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

Avantika S Chitre, Michael G Kattah, Yenny Y Rosli, Montha Pao, Monika Deswal, Steven G Deeks, Peter W Hunt, Mohamed Abdel-Mohsen, Luis J Montaner, Charles C Kim, Averil Ma, Ma Somsouk, Joseph M McCune, Avantika S Chitre, Michael G Kattah, Yenny Y Rosli, Montha Pao, Monika Deswal, Steven G Deeks, Peter W Hunt, Mohamed Abdel-Mohsen, Luis J Montaner, Charles C Kim, Averil Ma, Ma Somsouk, Joseph M McCune

Abstract

ClinicalTrials.gov Clinical Trial NCT00594880.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig 1. IEC expression pattern from ART-treated…
Fig 1. IEC expression pattern from ART-treated participants is consistent with upregulated A20 (TNFAIP3) levels.
(A) 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) Spearman correlation of A20 transcript levels and NFKBIA expression, as measured by RNAseq, in ART-treated participants (n = 19). Spearman correlation of A20 mRNA or NFKBIA gene expression, as indicated on the x-axis, to transcript levels of (C) proliferation-associated β-catenin, CTNNB1, or the tight junction genes (D) CLDN4 and (E) TJP1.
Fig 2
Fig 2
Intestinal epithelial A20 is downregulated after exposure to IFNα (A) Protein levels of A20, measured by western blot, in murine organoids after treatment with indicated dose of IFNα for 48 hours. (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.
Fig 3. A20 deletion is associated with…
Fig 3. A20 deletion is associated with enhanced IFNγ-mediated inhibition of tight junction and mucin gene expression.
(A) Western blot for A20 protein levels in A20FL/FL villin-ERCre+ or Cre- organoids after treatment with 4-hydroxytamoxifen (4OHT) for 24 hours. Gene expression by qPCR of (B) the proinflammatory chemokines Cxcl1 and Cxcl2, (C) the mucins Muc2, Muc4, Muc13, and Muc1, (D) the tight junction proteins Tjp1, Ocln, and Cldn4, and (E) the antimicrobial peptide Defb1 after stimulation with IFNγ for 12 hours. Statistical significance within Cre+ and Cre- conditions was first tested by ANOVA and specific pairwise comparisons were made using t-tests corrected for multiple comparisons by the Scheffe method (see also S1 Table). Stars above each column indicate statistical significance relative to the media condition. Bars with stars indicate statistically significant differences between the two indicated conditions. * P<0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.
Fig 4. Deletion of A20 enhances the…
Fig 4. Deletion of A20 enhances the proinflammatory effects of IL-17A.
Gene expression of (A) Lcn2, (B) Cxcl1, (C) Cxcl2, (D) Muc1 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 S2 Table). 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.
Fig 5. A20 deletion enhances the cytotoxic…
Fig 5. A20 deletion enhances the cytotoxic effects of IFNα and IFNγ.
(A) 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) 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.
Fig 6. Five week pegylated-IFNα2a immunotherapy of…
Fig 6. Five week pegylated-IFNα2a immunotherapy of ART-suppressed HIV-infected individuals leads to reduction in A20 expression.
(A) Schematic showing duration and frequency of pegylated-IFNα2a administration in participants. Transcript levels of (B) IFN-stimulated gene ISG15 and (C) 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.
Fig 7. A20 modulation of epithelial function…
Fig 7. A20 modulation of epithelial function during HIV infection and treatment.
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.

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