Article9 October 2020Open Access Transparent process IL-10 producing type 2 innate lymphoid cells prolong islet allograft survival Qingsong Huang Qingsong Huang Henan Key Laboratory of Immunology and Targeted Drugs, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang, China Search for more papers by this author Xiaoqian Ma Xiaoqian Ma Centre for Transplant and Renal Research, Westmead Institute for Medical Research, The University of Sydney, Sydney, NSW, Australia The Institute for Cell Transplantation and Gene Therapy, The Third Xiangya Hospital of Central South University, Changsha, China Search for more papers by this author Yiping Wang Yiping Wang Centre for Transplant and Renal Research, Westmead Institute for Medical Research, The University of Sydney, Sydney, NSW, Australia Search for more papers by this author Zhiguo Niu Zhiguo Niu Henan Key Laboratory of Immunology and Targeted Drugs, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang, China Search for more papers by this author Ruifeng Wang Ruifeng Wang Centre for Transplant and Renal Research, Westmead Institute for Medical Research, The University of Sydney, Sydney, NSW, Australia Search for more papers by this author Fuyan Yang Fuyan Yang The Department of Nephrology, First People's Hospital of Xinxiang Medical University, Xinxiang, China Search for more papers by this author Menglin Wu Menglin Wu Henan Key Laboratory of Immunology and Targeted Drugs, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang, China Search for more papers by this author Guining Liang Guining Liang The Department of Physiology, Guangxi Medical University, Nanning, China Search for more papers by this author Pengfei Rong Pengfei Rong The Institute for Cell Transplantation and Gene Therapy, The Third Xiangya Hospital of Central South University, Changsha, China Search for more papers by this author Hui Wang Hui Wang Henan Key Laboratory of Immunology and Targeted Drugs, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang, China Search for more papers by this author David CH Harris David CH Harris Centre for Transplant and Renal Research, Westmead Institute for Medical Research, The University of Sydney, Sydney, NSW, Australia Search for more papers by this author Wei Wang Corresponding Author Wei Wang [email protected] The Institute for Cell Transplantation and Gene Therapy, The Third Xiangya Hospital of Central South University, Changsha, China Search for more papers by this author Qi Cao Corresponding Author Qi Cao [email protected] orcid.org/0000-0002-3546-0766 Henan Key Laboratory of Immunology and Targeted Drugs, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang, China Centre for Transplant and Renal Research, Westmead Institute for Medical Research, The University of Sydney, Sydney, NSW, Australia Search for more papers by this author Qingsong Huang Qingsong Huang Henan Key Laboratory of Immunology and Targeted Drugs, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang, China Search for more papers by this author Xiaoqian Ma Xiaoqian Ma Centre for Transplant and Renal Research, Westmead Institute for Medical Research, The University of Sydney, Sydney, NSW, Australia The Institute for Cell Transplantation and Gene Therapy, The Third Xiangya Hospital of Central South University, Changsha, China Search for more papers by this author Yiping Wang Yiping Wang Centre for Transplant and Renal Research, Westmead Institute for Medical Research, The University of Sydney, Sydney, NSW, Australia Search for more papers by this author Zhiguo Niu Zhiguo Niu Henan Key Laboratory of Immunology and Targeted Drugs, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang, China Search for more papers by this author Ruifeng Wang Ruifeng Wang Centre for Transplant and Renal Research, Westmead Institute for Medical Research, The University of Sydney, Sydney, NSW, Australia Search for more papers by this author Fuyan Yang Fuyan Yang The Department of Nephrology, First People's Hospital of Xinxiang Medical University, Xinxiang, China Search for more papers by this author Menglin Wu Menglin Wu Henan Key Laboratory of Immunology and Targeted Drugs, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang, China Search for more papers by this author Guining Liang Guining Liang The Department of Physiology, Guangxi