Amar Singh

FoxP3+ Regulatory T Cells Suppress Effector T-Cell Function at Pathologic Site in Miliary Tuberculosis

RATIONALE The inadequacy of effector T-cell response in containment of tubercle bacilli is believed to result in the development of disseminated forms of tuberculosis (TB), such as miliary tuberculosis (MTB). Regulatory T cells (Treg) plausibly play a critical role in the immunopathogenesis of disseminated TB by suppression of effector immune response against Mycobacterium tuberculosis at the pathologic site(s). To understand the role of Treg cells in disseminated tuberculosis, we studied the frequency and function of Treg cells derived from the local disease site specimens (LDSS) of patients with TB pleural effusion and MTB as clinical models of contained and disseminated forms of disease, respectively. OBJECTIVES To (1) enumerate the frequency of Treg cells in bronchoalveolar lavage (BAL) fluid of patients with MTB and compare with that of peripheral blood, (2) study the role of Treg cells in suppression of local T-cell response, and (3) study the selective recruitment of Treg cells at the local disease site(s). METHODS Flow cytometry, reverse transcriptase polymerase chain reaction, and 3-(4,5-dimethylthythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)-based cell proliferation assay. MEASUREMENTS AND MAIN RESULTS Frequency of Treg cells (CD4(+)CD25(+)FoxP3(+)) was significantly higher in LDSS in MTB along with higher levels of FoxP3 mRNA. Importantly, FoxP3(+) Treg cells obtained from the BAL of patients with MTB predominantly produced IL-10 and could suppress the autologous T-cell proliferation in response to M. tuberculosis antigen. CONCLUSIONS Our results highlight the importance of Treg cells in suppression of effector immune response and their influence on bacillary dissemination, disease manifestation, and severity.

Inhibiting the Programmed Death 1 Pathway Rescues Mycobacterium tuberculosis–Specific Interferon γ–Producing T Cells From Apoptosis in Patients With Pulmonary Tuberculosis

BACKGROUND Overexpression of programmed death 1 (PD-1) receptor is thought to inhibit the effector T-cell response in human tuberculosis. However, the precise mechanism of such inhibition remains unclear. The present study addresses the role of PD-1 in dampening host T-cell function among patients with pulmonary tuberculosis. METHODS Expression of PD-1 and its ligands (PD-L1/L2) on T cells, B cells, and monocytes was evaluated by flow cytometry (FACS). In vitro stimulation of peripheral blood mononuclear cells in the presence of Mycobacterium tuberculosis antigens was performed with and without blocking PD-1, and intracellular cytokine production was measured by FACS. RESULTS We showed higher frequencies of T cells, monocytes, and B cells expressing PD-1 and its ligand(s) among patients with pulmonary tuberculosis. Infections with live M. tuberculosis upregulated PD-L1 expression on monocytes. In vitro PD-1 blocking rescued M. tuberculosis-specific interferon γ (IFN-γ)-producing T cells from undergoing apoptosis. The number of PD-1-expressing T cells decreased significantly during therapy and inversely correlated with IFN-γ-dominant T-cell response against M. tuberculosis. CONCLUSIONS Manipulation of PD-1 signaling may restore the host T-cell response and thus may have therapeutic potential. PD-1 also may serve as a biomarker to monitor host immunity among patients with tuberculosis during therapy and vaccine studies.

Foxp3+ regulatory T cells among tuberculosis patients: impact on prognosis and restoration of antigen specific IFN-γ producing T cells.

