL STZ (n = 14) 100 80 60 40 Diabetes incidence (%) Diabetes incidence (%) 20 0 100 [H3] Thymidine incorporation (fold change as compared to mock control) 3 * 2 1 4 wks 8 wks 0 C (n = 26) C (n = 5) BL (n = 26) IMN (n = 50) IMN (n = 12) BL IMN (n = 50) BL IMN (n = 12) 80 60 40 20 0 –1 12345678 Weeks 10 Pdx1+ cells (per islet and pancreatic area) (×109) 8 6 4 2 0IMN BL IMN (HG) 01234 Weeks 120 100 80 60 40 Diabetes incidence (%) 20 0 01234 Weeks 1.5 ns ** n = 6 1.0 Cell death (A405-A490) 0.5 siSC siLRH1 siLRH1 + BL siSC + BL 0.0 1.5 1.0 Rel. LRH-1 mRNA levels 0.5 0.0 siSC siLRH1 bc d e j l f k g m h n CTRLBLIMNBL IMN Insulin-Glucagon+ cells (per islet and pancreatic area) (×1011) 4 * 3 2 1 0 STZ STZ post-BL STZ pre-BL i n = 8 n = 5 * 0 IMN (n = 38) C (n = 14) BL (n = 8) BL IMN (n = 38) Diabetes incidence (%) Diabetes incidence (%) 100 80 60 40 100 80 60 40 20 0 20 0 –1 12345678 Weeks Weeks 812 16 20 24 o p 0 NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03943-0 ARTICLE NATURE COMMUNICATIONS | (2018) 9:1488 |DOI: 10.1038/s41467-018-03943-0 |www.nature.com/naturecommunications 3 0.0913 versus 0.3461 ± 0.0579 p=0.1092, unpaired Student’s ttest) and IL2 (1.306 ± 0.181 versus 1.111 ± 0.154 p=0.4221, unpaired Student’sttest) between immunized untreated and BL001-treated mice at 8 weeks. BL001 elicits a tolerogenic environment within the pancreas. To analyze how BL001 remodeled the immune cell environment, we performed flow cytometry studies on pancreatic cell suspensions (gating strategy shown in Supplementary Fig. 7). The proportions of CD4+/CD25+/FoxP3+Tregs and CD4+/IL4+Th2 cells were higher in the pancreas of the immunized and BL001- treated RIP-B7.1 mice than in the cognate controls (Fig. 3a, c), whereas CD4+/IFN gamma+Th1 cells were decreased, and CD4 +/IL17+Th17 cells were unaffected (Fig. 3b, d). Furthermore, the number of CD103+/IDO+tolerogenic dendritic cells, F4/80 +/CD11b+macrophages, and their M2-like anti-inflammatory subtype (F4/80+/CD11b+/CD206+) were also increased in BL001-treated mice (Fig. 3e–g). Accordingly, transcriptome profiling revealed that BL001 significantly increased the expression of key genes associated with the M2 genetic signature, including, Clec7a (Dectin-1), Clec10a (CD301), Chst7,Tiam1, Cd300ld, and Olfm1 (Noelin-1 or Pancortin)21 (Fig. 3h). BL001 favors a M2 macrophage phenotype via LRH-1.To further delineate the role of BL001 in promoting a M2 macrophage phenotype resulting in increased IL10 secretion, peritoneal macrophages were isolated and treated in vitro with the compound. BL001 dose-dependently increased transcript levels and the secretion of IL10 and TGFbeta (Fig. 4a–d). Exposure to LPS, also enhanced the expression and secretion of IL10, but not of TGF beta by these cells (Fig. 4e–h). To substantiate that BL001- mediated M2 polarization was conveyed by direct LRH-1 activation, the latter receptor was knock down using siRNA. Silencing of LRH-1 transcript and protein levels by ~70% in primary macrophages (Fig. 4i, j), significantly blunted the BL001-mediated increase in expression of the M2 signature genes, Clec7a,Clec10a, and Olfm1 as well as Il10 (Fig. 4k, l, p, q), independent of LPS treatment. Transcript levels of Chst7,Tiam1, and Cd300ld were also repressed, albeit in macrophages either treated or not with LPS (Fig. 4m–o). In contrast, TGFbeta expression was not altered in LRH-1 silenced cells (Fig. 4r). As IL10 biosynthesis and secretion in alternatively activated macrophage has been linked to Clec7a stimulation22,23, secretion levels of this cytokine was next evaluated in LRH-1 knockdown cells. Accordingly, IL10 secretion was decreased in BL001-treated macrophages independent of LPS treatment (Fig. 4s) indicating that LRH-1 is involved in the BL001-dependent