Nitric oxide brings innate immune resistance to M. tuberculosis infection among high-risk household contacts of pulmonary tuberculosis patients.

Nitric oxide (NO) and iNOS are crucial host factors in innate immunity against intracellular pathogens. However, the role of NO in Mycobacterium tuberculosis (M. tb) infection in humans remains controversial, unlike in the murine model of TB. To investigate this, levels of NO, iNOS, and L-arginine, as well as the NOS2A gene polymorphism rs57234985 at the promoter region of NOS2A, were evaluated in pulmonary TB (PTB) patients and their household contacts (HHCs). Increased levels of NO and iNOS expression in HHCs indicated exposure to M. tb infection which was confirmed by higher levels of iNOS and NO in Mantouxpositive individuals. Furthermore, higher levels of arginine were detected in HHCs, suggesting its potential role in promoting optimal NO synthesis. PTB patients had higher levels of these analytes due to ongoing active infection. Interestingly, iNOS and NO levels were inversely related to bacterial burden, suggesting their antimicrobial role. NOS2A gene polymorphism was found to be associated with disease susceptibility, with the TT genotype linked to increased iNOS expression. To conclude, iNOS plays a crucial role in controlling early M. tb infection in HHCs by inducing optimal NO production with help of L-arginine. Further longitudinal studies are needed to better understand the role of these host factors upon disease activation.

Protective Role of Vitamin D Against Development of Active Tuberculosis in Close Household Contacts of Pulmonary Tuberculosis Patients: A Prospective Cohort Study.

Vitamin-D is known to promote innate immune responses by acting as a cofactor of VDR for induction of antimicrobial peptides like cathelicidin. Close household contacts of pulmonary tuberculosis patients are at high risk of active infection, Therefore, possible role of vitamin-D in TB prevention through cathelicidin production was studied in high-risk household contacts (HHCs) of pulmonary tuberculosis (PTB) patients. 20 HHCs of PTB patients were recruited and followed up for one year. Levels of vitamin-D (25(OH)D) and its associated molecules were evaluated at 3-months intervals for one year or until the development of active TB. 25(OH)D was measured using chemiluminescence method. Serum VDR and cathelicidin levels were measured by ELISA and VDR mRNA expression by qPCR. Throughout the study period mean range of serum 25(OH)D levels was 20.51 ± 5.12 ng/ml. VDR and cathelicidin levels however showed significant decline after six months suggesting decrease in bacterial exposure. None of the HHCs developed active infection even with high exposure to 2 + to 3 + AFB positive index cases. Mantoux positive household contacts had high levels of VDR and cathelicidin, suggestive of an early or latent phase of infection, did not develop active TB plausibly due to maintenance of adequate serum levels of vitamin-D. Optimal levels of 25(OH)D and its associated molecules during early stages of infection may serve as protective factor against development of active TB. Cohort of HHCs with severely deficient vitamin-D levels (10 ng/ml) could be followed up for a better risk assessment.

From antigens to immune responses: Shaping the future of TB detection and prevention.

OBJECTIVES: Tuberculosis (TB) remains a global health challenge due to various factors, including delayed diagnoses leading to the spread of infection, limited efficacy of current vaccination strategies, and emergence of drug-resistant strains. Here, we explore the significance of Mycobacterium tuberculosis (Mtb)-specific antigens to overcome these challenges. METHODS: A narrative review exploring the dynamics of Mtb-specific antigens and the related T cell immune responses across the TB spectrum. RESULTS: A variety of antigens are expressed at different stages of Mtb infection, driving its diverse antigenic landscape and associated T cell functional heterogeneity. Recent advances in high-coverage genomic and proteomic approaches may lead to the identification and characterization of antigens/epitopes within the context of TB. CONCLUSION: Factors such as magnitude of memory response, cytokine profile, immunodominance, and conservation of epitopes should be emphasized as crucial parameters in assessing the potential efficacy of these antigens in diagnostics or vaccine research. Recognizing the antigenic repertoire of Mtb changes with the infection stage, it is important to assess the availability of different subsets of Mtb antigens across the spectrum of infection for more precise disease classifications. Targeting specific antigens holds promise as a pathway for developing specific immunological biomarkers to predict TB reactivation in populations.