Medical University, Nanning, China Search for more papers by this author Pengfei Rong Pengfei Rong The Institute for Cell Transplantation and Gene Therapy, The Third Xiangya Hospital of Central South University, Changsha, China Search for more papers by this author Hui Wang Hui Wang Henan Key Laboratory of Immunology and Targeted Drugs, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang, China Search for more papers by this author David CH Harris David CH Harris Centre for Transplant and Renal Research, Westmead Institute for Medical Research, The University of Sydney, Sydney, NSW, Australia Search for more papers by this author Wei Wang Corresponding Author Wei Wang [email protected] The Institute for Cell Transplantation and Gene Therapy, The Third Xiangya Hospital of Central South University, Changsha, China Search for more papers by this author Qi Cao Corresponding Author Qi Cao [email protected] orcid.org/0000-0002-3546-0766 Henan Key Laboratory of Immunology and Targeted Drugs, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang, China Centre for Transplant and Renal Research, Westmead Institute for Medical Research, The University of Sydney, Sydney, NSW, Australia Search for more papers by this author Author Information Qingsong Huang1,‡, Xiaoqian Ma2,3,‡, Yiping Wang2, Zhiguo Niu1, Ruifeng Wang2, Fuyan Yang4, Menglin Wu1, Guining Liang5, Pengfei Rong3, Hui Wang1, David CH Harris2, Wei Wang *,3 and Qi Cao *,1,2 1Henan Key Laboratory of Immunology and Targeted Drugs, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang, China 2Centre for Transplant and Renal Research, Westmead Institute for Medical Research, The University of Sydney, Sydney, NSW, Australia 3The Institute for Cell Transplantation and Gene Therapy, The Third Xiangya Hospital of Central South University, Changsha, China 4The Department of Nephrology, First People's Hospital of Xinxiang Medical University, Xinxiang, China 5The Department of Physiology, Guangxi Medical University, Nanning, China ‡These authors contributed equally to this work *Corresponding author. Tel: +86 731 88618411; E-mail: [email protected] *Corresponding author. Tel: +61 02 86273512; E-mail: [email protected] EMBO Mol Med (2020)12:e12305https://doi.org/10.15252/emmm.202012305 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Type 2 innate lymphoid cells (ILC2s) are a subset of ILCs with critical roles in immunoregulation. However, the possible role of ILC2s as immunotherapy against allograft rejection remains unclear. Here, we show that IL-33 significantly prolonged islet allograft survival. IL-33-treated mice had elevated numbers of ILC2s and regulatory T cells (Tregs). Depletion of Tregs partially abolished the protective effect of IL-33 on allograft survival, and additional ILC2 depletion in Treg-depleted DEREG mice completely abolished the protective effects of IL-33, indicating that ILC2s play critical roles in IL-33-mediated islet graft protection. Two subsets of ILC2s were identified in islet allografts of IL-33-treated mice: IL-10 producing ILC2s (ILC210) and non-IL-10 producing ILC2s (non-ILC10). Intravenous transfer of ILC210 cells, but not non-ILC10, prolonged islet allograft survival in an IL-10-dependent manner. Locally transferred ILC210 cells led to long-term islet graft survival, suggesting that ILC210 cells are required within the allograft for maximal suppressive effect and graft protection. This study has uncovered a major protective role of ILC210 in islet transplantation which could be potentiated as a therapeutic strategy. Synopsis This study reveals a major protective role of the IL-33/ILC2 axis in islet transplantation that could be potentiated as a therapeutic strategy. Adoptive transfer of ex vivo expanded IL-10-producing ILC2s (ILC210) significantly prolonged allograft survival. IL-33 significantly prolonged islet allograft survival by increasing numbers of ILC2s and regulatory T cells in vivo. IL-33 treatment induced a long-term accumulation of ILC2s in islet graft associated with a persistent increase of IL-33 in islet graft tissue. Expansion of ILC210 was induced by IL-33 and IL-2/IL-2 antibody complex in vivo and in vitro. ILC210 prolonged islet allograft survival in an IL-10-dependent manner. ILC210 migration into the islet allograft was necessary for maximum graft