We previously reported that, in chronically SIVagm-infected AGMs, normal levels of immune activation are associated with normal levels of T cell apoptosis and proliferation, as well as a lack of enteropathy and plasma LPS levels that are similar to those in uninfected monkeys (3
). To provide proof-of-concept data showing that release of bacterial components into the systemic circulation results in increased immune activation and viral replication, we initially administered a single dose of LPS to six chronically SIVagm-infected AGMs. This resulted in a transient increase in the frequency of activated CD4+
T cells () (p
= 0.02) and was accompanied by a transient but very rapid increase in VL (). One of the animals had VL comparable to that observed at the peak of viral replication during acute infection (). Moreover, the LPS was bioactive in vivo as indicated by increases in plasma levels of sCD14 (). To confirm that these increases in immune activation and viral replication were specifically induced by the LPS administration, we then treated four of the animals with saline alone and observed no effect ().
FIGURE 1 In vivo LPS and saline administration in chronically SIVagm-infected AGMs. A single dose of LPS induces a transient but significant increase in the activation of CD4+ T cells (a), a transient increase in the VL (b), and an increase in plasma sCD14 (c (more ...)
A second mechanism that may explain the lack of immune activation in natural SIV infections involves regulatory T cells (Tregs) (6
). Previously we determined that the ratio between the total number of Tregs and conventional CD4+
T cells is higher in uninfected AGMs compared with uninfected Rh in both the inductive and effector lymphoid sites of the intestine (10
). Furthermore, the level of FoxP3 in CD4+
T cells sorted from intestine was higher in AGMs compared with Rh (10
). Additionally, we reported differences in the dynamics of Tregs in SIVagm-infected AGMs compared with Rh infected with SIV of macaques (SIVmac), such that FoxP3 expression was increased at day 1 after SIVagm infection in AGMs but at a later time point in SIV-mac-infected Rh (6
). The same applies to cytokines associated with Tregs in that TGFβ
and IL-10 are increased very early in SIVagm infection but only late in SIVmac infection (6
). Thus, the mobilization of Tregs may be of insufficient rapidity and quantity to be effective in controlling immune activation in SIVmac-infected Rh.
To examine Treg involvement in the suppression of immune activation in AGMs, we conducted an in vivo Treg-depletion study in SIVagm-infected AGMs. We used Ontak (denileukin diftitox), a U.S. Federal Drug Administration-approved drug that depletes CD25+
cells in humans. Ontak is a fusion protein that combines a rIL-2 and a cytocidal diphtheria toxin moiety. Three Ontak treatments (of five daily injections) were administered at 2-mo intervals to chronically SIV-infected AGMs. Ontak did not change the total numbers of CD4+
T cells (p
= 0.79) (), but it reduced the percentage of these cells (p
= 0.0001) (supplementary figure 1 and supplemental table I).4
The absolute numbers (p
= 0.28) and percentages (p
= 0.48) of Tregs defined by FoxP3 expression were also not decreased; however, a transient increase in FoxP3 expression was observed by quantitative PCR (p
= 0.006 at day 4) during On-tak administration (supplemental figure 2). Ontak also failed to induce a decrease in Treg function, as sorted Tregs from treated AGMs were as capable of inhibiting the cell proliferation of conventional CD4+
T cells as Tregs purified from nontreated AGMs (supplemental figure 3).
FIGURE 2 Impact of Ontak administration in chronically SIVagm-infected AGMs. Although Ontak did not induce significant depletion of CD25+CD4+ T cells (Tregs) (a), at the end of the treatment mucosal CD4+ T cells were significantly depleted (b). Significant increases (more ...)
However, T cell functional measurements revealed increased proliferation of conventional CD4+ T cells in the Ontak-treated AGMs, which was confirmed by significant increases in the frequency of T cells expressing the proliferation marker Ki-67 (p = 0.003 for CD4+ and p = 0.0002 for CD8+; ). Over the 5 days of Ontak administration we also found significant increases of both the percentages (p < 0.00005) and the absolute numbers (p = 0.02) of peripheral CD4+ T cells (supplemental figure 4), as well of effector memory CD4+ T cells (p = 0.006), which are preferentially depleted in SIV/HIV infections (supplemental figure 5). It is therefore possible that this increase in overall T cell proliferation masked any partial depletion of both CD4+ CD25+ T cells and Tregs. Importantly, Ontak administration induced increases in T cell immune activation as measured by HLA-DR expression on both CD4+ (p = 0.07) and CD8+ T cells (p = 0.002) (), as well as by the dynamics of the proinflammatory cytokines IL-1 (p = 0.004), IL-8 (p = 0.02), and IL-12 (p = 0.01) (). As can be seen in the figures, these had different dynamics; IL-1 and IL-12 showed an early increase (<5 days), whereas DR expression and IL-8 had a slower increase (over the first 8 –11 days). The levels of CCR5 by RT-PCR were also increased (p = 0.005 at day 4), similar to primary infection, confirming an increase of immune activation after the Ontak treatment (supplemental figure 6). Ontak administration resulted in significant up-regulation of genes associated with immune activation such as CD80, HLA-DR, IL-3, IL-4, Toll-like receptors 4 and 6, IL-12R, and IL-17R as well as down-regulation of TGF-β1, CD3z, CD3d, CD3g, and T-box21 genes (data not shown). Finally, this marked increase in immune activation resulted in a significant increase in viral replication in all treated chronically SIVagm-infected AGMs (p = 0.0001; ) These increases in viral replication were up to the levels of the peak VLs. Moreover, in two animals increased viral replication was maintained between the first two treatments. Consequently, at the end of the Ontak treatment CD4+ T cells were significantly depleted in the intestine (p = 0.0001; ).
Taken together, our data provide proof-of-concept that experimental induction of immune activation in natural hosts of SIV infection drives increases in viral replication and mucosal CD4+
T cell depletion. In the first set of the experiments we observed that a single low-dose LPS administration results in increased immune activation and a consequent increase in viral replication, thus adding weight to the concept that microbial translocation contributes to systemic immune activation and disease progression. In the second set of experiments, a more persistent and significant increase in immune activation, and consequently a more robust and sustained increase in VL and significant depletion of mucosal CD4+
T cells, was obtained after Ontak administration in chronically SIVagm-infected AGMs. Although Ontak did not induce significant Treg depletion, the drug has potential to induce immune activation as it contains an IL-2 moiety combined with a TLR ligand (the diphtheria toxin) that interacts with the innate immune system. The increases in immune activation in both experiments through TLR ligand stimulation demonstrate that interaction between innate and adaptive immune systems is functional in the natural hosts and can induce significant increase of viral replication and CD4+
T cell depletion in this animal model that otherwise is characterized by very stable chronic lentiviral infection. Our results are in agreement with a recent study showing augmentation of viral replication at mucosal sites during early SIVmac infection of Rh due to increased immune activation driven by CTLA-4 blockade (11
) and with data reporting that the levels of VL in sooty mangabeys depends on the availability of activated CD4+
T cells (12
Therefore, our results demonstrate that immune activation and proliferation of the target cells for the virus are key factors in AIDS pathogenesis. These data suggest that therapeutic strategies to reduce immune activation should be explored, in addition to the classic antiretroviral therapies, in preventing progression to AIDS in chronically HIV-infected individuals.