Multisystem inflammatory syndrome in children characterized by enhanced antigen-specific T-cell expression of cytokines and its reversal following recovery.

Background: Multisystem inflammatory syndrome (MIS) in children is considered to be a post-infectious complication of COVID-19. T-cell responses in children with this condition have not been well-studied. Methods: We aimed to study the immune responses in children with MIS in comparison to children with acute COVID-19 and children with other infections. Whole blood was stimulated with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-specific antigens and flow cytometry was performed to examine CD4+ and CD8+ T-cell responses. Results: Children with MIS had higher frequencies of CD4+ and CD8+ T cells expressing cytokines at baseline and upon SARS-CoV-2 antigen-specific stimulation in comparison to children with COVID-19 and/or other infections. Children with COVID-19 also exhibited higher frequencies of CD4+ and CD8+ T cells expressing cytokines at baseline and upon SARS-CoV-2 antigen-specific stimulation in comparison to children with other infections. At 6-9 months following treatment and recovery, this enhanced response against SARS-CoV-2 antigens was down modulated in children with MIS. Conclusion: Our study, therefore, provides evidence of enhanced activation of CD4+ and CD8+ T-cell responses in children with MIS and reversal following recovery.

Role of matrix metalloproteinases in multi-system inflammatory syndrome and acute COVID-19 in children.

Introduction: Multisystem Inflammatory Syndrome in children (MIS-C) is a serious inflammatory sequela of SARS-CoV2 infection. The pathogenesis of MIS-C is vague and matrix metalloproteinases (MMPs) may have an important role. Matrix metalloproteinases (MMPs) are known drivers of lung pathology in many diseases. Methods: To elucidate the role of MMPs in pathogenesis of pediatric COVID-19, we examined their plasma levels in MIS-C and acute COVID-19 children and compared them to convalescent COVID-19 and children with other common tropical diseases (with overlapping clinical manifestations). Results: Children with MIS-C had elevated levels of MMPs (P < 0.005 statistically significant) in comparison to acute COVID-19, other tropical diseases (Dengue fever, typhoid fever, and scrub typhus fever) and convalescent COVID-19 children. PCA and ROC analysis (sensitivity 84-100% and specificity 80-100%) showed that MMP-8, 12, 13 could help distinguish MIS-C from acute COVID-19 and other tropical diseases with high sensitivity and specificity. Among MIS-C children, elevated levels of MMPs were seen in children requiring intensive care unit admission as compared to children not needing intensive care. Similar findings were noted when children with severe/moderate COVID-19 were compared to children with mild COVID-19. Finally, MMP levels exhibited significant correlation with laboratory parameters, including lymphocyte counts, CRP, D-dimer, Ferritin and Sodium levels. Discussion: Our findings suggest that MMPs play a pivotal role in the pathogenesis of MIS-C and COVID-19 in children and may help distinguish MIS-C from other conditions with overlapping clinical presentation.

Enhanced SARS-CoV-2-Specific CD4+ T Cell Activation and Multifunctionality in Late Convalescent COVID-19 Individuals.

Background: Examination of CD4+ T cell responses during the natural course of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection offers useful information for the improvement of vaccination strategies against this virus and the protective effect of these T cells. Methods: We characterized the SARS-CoV-2-specific CD4+ T cell activation marker, multifunctional cytokine and cytotoxic marker expression in recovered coronavirus disease 2019 (COVID-19) individuals. Results: CD4+ T-cell responses in late convalescent (>6 months of diagnosis) individuals are characterized by elevated frequencies of activated as well as mono, dual- and multi-functional Th1 and Th17 CD4+ T cells in comparison to early convalescent (<1 month of diagnosis) individuals following stimulation with SARS-CoV-2-specific antigens. Similarly, the frequencies of cytotoxic marker expressing CD4+ T cells were also enhanced in late convalescent compared to early convalescent individuals. Conclusion: Our findings from a low-to middle-income country suggest protective adaptive immune responses following natural infection of SARS-CoV-2 are elevated even at six months following initial symptoms, indicating the CD4+ T cell mediated immune protection lasts for six months or more in natural infection.

Characterization of memory T cell subsets and common γ-chain cytokines in convalescent COVID-19 individuals.

