LncRNA29RIK in macrophages promotes LPS-mediated sensitivity to obesity

Figures

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  Figure 1. LPS promotes expression of Lnc29rik (29RIK) in macrophages. (A) LncRNA microarray of myeloid-derived suppressor cells (MDSCs). MDSCs were generated, and lncRNA expression was then evaluated using an lncRNA expression microarray. NC, bone marrow cells. (B) QRT-PCR of lncRNA29RIK in MDSCs. NC, bone marrow cells. R. E, relative expression. (C) qRT-PCR of lncRNA29RIK in the spleen, and CD4, CD8, B cell, dendritic cells (DC), MDSC, and macrophages sorted from the spleen by flow cytometry. R. E, relative expression. (D) Fluorescence in situ hybridization of lncRNA29RIK in mouse macrophage upon exposure to LPS (1 μg/mL) for 24 h Nuclei were stained with DAPI (blue); green, lncRNA29RIK. Scale bar, 2.5 μM. (E) qRT-PCR of lncRNA29RIK in the macrophages after exposure to IAA, DCA, or LPS for 24 h (F) qRT-PCR of lncRNA29RIK in the macrophages at the different times after exposure to LPS (1 μg/mL). R. E, relative expression. (G) qRT-PCR of lncRNA29RIK in the macrophages after exposure to different concentrations of LPS. R. E, relative expression. (H) QRT-PCR of lncRNA29RIK in the TLR4 KO macrophages. Ctr., bone marrow cells. R. E, relative expression. One-way ANOVA Bonferroni’s multiple…

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  Figure 2. LncRNA29RIK (29RIK) affects mature IL-1β and pyroptosis in the macrophages. (A) ELISA of IL-1β in the supernatants of lncRNA29RIK siRNA (si29RIK) or exogenous lncRNA29RIK (oe29RIK) transfected macrophages after exposure to LPS/dotap. NC, control. (B) Immunoblotting of pro-IL-1β, mature (m) IL-1β, GSDMD, and cleaved-GSDMD (GSDMD-N) in exogenous lncRNA29RIK (oe29Rik) transfected macrophages after exposure to LPS/dotap. NC, no stimulator. (C) Analyses of LDH in the supernatants of lncRNA29RIK siRNA (si29RIK) transfected macrophages after exposure to LPS/dotap. siNC, siRNA control; NC, no stimulator. (D) ELISA of IL-1β in the supernatants of lncRNA29RIK KO (29RIK KO) macrophages after exposure to LPS/dotap. NC, control. (E) Immunoblotting of pro-IL-1β and mIL-1β in lncRNA29RIK KO macrophages after exposure to LPS/dotap. NC, control. (F) Analyses of LDH in the supernatants of lncRNA29RIK KO macrophages after exposure to LPS/dotap. NC, control. (G) Immunoblotting of GSDMD and cleaved-GSDMD (GSDMD-N) in lncRNA29RIK KO macrophages after exposure to LPS/dotap.NC, control. (H) flow cytometry of lncRNA29RIK KO macrophages after FLICA staining on exposure to LPS/dotap; NC, control. Student’s…

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  Figure 3. LncRNA29RIK (29RIK) KO mice are resistance to HFD-mediated obesity. (A) Body weight increases in male WT and lncRNA29RIK KO mice fed high-fat diet (HFD) (n=14). These mice are no differences at baseline before feeding HFD. (B) Fat-pad in male WT and lncRNA29RIK KO mice fed HFD for 3 months. (C) Glucose tolerance and insulin sensitivity of WT and lncRNA29RIK KO mice fed HFD for 3 months (n=6). (D) H/E staining of adipose tissues of WT and lncRNA29RIK KO mice fed HFD. One representative. (E) ELISA of mIL-1β and qRT-PCR of TNFα, IL-6 and MCP-1 in the adipose tissues of WT and lncRNA29RIK KO mice fed HFD (n=14). (F) Flow cytometry of F4/80 CD11b. F4/80 CD11C and F4/80 CD206 in adipose tissues of WT and lncRNA29RIK KO mice fed HFD. (G) Concentration of LPS in the sera of WT and lncRNA29RIK KO mice fed HFD (n=14). Analysis of variance test in parts (A, C); Student’s t-test in other panels, mean ± SD. R. E, relative expression. *p < 0.05, **p < 0.01, and ***p < 0.001. Data are a representative of three independent experiments.

