HIV-1 infection develops with acute viremia and rapid depletion of CD4 T cells within mucosal-associated lymphoid tissues (MALT), particularly in gut lymphoid compartments [1–3]. HIV-1-induced disruption of MALT results in intestinal fibrosis and translocation of microbial products across the intestinal mucosa into the peripheral circulation producing high plasma levels of lipopolysaccharide (LPS) and bacterial DNA [4–6] that persist throughout chronic HIV-1 infection . Microbial translocation appears related to systemic leukocyte activation and elevated plasma levels of inflammatory proteins [5,8,9], although whether or not microbial translocation and consequent inflammation are direct causes of progression to AIDS or merely sequelae of HIV-1 infection seems to differ among cohorts [5,7–9].
Chronic HIV-1 infection is characterized by progressive activation of CD4 and CD8 T cells, as well as CD14 monocyte/macrophages [5,10–14], and gradual depletion of peripheral naive and memory CD4 T cells. Microbial translocation is implicated in systemic T-cell activation associated with HIV-1 infection  and idiopathic CD4 lymphocytopenia , providing evidence that circulating LPS or other microbial products may contribute to acquired and/or primary immunodeficiency. The premise that microbial translocation is a causative factor for systemic T-cell activation, CD4 T-cell decline, and disease progression is supported by studies of natural simian immunodeficiency virus (SIV) infection of sooty mangabeys that develop high levels of viral replication in the absence of systemic T-cell activation, CD4 T-cell decline, or microbial translocation [16–18].
In HIV-1 infection, systemic immune activation drives viral replication generating a vicious cycle of viremia, immune activation, and CD4 T-cell attrition that ultimately results in AIDS . Effective combination antiretroviral treatment (ART) rapidly suppresses viremia, restores peripheral CD4 T cells, reduces lymphocyte activation, and restores most immune functions . However, ART fails to fully restore memory T cells in MALT [20,21] or completely reverse microbial translocation even following complete suppression of viremia . A unique group of individuals receiving ART reconstitute T-cell immunity but fail to control viral replication due to the development of drug-resistant virus . This discordant outcome is not uncommon among HIV-1-infected children who develop improved T-cell function despite viral rebound [23,24]. Persistent viral replication following ART in conjunction with immune reconstitution in HIV-1-infected children provides an opportunity to study the correlates of pathogenesis, including systemic immune activation and microbial translocation, in the presence of high viral replication in a human host that might be modeled by SIV infection in sooty mangabeys [22–25].
Effects of microbial translocation in HIV-1-infected infants and children may be unique due to inherent differences in gut flora and LPS responses during early development [26–28]. Robust thymic output in younger HIV-1-infected individuals  may compensate for CD4 T-cell activation and attrition induced by microbial translocation, although systemic activation of monocytes, macrophages, microglia, or other cell types potentially contribute to HIV-1-associated illnesses such as neurological disorders, dementia, cardiovascular events, or cancer [13,30–32]. Determining the causes and consequences of inflammatory HIV-1 complications is particularly critical for infants and children who may survive, due to effective antiretroviral therapies, for several decades with chronic inflammation.
Prospectively stored plasma and cryopreserved peripheral blood mononuclear cells were obtained from a cohort of HIV-infected infants and children enrolled in a clinical trial of protease inhibitors and two nucleotide reverse transcriptase inhibitors as previously described. [23,24,33]. Briefly, all HIV-1-infected patients were infected perinatally and viral and immune outcomes were known. The current study included 33 of the 40 patients who displayed T-cell immune reconstitution and completed 96 weeks on the study. The protocol was approved by University of Florida and University of South Florida/All Children's Hospital Institutional Review Boards. Control participants were volunteer healthy infants and children with no underlying medical conditions, recruited over the same time frame according to a separate protocol approved by both institutions. Volunteers with gastrointestinal conditions were excluded. Whole blood samples were collected, using phlebotomy, in sterile Vacutainer (Becton Dickinson, Franklin Lakes, New Jersey, USA) acid citrate dextrose tubes, and processed within 12 h [34–36]. Peripheral blood mononuclear cell and plasma samples were stored at −180°C in liquid nitrogen or at −80°C, respectively, in nonpyrogenic polypropylene cyrovials (Nunc Cryotubes; Thermo Fisher Scientific, Waltham, Massachusetts, USA).
