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Pre-HAART Immune Activation Associated with Developing Cancers & Death
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"In the absence of highly active antiretroviral therapy (HAART), markers of T cell activation (defined by CD70 or CD38 expression on CD4 or CD8 cells) and diverse plasma markers of immune activation, including soluble tumor necrosis factor receptor (sTNFR)-2, soluble (s) CD27, neopterin, and β2-microglobulin, predicted a CD4 cell count decrease, AIDS, or death, independently of the contribution of HIV type 1 (HIV-1) replication to these end points....HAART was associated with reductions in generalized immune activation, and the magnitude of reductions in CD8 T cell activation was associated with the magnitude of CD4 cell increases associated with HAART.....In this study of treatment-naive subjects who initiated HAART as part of 2 clinical trials, higher pretreatment baseline plasma concentrations of sTNFR-1, sCD27, sCD40L, and IL-6 and the latest CD8 T cell activation level were each associated with a new AIDS-defining illness or death in models that adjusted for other important predictors and correlates of HIV disease progression....sTNFR-1, sCD27, sCD40L, and the latest CD8 T cell activation level were associated with disease progression in the subset of subjects who developed an AIDS-defining malignancy or death, complications that developed later among subjects whose CD4 cell counts were often >200 cells/cm3 and who had achieved excellent suppression of viral replication.....In the subset of cases who developed a new opportunistic infection, a trend was evident in the association between higher infection risk with higher pretreatment plasma levels of sTNFR-1, sCD40L, and IL-6"
Pretreatment Levels of Soluble Cellular Receptors and Interleukin-6 Are Associated with HIV Disease Progression in Subjects Treated with Highly Active Antiretroviral Therapy
The Journal of Infectious Diseases 2010 May 6
Robert C. Kalayjian,1 Rhoderick N. Machekano,1Nesrine Rizk,1 Gregory K. Robbins,3 Rajesh T. Gandhi,3 Benigno A. Rodriguez,2 Richard B. Pollard,4 Michael M. Lederman,2 and Alan Landay5
1MetroHealth Medical Center and 2University Hospitals-Case Medical Center, Case Western Reserve University School of Medicine, Cleveland, Ohio; 3Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts; 4University of California-Davis Medical Center, Sacramento; 5Rush Medical College, Chicago, Illinois
ABSTRACT
Background. To identify inflammatory pathways that may contribute to the pathogenesis of human immunodeficiency virus (HIV) disease, we explored associations between AIDS or death and different inflammatory markers, including selected soluble tumor necrosis factor superfamily receptors (sTNFRs) and ligands, interleukin (IL)-6, and CD8 T cell activation, in individuals treated with highly active antiretroviral therapy (HAART).
Methods. A case-control study of subjects in AIDS Clinical Trials Group (ACTG) protocols 384 and 5015, who were matched according to the CD4 cell count and plasma viral load at baseline, was performed using conditional logistic regression.
Results. Higher pretreatment concentrations of sTNFR-1, sCD27, sCD40L, and plasma IL-6 were associated with a new AIDS-defining illness or death in separate models adjusted for age, sex, hemoglobin, and the latest CD4 cell counts. In additional models that excluded case patients with opportunistic infections, sTNFR-1, sCD27, and sCD40L were each associated with a new AIDS-defining malignancy or death that developed at a median of 51 weeks after initiation of HAART, by which time the majority of subjects had a CD4 cell count of >200 cells/cm3 and had achieved a plasma viral load of <50 copies/mL.
Conclusion. These data are compatible with a model in which these soluble inflammatory markers identify pathways that may contribute to the pathogenesis of HIV disease progression, pathways that might not be a direct consequence of ongoing HIV type 1 replication.