Hyperglycemia modulates M1/M2 macrophage polarization in chronic diabetic patients with pulmonary tuberculosis infection.

Increased susceptibility to bacterial infections like tuberculosis (TB) is one of the complications of type 2 diabetes, however the underlying mechanisms remains poorly characterized. To explore how chronic hyperglycemia in diabetes affects progression of active TB, we examined mRNA expression of M1 (proinflammatory) and M2 (anti-inflammatory) cytokines/markers, in monocyte-derived macrophages obtained from patients with PTB + DM (pulmonary TB + diabetes mellitus type 2), patients with DM alone, patients with PTB alone, and healthy individuals (controls). Our findings indicate a dysregulated cytokine response in patients with both PTB and DM, characterized by decreased expression levels of interferon-gamma (IFN-γ) and inducible nitric oxide synthase (iNOS), along with increased expression levels of interleukin-1 beta (IL-1ß) and CD206. Furthermore, we observed a positive correlation of IL-1ß and CD206 expression with levels of glycosylated hemoglobin (HbA1c) in both PTB + DM and DM groups, while IFN-γ showed a positive correlation with HbA1c levels, specifically in the PTB + DM group. Additionally, M1 cytokines/markers, IL-1ß and iNOS were found to be significantly associated with the extent of sputum positivity in both PTB and PTB + DM groups, suggesting it to be a function of increased bacterial load and hence severity of infection. Our data demonstrates that tuberculosis in individuals with PTB + DM is characterized by altered M1/M2 cytokine responses, indicating that chronic inflammation associated with type 2 diabetes may contribute to increased immune pathology and inadequate control of tuberculosis infection.

Identification of differentially recognized T cell epitopes in the spectrum of tuberculosis infection.

There is still incomplete knowledge of which Mycobacterium tuberculosis (Mtb) antigens can trigger distinct T cell responses at different stages of infection. Here, a proteome-wide screen of 20,610 Mtb-derived peptides in 21 patients mid-treatment for active tuberculosis (ATB) reveals IFNγ-specific T cell responses against 137 unique epitopes. Of these, 16% are recognized by two or more participants and predominantly derived from cell wall and cell processes antigens. There is differential recognition of antigens, including TB vaccine candidate antigens, between ATB participants and interferon-gamma release assay (IGRA + /-) individuals. We developed an ATB-specific peptide pool (ATB116) consisting of epitopes exclusively recognized by ATB participants. This pool can distinguish patients with pulmonary ATB from IGRA + /- individuals from various geographical locations, with a sensitivity of over 60% and a specificity exceeding 80%. This proteome-wide screen of T cell reactivity identified infection stage-specific epitopes and antigens for potential use in diagnostics and measuring Mtb-specific immune responses.

Antigen-specificity measurements are the key to understanding T cell responses.

Antigen-specific T cells play a central role in the adaptive immune response and come in a wide range of phenotypes. T cell receptors (TCRs) mediate the antigen-specificities found in T cells. Importantly, high-throughput TCR sequencing provides a fingerprint which allows tracking of specific T cells and their clonal expansion in response to particular antigens. As a result, many studies have leveraged TCR sequencing in an attempt to elucidate the role of antigen-specific T cells in various contexts. Here, we discuss the published approaches to studying antigen-specific T cells and their specific TCR repertoire. Further, we discuss how these methods have been applied to study the TCR repertoire in various diseases in order to characterize the antigen-specific T cells involved in the immune control of disease.

Identification of differentially recognized T cell epitopes in the spectrum of Mtb infection.