T cells are thought to be an important correlates of protection against SARS-CoV2 infection. However, the composition of T cell subsets in convalescent individuals of SARS-CoV2 infection has not been well studied. The authors determined the lymphocyte absolute counts, the frequency of memory T cell subsets, and the plasma levels of common γ-chain in 7 groups of COVID-19 individuals, based on days since RT-PCR confirmation of SARS-CoV-2 infection. The data show that both absolute counts and frequencies of lymphocytes as well as, the frequencies of CD4+ central and effector memory cells increased, and the frequencies of CD4+ naïve T cells, transitional memory, stem cell memory T cells, and regulatory cells decreased from Days 15-30 to Days 61-90 and plateaued thereafter. In addition, the frequencies of CD8+ central memory, effector, and terminal effector memory T cells increased, and the frequencies of CD8+ naïve cells, transitional memory, and stem cell memory T cells decreased from Days 15-30 to Days 61-90 and plateaued thereafter. The plasma levels of IL-2, IL-7, IL-15, and IL-21-common γc cytokines started decreasing from Days 15-30 till Days 151-180. Severe COVID-19 patients exhibit decreased levels of lymphocyte counts and frequencies, higher frequencies of naïve cells, regulatory T cells, lower frequencies of central memory, effector memory, and stem cell memory, and elevated plasma levels of IL-2, IL-7, IL-15, and IL-21. Finally, there was a significant correlation between memory T cell subsets and common γc cytokines. Thus, the study provides evidence of alterations in lymphocyte counts, memory T cell subset frequencies, and common γ-chain cytokines in convalescent COVID-19 individuals.

Multi-Dimensionality Immunophenotyping Analyses of MAIT Cells Expressing Th1/Th17 Cytokines and Cytotoxic Markers in Latent Tuberculosis Diabetes Comorbidity.

Mucosal-associated invariant T (MAIT) cells are innate like, and play a major role in restricting disease caused by Mycobacterium tuberculosis (Mtb) disease before the activation of antigen-specific T cells. Additionally, the potential link and synergistic function between diabetes mellitus (DM) and tuberculosis (TB) has been recognized for a long time. However, the role of MAIT cells in latent TB (LTB) DM or pre-DM (PDM) and non-DM (NDM) comorbidities is not known. Hence, we examined the frequencies (represented as geometric means, GM) of unstimulated (UNS), mycobacterial (purified protein derivative (PPD) and whole-cell lysate (WCL)), and positive control (phorbol myristate acetate (P)/ionomycin (I)) antigen stimulated MAIT cells expressing Th1 (IFNγ, TNFα, and IL-2), Th17 (IL-17A, IL-17F, and IL-22), and cytotoxic (perforin (PFN), granzyme (GZE B), and granulysin (GNLSN)) markers in LTB comorbidities by uniform manifold approximation (UMAP) and flow cytometry. We also performed a correlation analysis of Th1/Th17 cytokines and cytotoxic markers with HbA1c, TST, and BMI, and diverse hematological and biochemical parameters. The UMAP analysis demonstrated that the percentage of MAIT cells was higher; T helper (Th)1 cytokine and cytotoxic (PFN) markers expressions were different in LTB-DM and PDM individuals in comparison to the LTB-NDM group on UMAP. Similarly, no significant difference was observed in the geometric means (GM) of MAIT cells expressing Th1, Th17, and cytotoxic markers between the study population under UNS conditions. In mycobacterial antigen stimulation, the GM of Th1 (IFNγ (PPD and WCL), TNFα (PPD and WCL), and IL-2 (PPD)), and Th17 (IL-17A, IL-17F, and IL-22 (PPD and/or WCL)) cytokines were significantly elevated and cytotoxic markers (PFN, GZE B, and GNLSN (PPD and WCL)) were significantly reduced in the LTB-DM and/or PDM group compared to the LTB-NDM group. Some of the Th1/Th17 cytokines and cytotoxic markers were significantly correlated with the parameters analyzed. Overall, we found that different Th1 cytokines and cytotoxic marker population clusters and increased Th1 and Th17 (IL-17A, IL-22) cytokines and diminished cytotoxic markers expressing MAIT cells are associated with LTB-PDM and DM comorbidities.

Effect of SARS-CoV-2 seropositivity on antigen - specific cytokine and chemokine responses in latent tuberculosis.