CD4+CD25+Foxp3+ Regulatory T cells (Treg) and programmed death-1 (PD-1) molecules have emerged as pivotal players in immune suppression of chronic diseases. However, their impact on the disease severity, therapeutic response and restoration of immune response in human tuberculosis remains unclear. Here, we describe the possible role of Treg cells, their M. tuberculosis driven expansion and contribution of PD-1 pathway to the suppressive function of Treg cells among pulmonary tuberculosis (PTB) patients. Multicolor flow cytometry, cell culture, cells sorting and ELISA were employed to execute the study. Our results showed significant increase in frequency of antigen-reactive Treg cells, which gradually declined during successful therapy and paralleled with decline of M. tuberculosis–specific IL-10 along with elevation of IFN-γ production, and raising the IFN-γ/IL-4 ratio. Interestingly, persistence of Treg cells tightly correlated with MDR tuberculosis. Also, we show that blocking PD-1/PD-L1 pathway abrogates Treg-mediated suppression, suggesting that the PD-1/PD-L1 pathway is required for Treg-mediated suppression of the antigen-specific T cells. Treg cells possibly play a role in dampening the effector immune response and abrogating PD-1 pathway on Treg cells significantly rescued protective T cell response, suggesting its importance in immune restoration among tuberculosis patients.

Clinical significance of sperm DNA damage threshold value in the assessment of male infertility

Sperm DNA integrity is a prerequisite for normal spermatozoal function. The aim of the study was to evaluate the role of sperm chromatin damage, its cut-off level and its effect on sperm parameters in men with idiopathic infertility by analyzing 100 idiopathic infertile men and 50 fertile controls. Semen samples were analyzed as per WHO 1999 guidelines and sperm chromatin structure assay (SCSA) was applied to measure DNA fragmentation index (DFI) in sperm. The mean DFI of infertile men (35.75) was significantly (P < .0001) higher as compared to controls (26.22). The threshold level of 30.28% was obtained as cut-off value to discriminate infertile men from fertile controls. Sperm count, forward motility, and normal morphology found to be negatively associated with DFI in overall study subjects. Infertile men with severe oligozoospermia had higher mean DFI (40.01 ± 11.31) than infertile men with oligozoospermia (35.11 ± 10.05) and normal sperm count (33.99 ± 9.96). Moreover 64% of infertile men have DFI > 30 against 6% of fertile controls (P < .0001). Higher sperm DNA fragmentation may be the underlying cause for poor semen quality in idiopathic infertile men and the threshold value of 30.28% is a clear discriminator to distinguish infertile men from fertile men of Indian population. Thus, DFI is a good prognostic marker as cases with higher sperm DFI may have poor success rate even after assisted conception and may experience recurrent pregnancy loss (RPL) and should be counseled accordingly.

Regulatory T cells suppress T cell activation at the pathologic site of human visceral leishmaniasis.

Suppression of T cell response is thought to be involved in the pathogenesis of visceral leishmaniasis (VL). Regulatory T cell (Treg) mediated immune-suppression is reported in animal models of Leishmania infection. However, their precise role among human patients still requires pathologic validation. The present study is aimed at understanding the frequency dynamics and function of Treg cells in the blood and bone marrow (BM) of VL patients. The study included 42 parasitologically confirmed patients, 17 healthy contact and 9 normal bone marrow specimens (NBM). We show i) the selective accumulation of Treg cells at one of the disease inflicted site(s), the BM, ii) their in vitro expansion in response to LD antigen and iii) persistence after successful chemotherapy. Results indicate that the Treg cells isolated from BM produces IL-10 and may inhibit T cell activation in IL-10 dependent manner. Moreover, we observed significantly higher levels of IL-10 among drug unresponsive patients, suggesting their critical role in suppression of immunity among VL patients. Our results suggest that IL-10 plays an important role in suppression of host immunity in human VL and possibly determines the efficacy of chemotherapy.

Structural Basis of Recognition of Pathogen-associated Molecular Patterns and Inhibition of Proinflammatory Cytokines by Camel Peptidoglycan Recognition Protein