protection. The phenotypic stability of locally transferred ILC210 was an important factor in determining the fate of islet grafts. The paper explained Problem Pancreatic islet transplantation is a promising treatment option for patients with type 1 diabetes. However, islet graft rejection remains one of the main obstacles to successful transplantation. Clinically applicable strategies for immunomodulation need to be developed to achieve long-term graft tolerance. One attractive therapy to prevent allograft rejection relies on harnessing the potential of regulatory immune cells. Mounting evidence indicates that ILC2s play immune regulatory roles in acute and chronic inflammatory diseases. The current study explores whether ILC2s could suppress allograft rejection in an islet transplantation model. Results Here, we show that IL-33 treatment significantly prevented islet allograft rejection and improved islet function. This remarkable therapeutic benefit was shown to be mediated by inducing regulatory immune cells including Tregs and ILC2s. A short course of IL-33 treatment induced a sustained increase in ILC2 abundance in islet graft which was associated with IL-33-mediated islet graft protection. Importantly, we further demonstrated that IL-10-producing ILC2s (ILC210), a subset of ILC2, are important inhibitors of islet graft rejection. Co-transplantation of ILC210 with islets led to long-term allograft survival, suggesting that ILC210 cells are required within the allograft for maximal suppressive effect and graft protection. Impact Our study offers new insights into the role of IL-33 and ILC210 in islet allograft survival. We propose administration of IL-33 and ILC210 as an adjunctive therapy to prevent allograft rejection, bringing potential novel therapeutics to the field of transplantation. Introduction Innate lymphoid cells (ILCs) are a novel group of immune cells with critical roles in immunity, tissue homeostasis, and pathological inflammation (Eberl et al, 2015; Sonnenberg & Artis, 2015; Vivier et al, 2018). ILCs are subdivided into three groups: ILC1, ILC2, and ILC3, based on their cytokine profiles and expression of specific transcription factors, mirroring the classification of CD4+ T helper cell subsets into TH1, TH2, and TH17 cells. Type 2 ILCs (ILC2s) resemble TH2 cells as they require the transcription factor GATA-3; produce type 2 cytokines IL-4, IL-5, IL-9, and IL-13; and play important roles in immunity against pathogens and type 2 inflammation (Krabbendam et al, 2018). ILC2s also promote tissue recovery following acute injury in multiple organs, such as lung, intestine, and kidney (Monticelli et al, 2011; Cao et al, 2018). For example, in influenza virus infection of mice, ILC2s were activated by lung epithelial cell-derived IL-25 and IL-33 and promoted repair of the airway epithelium via producing amphiregulin (Areg) (Monticelli et al, 2011). More recently, IL-10 producing ILC2s, namely ILC210, have been identified in lung where they play important roles in resolution of lung inflammation (Seehus et al, 2017), indicating that ILC2s include different functional subsets. However, the possible immunoregulatory role of ILC2 in transplant rejection has not been addressed so far. Modulation of ILC2 activity may provide a therapeutic approach to maintain allograft tolerance. Type 1 diabetes mellitus (T1DM) is an autoimmune disease in which pancreatic β cells are destroyed by autoreactive T cells, resulting in lifelong insulin dependency (Burrack et al, 2017; Paschou et al, 2018). Pancreatic islet transplantation is a promising treatment option for patients with type 1 diabetes that restores both endogenous insulin production and glycemic stability (Anazawa et al, 2019). However, islet graft rejection caused by immune cells remains one of the main obstacles to successful transplantation. Although advances in immunosuppressive therapies have promoted excellent short-term graft survival after islet transplantation, immunosuppressive drugs with severe side effects remain ineffective at preventing late-stage allograft rejection (Gibly et al, 2011; Anazawa et al, 2019). Therefore, it is critical to develop applicable strategies that specifically target anti-islet immune responses to achieve long-term graft tolerance without use of immunosuppressive