Differential CD4+ cell count increase and CD4+ :  CD8+ ratio normalization with maraviroc compared with tenofovir.

OBJECTIVE: Studies exploring the immunologic effects of maraviroc (MVC) have produced mixed results; hence, it remains unclear whether MVC has unique immunologic effects in comparison with other antiretroviral drugs. We sought to determine whether MVC has differential effects compared with tenofovir disoproxil fumarate (TDF) during initial antiretroviral therapy. DESIGN: Prospective study in AIDS Clinical Trials Group A5303, a double-blind, placebo-controlled trial (N = 262) of MVC vs. TDF, each combined with boosted darunavir and emtricitabine. METHODS: A total of 31 cellular and soluble biomarkers were assayed at weeks 0 and 48. Polychromatic flow cytometry was performed on cryopreserved peripheral blood mononuclear cells. Soluble markers were assayed in plasma using ELISA kits. Analyses were as treated. RESULTS: Analyses included 230 participants (119 in MVC arm and 111 in TDF arm). Over 48 weeks of treatment, no significant differences were detected in declines in markers of inflammation and activation with MVC vs. TDF. A greater CD4 T-cell count increase (median +234 vs. +188 cells/µl, P = 0.036), a smaller CD8 T-cell count decrease (-6 vs. -109 cells/µl, P = 0.008), and a smaller CD4 : CD8 ratio increase (0.26 vs. 0.39, P = 0.003) occurred with MVC. Among participants with a baseline CD4 : CD8 ratio less than 1, a smaller proportion of MVC group normalized to a ratio greater than 1 at week 48 (15 and 36%, P < 0.001). CONCLUSION: MVC resulted in less improvement in the CD4 : CD8 ratio driven by greater increase in CD4 cell count but smaller decline in CD8 cell count. Changes in soluble or cellular biomarkers of inflammation and immune activation were not different between MVC and TDF.

The Effect of Chloroquine on Immune Activation and Interferon Signatures Associated with HIV-1.

Immune activation associated with HIV-1 infection contributes to morbidity and mortality. We studied whether chloroquine, through Toll-like receptor (TLR) antagonist properties, could reduce immune activation thought to be driven by TLR ligands, such as gut-derived bacterial elements and HIV-1 RNAs. AIDS Clinical Trials Group A5258 was a randomized, double-blind, placebo-controlled study in 33 HIV-1-infected participants off antiretroviral therapy (ART) and 37 participants on ART. Study participants in each cohort were randomized 1:1 to receive chloroquine 250 mg orally for the first 12 weeks then cross over to placebo for 12 weeks or placebo first and then chloroquine. Combining the periods of chloroquine use in both arms of the on-ART cohort yielded a modest reduction in the proportions of CD8 T cells co-expressing CD38 and DR (median decrease = 3.0%, p = .003). The effect on immune activation in the off-ART cohort was likely confounded by increased plasma HIV-1 RNA during chloroquine administration (median 0.29 log10 increase, p < .001). Transcriptional analyses in the off-ART cohort showed decreased expression of interferon-stimulated genes in 5 of 10 chloroquine-treated participants and modest decreases in CD38 and CCR5 RNAs in all chloroquine-treated participants. Chloroquine modestly reduced immune activation in ART-treated HIV-infected participants. Clinical Trials Registry Number: NCT00819390.

Characterization of IL-22 and IL-17 Expressing Leukocytes in the Cervix.

PROBLEM: Identification of the types of cells that produce IL-17 and IL-22 in the genital tract can clarify the roles that these cytokines play in responses to pathogens. METHOD OF STUDY: We isolated and stimulated cells from cervical tissue to identify and characterize cytokine-producing cells. RESULTS: Upon stimulation of CD3+ CD4+ endocervical cells, 1.6, 3.4, and 1.5% were induced to produce IL-22, IL-17, and both cytokines, respectively. Stimulation of CD3+ CD4+ ectocervical cells resulted in 3.3% IL-22+, 5.5% IL-17(+) and 2.6% IL-22(+) IL17+ cells. CD45+ CD3- cells had relatively high endogenous levels of cytokine expression that did not increase upon stimulation. Innate lymphoid cells (ILCs) made up 5.7-8% of CD45+ cervical cells and stimulation caused increases in IL-17 and IL-22. CONCLUSION: These studies show that the majority of the CD45+ leukocytes that can be induced to produce IL-22 and IL-17 in cervix are CD3+ CD4+, but ILCs are also present and can make both cytokines.