Peptidoglycan recognition proteins (PGRPs) are involved in the recognition of pathogen-associated molecular patterns. The well known pathogen-associated molecular patterns include LPS from Gram-negative bacteria and lipoteichoic acid (LTA) from Gram-positive bacteria. In this work, the crystal structures of two complexes of the short form of camel PGRP (CPGRP-S) with LPS and LTA determined at 1.7- and 2.1-Å resolutions, respectively, are reported. Both compounds were held firmly inside the complex formed with four CPGRP-S molecules designated A, B, C, and D. The binding cleft is located at the interface of molecules C and D, which is extendable to the interface of molecules A and C. The interface of molecules A and B is tightly packed, whereas that of molecules B and D forms a wide channel. The hydrophilic moieties of these compounds occupy a common region, whereas hydrophobic chains interact with distinct regions in the binding site. The binding studies showed that CPGRP-S binds to LPS and LTA with affinities of 1.6 × 10−9 and 2.4 × 10−8 m, respectively. The flow cytometric studies showed that both LPS- and LTA-induced expression of the proinflammatory cytokines TNF-α and IL-6 was inhibited by CPGRP-S. The results of animal studies using mouse models indicated that both LPS- and LTA-induced mortality rates decreased drastically when CPGRP-S was administered. The recognition of both LPS and LTA, their high binding affinities for CPGRP-S, the significant decrease in the production of LPS- and LTA-induced TNF-α and IL-6, and the drastic reduction in the mortality rates in mice by CPGRP-S indicate its useful properties as an antibiotic agent.

Association of Intracellular TH1-TH2 Balance in CD4+ T-cells and MIP-1α in CD8+ T-cells with Disease Severity in Adults with Dengue

Background We tested the hypothesis that dengue haemorrhagic fever (DHF) is associated with a TH1-skewed immune response as opposed to dengue fever (DF). Methods We estimated intracellular (in T-cells) and serum levels of designate TH1/TH2 cytokines [interferon-γ (IFN-γ), interleukin-4 (IL-4), and tumor necrosis factor-α] and macrophage inflammatory protein-1α (MIP-1α) at admission, 48 h, and day 5 in 20 adults with dengue (DF=10, DHF=10) and 10 dengue-naïve healthy controls. Results At admission, intracellular IFN-γ/IL-4 ratio in CD4+ T-cells and proportion of MIP-1α-positive CD8+ T-cells were significantly higher in patients with DHF [7.21 (5.36~10.81) vs. 3.04 (1.75~4.02); p=0.011 and 6.2% (3.2~8.2%) vs. 2.4% (2.0~3.6%); p=0.023]. The latter showed a significant positive correlation with IFN-γ/IL-4 ratio in CD4+ T-cells (Spearmans rho=0.64; p=0.003), percentage-change in haematocrit (rho=0.47; p=0.048), and serum alanine aminotransferase level (rho=0.61; p=0.009). Conclusion We conclude that DHF is associated with a TH1-skewed immune response. Further, MIP-1α in CD8+ T-cells is an important immunologic correlate of disease severity in dengue.

Tregs Modulate Lymphocyte Proliferation, Activation, and Resident-Memory T-Cell Accumulation within the Brain during MCMV Infection

Accumulation and retention of regulatory T-cells (Tregs) has been reported within post viral-encephalitic brains, however, the full extent to which these cells modulate neuroinflammation is yet to be elucidated. Here, we used Foxp3-DTR (diphtheria toxin receptor) knock-in transgenic mice, which upon administration of low dose diphtheria toxin (DTx) results in specific deletion of Tregs. We investigated the proliferation status of various immune cell subtypes within inflamed central nervous system (CNS) tissue. Depletion of Tregs resulted in increased proliferation of both CD8+ and CD4+ T-cell subsets within the brain at 14 d post infection (dpi) when compared to Treg-sufficient animals. At 30 dpi, while proliferation of CD8+ T-cells was controlled within brains of both Treg-depleted and undepleted mice, proliferation of CD4+ T-cells remained significantly enhanced with DTx-treatment. Previous studies have demonstrated that Treg numbers within the brain rebound following DTx treatment to even higher numbers than in untreated animals. Despite this rebound, CD8+ and CD4+ T-cells proliferated at a higher rate when compared to that of Treg-sufficient mice, thus maintaining sustained neuroinflammation. Furthermore, at 30 dpi we found the majority of CD8+ T-cells were CD127hi KLRG1- indicating that the cells were long lived memory precursor cells. These cells showed marked elevation of CD103 expression, a marker of tissue resident-memory T-cells (TRM) in the CNS, in untreated animals when compared to DTx-treated animals suggesting that generation of TRM is impaired upon Treg depletion. Moreover, the effector function of TRM as indicated by granzyme B production in response to peptide re-stimulation was found to be more potent in Treg-sufficient animals. Taken together, our findings demonstrate that Tregs limit neuroinflammatory responses to viral infection by controlling cell proliferation and may direct a larger proportion of lymphocytes within the brain to be maintained as TRM cells.

