Anna C. Hearps

Aging is associated with chronic innate immune activation and dysregulation of monocyte phenotype and function

Chronic inflammation in older individuals is thought to contribute to inflammatory, age‐related diseases. Human monocytes are comprised of three subsets (classical, intermediate and nonclassical subsets), and despite being critical regulators of inflammation, the effect of age on the functionality of monocyte subsets remains to be fully defined. In a cross‐sectional study involving 91 healthy male (aged 20–84 years, median 52.4) and 55 female (aged 20–82 years, median 48.3) individuals, we found age was associated with an increased proportion of intermediate and nonclassical monocytes (P = 0.002 and 0.04, respectively) and altered phenotype of specific monocyte subsets (e.g. increased expression of CD11b and decreased expression of CD38, CD62L and CD115). Plasma levels of the innate immune activation markers CXCL10, neopterin (P < 0.001 for both) and sCD163 (P = 0.003) were significantly increased with age. Whilst similar age‐related changes were observed in both sexes, monocytes from women were phenotypically different to men [e.g. lower proportion of nonclassical monocytes (P = 0.002) and higher CD115 and CD62L but lower CD38 expression] and women exhibited higher levels of CXCL10 (P = 0.012) and sCD163 (P < 0.001) but lower sCD14 levels (P < 0.001). Monocytes from older individuals exhibit impaired phagocytosis (P < 0.05) but contain shortened telomeres (P < 0.001) and significantly higher intracellular levels of TNF both at baseline and following TLR4 stimulation (P < 0.05 for both), suggesting a dysregulation of monocyte function in the aged. These data show that aging is associated with chronic innate immune activation and significant changes in monocyte function, which may have implications for the development of age‐related diseases.

HIV infection induces age-related changes to monocytes and innate immune activation in young men that persist despite combination antiretroviral therapy.

Objectives:To compare the impact of HIV infection and healthy ageing on monocyte phenotype and function and determine whether age-related changes induced by HIV are reversed in antiretroviral treated individuals. Design:A cross sectional study of monocyte ageing markers in viremic and virologically suppressed HIV-positive males aged 45 years or less and age-matched and elderly (≥65 years) HIV-uninfected individuals. Methods:Age-related changes to monocyte phenotype and function were measured in whole blood assays ex vivo on both CD14++CD16− (CD14+) and CD14variableCD16+ (CD16+) subsets. Plasma markers relevant to innate immune activation were measured by ELISA. Results:Monocytes from young viremic HIV-positive males resemble those from elderly controls, and show increased expression of CD11b (P < 0.0001 on CD14+ and CD16+subsets) and decreased expression of CD62L and CD115 (P = 0.04 and 0.001, respectively, on CD14+ monocytes) when compared with young uninfected controls. These changes were also present in young virologically suppressed HIV-positive males. Innate immune activation markers neopterin, soluble CD163 and CXCL10 were elevated in both young viremic (P < 0.0001 for all) and virologically suppressed (P = 0.0005, 0.003 and 0.002, respectively) HIV-positive males with levels in suppressed individuals resembling those observed in elderly controls. Like the elderly, CD14+ monocytes from young HIV-positive males exhibited impaired phagocytic function (P = 0.007) and telomere-shortening (P = 0.03) as compared with young uninfected controls. Conclusion:HIV infection induces changes to monocyte phenotype and function in young HIV-positive males that mimic those observed in elderly uninfected individuals, suggesting HIV may accelerate age-related changes to monocytes. Importantly, these defects persist in virologically suppressed HIV-positive individuals.

Age-associated changes in monocyte and innate immune activation markers occur more rapidly in HIV infected women

Background Aging is associated with immune dysfunction and the related development of conditions with an inflammatory pathogenesis. Some of these immune changes are also observed in HIV infection, but the interaction between immune changes with aging and HIV infection are unknown. Whilst sex differences in innate immunity are recognized, little research into innate immune aging has been performed on women. Methods This cross-sectional study of HIV positive and negative women used whole blood flow cytometric analysis to characterize monocyte and CD8+ T cell subsets. Plasma markers of innate immune activation were measured using standard ELISA-based assays. Results HIV positive women exhibited elevated plasma levels of the innate immune activation markers CXCL10 (p<0.001), soluble CD163 (sCD163, p = 0.001), sCD14 (p = 0.022), neopterin (p = 0.029) and an increased proportion of CD16+ monocytes (p = 0.009) compared to uninfected controls. Levels of the innate immune aging biomarkers sCD163 and the proportion of CD16+ monocytes were equivalent to those observed in HIV negative women aged 14.5 and 10.6 years older, respectively. CXCL10 increased with age at an accelerated rate in HIV positive women (p = 0.002) suggesting a synergistic effect between HIV and aging on innate immune activation. Multivariable modeling indicated that age-related increases in innate immune biomarkers CXCL10 and sCD163 are independent of senescent changes in CD8+ T lymphocytes. Conclusions Quantifying the impact of HIV on immune aging reveals that HIV infection in women confers the equivalent of a 10–14 year increase in the levels of innate immune aging markers. These changes may contribute to the increased risk of inflammatory age-related diseases in HIV positive women.