Cumulative evidence supports the importance of generalized immune activation in the pathogenesis of human immunodeficiency virus (HIV) disease, although the mechanisms by which this may occur remain incompletely understood [1]. In the absence of highly active antiretroviral therapy (HAART), markers of T cell activation (defined by CD70 or CD38 expression on CD4 or CD8 cells) and diverse plasma markers of immune activation, including soluble tumor necrosis factor receptor (sTNFR)-2, soluble (s) CD27, neopterin, and β2-microglobulin, predicted a CD4 cell count decrease, AIDS, or death, independently of the contribution of HIV type 1 (HIV-1) replication to these end points [2-10].
HAART was associated with reductions in generalized immune activation, and the magnitude of reductions in CD8 T cell activation was associated with the magnitude of CD4 cell increases associated with HAART [11]. Differences in the magnitude of CD4 cell increases also were associated with single-nucleotide haplotype differences in genes that encode tumor necrosis factor (TNF)-α, tumor necrosis factor receptor (TNFR)-1, and TNF-related apoptosis-inducing ligand (TRAIL), among others, suggesting a possible role of TNF-related apoptosis pathways in T cell reconstitution with HAART [12]. Finally, higher plasma concentrations of interleukin (IL)-6 predicted a greater risk of death due to all causes and opportunistic infections in the Strategies for Management of Anti-Retroviral Therapy (SMART) trial, both in subjects who were randomized to interrupt therapy and in those who were randomized to continue antiretroviral therapy [13, 14]. These observations suggest that CD4 cell restoration and improved survival with HAART may be mediated in part by reductions in generalized immune activation.
To better characterize the activation pathways by which this may occur, we explored, in a case-control study, the contributions of different immune activation markers, including CD8 T cell activation markers, plasma levels of IL-6, and selected soluble surface receptors and ligands within the tumor necrosis superfamily, to the risk of AIDS or death among subjects who began their first HAART regimen through 1 of 2 AIDS Clinical Trials Group (ACTG) studies (protocols 384 and 5015).
Results
Baseline pretreatment plasma samples were available from 42 of 49 subjects who experienced a new AIDS-defining clinical event or death during up to 192 weeks of observation while participating in ACTG protocols 384 and 5015, respectively. One death, which was due to a motor vehicle accident, was excluded. Baseline and time-updated characteristics among the cases and the 111 matched controls are summarized in Table 1. Included among the clinical events for these cases were 24 opportunistic infections, 6 malignancies, and 11 deaths (Table 2). Among the listed causes of death were included 2 cardiac-related deaths and 1 death each from adenocarcinoma of the lung, gastrointestinal bleeding, disseminated intravascular coagulation, pneumonia, meningitis, and progressive wasting with AIDS dementia complex. A cause of death was not provided for 3 cases. The median onset of a clinical event or death was 12 weeks after HAART initiation (Table 1 and Figure 1). The median CD4 cell count and plasma viral load at the time of a new AIDS-defining illness or death were 66 cells/mm3 and 2.86 log10 copies/mL, respectively.
Correlations between the immune activation markers and other variables at baseline among all subjects are summarized in Table 3. Notably, higher levels of sTNFR-1 were associated with older age, lower hemoglobin level, lower CD4 cell count, lower estimated glomerular filtration rate, higher plasma viral load, and higher levels of each of the other activation markers, except TRAIL and CD8 T cell activation. Neither sex nor hepatitis C virus antibody status was associated with any baseline inflammatory marker, but women exhibited a trend toward having fewer CD4 cells at baseline (median, 25 vs. 65 cells/mm3 for women vs. men, respectively; p=.09).
Older age, female sex, lower hemoglobin level at baseline, and higher baseline levels of sTNFR-1, sCD27, sCD40L and IL-6 were each associated with the composite end point of a new AIDS-defining illness or death, on univariate analyses (Table 4). Neither race/ethnicity, CD8 T cell activation levels at baseline, baseline sTNFR-2 level, sTRAIL level, estimated glomerular filtration rate, basal metabolic index, nor hepatitis C virus serostatus was associated with this end point. Consistent with the matching strategy, neither CD4 cell count at baseline nor plasma viral load at baseline was associated with this end point. Lower latest CD4 cell count and higher latest plasma viral load each was associated with this end point, however, as was the higher most recent CD8 T cell activation level.