drugs. One attractive alternative therapy to prevent allograft rejection relies on harnessing the potential of regulatory T cells (Treg) (Gagliani et al, 2010; Lam et al, 2017). Recent studies have shown that ex vivo-expanded human Treg can prevent the development of islet and skin allograft rejection in a humanized mouse model (Issa et al, 2010; Yi et al, 2012). IL-33 significantly prolonged allograft survival in organ transplantation partially via increasing numbers of Tregs (Turnquist et al, 2011; Matta et al, 2016). We recently reported that IL-33-expanded kidney resident ILC2s prevented renal ischemia-reperfusion injury (IRI) via production of Areg (Cao et al, 2018). In the present study, we sought to determine the role of the IL-33-ILC2 pathway in islet allograft survival. Here, we report that IL-33 induced long-term survival of islet allografts via increasing both Tregs and ILC2s in vivo. Importantly, we further demonstrated that ex vivo-expanded ILC2s significantly prolonged allograft survival in an IL-10-dependent manner. Results IL-33 prevented rejection of islet allograft To determine whether IL-33 could prolong islet graft survival, we treated diabetic C57BL/6 mice with mouse recombinant IL-33 (0.3 μg/mouse/day, intraperitoneally) for five consecutive days prior to islet transplantation (Fig 1A). As expected, blood glucose measurement showed that PBS-treated control mice all rejected their grafts rapidly, with a mean survival time of 12 days. In contrast, pretreatment with IL-33 led to prominent long-term graft survival (Fig 1B). In this case, a small proportion of the mice rejected their grafts around days 14–20, but the remaining 75% (n = 9 out of 12) of the mice retained their grafts indefinitely (> 80 days) (Fig 1C). Intraperitoneal glucose tolerance tests (IPGTTs) were performed to investigate islet graft function in vivo. The glucose tolerance in islet transplant mice treated with IL-33 was significantly improved compared with mice receiving islets alone (Fig 1D). The values of the area under the glucose curve (AUC) for IPGTT in the group with IL-33 treatment were significantly smaller than in those receiving islets alone (Fig 1E). Histology in PBS-treated control mice showed that islet architecture was lost, with only a few remaining insulin-positive cells and massive leukocyte infiltration. In contrast, the group with IL-33 treatment showed well-preserved islet morphology with reduced/minimal leukocyte infiltration (Fig 1F). These results showed that IL-33 treatment prevented islet allograft rejection and improved islet function. We also found that IL-33 treatment in STZ-induced diabetic mice without islet transplantation improved fasting and non-fasting glycemia at day 15 and 18 post-STZ injection, but did not enhance survival of STZ-induced diabetic mice (Appendix Fig S1). The improved glycemia status of diabetic mice with IL-33 treatment could possibly contribute to prolonged islet allograft survival. Figure 1. IL-33 prolonged islet allograft survival A. Streptozotocin-induced diabetic C57BL/6 (H2b) mice were treated with mouse recombinant IL-33 daily for 5 consecutive days before islet transplantation. On day 0, mice were transplanted with BALB/c (H2d) islets. Mice were sacrificed at day 80 post-islet transplantation or at the day when grafts were considered rejected after two consecutive BGLs > 16 mmol/l (mM) after a period of normoglycemia. B. Islet graft survival of mice receiving vehicle (PBS) or IL-33 was assessed by monitoring blood glucose and calculated using the Kaplan–Meier method. Cumulative data from two independent experiments are shown. Statistical analysis was performed with a log-rank test. ***P < 0.001 vs. islet+vehicle. C. Blood glucose level of mice treated with IL-33 (the horizontal black line indicates a BGL of 16 mmol/l, the threshold for rejection). Each line represents one mouse. D. Intraperitoneal glucose tolerance test (IPGTT) was assessed in normal mice, islet transplant mice receiving vehicle (on the day when grafts were considered rejected), and islet transplant mice treated with IL-33 (at day 30 and day 80 post-islet transplantation). Data shown are the mean ± SEM (n = 6–9 per group). E. Area under the curve (AUC) for IPGTT was assessed. Data shown are the mean ± SEM (n = 6–9 per group), and a one-way