Intracellular granzyme A expression of peripheral blood lymphocyte subsets in pulmonary tuberculosis

Cell-mediated immunity is a key weapon of host defence against tuberculosis (TB). Granzyme A (GzmA), a serine protease, present in the granules of cytotoxic cells induces caspase-independent cell death. We estimated the proportion of GzmA producing lymphocyte subsets in peripheral blood from 59 normal healthy volunteers and 48 pulmonary TB (PTB) patients using flow cytometry. When compared with normal healthy subjects, we observed a significantly higher percentage of GzmA-positive CD56+ cells (P = 0.01) in PTB patients. However, when the absolute number was compared between the two groups, a significantly decreased number of GzmA-expressing CD16+ (P = 0.01) and CD56+ (P = 0.0001) cells was observed in patients and this could be explained by the significantly reduced number of total lymphocytes (P = 0.0009) seen in the patients. There was no significant difference in the number of CD4+ and CD8+ GzmA double-positive cells between the two study groups. CD56 is a natural killer cell marker and these cells represent innate immune response to TB. We report an increased percentage of CD56+ cells expressing GzmA in TB patients, which shows the relevance of the GzmA-mediated pathway of apoptosis in immunity against Mycobacterium tuberculosis

Defective dendritic cell response to toll-like receptor 7/8 agonists in perinatally HIV-infected children

Understanding the defects in innate immunity associated with perinatal HIV infection is a prerequisite for effective antiretroviral treatment. We therefore compared the innate immune response [dendritic cell (DC) phenotype and function] in peripheral blood by flow cytometry at baseline and 12 months in HIV-infected children to determine the defect associated with perinatal HIV infection. As compared with controls, patients had decreased numbers of total DCs including plasmacytoid (p)DCs and myeloid (m)DCs and impaired function based on induction of maturation markers (CD83, CD80, CCR7) and cytokines tumor necrosis factor-α and interferon-α (exclusive to pDC) upon stimulation with the TLR7/8 agonist Resiquimod. These abnormalities were evident in all three CD4 immune categories and persisted over 12 months; pDC function worsened in HIV+ children without treatment and improved slightly in those on highly active antiretroviral therapy (HAART). In conclusion, a majority of perinatally HIV-infected older children without HAART remain clinically stable in the short term, but have demonstrable immunologic abnormalities indicative of defects in the innate immune system. Children initiated on HAART showed improvement in CD4 counts but did not show improvement in DC function over the short term

Immune Activation Is Associated With Increased Gut Microbial Translocation in Treatment-Naive, HIV-Infected Children in a Resource-Limited Setting

BACKGROUND: Gut damage resulting in microbial translocation (MT) is considered a major cause of immune activation (IA) in HIV infection, but data in children are limited, particularly in the absence of antiretroviral therapy. METHODS: Sixty perinatally HIV-infected, antiretroviral therapy–naive children, aged 2–12 years, were evaluated for plasma levels of lipopolysaccharide, DNA sequences encoding bacterial ribosomal 16 second (16S) RNA (16S rDNA) and soluble CD14 concurrently with markers of CD4 and CD8 T-cell IA and immune exhaustion (IE), CD4 counts, and plasma viral load. At study entry, participants were classified into immune categories (ICs): IC1 (CD4% > 25), IC2 (CD4% 15–25), and IC3 (CD4% < 15). Age-matched HIV-uninfected children served as controls. Data were evaluated at study entry and at 12 months. RESULTS: Levels of MT, IA, and IE were increased in patients as compared with controls, were highest in patients in IC3 group, and did not change over 12 months. MT products lipopolysaccharide and 16S rDNA correlated with each other and each correlated with plasma viral load, soluble CD14, and T-cell IA and IE. There was a correlation of IA with IE. CD4 counts and percentage were inversely correlated with MT products and underlying CD4 activation. CONCLUSIONS: In a natural history cohort of HIV-infected children not on therapy, MT was more pronounced in the most severely immunocompromised patients and was associated with IA. Strategies to reduce MT may help to reduce IA and prevent CD4 depletion

Dynamics of T-Lymphocyte Activation Related to Paradoxical Tuberculosis-Associated Immune Reconstitution Inflammatory Syndrome in Persons With Advanced HIV