SARS-CoV-2 and latent Mycobacterium tuberculosis infection are both highly co-prevalent in many parts of the globe. Whether exposure to SARS-CoV-2 influences the antigen specific immune responses in latent tuberculosis has not been investigated. We examined the baseline, mycobacterial antigen and mitogen induced cytokine and chemokine responses in latent tuberculosis (LTBI) individuals with or without SARS-CoV-2 seropositivity, LTBI negative individuals with SARS-CoV-2 seropositivity and healthy control (both LTBI and SARS-CoV-2 negative) individuals. Our results demonstrated that LTBI individuals with SARS-CoV-2 seropositivity (LTBI+/IgG +) were associated with increased levels of unstimulated and TB-antigen stimulated IFNγ, IL-2, TNFα, IL-17, IL-1ß, IL-6, IL-12, IL-4, CXCL1, CXCL9 and CXCL10 when compared to those without seropositivity (LTBI+/IgG-). In contrast, LTBI+/IgG+ individuals were associated with decreased levels of IL-5 and IL-10. No significant difference in the levels of cytokines/chemokines was observed upon mitogen stimulation between the groups. SARS-CoV-2 seropositivity was associated with enhanced unstimulated and TB-antigen stimulated but not mitogen stimulated production of cytokines and chemokines in LTBI+ compared to LTBI negative individuals. Finally, most of these significant differences were not observed when LTBI negative individuals with SARS-CoV-2 seropositivity and controls were examined. Our data clearly demonstrate that both baseline and TB - antigen induced cytokine responses are augmented in the presence of SARS-CoV-2 seropositivity, suggesting an augmenting effect of prior SARS-CoV-2 infection on the immune responses of LTBI individuals.

Effect of BCG vaccination on proinflammatory responses in elderly individuals.

We investigated the influence of Bacillus Calmette-Guérin (BCG) vaccination on the unstimulated plasma levels of a wide panel of cytokines, chemokines, acute-phase proteins (APPs), matrix metalloproteinases (MMPs), and growth factors in a group of healthy elderly individuals (age, 60 to 80 years) at baseline (before vaccination) and 1 month after vaccination as part of our clinical study to examine the effect of BCG on COVID-19. Our results demonstrated that BCG vaccination resulted in diminished plasma levels of types 1, 2, and 17 and other proinflammatory cytokines and type 1 interferons. BCG vaccination also resulted in decreased plasma levels of CC, CXC chemokines, APPs, MMPs, and growth factors. Plasma levels of the aforementioned parameters were significantly lower in vaccinated individuals when compared to unvaccinated control individuals. Thus, our study demonstrates the immunomodulatory properties of BCG vaccination and suggests its potential utility in nonspecific vaccination of COVID-19 by down-modulating pathogenic inflammatory responses.

Plasma Eicosanoid Levels in Tuberculosis and Tuberculosis-Diabetes Co-morbidity Are Associated With Lung Pathology and Bacterial Burden.

Host eicosanoids are lipid mediators of inflammation that are commonly accepted as important modulators of the host immune response in Mycobacterium tuberculosis infection. During active tuberculosis (TB), eicosanoids may play an important role in the regulation of inflammatory responses. However, a detailed investigation of the relationship of eicosanoids in TB and TB-diabetes comorbidity (TB-DM) and association to disease pathology or bacterial burdens has not been studied. To study this, we examined the plasma levels of Lipoxin A4 (LXA4), 15-epi-LXA4, Leukotriene B4 (LTB4), and Prostaglandin E2 (PGE2) in individuals with either TB-DM, TB, diabetes mellitus (DM) or healthy controls (HC). Plasma levels of LXA4, 15-epi-LXA4, and PGE2 were significantly increased while the levels of LTB4 were significantly decreased in TB-DM and TB group compared to DM and HC. The ratio of LXA4 to LTB4 and 15-epiLXA4 to LTB4 was significantly enhanced in TB-DM compared to TB. Moreover, the levels of LXA4, 15-epi-LXA4 and the ratios of LXA4 to LTB4 and 15-epiLX4 to LTB4 were significantly increased in TB individuals with bilateral or cavitary disease and these markers also revealed a significant positive relationship with bacterial burden. At the completion of anti-tuberculosis therapy (ATT), levels of LXA4, 15-epi-LXA4, and PGE2 in TB-DM and TB groups were diminished and levels of LTB4 were enhanced in the TB group compared to pre-treatment. Our data imply that alteration and upregulation of eicosanoids are standard characteristics of TB-DM co-morbidity. Our data also demonstrate that modulation in the eicosanoid levels reflect disease severity and extent in TB and TB-DM and are modulated by ATT.

Systems Immunology of Diabetes-Tuberculosis Comorbidity Reveals Signatures of Disease Complications.

