The repertoire of viruses to which research primates are exposed, even in the absence of clinical disease, may contribute to experimental confounding, particularly when measuring immunologic endpoints. In this study we compared two populations of rhesus macaques, those with a limited exposure to viral agents (expanded SPF macaques) and those exposed to a wider spectrum of viral agents (standard SPF macaques). Surprisingly, the proinflammatory cytokine IL-15 circulated at greater levels in the serum of macaques from the expanded SPF group. This group also demonstrated decreased CD4+ and CD8+ memory T cell counts including significant reductions of both TTrM and TEM cell populations. Cultured PBMCs isolated from expanded SPF macaques demonstrated a greater capacity to support SIV replication compared to cultures of PBMCs from standard SPF macaques and, although SIV replication did not correlate with CD4+ TEM cell counts, in vitro virus production was highly correlated with serum IL-15 levels in the subset of animals examined.
Following antigen exposure, TEM
cells differentiate from TCM
cells to gain cytotoxic potential, polarized cytokine production, and the ability to home to effector sites.19,20
It is conceivable that the degree of antigenic stimulation associated with SPF status contributes to the observed difference in TEM
cell counts. This is consistent with the observation by Pitcher et al
. who ascribed the attainment of memory cell frequencies in juvenile macaques comparable to those of adult humans to an early and more frequent exposure to infectious organisms.20
It was counter to our expectations that both lower TEM
cell counts and increased serum IL-15 were observed within the expanded SPF group. Constitutive production of IL-15 allows for homeostatic proliferation and differentiation of TEM
cells independent of T cell receptor-antigen interaction.21–23
Administration of recombinant IL-15 (rIL-15) to rhesus macaques has been shown to induce proliferation of CD4+
However, the doses administered were greater than physiologic levels and repeated administration resulted in refractoriness to IL-15 signaling.22
It is possible that the degree of antigen exposure associated with SPF status rather than the amount of circulating IL-15 plays an equal or greater role in the expansion of the TEM
pool. Unmeasured cytokines, such as IL-7, may also impact the magnitude of this cell population. Lastly, we did not examine TEM
populations in lymphoid or effector sites. TEM
cells rapidly migrate to lymph nodes, lung, liver, and gut raising the possibility that circulating TEM
cell counts are not reflective of cell frequencies in extravascular sites.22
Members of the CD4+
population are the initial cellular targets of acute SIV infection. In this study, SIV replication in PBMC cultures was not correlated with circulating TEM
cell numbers and but instead was highly correlated with serum IL-15 levels. In vivo
studies support these findings and have shown that expansion of the TEM
target cell pool in rIL-15-treated macaques was not associated with increased SIV plasma viral loads.21,22
Others have also shown that coincubation of infected PBMCs with IL-15 results in increased HIV replication and that increased susceptibility of IL-15-treated CD4+
T cell cultures to SIV infection was associated with an IL-15 dependent increase in CD4 expression.24,25
Although the utility of IL-15 has been explored in the SIV model as an immunomodulatory therapeutic and vaccine adjuvant,26–29
a recent report suggests that treatment with IL-15 may augment CD4+
T cell proliferation and activation resulting in a 3-log increase in viral set point and accelerated onset to simian AIDS.30
Our findings suggest that the endogenous level of circulating serum IL-15 associated with SPF status may serve as a host factor with the capacity to modulate SIV pathogenesis.
The enhancement of cytokine production following mitogen stimulation of cultured PBMCs from expanded SPF macaques suggests an increased responsiveness to immune stimulation in this group. Cytokines elaborated at greater levels included IL-10, GM-CSF, IL-12, MIP-1b, IL1-ra and VEGF. A number of these are reported to play critical roles in SIV/HIV pathogenesis. For instance, genetic polymorphisms resulting in decreased IL-10 production have been linked to increased HIV susceptibility and accelerated CD4+
T cell loss.31,32
IL-10 production also contributes to the anti-inflammatory cytokine profile in African green monkeys with apathogenic SIV infection.33
Suppressive effects of IL-10 on HIV replication in vitro
have also been reported.34,35
While these findings would suggest that increased IL-10 elaborated in expanded SPF macaque PBMC cultures would not contribute to the observed increased in vitro
SIV replication, there have also been numerous contradictory reports indicating an IL-10 synergizes with other cytokines to enhance HIV production.36–38
Similar contradictory reports exist in the literature for GM-CSF and IL-12, illustrating the importance of cell type, culture conditions, and cytokine milieu.
