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

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

Analysis of the immunological biomarker profile during acute zika virus infection reveals the overexpression of CXCL10, a chemokine linked to neuronal damage

BACKGROUND Infection with Zika virus (ZIKV) manifests in a broad spectrum of disease ranging from mild illness to severe neurological complications and little is known about Zika immunopathogenesis. OBJECTIVES To define the immunologic biomarkers that correlate with acute ZIKV infection. METHODS We characterized the levels of circulating cytokines, chemokines, and growth factors in 54 infected patients of both genders at five different time points after symptom onset using microbeads multiplex immunoassay; comparison to 100 age-matched controls was performed for statistical analysis and data mining. FINDINGS ZIKV-infected patients present a striking systemic inflammatory response with high levels of pro-inflammatory mediators. Despite the strong inflammatory pattern, IL-1Ra and IL-4 are also induced during the acute infection. Interestingly, the inflammatory cytokines IL-1β, IL-13, IL-17, TNF-α, and IFN-γ; chemokines CXCL8, CCL2, CCL5; and the growth factor G-CSF, displayed a bimodal distribution accompanying viremia. While this is the first manuscript to document bimodal distributions of viremia in ZIKV infection, this has been documented in other viral infections, with a primary viremia peak during mild systemic disease and a secondary peak associated with distribution of the virus to organs and tissues. MAIN CONCLUSIONS Biomarker network analysis demonstrated distinct dynamics in concurrence with the bimodal viremia profiles at different time points during ZIKV infection. Such a robust cytokine and chemokine response has been associated with blood-brain barrier permeability and neuroinvasiveness in other flaviviral infections. High-dimensional data analysis further identified CXCL10, a chemokine involved in foetal neuron apoptosis and Guillain-Barré syndrome, as the most promising biomarker of acute ZIKV infection for potential clinical application. © 2018, Fundacao Oswaldo Cruz. All rights reserved

Disparate Immunoregulatory Potentials for Double-Negative (CD4(−) CD8(−)) αβ and γδ T Cells from Human Patients with Cutaneous Leishmaniasis

Although most T lymphocytes express the αβ T-cell receptor and either CD4 or CD8 molecules, a small population of cells lacking these coreceptors, CD4(−) CD8(−) (double negative [DN]) T cells, has been identified in the peripheral immune system of mice and humans. To better understand the role that this population may have in the human immune response against Leishmania spp., a detailed study defining the activation state, cytokine profile, and the heterogeneity of DN T cells bearing αβ or γδ T-cell receptors was performed with a group of well-defined cutaneous leishmaniasis patients. Strikingly, on average 75% of DN T cells from cutaneous leishmaniasis patients expressed the αβ T-cell receptor, with the remainder expressing the γδ receptor, while healthy donors displayed the opposite distribution with ∼75% of the DN T cells expressing the γδ T-cell receptor. Additionally, αβ DN T cells from cutaneous leishmaniasis patients are compatible with previous antigen exposure and recent activation. Moreover, while αβ DN T cells from Leishmania-infected individuals present a proinflammatory cytokine profile, γδ DN T cells express a regulatory profile exemplified by interleukin-10 production. The balance between these subpopulations could allow for the formation of an effective cellular response while limiting its pathogenic potential

Evaluation of the interference of platelets in reticulocyte counting by flow cytometry using thiazole orange

ABSTRACT Introduction: The reticulocyte count by flow cytometry (FC) - an automated counting method - can present errors due to the presence of interfering factors, contributing to a slight increase in results. However, automated methods have large advantages over the manual method, taken as reference, what justifies efforts to improve their quality. Objective: Evaluate platelet interference with the reticulocyte count by FC, using thiazole orange (TO) (FC/TO). Materials and methods: The method of reticulocyte count by FC/TO and a modified automated equivalent method, which excluded CD61-positive cells (platelets) from analysis (FC/TO/MOD), were compared to the manual method. Conclusion: Results were analyzed according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) to assess interchangeability between the methods, by linear regression analysis and paired t-test. The exclusion of interfering fragments from result analysis by the modified method produced results in closer proximity to those of the reference method

The Immunomodulatory Action of Sialostatin L on Dendritic Cells Reveals Its Potential to Interfere with Autoimmunity

Sialostatin L (SialoL) is a secreted cysteine protease inhibitor identified in the salivary glands of the Lyme disease vector Ixodes scapularis. In this study, we reveal the mechanisms of SialoL immunomodulatory actions on the vertebrate host. LPS-induced maturation of dendritic cells from C57BL/6 mice was significantly reduced in the presence of SialoL. Although OVA degradation was not affected by the presence of SialoL in dendritic cell cultures, cathepsin S activity was partially inhibited, leading to an accumulation of a 10-kDa invariant chain intermediate in these cells. As a consequence, in vitro Ag-specific CD4(+) T cell proliferation was inhibited in a time-dependent manner by SialoL, and further studies engaging cathepsin S(-/-) or cathepsin L(-/-) dendritic cells confirmed that the immunomodulatory actions of SialoL are mediated by inhibition of cathepsin S. Moreover, mice treated with SialoL displayed decreased early T cell expansion and recall response upon antigenic stimulation. Finally, SialoL administration during the immunization phase of experimental autoimmune encephalomyelitis in mice significantly prevented disease symptoms, which was associated with impaired IFN-gamma and IL-17 production and specific T cell proliferation. These results illuminate the dual mechanism by which a human disease vector protein modulates vertebrate host immunity and reveals its potential in prevention of an autoimmune disease. The Journal of Immunology, 2009, 182: 7422-7429.Intramural Research Program of the Division of Intramural ResearchIntramural Research Program of the Division of Intramural ResearchU.S. National Institutes of Health (NIH)National Institute of Allergy and Infectious Diseases (NIAID/NIH)U.S. National Institutes of Health (NIH)National Institutes of Health (NIH)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)CNPq Conselho Nacional de Pesquisas[472477/2007-2]Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)CNPq Conselho Nacional de Pesquisas[565496/2008-5]Fundacao de Apoio a Pesquisa Cientifica e Tecnologica do Estado de Santa Catarina-FAPESC[04524/2008-1]Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina (FAPESC)WHO World Health Organization/TDR[2008-8734-0]WHO World Health Organization/TD