ANOVA was performed, ***P < 0.001. F. Representative immunohistochemical staining for insulin in graft samples from mice receiving vehicle or IL-33. Scale bar = 100 μm. Download figure Download PowerPoint IL-33 induced Th2 cytokine, ILC2s, and regulatory T cells in vivo We then investigated the mechanism by which IL-33 prevents rejection of islet allografts. In islet transplant mice treated with IL-33, the serum levels of the Th1-related cytokines IFN-γ and IL-6 were significantly reduced when compared with those of PBS-treated control mice (Appendix Fig S2A). Meanwhile, IL-33 treatment markedly reduced the expression of Th1-related cytokines IFN-γ and IL-6 in allografts (Appendix Fig S2B). In contrast, IL-33 treatment enhanced the serum levels of the Th2-related cytokines IL-4 and IL-13 and the expression of IL-4 and IL-13 in allografts (Appendix Fig S2C and D). Regarding serum levels of IL-10, we found no significant differences between the IL-33-treatment group and the controls. However, IL-33 treatment markedly increased the expression of IL-10 in allografts (Appendix Fig S2). These experiments suggest that IL-33 treatment has large effects on the systemic and local Th1/Th2 response, promoting polarization of the immune response toward a Th2 phenotype, and simultaneously inhibiting Th1 reactivity. Moreover, we observed a significant increase in the ratio of CD4 T cells/CD8 T cells in islet grafts of mice treated with IL-33 (Appendix Fig S2E and F), suggesting that IL-33 treatment may prevent islet graft rejection through modulating CD4 and CD8 T-cell responses. We and others have previously shown that IL-33 can induce expansion of regulatory T cells (Tregs) and ILC2s in multiple anatomical sites where they display an immunosuppressive role in various disease conditions (Turnquist et al, 2011; Schiering et al, 2014; Matta et al, 2016; Cao et al, 2018). Here, we aimed to examine whether short-term IL-33 treatment can induce long-term accumulation of Tregs and ILC2s in multiple anatomical sites. Firstly, we analyzed the effect of IL-33 on Tregs and ILC2s in spleen and kidney of normal C57BL/c mice at different time points after IL-33 administration. Analysis of leukocytes isolated from the spleen and kidneys of C57BL/6 mice 3 days after short-term IL-33 treatment showed a moderate increase in CD4+Foxp3+Treg frequencies (Fig 2A and B) and a massive increase in Lin(−)GATA-3+ ILC2 frequencies (Fig 2C and D) as compared with PBS-treated control mice. IL-33-induced Treg accumulation in the spleen and kidney was only maintained for 1 week after a single course of five IL-33 injections (Fig 2E), whereas IL-33-induced ILC2 accumulation in the kidney was maintained at a high level for up to 8 weeks and the ILC2 increase was more transient in the spleen (Fig 2F). Furthermore, we examined the Tregs and ILC2 accumulation in spleen, kidney, and islet graft of islet transplant mice at different time points after IL-33 administration (Fig 3A). As expected, IL-33 treatment induced the accumulation of Tregs in spleen, kidney, and islet graft at day 7 post-islet transplantation, but not at day 30 and day 80 (Fig 3B–D), indicating IL-33 induced only a short-term increase in Tregs in different tissues, especially islet graft. In contrast, IL-33 treatment induced a long-term increase in ILC2s in kidney and islet graft, but not in the spleen (Fig 3E–I). A greater amount of ILC2s were found in islet graft than in kidney and liver, which indicates that ILC2s tend to migrate to islet graft undergoing immune response (Appendix Fig S3). Moreover, we observed a consistent increase in IL-33, but not IL-25 or thymic stromal lymphopoietin (TSLP), in islet graft tissue of mice treated with IL-33 (Fig 3J). These data could partly explain why ILC2s are found within the graft for so long. Taken together, a short course of IL-33 treatment induced a sustained increase in ILC2 abundance in kidney and islet graft which may be involved in IL-33-mediated islet graft protection. Figure 2. IL-33 induced Tregs and ILC2s in vivo . C57BL/6 mice were treated with mouse recombinant IL-33 or PBS daily for 5 consecutive days. A, B. Representative FACS analysis showing the proportion of Tregs (CD4+Foxp3+) in the CD4+T-cell compartment from the spleens (A) and kidneys (B) at day 3 after treatment in