In HIV-infected adults with a history of sustained viral suppression on ART, we measured levels of HIV RNA, DNA, RNA/DNA ratios, CD4+T-cells, and T-cell activation in blood and four different regions of gut. HIV DNA levels per CD4+T-cell were on average 5–10 fold higher in the gut compared to the peripheral blood, in agreement with Chun, et al [6
]. If the gut CD4+T-cells have on average five times more HIV DNA than CD4+T-cells in the blood (and possibly the rest of the body), and the gut contains 50–80% of total body CD4+T-cells, then the gut harbors 83% to >95% of all infected cells in the body. Based on the average (across sites and participants) HIV DNA level of 20,000 DNA copies per 106
CD4+T-cells, an estimate of 1.2×1011
total body CD4+T-cells, and an estimate of 50% of total body CD4+T-cells residing in the gut, the gut contains 1.2×109
infected CD4+T-cells after a median of 7 years of suppressive ART. If 1 in 100 infected cells carries replication-competent HIV, the gut alone contains 1.2×107
latently-infected HIV genomes. This estimate exceeds by an order of magnitude earlier estimates of the latent reservoir size in PBMC and central lymphoid tissues [32
It is unclear why the gut harbors such a disproportionate concentration of infected CD4+T-cells. Possible explanations include greater rates of initial infection (especially in the rectum, which may be a site of transmission), differences in the content of memory CD4+ T cell subpopulations (such as transitional memory cells) that may harbor more HIV DNA [34
], higher rates of replication of integrated HIV DNA by cell division, increased establishment of latent infection, reduced reactivation from latency, slower clearance, trafficking from the blood, or ongoing replication (the latter was suggested by Chun et al [6
While ISH for HIV RNA was negative in all gut sites, UsRNA was detectable in the majority of gut samples using qRT-PCR. ISH can detect as few as 2–5 genomic HIV equivalents per cell but has a detection limit of 104
copies/g tissue for dispersed HIV RNA, whereas PCR provides very sensitive detection of HIV RNA pooled from many cells but cannot discriminate RNA produced from few or many cells. By qRT-PCR, UsRNA levels per CD4+T-cell were higher in all four gut sites compared to blood. It is unclear whether this RNA represents reactivation of latently-infected cells, stable chronically-infected cells, or cells newly-infected as a result of ongoing replication. Based on the absence of HIV RNA+ cells by ISH, we suspect that productively-infected cells in the gut, if present, must be very infrequent or exhibit very attenuated production, and that the HIV RNA detected by qRT-PCR represents modest viral transcription distributed across many HIV DNA+ cells. This assumption is in agreement with the observation that MsRNAs, which may be found in latently-infected cells but are expressed at high level in productive infection [13
], were rarely detected in the gut.
The RNA/DNA ratio peaked in the ileum, where the median ratio tended to be higher than that of the PBMC, suggesting that this site may have a greater ratio of productive to latent infection and should be sampled in studies aimed at detecting ongoing replication. In contrast, in the large bowel, the median HIV RNA/DNA ratio tended to be lower than in the PBMC, suggesting that more of these cells behave as if they are “latently” infected. However, since most gut lymphocytes display markers of T-cell activation, “latent” infection of these cells may differ from the classic latent infection in the blood, which was originally described in resting CD4+T-cells. Differences between gut sites could reflect differences in T-cell activation, memory CD4+T-cell subsets, or the proportion of lymphocytes from lymphoid aggregates.
The infrequent detection of MsRNA and the trend towards lower HIV RNA/DNA ratio in most gut sites suggest lower levels of HIV transcription. Given the high degree of T-cell activation in the gut, it is very surprising that gut cells have such low levels of HIV transcription, suggesting that they are hyporesponsive to activating stimuli, or that T-cell activation has different consequences (or activation markers have different meanings) in the gut compared to the blood. Gut lymphocytes may have a reduced ability to respond to antigens and may resemble more immunotolerant or “anergic” T-cells. Previous reports have shown that CD4+T-cell tolerance and anergy can be caused by epigenetic modification [35
]. Given that epigenetic modification of the LTR has also been implicated as a feature of latent infection with HIV [38
], it is tempting to hypothesize that the unique environment of the gut favors both induction of CD4+T-cell tolerance and HIV latency through epigenetic modification. It is not clear whether these cells would respond to therapies that may reduce latently-infected cells in the blood.
Whereas HIV DNA levels in PBMC tended to correlate positively with T-cell activation, in the gut, we found a surprising trend towards a negative correlation between HIV DNA and T-cell activation. Immune activation could have divergent effects on HIV infection. Systemic immune activation may increase the susceptibility of CD4+T-cells to infection, cause replication of proviral DNA by cell division, or serve as a marker for spread of infection, thus explaining the positive correlation between activation and HIV DNA seen in the blood. On the other hand, activation of HIV-specific T-cells can lead to death of virally-infected cells, and HIV-nonspecific activation can reduce the number of susceptible target cells (by apoptosis) or lead to reactivation and clearance of latently infected cells, thus explaining the negative correlation seen in the gut. If verified, the opposing directions for the correlations seen in the gut and the peripheral blood further suggest that activation may have different consequences for HIV persistence in these two sites.
Potential limitations of the study should be noted. First, the number of participants was relatively small, thus limiting generalizability and the power to detect small differences. Second, even with multiple biopsies, there remains the possibility of insufficient sampling. Previous studies have shown that HIV DNA and RNA can be reproducibly quantified from a single endoscopic biopsy [5
], and we pooled 6 to 9 biopsies from each site. While in situ studies confirmed the presence of lymphoid aggregates (in 50% of ileal biopsies), additional sampling error may have been introduced by the tissue digestion, which resulted in some cell loss and death (AARD staining showed that 75–80% of gut cells were viable). Third, the normalization per CD4 cell assumes that all of the HIV is in CD4 cells. Finally, the PCR detection methods, while sensitive, do not overcome confounding effects of sampling that occur when target nucleic acids are present at low copy numbers, so that Poissonian effects have a greater influence on results.
Nevertheless, the findings here confirm and extend the important role for GALT as a reservoir for HIV in patients on suppressive ART. Additional studies are needed to better define and distinguish the modes of viral persistence in blood and different regions of gut, and to investigate whether site-specific differences result in different responses to therapies designed to “re-activate” HIV from latently-infected cells.