Healthy and HIV-1-infected patients ranged in age from 0.4 to 15 years (median = 1.4; 25th/75th quartiles = 0.4/13.0) and 0.2 to 17.7 years (median = 6.6; 25th/75th quartiles = 4.5/11.6), respectively (P > 0.05, Mann–Whitney U-test). Viral load, CD4 cell percentage, and CD4 CD45RO percentage were analyzed at 0, 4, 12, 48, and 96 weeks post-ART. Prior to ART, HIV-1-infected patients had plasma viral loads ranging from 2.9 to 6.1 log10 HIV-1 RNA copies/ml (median = 4.6 log10). By 24 weeks of ART, all patients experienced increased CD4 cell percentage either with viral suppression [<400 viral RNA copies/ml plasma] that was sustained for 96 weeks or viral rebound [>400 viral RNA copies/ml plasma] (24-week median = 3.9 log10 copies/ml; 96-week median = 4.0 log10 copies/ml) [23,24,33].
Measurement of CD4 and CD8 T-cell activation
Polychromatic flow cytometry analysis was performed with fluorescence-tagged monoclonal antibodies described previously [29,37] and a multiparameter LSR2 flow cytometer incorporating the cytometer setup and tracking program and Diva software (BD Biosciences, San Jose, California, USA). Activated effector/memory CD4 T cells were defined as CD3 CD4 cells expressing CD45RO. . Activated CD3 CD8 T cells were defined as CD45RA+ CD27− CD11a++ (bright), a phenotype associated with HIV-1 disease progression [29,39–41]. The frequency of recent thymic emigrant T cells was determined by performing T-cell receptor excision circle (TREC) analysis as previously described .
Measurement of soluble markers for immune activation and microbial translocation
Plasma samples were diluted 1: 4 in 0.15 mol/l NaCl, heat inactivated at 65°C for 30 min, then used to measure soluble CD27 (sCD27) by PeliKine human soluble CD27 ELISA kit (RDI Fitzgerald Industries, Acton, Massachusetts, USA); soluble CD14 by Human sCD14 Immunoassay (R&D Systems, Minneapolis, Minnesota, USA); LPS levels by limulus amebocyte lysate assay (LAL) Chromogenic Endpoint Assay (Lonza Group, Ltd, Allendale, New Jersey, USA); tumor necrosis factor (TNF) (TNF superfamily, member 2; TNFα) levels by human TNF ELISA (BD Biosciences); and antiendotoxin core antigen antibodies [EndoCAb] IgM levels by EndoCAb ELISA (Cell Sciences Inc., Canton, Massachusetts, USA). Specimens were handled in an endotoxin-free environment, and controls were performed with all LAL assays to rule out endotoxin contamination.
Pearson's correlation and simple linear regression analyses were performed to determine the relationships between two variables. Cross-sectional comparisons of median clinical values between different groups of study volunteers were performed by Mann–Whitney U test. Longitudinal comparison between pre-ART and post-ART values was performed by Wilcoxon matched pairs test. Power analysis was performed based on a one-way ANOVA for three group comparisons. Sample size has an 80% power to identify a minimum detectable effect size of 0.95 SD in the group differences. Effects of continuous predictors and their interactions on CD4 cell percentage were measured and tested for significance by using a multiple regression model. Analyses were performed with GraphPad Prism 5.0 software (La Jolla, California, USA) or SAS software (Cary, North Carolina, USA) and considered significant when P value was less than 0.05.
