PATHOGENESIS OF POOR IMMUNE RECOVERY ON COMBINATION ANTIRETROVIRAL THERAPY (CART): THE ROLE OF THE GASTROINTESTINAL TRACT AND MICROBIAL TRANSLOCATION

In the era of combination antiretroviral therapy (cART), a remarkable reduction in AIDS-related morbidity and mortality rates has been described. However, 15%\u201330% of treated individuals display a discordant response to cART, which consists of inefficient CD4+ T-cell recovery despite effective virological control. These subjects are referred to as \u201cImmunological Non Responders\u201d (INRs) and remain at a considerable higher risk of HIV progression and mortality from both AIDS and non-AIDS events and poorly responsive to experimental treatments. It is thus critical to investigate the underlying mechanisms of poor immune recovery on effective cART and elaborate targeted interventional strategies for this population in a timely manner. T-cell activation has been described an independent factor of poor CD4+ T-cell recovery on cART and INR have been shown to present significant higher levels of peripheral immune activation compared to Full Responders (FR). Building on prior evidence of the translocation of microbial bioproducts through the gastrointestinal (GI) tract as a cause of immune activation in HIV disease, studies addressing the pathogenesis of inefficient CD4+ T-cell recovery in INR have shown increased levels of circulating lipopolysaccharide (LPS) in this setting. Despite evidence of an association between microbial translocation parameters and expression of activation markers in INR, whether stimulation with microbial components per se results in the induction of T-cell activation markers in this population is unknown. Further, a biological model explaining the precise mechanisms by which exposure to microbial components causes T-cell activation in HIV disease is currently lacking. Finally, literature has so far not disentangled the possible links between GI barrier damage and poor immune reconstitution in the course of effective cART in INR. The overall objective of the present study was to understand whether damage of the GI tract and microbial-induced T-cell activation feature HIV-infected individuals with poor immune recovery on cART. Specific Aim 1: Comparative study of gut junctional complexes in HIV-infected individuals with different CD4+ T-cell recovery on cART. We aimed to analyze the structure and function of gut JC in Immunological Non Responder (INR) and Full Responder (FR) and to assess whether the fecal microbiome and/or HIV reservoirs may represent underlying causes of gut epithelial barrier dysfunction in course of treated HIV disease. Specific Aim 2: Expression of activation markers on immune cells following stimulation with microbial components in HIV-infected individuals with different CD4+ T-cell recovery on cART. We aimed to study the expression of activation markers on immune cells following stimulation with microbial components in HIV-infected individuals with different CD4+ T-cell recovery on cART. We analyzed the effect of LPS in vitro stimulation on T-cell activation markers (CD38 and HLA-DR) in HIV-infected patients with different CD4+ recovery on cART and then set up an in vitro model to assess the TLR-mediated signalling pathways in monocyte-derived macrophages (MDM) and PBMCs in a similar study population. Our experiments revealed: i) Immunohistochemical and statistical evidence of INR presenting the lowest expression of junctional complex (JC) proteins at mucosal (ileum and colon) sites, with electron microscopy proof of dilated intercellular spaces; ii) A negative correlation of CD4+ T-cell counts with intestinal JC protein expression as well as HIV reservoirs in the gut and peripheral blood; iii) A higher proportion of HLA-DR-expressing CD4+ and CD8+ T-cells in INR following lipopolysaccharide (LPS) in vitro stimulation, yet the CD38+CD8+ pool only is significantly expanded according to the degree of immunological impairment; iv) Up-regulation of T-cell activation markers following broad microbial challenge in INR, as well as heightened expression of effector and pathogen specific response genes prior to stimulation and selective upregulation of type I interferons following ssRNA stimulation; v) Preserved response of monocyte-derived macrophages (MDM) from INR following broad microbial challenge. Our findings show that incomplete immunological response in the course of effective cART associates with severe damage of the GI epithelial barrier and increased size of the HIV reservoir both at mucosal sites and in circulating T-cells, thus suggesting to target the GI tract in the elaboration of interventional strategies for INR. We also demonstrate the uniqueness of the CD8+CD38+ T-cells subset in depicting T-cell activation following LPS stimulation in individuals with poor CD4+ T-cell recovery, strengthening its possible exploitation in the clinic to monitor the immune response to cART. Consistently with these findings, we show the up-regulation of activation markers on T-cells from INR following ssRNA which appears to be involved in TLR-mediated signaling of non-CD14-dependent pathways, highlighting the importance of low-level viremia/HIV reservoirs as sources of persistent antigenic stimulation in this setting

