Epicardial fat density, coronary artery disease and inflammation in people living with HIV.

Studies have shown an increased risk of coronary artery disease (CAD) in the human immunodeficiency virus (HIV) population. Epicardial fat (EF) quality may be linked to this increased risk. In our study, we evaluated the associations between EF density, a qualitative characteristic of fat, and inflammatory markers, cardiovascular risk factors, HIV-related parameters, and CAD. Our study was cross-sectional, nested in the Canadian HIV and Aging Cohort Study, a large prospective cohort that includes participants living with HIV (PLHIV) and healthy controls. Participants underwent cardiac computed tomography angiography to measure volume and density of EF, coronary artery calcium score, coronary plaque, and low attenuation plaque volume. Association between EF density, cardiovascular risk factors, HIV parameters, and CAD were evaluated using adjusted regression analysis. A total of 177 PLHIV and 83 healthy controls were included in this study. EF density was similar between the two groups (-77.4 ± 5.6 HU for PLHIV and -77.0 ± 5.6 HU for uninfected controls, P = .162). Multivariable models showed positive association between EF density and coronary calcium score (odds ratio, 1.07, P = .023). Among the soluble biomarkers measured in our study, adjusted analyses showed that IL2Rα, tumor necrosis factor alpha and luteizing hormone were significantly associated with EF density. Our study showed that an increase in EF density was associated with a higher coronary calcium score and with inflammatory markers in a population that includes PLHIV.

Overt IL-32 isoform expression at intestinal level during HIV-1 infection is negatively regulated by IL-17A.

OBJECTIVES: Untreated HIV infection was previously associated with IL-32 overexpression in gut/intestinal epithelial cells (IEC). Here, we explored IL-32 isoform expression in the colon of people with HIV (PWH) receiving antiretroviral therapy (ART) and IL-32 triggers/modulators in IEC. DESIGN: Sigmoid colon biopsies (SCB) and blood were collected from ART-treated PWH (HIV + ART; n = 17; mean age: 56 years; CD4+ cell counts: 679 cells/µl; time on ART: 72 months) and age-matched HIV-uninfected controls (HIVneg; n = 5). The IEC line HT-29 was used for mechanistic studies. METHODS: Cells from SCB and blood were isolated by enzymatic digestion and/or gradient centrifugation. HT-29 cells were exposed to TLR1-9 agonists, TNF-α, IL-17A and HIV. IL-32α/ß/γ/D/ε/θ and IL-17A mRNA levels were quantified by real-time RT-PCR. IL-32 protein levels were quantified by ELISA. RESULTS: IL-32ß/γ/ε isoform transcripts were detectable in the blood and SCB, with IL-32ß mRNA levels being predominantly expressed in both compartments and at significantly higher levels in HIV + ART compared to HIVneg. IL-17A transcripts were only detectable in SCB, with increased IL-17A levels in HIVneg compared with HIV + ART and negatively correlated with IL-32ß mRNA levels. IL-32ß/γ/ε isoform mRNA were detected in HT-29 cells upon exposure to TNF-α, Poly I:C (TLR3 agonist), Flagellin (TLR-5 agonist) and HIV. IL-17A significantly decreased both IL-32 ß/γ/ε mRNA and cell-associated IL-32 protein levels induced upon TNF-α and Poly I:C triggering. CONCLUSION: We document IL-32 isoforms abundant in the colon of ART-treated PWH and reveal the capacity of the Th17 hallmark cytokine IL-17A to attenuate IL-32 overexpression in a model of inflamed IEC.

Brief Report: Subclinical Carotid Artery Atherosclerosis Is Associated With Increased Expression of Peripheral Blood IL-32 Isoforms Among Women Living With HIV.

BACKGROUND: Persistent inflammation in HIV infection is associated with elevated cardiovascular disease (CVD) risk, even with viral suppression. Identification of novel surrogate biomarkers can enhance CVD risk stratification and suggest novel therapies. We investigated the potential of interleukin 32 (IL-32), a proinflammatory multi-isoform cytokine, as a biomarker for subclinical carotid artery atherosclerosis in virologically suppressed women living with HIV (WLWH). METHODS AND RESULTS: Nested within the Women's Interagency HIV Study, we conducted a cross-sectional comparison of IL-32 between 399 WLWH and 100 women without HIV, followed by a case-control study of 72 WLWH (36 carotid artery plaque cases vs. 36 age-matched controls without plaque). Plasma IL-32 protein was measured by ELISA, and mRNA of IL-32 isoforms (IL-32α, ß, γ, D, ε, and θ) was quantified by reverse transcription polymerase chain reaction from peripheral blood mononuclear cells. Plasma IL-32 protein levels were higher in WLWH compared with women without HIV (P = 0.02). Among WLWH, although plasma IL-32 levels did not differ significantly between plaque cases and controls, expression of IL-32 isoforms α, ß, and ε mRNA was significantly higher in peripheral blood mononuclear cells from cases (P = 0.01, P = 0.005, and P = 0.018, respectively). Upregulation of IL-32ß and IL-32ε among WLWH with carotid artery plaque persisted after adjustment for age, race/ethnicity, smoking, systolic blood pressure, body mass index, and history of hepatitis C virus (P = 0.04 and P = 0.045); the adjusted association for IL-32α was marginally significant (P = 0.07). CONCLUSIONS: IL-32 isoforms should be studied further as potential CVD biomarkers. This is of particular interest in WLWH by virtue of altered IL-32 levels in this population.

