We undertook the present study to explore putative virological and immunological factors associated with selective depletion of mucosal gastrointestinal CD4+ T cells during acute and early HIV-1 infection.
We demonstrate conclusively here that the HIV-1 viral burden is consistently greater in the GI CD4+ T-cell population than in the PB. This was demonstrated for both viral DNA and HIV-1 RNA by using PCR-based methods. By combining in situ hybridization and immunohistochemistry, we found both activated and nonactivated cells expressing HIV-1 mRNA at (estimated) days 18 to 19 postinfection. Furthermore, we observed that high levels of virus in the GI tract are associated with activation and proliferation of mucosal lymphocytes and a rather dramatic increase in cytotoxic cells, both CD8+ and CD8−. Based on these observations, we propose that mucosal CD4+ T-cell depletion during AEI is multifactorial and is due to, though not limited to, a combination of direct viral infection, activation induced cell death and host-derived cytotoxic cellular response.
Our data are consistent with the SIV-macaque experiments described above (16
) in that, during AEI with HIV-1, GI CD4+
lymphocytes are preferentially infected and have a greater viral burden than PB CD4+
lymphocytes. The results presented here show higher levels of both HIV-1 viral DNA and RNA content within intestinal derived CD4+
T cells than within PB CD4+
T cells. Although our studies represent snapshots of post-peak viremia events, the fact that up to 102-fold-greater HIV-1 RNA levels were observed in GI CD4+
T cells than in PB CD4+
T cells supports the concept that the GI tract preferentially supports early events during acute infection. This is likely to be due to the presence of densely clustered memory CD4+
T cells expressing high levels of CCR5 in the GI tract (21
). Finally, we believe that the high correlation between cell-associated HIV-1 RNA and plasma RNA both validates our assay and is consistent with established models of HIV-1 viral dynamics confirming that the plasma viral load is a reflection of the numbers of infected cells (20
Although prior studies have documented the presence of virus in the GI tract during acute (3
) and chronic (5
) stages of HIV-1 infection, viral quantification was performed here in separated CD4+
T cells and not biopsy tissue to avoid confounding variables such as quantification of trapped virus in mucosal inductive sites (4
) and to have the same denominator in comparing the GI tract and PB.
We determined the phenotype of infected mucosal cells in two patients who were biopsied earliest in order to compare our findings to what has been observed in the SIV model. We observed that there is no clear difference in virus production between “activated” and “nonactivated cells” at (estimated) days 18 to 19 postinfection, and this is consistent with the notion put forth by Li et al. of a switch from nonactivated to activated cells accounting for viral production as acute infection evolves (16
). It should be noted that in both the present study and the study by Li et al. the assessment of activated versus nonactivated cells was done on formalin-fixed tissues (and not fresh-frozen tissue, which could potentially produce different results).
HIV-1 infection is known to trigger a brisk innate and adaptive cytotoxic immune response in the infected host (14
). We document a robust cytotoxic response in the GI tract during AEI, visible as early as day 18 postinfection. As part of this response, we observed significantly elevated levels of perforin expression in the GI tract during AEI, which is evident in both the immune inductive and the effector sites. These findings differ from previous reports describing a deficiency of perforin production in mucosal CD8+
T cells (1
). The precise nature of these mucosal cytotoxic cells is unclear (we did not determine whether the cytotoxic cells were HIV-1 specific or not) and will be characterized in subsequent studies. However, we do believe that HIV-1-specific cells are likely to be contained within the overall cytotoxic cellular pool. Importantly, we also identified CD8-negative cytotoxic cells in the GI mucosa during acute HIV-1 infection. Although not yet characterized, these cells are likely to be innate immune cells, such as natural killer cells. The role of the cytotoxic T-cell response in the pathogenesis of HIV-1 infection and the observed mucosal CD4+
T-cell depletion need to be further defined.
Immune activation is a characteristic feature of untreated HIV-1 infection and is associated with a progressive depletion of CD4+
T cells (9
). Here we demonstrate that during AEI there is a significant increase in activated memory cells (CD45RO+
) within the GI tract. While not in itself surprising, this observation has two potential consequences. First, the majority of mucosal cells are terminally differentiated effector cells(23
) and are much more likely to apoptose when activated than are PB-derived naive cells. Therefore, given that about two-thirds of mucosal CD4+
T cells express markers of activation (CD38) during AEI (Fig. ), it is likely that activation-induced cell death plays an important role in GI CD4+
T-cell depletion during acute infection. Second, activated CD4+
T cells represent the “preferred cellular targets” for HIV-1(24
). Thus, HIV-1 infection generates an expanding population of cellular targets within the GI tract by triggering activation of densely packed mucosal mononuclear cells. There have been conflicting reports regarding the relationship between proliferation and CD4+
T-cell depletion during HIV infection. Using ex vivo labeling with bromodeoxyuridine, Lane and coworkers demonstrated a significant increase in dividing CD4+
T cells during untreated HIV-1 infection(15
). In contrast, Pantaleo and coworkers, using Ki67 as a marker of proliferation, suggested that the total number of proliferating CD4+
T cells is not significantly different between HIV-infected and uninfected subjects (6
). In studies of the GI tract during AEI, some groups have demonstrated an increase in lymphocyte proliferation (8
), whereas others have not (3
). To address this issue conclusively, we examined AEI-associated GI lymphocytic proliferation by two different and complementary techniques: flow cytometry and immunohistochemistry. We conclude that there is a significant increase in proliferating lymphocytes (as measured by the absolute number and percentage of Ki67+
cells) in the mucosa during AEI and that decreased lymphocyte proliferation is not a factor in mucosal CD4+
Based on the present study, we propose that mucosal CD4+ T-cell depletion is multifactorial. It is likely that direct viral infection is responsible for the earliest loss of CD4+ T cells demonstrated by the increased viral burden. Moreover, we believe that ongoing infection of susceptible CD4+ T cells, along with activation-induced cellular death and a host-derived cytotoxic cellular response, is responsible for the persistence of the lesion. We cannot exclude alterations in mucosal cell recruitment and homing that may contribute to this process, an area of ongoing investigation.
The present findings extend our understanding of the early events within the human GI tract during HIV-1 infection. It is plausible that disruption of these events could have a significant impact on viral pathogenesis. It is important that such interventions be the focus of future research.