Tuberculosis caused by Mycobacterium tuberculosis is one of the leading causes of death from a single infectious agent. Identifying dominant epitopes and comparing their reactivity in different tuberculosis (TB) infection states can help design diagnostics and vaccines. We performed a proteome-wide screen of 20,610 Mtb derived peptides in 21 Active TB (ATB) patients 3-4 months post-diagnosis of pulmonary TB (mid-treatment) using an IFNγ and IL-17 Fluorospot assay. Responses were mediated exclusively by IFNγ and identified a total of 137 unique epitopes, with each patient recognizing, on average, 8 individual epitopes and 22 epitopes (16%) recognized by 2 or more participants. Responses were predominantly directed against antigens part of the cell wall and cell processes category. Testing 517 peptides spanning TB vaccine candidates and ESAT-6 and CFP10 antigens also revealed differential recognition between ATB participants mid-treatment and healthy IGRA+ participants of several vaccine antigens. An ATB-specific peptide pool consisting of epitopes exclusively recognized by participants mid-treatment, allowed distinguishing participants with active pulmonary TB from healthy interferon-gamma release assay (IGRA)+/- participants from diverse geographical locations. Analysis of longitudinal samples indicated decreased reactivity during treatment for pulmonary TB. Together, these results show that a proteome-wide screen of T cell reactivity identifies epitopes and antigens that are differentially recognized depending on the Mtb infection stage. These have potential use in developing diagnostics and vaccine candidates and measuring correlates of protection.

Chronic hyperglycemia drives alterations in macrophage effector function in pulmonary tuberculosis.

Diabetes mellitus (DM) alters immune responses and given the rising prevalence of DM in tuberculosis (TB) endemic countries; hyperglycemia can be a potential risk factor for active TB development. However, the impact of hyperglycemia on TB-specific innate immune response in terms of macrophage functions remains poorly addressed. We assessed macrophage effector functions in uncontrolled DM patients with or without TB infection (PTB+DM and DM), non-diabetic TB patients (PTB), and non-diabetic-uninfected controls. Phagocytic capacity against BCG and surface expression of different pattern recognition receptors (PRRs) (CD11b, CD14, CD206, MARCO, and TLR-2) were measured via flow cytometry. Effector molecules (ROS and NO) required for bacterial killing were assessed via DCFDA and Griess reaction respectively. A systematic dysregulation in phagocytic capacity with concurrent alterations in the expression pattern of key PRRs (CD11b, MARCO, and CD206) was observed in PTB+DM. These altered PRR expressions were associated with decreased phagocytic capacity of macrophages. Similarly, ROS was aberrantly higher while NO was lower in PTB+DM. These altered macrophage functions were positively correlated with increasing disease severity. Our results highlight several key patterns of immune dysregulation against TB infection under hyperglycemic conditions and highlight a negative impact of hyperglycemia with etiology and progression of TB.

Role of Regulatory Proteins Involved in Iron Homeostasis in Pulmonary Tuberculosis Patients and Their Household Contacts.

Early identification and treatment of active tuberculosis disease among high risk household contacts could limit new transmission and better clinical outcome, thus decreasing TB burden. Host iron homeostasis is an important yet underevaluated factor in pathophysiology of tuberculosis (TB). One such protein is hepcidin which internalizes ferroportin (membrane iron transporter), thus inhibiting iron export from macrophages which is utilised by bacteria leading to disease severity. Iron homeostasis markers were evaluated in 50 pulmonary tuberculosis patients (PTB) and their household contacts to assess their utility as biomarkers for TB development. Altered iron homeostasis with significantly lower haemoglobin levels despite optimum serum iron levels was observed in PTB compared to household contacts and healthy controls pointing towards anaemia of inflammation. Higher serum hepcidin with lower ferroportin expression and hence higher ferritin levels was seen in PTB compared to both household contacts and healthy controls due to IL-6 induced hepcidin production in TB. Transferrin levels were found to be significantly lower in PTB and household contacts as compared to healthy controls owing to higher ferritin levels in PTB group. Upon infection, regulation of iron absorption is disturbed via increased hepcidin levels leading to ferroportin internalization and thus inhibition of iron export from macrophages which may lead to favourable M.tb. survival and multiplication leading to tuberculosis. Some of these markers could be assessed for early identification and treatment of active tuberculosis among high risk household contacts limiting new transmission and better clinical outcome, thus decreasing TB burden.