Pretherapy CD4 CD45RO T-cell frequency and viremia differentially correlate with CD4 T-cell decline
Increased frequency of effector/memory CD4 CD45RO T cells, reduced frequency of CD4 CD45RA naive T cells, and activation of CD8 T cells are associated with CD4 T-cell decline and progression to AIDS in HIV-1 infection [29,42]. As expected, prior to ART, HIV-1-infected patients displayed significant inverse correlations between overall CD4 T-cell percentage and frequency of CD4 CD45RO T cells (P < 0.0001), frequency of CD8 CD45RA T cells with an activated CD27−CD11a bright phenotype (P < 0.05), or plasma viral load (P < 0.01) (Fig. 1a, c, and e). Pre-ART measures of T-cell frequency, T-cell activation, or viral load were similar between groups who ultimately suppressed, (viral success) or failed to suppress (viral failure) viral burden following ART (Fig. 1b, d, and f).
To determine the effects of length of infection (LOI), which was identical to age as all patients were infected perinatally, a multiple regression model was applied. CD4 cell decline was related inversely to the frequency of CD4 CD45RO cells, a correlation more pronounced in infants than children and adolescents (Fig. 1g, P < 0.01). Conversely, a trend in the relationship between viral load and CD4 T-cell attrition was more pronounced among older rather than younger children (Fig. 1h, P = 0.097). These analyses indicated that age/LOI is a determining factor in the relationship between viral load, CD4 T-cell activation, and CD4 T-cell decline.
Microbial translocation develops concomitant with multilineage leukocyte activation in HIV-1 infection
Microbial translocation develops coincident to systemic inflammation in some, but not all HIV-1-infected patients [5,7,8]. Microbial translocation (LPS) was evident among all therapy naive HIV-1-infected patients, as well as a subset of healthy children (Fig. 2a, P < 0.05). Among healthy volunteers, microbial translocation was more pronounced in infants (0–2 years), than in children or adolescents (>2–18 years) (Fig. 2b, P < 0.05). Levels of LPS in HIV-1-infected infants were comparable with healthy infants, whereas infected children had higher levels than healthy children (P < 0.01). The monocyte/macrophage soluble activation marker sCD14 was detected in the plasma of all healthy and HIV-1-infected pediatric patients. In our study sCD14 levels (median sCD14 = 20.4 ng/ml) were markedly lower in healthy pediatric volunteers, including infants who had microbial translocation, than sCD14 levels reported for healthy adults [5,7] (Fig. 2b and c). In contrast, sCD14 levels were significantly elevated (median sCD14 = 931.8 ng/ml) in HIV-1-infected pediatric patients compared with healthy volunteers (Fig. 2c, P < 0.0001). EndoCAb levels were significantly reduced in HIV-1-infected patients (Fig. 2d, P < 0.05), although unrelated to LPS levels in HIV-1-infected or healthy children (Fig. 2e or data not shown). Levels of sCD27, reflecting generalized lymphocyte activation, were elevated in HIV-1-infected patients compared with healthy controls (Fig. 2f, P < 0.0001).
The cytokine TNF, a key mediator of systemic inflammation, was undetectable among healthy volunteers, but detected in 8 of 33 HIV-1-infected patients prior to ART (Fig. 3a). No difference in LPS levels between TNF(−) and TNF(+) patients was identified (Fig. 3b). Microbial translocation displayed a trend toward a positive correlation with activation of monocyte/macrophages (Fig. 3c, P = 0.055). In contrast, LPS levels showed no relationship to general lymphocyte activation measured by sCD27 levels or frequency of CD4 CD45RO T cells or activated CD8 T cells (Fig. 3d, e, and f). Together, these results indicate that microbial translocation was independent of lymphocyte activation.
Microbial translocation or systemic inflammation may contribute to CD4 T-cell attrition in HIV-1 infection. Prior to ART, plasma levels of LPS were unrelated to CD4 T-cell levels (Fig. 4a). Likewise, neither sCD14 nor sCD27 levels were associated with CD4 cell percentage (Fig. 4b and c). Overall, pre-ART levels of markers for microbial translocation or multilineage leukocyte activation were unrelated to CD4 T-cell decline and similar between patients who developed viral success or failure following ART (Fig. 4d, e, and f).