Virologically Suppressed HIV Patients Show Activation of NK Cells and Persistent Innate Immune Activation

FcRγ is an ITAM-containing adaptor required for CD16 signaling and function in NK cells. We have previously shown that NK cells from HIV patients receiving combination antiretroviral therapy (cART) have decreased FcRγ expression, but the factors causing this are unknown. We conducted a cross-sectional study of cART-naive viremic patients (ART−), virologically suppressed patients receiving cART (ART+), and HIV-uninfected controls. CD8+ T cells were activated, as assessed by CD38+HLA-DR+ expression, in ART− patients (p < 0.0001), which was significantly reduced in ART+ patients (p = 0.0005). In contrast, CD38+HLA-DR+ NK cells were elevated in ART− patients (p = 0.0001) but did not decrease in ART+ patients (p = 0.88). NK cells from both ART− and ART+ patients showed high levels of spontaneous degranulation in ex vivo whole blood assays as well as decreased CD16 expression (p = 0.0001 and p = 0.0025, respectively), FcRγ mRNA (p < 0.0001 for both groups), FcRγ protein expression (p = 0.0016 and p < 0.0001, respectively), and CD16-dependent Syk phosphorylation (p = 0.0001 and p = 0.003, respectively). HIV-infected subjects showed alterations in NK activation, degranulation, CD16 expression and signaling, and elevated plasma markers of inflammation and macrophage activation, that is, neopterin and sCD14, which remained elevated in ART+ patients. Alterations in NK cell measures did not correlate with viral load or CD4 counts. These data show that in HIV patients who achieve viral suppression following cART, NK cell activation persists. This suggests that NK cells respond to factors different from those driving T cell activation, but which are associated with inflammation in HIV patients.

Inhibition of Telomerase Activity by Human Immunodeficiency Virus (HIV) Nucleos(t)ide Reverse Transcriptase Inhibitors: A Potential Factor Contributing to HIV-Associated Accelerated Aging

BACKGROUND Human immunodeficiency virus (HIV)-infected patients on combination active antiretroviral therapy (cART) are at increased risk of age-related complications. We hypothesized that nucleos(t)ide reverse transcriptase inhibitors (NRTI) may contribute to accelerated aging in HIV-infected individuals on cART via inhibition of telomerase activity. METHODS Telomerase activity and telomere length (TL) were measured by quantitative polymerase chain reaction in vitro in activated peripheral blood mononuclear cells (PBMCs) cultured with NRTI and ex vivo in PBMCs from uninfected patients exposed to NRTI and from HIV-infected patients on NRTI-containing cART. RESULTS Lamivudine, abacavir, zidovudine, emtricitabine, and tenofovir significantly inhibited telomerase activity in activated PBMCs in vitro. Tenofovir was the most potent inhibitor of telomerase activity and caused greatest shortening of TL in vitro at the therapeutic concentration of 0.3 μM. PBMCs from HIV-infected patients receiving NRTI-containing cART (n = 39) had significantly lower telomerase activity than HIV-uninfected patients (n = 47; P = .011) and HIV-infected patients receiving non-NRTI-containing cART (n = 11; P < .001). TL was significantly inversely associated with age (P = .009) and the total duration on any NRTI (P = .01). CONCLUSIONS NRTIs and, specifically tenofovir at therapeutic concentrations, inhibit telomerase activity leading to accelerated shortening of TL in activated PBMCs. The relationship between NRTI, reduced telomerase activity, and accelerated aging requires further investigation in HIV-infected individuals on cART.

Antimicrobial and immune modulatory effects of lactic acid and short chain fatty acids produced by vaginal microbiota associated with eubiosis and bacterial vaginosis.

Lactic acid and short chain fatty acids (SCFAs) produced by vaginal microbiota have reported antimicrobial and immune modulatory activities indicating their potential as biomarkers of disease and/or disease susceptibility. In asymptomatic women of reproductive-age the vaginal microbiota is comprised of lactic acid-producing bacteria that are primarily responsible for the production of lactic acid present at ~110 mM and acidifying the vaginal milieu to pH ~3.5. In contrast, bacterial vaginosis (BV), a dysbiosis of the vaginal microbiota, is characterized by decreased lactic acid-producing microbiota and increased diverse anaerobic bacteria accompanied by an elevated pH>4.5. BV is also characterized by a dramatic loss of lactic acid and greater concentrations of mixed SCFAs including acetate, propionate, butyrate, and succinate. Notably women with lactic acid-producing microbiota have more favorable reproductive and sexual health outcomes compared to women with BV. Regarding the latter, BV is associated with increased susceptibility to sexually transmitted infections (STIs) including HIV. In vitro studies demonstrate that lactic acid produced by vaginal microbiota has microbicidal and virucidal activities that may protect against STIs and endogenous opportunistic bacteria as well as immune modulatory properties that require further characterization with regard to their effects on the vaginal mucosa. In contrast, BV-associated SCFAs have far less antimicrobial activity with the potential to contribute to a pro-inflammatory vaginal environment. Here we review the composition of lactic acid and SCFAs in respective states of eubiosis (non-BV) or dysbiosis (BV), their effects on susceptibility to bacterial/viral STIs and whether they have inherent microbicidal/virucidal and immune modulatory properties. We also explore their potential as biomarkers for the presence and/or increased susceptibility to STIs.

