Endocannabinoid-activated Nlrp3 inflammasome in infiltrating macrophages mediates β- cell loss in type 2 diabetes

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Endocannabinoid-activated Nlrp3 inflammasome in infiltrating macrophages mediates β- cell loss in type 2 diabetes T Jourdan, G Godlewski, R Cinar, A Bertola, G Szanda, J Liu, J Tam, T Han, B Mukhopadhyay, M.C Skarulis, C Ju, M Aouadi, M.P Czech and G Kunos. Fig. S1 Effects of JD5037 and its inactive diastereomer JD5037i on blood glucose, insulin and c- peptide levels in ZDF rats. Vehicle-treated lean (open columns), ZDF (gray columns), JD5037-treated ZDF (black columns) and JD5037i-treated ZDF rats (hatched columns) were sacrificed after 1 week of treatment. Blood glucose, plasma insulin and c-peptide concentrations were determined as described in Methods. Columns and bars represent means±sem from 10 animals/group; *P<0.001. Note that only JD5037, and not its inactive diastereomer JD5037i, was effective in improving glycemic control in ZDF rats.

Fig. S2 Effects of JD5037 treatment of ZDF rats on pro- and anti-apoptotic gene expression (a) β- cell mass (b), gene expression of beta cell proliferation markers (c), and beta cell proliferation (d). Vehicle-treated lean (open columns), ZDF (gray columns) and JD5037-treated ZDF rats (black columns) were sacrificed after 4 weeks of treatment. Pancreatic islets were isolated and analyzed for gene expression. Beta cell proliferation was assessed by quantifying cells double positive for insulin and Ki67 staining. Columns and bars represent means ± s.e.m. from 10 animals/group; *P<0.05, ** P<0.01, ***P<0.001. Scale bars, 80 μm.

Fig. S3 Expression of various inflammasomes in islets of lean, ZDF and JD5037-treated ZDF rats. (a) Islets from vehicle-treated lean (open columns) and ZDF rats (gray columns) or JD5037- treated ZDF rats (black columns) were isolated and analyzed for mrnas encoding various inflammasomes; (b) Effects of anandamide (1 μm) and LPS (10 ng/ml) on ASC protein levels and active, cleaved forms of caspase-1 (p10, p45) in islets isolated from lean rats; (c) Adiponectin receptor-1 and 2 gene expression in islets from lean, vehicle-treated ZDF and JD5037-treated ZDF rats. Columns and bars represent means ± s.e.m. from 10 animals/group; *P<0.05, ** P<0.01, ***P<0.001 relative to lean group.

Fig. S4 Analyses of co-localization of CB 1 R and Nlrp3 with CD68+ macrophages and insulin+ beta cells in islets of ZDF rats. Double immunohistochemistry was performed for insulin with CB 1 R, and for CD68 with CB 1 R or with Nlrp3. Representative sections from 8 separate experiments are shown. Co-localization is indicated by the yellow color in the merged pictures, with doublepositive cells marked by arrows. Note that co-localization is evident for CD68 with both CB 1 R and Nlrp3, but not for insulin with CB 1 R. Scale bar, 90 μm.

Fig. S5 Effects of treatment of ZDF rats with GeRPs containing CB 1 R sirna on CB 1 R expression in macrophages, monocytes and lymphocytes. (a) Thioglycollate-induced PECs were treated 48h with 40, 80 or 120 nm of either scrambled (gray columns) or CB 1 R SiRNA (black columns). CB 1 R expression was assessed by RT-qPCR. Columns and bars represent means±sem from 4 independent experiments; *P<0.05, ***P<0.001 relative to vehicletreated controls. (b) Isolated leukocytes from GeRP-treated ZDF rats were analyzed by FACS after staining for CD68 and CD3. No GeRPs positive cells were detected and the numbers of cells were comparable between scrambled and CB 1 R SiRNA groups. (c) PECs from GeRP-treated ZDF rats were analyzed by FACS after staining for CD68 and CD3 proteins; only CD68 + macrophages were also FITC + (GeRPs). The numbers of CD3 + and CD68 + cells were comparable between the 2 treatments. Blood and PECs from scrambled GeRP (gray) and CB 1 R-GeRP-treated ZDF rats (black columns) were collected. after 9-10 days of treatment. (d-e) Selective knockdown of Cnr1 but not Cnr2 expression in CD68 + (d) but not in CD3 + cells (e). **P<0.01 relative to scrb-gerp-treated group.

Fig. S6 Levels of anandamide and 2-AG in islets from lean, vehicle-treated ZDF and JD5037- treated ZDF rats, and the effects of high glucose and palmitic acid. (a) Higher anandamide in ZDF (gray) compared to lean rats (white) is reversed by chronic JD5037 treatment (black) and is associated with increased expression of the biosynthetic enzyme NAPE-PLD and decreased activity of the anandamide degrading enzyme FAAH. Unchanged 2- AG levels are associated with no change in MAGL activity of Daglb expression and decreased- Dagla expression; columns and bars are means ± s.e.m. from 6-9 preparations, *P<0.05; **P<0.01, ***P<0.001 relative to values in lean rats. (b) NAPE-PLD in the ZDF islet does not co-localize with insulin-containing beta cells (double immunostaining); (c) islet anandamide content is increased by high glucose (33 mm) and less so by palmitate (250 μm).