polarization and cytokine secretory function of M2 macrophages. BL001 increases the number of bi-hormonal cells in islets.To assess whether the BL001 treatment favored the survival and replenishment of islet cells, we morphometrically evaluated the mass of beta and alpha cells, as well as islet numbers and sizes. Eight weeks after immunization, all these parameters were increased in the BL001-treated mice (Fig. 5a–d, Supplementary Fig. 8a–c). This increase was not associated to enhanced proliferation or decreased apoptosis of beta cells, as assessed using different methods (Fig. 5e–h, Supplementary Fig. 9a, b). In contrast, the immunized mice, which did not receive BL001, featured higher levels of beta cell proliferation and apoptosis as a result of the ongoing autoimmune attack (Fig. 5e–h). Eight weeks after immunization, the mass of alpha cells was also increased in BL001-treated mice in the absence of enhanced proliferation (Fig. 5c, d, i, j, Supplementary Fig. 9c). At this time point, islets of these mice, also contained increased numbers of cells coexpressing insulin and glucagon, as well as PDX1 and glucagon (Fig. 5k–n and supplementary Fig. 10). Since the data suggest the potential activation of a genetic beta cell program in alpha cells, we investigated whether a 48-h BL001 treatment could suppress the expression of ARX, whose down-regulation triggers beta-to- alpha cell trans-differentiation24. Accordingly, a 2-day exposure to BL001 decreased the expression of ARX, glucagon, and MafB transcripts in the alpha cell-derived TC1–6 cell line (Fig. 5o). BL001 protects human islet from apoptosis improving function. Having validated the proof-of-concept in mice that BL001 protects islets and prevents both chemically and autoimmuneinduced diabetes, we next sought to determine whether these beneficial effects were translated to human islets under stress conditions. Ten to twenty μM BL001 did not reveal any cytotoxic effects on islets obtained from normoglycaemic donors (Supplementary Fig. 11a). In the same islets, the expression levels of LRH-1 were also unchanged, whereas that of its target gene shp was increased (Supplementary Fig. 11b). However, 10 μM BL001 decreased the apoptosis of islet beta cells, 24 and 72 h after an exposure to either cytokines (Fig. 6a, c) or streptozotocin (Fig. 6b, d). Glucose-stimulated insulin secretion (GSIS) of islets from non diabetic donors (Supplementary Table 1) was not modified by BL001 (Fig. 6e). In contrast, GSIS of islets from type 2 diabetic donors (Supplementary Table 2) was significantly increased after exposure to BL001 (Fig. 6f). Under these conditions, the proportion of apoptotic beta cells was significantly decreased (Fig. 6g, Fig. 1 BL001 improves islet survival and blunts development of diabetes in three mouse models. aExposure to 10 μM BL001 (BL) for 72 h blocked cytokineinduced apoptosis (CK; 2 ng/ml IL1beta, 28 ng/ml TNFalpha and 833 ng/ml IFNgamma) in mouse islets. Cell death was assessed by ELISA quantification of mono and oligonucleosomes released by apoptotic cells. This blockade was blunted in islets transfected with siRNAs (d) which decreased LRH-1 transcript (b) and protein levels (LRH1, red; scale bar: 25 µM) (c). Data are means +s.e.m. values of six independent experiments using six different islet preparations and performed in triplicates. siSC, pool of siRNAs against scrambled sequence. BL001 (10 mg/kg b.w.) also decreased the incidence of diabetes in C57Bl/6 male mice treated with a single dose of streptozotocin (STZ;150 mg/kg b.w.), irrespective of whether the former drug was given prior to (e) or 1 week after (f) STZ. Values are means ± s.e.m. of n=5 (STZ)-14 mice (BL/STZ). After these 2 protocols, insulin-containing beta cells (red) and