In the present study, we investigated the effects of HAART on the restoration of the gut mucosal immune system compared to the peripheral blood in HIV-1-infected patients. A longitudinal analysis of virologic, immunologic, and molecular characteristics of patients initiating HAART during primary or chronic HIV infection indicated that the degree of mucosal CD4+ T-cell restoration was associated with the level of viral suppression and the severity of disruption of the gut microenvironment, independent of the time of the initiation of therapy. Our findings suggest that initiation of HAART prior to the manifestation of severe enteropathy may support effective restoration of the mucosal immune system. Thus, monitoring of gut lymphoid tissue will be important for an accurate assessment of disease progression and the efficacy of antiretroviral therapy. Although the numbers of patients in our patient cohorts were relatively small, the strength of this investigation lies in the longitudinal evaluation of the patients in the study. Future studies involving larger number of patients in the study groups will increase the statistical significance of our observations and determine the variation inherent within different patient groups.
Our previous studies have shown that the severity of CD4+
T-cell depletion and viral burden in GALT are not adequately reflected in the peripheral blood of HIV-infected patients (13
). In this study, we also showed that the course of CD4+
T-cell restoration in GALT during HAART was slow with biphasic kinetics compared to the peripheral blood compartment of both PHI and CHI patients (Fig. ). Since impaired thymic functions and disruption of lymph node architecture have been well documented in patients with chronic HIV infection (18
), it was not surprising to observe the delay in CD4+
T-cell restoration in GALT of CHI patients. On the other hand, the delay in CD4+
T restoration was observed in GALT of patients beginning HAART during primary HIV infection, despite having normal CD4+
T-cell numbers and undetectable viral loads in peripheral blood. A possible explanation for this delay could be attributed to the severe pathological changes occurring in GALT very early during primary HIV infection. Gene expression studies indicated that patients displaying modest CD4+
T-cell restoration during HAART also displayed increased expression of genes involved in inflammation, innate and cell-mediated immune responses, and response to wounding prior to HAART (Fig. ). Thus, a significant time period may be required to repair local tissue damage in the intestinal mucosal microenvironment before substantial CD4+
T-cell restoration could occur. Supporting evidence is provided by studies with the SIV-infected rhesus macaque model which demonstrated severe CD4+
T-cell depletion and histopathologic and functional changes in GALT during the early stages of SIV infection (14
). Our previous studies have also reported acute enteropathy in SIV-infected rhesus macaques during primary SIV infection accompanied by decreased nutrient digestive and absorptive functions that correlated with a high level of viral replication in GALT (14
). Loss of epithelial growth and repair-associated gene expression was also observed in GALT during primary SIV infection (12
). These findings suggested that enteropathogenic changes occurring early in viral infection might lead to a delay in the restoration during therapy.
Multiple mechanisms could contribute to the discordant results of CD4+
T-cell restoration between GALT and peripheral blood during therapy. These include incomplete suppression of viral replication and impaired immune functions in GALT compared to peripheral blood. Anton and colleagues have previously reported ongoing viral replication in GALT of some HIV-infected patients receiving HAART, despite undetectable plasma viral loads (3
). Thus, the ongoing viral replication may lead to infection and killing of CD4+
T cells trafficking to the gut mucosa during HAART. CD4+
T lymphocytes in GALT have dual expression of the HIV coreceptors CCR5 and CXCR4 and are naturally permissive to HIV infection by both CCR5-tropic and CXCR4-tropic variants (17
). In this study, we demonstrated that repopulating CD4+
T cells in GALT during HAART expressed low levels of the HIV coreceptors CCR5 and CXCR4, thus becoming potential targets for HIV infection. This poses a major challenge for the control of viral replication and immune restoration of the gut mucosa during HAART. The incomplete suppression of HIV replication in GALT following HAART may contribute to the ongoing infection of the repopulating CD4+
T cells, causing the delay in the restoration of T-cell homeostasis in GALT.
In the SIV model, initiation of antiretroviral therapy during early SIV infection led to effective viral suppression and near complete restoration of CD4+
T cells in GALT that correlated with increased expression of genes associated with mucosal regeneration and repair (11
). In animals with incomplete suppression of viral replication in GALT during therapy, mucosal CD4+
T cells were found to be functionally impaired (23
). Thus, suppression of viral replication and dampening of immune activation and inflammatory processes in GALT during therapy may be crucial to achieve effective restoration of the gut mucosal immune system in HIV and SIV infections. Our recent studies showed that decreased expression of genes associated with absorptive and digestive functions and drug metabolism were detected in chronically HIV-infected patients (11
). The access to antiretrovirals in the gut mucosal tissue may be less efficient than in peripheral blood or other lymphoid compartments, and this may partly contribute to the incomplete suppression of viral replication in GALT during therapy.