Microbial translocation and monocyte/macrophage activation both persist independent of antiretroviral treatment-related CD4 T-cell reconstitution
ART results in significant immune reconstitution and improved immune function in individuals who optimally control viral replication, as well as in patients who develop viral rebound with drug-resistant virus . In longitudinal analyses, viral success patients suppressed viremia below limits of detection by 24 weeks of ART (Fig. 5a, P < 0.0001) with concomitant improvement in CD4 T-cell frequency (Fig. 5b, P < 0.01). In contrast, viral failure patients experienced transiently reduced viral loads at 4 weeks post-ART that rebounded by 24 weeks to an average >4.0 log10 copies/ml (Fig. 5a), even though CD4 T-cell frequencies improved significantly and persisted at levels >25% (Fig. 5b, P < 0.001).
The frequency of CD4 CD45RO T cells declined by 24 weeks of ART in both outcome groups to levels approaching those in healthy volunteers (Fig. 5c). Decline in CD4 CD45RO T-cell frequency was significantly associated with increasing TRECs (Fig. 5d, P < 0.0001). Plasma sCD27 levels remained unchanged by ART in viral failure patients, but were reduced in viral success patients (Fig. 5e, P < 0.05).
Pretherapy levels of sCD14 were unchanged at 24 weeks of ART, but declined by 96 weeks to significantly lower levels in both viral success (P < 0.001) and viral failure (P < 0.05) groups. Nonetheless, elevated sCD14 persisted among HIV-1-infected individuals for 2 years after initiation of ART at levels about 20-fold higher than healthy controls (Fig. 5f). Likewise, microbial translocation was unchanged over 96 weeks of ART in viral success or viral failure patients and remained well above levels detected in healthy volunteers (Fig. 5g). No patient with undetectable TNF pre-ART developed measurable levels post-ART, whereas pretherapy plasma TNF, detected among eight patients, persisted for 96 weeks of ART (Fig. 5h). Elevated TNF had no association with CD4 cell percentage, microbial translocation or monocyte macrophage activation and was independent of viral success or viral failure outcome (data not shown).
Longitudinal changes in viremia, immune activation, and microbial translocation were compared over the course of treatment by linear regression to contemporaneous changes in CD4 cell percentage. No significant associations between changes in CD4 cell percentage and viral load, sCD27, sCD14, or LPS following 24 or 96 weeks of ART were identified. Decreasing frequency of CD4 CD45RO T cells was the only parameter related to sustained improvement in CD4 T-cell frequency during ART in viral success and viral failure patients (24 weeks: P < 0.05; 96 weeks: P < 0.001). (See Table S2, http://www.links.lww.com/QAD/A24, Supplemental Digital Content.)
HIV-1 infection results in systemic immune activation, which depends on virus replication and/or microbial translocation, and is resolved to an extent by antiretroviral therapy [5,9,14]. Systemic immune activation drives viral replication creating a cycle of T-cell attrition. When combined with viral effects on thymic T-cell output, dissecting the contribution of each disease variable to immune activation poses significant challenges. Our studies show that during the natural history of pediatric HIV-1 infection, T-cell activation and CD4 T-cell attrition are related to levels of viral replication depending on age/LOI. In contrast, macrophage activation is related to both microbial translocation and effects of HIV-1 infection that persist even when viremia is suppressed below limits of detection. Sustained immune reconstitution by ART, independent of viral outcomes, induced concordant attenuation of CD4 and CD8 T-cell activation , even though microbial translocation or macrophage activation persisted indicating that systemic immune activation in T-cell versus monocyte/macrophage compartments can be dissociated based on distinct mechanisms.