glucagon-containing alpha cells (green) were preserved in islets of STZ and BL001-treated mice (g), which also featured an increase number of cells containing both hormones (h, arrowheads). Nuclei were stained with DAPI (blue). Scale bars: 25 µM. iQuantification of insulin- and glucagon-positive cells. jA 5-day BL001 pre-treatment of RIP-B7.1 mice prior to immunization decreased the diabetes incidence. Values are means ± s.e.m. C (vehicle treated, n=26 male and female mice); BL (BL001, n=26 male and female mice); IMN (immunized, n=50 male and female mice), and BL IMN (BL001-treated and immunized, n=50 male and female mice). kImmunization increased the proliferation of RIP-B7.1 splenocytes, an effect that was not modified by the BL001 treatment. Values are median (lines in the boxes) ± median errors of 5–12 preparations. lThe 8-week-long BL001 treatment preserved insulin- (red) and glucagon-containing cells (green) in islets of immunized RIP-B7.1 mice (scale bar: 25 µM). m,nBL001-treated and immunized RIP-B7.1 mice that developed hyperglycemia contained a higher number of PDX1+cells as compared to untreated and immunized animals (scale bar: 25 µM). oDiabetes incidence of RIP-B7.1 mice that were immunized at days 0 and 7 and subsequently injected daily, starting at week 1 and for up to 8 weeks, with either vehicle (IMN, n=38 male and female mice) or BL001 (BL IMN, n=38 male and female mice). pDiabetes incidence in female NOD mice receiving (BL, n =8) or not (C, n=14) BL001 from 12 weeks. *p< 0.05, **p< 0.01, ***p< 0.001, and ****p< 0.0001, one-way ANOVA (a,d,k,i) or Student’sttest (b,n) ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03943-0 4NATURE COMMUNICATIONS | (2018) 9:1488 |DOI: 10.1038/s41467-018-03943-0 |www.nature.com/naturecommunications ab cd e j f g h i 4 Weeks CTRL IMN BL001 IMNBL001 CTRL IMN BL001 IMNBL001 8 Weeks Grade 0 Grade 1 Grade 2 Grade 3 Grade 4 IL-4 IL-5 IL-10IL-6 IL-4 IL-5 IL-10IL-6 C BL IMN BL IMN CCL2 CCL4 CCL2 CCL4 C BL IMN BL IMN C BL IMN BL IMN TGFβ TGFβ 4 Weeks pg/ml (n = 6–11) 8 Weeks pg/ml (n = 12–20) 4 Weeks pg/ml (n = 6–12) 8 Weeks pg/ml (n = 12) 4 Weeks ×103 pg/ml (n = 6–11) 8 Weeks ×103 pg/ml (n =12) 120 n = 6 100 80 Islet insulitis grade (%) 60 40 20 0 C BL IMN BL IMN 120 n = 6 100 80 Islet insulitis grade (%) 60 40 20 0 1.6 1.2 0.8 0.4 0.0 30 20 10 0 400 ** 300 200 100 0 150 100 50 0 1.6 1.2 0.8 0.4 0.0 1500 1000 500 0 1500 1000 500 0 30 20 10 0 30 40 20 10 0 30 40 20 10 0 30 50 40 20 10 0 30 50 40 20 10 0 400 300 200 100 0 150 100 50 0 C BL IMN BL IMN (NG) BL IMN (HG) *** *** *** * ** * ** ** ** ** ** ** *** Fig. 2 BL001 decreases insulitis and modulates the blood cytokine profile. Histology (representative images, scale bar: 25 µM) and morphometry at a,b4 and c,d8 weeks revealed that treatment with 10 mg/kg b.w. BL001 decreased the incidence and severity of insulitis in immunized RIP-B7.1 mice. At 8 weeks, BL001-treated and immunized mice that developed hypeglycemia exhibited a similar degree of insulitis to the BL001-treated and immunized mice that were normoglycemic at 4 weeks. Impact of BL001 treatment on the profile of cytokines, chemokines and TGFbeta at 4 weeks (e,g,i) and 8 weeks (f, h,j). Plots show individual mice values. *p< 0.05, **p< 0.01, and ***p< 0.001, one-way ANOVA NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03943-0 ARTICLE NATURE COMMUNICATIONS | (2018) 9:1488 |DOI: 10.1038/s41467-018-03943-0 |www.nature.com/naturecommunications 5 abcd h Th1 Th2 Th17 ef g Dendritic Macrophages M2 Egr2 Mrc1 Myc Il6st Lmna Clec10a Plk2 Clec7a Atp6v0a1 Rhoj Cd300ld Mmp12 Bcar3 Matk Olfm1 Tanc2 Amz1 Actn1 Ptgs1 Tiam1 Emp2 Mmp9 Chst7 Gnb4 Vwf Flrt2 P2ry1 Socs6 5.73e–01 8.28e–01 8.62e–01 4.30e–02 7.78e–01 1.60e–02 5.43e–01 1.00e–03 5.72e–01 7.21e–01 