Levels of sTNFR-1, sCD27, sCD40L, and IL-6 at baseline and the latest CD8 T cell activation level remained significantly associated with this composite end point in separate adjusted models in which the latest CD4 cell count also contributed significantly to each of these models, as did age (in models testing sCD27, sCD40L, and IL-6), sex (in models testing sTNFR-1, sCD27, sCD40L, and IL-6), and pretreatment hemoglobin (in models testing sCD27 and latest T cell activation levels) (Table 4); latest plasma viral load did not contribute to any of these adjusted models. Pretreatment levels of sTNFR-1, sCD27, and sCD40L also remained significantly associated with this composite end point in a multivariable model that simultaneously tested all of these inflammatory markers, adjusting only for the latest CD4 cell count.
The associations between these immune activation markers and clinical outcomes are graphically depicted in Figure 2. In additional models, which were fitted on data that excluded opportunistic infections, higher levels of sTNFR-1 and sCD27 at baseline and higher latest CD8 T cell activation levels were significantly associated with an AIDS-defining malignancy or death after adjusting for the latest CD4 cell count (OR for sTNFR-1, 5.03 [95% CI, 1.42-17.7] [p=.01]; OR for sCD27, 3.37 [95% CI, 1.31-8.64] [p=.01]; OR for the latest CD8 T cell activation level, 2.62 [95% CI, 1.03-6.67] [p=.04]), and a trend was evident in association with a higher sCD40L level (OR, 1.90 [95% CI, 0.92-3.91] [p=.08]). In this subset, the median onset of an AIDS-defining malignancy or death was 51 weeks after HAART initiation (interquartile range [IQR], 27-69 weeks), with 59%, and 70% of these individuals achieving a plasma viral load of <50 and <200 copies/mL, respectively, according to the latest antecedent plasma viral load measurement. The median latest CD4 cell count was 232 cells/mm3 (IQR, 103-378 cells/mm3) among these cases and 303 cells/mm3 (IQR, 198-423 cells/mm3) among corresponding controls.
In the subset of cases who developed a new opportunistic infection, a trend was evident in the association between higher infection risk with higher pretreatment plasma levels of sTNFR-1, sCD40L, and IL-6 in separate models that also were adjusted for the latest CD4 cell count (OR for sTNFR-1, 1.50 [95% CI, 0.93-2.41] [p=.10]; OR for sCD40L, 1.87 [95% CI, 0.92-3.79] [p=.08]; and OR for IL-6, 1.60 [95% CI, 0.99-2.58] [p=.06]).
Discussion
In this study of treatment-naive subjects who initiated HAART as part of 2 clinical trials, higher pretreatment baseline plasma concentrations of sTNFR-1, sCD27, sCD40L, and IL-6 and the latest CD8 T cell activation level were each associated with a new AIDS-defining illness or death in models that adjusted for other important predictors and correlates of HIV disease progression that were identified from previous studies, including age, sex, hemoglobin level, and the latest CD4 cell count [19-22]. Although many of the clinical events in this study included opportunistic infections that presented soon after HAART initiation, pretreatment sTNFR-1, sCD27, and sCD40L levels remained associated with an AIDS-defining malignancy or death, clinical events that presented a median of 51 weeks after HAART initiation, by which time the majority of subjects had >200 circulating CD4 cells/mm3 and had suppressed plasma viremia to <50 copies/mL.
TNFR-1 is expressed on virtually all somatic cell types and mediates most of the known cellular responses that are attributed to TNF, including cellular proliferation via nuclear factor-κB activation, and apoptosis via the activation of caspases [23]. Receptor activation by various agonists initiates proteolytic cleavage of its extracellular domain, with plasma shedding as sTNFR-1, and down-regulation of cell surface receptor expression [24]. sTNFR-1 modulates TNF activity by competing for circulating TNF, but it also may activate caspase-mediated apoptosis by interacting with cell membrane-associated TNF [25].