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  Figure 4. LncRNA29RIK (29RIK) promote the binding of LPS with caspase-11. (A) Schematic illustration showing the LPS mediated mIL-1β and pyroptosis. (B) Immunostaining of caspase-11 and LncRNA29RIK in the macrophages after exposure to LPS/dotap. Pearson’s correlation coefficient indicating co-localization of caspase-11 (cas-11) and LncRNA29RIK (29rik). DAPI, nuclear staining; NC, control; (C) Predicting binding of mouse lncRNA29RIK with caspase-11. The prediction results showed that there were two domains binding to lncRNA29RIK in caspase-11 protein, and the amino acid start and end site of the domain was given. (D) Immunostaining of LPS, GBP1, and caspase-11 (Cas-11) in the WT, LncRNA29RIK (29Rik) KO, and caspase-1/caspase-11 (Casp1/11) KO macrophages. Pearson’s correlation coefficient indicating co-localization of LPS and GBP1, LPS/Casp11, and Cas-11/GBP in 29 RIK KO and Cas-1/11 KO macrophages; WT, control. (E) Immunoblotting of caspase-11 (Cas-11) in the biotin-labeled LPS pull-down lysis of the WT and lncRNA29RIK KO microphages. (F) RIP of V5-tagged caspase-11 derivatives and lncRNA29RIK cotransfected HEK293T cells. RIP was performed using anti-V5. Caspase-11 and its derivatives were…

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  Figure 5. There has a similar function among lncRNA29RIK (29RIK) KO, caspase-1/11 and NLRC3 KO macrophages. (A) ELISA of IL-1β in the supernatants of lncRNA29RIK KO, caspase-1/11, and NLRC3 KO macrophages after exposure to LPS/dotap. NC, control. (B) Analyses of LDH in the supernatants of lncRNA29RIK KO, caspase-1/11, and NLRC3 KO macrophages after exposure to LPS/dotap. NC, control. (C) Immunoblotting of pro-IL-1β, IL-1β, GSDMD, and cleaved GSDMD (GSDMD-N) in the lncRNA29RIK KO, caspase-1/11, and NLRC3 KO macrophages after exposure to LPA/dotap. (D) Pyroptosis of lncRNA29RIK KO, caspase-1/11, and NLRC3 KO macrophages after exposure to LPS/dotap. NC, control. (E) ELISA of IL-1β in the supernatants of caspase-11 overexpressed lncRNA29RIK KO macrophages after exposure to LPS/dotap; WT, control macrophages. (F) Schematic illustration showing a mechanism for lncRNA29RIK to induce production of IL-1β and pyroptosis of macrophages. Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001; NS, not significant.

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  Figure 6. lncRNA29RIK (29RIK) promotes LPS-mediated inflammation in vivo. (A) Survival rate of WT and lncRNA29RIK KO mice with or without intraperitoneal injection LPS (52 mg/kg) (n=8). (B) ELISA of IL-1β in the sera of WT and lncRNA29RIK KO mice with or without intraperitoneal injection LPS (52 mg/kg) (n=8). (C) Survival rate and body weight of WT and lncRNA29RIK KO mice with or without S. typhimurium infection (200 CFUs/mouse, n=8). (D) Body weight of WT and lncRNA29RIK KO mice with or without S. typhimurium infection (200 CFUs/mouse, n=8). (E) ELISA of IL-1β in the sera of WT and lncRNA29RIK KO mice with or without S. typhimurium infection (200 CFUs/mouse, n=8). (F) Follow cytometry of CD11B+Ly6G+ and CD11b+ Ly6C+ cells in the colon tissues of WT and lncRNA29RIK KO mice with or without S. typhimurium infection (200 CFUs/mouse, n=8). One-way ANOVA Bonferroni’s multiple comparison test in part (D); Wilcoxon’s test in parts (A, C); two-sided Student’s t-test in (B, E, F) **p < 0.01, ***p < 0.001; NS, not significant.

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  Figure 7. Human lncRNA29RIK (29RIK) have similar function with mice. (A) Schematic illustration showing mouse caspase-11 and human caspase-4 structures. (B) Predicting binding of human lncRNA29RIK with caspase-4. The prediction results showed that there were one domain binding to human lncRNA29RIK in caspase-4 protein, and the amino acid start and end site of the domain was given. (C) RIP of V5-tagged caspase-4 and human lncRNA29RIK co-transfected HEK293T cells. RIP was performed using anti-V5. % input of human lncRNA29RIK was analyzed. Caspase-4 were cloned into pcDNA3.1/V5 to generate V5-tagged caspase-4, and then individually transfected into HEK293T cells. (D) Immunostaining of caspase-4 (Cas-4) in human-monocyte-derived macrophages after exposure to LPS. (E) Analyses of LDH in the supernatants of human lncRNA29RIK KO macrophages (h29RIK KO) after exposure to LPS/dotap. NC, control. (F) ELISA of IL-1β in the supernatants of human lncRNA29 RIK KO macrophages (h29RIK KO) after exposure to LPS/dotap. NC, control. (G) Immunoblotting of cleaved GSDMD (GSDMD-N) in the human lncRNA29RIK KO macrophages (h29RIK KO) after exposure to LPS/dotap or LPS+ Nigericin (LPS+Nig). (H) Flow cytometry of…