T. vaginalis Infection Is Associated with Increased IL-8 and TNFr1 Levels but with the Absence of CD38 and HLADR Activation in the Cervix of ESN.

INTRODUCTION: Trichomonas vaginalis infection is associated with an increased risk of HIV infection in exposed-seronegative women (ESN) despite their unique immune quiescent profile. It is important to understand possible mechanisms, such as recruitment of activated T cells, by which T. vaginalis could facilitate HIV infection in this population. METHODS: We conducted a cross-sectional study exploring the relationships between T. vaginalis infection, inflammatory markers and T cell activation in the cervix of ESN. During scheduled study visits, participants completed a behavioral questionnaire and physical exam, including sexually transmitted infection (STI) screening and collection of endocervical sponge and cytobrush specimens. T cell and monocyte phenotypes were measured in cervical cytobrush specimens using multi-parameter flow cytometry. Cervical sponge specimens were used to measure cytokines (IL-6, IL-8,IL-10, IP-10, RANTES) using Luminex immunoassays and the immune activation marker soluble TNF receptor 1 using ELISA. RESULTS: Specimens of 65 women were tested. Twenty-one of these women were infected with T. vaginalis. T. vaginalis infection was associated with significantly increased concentrations of IL-8 (1275pg/ml vs. 566pg/ml, p=.02) and sTNFr1 (430 pg/ml vs. 264 pg/ml, p=.005). However, T. vaginalis infection was not associated with increased percent expression of CCR5+ T cells nor increased CD38 and HLADR activation compared to uninfected women. It was also not associated with increased expression of CCR5+ monocytes. CONCLUSIONS: Among ESN T. vaginalis infection is associated with increased levels of genital pro-inflammatory/immune activation markers IL-8 and TNFr1, but was not associated with an increased percentage of activated endocervical T cells along the CD38 and HLADR pathways. Thus, while T.vaginalis infection may result in some reversal of the immune quiescent profile of ESN, enhanced recruitment of activated CD38 and HLADR expressing CD4+ cells into the endocervix may not be part of the mechanism by which Trichomonas infection alters HIV susceptibility in this unique subset of women.

Differential levels of soluble inflammatory markers by human immunodeficiency virus controller status and demographics.

Background. Human immunodeficiency virus (HIV)-1 elite controllers (ECs) represent an ideal population to study the effects of HIV persistence on chronic inflammation in the absence of antiretroviral therapy (ART). Methods. Twenty inflammatory markers measured in cohorts of ECs, HIV suppressed noncontrollers, and HIV-uninfected controls were compared using rank-based tests and partial least squares discriminant analysis (PLSDA). Spearman correlations were determined among the inflammatory markers, residual viremia by the single-copy assay, and CD4(+) T cell slope. Results. Significant differences were seen between cohorts in 15 of the soluble inflammatory markers. Human immunodeficiency virus-1 ECs were found to have the highest levels for all of the markers with the exception of RANTES. In particular, median levels of 7 inflammatory markers (soluble CD14 [sCD14], interferon [IFN]-γ, IFN-γ-inducible protein [IP]-10, interleukin [IL]-4, IL-10, sCD40L, and granulocyte-macrophage colony-stimulating factor) were twice as high in the HIV-1 ECs compared with either of the HIV-suppressed or uninfected groups. Multivariate PLSDA analysis of inflammatory markers improved differentiation between the patient cohorts, discerning gender differences in inflammatory profile amongst individuals on suppressive ART. Soluble markers of inflammation in ECs were not associated with either levels of residual HIV-1 viremia or CD4(+) T cell decline. Conclusions. Despite maintaining relatively low levels of viremia, HIV-1 ECs had elevated levels of a set of key inflammatory markers. Additional studies are needed to determine whether ECs may benefit from ART and to further evaluate the observed gender differences.