Biomarkers of aging in HIV: inflammation and the microbiome

Purpose: HIV-infected subjects present increased levels of inflammatory cytokines and T cell activation in the peripheral blood despite suppressive combination antiretroviral therapy which renders them susceptible to premature aging. The purpose of the present work was to review existing evidence on the ways in which the anatomical and microbiological abnormalities of the gastrointestinal tract can represent a major cause of organ disease in HIV infection. Methods: We conducted a systematic review of the Pubmed database for articles published from 2014 to 2018. We included studies on inflammatory/activation biomarkers associated with cardiovascular and bone disease, neurocognitive impairment and serious non-AIDS events in HIV-infected subjects. We also included researches which linked peripheral inflammation/activation to the anatomical, immune and microbiological alterations of the gastrointestinal tract. Results: Recent literature data confirm the association between non-infectious comorbidities and inflammation in HIV infection which may be driven by gastrointestinal tract abnormalities, specifically microbial translocation and dysbiosis. Furthermore, there is mounting evidence on the possible role of metabolic functions of the microbiota in the pathogenesis of premature aging in the HIV-infected population. Conclusions: Biomarkers need to be validated for their use in the management of HIV infection. Compounds which counteract microbial translocation, inflammation and dysbiosis have been investigated as alternative therapeutic strategies in viro-suppressed HIV-infected individuals, but appear to have limited efficacy, probably due to the multifactorial pathogenesis of non-infectious comorbidities in this setting

Microbial translocation in the pathogenesis of HIV infection and AIDS

In pathogenic simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) infections, the translocation of microbial products from the gastrointestinal (GI) tract to portal and systemic circulation has been proposed as a major driver of the chronic immune activation that is associated with disease progression. Consistently, microbial translocation is not present in nonpathogenic SIV infections of natural host species. In vivo studies demonstrated that HIV/SIV-associated microbial translocation results from a series of immunopathological events occurring at the GI mucosa: (i) early and severe mucosal CD4+ depletion, (ii) mucosal immune hyperactivation/persistent inflammation; (iii) damage to the integrity of the intestinal epithelium with enterocyte apoptosis and tight junction disruption; and (iv) subverted the gut microbiome, with a predominance of opportunistic bacteria. Direct in situ evidence of microbial translocation has been provided for SIV-infected rhesus macaques showing translocated microbial products in the intestinal lamina propria and distant sites. While the mechanisms by which microbial translocation causes immune activation remain controversial, a key pathogenic event appears to be innate immunity activation via Toll-like receptors and other pathogen recognition receptors. Accumulating clinical observations suggest that microbial translocation might affect HIVdisease progression, response to therapy, and non-AIDS comorbidities. Given its detrimental effect on overall immunity, several interventions to prevent/block microbial translocation are currently under investigation as novel therapeutic agents for HIV/AIDS

Distinguishing Latent from Active Mycobacterium tuberculosis Infection Using Elispot Assays: Looking Beyond Interferon-gamma

Mycobacterium tuberculosis (MTB) is a global heath epidemic, its threat amplified by HIV infection and the emergence of multidrug-resistant tuberculosis (MDR-TB). Interferon (IFN)-gamma release assays (IGRAs) have improved the accuracy of detection of MTB exposure in some subject groups as compared to the Tuberculin Skin Test (TST). However, as IFN-gamma is produced by both fully rested and more recently activated populations of memory T cells, it is not surprising that the measurement of this cytokine alone cannot accurately distinguish Latent TB Infected (LTBI) subjects from those with active (infectious) disease. Accurate and rapid diagnosis of infectious individuals would allow medication to be properly allocated and other actions taken to more effectively curtail MTB spread. Analysis of multi-cytokine profiles ex vivo after stimulation of PBMCs from LTBI and active MTB subjects indicate the real possibility of successfully discerning these two disease states within 24 hours of a subject's blood draw. Due to the unparalleled sensitivity, low cost, and ease of use of Elispot assays, we propose that via a multiplex Elispot platform the accurate distinction of LTBI from active MTB-infected individuals is within reach

Gut-dependent inflammation and alterations of the intestinal microbiota in individuals with perinatal HIV exposure and different HIV serostatus