C57BL/6 mice receiving PBS (n = 4) or IL-33 (n = 6). C, D. Representative FACS analysis showing the proportion of ILC2s (Lin-GATA-3+) in the CD45+leukocyte compartment from the spleens (C) and kidneys (D) at day 3 after treatment in C57BL/6 mice receiving PBS (n = 4) or IL-33 (n = 6). E, F. proportion of Tregs (E) and ILC2s (F) in the spleen and kidney in PBS-injected controls (n = 4) and at weeks 1–12 after IL-33 treatment (n = 4–6 per IL-33–treated group). Data information: Data shown are the mean ± SEM; statistical analysis was performed with an unpaired t-test, **P < 0.01, ***P < 0.001. Download figure Download PowerPoint Figure 3. IL-33 induced Tregs and ILC2s in mice with islet transplantation A. Streptozotocin-induced diabetic C57BL/6 (H2b) mice with IL-33 treatment were transplanted with BALB/c (H2d) islets. Mice were sacrificed at day 7, 30, and 80 post-islet transplantation. B–D. Proportion or numbers of CD4+Foxp3+Tregs in the spleens, kidneys, and islet grafts of normal, islet transplant mice receiving vehicle and islet transplant mice with IL-33 treatment (at day 7, 30, and 80 post-islet transplantation). Data shown are the mean ± SEM (n = 4–6 per group), and a one-way ANOVA was performed; ***P < 0.001. E. Representative confocal microscopy images of immunostaining for CD4, Foxp3, and insulin in islet grafts. Scale bar = 50 μm. F–H. Proportion or numbers of ILC2s in the spleens, kidneys, and islet graft of mice with or without IL-33 treatment. Data shown are the mean ± SEM (n = 4–6 per group), and a one-way ANOVA was performed; **P < 0.01, ***P < 0.001. I. Representative confocal microscopy images of immunostaining for CD127, ST2, CD3, and insulin in islet grafts. Scale bar = 50 μm. J. The mRNA expression of IL-25, IL-33, and TSLP in islet grafts of mice with or without IL-33 treatment was examined by qPCR, and expressed relative to the control of each experiment. Data shown are the mean ± SEM (n = 4–6 per group), and a one-way ANOVA was performed; **P < 0.01, ***P < 0.001. Download figure Download PowerPoint Tregs and ILC2s played critical roles in IL-33-mediated islet graft protection Previous studies have shown that ex vivo-expanded Tregs protected against islet graft rejection (Shi et al, 2012; Yi et al, 2012). IL-33-mediated cardiac allograft survival and acute graft-versus-host disease (GVHD) protection was dependent on Tregs (Turnquist et al, 2011; Matta et al, 2016). To examine whether Treg accumulation in vivo contributes to IL-33-mediated islet graft protection, depletion of regulatory T cells (DEREG) mice was administered diphtheria toxin (DT) to selectively deplete Tregs in vivo (Fig 4A). Treg depletion in DEREG mice was confirmed in the spleen and kidney by flow cytometry at day 5 post-islet transplantation (Fig 4B). Treg depletion significantly reduced the survival rate of islet graft in IL-33-treated mice, suggesting that Treg accumulation is an important mechanism in IL-33-mediated islet graft protection (Fig 4D and E). ILC2s play important roles in tissue repair and immunoregulation (Sonnenberg & Artis, 2015). We and other groups found that IL-33-activated ILC2s expressed significantly higher levels of CD25 in vivo (Appendix Fig S4; Roediger et al, 2015), suggesting that administration of anti-CD25 antibodies could be utilized to deplete ILC2s in vivo. However, administration of anti-CD25 antibody has been used to successfully deplete Tregs in vivo as CD25 is also highly expressed on Tregs (Lu et al, 2013). Here, the potential contribution of ILC2s to IL-33-mediated islet graft protection was assessed in Treg-depleted mice (DEREG mice treated with DT), in which administration of anti-CD25 antibodies (PC61) leads to depletion of ILC2s (Fig 4A). Flow cytometric analysis of Lin-GATA3+ ILC2s in spleen and kidney confirmed significant depletion of ILC2s in mice with anti-CD25 antibody treatment compared with Treg-depleted DEREG mice (Fig 4C). Additional ILC2 depletion in Treg-depleted DEREG mice completely abolished the protective effects of IL-33 on islet transplantation (Fig 4D and E). Taken together, these results demonstrate that both Tregs and ILC2s play critical roles in IL-33-mediated islet graft protection. Figure 4. ILC2s and Tregs contributed to IL-33-mediated islet protection in vivo A. Streptozotocin-induced diabetic DEREG