CD27 is induced on B cells after antigenic stimulation and is constitutively expressed on T cells as a costimulatory molecule for T cell activation through binding to its ligand CD70 that is found on activated T cells, B cells, and other antigen-presenting cells. Plasma shedding of sCD27 on T cells follows T cell receptor-mediated activation [26]. Although essential for effector T cell maturation, CD27-CD70 costimulation also may impair B cell activation and function by inducing interferon-γ- and TNF-α-mediated disruption of germinal centers [27]. Elevated plasma levels of sCD27 predicted an increased risk of AIDS-associated non-Hodgkin lymphoma in the Multicenter AIDS Cohort [28] and CD4 cell decrease in HIV-1-infected Ethiopians in the absence of HAART [7].
CD40L is predominately expressed by activated CD4 cells and induces B cell proliferation, while promoting B cell survival and antigen-presenting cells maturation upon interacting with its receptor CD40 [29]. HIV infection is associated with reduced cell-associated CD40L but increased sCD40L in plasma [30], which in one study did not normalize with HAART [31].
IL-6 is a major mediator of the acute-phase response that is expressed by antigen-presenting cells and nonhematopoietic cells. It is an important growth factor for B cells while also promoting CD4 T cell proliferation and survival [32]. Elevated IL-6 plasma levels preceded the onset of Burkitt and small noncleaved cell lymphoma in HIV-infected subjects, and non-Hodgkin lymphomatous cells from both HIV-infected and HIV-uninfected patients expressed heightened levels of IL-6 in situ, suggesting that IL-6 may act as a paracrine growth factor in these malignancies [33, 34]. Although IL-6 was not associated with the risk of a malignancy or death in the present study, it is likely that the small sample size may have precluded our ability to detect any differences should they exist. IL-6 expression was substantially reduced in TNFR-1-deficient mice [35], and elevated IL-6 levels were associated with an increased risk of death or an opportunistic infection in the SMART study and with a greater risk of coronary heart disease among HIV-uninfected participants in a large prospective cohort study [13, 14, 36].
sTNFR-1, sCD27, and CD40 belong to the TNF superfamily of receptors that regulate diverse pathways involving mechanisms of B and T cell proliferation, differentiation, and survival. Among the possible sources of the heightened immune activation that these markers represent are Toll-like receptor-7/8 activation, which occurs via single-stranded HIV-1 RNA or activation mediated by HIV proteins [37, 38], or Toll-like receptor-4 activation, which occurs via lipopolysaccharide or other bacterial products arising from translocation through the disrupted gut mucosa [39]. HIV-1 regulatory proteins Nef, Tat, and Vpr also can activate TNFR-1 and TNFR-2 signaling pathways [40].
In contrast to previous studies from the pre-HAART era, we did not observe an association between the sTNFR-2 level at baseline and HIV disease progression [3, 6]. TNFR-2 enhances TNFR-1-induced apoptosis and plays an important role in TNF-induced cellular activation [41]. TNFR-2 has a significantly lower affinity for circulating TNF than TNFR-1 [42] and is a less potent inducer of nuclear factor-κB activation [43]. It is possible that sTNFR-2 may identify inflammatory processes that are more readily reversed by HAART than are those that are associated with sTNFR-1 and the other activation markers that we identify, to account for this discrepancy. This also might explain similar discrepancies involving CD8 T cell activation, which predicted decreases in the CD4 cell count and clinical outcomes in previous studies of patients who did not receive HAART [2, 5], but it was associated with CD4 cell changes and clinical outcomes (in the present study) only when using time-updated levels in HAART-treated individuals [11, 44].