MIP-1β production was also increased in mitogen stimulated PBMC from expanded SPF macaques. This chemokine is secreted by a variety of cells and is involved in recruitment of immunocompetent cells. As a ligand for the CCR5 coreceptor, MIP-1β, like MIP-1α and RANTES, is an endogenous inhibitor of viral entry and capable of inducing a dose dependent suppression of HIV-1, HIV-2 and SIV.39,40
Higher levels of MIP-1α and MIP-1β have been reported in asymptomatic HIV positive subjects compared to patients that progressed to AIDS.41
While this literature would argue against the increase in viral replicative capacity observed in PBMC cultures from expanded SPF macaques, effects of elevated MIP-1β may be negated by alterations in the remaining cytokines such as the observed trend towards decreased MIP-1α production in expanded SPF macaque PBMC cultures. It is unlikely that specific cytokines contribute to the variation in viral replicative capacity but rather the overall shift in cytokine environment combined with other factors.
The immunologic and viral milieu established during the acute stages of HIV/SIV infection, in part, defines the pathogenic potential of infection in a given individual.42–44
A number of host factors are thought to contribute to these early set points. One such host factor postulated to result in a poor prognosis for HIV-infected populations in developing countries is increased exposure to bacterial, viral, and parasitic agents.45,46
In this study we suggest the opposite. Macaques exposed to fewer viral agents via maintenance under an increased level of bioexclusion had increased levels of serum IL-15, greater PBMC stimulability, and an increased capacity to support in vitro
SIV replication. Although counter to our expectations, this finding may be explained by the “hygiene hypothesis.” This theory suggests that the reduction in bacterial, viral, and parasitic infections associated with antibiotic use, vaccination programs, and improved living conditions in developed countries may explain an increased incidence of allergic and autoimmune disease.47,48
Animal models have provided some proof of this hypothesis. Elimination of environmental viruses results in an increased frequency and rapidity of onset of diabetes and an increased incidence and severity of adjuvant induced arthritis in rodent models.49,50
Adoptive transfer of lymphocytes from virus-exposed animals or the introduction of specific rodent viruses has been shown to provide a protective effect from the induction of autoimmune disease.51–53
In rhesus macaques, animals with reduced pathogen burden as a result of indoor housing display skin pathology consistent with chronic hypersensitivity dermatitis and animals with a prior history of outdoor housing or exposure to lung mites had a reduced incidence of dermatitis.54
A mechanistic explanation for the “hygiene hypothesis” is not completely elucidated, although shifts in cytokine profiles, down-regulation of CD25+
T regulatory cells, and antigenic competition have been suggested as contributing factors.47
The examination of hematologic parameters and cellular immunophenotypes revealed an increased percentage of CD25+
B lymphocytes in standard SPF macaques. CD25 expression indicates a mature phenotype with increased expression in memory B cells and decreased expression in naive and precursor populations.55
B cells are also described as having an activated phenotype with improved antigen presentation capabilities and the ability to trigger CD4+
T cell proliferation in mixed lymphocyte reaction cultures.56
Alternately and depending on the cytokine environment, CD25+
B cells may be tolerogenic, participating in inhibition of the T cell proliferation and cytokine elaboration.57
Similar to the increase in the memory T cell pool, a more mature B cell phenotype in standard SPF animals may be attributed to an early and recurring exposure to viral pathogens to which expanded SPF animals are not subject. A complete characterization of the B cell phenotype, including an assessment of additional markers of memory populations, in expanded and standard SPF macaques and the potential contribution to alterations in T cell function require further investigation. Such further studies should also utilize additional markers of T cell activation and proliferation such as HLA-DR and Ki67.
There are limitations to this study. Although randomly selected animals were age and gender matched for each experiment, sampling was restricted to those animals not currently assigned to other studies and not currently in active breeding arrangements. Because samples from all animals were not able to be used for all assays, only a limited examination of correlations between in vitro SIV replicative capacity, cytokine production, and cellular immunophenotype was able to be performed. These animals were also not MHC haplotyped, although the random selection of unrelated animals likely limits this as a study confound. Lastly, we do not have data on the response of expanded and standard SPF animals to in vivo SIV inoculation. Despite these limitations we have been able to demonstrate the profound impact of SPF status on memory T cell populations and the capacity to support in vitro SIV replication. Based on the findings presented here, performance of a subsequent study in which SIV inoculation is performed in MHC haplotyped expanded and standard SPF animals subject to a complete battery of baseline phenotyping may further elucidate the contribution of environmental infectious agent exposure to HIV/SIV progression.
In this study we report significant differences in immune cell populations, cytokine production, and the capacity to support in vitro SIV virus replication between cohorts of standard and expanded SPF macaques. Although additional experiments are required, findings here suggest that an altered immunophenotype associated with housing of animals under differential levels of bioexclusion has the potential to impact the outcome of studies using the SIV model. This risk of experimental confounding associated with SPF status may extend beyond nonhuman primate models employed in HIV research to other studies for which the measurement of immunologic endpoints is critical, including models of solid organ transplantation, drug discovery safety assessment, and vaccine development. Results of this study suggest that critical consideration be given to virus exposure history when selecting animals for study inclusion and allocating animals to experimental groups.