Most persons living with HIV (PLWH) experience a significant restoration of their immunity associated with successful inhibition of viral replication after antiretroviral therapy (ART) initiation. Nevertheless, with the robust quantitative and qualitative restoration of CD4(+) T-lymphocytes, a fraction of patients co-infected with tuberculosis develop immune reconstitution inflammatory syndrome (TB-IRIS), a dysregulated inflammatory response that can be associated with significant tissue damage. Several studies underscored the role of adaptive immune cells in IRIS pathogenesis, but to what degree T lymphocyte activation contributes to TB-IRIS development remains largely elusive. Here, we sought to dissect the phenotypic landscape of T lymphocyte activation in PLWH coinfected with TB inititating ART, focusing on characterization of the profiles linked to development of TB-IRIS. We confirmed previous observations demonstrating that TB-IRIS individuals display pronounced CD4(+) lymphopenia prior to ART initiation. Additionally, we found an ART-induced increase in T lymphocyte activation, proliferation and cytotoxicity among TB-IRIS patients. Importantly, we demonstrate that TB-IRIS subjects display higher frequencies of cytotoxic CD8(+) T lymphocytes which is not affected by ART. Moreover, These patients exhibit higher levels of activated (HLA-DR(+)) and profilerative (Ki-67(+)) CD4(+) T cells after ART commencenment than their Non-IRIS counterparts. Our network analysis reveal significant negative correlations between Total CD4(+) T cells counts and the frequencies of Cytotoxic CD8(+) T cells in our study population which could suggest the existance of compensatory mechanisms for Mtb-infected cells elimination in the face of severe CD4(+) T cell lymphopenia. We also investigated the correlation between T lymphocyte activation profiles and the abundance of several inflammatory molecules in plasma. We applied unsupervised machine learning techniques to predict and diagnose TB-IRIS before and during ART. Our analyses suggest that CD4(+) T cell activation markers are good TB-IRIS predictors, whereas the combination of CD4(+) and CD8(+) T cells markers are better at diagnosing TB-IRIS patients during IRIS events Overall, our findings contribute to a more refined understanding of immunological mechanisms in TB-IRIS pathogenesis that may assist in new diagnostic tools and more targeted patient management

Mycobacterial antigen driven activation of CD14++ CD16-monocytes is a predictor of tuberculosis-associated immune reconstitution inflammatory syndrome

Paradoxical tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is an aberrant inflammatory response occurring in a subset of TB-HIV co-infected patients initiating anti-retroviral therapy (ART). Here, we examined monocyte activation by prospectively quantitating pro-inflammatory plasma markers and monocyte subsets in TB-HIV co-infected patients from a South Indian cohort at baseline and following ART initiation at the time of IRIS, or at equivalent time points in non-IRIS controls. Pro-inflammatory biomarkers of innate and myeloid cell activation were increased in plasma of IRIS patients pre-ART and at the time of IRIS; this association was confirmed in a second cohort in South Africa. Increased expression of these markers correlated with elevated antigen load as measured by higher sputum culture grade and shorter duration of anti-TB therapy. Phenotypic analysis revealed the frequency of CD14++CD16− monocytes was an independent predictor of TB-IRIS, and was closely associated with plasma levels of CRP, TNF, IL-6 and tissue factor during IRIS. In addition, production of inflammatory cytokines by monocytes was higher in IRIS patients compared to controls pre-ART. These data point to a major role of mycobacterial antigen load and myeloid cell hyperactivation in the pathogenesis of TB-IRIS, and implicate monocytes and monocyte-derived cytokines as potential targets for TB-IRIS prevention or treatment

Paradoxical tuberculosis immune reconstitution inflammatory syndrome (TB-IRIS) in HIV patients with culture confirmed pulmonary tuberculosis in India and the potential role of IL-6 in prediction

Background: The incidence, manifestations, outcome and clinical predictors of paradoxical TB-IRIS in patients with HIV and culture confirmed pulmonary tuberculosis (PTB) in India have not been studied prospectively. Methods: HIV+ patients with culture confirmed PTB started on anti-tuberculosis therapy (ATT) were followed prospectively after anti-retroviral therapy (ART) initiation. Established criteria for IRIS diagnosis were used including decline in plasma HIV RNA at IRIS event. Pre-ART plasma levels of interleukin (IL)-6 and C-reactive protein (CRP) were measured. Univariate and multivariate logistic regression models were used to evaluate associations between baseline variables and IRIS. Results: Of 57 patients enrolled, 48 had complete follow up data. Median ATT-ART interval was 28 days (interquartile range, IQR 14–47). IRIS events occurred in 26 patients (54.2%) at a median of 11 days (IQR: 7–16) after ART initiation. Corticosteroids were required for treatment of most IRIS events that resolved within a median of 13 days (IQR: 9–23). Two patients died due to CNS TB-IRIS. Lower CD4+ T-cell counts, higher plasma HIV RNA levels, lower CD4/CD8 ratio, lower hemoglobin, shorter ATT to ART interval, extra-pulmonary or miliary TB and higher plasma IL-6 and CRP levels at baseline were associated with paradoxical TB-IRIS in the univariate analysis. Shorter ATT to ART interval, lower hemoglobin and higher IL-6 and CRP levels remained significant in the multivariate analysis. Conclusion: Paradoxical TB–IRIS frequently complicates HIV-TB therapy in India. IL-6 and CRP may assist in predicting IRIS events and serve as potential targets for immune interventions