Structural Studies on Molecular Interactions between Camel Peptidoglycan Recognition Protein, CPGRP-S, and Peptidoglycan Moieties N-Acetylglucosamine and N-Acetylmuramic Acid

Background: PGRP-S is an innate immunity protein that protects hosts from invading microbes. Results: CPGRP-S forms linear polymers with alternating A-B and C-D contacts, and both GlcNAc and MurNAc bind at the same subsite. Conclusion: The mode of binding of camel PGRP-S is different from other PGRPs. Significance: The better antibacterial properties of camel PGRP-S can be exploited for therapeutic applications. Peptidoglycan (PGN) consists of repeating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), which are cross-linked by short peptides. It is well known that PGN forms a major cell wall component of bacteria making it an important ligand for the recognition by peptidoglycan recognition proteins (PGRPs) of the host. The binding studies showed that PGN, GlcNAc, and MurNAc bind to camel PGRP-S (CPGRP-S) with affinities corresponding to dissociation constants of 1.3 × 10−9, 2.6 × 10−7, and 1.8 × 10−7 m, respectively. The crystal structure determinations of the complexes of CPGRP-S with GlcNAc and MurNAc showed that the structures consist of four crystallographically independent molecules, A, B, C, and D, in the asymmetric unit that exists as A-B and C-D units of two neighboring linear polymers. The structure determinations showed that compounds GlcNAc and MurNAc bound to CPGRP-S at the same subsite in molecule C. Both GlcNAc and MurNAc form several hydrogen bonds and extensive hydrophobic interactions with protein atoms, indicating the specific nature of their bindings. Flow cytometric studies showed that PGN enhanced the secretions of TNF-α and IL-6 from human peripheral blood mononuclear cells. The introduction of CPGRP-S to the PGN-challenged cultured peripheral blood mononuclear cells reduced the expressions of proinflammatory cytokines, TNF-α and IL-6. This showed that CPGRP-S inhibited PGN-induced production of proinflammatory cytokines and down-regulated macrophage-mediated inflammation, indicating its potential applications as an antibacterial agent.

Differential Role of B Cells and IL-17 Versus IFN-γ During Early and Late Rejection of Pig Islet Xenografts in Mice

Background Xenogeneic islet transplantation is an emerging therapeutic option for diabetic patients. However, immunological tolerance to xenogeneic islets remains a challenge. Methods The current study used a pig-to-mouse discordant xenogeneic islet transplant model to examine antidonor xenogeneic immune responses during early and late rejection and to determine experimental therapeutic interventions that promote durable pig islet xenograft survival. Results We found that during early acute rejection of pig islet xenografts, the rejecting hosts exhibited a heavy graft infiltration with B220+ B cells and a robust antipig antibody production. In addition, early donor-stimulated IL-17 production, but not IFN-&ggr; production, dominated during early acute rejection. Recipient treatment with donor apoptotic 1-ethyl-3-(3′-dimethylaminopropyl)-carbodiimide-treated splenocytes significantly inhibited antidonor IL-17 response, and when combined with B cell depletion and a short course of rapamycin led to survival of pig islet xenografts beyond 100 days in approximately 65% recipients. Interestingly, treated recipients in this model experienced late rejection between 100 and 200 days posttransplant, which coincided with B cell reconstitution and an ensuing emergence of a robust antidonor IFN-&ggr;, but not IL-17, response. Conclusions These findings reveal that early and late rejection of pig islet xenografts may be dominated by different immune responses and that maintenance of long-term xenogeneic tolerance will require strategies that target the temporal sequence of antixenogeneic immune responses.