C57BL/6 mice were treated with mouse recombinant IL-33 daily for 5 consecutive days, as well as diphtheria toxin (DT), PC61 or DT+PC61 on days −4 and sletallogr1 prior to and on day 2 post-islet transplantation. Mice were sacrificed at day 80 post-islet transplantation or at the day when grafts were considered rejected. B, C. Proportion of CD4+Foxp+Tregs (B) and Lin-GATA-3+ILC2s (C) from the spleens and kidneys of islet transplant mice receiving vehicle, IL-33, IL-33/DT, IL-33/PC61, or IL-33/DT/PC61 at day 5 post-islet transplantation. Data shown are the mean ± SEM (n = 4–5 per group), and a one-way ANOVA was performed; NS: non-significant, ***P < 0.001. D. Islet graft survival of five groups of mice was assessed by monitoring blood glucose and calculated using the Kaplan–Meier method. Cumulative data from two independent experiments are shown. Statistical analysis was performed with a log-rank test. *P < 0.05, **P < 0.01. E. Data are shown as blood glucose measurement in islet transplant mice treated with IL-33/DT, IL-33/PC61, or IL-33/DT/PC61. The horizontal black line indicates a BGL of 16 mmol/l, the threshold for rejection. Each line represents one mouse. Download figure Download PowerPoint IL-33 and IL-2/anti-IL-2 antibody complex induced ILC210 IL-10 producing ILC2s (ILC210) have been described in lung and intestine (Seehus et al, 2017; Wang et al, 2017). IL-33 and IL-2 have been shown to induce ILC210 in vivo (Seehus et al, 2017). Here, we identified two subsets of ILC2s in islet graft and kidney of islet transplant mice treated with IL-33, ILC210, and non-IL-10 producing ILC2s (Fig 5A and B). The IL-2/anti-IL-2 antibody complex (IL-2C) has been found to directly induce expansion of ILC2 that express the high-affinity IL-2 receptor CD25 (Seehus et al, 2017; Cao et al, 2020). Using IL-10 reporter mice, we further demonstrated that combined IL-33 and IL-2C treatment markedly enhanced the ILC210 expansion in the kidney when compared with IL-33 treatment alone (Fig 5C). In vitro, ILC2s isolated from kidneys were treated with IL-33 and IL-2C and analyzed for IL-10 by flow cytometry and ELISA. IL-33 and IL-2C treatment induced more ILC210 expansion in cultured ILC2s (Fig 5D). ILC2s when cultured with IL-33 and IL-2C produced a high level of IL-10 in the supernatant (Fig 5E). IL-2 is known to activate STAT5 which has been shown to promote IL-10 expression in T cells (Polhill et al, 2012). Therefore, we evaluated the degree of phosphorylation of STAT5 (p-STAT5) in ILC2s. As expected, kidney ILC2s treated with IL-33 and IL-2C rapidly increased the expression of p-STAT5 in vitro (Fig 5F). Moreover, inhibition of STAT5 significantly reduced IL-10 production by ILC2s cultured with IL-33 and IL-2C (Fig 5G). Figure 5. IL-33 and IL-2C induced ILC210 A, B. ILC210 and non-IL-10 producing ILC2s were assessed in islet graft (A) and kidney (B) by intracellular IL-10 staining at day 5 post-islet transplantation. Data shown are the mean ± SEM (n = 5 per group), and an unpaired t-test was performed; ***P < 0.001. ST2: suppression of tumorigenicity 2. C. IL-10 reporter C57BL/6 mice were treated with PBS, IL-33 alone or IL-33 and IL-2C daily for 5 consecutive days. Proportion of lin-CD127+ST2+IL-10-GFP ILC210 from the kidneys at day 3 after treatment in IL-10 reporter C57BL/6 mice. Data shown
Objective To study the effects of the Yinlingtong capsules on intracellular triglyceride,fatty acid oxidation levels and CPT-1/ACC-β in HHL-5 cells.Methods After treated HHL-5 cells with different doses Yinlingtong capsules,intracellular triglyceride content and the fatty acid oxidation level were detected by the kits.The CPT-1β and ACC-β mRNA levels were detected by Q-PCR.Results Yinlingtong capsules could reduce triglyceride content (compared with the control group,triglyceride content of0.5,1 and 2 mg/ml dose group were reduced by 9.5％,14.3％ and 19.4％,respectively),enhance the level of fatty acid oxidation by a dose-response relationship(compared with the control group,the level of fatty acid oxidation of 0.5,1and 2 mg/ml dose group were increased 4.3％,6.9％ and 11.2％,respectively),but could not affect the CPT-1βand ACC-β mRNA levels.Conclusion Yinlingtong capsules reduced the accumulation of fat in HHL-5 hepatocytes,and the specific impact mechanism needs to be further explored.