A matched cross-sectional study of the association between circulating tissue factor activity, immune activation and advanced liver fibrosis in hepatitis C infection.

BACKGROUND: Tissue factor (TF) is a protein that mediates the initiation of the coagulation cascade. TF expression is increased in patients with poorly-controlled HIV, and may be associated with increased immune activation that leads to cardiovascular morbidity. The role of TF in immune activation in liver disease in hepatitis C virus (HCV)-monoinfection and HIV/HCV-coinfection has not been explored. METHODS: Fifty-nine patients were stratified: A) HIV-monoinfection (N = 15), B) HCV-monoinfection with chronic hepatitis C (CHC) (N = 15), C) HIV/HCV-coinfection with CHC (N = 14), and D) HIV/HCV-seropositive with cleared-HCV (N = 15). All HIV+ patients had undetectable HIV viremia. Whole blood was collected for CD4/CD8 immune activation markers by flow cytometry and plasma was assayed for microparticle TF (MPTF) activity. Subjects underwent transient elastography (TE) to stage liver fibrosis. Undetectable versus detectable MPTF was compared across strata using Fisher's Exact test. RESULTS: MPTF activity was more frequently detected among patients with HCV-monoinfection (40%), compared to HIV-monoinfection and HIV/HCV-seropositive with cleared HCV (7%) and HIV/HCV-coinfection with CHC (14%) (p = 0.02). Mean TE-derived liver stiffness score in kPa was higher in patients with detectable MPTF (12.4 ± 8.5) than those with undetectable MPTF (6.4 ± 3.0) (p = 0.01). Mean CD4 + HLADR+ and CD4 + CD38-HLADR+ expression were higher in those with detectable MPTF (44 ± 9.8% and 38 ± 8.7%, respectively) than those with undetectable MPTF (36 ± 11% and 31 ± 10.4% respectively) (p = 0.05 and 0.04 respectively). CONCLUSIONS: HCV-monoinfection and HIV/HCV-coinfection with CHC were associated with MPTF activity. MPTF activity is also associated with advanced liver fibrosis and with CD4 + HLADR+ immune activation.

Modulation of TIM-3 expression on NK and T cell subsets in HIV immunological non-responders.

Highly active antiretroviral therapy (HAART) for the treatment of HIV infection sustains viral suppression and increases CD4(+) T cells in HIV patients. However, in 10-25% of subjects, known as immunological non-responders (INRs), HAART does not increase CD4 count. We investigated a potential role for galectin-9 and TIM-3 in INRs as galectin-9 and TIM-3 have been described to modulate NK and T cell function. PBMCs were isolated from healthy controls, HIV immunological responders (IRs, >350CD4(+) cells/mm(3)) and HIV INRs (<350CD4(+) cells/mm(3)) and TIM-3 and galectin-9 expressions on NK cell subsets and CD4(+) T cells were assessed. HIV INRs and HIV IRs showed increased galectin-9 expression on CD16(-)CD56(bright) and CD16(+)CD56(+) NK cells and CD4(+) T cells. Only HIV INRs showed a reduced frequency of TIM-3-expressing CD16(+)CD56(+), CD16(+)CD56(-) and CD4(+) cells, which correlated with low peripheral CD4 counts. These data suggest that TIM-3 expression may be characteristic for HIV INRs.

Soluble markers of inflammation and coagulation but not T-cell activation predict non-AIDS-defining morbid events during suppressive antiretroviral treatment.

