Front Immunol. 2026 May 26;17:1769388. doi: 10.3389/fimmu.2026.1769388. eCollection 2026.
ABSTRACT
BACKGROUND: The crosstalk between intestinal epithelial cells (IEC) and CD4+ T-cells is essential for the maintenance of mucosal homeostasis, but its role in governing HIV-1 latency versus reactivation in gut-homing/resident T-cells remains poorly documented. We previously demonstrated that the Th17-lineage cytokine IL-17A transcriptionally reprograms IEC for promoting viral outgrowth in CD4+ T-cells of antiretroviral therapy (ART)-treated people with HIV-1 (PWH). These effects coincided with the downregulation of IL-32, a cytokine with documented antiviral properties and identified as a marker for HIV-1 disease progression and cardiovascular disease risk. Here, we aimed to identify modulators of IL-32 expression and to define the impact of IEC expressing IL-32 on HIV-1 outgrowth in neighboring CD4+ T-cells carrying viral reservoirs.
METHODS: HT-29 IEC were activated with TNF and/or IL-22, IL-26, all-trans retinoic acid (ATRA) and rosiglitazone (RGZ). CRISPR/Cas9 gene editing was used to knockout (KO) IL32 in IEC, with efficiency/off-target effect assessments performed using TIDE and whole-genome RNA-Sequencing. IL-32β/γ/ϵ mRNA/protein were quantified by RT-PCR/ELISA. An IEC-based viral outgrowth assay (VOA) was performed with CD4+ T-cells of ART-treated PWH. Soluble/intracellular HIV-p24 levels were measured by ELISA/flow cytometry.
RESULTS: In combination with TNF, IL-22 upregulated IL-32β/ϵ, RGZ increased IL-32β, ATRA decreased IL-32β/γ/ϵ, while IL-26 had no impact in IEC. HIV-1 outgrowth in IEC:T-cell co-cultures was not affected by IL-22/RGZ/ATRA-mediated changes in IL-32 expression. The analysis of differentially expressed genes in control versus IL32KO IEC revealed minor differences in transcriptional profiles before/after exposure to TNF, while the IL-32 mRNA/protein expression was induced by TNF in control but not IL32KO IEC. Finally, TNF-activated control versus IL32KO IEC supported with similar efficacy HIV-1 outgrowth in CD4+ T-cells of PWH.
CONCLUSIONS: We identified IL-22, ATRA and RGZ as novel regulators of IL-32 expression in TNF-primed IEC and demonstrated that pharmacological and genetic modulation of IL-32 expression in IEC has no major impact on HIV-1 outgrowth from co-cultured CD4+ T-cells of ART-treated PWH carrying viral reservoirs. By excluding its role in modulating HIV reservoir latency/reactivation at intestinal barrier level, these results support the idea of testing the potential use of IL-32 as a novel therapeutic target to reduce comorbidities in ART-treated PWH.
PMID:42273706 | PMC:PMC13247358 | DOI:10.3389/fimmu.2026.1769388