Mechanistic differences underlying HIV latency in the gut and blood contribute to differential responses to latency-reversing agents

Abstract

Objective: While latently HIV-infected cells have been described in the blood, it is unclear whether a similar inducible reservoir exists in the gut, where most HIV-infected cells reside. Tissue-specific environments may contribute to differences in the mechanisms that govern latent HIV infection and amenability to reactivation. We sought to determine whether HIV-infected cells from the blood and gut differ in their responses to T-cell activation and mechanistically distinct latency reversing agents (LRAs).

Design: Cross sectional study using samples from HIV-infected individuals (n = 11).

Methods: Matched peripheral blood mononuclear cells (PBMC) and dissociated total cells from rectum ± ileum were treated ex vivo for 24 h with anti-CD3/CD28 or LRAs in the presence of antiretrovirals. HIV DNA and 'read-through', initiated, 5' elongated, completed, and multiply-spliced HIV transcripts were quantified using droplet digital PCR.

Results: T-cell activation increased levels of all HIV transcripts in PBMC and gut cells, and was the only treatment that increased multiply-spliced HIV RNA. Disulfiram increased initiated HIV transcripts in PBMC but not gut cells, while ingenol mebutate increased HIV transcription more in gut cells. Romidepsin increased HIV transcription in PBMC and gut cells, but the increase in transcription initiation was greater in PBMC.

Conclusion: The gut harbors HIV-infected cells in a latent-like state that can be reversed by T-cell activation involving CD3/CD28 signaling. Histone deacetylation and protein kinase B may contribute less to HIV transcriptional initiation in the gut, whereas protein kinase C may contribute more. New LRAs or combinations are needed to induce multiply-spliced HIV and should be tested on both blood and gut.

Figure 1.. The HIV genome and the targets for transcription profiling assays.

This schematic shows the genetic organization of proviral HIV DNA and the HIV ‘transcription profiling’ assays targeting specific RNA sequence regions that provide insight into blocks to transcription.

Figure 2.. Effect of activation and LRAs on PBMC from ART-suppressed individuals.

PBMC (5×10⁶ cells/condition; n=11 participants) were treated for 24h with DMSO alone or (A) disulfiram (1μM); (B) HDAC inhibitors, panobinostat (30nM) and romidepsin (40nM); (C) chaetocin (50nM); (D) JQ1 (2μM); (E) ingenol derivatives, ingenol 3,20 dibenzoate (20nM) and ingenol mebutate (12nM); or (F) αCD3/αCD28 antibodies. HIV RNA levels were measured using RT-ddPCR^[9] and normalized to cellular RNA input (copies/μg RNA).

Figure 3.. Effect of activation and LRAs on PBMC vs. total rectal cells.

Matched PBMC and rectal cells were treated with DMSO or (A) αCD3/αCD28, n=9; (B) disulfiram, n=7; (C) romidepsin, n=9; and (D) ingenol mebutate, n=9. HIV RNA levels were normalized to copies/μg RNA. * denotes P<0.05 and ** denotes P<0.01.

Figure 4.. Effect of activation and LRAs on PBMC vs. total ileal cells.

Matched PBMC and ileal cells were treated with DMSO or (A) αCD3/αCD28, n=7; (B) disulfiram, n=3; (C) romidepsin, n=7; and (D) ingenol mebutate, n=6. HIV RNA levels were normalized to copies/μg RNA. * denotes P<0.05.

Figure 5.. Fold change in HIV transcripts relative to DMSO in PBMC vs. total rectal cells.

The fold changes in HIV transcripts (relative to DMSO) are plotted for matched PBMC and total rectal cells treated with (A) αCD3/αCD28, n=9; (B) disulfiram, n=7; (C) romidepsin, n=9; and (D) ingenol mebutate, n=9. * denotes P<0.05. Colors denote individual participants. Dotted line represents baseline (DMSO; fold change =1).