BACKGROUND: Defining the association of non-AIDS-defining events with inflammation and immune activation among human immunodeficiency virus (HIV)-infected persons with antiretroviral therapy (ART)-associated virological suppression is critical to identifying interventions to decrease the occurrence of these events. METHODS: We conducted a case-control study of HIV-infected subjects who had achieved virological suppression within 1 year after ART initiation. Cases were patients who experienced non-AIDS-defining events, defined as myocardial infarction, stroke, non-AIDS-defining cancer, non-AIDS-defining serious bacterial infection, or death. Controls were matched to cases on the basis of age, sex, pre-ART CD4(+) T-cell count, and ART regimen. Peripheral blood mononuclear cells and plasma specimens obtained at the visit before ART initiation (hereafter, baseline), the visit approximately 1 year after ART initiation (hereafter, year 1), and the visit immediately preceding the non-AIDS-defining event (hereafter, pre-event) were analyzed for activated CD4(+) and CD8(+) T cells, plasma interleukin 6 (IL-6) level, soluble tumor necrosis factor receptor I (sTNFR-I) level, sTNFR-II level, soluble CD14 level, kynurenine-to-tryptophan (KT) ratio, and D-dimer level. Conditional logistic regression analysis was used to study the association between biomarkers and outcomes, with adjustment for potential confounders. RESULTS: Higher IL-6 level, sTNFR-I level, sTNFR-II level, KT ratio, and D-dimer level at year 1 were associated with the occurrence of a non-AIDS-defining event. Significant associations were also seen between non-AIDS-defining events and values of these biomarkers in specimens obtained at baseline and the pre-event time points. Effects remained significant after control for confounders. T-cell activation was not significantly related to outcomes. CONCLUSIONS: Interventional trials to decrease the incidence of non-AIDS-defining events among HIV-infected persons with virological suppression should consider targeting the pathways represented by these soluble markers. Clinical Trials Registration. NCT00001137.

Macrophage inflammatory markers are associated with subclinical carotid artery disease in women with human immunodeficiency virus or hepatitis C virus infection.

OBJECTIVE: Infection with hepatitis C virus (HCV) or human immunodeficiency virus (HIV) may be associated with atherosclerosis and vascular disease. Macrophages are a major component of atherosclerotic plaque, and classically activated (M1) macrophages contribute to plaque instability. Our goal was to identify plasma biomarkers that reflect macrophage inflammation and are associated with subclinical atherosclerosis. APPROACH AND RESULTS: We tested whether M1 macrophages produce galectin-3-binding protein in vitro. Then, we measured galectin-3-binding protein and the soluble macrophage biomarkers soluble cluster of differentiation (CD) 163 and soluble CD14 in 264 participants in the Women's Interagency HIV Study. Women were positive for HIV, HCV, both, or neither (66 in each group, matched for age, race/ethnicity, and smoking status). Carotid artery disease was assessed by ultrasound measurement of right distal common carotid artery intima-media thickness, distensibility, and presence of atherosclerotic lesions (intima-media thickness >1.5 mm). Plasma galectin-3-binding protein was higher in HCV+ than HCV- women (P<0.01) but did not differ by HIV status. The 3 inflammatory macrophage markers were significantly correlated with each other and negatively correlated with CD4+ counts in HIV-infected women. We defined a macrophage score as 1, 2, or 3 biomarkers elevated above the median. In models adjusted for traditional risk factors, higher macrophage scores were significantly associated with increased atherosclerotic lesions and lower carotid distensibility. Receiver-operator curve analysis of lesions revealed that the markers added predictive value beyond traditional risk factors and C-reactive protein. CONCLUSIONS: The macrophage inflammatory markers galectin-3-binding protein, soluble CD163, and soluble CD14 are significantly associated with carotid artery disease in the setting of HIV and HCV infection.

Optimizing viable leukocyte sampling from the female genital tract for clinical trials: an international multi-site study.

