miR-422a is modulated by IFN-α treatment.

<p>(A) Expression of miR-422a in PBMC <i>in vivo</i> before, during and after IFN-α/RBV treatment (labeled as “Rx”). Error bars represent SEM. P-value was obtained using a paired t-test and FDR is reported. (B) Effects of IFN-α on the expression of miR-422a in CD4+ T cells <i>in vitro</i>; cells were plated at a million cells per well and treated with either 5 U/ml of IFN-α or media as a negative control. (C) Expression of MLH1 in PBMC <i>in vivo</i> before, during, and after IFN-α/RBV treatment. (D) Expression of TP53 in PBMC <i>in vivo</i> before, during, and after IFN-α/RBV treatment. Error bars represent SEM. P-values were obtained using paired Wilcoxon tests. (E) Correlation between TP53 fold induction and MLH1 fold induction in PBMC <i>in vivo</i> during IFN-α/RBV treatment. P-value was obtained using Spearman’s rank test.</p

Correlations between miRNA and HIV-1 viral load.

<p>Correlations between miRNA and HIV-1 viral load.</p

Global network of putative regulatory interactions between miRNAs and anti-HIV-1 restriction factor mRNAs.

<p>miRNA and mRNA expression data were measured in longitudinal PBMC samples from IFN-α/RBV-treated patients enrolled in the SHCS. Two variables were used to generate the network: 1) inverse expression relationships between a given miRNA-mRNA pair (represented as a dashed line drawn between a miRNA – mRNA pair), and 2) Significant sequence homology between a given miRNA seed region and a restriction factor 3′ UTR (word size 4, alignment length > = 5, e-value<1.0). Significant sequence homology is represented by a solid line. miRNAs are listed in blue; restriction factors are listed in pink.</p

Heat map representing effects of IFN-α/RBV treatment on the expression of individual miRNAs.

<p>45 miRNAs were significantly modulated by IFN-α/RBV <i>in vivo</i>, based on a paired t test and an uncorrected significance cutoff of p<0.05. Relative copy numbers of each miRNA are reported. Blue color indicates ≤−3 SD from the mean, red indicates ≥3 SD from the mean, and white represents the mean. Asterisks indicate previously established association with HIV-1.</p

Visualization of miRNA and mRNA regulatory networks.

<p>(A) Plot of global miRNA and anti-HIV-1 restriction factor responses to IFN-α/RBV treatment. Numbers in blue and red represent tallies of microRNAs and restriction factor mRNAs in each quadrant, respectively. P-values were obtained using Fisher’s exact tests. (B) Integrative bioinformatic analyses of our miRNA data, restriction factor, MLH1 and TP53 mRNA profiles within the context of gene regulatory networks. Ingenuity Pathway Analysis (IPA) software was implemented to create the network map.</p

A20 upregulation during treated HIV disease is associated with intestinal epithelial cell recovery and function

<div><p>Untreated Human Immunodeficiency Virus (HIV) infection is characterized by intestinal epithelial barrier dysfunction and chronic inflammation, related features that are attenuated to variable degrees by suppressive antiretroviral therapy (ART). Specific mediators of intestinal epithelial cell (IEC) dysfunction and restoration during HIV disease and treatment have yet to be identified. We studied IECs isolated from intestinal biopsies by RNAseq and found that mRNA levels for the ubiquitin-modifying enzyme, A20, are upregulated in ART-treated individuals and are positively correlated with markers of epithelial function (e.g., <i>CTNNB</i>, <i>CLDN4</i>, and <i>TJP1</i>). In a murine intestinal organoid model, A20 expression was suppressed by interferon-alpha (IFNα), which is highly expressed during HIV viremia and induces IFN-mediated signaling. Notably, A20 deletion rendered intestinal organoids more susceptible to cell death and inhibition of barrier-related genes mediated by interferon-gamma (IFNγ), a cytokine also present at elevated levels during untreated infection. Furthermore, A20 specifically restricted expression of IL-17A-induced inflammatory genes in organoids. Finally, ART-suppressed chronically infected individuals treated with pegylated IFNα2a for five weeks demonstrated reduced expression of A20 in peripheral blood mononuclear cells. Our results are thus consistent with a model in which enhanced type I interferons suppress A20 levels, leading to IFNγ-mediated dysfunction. As such, variation in A20 expression during the course of HIV infection could underlie both the development of epithelial dysfunction before the initiation of ART and the recovery of intestinal epithelial integrity thereafter.</p><p>Trial registration</p><p>ClinicalTrials.gov <a href="https://clinicaltrials.gov/ct2/show/Clinical Trial NCT00594880" target="_blank">Clinical Trial NCT00594880</a></p></div

A20 upregulation during treated HIV disease is associated with intestinal epithelial cell recovery and function - Fig 2

<p><b>Intestinal epithelial A20 is downregulated after exposure to IFN</b>α (<b>A</b>) Protein levels of A20, measured by western blot, in murine organoids after treatment with indicated dose of IFNα for 48 hours. (<b>B</b>) Quantification of A20 levels normalized to GAPDH in three independent experiments conducted as in (a). Statistical significance was assessed using a t-test. * P<0.05, **P≤0.01, ***P≤0.001.</p

Five week pegylated-IFNα2a immunotherapy of ART-suppressed HIV-infected individuals leads to reduction in A20 expression.

<p>(<b>A</b>) Schematic showing duration and frequency of pegylated-IFNα2a administration in participants. Transcript levels of (<b>B</b>) IFN-stimulated gene ISG15 and (<b>C</b>) A20 in PBMCs at baseline and after five weeks of IFNα treatment as assessed by qPCR. Statistical significance was determined by paired t-test. * P<0.05, ****P≤0.0001.</p

IEC expression pattern from ART-treated participants is consistent with upregulated A20 (<i>TNFAIP3</i>) levels.

<p>(<b>A</b>) Pairwise comparison of IEC gene expression between viremic and ART-treated participants by RNAseq as analyzed by DESeq2 workflow. Each dot represents a sequenced gene. The right side of the plot indicates relative enrichment in ART-treated relative to viremic individuals, whereas the left side indicates the inverse, enrichment in viremics relative to ART-treated participants. Purple signifies greater than two-fold change expression relative to the comparator in either direction. Red indicates a significant p-value (<0.05) after false discovery rate correction. Green indicates fulfillment of both of these criteria. Genes of particular interest are highlighted in bold. (<b>B</b>) Spearman correlation of A20 transcript levels and <i>NFKBIA</i> expression, as measured by RNAseq, in ART-treated participants (n = 19). Spearman correlation of A20 mRNA or <i>NFKBIA</i> gene expression, as indicated on the x-axis, to transcript levels of (<b>C</b>) proliferation-associated β-catenin, <i>CTNNB1</i>, or the tight junction genes (<b>D</b>) <i>CLDN4</i> and (<b>E</b>) <i>TJP1</i>.</p

Clinical characteristics of the cohort.

<p>Clinical characteristics of the cohort.</p