Anemia and older age are long-standing predictors of accelerated HIV disease progression that were demonstrated both among antiretroviral-naive and HAART-treated individuals [19, 22, 45]. In the present study, the hemoglobin concentration at baseline was correlated with both sTNFR-1 and IL-6, and hemoglobin did not contribute to adjusted models that examined either of these 2 inflammatory markers or sCD40L, suggesting that heightened immune activation may underlie the association between anemia and accelerated HIV disease progression. Because of the correlation that we observed between older age and baseline sTNFR-1, and because age did not significantly contribute to the adjusted model that included sTNFR-1, this suggests that heightened TNFR-1-associated pathways may contribute to the accelerated HIV disease progression that is associated with aging. Consistent with this possibility are previous observations of elevated sTNFR-1 levels in association with normal aging [46, 47] and recent evidence of an important role of TNF-α and TNFR-1 in replicative senescence of CD8 T cells [48], a process of terminal differentiation that is a characteristic of both aging and HIV disease following prolonged, or enhanced, cellular proliferation.
Consistent with some previous studies among pre-HAART era and HAART era cohorts, women exhibited a greater risk of HIV disease progression [19, 49]. In a recent study, plasmacytoid dendritic cells from women exhibited higher IFN-α production after in vitro Toll-like receptor-7/8 activation by HIV RNAs, and higher CD8 T cell activation levels also were observed in HIV-infected women, when compared with HIV-infected men, for the same level of plasma viral load [50]. Consistent with this observation, sex did not significantly contribute to the adjusted model that examined the latest CD8 T cell activation levels in the present study, but women had a higher risk of AIDS or death in models that examined each of the other pretreatment inflammatory markers. This implies that CD8 T cell activation-associated additional activation pathways, but not those associated with the other markers, may contribute to differences in HIV disease progression among women.
This study is limited by a small number of clinical events and the relatively short delay between HAART initiation and these events, which often developed before the maximal effects on HIV-1 suppression might have been achieved. Although we examined soluble plasma immune activation markers only at baseline, it would also be of interest to examine the effect of HAART on the time-updated levels of these markers and to assess the variability of these markers. We also cannot exclude the possibility that these associations merely reflected the host immune response to infections or malignancies that were clinically latent at the start of therapy. Although both baseline and latest plasma IL-6 levels were associated with an increased risk of opportunistic infection in the SMART study, only the baseline levels remained significant when both were included in the same model, supporting a true predictive association with this cytokine. Finally, although hepatitis C virus serostatus was available for only 98 of our subjects, hepatitis C virus infection did not appear to influence our results.
Despite the small number of cases, however, our models identified many consistent associations among previously described predictors and correlates of HIV disease progression in HAART-treated subjects, while also identifying sTNFR-1 and sCD40L as possible new correlates of disease progression. sTNFR-1, sCD27, sCD40L, and the latest CD8 T cell activation level were associated with disease progression in the subset of subjects who developed an AIDS-defining malignancy or death, complications that developed later among subjects whose CD4 cell counts were often >200 cells/cm3 and who had achieved excellent suppression of viral replication. These inflammatory markers may help to identify cellular pathways that are important in the pathogenesis of HIV disease but may not depend on ongoing viral replication and might represent future targets of immune-based therapies.
Methods
HAART-naive participants in ACTG protocol 384 were randomized to receive 1 of 6 initial HAART regimens that included 2 nucleosides: either stavudine and didanosine or lamivudine and zidovudine, in combination with either nelfinavir, efavirenz, or both [15]. HAART-naive participants in ACTG protocol 5015 began receiving a uniform regimen of stavudine and emtricitabine with ritonavir-boosted lopinavir [16]. Follow-up visits occurred every 4 weeks to week 24 and then every 8 or every 12 weeks thereafter, for protocols 384 and 5015, respectively. In both protocols, participants were required to switch to a different HAART regimen if they had a plasma viral load of >200 copies/mL on 2 consecutive occasions after week 24. All participants provided written, informed consent to participate in these studies, which were approved by the institutional review board of each participating site.