1.30e–02 5.50e–01 7.50e–02 3.06e–01 6.00e–03 1.00e–01 3.32e–01 6.58e–01 7.75e–01 1.40e–02 5.74e–01 1.56e–01 1.70e–02 5.80e–01 4.26e–01 1.65e–01 3.10e–02 9.57e–01 IMN BL IMN Genes p-values logFC –0.5 0 0.5 1 1.5 log2 expr 12108642 15 Tregs 12 10 8 6 4 2 0 **** *** ** ** * ** ** *** * * n = 10–16 n = 5 n = 7 n = 7 n = 5–11 n = 5–11 n = 5–11 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 1.5 1.0 0.5 0.0 10 CD4 + CD25 + Foxp3 + (%)×10 –1 CD4 + IFNγ + (%) CD103 + IDO + (%) F4/80 + CD11b + (%) F4/80 + CD11b + CD206 + (%) CD4 + IL4 + (%) CD4 + IL17 + (%) 5 0 25 20 15 10 5 0 6 5 4 3 2 1 0 5 4 3 2 1 0 C BL IMN BL IMN CBL IMN BL IMN C BL IMN BL IMN CBL IMN BL IMN C BL IMN BL IMN CBL IMN BL IMN CBL IMN BL IMN Fig. 3 BL001 promotes a pancreatic anti-inflammatory/tolerogenic environment. RIP-B7.1 male and female mice were pre-treated or not with BL001 for 5 days and then immunized. Treatment with BL001 was pursued for an additional 8 weeks at which point animals were sacrificed and pancreas extracted and processed for flow cytometry quantification of aTregs (n=10–16 mice), bTh1 (n=5–11 mice), cTh2 (n=5–11 mice), dTh17 (n=5–11 mice), e dentritic cells (n=5 mice), fmacrophages (n=7 mice), and gM2-like macrophages (n=7 mice). hHeatmap displaying expression values of a subset of M2 macrophage genes in F4/80+/CD11b+/CD206+pancreatic subpopulations isolated from immunized mice treated or nor with BL001 (IMN and IMN BL). Also depicted are raw p-values and the graphical representation of logFC from the differential expression analysis (IMN BL versus IMN). *p< 0.05, **p< 0.01, ***p< 0.001, and ****p< 0.0001, one-way ANOVA (a–g) ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03943-0 6NATURE COMMUNICATIONS | (2018) 9:1488 |DOI: 10.1038/s41467-018-03943-0 |www.nature.com/naturecommunications h) and paralleled by reduced cleaved caspase-3 activity (Fig. 6i, j and Supplementary Fig. 12). To assess whether comparable effects could be observed during an ongoing, in vivo immune attack, mimicking a T1DM environment, we transplanted human islets under the kidney capsule of immune competent C57BL/6 mice, treated or not with BL001. Two days after xenotransplantation, mice were treated with daily injections of BL001 or vehicle for 7 days. At this time point, rejection of the human islets is anticipated25,26. Consistent with the protective effect of BL001, grafts from BL001-treated abcd e jl f k g m h i op n DAPI LRH1 siLuc siLRH1 q siLuc-BL001/siLuc siLRH1-BL001/siLRH1 siLuc-BL001/siLuc siLRH1-BL001/siLRH1 r s siLuc-BL001/siLuc siLRH1-BL001/siLRH1 siLuc-BL001/siLuc siLRH1-BL001/siLRH1 LPS LPS LPS LPS 4 4 6 2 0 4 6 2 0 4 6 8 2 0 n=9 n=9 n=9n=9n=9n=9 n=5 n=5 n=5 n=4 n=4 n=4 n=4n=4 n=4 n=4 n=9 3 n=9 2 Rel. IL10 mRNA levels Rel. IL10 mRNA levels Rel. LRH1 mRNA levels (fold change vs siLuc) Secreted IL-10 (rel. to C) Secreted IL-10 (rel. to C) Rel. TGFβ mRNA levels Rel. TGFβ mRNA levels Rel. TGFβ mRNA levels Rel. Tiam1 mRNA levels Secreted IL-10 Rel. Cd300ld mRNA levels Rel. Olfm1 mRNA levels Rel. Clec7a mRNA levels Rel. IL10 mRNA levels Secreted TGFβ (rel. to C) Secreted TGFβ (rel. to C) 1 0 4 3 2 1 0 4 3 2 1 0 4 3 2 1 0 3 2 1 0 3 2 1 0 C 0.1 1 10 * (μM BL) C ** ** * * **** ** ** * *** ** ** * * * *** * 0.1 1 10 (μM BL) C 0.1 1 10 (μM BL) C 0.1 1 10 (μM BL) C 0.1 1 10 (μM BL) C0.1 1 10 (μM BL) C 0.1 1 10 (μM BL) C 1.2 2.5 2.0 1.5 1.0 0.5 0.0 Rel. Chst7 mRNA levels 2.5 2.0 1.5 1 2 0 1.0 0.5 0.0 2.5 2.0 1.5 1.0 0.5 0.0 2.5 2.0 1.5 1.0 0.5 0.0 2.0 1.5 1.0 0.5 0.0 LPS – – + + LPS – – + + LPS ––++LPS – –++ LPS – – + + LPS ––++ LPS – – + + LPS – – + + LPS – –++ Rel. Clec10a mRNA levels 2.5 2.0 1.5 1.0 0.5 0.0 2.0 1.5 1.0 0.5 0.0 1.0 0.8 0.6 0.4 0.2 0.0 siLuc siLRH1 0.1 1 10 (μM BL) Fig. 4 LRH-1 is required for BL001-mediated M2 