Upregulated IL-32 Expression And Reduced Gut Short Chain Fatty Acid Caproic Acid in People Living With HIV With Subclinical Atherosclerosis.

Despite the success of antiretroviral therapy (ART), people living with HIV (PLWH) are still at higher risk for cardiovascular diseases (CVDs) that are mediated by chronic inflammation. Identification of novel inflammatory mediators with the inherent potential to be used as CVD biomarkers and also as therapeutic targets is critically needed for better risk stratification and disease management in PLWH. Here, we investigated the expression and potential role of the multi-isoform proinflammatory cytokine IL-32 in subclinical atherosclerosis in PLWH (n=49 with subclinical atherosclerosis and n=30 without) and HIV- controls (n=25 with subclinical atherosclerosis and n=24 without). While expression of all tested IL-32 isoforms (α, ß, γ, D, ϵ, and θ) was significantly higher in peripheral blood from PLWH compared to HIV- controls, IL-32D and IL-32θ isoforms were further upregulated in HIV+ individuals with coronary artery atherosclerosis compared to their counterparts without. Upregulation of these two isoforms was associated with increased plasma levels of IL-18 and IL-1ß and downregulation of the atheroprotective protein TRAIL, which together composed a unique atherosclerotic inflammatory signature specific for PLWH compared to HIV- controls. Logistic regression analysis demonstrated that modulation of these inflammatory variables was independent of age, smoking, and statin treatment. Furthermore, our in vitro functional data linked IL-32 to macrophage activation and production of IL-18 and downregulation of TRAIL, a mechanism previously shown to be associated with impaired cholesterol metabolism and atherosclerosis. Finally, increased expression of IL-32 isoforms in PLWH with subclinical atherosclerosis was associated with altered gut microbiome (increased pathogenic bacteria; Rothia and Eggerthella species) and lower abundance of the gut metabolite short-chain fatty acid (SCFA) caproic acid, measured in fecal samples from the study participants. Importantly, caproic acid diminished the production of IL-32, IL-18, and IL-1ß in human PBMCs in response to bacterial LPS stimulation. In conclusion, our studies identified an HIV-specific atherosclerotic inflammatory signature including specific IL-32 isoforms, which is regulated by the SCFA caproic acid and that may lead to new potential therapies to prevent CVD in ART-treated PLWH.

Upregulation of IL-32 Isoforms in Virologically Suppressed HIV-Infected Individuals: Potential Role in Persistent Inflammation and Transcription From Stable HIV-1 Reservoirs.

BACKGROUND: Human IL-32 is a polyfunctional cytokine that was initially reported to inhibit HIV-1 infection. However, recent data suggest that IL-32 may enhance HIV-1 replication by activating the HIV-1 primary targets, CD4 T-cells. Indeed, IL-32 is expressed in multiple isoforms, some of which are proinflammatory, whereas others are anti-inflammatory. SETTING AND METHODS: Here, we aimed to determine the relative expression of IL-32 isoforms and to test their inflammatory nature and potential to induce HIV-1 production in latently infected cells from virologically suppressed HIV-infected individuals. IL-32 and other cytokines were quantified from plasma and supernatant of CD4 T-cells by ELISA. Transcripts of IL-32 isoforms were quantified by qRT-PCR in peripheral blood mononuclear cells. The impact of recombinant human IL-32 isoforms on HIV-1 transcription was assessed in CD4 T-cells from HIV-1cART individuals by qRT-PCR. RESULTS: All IL-32 isoforms were significantly upregulated in HIV-1cART compared to HIV individuals with IL-32ß representing the dominantly expressed isoform, mainly in T-cells and NK-cells. At the functional level, although IL-32γ induced typical proinflammatory cytokines (IL-6 and IFN-γ) in TCR-activated CD4 T-cells, IL-32α showed an anti-inflammatory profile by inducing IL-10 but not IL-6 or IFN-γ. However, IL-32ß showed a dual phenotype by inducing both pro- and anti-inflammatory cytokines. Interestingly, consistent with its highly pro-inflammatory nature, IL-32γ, but not IL-32α or IL-32ß, induced HIV-1 production in latently infected CD4 T-cells isolated from combined antiretroviral therapy-treated individuals. CONCLUSIONS: Our data report on the differential expression of IL-32 isoforms and highlight the potential role of IL-32, particularly the γ isoform, in fueling persistent inflammation and transcription of viral reservoir in HIV-1 infection.