Association of Fok1 VDR polymorphism with Vitamin D and its associated molecules in pulmonary tuberculosis patients and their household contacts.

Status of Fok I VDR polymorphism along with vitamin D, Vitamin D receptor (VDR), and cathelicidin levels in Tuberculosis (TB) patients compared to household contacts and implication of these findings in susceptibility to TB is not known. 150 active TB patients, 150 household contacts and 150 healthy controls were recruited from North Indian population. Fok1 VDR polymorphism was studied by polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP).VDR mRNA and protein levels were studied using quantitative real time PCR (q rt PCR) and enzyme linked immunosorbent assay (ELISA) respectively. Cathelicidin and Vitamin D levels were measured using ELISA and chemiluminescence immunoassay (CLIA) respectively. Significant association was found between Fok1 polymorphism and susceptibility to TB (P < 0.0005). VDR mRNA, VDR protein and vitamin D levels were significantly lower in active TB group when compared to household contacts and healthy controls (P < 0.0001, 0.0001 and 0.0005 respectively). Cathelicidin levels were higher in active TB patients compared to other groups (P < 0.0001). Expression of VDR and cathelicidin was significantly higher among 'FF' genotypes of VDR (more active form of VDR) compared to 'ff' genotype (less active form of VDR). 'f' allele was associated with increased susceptibility to TB. Higher frequency of 'F' allele, increased VDR expression along with increased vitamin D levels in household contacts compared to active TB group might be responsible for protection against active TB.

Status of vitamin D and the associated host factors in pulmonary tuberculosis patients and their household contacts: A cross sectional study.

Innate immunity plays an important role in pathophysiology of tuberculosis which is influenced by various host factors. One such factor is vitamin D which, along with its associated molecule, can alter the host defense against Mycobacterium Tuberculosis (M.Tb.) via altered production of cathelicidin and nitric oxide, both having bactericidal effect. Therefore, assessment of vitamin D and its associated molecules in tuberculosis patients and household contacts as compared to healthy controls were done and the implication of these findings in susceptibility to tuberculosis (TB) was studied. 80 active TB patients, 75 household contacts and 70 healthy controls were included. Vitamin D receptor (VDR), vitamin D binding protein (VDBP) and inducible nitric oxide synthase (iNOS) mRNA levels were studied using quantitative PCR. Serum VDR, cathelicidin, and iNOS levels were measured using ELISA. Vitamin D and NO levels were measured in serum using chemiluminescence based immunoassay and greiss reaction based colorimetry kit respectively. Decreased serum levels of vitamin D were observed in active TB patients as compared to healthy controls (p < 0.001). VDR and iNOS mRNA levels were found to be significantly lower in active TB patients compared to household contacts and healthy controls (p < 0.0001 and 0.005 respectively). VDBP mRNA expression was found to be lower in active TB group as compared to household contacts and healthy controls however the difference was not found to be significant (p > 0.21). Although, mRNA expression of VDR, VDR protein and iNOS along with vitamin D levels were significantly (p < 0.05) higher in household contacts compared to active TB group. However, levels of iNOS, NO and cathelicidin were found to be higher in TB patients as compared to household contacts and healthy controls (p < 0.01, 0.05 and 0.01 respectively). Higher levels of Vitamin D along with VDR and iNOS expression in household contacts as compared to active TB patients suggest vitamin D might have a protective role against TB plausibly decreasing disease susceptibility. Low vitamin D levels in active TB patients warrants further studies to determine the role of vitamin D supplementation in prevention and treatment of TB.