Fig. S7. Effects of anandamide and IL-1β on inflammasome expression and activity in RAW264.7 macrophages, MIN6 beta cells and human isolated islets. (a) Effects of anandamide (AEA) on Nlrp3 inflammasome and its downstream targets in RAW264.7 macrophages and in MIN6 insulinoma cells. Cells were incubated for 4 h with vehicle or the indicated concentrations of AEA, followed by analyses of cellular mrna levels and cytokines secreted into the medium; (b) Comparative effects of IL-1β and AEA on the expression of pro- and anti-apoptotic genes in MIN6 β-cells. MIN6 cells were incubated with maximally effective concentrations of AEA (0.5 μm) or IL-1β (30 ng/ml); (c) Effects of IL-1β and AEA on apoptotic markers in non-diabetic human isolated islets. (d) IL-1β down-regulates Cnr1 mrna and protein in MIN6 cells via the IL-1R, as indicated by blockade of this effect by IL-1R antagonist (10 ng/ml); Points/columns and vertical bars represent means ± s.e.m. from 6-8 independent experiments, statistics as in Figure 1.

Fig. S8 Effect of anandamide on inflammasome gene expression and activation in PECs from CB 1 R / (a) or Nlrp3 / mice (b) and their wild-type littermates. (c): Effect of acute in vivo treatment of mice with anandamide (10 mg/kg ip) on insulin-induced hypoglycemia in wild-type and Nlrp3 / - mice. Note that Nlrp3 / mice have increased baseline insulin sensitivity which remains unaffected by anandamide. Insulin sensitivity test in 4-6 mice/group was done as described in Methods.

Table. S1 Lean ZDF+Veh ZDF+JD5037 ZDF+Clodronate Leptin ng/ml 3.46 ± 0.15 12.91 ± 0.48 *** 9.48 ± 0.31 ***### 11.97 ± 0.98 *** Adiponectin µg/ml 4.60 ± 0.29 2.44 ± 0.18 *** 5.70 ± 0.07 **### 2.35 ± 0.23 *** TG mg/dl 63.2 ± 10.14 1196 ± 106.67 *** 817.6 ± 31.36 ***### 1136 ± 114 *** Cholesterol mg/dl 88 ± 7.35 164.8 ± 3.85 *** 104.8 ± 3.85 ### 159 ± 17 *** Uric acid µg/ml 2.27 ± 0.13 4.84 ± 0.36 *** 2.57 ± 0.04 ### 5.01 ± 0.44 *** IL-1β ng/ml 4.06 ± 0.41 14.05 ± 0.88 *** 7.63 ± 0.78 **### 8.42 ± 0.45 **### TNF-α pg/ml 1.71 ± 0.25 3.50 ± 0.74 *** 0.83 ± 0.07 **### Effects of peripheral CB 1 R antagonism on plasma levels of hormonal/metabolic variables. ZDF rats were treated daily for 28 d with vehicle or 3 mg/kg/day of JD5037. Figures in table represent means ± s.e.m. from 20 animals/group, * P< 0.05, ** P<0.01, *** P<0.001 relative to values in lean littermate rats; # P<0.05, ## P<0.01, ### P<0.001 relative to values in ZDF + Veh group.

Table. S2: List of antibodies used Protein reference specificity Supplier dilution Primary Antibodies Insulin A0564 Guinea Pig anti-human Dako 1/100 CB 1 R PA1-743 Rabbit anti-human/rat Thermo scientific 1/200 NLRP3 LS-B1766 Goat anti-human Lifespan Biosciences 1/200 CD68 ab31630 Mouse anti-rat/mouse Abcam 1/100 NAPE-PLD LS-B6951 Rabbit anti-human/rat Lifespan Biosciences 1/125 Ki67 AB9260 Rabbit anti-human/rat/mouse Millipore 1/100 Secondary Antibodies ab6904 Goat anti-guinea Pig (FITC) Abcam 1/200 ab7007 Donkey anti-rabbit (PE) Abcam 1/200 ab8517 Rabbit anti-mouse (FITC) Abcam 1/100 ab7004 Donkey anti-goat (PE) Abcam 1/100 Table. S3: List of primers used for rat, mouse and human tissues Rat primers Mouse Primers Human Primers Gene Reference Gene Reference Gene Reference Gene Reference 18S QT00199374 IL-18 QT00183071 L19 QT01779218 18S QT00199367 AdipoR1 QT01811922 IL-1R QT00178920 TBP QT00198443 ASC QT00232134 AdipoR2 QT01595580 IL-1Ra QT00193970 18S QT01036875 CB1 QT02305702 AIM-2 QT02376171 IL-1β QT00181657 Nlrp3 QT00122458 CB2 QT00012376 Arginase 1 QT00177611 insulin QT00373303 Caspase 1 QT00199458 FAAH QT00192444 ASC QT00368676 L19 QT01810704 IL-1β QT01048355 IL-18 QT00014560 BAK QT02346736 L38 QT01612625 TNF-α QT00104006 IL-1β QT00021385S BAX QT01081752 Mafa QT00457513 MCP-1 QT00167832 L19 QT01869742 Bcl-2 QT00184863 MCP-1 QT00183253 CB1 QT01748831 MAGL QT00189742 Bcl-xl QT01081346 Neurogenin3 QT00408779 CB2 QT00159558 NAPE-PLD QT00424928 CB1 QT00191737 Nlrp12 QT01591744 insulin QT01660855 Nlrp3 QT00029771 CB2 QT0013004 Nlrp3 QT01568448 BAK QT00246988 RPLP0 QT01839887 CD68 QT00372204 Nlrp6 QT01080401 BAX QT00102536 TBP QT00000721 FAS QT00196595 NOS QT00178325 Bcl-2 QT02392292 Fas QT00196595 PDX-1 QT00405328 Bcl-xl QT00149254 FasL QT00178171 SCD-1 QT02285493 Fat-CD36 QT01830395 TBP QT01605632 glucokinase QT00182966 TGF-β QT00187796 Glut 2 QT00192822 TNF-R1 QT01599738 ifi16 QT00463393 TNF-α QT00178717 IFN-g QT00184982 Txnip QT00368984

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