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

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

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

Clinical characteristics of the cohort.

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

A20 deletion enhances the cytotoxic effects of IFNα and IFNγ.

<p>(<b>A</b>) Quantification of cell viability by CellTiter Glo 3D assay in organoid cultures after stimulation with three doses of rIFNγ, rIFNα, or rIL-17A for 24 hours. Significance of Cre+ or Cre- was assessed separately by ANOVA. Stars above data points represent statistical significance of cytokine treatment relative to the media control within either the Cre+ (red) or Cre- (black) line after post-hoc comparisons were made by t-test, correcting for multiple comparisons by Scheffe method. Stars above brackets indicate a significant difference between A20-sufficient (Cre-, black) and A20-deficient (Cre+, red) culture within the indicated media condition by t-test. *P<0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001. (<b>B</b>) Representative confocal images of propidium iodide (PI)-stained organoids treated for 24 hours with 4OHT followed by 10 ng/ml of indicated cytokine for 24 hours. Two organoids representing the range of PI staining are shown for each condition.</p

A20 modulation of epithelial function during HIV infection and treatment.

<p>Model demonstrating proposed relationship of A20 and relevant cytokines (Type I IFNs, IFNγ, and IL-17A) in the context of untreated (viremic) (left) and ART-treated (right) HIV disease.</p

Deletion of A20 enhances the proinflammatory effects of IL-17A.

<p>Gene expression of (<b>A</b>) <i>Lcn2</i>, (<b>B</b>) <i>Cxcl1</i>, (<b>C</b>) <i>Cxcl2</i>, (<b>D</b>) <i>Muc1</i> by qPCR of organoid cultures after 24 hours of 4OHT treatment and/or a 12 hour stimulation with recombinant IL-17A. Cre+ or Cre- conditions were tested separately by ANOVA. Pairwise comparisons were completed post-hoc using t-tests and correcting for multiple comparisons by the Scheffe method (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006806#ppat.1006806.s006" target="_blank">S2 Table</a>). Stars above each column indicate statistical significance relative to the media condition of the same organoid line (Cre⁺ or Cre^-) by a t-test. Bars with stars indicate statistically significant differences between the two indicated conditions. **P≤0.01, ***P≤0.001, ****P≤0.0001.</p