Although viral replication is the principal factor driving lymphocyte activation, microbial translocation is also implicated in systemic T-cell activation in some, but not all, natural history studies of HIV-1 infection [5,7,8]. In contrast, our study of perinatally infected children found no association between microbial translocation and T-cell activation or immune deficiency pre-ART, highlighting the complex relationships between different causes and consequences of immune activation. Following ART, reconstitution of CD4 T cells and increased TREC occurred in our cohort despite persistent microbial translocation indicating that thymic output, a major contributor to post-ART lymphocyte reconstitution [29,43,44], is independent of LPS-driven inflammation. Failure of ART to attenuate microbial translocation and LPS levels in children, unlike ART treatment of HIV-1-infected adults , may reflect irreversible intestinal fibrosis induced by early viremia during infancy . HIV-1-infection during infancy, compared with infection during adolescence or adulthood may have different effects on MALT due to normal processes of gut remodeling that occur during growth  when intestinal permeability is increased and adaptive immunity is still developing. Indeed, transient microbial translocation found in our study of healthy and HIV-1-infected infants resolved after 2 years of age only in healthy children, indicating that HIV-1 disrupted normal gut development. It would be interesting to further investigate the nature of microbial translocation in early infancy versus older HIV-1-infected children to determine if disruption of gut epithelial and endothelia tissues differs by age and illness. Microbial translocation, as a consequence of HIV-1 pathogenesis in adults, is inversely correlated with EndoCAb levels [5,47]. In our study, EndoCab levels were reduced among HIV-1-infected children, but no relationship between EndoCab and LPS levels was apparent. Developmental regulation of natural antibody production could impair the ability of perinatally infected children to neutralize and clear circulating LPS during early infection.
The LPS detected in plasma of HIV-1-infected patients is biologically active and inflammatory in vivo, reflected by the link with monocyte/macrophage activation, although healthy infants with similarly elevated LPS levels displayed no concomitant increase in monocyte/macrophage activation. HIV-1 might function as a cofactor for LPS-induced inflammation in infants. The overall extent of monocyte/macrophage activation found with HIV-1 infection could reflect direct activating effects of HIV-1 on monocytes [6,48,49] or priming by HIV-1, which sensitizes macrophages to subsequent activation with toll-like receptor ligands including LPS . Whether sCD14 detected in plasma is derived from monocytes or differentiated macrophages is unclear, but both scenarios are plausible. In vivo, macrophages are efficient targets for productive HIV-1 infection, whereas monocytes are rarely infected [51,52], although binding of HIV-1 envelope proteins to CCR5 on monocytes induces cell signaling that enhances monocyte survival  and may induce activation and shedding of sCD14.
Lack of monocyte/macrophage responsiveness to LPS in healthy infants is characteristic of normal immune development [26,27]. For example, in atopic allergy, an immune hypersensitivity disorder, sCD14 levels are lower in neonates versus older children, even though neonates have increased frequency of CD14+ cells . Breast-feeding is another variable that regulates inflammatory versus tolerogenic responses to microbial translocation. Secretory IgA in breast milk can have significant effects on gut microbial flora through alterations in colonization and prevention of transmucosal sepsis, and other soluble milk components can mediate anti-inflammatory effects . None of the neonates in our study was breast-fed, which may itself contribute to immune activation. Clearly, systemic monocyte/macrophage activation is less pronounced during childhood, and HIV-1 infection significantly disrupts normal immune homeostasis. Although ART for HIV-1-infected patients in this study improved health with significant increases in height and weight and decreased prevalence of AIDS-defining illnesses , inability of therapy to resolve either microbial translocation or monocyte/macrophage activation may contribute to accelerated immune senescence with increased long-term morbidity and mortality from non-AIDS-related inflammatory conditions .
CD27, a member of the tumor necrosis factor receptor family and its ligand, CD70, are predominantly expressed by lymphocytes . Lymphocyte activation in both the T-cell and B-cell compartments results in high levels of the soluble protein in the plasma and can be used to measure systemic immune activation in HIV-1 infection . The divergence in sCD27 levels in post-ART viral success versus viral failure patients may be linked to qualitative differences in HIV-1 phenotype and coreceptor utilization. HIV-1 isolated from patients who suppress viremia primarily use only CCR5 coreceptor for target cell entry, whereas HIV-1 from viral failure patients use both CCR5 and CXCR4 coreceptors before and after ART . Persistently elevated sCD27 demonstrates that replication of drug-resistant HIV-1 induces some component of lymphocyte activation in spite of improved CD4 T-cell counts and normal immune function , indicating that systemic immune activation and its consequences are complex and multifactorial.