Comorbid diabetes mellitus (DM) increases tuberculosis (TB) risk and adverse outcomes but the pathological interactions between DM and TB remain incompletely understood. We performed an integrative analysis of whole blood gene expression and plasma analytes, comparing South Indian TB patients with and without DM to diabetic and non-diabetic controls without TB. Luminex assay of plasma cytokines and growth factors delineated a distinct biosignature in comorbid TBDM in this cohort. Transcriptional profiling revealed elements in common with published TB signatures from cohorts that excluded DM. Neutrophil count correlated with the molecular degree of perturbation, especially in TBDM patients. Body mass index and HDL cholesterol were negatively correlated with molecular degree of perturbation. Diabetic complication pathways including several pathways linked to epigenetic reprogramming were activated in TBDM above levels observed with DM alone. Our data provide a rationale for trials of host-directed therapies in TBDM, targeting neutrophilic inflammation and diabetic complication pathways to address the greater morbidity and mortality associated with this increasingly prevalent dual burden of communicable and non-communicable diseases.

Type 2 diabetes mellitus coincident with pulmonary or latent tuberculosis results in modulation of adipocytokines.

Type 2 diabetes mellitus (T2DM) is recognized as major risk factor for the progress of active pulmonary tuberculosis (PTB), although the mechanistic link between diabetes and tuberculosis remains poorly characterized. Moreover, the influence of poorly controlled diabetes on the baseline levels of adipocytokines in the context of tuberculosis has not been explored in detail. To characterize the influence of coexistent DM on adipocytokine levels in pulmonary or latent TB (LTB), we examined circulating levels of adipocytokines in the plasma of individuals with PTB-DM or LTB-DM and compared them with those without DM (PTB or LTB). PTB-DM or LTB-DM is characterized by diminished circulating levels of adiponectin and adipsin and/or heightened circulating levels of leptin, visfatin and PAI-1. In addition, adiponectin and adipsin exhibit a significant negative correlation, whereas leptin, visfatin and PAI-1 display a significant positive correlation with HbA1C levels and random blood glucose levels. Therefore, our data reveal that PTB-DM or LTB-DM is characterized by alterations in the systemic levels of adipocytokines, indicating that altered adipose tissue inflammation underlying Type 2 diabetes potentially contributes to pathogenesis of TB disease.

Modulation of pro- and anti-inflammatory cytokines in active and latent tuberculosis by coexistent Strongyloides stercoralis infection.

Helminth infections are known to induce modulation of both innate and adaptive immune responses in active and latent tuberculosis (TB). However, the role of helminth infections in modulating systemic cytokine responses in active and latent tuberculosis (LTB) is not known. To define the systemic cytokine levels in helminth-TB coinfection, we measured the circulating plasma levels of Type 1, Type 2, Type 17, other pro-inflammatory and regulatory cytokines in individuals with active TB (ATB) with or without coexistent Strongyloides stercoralis (Ss) infection by multiplex ELISA. Similarly, we also measured the same cytokine levels in individuals with LTB with or without concomitant Ss infection in a cross-sectional study. Our data reveal that individuals with ATB or LTB and coexistent Ss infection have significantly lower levels of Type 1 (IFNγ, TNFα and IL-2) and Type 17 (IL-17A and IL-17F) cytokines compared to those without Ss infection. In contrast, those with ATB and LTB with Ss infection have significantly higher levels of the regulatory cytokines (IL-10 and TGFß), and those with LTB and Ss infection also have significantly higher levels of Type 2 cytokines (IL-4, IL-5 and IL-13) as well. Finally, those with LTB (but not ATB) exhibit significantly lower levels of other pro-inflammatory cytokines (IFNα, IFNß, IL-6, IL-12 and GM-CSF). Our data therefore reveal a profound effect of Ss infection on the systemic cytokine responses in ATB and LTB and indicate that coincident helminth infections might influence pathogenesis of TB infection and disease.

Heme Oxygenase-1 Regulation of Matrix Metalloproteinase-1 Expression Underlies Distinct Disease Profiles in Tuberculosis.

