CB2 stimulation of adipose resident ILC2s orchestrates immune balance and ameliorates type 2 diabetes mellitus

Abstract

Development of type 2 diabetes mellitus (T2DM) is associated with low-grade chronic type 2 inflammation and disturbance of glucose homeostasis. Group 2 innate lymphoid cells (ILC2s) play a critical role in maintaining adipose homeostasis via the production of type 2 cytokines. Here, we demonstrate that CB₂, a G-protein-coupled receptor (GPCR) and member of the endocannabinoid system, is expressed on both visceral adipose tissue (VAT)-derived murine and human ILC2s. Moreover, we utilize a combination of ex vivo and in vivo approaches to explore the functional and therapeutic impacts of CB₂ engagement on VAT ILC2s in a T2DM model. Our results show that CB₂ stimulation of ILC2s protects against insulin-resistance onset, ameliorates glucose tolerance, and reverses established insulin resistance. Our mechanistic studies reveal that the therapeutic effects of CB₂ are mediated through activation of the AKT, ERK1/2, and CREB pathways on ILC2s. The results reveal that the CB₂ agonist can serve as a candidate for the prevention and treatment of T2DM.

Keywords: CB2; CP: Immunology; CP: Metabolism; ILC2; T2DM; adipose inflammation; glucose tolerance; immunotherapy; insulin resistance.

Figure 1.. CB₂ engagement enhances activity and proliferation of activated ILC2s

(A) C57BL/6J mice were assigned to receive a normal chow diet (NCD) for 14 weeks, followed by intraperitoneal (i.p.) administration of recombinant mouse (rm)IL-33 (0.5 μg in 50 μL) or PBS for 3 days. On the 4^th day, the mice were euthanized, and visceral adipose tissue (VAT) was collected. n = 6. (B) Gating strategy of Lin⁻CD45⁺IL-7R⁺ST2⁺ILC2s. (C) CB₂ expression in naive and IL-33-activated murine VAT-derived ILC2s versus the vehicle control. CB₂ expression is quantified as mean fluorescence intensity (MFI). (D–F) Naive and activated VAT ILC2s were cultured with rmIL-2 and rmIL-7 and stimulated with CB₂ agonist (10 μg/mL) or vehicle control (2% dimethyl sulfoxide) for 48 h. Levels of IL-5, IL-6, and IL-13 in culture supernatants were assessed by LEGENDplex (D). Intranuclear expression of Ki67 (E) and GATA-3 (F) were quantified as MFI. Data are representative of 3 independent experiments, and bar graphs are shown as mean ± SEM. Statistical analyses involved one-way ANOVA (C) and two-tailed Student’s t test (D–F); n.s.: p > 0.05. Mouse image used with permission from Servier Medical Art.

Figure 2.. CB₂ stimulation protects from the onset of type 2 diabetes in WT mice

(A) Cohorts of C57BL/6J mice were assigned to receive either a NCD or a high-fat diet (HFD) and treated with either CB₂ agonist (1 mg/kg/mouse) or vehicle control (2% dimethyl sulfoxide) via i.p. injections every 4 days. n = 6. (B and C) Mice weight (B) and fasting blood glucose levels (C) were measured every 2 weeks for 14 weeks. (D and E) Glucose tolerance test (D) and insulin tolerance test (E) were conducted on the 14^th and 15^th week of the treatment, respectively. Data are representative of 3 independent experiments, and bar graphs are shown as mean ± SEM. Statistical analysis involved one-way ANOVA (B and C) and two-way ANOVA (D and E); n.s.: p > 0.05. Mouse image used with permission from Servier Medical Art.

Figure 3.. CB₂ engagement provides preventive protection against type 2 diabetes in alymphoid mice

(A) Cohorts of Rag2^−/− mice were fed an HFD and i.p. treated with either CB₂ agonist (1 mg/kg/mouse) or vehicle (2% dimethyl sulfoxide) every 4 days. n = 6. (B and C) Mice weights (B) and fasting blood glucose levels (C) were measured every 2 weeks for 14 weeks. (D) Plasma insulin concentrations were quantified using ELISA. (E and F) Glucose tolerance test (E) and insulin tolerance test (F) were performed at the end of the 14^th and 15^th weeks, respectively. The corresponding area under the curve (AUC) was calculated for each group. (G) Relative expression of Ucp1 mRNA in VAT lysates. (H) Relative amount of CD45⁺CD11b^hiF4/80^hiCD206⁺CD11c⁺ alternatively activated macrophages (AAM) in VAT after 14 weeks of treatment. n = 6. (I) The mRNA expression of VAT lysates after 14 weeks was determined by real-time qPCR using specific primers for Cidea, Prdm16, Pgc1a, Cox7a, Dio2, and hypoxanthine-guanine phosphoribosyltransferase (Hprt). (J) Epididymal adipose tissue sections stained with hematoxylin and eosin (×400, scale bars: 100 μm). (K and L) Quantitation of the mean adipocyte area (K) and the number of adipocytes per milligram of VAT (L). Data are representative of 3 independent experiments, and bar graphs are shown as mean ± SEM. Statistical analysis involved one-way ANOVA (B and C), two-way ANOVA (E and F), and two-tailed Student’s t test (D, G–I, and J–K). Mouse image used with permission from Servier Medical Art.