BACKGROUND: Functional analysis of mononuclear leukocytes in the female genital mucosa is essential for understanding the immunologic effects of HIV vaccines and microbicides at the site of HIV exposure. However, the best female genital tract sampling technique is unclear. METHODS AND FINDINGS: We enrolled women from four sites in Africa and the US to compare three genital leukocyte sampling methods: cervicovaginal lavages (CVL), endocervical cytobrushes, and ectocervical biopsies. Absolute yields of mononuclear leukocyte subpopulations were determined by flow cytometric bead-based cell counting. Of the non-invasive sampling types, two combined sequential cytobrushes yielded significantly more viable mononuclear leukocytes than a CVL (p<0.0001). In a subsequent comparison, two cytobrushes yielded as many leukocytes (∼ 10,000) as one biopsy, with macrophages/monocytes being more prominent in cytobrushes and T lymphocytes in biopsies. Sample yields were consistent between sites. In a subgroup analysis, we observed significant reproducibility between replicate same-day biopsies (r = 0.89, p = 0.0123). Visible red blood cells in cytobrushes increased leukocyte yields more than three-fold (p = 0.0078), but did not change their subpopulation profile, indicating that these leukocytes were still largely derived from the mucosa and not peripheral blood. We also confirmed that many CD4(+) T cells in the female genital tract express the α4ß7 integrin, an HIV envelope-binding mucosal homing receptor. CONCLUSIONS: CVL sampling recovered the lowest number of viable mononuclear leukocytes. Two cervical cytobrushes yielded comparable total numbers of viable leukocytes to one biopsy, but cytobrushes and biopsies were biased toward macrophages and T lymphocytes, respectively. Our study also established the feasibility of obtaining consistent flow cytometric analyses of isolated genital cells from four study sites in the US and Africa. These data represent an important step towards implementing mucosal cell sampling in international clinical trials of HIV prevention.

Characterization of CD4⁺ T-cell immune activation and interleukin 10 levels among HIV, hepatitis C virus, and HIV/HCV-coinfected patients.

BACKGROUND: HIV/hepatitis C virus (HCV)-coinfected patients have accelerated liver disease compared with HCV monoinfection. In HIV-positive patients with viral suppression, data comparing inflammatory cytokines and immune activation between HIV/HCV coinfection with chronic hepatitis C (CHC) to HIV/HCV-seropositive patients with cleared HCV are limited. METHODS: Fifty-nine age- and sex-matched patients were stratified: (1) HIV monoinfection (n = 15); (2) HCV monoinfection with CHC (n = 15); (3) HIV/HCV coinfection with CHC (n = 14); and (4) HIV/HCV seropositive with cleared HCV (n = 15). All HIV-positive patients had undetectable HIV viremia, and median CD4 was 420 cells per microliter. Liver fibrosis was assessed in each subject using transient elastography. Cells were collected for CD4 and CD8 immune activation (CD38/HLA-DR) markers via flow cytometry and plasma for luminex-multiplex cytokine assays. RESULTS: CD38⁺HLA-DR⁺ expression on CD4⁺ T cells was significantly increased in HIV/HCV coinfection with CHC (7%) versus HCV monoinfection (4%) (P = 0.012). CD4⁺ total HLA-DR⁺ expression was significantly increased in HIV/HCV coinfection with CHC (43%) versus HIV monoinfection (31%) (P = 0.010) and HIV/HCV seropositive with cleared HCV (38%) (P = 0.046). Total CD4⁺CD38⁺ and CD4⁺CD38⁺HLA-DR⁻ expression was significantly higher in HIV monoinfection (23% and 18%) than HCV moninfection (13%, P = 0.002% and 9%, P = 0.001, respectively). Interleukin 10 levels were significantly lower in HIV monoinfection versus HIV/HCV coinfection with CHC (P = 0.0002). In multivariate analysis, severe fibrosis was associated with lower expression of CD4⁺CD38⁺HLA-DR⁺ and CD4⁺ total CD38⁺ than mild-moderate fibrosis (P = 0.03 and 0.03, respectively). CONCLUSIONS: CD4 immune activation with HLA-DR⁺ expression in HIV/HCV coinfection with well-controlled HIV may arise from chronic HCV viremia. Conversely, CD4⁺CD38⁺ expression may be driven by underlying HIV infection. CD4 immune activation was unexpectedly found to be associated with decreased liver fibrosis.