Fig 1: Caspase-4/11 accelerates lipid-promoted atherosclerosis and ox-LDL-treated macrophage pyroptosis. (A) Left, western blot analysis and quantification of caspase-11 and its cleaved fragment expression in aortic arteries from WT, ApoE−/−, and ApoE−/−Caspase-11−/− mice fed with chow diet and HFHC diet for twelve weeks (n = 4 per group). Middle, quantification results of the level of cleaved caspase-11 in indicated groups. Right, quantification results of the level of pro-caspase-11 in indicated groups. (B) Left, representative images of caspase-11 (green), F4/80 (red) and DAPI (blue) in immunofluorescent staining of aortic root sections of indicated group, and quantification of the F4/80 and caspase-11 positive area of aortic root sections (n = 4 per group). Right, quantification results of the level of F4/80 and caspase-11 positive area of aortic root sections in the indicated groups. (C) Left, representative immunoblots of cleaved caspase-11 in peritoneal macrophage primed with 16-h ox-LDL stimulation in the indicated groups (n = 4 per group). Right, quantification results of the level of cleaved caspase-11 in indicated groups. (D) IL-1β (Left), IL-18 (Middle) and LDH release (Right) level secreted in peritoneal macrophage primed with LPS for 5 h and then treated with ox-LDL for 16 h after LPS administration was measured via enzyme-linked immunosorbent assay (ELISA) kits (n = 4 in each group). (E) Quantification results of the mRNA levels of caspase-4 genes in peripheral blood monocytes in patients with coronary heart diseases and the control group (n = 10 in each group). (F) Pearson comparison analyses of the correlations between caspase-4 mRNA levels in PBMC and SYNTAX score of patients with coronary heart diseases (n = 25). p < 0.05 for all of these correlations, by Pearson's rank correlation coefficient analysis. Data shown are mean ± SD (A–F). Data were first analyzed and passed normality test [Shapiro-Wilk test in (A–E), D’Agostino and Pearson test in (F)]. p values were shown and assessed by one-way ANOVA with Tukey’s test (A, B), by Mann-Whitney test (C) and by unpaired two-tailed t test (D, E). All of the p values were labeled on the pictures and p < 0.05 was considered to indicate statistical significance. Ox-LDL, Oxidized low-density lipoprotein; PBMC, peripheral blood monoculear cell; SYNTAX, Synergy Between Percutaneous Coronary Intervention with Taxus and Cardiac Surgery.
Fig 2: DHX15 is essential for producing IFN-β, IFN-λ3, and IL-18 by human HT-29 IECs in response to poly I:C(A) Immunoblot (IB) showing the knockdown efficiency of shRNAs targeting the indicated genes in HT-29 IECs. Nontargeting shRNA served as a control (sh-Ctrl). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) blots are shown as loading controls. The position of protein markers (shown in kDa) is indicated at right. (B–G) ELISA of IFN-β (B and E), IFN-λ3 (C and F), and IL-18 (D and G) production from human HT-29 IECs with the indicated shRNA after a 20-h stimulation with 5 μg/mL poly I:C (B–D) or 2.5 μg/mL poly dG:dC (E–G) delivered by Lipofectamine 3000. N-STM, scrambled shRNA-treated HT-29 IECs without stimulation. Each circle represents an individual independent experiment, and small solid black lines indicate the average of triplicates. NS, not significant; p > 0.05, ***p < 0.001 (unpaired t test).