Key words:
Triglyceride; Fatty acid oxidation level; CPT-1β; ACC-β
This paper introduces a simple method to make a rat trachea tube. It is easy to find cheap materials to make this kind of good-looking tube, which can be inserted into the windpipe of animals and changed as necessary, in order to reduce experimental expenditure. This kind of tube can also be used on guinea pigs or small rabbits.
Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by gradual loss of cognitive function. Understanding the molecular mechanisms is crucial for developing effective therapies.
Caveolin-3 (CAV3) is a muscle-specific protein present within the muscle cell membrane that affects signaling pathways, including the insulin signaling pathway. A previous assessment of patients with newly developed type 2 diabetes (T2DM) demonstrated that CAV3 gene mutations may lead to changes in protein secondary structure. A severe CAV3 P104L mutation has previously been indicated to influence the phosphorylation of skeletal muscle cells and result in impaired glucose metabolism. In the present study, the effect of CAV3 K15N gene transfection in C2C12 cells was assessed. Transfection with K15N reduced the expression of total CAV3 and AKT2 proteins in the cells, and the translocation of glucose transporter type 4 to the muscle cell membrane, which resulted in decreased glucose uptake and glycogen synthesis in myocytes. In conclusion, these results indicate that the CAV3 K15N mutation may cause insulin-stimulated impaired glucose metabolism in myocytes, which may contribute to the development of T2DM.
The caveolin-3 (CAV3) protein is known to be specifically expressed in various myocytes, and skeletal muscle consumes most of the blood glucose as an energy source to maintain normal cell metabolism and function. The P104L mutation in the coding sequence of the human CAV3 gene leads to autosomal dominant disease limb-girdle muscular dystrophy type 1C (LGMD-1C). We previously reported that C2C12 cells transiently transfected with the P104L CAV3 mutant exhibited decreased glucose uptake and glycogen synthesis after insulin stimulation. The present study aimed to examine whether the P104L mutation affects C2C12 cell glucose metabolism, growth, and proliferation without insulin stimulation. C2C12 cells stably transfected with CAV3-P104L were established, and biochemical assays, western blot analysis and confocal microscopy were used to observe glucose metabolism as well as cell growth and proliferation and to determine the effect of the P104L mutation on the PI3K/Akt signaling pathway. Without insulin stimulation, C2C12 cells stably transfected with the P104L CAV3 mutant exhibited decreased glucose uptake and glycogen synthesis, decreased CAV3 expression and reduced localization of CAV3 and GLUT4 on the cell membrane. The P104L mutant significantly reduced the cell diameters, but accelerated cell proliferation. Akt phosphorylation was inhibited, and protein expression of GLUT4, p-GSK3β, and p-p70s6K, which are molecules downstream of Akt, was significantly decreased. The CAV3-P104L mutation inhibits glycometabolism and cell growth but accelerates C2C12 cell proliferation by reducing CAV3 protein expression and cell membrane localization, which may contribute to the pathogenesis of LGMD-1C.
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