18 research outputs found

    Risk Factors for Fatty Liver in the Multicenter AIDS Cohort Study

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    ObjectivesHuman immunodeficiency virus (HIV) infection and antiretroviral therapy (ART) may increase the risk of fatty liver disease. We determined the prevalence of and risk factors for fatty liver by comparing HIV-infected men with HIV-uninfected men who have sex with men in the Multicenter AIDS Cohort Study (MACS).MethodsIn 719 MACS participants who consumed less than three alcoholic drinks daily, fatty liver was defined as a liver-to-spleen attenuation ratio <1 on noncontrast computed tomography (CT). We genotyped single nucleotide polymorphisms in the patatin-like phospholipase domain-containing 3 (PNPLA3) gene and in other genes previously associated with nonalcoholic fatty liver disease. Risk factors for fatty liver were determined using multivariable logistic regression.ResultsAmong 254 HIV-uninfected men and 465 HIV-infected men, 56% were White with median age 53 years and median body mass index 25.8 kg/m(2). The vast majority of HIV-infected men (92%) were on ART, and 87% of the HIV-infected men were treated with a nucleoside reverse transcriptase inhibitor for a median duration of 8.5 years. Overall, 15% of the cohort had fatty liver, which was more common in the HIV-uninfected compared with the HIV-infected men (19 vs. 13%, P=0.02). In multivariable analysis, HIV infection was associated with a lower prevalence of fatty liver (odds ratio (OR)=0.44, P=0.002), whereas a higher prevalence of fatty liver was seen in participants with PNPLA3 (rs738409) non-CC genotype (OR=2.06, P=0.005), more abdominal visceral adipose tissue (OR=1.08 per 10 cm(2), P<0.001), and homeostatic model assessment of insulin resistance (HOMA-IR) ≥4.9 (OR=2.50, P=0.001). Among HIV-infected men, PNPLA3 (rs738409) non-CC genotype was associated with a higher prevalence of fatty liver (OR=3.30, P=0.001) and cumulative dideoxynucleoside exposure (OR=1.44 per 5 years, P=0.02).ConclusionsCT-defined fatty liver is common among men at risk for HIV infection and is associated with greater visceral adiposity, HOMA-IR, and PNPLA3 (rs738409). Although treated HIV infection was associated with a lower prevalence of fatty liver, prolonged exposure to dideoxynucleoside analogs is associated with higher prevalence

    HIV and HCV Activate the Inflammasome in Monocytes and Macrophages via Endosomal Toll-Like Receptors without Induction of Type 1 Interferon

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    <div><p>Innate immune sensing of viral infection results in type I interferon (IFN) production and inflammasome activation. Type I IFNs, primarily IFN-α and IFN-β, are produced by all cell types upon virus infection and promote an antiviral state in surrounding cells by inducing the expression of IFN-stimulated genes. Type I IFN production is mediated by Toll-like receptor (TLR) 3 in HCV infected hepatocytes. Type I IFNs are also produced by plasmacytoid dendritic cells (pDC) after sensing of HIV and HCV through TLR7 in the absence of productive pDC infection. Inflammasomes are multi-protein cytosolic complexes that integrate several pathogen-triggered signaling cascades ultimately leading to caspase-1 activation and generation pro-inflammatory cytokines including interleukin (IL)-18 and IL-1β. Here, we demonstrate that HIV and HCV activate the inflammasome, but not Type I IFN production, in monocytes and macrophages in an infection-independent process that requires clathrin-mediated endocytosis and recognition of the virus by distinct endosomal TLRs. Knockdown of each endosomal TLR in primary monocytes by RNA interference reveals that inflammasome activation in these cells results from HIV sensing by TLR8 and HCV recognition by TLR7. Despite its critical role in type I IFN production by pDCs stimulated with HIV, TLR7 is not required for inflammasome activation by HIV. Similarly, HCV activation of the inflammasome in monocytes does not require TLR3 or its downstream signaling adaptor TICAM-1, while this pathway leads to type I IFN in infected hepatocytes. Monocytes and macrophages do not produce type I IFN upon TLR8 or TLR7 sensing of HIV or HCV, respectively. These findings reveal a novel infection-independent mechanism for chronic viral induction of key anti-viral programs and demonstrate distinct TLR utilization by different cell types for activation of the type I IFN vs. inflammasome pathways of inflammation.</p></div

    Differential importance of cytoplasmic sensors in inflammasome response to HIV and HCV.

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    <p>Functional knockdowns of the cytoplasmic sensors NLRP3, RIG-I and AIM2 in monocytes were created as previously described for TLRs. (<b>A</b>) Knockdown monocytes were cultured with HIV<sub>BaL</sub>, HIV<sub>RF</sub>, HIV<sub>IIIB</sub>, or HIV<sub>RF</sub> and IL-18 measured after 24 h. IL-18 produced relative to that of mock transfected cells is shown. (<b>B</b>) Similarly prepared monocytes were cultured with a panel of plasma from HCV infected individuals (HCV<sub>Subject 117</sub>, HCV<sub>Subject 54</sub>, HCV<sub>Subject 16</sub>, HCV<sub>Subject 180</sub>) and IL-18 measured at 24 h. Bars represent the mean ± S.D. for <i>n = 6–9</i> independent transfection experiments.</p

    HCV and HIV virions stimulate monocytes to produce IL-18.

