This study showed that serum levels of the innate immune system pro-inflammatory modulators TNF-α and IP-10 were elevated, increased among untreated HIV patients, compared with uninfected comparisons, a finding that is consistent with a broad inflammatory response. Additionally, serum IL-12(p40) and IL-15, important for T cell homeostasis and function, were decreased in chronic, untreated HIV infection compared to otherwise similar HIV-uninfected women. When compared with untreated women the HAART recipients showed fewer detectable differences in cytokines from HIV uninfected persons (2 vs. 5 analytes), though TNF-α and FGF-2 were still different in the HAART compared to NEG group. Finally, while as expected many factors correlated with both plasma viral burden and CD4+ T cell counts, some factors correlated with one or the other. Interestingly, the growth factors VEGF, EGF, and FGF-2 showed a positive correlation with higher CD4+ T cell counts.
Analytes found to be elevated in chronic infection in this study have been shown to be elevated using separate test methodologies for TNF-α [19
] and IP-10 [34
]. During primary infection IP-10 and TNF-α correlated positively with quantitative viral load [35
]. We found that the only factor to show a significant positive correlation with viremia in untreated women during chronic HIV infection was IP-10, underlining the importance of this chemokine in the response to HIV and consistent with in vitro
experiments demonstrating its ability to stimulate HIV replication [36
]. Elevated IP-10 also has been detected in multiple viral infections, including acute West Nile virus [37
], severe influenza infection [38
] acute HCV [26
] and chronic, persistent HCV in this study, suggesting a general role for this chemokine in the immune response to viral infections. Elevated IP-10 levels in chronic HIV infection could be deleterious and contribute to ongoing immune activation and T cell depletion, supported by the strong negative correlation between IP-10 levels and CD4+ T cell count we found in this study (). Finally, the suppression of plasma IL-12 levels in untreated HIV-infected participants is consistent with published work demonstrating cellular defects in production of these cytokines in chronic HIV infection [40
While most of the cytokine changes previously described to be elevated or reduced during chronic HIV infection were confirmed in our cohort of women with uncontrolled HIV replication, we did not find significant elevations reported by others in predominantly male populations using ELISA tests for or IL-6 [42
], IL-10 [44
], or FGF-2 [46
]. Although median IL-10 levels in our untreated HIV-infected group were nearly two-fold higher compared to the HIV-negative participants, this difference was not statistically significant. In contrast, median IL-6 and FGF-2 values were lower in the NC than NEG group. The IL-6 data are consistent with our recent study of acute HIV infection, where only a subset of participants showed elevations in IL-6 levels [26
], though the different assay format (ELISA vs. Luminex) may have been responsible for the discrepant results. We re-tested the samples in the current study using an FGF-2 ELISA kit from the same manufacturer used in the prior study and the significant differences in FGF-2 levels between the clinical groups seen by Luminex testing was not seen using the ELISA kit (data not shown). The discrepancy between the ELISA test and Luminex assay for FGF-2 is potentially due to the different dynamic ranges of the two assays or to the smaller sample size of the current study.
The current study has a number of limitations, including a relatively small sample size, which made detection of only relatively large differences in cytokine concentrations between clinical groups possible. Another potential limitation of the study is the fact that our participants were female and primarily African American, therefore the results may or may not be generalizable to other HIV-infected populations. Data are conflicting regarding the extent to which race affects cytokine responses, with African Americans showing higher baseline levels of inflammatory cytokines [47
], but race having no effect on reactivity of immune cells to IFN-α [48
] or on the association between heart failure and inflammatory markers [49
]. Finally, we did not formally demonstrate that women initiating HAART showed normalization of perturbed cytokine levels. We also did not test cytokine levels in individuals with high viral load who were taking HAART. However, there are indications from the literature that HAART initiation can normalize or partially normalize inflammatory and coagulation markers [32
]. This, along with the fact that our participants were matched on potential confounders, makes it likely that the differences that we observed between NC and HAART groups were in fact due to HAART.
Recent work by Roberts et al. identified a number of factors that were associated with viral load set point and the rate of CD4+ T cell decline during primary HIV infection [35
]. IL-12, IFN-γ, and GM-CSF were associated with lower viral load set point at 12 months and/or slower CD4+ T cell decline, while IL-1α, IL-7, IL-15 and eotaxin showed the opposite effect. In our study none of these factors except IL-1α showed a correlation with viral load or CD4+ T cell count, and the association between IL-1α and CD4+ T cell count in chronic HIV infection was opposite to what was seen in primary infection. These differences highlight the fact that the interaction between HIV and the immune system is likely very different in primary and chronic HIV. For example, biasing the immune system toward a Th1 response with higher IL-12 levels in primary HIV infection may be of benefit to establish a lower viral load set point, whereas after the set point has been reached many individuals have lost detectable IL-12 secretion (). Similarly, high IP-10 levels during primary HIV infection did not appear to increase subsequent viral load set point or the rate of CD4+ T cell decline [35
], while they were associated with lower CD4+ T cell counts during chronic HIV infection.
In summary, we found that compared to HIV-negative women, untreated chronic HIV infection was associated with defects in T cell signaling pathways, coupled with evidence of activation of the innate immune system, and these differences were less apparent or absent in HAART-treated women with undetectable viral load. By examining a broad array of soluble immune mediators we were able to identify specific analytes that behaved differently from the majority of immune markers. For example, TNF-α and FGF-2 were different in the HAART compared to HIV-uninfected group, as opposed to the majority of other analytes measured. This finding suggests that the pathways driving the dysregulation of these two factors may require very low levels of virus for stimulation (or suppression), are influenced by antiretroviral drugs themselves, or are dependent on immune activation induced by HIV in an indirect fashion. Finally, while T cell homeostasis factors such as IL-2, IL-7, and IL-15 did not appear to be associated with higher CD4+ T cell counts, the growth factors VEGF, EGF, and FGF-2 were associated with higher CD4+ T cell counts. Given that the causal role of the relationship is unknown, it is unclear if these growth factors would have therapeutic potential to ameliorate the immune deficiency associated with HIV infection or would serve as useful biomarkers of a more preserved or restored immune system.