polarization. Primary macrophages were exposed in culture to increasing concentration of BL001 for 24 h. Media was collected and RNA isolated to perform QT-PCR. Transcript and secreted levels of a,bIl10 and c,dTgfb were then determined (n=9 mice). e–h Primary macrophages were incubated for 24 h with increasing concentrations of BL001 alone or in combination with 1.0 µg/ml lipopolysaccharide (LPS). Transcript and secreted levels of e,fIL10 and g,hTGFbeta were then measured (n=9 mice). isiRNA-mediated repression of LRH-1 transcript and j protein levels in mouse primary macrophages as assessed by QT-PCR and immunostaining (LRH-1, green; scale bar: 25 µM). Relative expression levels of M2 signature genes, kClec7a,lClec10a,mChst7,nTiam1,oCd300ld, and pOlfm1 in control or LRH-1-silenced macrophages treated or not with 10 µM BL001 and LPS (n=4). qIl10 and rTgfb transcript levels in siLRH1-treated or not macrophages in the presence or absence of 10 µM BL001 and LPS (n=5). sSecreted levels of IL10 in silenced or not macrophages. Results are expressed as the averages +s.e.m. (a–iand k–s). *p< 0.05, **p< 0.01, ***p< 0.001, and ****p< 0.0001, one-way ANOVA (a–h) and Student’sttest (i,k–s) NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03943-0 ARTICLE NATURE COMMUNICATIONS | (2018) 9:1488 |DOI: 10.1038/s41467-018-03943-0 |www.nature.com/naturecommunications 7 mice showed greater numbers of beta cells than the controls (Fig. 6k, l). Immune-related biological processes are targeted by BL001.To evaluate the effects of BL001 on a larger scale, we profiled gene expression via DNA microarray analysis in mouse islets exposed to BL001 for 2 days. We found that 277, 23, and 195 gene ontology (GO) terms were enriched in islets exposed to 0.1, 1.0, and 10 µM BL001, respectively (Fig. 7a). Seven of these GO terms were common to all conditions, whereas no common downregulated GO terms was found in the same samples (Fig. 7a). The seven enriched GO terms were related to genes involved in abcd e jl k m i on DAPI INS Ki67 CTRLBLIMNBL IMN DAPI INS Tunel CTRLBLIMNBL IMN DAPI GLUC Ki67 CTRLBLIMNBL IMN DAPI INS GLUC CTRLBLBL IMN f g h DAPI PDX1 GLUC ## ** 4 weeks 4 weeks8 weeks 8 weeks 4.0 n=5 n=5 n=5 n=6 n=15 120 100 n=8 n=16 n=8 n=16 80 60 40 20 0 C *** **** **** *** *** ** ** ** ** **** *** **** **** ** * * * * * * ** BL IMN BL IMN CBL IMN BL IMN CBL IMN BL IMN CBL IMN BL IMN C BL IMN BL IMN CBL IMN BL IMN C BL IMN BL IMN C BL IMN BL IMN C BL IMN BL IMN β cell mass (area: Ins/islet /pancreas) (×10–9) 120 40 30 20 10 0 40 30 20 10 0 100 80 60 40 20 0 β cell mass (area: Ins/islet /pancreas) (×10–9) α cell mass (area: Gluc/islet /pancreas) (×10–9) α cell mass (area: Gluc/islet /pancreas) (×10–9) 3.5 10 2.0 140 120 100 80 60 40 20 0 Arx Gcg MafB C 10 μM BL 1.5 1.0 0.5 0.0 9 8 7 6 5 2.0 1.5 1.0 0.5 0.0 3.0 2.5 2.0 1.5 Ki67-ins+/ins+ (%) Pdx1-glucagon+ cells (per islet and pancreatic area (×1011) mRNA level (relative to β-actin) Tunel-ins+/ins+ (%) 1.0 0.5 0.0 4.0 16 12 8 4 0 3.5 3.0 2.5 2.0 1.5 Ki67-glu+/glu+ (%) Insulin-glucagon+ cells (per islet and pancreatic area) (x1011) 1.0 0.5 0.0 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03943-0 8NATURE COMMUNICATIONS | (2018) 9:1488 |DOI: 10.1038/s41467-018-03943-0 |www.nature.com/naturecommunications immune functions (Fig. 7b). Heat maps of those genes which were consistently modified, identified 33 genes (Fig. 7c). Those encoding for the CCL2, CCL3, CCL7 chemokines, the cytokine IL6 and the prostaglandin-endoperoxide synthase-2 (PTGS2) were the most induced by the BL001 exposure, whereas the gene coding for the IL1 beta receptor (IL1R1) was the most downregulated (Fig. 7c). Quantitative PCR of these genes, confirmed that the CCL2, CCL3, and PTGS2 transcripts were upregulated by the BL001 