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IFN-a-induced monocyte phenotype in chronic unsuppressed HIV infection

ABSTRACT: Abstract Objectives: HIV infection dysregulates the innate immune system and alters leukocyte gene expression. The objectives were two fold: to characterize the impact of HIV infection on peripheral monocyte gene expression and to identify the predominant factor(s) responsible for altered gene expression. Design and methods: In a cross-sectional study, CD14+ monocytes were isolated from 11 HIV seronegative controls, 22 HIV seropositive subjects with low viral loads (LVL, <= 10,000 RNA copies/ml) and 22 HIV seropositive subjects with high viral loads (HVL, > 10,000 RNA/copies/ml). Total monocyte RNA was hybridized to 55K probes on high-density microarrays to obtained detailed gene expression profiles from control, LVL and HVL subjects. As potential candidates for immune disruption, we evaluated three HIV-related peripheral factors, interferon (IFN)-a, IFN-a and lipopolysaccharide (LPS) by treating HIV seronegative CD14+ monocytes for 48h and then analyzing gene expression. Plasma collected from the HIV subjects were quantified for LPS using a Pyrogene assay and for soluble (s) CD14 by ELISA. Results: In this cross-sectional study of HIV subjects (n=44), viral loads above 10,000 RNA copies/ml were associated with an activated phenotype. Characterization of the gene expression pattern by Gene Ontology reveals an ongoing immune response to viral infection including inflammation and chemotaxis. Gene expression analysis of in vitro treated HIV seronegative monocytes with IFN-a, IFN-g or LPS demonstrated that IFN-a most accurately recapitulated the HIV HVL profile. There was no detectable LPS signature even in those HIV subjects with the highest LPS and sCD14 levels. Conclusions: Monocyte gene expression in subjects with viremia is predominantly due to IFN-a, while subjects with LVL have a non-activated phenotype. In monocytes, there was no discernible expression profile linked to LPS exposure.

ORGANISM(S): Homo sapiens

PROVIDER: GSE18464 | GEO | 2009/10/09

SECONDARY ACCESSION(S): PRJNA118189

REPOSITORIES: GEO

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Project description:Chronic immune activation is a hallmark of human immunodeficiency virus (HIV) infection and the best prognostic indicator of disease progression. Suppressing HIV viremia by antiretroviral therapy (ART) restores normal immune response and effectively prolongs life. In HIV-infected individuals who are coinfected with hepatitis C virus (HCV) the immune system is activated despite effective HIV antiretroviral therapy controlling viral load. Here we examined CD14+ monocyte gene expression by high-density microarray analysis and T cell subsets, CD4 and CD8, by flow cytometry to characterize immune activation in monoinfected HCV, monoinfected HIV and HIV/HCV coinfected subjects with undetected HIV viral load. To determine the impact of coinfection on cognition, subjects were evaluated in 7 domains for neuropsychological (NP) performance, which was summarized as global deficit scores (GDS). Gene expression analysis of CD14+ monocytes from coinfected subjects revealed an elevated type 1 interferon (IFN) response profile unique to coinfection. For both CD4 and CD8 T cells, coinfection triggered significantly increased expression of activation markers CD38 and HLA-DR. In the coinfected group, mild cognitive impairment was associated with a type 1 IFN monocyte response but not plasma lipopolysaccharide. These observations raise the possibility that cognitive impairment evident in the HIV/HCV population is associated with the IFN response detected in coinfected individuals. Monocytes isolated from healthy controls (n=17), HCV monoinfected (n=19) and HIV/HCV coinfected (n=17) were analyzed for gene expression using high-density microarrays. Whole blood was collected in Vacutainer CPT tubes (BD Biosciences) and PBMCs were enriched by centrifugation. Typically, three million CD14+ monocytes were isolated from 30 ml of whole blood using an anti-CD14 monoclonal antibody immunomagnetic - ferrous bead conjugate according to the manufacturer’s instructions (Miltenyi Biotech). Monocyte purity exceeded 97% with <1% T or B cell contamination as determined by flow cytometry. Monocyte RNA was isolated using a Qiagen RNeasy Micro Kit with an RNA integrity value exceeding 9. Complementary DNA was synthesized and labeled with biotin (iExpress iAmplify kit, Applied Microarrays) and hybridized to Codelink Whole Human Genome Bioarrays (55K probes, Applied Microarrays). Slides were scanned (Axon GenePix 4000B, Molecular Devices), analyzed (CodeLink Expression Software Kit v4.1) and microarray data were normalized with loess normalization using R and Bioconductor package. Determination of differential gene expression and multiple testing correction / false discovery rate adjustments were performed using GeneSpring GX 7.3 software package (Agilent). Microarray data was analyzed using a variety of custom data analytic techniques for gene expression profile identification as described previously. Correlations were determined by Spearman rank correlation coefficient.

Project description:Persistent and elevated systemic inflammation contributes to the increased risk of cardiovascular and metabolic diseases in persons with HIV (PWH). , but aspects of the innate and adaptive immune response to HIV and other chronic co-infections that promote the development of these comorbid conditions remain unclear. We used mass cytometry and the tracking responders expanding (T-REX) workflow to define differences in circulating cells of the innate and adaptive immune arms, which differentiate non-diabetic and prediabetic/diabetic persons with HIV (PWH) on long-term antiretroviral therapy (ART). We found a significantly higher proportion of circulating CD14+ monocytes complexed with T cells, B cells, and NK cells among PWH with diabetes. The CD3+ T cells in these complexes were predominantly CD8+ memory T cells. The CD3+ CD14+ T cell-monocyte complexes are pro-inflammatory and negatively associated with plasma interleukin-10 levels and circulating CD4+ T regulatory cells. Using transmission electron microscopy, we observed heterogeneous interactions between CD3+ T cells and monocytes within the complexes, including formation of immune synapses and phagocytosis. 10x Chromium single-cell sequencing RNA transcriptomic analysis of CD3+ CD14+ doublets showed differential expression of genes involved in T cell activation and the adaptive immune response compared to monocytes that were not complexed with other immune cells. Notably, there were significantly more copies of HIV RNA per cell in the CD3+ CD14+ T cell-monocyte complexes compared to CD14+ monocytes or CD3+ CD4+ T cells alone, and HIV virions were observed in both T cells and monocytes within the doublets?. Metabolically, CD3+ CD14+ T cell-monocyte complexes had high glucose-dependence with minimal mitochondrial dependence. Taken together, CD3+ CD14+ T cell-monocyte pairs are functional and physically interacting immune cell complexes which might be enriched with HIV. These complexesare increased among individuals with diabetes and may be a target for future HIV cure studies and diseases of aging.

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IFN-a-induced monocyte phenotype in chronic unsuppressed HIV infection

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