Fig 1: Difference in microbiome composition in HIV+ participants with or without subclinical atherosclerosis. (A) Heat tree analysis showing group-wise relative abundance for microbial communities using the hierarchical structure of metaphlan2 taxonomic classifications in HIV+ (n=12) compared to HIVneg participants (n=4) (left panel) and HIV+ with (n=7) compared to HIV+ participants without subclinical atherosclerosis (n=5) (right panel). (B) Bar graphs showing mean ± standard deviation for the significantly decreased bacterial species (left panels) and the significantly abundant species (right panels) in HIV+ participants with (n=7) compared to their counterparts without subclinical atherosclerosis (n=5). (C) Correlations between the bacterial species Eggerthella and IL-32?, IL-1ß, IL-18 and TNF-a in the HIV+ group (with and without subclinical atherosclerosis). Non-parametric Mann-Whitney test was used to compare metaphlan2 inferred genera between Plaqueneg and Plaque+ groups in (B) and non-parametric Spearman was used to test correlations in (C).
Fig 2: 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-?3 (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 3: DHX15 is required for IFN-ß, IFN-?3, and IL-18 production in human HT-29 IECs upon enteric RNA virus infection(A–I) ELISA of IFN-ß (A, D, and G), IFN-?3 (B, E, and H), and IL-18 (C, F, and I) production from human HT-29 IECs with the indicated shRNA after a 20-h infection with enteric RNA viruses, including simian rotavirus SA-11 strain (A–C) and reovirus T3D strain (D–F), or DNA virus HSV-1 KOS strain (G–I) at a multiplicity of infection (MOI) of 10. Mock, scrambled shRNA (sh-Ctrl)-treated human HT-29 IECs 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 human HT-29 IECs 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 4: Flagellin activates the canonical NLRP3 inflammasome in THP-1 cells. (A) Schematic figure of canonical NLRP3 activation is shown in black; noncanoncial caspase-4/-5–mediated pathway is shown in gray. (B) THP-1 NLRC4 KO cells were transfected with flagellin or LPS or stimulated with extracellular LPS and EtOH or nigericin in the presence of the pan-caspase inhibitor z-VAD-fmk. ASC speck formation and IL-18 release were determined at 4 h poststimulation. Mean with SD of at least three individual experiments (two replicates/experiment) is displayed. Statistical analysis was performed using two-way ANOVA followed by a Bonferroni posttest. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 5: THP-1 cells and hMDM express similar levels of NLRC4 and NLRP3, but NAIP mRNA levels are higher in hMDM. THP-1 cells and hMDM were treated with LPS for 3 h or left unstimulated. Total RNA was isolated, cDNA generated, and a quantitative RT-PCR performed to assess levels of NAIP, NLRC4, NLRP3, pro–IL-1ß, and pro–IL-18. Levels were normalized against three housekeeping genes (ß-actin, RPL37A, and EIF2B2) and are shown as normalized ratio to THP-1–unstimulated cells. Mean with SD of three individual experiments (THP-1: three replicates/experiment; hMDM: two donors in triplicate/experiment) is displayed. Statistical analysis was performed using two-way ANOVA followed by a Bonferroni posttest. *p < 0.05, ****p < 0.0001.
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