Discordant outcomes following ART represent an important paradigm in HIV-1 pathogenesis, which may be modeled by high levels of SIV replication in its natural host . However, our study demonstrates fundamental differences between discordant therapy outcome in HIV-1 infection and natural SIV infection of sooty mangabeys in that CD4 T-cell pathogenesis is uncoupled from microbial translocation in HIV-1 infection. In sooty mangabeys, viremia fails to induce intestinal fibrosis or microbial translocation, which is related to the overall lack of CD4 T-cell pathogenesis or immune deficiency in this model [5,17]. In contrast, HIV-1-infected children experience increased naive CD4 T-cell counts and TREC levels after ART regardless of viral outcome, suggesting that persistently elevated LPS 2 years into therapy does not adversely impact thymic output [29,57,58]. After therapy viruses do not induce the same degree of T-cell activation as wild-type virus and are associated with higher TREC levels. In the discordant response group, after therapy drug-resistant viruses are less fit for replication in the thymus resulting in a high proportion of naive T cells in the peripheral blood [59,60]. Thus, improved CD4 T-cell frequency in post-ART discordant response patients is more likely a consequence of attenuated viral pathogenesis than changes in gut pathogenesis or microbial translocation. Similar to adults who continue failing ART regimens , discordant response in children and adolescents was associated with evidence of CD4 T-cell declines by 96 weeks, although the proportion of CD45RO CD4 T cells failed to increase, an indication that robust thymic output persisted.
Our finding that microbial translocation is not a major factor in HIV-1 immunodeficiency in infants, children, and adolescents is similar to a study of HIV-1 subtype A and D infections in African adults, who progressed to AIDS in the absence of HIV-1-specific microbial translocation and chronic HIV-1-associated inflammation . Nonetheless microbial translocation is clearly an important component of innate immune inflammation in some HIV-1-infected patients. In our study, both outcome groups developed post-ART declines in sCD14 levels, but monocyte/macrophage activation failed to normalize in either group, even following 2 years of ART, indicating that microbial translocation contributes to sustained macrophage activation that can be independent of high-level viral replication. Prolonged systemic monocyte/macrophage activation may contribute to HIV-1-associated inflammatory conditions including dementia, increased risk for cardiovascular events and thrombophilia or enhanced angiogenesis in malignancy [13,30–32]. The extent of monocyte/macrophage activation and resulting inflammatory conditions may differ in perinatally infected children compared with infected adults due to greater infectivity and HIV-1 replication in neonatal or cord blood-derived monocyte/macrophages [61,62]. Considering that perinatally infected neonates will likely survive several decades with ART, the implications of persistent microbial translocation and monocyte/macrophage activation should be a major focus of investigation in coming years.
The authors wish to thank the study volunteers for participating, the clinical coordinator Carla Duff, R.N., C.C.R.P. for enrollment and maintenance of the cohort and Brent Gardener and Amanda Lowe for managing the clinical specimen repository and clinical database. We also wish to thank Dr Grace Aldrovandi for critical reading of this manuscript.
M.A.W. conceived and designed the study, performed most experiments, collected and analyzed data and wrote the manuscript. C.A.R. enrolled patients and performed flow cytometry to assess T-cell activation and contributed to writing and revising the manuscript. L.Y. performed TREC assays and contributed to writing and revising the manuscript. S.S. performed EndoCAb ELISA. S.C. performed sCD14 ELISA. W.H. performed statistical analyses and contributed to revising the manuscript. J.W.S. oversaw the project including conception and study design, enrolled patients and healthy volunteers, specimen archiving and clinical data, and contributed to writing and revising the manuscript. M.M.G. oversaw the entire project including conception and study design, specimen archiving, clinical database management and contributed to writing and revising the manuscript.
M.A.W.: NIH T32 training grant fellowship AR007603 and the Laura McClamma Fellowship for Immune Deficiency. C.A.R., L.Y., and W.H.: Florida Center for AIDS Research. S.S.: Eleanor Dana Charitable Trust. J.W.S.: NIH grant AI047723. M.M.G.: NIH grants HD032259 and AI065265; Center for Research in Pediatric Immune Deficiency; and Stephany W. Holloway University Chair for AIDS Research.
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