Expression of mucosal homing markers on T lymphocytes is crucial for their trafficking to gut mucosal tissue. Previous studies have also demonstrated depletion of CD4+
T cells expressing αΕβ7 in GALT of chronically infected HIV patients (37
). In the SIV model, CD4 T cells expressing αΕβ7 have primarily a memory phenotype and become rapidly depleted, as early as 1 week postinfection, and remain depleted during chronic stages in the absence of antiviral therapy (22
). Evaluation of the phenotypic characteristics of CD4+
T cells repopulating GALT during HAART showed that the majority of these cells were of memory phenotype and expressed lymphocyte trafficking markers αΕβ7 and LFA-1. Our results demonstrated that the levels of memory CD4+
T cells expressing LFA-1 in PHI patients was higher than that observed in CHI patients. However, both CHI and PHI patients had decreased levels of LFA-1+
T cells in comparison to healthy uninfected individuals, demonstrating that impairment in mucosal homing may occur early in HIV infection. These data suggested that initiation of therapy before the immune system becomes severely compromised may help preserve the homing potential of memory T cells to mucosal tissues.
cytotoxic T-cell responses in mucosal tissues of HIV-infected patients have been shown to produce high levels of granzyme A but low levels of perforin (36
), suggesting that HIV-specific CD8+
T-cell responses may not be fully functional in chronically infected patients (4
). In agreement with previous studies, gene expression profiling indicated that although granzymes, T-cell receptors, and major histocompatibility complex class I molecules were up regulated, we could not detect up regulation of perforin in our HIV-infected patient cohorts. These data suggest that elevated, nonspecific CD8+
T-cell responses may contribute to the impairment of CD4+
T-cell restoration. We observed the presence of HIV Gag-specific CD8+
T-cell immune responses in both the peripheral blood and GALT of PHI and CHI patients during HAART. It was interesting that, in the PHI group, patient 116 did not have detectable HIV-specific CD8+
T-cell responses despite the complete restoration of mucosal CD4+
T cells and T-cell homeostasis (normal levels of CD8+
T cells) and undetectable viral loads in both peripheral blood and GALT. In contrast, patients 134 and 140 had relatively high Gag-specific CD8+
T-cell responses but did not experience CD4+
T-cell restoration and showed elevated CD8+
T-cell percentages in GALT. These data suggested that despite effective suppression of viral loads in the peripheral blood of PHI during HAART, a similar level of viral suppression might not have been achieved in GALT of these two patients, as reflected by the slow to modest increase in CD4+
T cells and high levels of viral antigen-specific responses. However, efficient viral suppression in GALT during HAART may lead to rapid restoration of mucosal CD4+
T cells, as was seen in patient 116. We hypothesized that a very low level of viral replication in GALT that was not detected by real-time reverse transcription-PCR was sufficient to induce antiviral responses in these patients. Thus, the high levels of HIV Gag-specific mucosal CD8+
T-cell responses might be indicative of incomplete viral suppression in GALT.
The gene expression analysis of mucosal samples in patients with efficient CD4+
T-cell restoration in GALT showed a correlation between decreased gene expression associated with inflammation and cell activation and better CD4+
T-cell restoration. In addition, the expression of genes involved in inflammatory responses, such as NF-κΒ, were largely normalized when HAART was initiated during PHI. Recent studies have demonstrated that control of HIV replication in the peripheral blood of HIV-infected patients receiving HAART was associated with high levels of HIV-specific interleukin-2- and IFN-γ-producing CD4+
T cells and low levels of T-cell activation (10
). The low level of activation state could support the expansion of the cell populations in response to HIV but not deplete them due to hyperimmune activation. Thus, a relatively low level of cell activation state may support the survival of the quiescent pool of T cells. Our recent studies showed that drug-naive long-term HIV-infected patients had maintained normal CD4+
T-cell percentages and undetectable viral loads in GALT, which correlated with control of gene expression related to inflammation and cell activation (34
). These results provided further evidence that chronic immune activation and inflammation and failure to control viral replication in mucosal tissues may be associated with impaired ability to restore the CD4+
T cells in GALT.
Our findings suggested that suppression of viral replication and control of inflammatory responses in GALT determine the level of restoration of the mucosal immune system during HAART. Previous studies in both peripheral blood and lymph nodes of HIV-infected patients have suggested that early HAART may avert the progressive immunologic damage associated with untreated HIV infection (1
). However, limited information is available about the effects of HAART on viral suppression and restoration of T-cell homeostasis in gut mucosal tissues during HIV infection. In this study, we have demonstrated that pathological processes that occur in GALT during primary HIV infection may profoundly influence the characteristics of host antiviral responses and the integrity of the gut microenvironment. This may play a significant role in the degree of mucosal CD4+
T-cell restoration during the course of therapy. Our results emphasize the importance of monitoring GALT in the evaluation of immune restoration during therapy as well as the need for developing novel therapeutic strategies to enhance the repair and regeneration of the mucosal immune system.