Background
People with HIV (PWH) experience compromised intestinal barrier integrity (gut leakage), which persists despite viral suppression by antiretroviral therapy (ART) and contributes to chronic inflammation. The mechanisms behind this ongoing disruption remain unclear and are likely multifactorial. We tested a novel hypothesis that living with HIV impairs the intestinal barrier’s ability to resist injurious agents, making it more susceptible to leakage when exposed to disruptors such as inflammatory cytokines, which are commonly elevated in PWH.
Methods
We generated 3D intestinal apical out organoids from the colon (colonoids) and ileum (enteroids) of 13 PWH on ART (<40 HIV copies/ml plasma) and 19 controls without HIV. Organoids were treated with or without a 3 ng/mL IFNγ/TNFα cocktail for 3 hours. Organoid permeability was assessed using the 4kDa FITC-dextran diffusion assay, where higher fluorescence indicates increased permeability. RNA sequencing of baseline (pre-treatment) organoids was performed to identify molecular pathways involved in intestinal leakage.
Results
Colonoids, but not enteroids, from PWH on ART showed a non-significant trend toward higher baseline permeability compared to controls (p=0.077). However, both colonoids and enteroids from PWH exhibited significantly lower resilience to cytokine stimulation, with increased dextran diffusion compared to controls (p=0.041 for colonoids (Fig), p=0.006 for enteroids). Pathway analysis at baseline revealed disrupted epithelial integrity pathways in PWH organoids, including epithelial-mesenchymal transition (FDR=0.016) and DNA repair (FDR=0.0003). Downregulation of key genes within these protective pathways, such as P3H1 and POLD1, significantly correlated with increased permeability (P3H1: p=0.003, rho=-0.53; POLD1: p=0.02, rho=-0.47). PWH organoids also showed upregulation of pathways detrimental to intestinal barrier, such as oxidative stress-related pathways, including oxidative phosphorylation (FDR=0.03), reactive oxygen species pathway (FDR=0.00002), and fatty acid metabolism (FDR=0.0006).
Conclusions
Even with comparable baseline intestinal permeability, intestines of PWH on ART exhibit less resilience to disruption by inflammatory cytokines than those of controls, likely contributing to the ongoing intestinal dysfunction in PWH. Disruption of specific intestinal molecular pathways may contribute to this phenomenon, highlighting potential targets for novel strategies to prevent inflammation-associated comorbidities in PWH.
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