Cannabis use is frequent in HIV-infected individuals for its appetite stimulation and anti-inflammatory effects. To identify the underlying molecular mechanisms associated with these effects, we simultaneously profiled micro-RNA (miRNA) and mRNA expression in the colon of chronically simian immunodeficiency virus (SIV)-infected rhesus macaques administered either vehicle (VEH/SIV; n = 9) or Δ 9 -tetrahydrocannabinol (Δ 9 -THC; THC/SIV; n = 8). Pro-inflammatory miR-130a, miR-222, and miR-29b, lipopolysaccharide-responsive miR-146b-5p and SIV-induced miR-190b were significantly upregulated in VEH/SIV rhesus macaques. Compared to VEH/SIV rhesus macaques, 10 miRNAs were significantly upregulated in THC/SIV rhesus macaques, among which miR-204 was confirmed to directly target MMP8, an extracellular matrix-degrading collagenase that was significantly downregulated in THC/SIV rhesus macaques. Moreover, THC/SIV rhesus macaques failed to upregulate pro-inflammatory miR-21, miR-141 and miR-222, and alpha/beta-defensins, suggesting attenuated intestinal inflammation. Further, THC/SIV rhesus macaques showed higher expression of tight junction proteins (occludin, claudin-3), anti-inflammatory MUC13 , keratin-8 (stress protection), PROM1 (epithelial proliferation), and anti-HIV CCL5 . Gomori one-step trichrome staining detected significant collagen deposition (fibrosis) in the paracortex and B cell follicular zones of axillary lymph nodes from all VEH/SIV but not in THC/SIV rhesus macaques, thus demonstrating the ability of Δ 9 -THC to prevent lymph node fibrosis, a serious irreversible consequence of HIV induced chronic inflammation. Furthermore, using flow cytometry, we showed that Δ 9 -THC suppressed intestinal T cell proliferation/activation (Ki67/HLA-DR) and PD-1 expression and increased the percentages of anti-inflammatory CD163 + macrophages. Finally, while Δ 9 -THC did not affect the levels of CD4 + T cells, it significantly reduced absolute CD8 + T cell numbers in peripheral blood at 14 and 150 days post-SIV infection. These translational findings strongly support a role for differential miRNA/gene induction and T cell activation in Δ 9 -THC-mediated suppression of intestinal inflammation in HIV/SIV and potentially other chronic inflammatory diseases of the intestine.

Intestinal epithelial barrier dysfunction is a well-known sequela of HIV/SIV infection that persists despite antiretroviral therapy. Although inflammation is a triggering factor, the underlying molecular mechanisms remain unknown. Emerging evidence suggests that epithelial barrier function is epigenetically regulated by inflammation-induced microRNAs (miRNAs). Accordingly, we profiled and characterized miRNA/mRNA expression exclusively in colonic epithelium and identified 46 differentially expressed miRNAs (20 upregulated and 26 downregulated) in chronically SIV-infected rhesus macaques (). We bioinformatically crossed the predicted miRNA targets to transcriptomic data and characterized miR-130a and miR-212 as both were predicted to interact with critical epithelial barrier-associated genes. Next, we characterized peroxisome proliferator-activated receptor γ (PPARγ) and occludin (OCLN), predicted targets of miR-130a and miR-212, respectively, as their downregulation has been strongly linked to epithelial barrier disruption and dysbiosis. Immunofluorescence, luciferase reporter, and overexpression studies confirmed the ability of miR-130a and miR-212 to decrease protein expression of PPARγ and OCLN, respectively, and reduce transepithelial electrical resistance. Because Δ-9-tetrahydrocannabinol exerted protective effects in the intestine in our previous studies, we successfully used it to reverse miR-130a- and miR-212-mediated reduction in transepithelial electrical resistance. Finally, ex vivo Δ-9-tetrahydrocannabinol treatment of colon tissue from chronically SIV-infected rhesus macaques significantly increased PPARγ expression. Our findings suggest that dysregulated miR-130a and miR-212 expression in colonic epithelium during chronic HIV/SIV infection can facilitate epithelial barrier disruption by downregulating OCLN and PPARγ expression. Most importantly, our results highlight the beneficial effects of cannabinoids on epithelial barrier function in not just HIV/SIV but potentially other chronic intestinal inflammatory diseases.

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Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in children in low- to middle-income countries. Previous studies identified heat-stable enterotoxin (ST)-producing ETEC as a prevalent diarrheal pathogen in children younger than 5 years. While many studies have evaluated the interaction of ETEC heat-labile enterotoxin (LT) with host epithelium and immunity, few investigations have attempted similar studies with ST. To further understand ST pathogenesis, we examined the impact of ST on cGMP localization, epithelial cell cytokine production, and antibody development following immunization. In addition to robust intracellular cGMP in T84 cells in the presence of phosphodiesterase inhibitors (PDEis) that prevent the breakdown of cyclic nucleotides, we found that prolonged ST intoxication induced extracellular cGMP accumulation in the presence or absence of PDEis. Further, ST intoxication induced luminal cGMP in vivo in mice, suggesting that secreted cGMP may have other cellular functions. Using transcriptome sequencing (RNA-seq) and quantitative PCR (qPCR), we demonstrated that ST intoxication, or treatment with the clinically used ST mimic linaclotide, altered inflammatory cytokine gene expression, including the interleukin 1 (IL-1) family member IL-33, which could also be induced by cell-permeative 8-Br-cGMP. Finally, when present during immunization, ST suppressed induction of antibodies to specific antigens. In conclusion, our studies indicate that ST modulates epithelial cell physiology and the interplay between the epithelial and immune compartments.