Pulmonary tuberculosis (TB) is characterized by oxidative stress and lung tissue destruction by matrix metalloproteinases (MMPs). The interplay between these distinct pathological processes and the implications for TB diagnosis and disease staging are poorly understood. Heme oxygenase-1 (HO-1) levels were previously shown to distinguish active from latent TB, as well as successfully treated Mycobacterium tuberculosis infection. MMP-1 expression is also associated with active TB. In this study, we measured plasma levels of these two important biomarkers in distinct TB cohorts from India and Brazil. Patients with active TB expressed either very high levels of HO-1 and low levels of MMP-1 or the converse. Moreover, TB patients with either high HO-1 or MMP-1 levels displayed distinct clinical presentations, as well as plasma inflammatory marker profiles. In contrast, in an exploratory North American study, inversely correlated expression of HO-1 and MMP-1 was not observed in patients with other nontuberculous lung diseases. To assess possible regulatory interactions in the biosynthesis of these two enzymes at the cellular level, we studied the expression of HO-1 and MMP-1 in M. tuberculosis-infected human and murine macrophages. We found that infection of macrophages with live virulent M. tuberculosis is required for robust induction of high levels of HO-1 but not MMP-1. In addition, we observed that CO, a product of M. tuberculosis-induced HO-1 activity, inhibits MMP-1 expression by suppressing c-Jun/AP-1 activation. These findings reveal a mechanistic link between oxidative stress and tissue remodeling that may find applicability in the clinical staging of TB patients.

Plasma heme oxygenase-1 levels distinguish latent or successfully treated human tuberculosis from active disease.

BACKGROUND: Tuberculosis (TB) is associated with oxidative stress and the induction of host anti-oxidants to counteract this response. Heme oxygenase-1 (HO-1) is a critical promoter of cytoprotection in diverse disease models including mycobacterial infection. Nevertheless, the pattern of expression of HO-1 in human tuberculosis has not been studied. Here, we examine expression of HO-1 in M. tuberculosis-exposed and -infected individuals and test its ability to distinguish active from latent and successfully treated TB cases. In addition, we assess correlations between plasma levels of HO-1 and cytokines closely associated with the immunopathogenesis of TB. METHODS: Cross-sectional and longitudinal analyses of levels of HO-1, acute phase proteins and pro-inflammatory cytokines were performed in plasma samples from individuals with active pulmonary, extra-pulmonary or latent TB infection and healthy controls as part of a prospective cohort study in South India. RESULTS: Systemic levels of HO-1 were dramatically increased in individuals with active pulmonary and extra-pulmonary tuberculosis and particularly those with bilateral lung lesions and elevated bacillary loads in sputum. HO-1 levels effectively discriminated active from latent tuberculosis with higher predictive values than either C-reactive protein or serum amyloid protein. Moreover, there was a marked reduction in HO-1 levels in active TB cases following anti-tuberculous therapy but not in those who failed treatment. Pulmonary TB patients displaying the highest concentrations of HO-1 in plasma exhibited significantly elevated plasma levels of interleukin (IL)-10, interferon (IFN)-γ and IL-17 and diminished levels of tumor necrosis factor (TNF)-α. CONCLUSION: These findings establish HO-1 levels as a potentially useful parameter for distinguishing active from latent or treated pulmonary tuberculosis, that is superior in this respect to the measurement of other acute inflammatory proteins.

Circulating biomarkers of pulmonary and extrapulmonary tuberculosis in children.

Tuberculosis (TB) in children is not only more likely to cause more severe disease than that seen in adults, it is also more likely to be extrapulmonary. Moreover, pediatric TB is very difficult to diagnose and suffers from a lack of understanding of host biomarkers for monitoring the progression of disease. Hence, we sought to identify the expression patterns of a variety of biomarkers in the plasma of children with pulmonary TB (PTB) and extrapulmonary TB (ETB), as well as in healthy control (HC) children. Thus, we examined a variety of circulating markers reflecting tissue inflammation, oxidative stress, innate immune activation, fibrosis, and the cytokine response. Children with active TB, compared to HC children, showed markedly elevated plasma levels of matrix metalloproteinases and their endogenous inhibitors. In addition, children with active TB had significantly elevated levels of C-reactive protein, α-2 macroglobulin, and haptoglobin, as well as hemoxygenase 1. Markers of innate immune activation (lipopolysaccharide [LPS] and lipopolysaccharide-binding protein [LBP]) were significantly lower in ETB than in PTB children. Although there were no significant differences between the two groups in their levels of cytokines (type 1 [gamma interferon (IFN-γ), tumor necrosis factor α (TNF-α), interleukin 2 (IL-2), and IL-12], type 2 [IL-4, IL-5, IL-13, and IL-33], and most type 17 [IL-17A, IL-22, IL-1ß, and IL-6] and type 1 interferons [IFN-α and IFN-ß]) or most of the cytokines associated with immune modulation (IL-10 and IL-20), pediatric TB was associated with elevated plasma transforming growth factor ß (TGF-ß), IL-21, and IL-23 levels. Thus, pediatric TB is characterized by elevated levels of a variety of biomarkers at homeostasis, suggesting that these responses may play a crucial role in disease pathogenesis.