Figure 4.. Therapeutic CB₂ treatment ameliorates established type 2 diabetes in alymphoid mice

(A) Cohorts of Rag2^−/− mice were fed an HFD for 14 weeks. After 8 weeks of diet, mice were assigned to i.p. treatments with either CB₂ agonist (1 mg/kg/mouse) or vehicle (2% dimethyl sulfoxide) every 4 days. n = 6. (B and C) Mice weights (B) and fasting blood glucose levels (C) were measured every 2 weeks for 14 weeks. (D) Plasma insulin concentrations were quantified using ELISA. (E and F) Glucose tolerance test (E) and insulin tolerance test (F) were performed at the end of the 14^th and 15^th weeks, respectively. The corresponding AUC was calculated for each group. (G) Epididymal adipose tissue sections stained with hematoxylin and eosin (×400, scale bars: 100 μm). (H and I) Quantitation of the mean adipocyte area (H) and the number of adipocytes per milligram of VAT (I). Data are representative of 3 independent experiments, and bar graphs are shown as mean ± SEM. Statistical analysis involved one-way ANOVA (B and C), two-way ANOVA (E and F), and two-tailed Student’s t test (D, H, and I). Mouse image used with permission from Servier Medical Art.

Figure 5.. Therapeutic effects of CB₂ engagement depend on CB₂ and IL-13 expression on ILC2s

(A) Cohorts of CB₂^−/− mice were either adoptively transferred with WT ILC2s isolated from C57BL/6J mice or left untreated. Mice were then treated with CB₂ agonist (1 mg/kg/mouse) or vehicle control every 4 days and fed an HFD for 14 weeks. n = 6. (B and C) Mice weights (B) and fasting blood glucose level (C) were measured every 2 weeks for 14 weeks. (D) Glucose tolerance test were measured after 14 weeks of treatment. (E) Cohorts of CB₂^−/− mice were adoptively transferred with ILC2s from either WT, IL-5^−/−, or IL-13^−/− mice and then treated with CB₂ agonist (1 mg/kg/mouse) or vehicle control and fed an HFD for 14 weeks. n = 6. (F and G) Mice weights (F) and fasting blood glucose level (G) were measured every 2 weeks for 14 weeks. (H) Glucose tolerance test was performed after 14 weeks of treatment. (I and J) VAT weight (I) was measured and the number of ILC2s per gram of VAT (J) was quantified 14 weeks after adoptive transfer. Data are representative of 3 independent experiments, and bar graphs are shown as mean ± SEM. Statistical analyses involved one-way ANOVA (B, C, F, and G), two-way ANOVA (D and H), and two-tailed Student’s t test (I and J). Mouse image used with permission from Servier Medical Art.

Figure 6.. CB₂ signaling induces the CREB pathway in activated VAT-derived ILC2s

WT and CB₂ KO mice received i.p. injections of rmIL-33 (0.5 μg in 50 μL) for 3 days. On the 4^th day, the mice were euthanized, and their VAT was collected. In vivo activated VAT-derived murine ILC2s (aILC2s) were isolated and cultured in the presence of rmIL-2 and rmIL-7 for 24 h. Total RNA was isolated for bulk RNA-seq. (A) Principal-component analysis of VAT-derived aILC2s from CB₂ KO (red) and WT mice (blue). (B) A volcano plot comparison illustrates whole-transcriptome gene expression in VAT-derived aILC2s from CB₂ KO and WT mice. Differentially expressed genes (identified as statistically significant with an adjusted p < 0.05) showing at least a 1.5-fold change (FC) are highlighted in red (upregulated) and blue (downregulated). Relevant differentially expressed genes are labeled. (C) A heatmap presents gene-specific analysis for RNA-seq expression, highlighting the genes that are differentially regulated in CB₂ KO ILC2s compared to WT ILC2s. Differentially expressed genes were selected based on p < 0.05. (D) Gene set enrichment analysis conducted through Ingenuity Pathway Analysis displays the top canonical pathways influenced in CB₂ KO aILC2s compared to WT aILC2s. (E) A schematic diagram exhibits the PI3K-AKT, protein kinase A (PKA), MEK, and CREB signaling pathways downstream of CB₂. (F) A heatplot shows the differentially expressed genes associated with the PI3K-AKT, PKA, MEK, ERK1/2, p38, and CREB signaling pathways. (G–I) Intranuclear expression of pERK1/2 (G), phospho-AKT (pAKT) (H), and pCREB (I) on CB₂ KO and WT ILC2s, quantified as MFI. Data are representative of 3 independent experiments, and bar graphs are shown as mean ± SEM. Statistical analysis, two-tailed Student’s t test (G-I); a p value of <0.05 was considered non-significant.

Figure 7.. CB₂ engagement enhances human ILC2 activity

(A) Human peripheral blood ILC2s were freshly isolated and cultured with 10 ng/mL of recombinant human (rh)IL-2, rhIL-7, and rhIL-33 for 48 h. (B) The gating strategy for Lin⁻CD45⁺CD127⁺CRTH2⁺ human ILC2s. (C) Flow cytometry analysis of CB₂ expression on naive and IL-33-activated ILC2s, quantified as MFI with SEM. (D) Naive and activated ILC2s were cultured with 10 ng/mL of rhIL-2, rhIL-7, and either CB₂ agonist or vehicle control for 48 h. Levels of IL-5, IL-6, and IL-13 in the culture supernatants were quantified by LEGENDplex. Data are representative of five individual blood donors, presented as mean ± SEM. Statistical analysis involved one-way ANOVA (C) or two-tailed Student’s t test (D); n.s.: p > 0.05. Human image used with permission from Servier Medical Art.