exposure (Fig. 7d–f), whereas the IL1R1 transcript was downregulated under the same conditions (Fig. 7g). Discussion In spite of major efforts, the available strategies to restore/preserve a functional beta cell mass through immunomodulation and/or beta cell regeneration/replacement treatments have so far shown limited efficacy for the long-term improvement of glycaemia in T1DM patients27. This frustrating situation calls for innovative approaches to this complex problem; identify novel “druggable”targets that could promote the regeneration of a functional beta cell mass, while attenuating the autoimmune attack, and preserving the anti-inflammatory locale that appears necessary for beta cell renewal. Here, we show that BL001, a small agonist of the LRH-1 receptor, has such characteristics, which prevent and revert diabetes in three different mouse models of T1DM. Our data first document that, by activating LRH-1, BL001 primes macrophages toward the anti-inflammatory M2 phenotype (as revealed by the enhanced M2 genetic signature) resulting in direct stimulation of IL10 expression and secretion23. BL001- treated mice also featured increased numbers of pancreatic Tregs, whose expansion is induced by IL1028, and which are essential in maintaining self-immune tolerance including in T1DM29. Circulating levels of CCL2 as well as its expression in islets were increased in BL001-treated and immunized RIP-B7.1 mice. Although the impact of CCL2 as a pro or anti-inflammatory chemokine is disease- and cell-dependent30, our results suggest that in the context of autoimmune diabetes, increased levels of local CCL2 appears to be associated with the recruitment of macrophages to the pancreas which, in turn are further polarized towards the M2-like subtype under the influence of IL10 production. CCL2 through enhanced IL4 production also fosters an environment that favors expansion of Th231, a cell type and cytokine that were increased in the pancreas and in the circulation of immunized, BL001-treated RIP-B7.1 mice. Immunized mice treated with BL001 also displayed increased circulating TGFbeta levels correlating with the presence of a larger number of CD103+/IDO+dentritic cells, which convey immunosuppressive and tolerogic functions32,33. Given that the adoptive transfer of M2 macrophages as well as the upregulation of CCL2 enhance beta cell survival in NOD mice34,35 and that the autologous transfer of Tregs improves islet survival and function in T1DM patients36, the immune changes we observed converge to demonstrate that BL001 significantly inhibits the aggressive autoimmune process favoring tolerance, which presumably contributes to its beneficial effects. In this context, BL001-treated and immunized mice that develop diabetes retained islets with a significant number of PDX1+cells and reduced insulitis at 8 weeks as compared to immunized mice. Although speculative, the latter results indicate that BL001 dosage or activity was suboptimal in favoring an anti-inflammatory and tolerogenic environment resulting in beta cell destruction and hyperglycemia in these animals. Blood analysis further showed that BL001 increased circulating levels of the IL5 and IL6 cytokines, and of the chemokine CCL4, which have anti-inflammatory actions. IL6 enhances insulin secretion via the release of GLP1 by alpha cells37,38 while IL5 and CCL4 shift pro-inflammatory Th1 cells towards the antiinflammatory Th2 subset and stimulate expansion of Tregs39,40. Accordingly, we found reduced numbers of Th1 cells but increased numbers of Th2 and Tregs cells in the pancreas of the immunized and BL001-treated mice. In contrast, circulating levels of IFNgamma, TNFalpha, IL1beta and IL2 remained constant suggesting that production of these cytokines were