Objective: HIV-exposed infected (HEI) and uninfected (HEU) children represent the two possible outcomes of maternal HIV infection. Modifications of the intestinal microbiome have been linked to clinical vulnerability in both settings, yet whether HEI and HEU differ in terms of gut impairment and peripheral inflammation/activation is unknown. Design: We performed a cross-sectional, pilot study on fecal and plasma microbiome as well as plasma markers of gut damage, microbial translocation, inflammation and immune activation in HIV-infected and uninfected children born from an HIV-infected mother. Methods: Fecal and plasma microbiome were determined by means of 16S rDNA amplification with subsequent qPCR quantification. Plasma markers were quantified via ELISA. Results: Forty-seven HEI and 33 HEU children were consecutively enrolled. The two groups displayed differences in fecal beta-diversity and relative abundance, yet similar microbiome profiles in plasma as well as comparable gut damage and microbial translocation. In contrast, monocyte activation (sCD14) and systemic inflammation (IL-6) were significantly higher in HEI than HEU. Conclusion: In the setting of perinatal HIV infection, enduring immune activation and inflammation do not appear to be linked to alterations within the gut. Given that markers of activation and inflammation are independent predictors of HIV disease progression, future studies are needed to understand the underlying mechanisms of such processes and elaborate adjuvant therapies to reduce the clinical risk in individuals with perinatal HIV infection

A Comprehensive Ex Vivo Functional Analysis of Human NKT Cells Reveals Production of MIP1-α and MIP1-β, a Lack of IL-17, and a Th1-Bias in Males

NKT cells contribute to the modulation of immune responses and are believed to be important in the pathogenesis of autoimmune and infectious diseases, as well as cancer. Variations in the composite NKT cytokine response may determine individual disease susceptibility or severity. Due to low frequencies in peripheral blood, knowledge of the breadth of ex vivo human NKT cell functions has been limited. To bridge this gap, we studied highly purified NKT cells from PBMC of healthy donors and assessed the production of 27 effector functions using sensitive Elispot and multiplex bead assays. We found the ex vivo human NKT cell response is predominantly comprised of the chemokines MIP1-α, and MIP1-β as well as the Th1 cytokines IFN-γ and TNF-α. Although lower in magnitude, there was also significant production of IL-2, IL-4, and perforin after mitogen stimulation. Surprisingly, little/no IL-5, IL-6, IL-10, or IL-13 was detected, and no subjects' NKT cells produced IL-17. Comparison of the NKT functional profiles between age-matched male and female subjects revealed similar IL-4 responses, but higher frequencies of cells producing IFN-γ and MIP1-α, from males. There were no gender differences in the circulating NKT subset distribution. These findings implicate chemokines as a major mechanism by which NKT cells control responses in humans. In addition, the panoply of Th2 and Th17 cytokine secretion by NKT cells from healthy donors may not be as pronounced as previously believed. NKT cells may therefore contribute to the gender bias found in many diseases

FoxP3 mRNA Expression in Regulatory T Cells from Patients with Tuberculosis

Comment on : V. Guyot-Revol, J.A. Innes, S. Hackforth, T. Hinks, A. Lalvani, Regulatory T cells are expanded in blood and disease sites in patients with tuberculosis. [Am J Respir Crit Care Med. 2006 Apr 1 173(7):803-810

Invariant Natural Killer T (iNKT) cells in HAART-treated, HIV-positive patients with bone and cardiovascular impairment

Background: Invariant Natural Killer T (iNKT) cells represent a determinant in the course of infections and diseases, however, their role in the pathogenesis of non-infectious co-morbidities in HIV-positive patients is unknown. Methods: Flow cytometry was used to investigate iNKT cell frequency, phenotype and function in HIV-infected patients on HAART with bone and/or cardiovascular disorders and in HIV-positive controls free from co-morbidities. Results: iNKT cells from subjects with bone and cardiovascular impairment expressed high levels of CD161 and predominantly secreted TNF. iNKT cells from individuals with bone disease alone did not show any distinctive phenotypical or functional characteristics. The functional capacity of iNKT cells in patients with cardiovascular disorder was impaired with no cytokine release upon stimulation. Conclusion: iNKT cells may have a role in non-infectious co-morbidities in treated HIV disease, possibly through the exacerbation of inflammation. Further studies are needed to investigate iNKT cells in the pathogenesis of non-communicable disorders in HIV infection