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    <p>HCV viremic plasma (<b>A</b>) or HCV<sub>JFH-1</sub> (<b>B</b>) was separated into fractions by sucrose gradient equilibrium ultracentrifugation. HCV RNA (black circles) for each fraction was determined by RT-PCR. Monocytes are maximally stimulated to secrete IL-18 (grey bars, n = 3) when cultured with fractions in the density range 1.09–1.16 g/mL (*), which are most enriched in HCV RNA. (<b>c</b>) HIV<sub>IIIB</sub>, HIV<sub>MN</sub>, HIV<sub>RF</sub> and HIV<sub>BaL</sub> were cultured in activated CD4<sup>+</sup> T-cells, p24 measured in supernatant, and supernatant transferred to monocytes. Monocytes cultured with HIV<sub>IIIB</sub> (black circles), HIV<sub>MN</sub> (white triangles), HIV<sub>RF</sub> (black squares) and HIV<sub>BaL</sub> (white diamonds) culture supernatant secrete IL-18 in a dose dependent manner. Symbols represent the mean ± S.D. of 6 experiments.</p

    Monocytes produce inflammasome cytokines in response to HCV and HIV.

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    <p>(<b>A</b>) HIV and HCV uninfected PBMC, sorted monocytes, or <i>in vitro</i> differentiated macrophages were cultured with plasma from HCV infected subjects (V, <i>n = 15, plasma vol. creates at the final concentration of 5×10<sup>5</sup> HCV RNA IU/mL in culture</i>) or plasma from the same subjects prior to infection (Pre, <i>n = 15, matched volume</i>). IL-18 and IL-1β were measured in duplicate after 24 h. Primary human hepatocytes were cultured with V and Pre plasma as well as the culture strain HCV<sub>JFH-1</sub> (<i>n = 5</i>). Monocytes and derived macrophages secrete IL-18 (<b>top panel</b>) and IL-1β (<b>lower panel</b>) in response to V but not Pre plasma. Hepatocytes fail to produce inflammasome cytokines. In (<b>B</b>), IL-18 and IL-1β were measured after HIV and HCV uninfected PBMC, sorted monocytes, T-cells or <i>in vitro</i> differentiated macrophages were cultured with plasma from subjects on HAART (H, <i>n = 6</i>), plasma from elite suppressors (ES, <i>n = 5</i>), or viremic HIV plasma (V, <i>n = 15</i>). Plasma with measurable HIV stimulated significant monocyte IL-18 and IL-1β secretion. IL-18 produced from cells cultured with individual plasma samples (white circles), means (horizontal dash) ± S.D. are shown.</p

    MyD88 is required for inflammasome sensing of HIV and HCV.

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    <p>Knockdowns of the TLR adaptors MyD88 (<b>A</b>, <b>B</b>) and TICAM-1 (TRIF, <b>E</b>, <b>F</b>) were generated and confirmed by previously described RNA interference techniques. (*) denotes comparisons with p≤0.05 compared to the mock transfected cells. Monocytes in which MyD88 was knocked down were cultured with HIV<sub>BaL</sub> (solid bars) or HCV<sub>Subject 180</sub> (hatched bars) and pro-IL-1β mRNA transcription measured at 6 h (<b>C</b>, <b>G</b>) and IL-18 secretion measured at 24 h (<b>D</b>, <b>H</b>). Shown are the relative production of pro-IL-1β mRNA and IL-18 in MyD88 (<b>C</b>, <b>D</b>) or TICAM-1 (<b>G</b>, <b>H</b>) knockdown monocytes normalized to mock transfected monocytes (no siRNA) stimulated with the same viruses. Bars represent the mean ± S.D. for <i>n = 6–9</i> independent transfection experiments, (**) denotes comparisons with p≤0.001 compared to the scramble siRNA transfected cells.</p

    Differential importance of endosomal TLRs in inflammasome sensing of HIV and HCV.

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    <p>Monocytes were cultured without stimulation or with HIV<sub>BaL</sub> or HCV<sub>Subject 180</sub> and IL-1β, IFNα and measured at multiple timepoints. Fold change in expression of IL-1β (black bar) and IFNα (grey bar) following HIV<sub>BaL</sub> or HCV<sub>Subject 180</sub> relative to stimulation with media alone is shown at 6 h (<b>A</b>). Functional knockdowns of the endosomal located TLRs in monocytes were generated by RNA interference. Monocytes were mock transfected or transfected with either siRNA targeting TLR7 or a non-targeting sequence (Scramble). After 24 h, cell lysates were prepared and efficacy of knockdown determined by (<b>B</b>) western blot and (<b>C</b>) qRT-PCR. (<b>D</b>) Specificity of the TLR7 siRNA was confirmed by qRT-PCR using primers for TLR3, TLR7, TLR8, and TLR9. (*) denotes comparisons with p≤0.05 compared to the mock transfected cells. Monocytes in which endosomal TLR knockdown were generated were cultured with HIV<sub>BaL</sub> (solid bars) or HCV<sub>Subject 180</sub> (hatched bars) and pro-IL-1β mRNA transcription measured at 6 h (<b>E–H</b>) and IL-18 secretion measured at 24 h (<b>I–L</b>). Shown are the relative production of pro-IL-1β mRNA and IL-18 in TLR8 (<b>E</b>, <b>I</b>), TLR7 (<b>F</b>, <b>J</b>), TLR3 (<b>G</b>, <b>K</b>) and TLR9 (<b>H</b>, <b>L</b>) knockdown monocytes normalized to mock transfected monocytes (no siRNA) stimulated with the same viruses. Bars represent the mean ± S.D. for <i>n = 6–9</i> independent transfection experiments, (**) denotes comparisons with p≤0.05 and (***) denoted p≤0.001 compared to the scramble siRNA transfected cells.</p
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