The importance of monocytes and macrophages in HIV pathogenesis, treatment, and cure.

Monocytes and macrophages play critical roles in HIV transmission, viral spread early in infection, and as a reservoir of virus throughout infection. There has been a recent resurgence of interest in the biology of monocyte subsets and macrophages and their role in HIV pathogenesis, partly fuelled by efforts to understand difficulties in achieving HIV eradication. This article examines the importance of monocyte subsets and tissue macrophages in HIV pathogenesis. Additionally, we will review the role of monocytes and macrophages in the development of serious non-AIDS events including cardiovascular disease and neurocognitive impairment, their significance in viral persistence, and how these cells represent an important obstacle to achieving HIV eradication.

Inflammatory Co-morbidities in HIV+ Individuals: Learning Lessons from Healthy Ageing

Increased life expectancy due to improved efficacy of cART has uncovered an increased risk of age-related morbidities in HIV+ individuals and catalyzed significant research into mechanisms driving these diseases. HIV infection increases the risk of non-communicable diseases common in the aged, including cardiovascular disease, neurocognitive decline, non-AIDS malignancies, osteoporosis, and frailty. These observations suggest that HIV accelerates immunological ageing, and there are many immunological similarities with the aged, including shortened telomeres, accumulation of senescent T cells and altered monocyte phenotype/function. However, the most critical similarity between HIV+ individuals and the elderly, which most likely underpins the heightened risk of non-communicable diseases, is chronic inflammation and associated immune activation. Here, we review the similarities between HIV+ individuals and the aged regarding the pathogenesis of inflammatory diseases, the current evidence for mechanisms driving these processes and discuss current and potential therapeutic strategies for addressing inflammatory co-morbidity in HIV+ infection.

An AlphaScreen®-Based Assay for High-Throughput Screening for Specific Inhibitors of Nuclear Import

Specific viral proteins enter the nucleus of infected cells to perform essential functions, as part of the viral life cycle. The integrase (IN) molecule of human immunodeficiency virus (HIV)–1 is of particular interest in this context due to its integral role in integrating the HIV genome into that of the infected host cell. Most IN-based antiviral compounds target the IN/DNA interaction, but since IN must first enter the nucleus before it can perform these critical functions, nuclear transport of IN is also an attractive target for therapeutic intervention. Here the authors describe a novel high-throughput screening assay for identifying inhibitors of nuclear import, particularly IN, based on amplified luminescent proximity homogeneous assay (AlphaScreen®) technology, which is high throughput, requires low amounts of material, and is efficient and cost-effective. The authors use the assay to screen for specific inhibitors of the interaction between IN and its nuclear transport receptor importin α/β, successfully identifying several inhibitors of the IN/importin α/β interaction. Importantly, they demonstrate that one of the identified compounds, mifepristone, is effective in preventing active nuclear transport of IN in transfected cells and hence may represent a useful anti-HIV therapeutic. The screen also identified broad-spectrum importin α/β inhibitors such as ivermectin, which may represent useful tools for nuclear transport research in the future. The authors validate the activity and specificity of mifepristone and ivermectin in inhibiting nuclear protein import in living cells, underlining the utility of the screening approach.

Increased glucose metabolic activity is associated with CD4+ T-cell activation and depletion during chronic HIV infection

Objectives:Glucose metabolism plays a fundamental role in supporting the growth, proliferation and effector functions of T cells. We investigated the impact of HIV infection on key processes that regulate glucose uptake and metabolism in primary CD4+ and CD8+ T cells. Design and methods:Thirty-eight HIV-infected treatment-naive, 35 HIV+/combination antiretroviral therapy, seven HIV+ long-term nonprogressors and 25 HIV control individuals were studied. Basal markers of glycolysis [e.g. glucose transporter-1 (Glut1) expression, glucose uptake, intracellular glucose-6-phosphate, and L-lactate] were measured in T cells. The cellular markers of immune activation, CD38 and HLA-DR, were measured by flow cytometry. Results:The surface expression of the Glut1 is up-regulated in CD4+ T cells in HIV-infected patients compared with uninfected controls. The percentage of circulating CD4+Glut1+ T cells was significantly increased in HIV-infected patients and was not restored to normal levels following combination antiretroviral therapy. Basal markers of glycolysis were significantly higher in CD4+Glut1+ T cells compared to CD4+Glut1− T cells. The proportion of CD4+Glut1+ T cells correlated positively with the expression of the cellular activation marker, HLA-DR, on total CD4+ T cells, but inversely with the absolute CD4+ T-cell count irrespective of HIV treatment status. Conclusion:Our data suggest that Glut1 is a potentially novel and functional marker of CD4+ T-cell activation during HIV infection. In addition, Glut1 expression on CD4+ T cells may be exploited as a prognostic marker for CD4+ T-cell loss during HIV disease progression.