Virologic and immunologic studies.
The plasma viral load was measured using quantitative HIV type 1 (HIV-1) RNA polymerase chain reaction (PCR) assays (Ultrasensitive Amplicor HIV-1 Monitor, version 1.0; Roche Molecular Systems) with a sensitivity of 50 copies/mL. Lymphocyte subsets and expression of CD38 and human leukocyte antigen (HLA)-DR were examined in freshly obtained whole blood, for all subjects at the aforementioned time points, by use of directly labeled murine monoclonal antibodies (PharmMingen) and 3-color flow cytometry.
Plasma cytokines and soluble receptors.
Plasma specimens were collected in ethylenediaminetetraacetic acid and stored at or below -70°C. Cytokines and soluble cellular receptors were measured in thawed plasma aliquots, with the use of commercial enzyme-lined immunosorbent assay kits used in accordance with the specifications of the manufacturer for sTNFR-1 (R&D Systems); for sTNFR-2, soluble TRAIL (sTRAIL), and IL-6 (BioSource); and for sCD27 (PeliKine). The lower limits of quantification for these assays were 7.8 pg/mL for sTNFR-1, 1.6 ng/mL for sTNFR-2, 1.56 U/mL for sCD27, 10 ng/mL for CD40L, 46.8 pg/mL for TRAIL, and 0.156 pg/mL for IL-6.
Statistical methods.
For each case, defined as a new AIDS-defining illness (a Centers for Disease Control and Prevention category C clinical event) or death, at least 2 and up to 3 controls were matched by CD4 cell count at baseline, plasma viral load at baseline, and duration of study follow-up, based on the availability of samples. Correlations among continuous baseline variables were assessed using the Spearman rank test. Among continuous variables, differences between groups were compared using Wilcoxon rank sum tests; among categorical variables, differences were compared using FisherŐs exact tests.
Associations between markers of inflammation and a new AIDS-defining clinical illness or death were assessed using conditional logistic regression models in which the exploratory variables included the following plasma concentrations at baseline-sTNFR-1, sTNFR-2, sCD27, sCD40L, sTRAIL, and IL-6-as well as CD8 T cell activation levels (defined by the coexpression of HLA-DR/CD38 on CD8 cells), which were measured both at baseline and during follow-up. Candidate adjusting variables in these models included age, race/ethnicity (white, black, Hispanic, and other), sex, plasma hemoglobin concentration at baseline, basal metabolic index, estimated glomerular filtration rate at baseline (determined using the simplified Modification of Diet in Renal Disease [MDRD] equation), hepatitis C virus infection determined by antibody detection, and total CD4 cell counts and plasma viral loads measured both at baseline and during follow-up. Time-updated variables for cases used the latest values antecedent to the date of the first AIDS-defining illness or death; these values for control subjects used data from the same study week or from the closest week antecedent to that used for their corresponding case.
Because the association between CD4 cell counts and the risk of an AIDS-defining clinical event is not linear, the baseline and latest CD4 cell counts were transformed to their cube root values [17]. All other continuous variables were transformed to a standardized score (z score) by subtracting the mean value from the original value and then dividing by its standard deviation to facilitate comparisons among these variables, which displayed vast differences in their ranges, and to minimize possible collinearity. Each exploratory variable that was identified by a univariate association (p<.1) was tested in a separate adjusted model that included age, sex, hemoglobin level at baseline, latest CD4 cell count, and latest plasma viral load. Model selection used forward, stepwise selection based on goodness-of-fit criteria that compared log likelihood ratios by a distribution, where the final model included only those variables that contributed significantly to the model (p<.05). These exploratory variables also were simultaneously examined in a multivariable model that adjusted only for the latest CD4 cell count. The strength of association was expressed as an odds ratio (OR) with 95% confidence intervals (CIs) for all variables in the final models. Statistical analyses were performed using R software (version 2.10.1) [18] and Stata (Foundation for Statistical Computing; StataCorp).
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