Fig 3: DHX15 is essential to control intestinal inflammation induced by enteric rotavirus infection in suckling mice in vivo(A) Diarrhea duration and percentage of mice with diarrhea (score ≥ 2) from 8-day-old wild-type Dhx15fl/fl and Dhx15IEC-KO suckling mice (n = 20 per strain) orally inoculated by gavage with 1 DD50 rotavirus EW strain.(B–F) The wild-type Dhx15fl/fl and Dhx15IEC-KO suckling mice (n = 5 per strain) were orally inoculated by gavage with 1 DD50 rotavirus EW strain. At day 1 post-inoculation, mice were euthanized, and intestine tissues were excised for qRT-PCR detection of Ifnb (B), Ifnl2/3 (C), and Il18 (D) expression. In addition, the excised intestine was homogenized in PBS for the detection of IFN-λ3 (E) and IL-18 (F) in intestine homogenates by ELISA. Data are represented as means ± SEMs.(G and H) The wild-type Dhx15fl/fl and Dhx15IEC-KO suckling mice (n = 20 per strain) were orally inoculated by gavage with 1 DD50 rotavirus EW strain. At day 5 post-inoculation, mice were euthanized, and intestine tissues (G) and feces (H) were collected for qRT-PCR detection of rotavirus levels. Mock, mouse without rotavirus infection. *p < 0.05, **p < 0.01, and ***p < 0.001 (unpaired t test).
Fig 4: DHX15 positively regulates production of IFN-β, IFN-λ3, and IL-18 in mouse primary IECs upon enteric RNA virus infection(A–I) ELISA of IFN-β (A, D, and G), IFN-λ3 (B, E, and H) and IL-18 (C, F, and I) production in mouse primary IECs from wild-type Dhx15fl/fl and Dhx15IEC-KO mice after a 20-h infection with enteric RNA viruses, including rotavirus EW strain (A–C) and reovirus T3D strain (D–F), or DNA virus HSV-1 KOS strain (G–I) at a MOI of 10. Mock, cells without virus infection. Each circle represents an individual independent experiment, and small solid black lines indicate the average of triplicates.(J–L) Quantification of expression of rotavirus NSP5 gene (J), reovirus S4 gene (K), and HSV-1 VP16 gene (L) relative to GAPDH in mouse primary IECs from wild-type Dhx15fl/fl and Dhx15IEC-KO mice infected by rotavirus (J), reovirus (K), or HSV-1(L) as in (A)–(I). Data are represented as means ± SEMs. NS, p > 0.05, ***p < 0.001 (unpaired t test).
Fig 5: DHX15 is required for control of intestinal inflammation induced by enteric reovirus infection in adult mice in vivo(A) Survival of 5-week-old wild-type Dhx15fl/fl and Dhx15IEC-KO adult mice (n = 10 per strain) after intragastric injection of reovirus T3D strain (1 × 108 plaque-forming units [PFUs] per mouse).(B–D) The wild-type Dhx15fl/fl and Dhx15IEC-KO mice (n = 5 per strain) were inoculated intragastrically with 1 × 108 PFUs reovirus T3D strain. At day 1 post-inoculation, mice were euthanized, and intestine tissues were excised and homogenized in PBS. Levels of IFN-β (B), IFN-λ3 (C), and IL-18 (D) in intestine homogenates were quantified by ELISA.(E and F) The wild-type Dhx15fl/fl and Dhx15IEC-KO mice (n = 15 per strain) were inoculated intragastrically with 1 × 108 PFU of reovirus T3D strain. At day 4 post-inoculation, mice were euthanized, feces were collected, and intestinal tissues were excised. The viral titers in intestine homogenates (E) and shedding in feces (F) were determined by plaque assay. Results are expressed as mean viral titers for 15 animals for each time point. Error bars indicate SEMs.(G) Flow cytometry analysis of CD4+ T cells, CD8+ T cells, B cells, and NK cells of intestine lamina propria lymphocytes (left panel) and mesenteric lymph nodes (mLN) (right panel) from wild-type Dhx15fl/fl and Dhx15IEC-KO mice infected with reovirus for 2 days using CD3-FITC, CD4-PE/cyanine7, CD8a-PerCP/cyanine5.5, CD19-APC, and NK1.1-PE antibodies.(H) The absolute cell numbers in intestine (left) and mLN (right) from wild-type Dhx15fl/fl and Dhx15IEC-KO mice (n = 3 mice) for representative flow cytometry data in (G).(I) Hematoxylin and eosin (H&E) staining of intestine sections from wild-type Dhx15fl/fl and Dhx15IEC-KO mice as in (E). Scale bars represent 200 μm.(J) Graph depicting histology scores for inflammation and tissue damage of intestine sections in (I). Data are represented as means ± SEMs. NS, p > 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001 (unpaired t test).
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