not directly impacted by the compound. BL001 treatment also stimulated the islet expression of PTGS2, an inducible prostaglandin synthase, whose PGE 2 metabolite also inhibits Th1 cells and protects against T1DM41–43. In addition, IL6 and the potential secretion of PTGS2-derived PGE 2 by islets along with IL4 and IL10 released by Th2 as well as Tregs, will further contribute to M2 polarization within the pancreas independent of the direct activation of LRH-1 in macrophages by BL00144–47. These data highlight that, in addition to a direct sizable action on the immune system, BL001 also promotes the release of pancreatic islet-derived factors favoring an anti-inflammatory environment that will further induce tolerogenecity. Future work will focus on dissecting the mechanism whereby the BL001/LRH-1 signaling pathway achieves these beneficial effects. Our study further documents that the LRH-1 receptor is essential for the proper organization of pancreatic islets. Thus, the beta cell loss of the LHR-1 receptor modifies the proportion of islet cells characterized by an increase in the number of alpha cells, and the retention of a sizable beta cell mass, which associate with a rapid, post-natal death of transgenic mice. Our parallel experiments also show that BL001-mediated activation of LRH-1 stimulates the regeneration of beta cells, in the islets of both control and diabetic mice, an effect which is not attributable to a change in the proliferation and apoptosis of these Fig. 5 BL001 promotes beta and alpha cell mass expansion in immunized RIP-B7.1 mice. The control (C) mass of beta (a,b) and alpha cells (c,d) of RIP- B7.1 mice was decreased after immunization (IMN), an alteration that was prevented by 10 mg/kg b.w. BL001 daily treatment for 8 weeks (BL IMN). Dot plots show the results from 8 (4 weeks) and 16 (8 weeks) pancreas per group, each dot corresponding to the cell mass of an entire pancreatic section. e–j Representative immunofluorescence images of islets co-stained and quantified for e,finsulin (INS, red) and Ki67 (green), g,hinsulin (red) and Tunel (green), i,jglucagon (GLUC, red) and Ki67 (green). All values are means of 5 mice per group. Scale bars: 25 µM. Right panels are enlargements of boxed area, and arrow heads point to example of cells simultaneously stained for 2 proteins. kRepresentative confocal images showing the distribution of the insulin- (red) and glucagon-bihormonal cells (green) as well as lquantification in islets of the different animal groups. Nuclei were stained with DAPI (blue). Scale bar: 25 µM. Right panel show enlargements of boxed areas. Arrow heads point to cells co-stained for the two depicted markers. mA representative image depicting co-immunostaining of PDX1 (red) and glucagon (green) in a pancreas of a normoglycemic immunized BL001-treated mouse, and nquantification of such cells in the various groups. Bottom left panels are enlargements of boxed area. Bottom right panels, are without DAPI staining. White arrows point to PDX1+/GLUC+cells, # identifies a PDX1-/GLUC+cell and * highlights a PDX1+/GLUC-cell. n=15 pancreatic slices obtained from five independent mice per group. Scale bar: 25 µM. oAfter a 24-h exposure to 10 μM BL001 (BL), alpha TC-1.6 cells featured decreased expression of the Arx, glucagon (Ggc) and MafB transcripts. Six independent experiments were performed and subsequent QT-PCR conducted in triplicates. Values are means +s.e.m. (f,h,j,l,n,o). *p< 0.05, **p< 0.01, ***p< 0.001, and ****p< 0.0001, one-way ANOVA (a–d,f,h,j,l,n) and the Student’sttest as compared to control (o) NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03943-0 ARTICLE NATURE COMMUNICATIONS | (2018) 9:1488 |DOI: 10.1038/s41467-018-03943-0 |www.nature.com/naturecommunications 9