Balwan Singh

Resveratrol (Trans-3,5,4′-trihydroxystilbene) Induces Silent Mating Type Information Regulation-1 and Down-Regulates Nuclear Transcription Factor-κB Activation to Abrogate Dextran Sulfate Sodium-Induced Colitis

Inflammatory bowel disease is a chronic, relapsing, and tissue-destructive disease. Resveratrol (3,4,5-trihydroxy-trans-stilbene), a naturally occurring polyphenol that exhibits beneficial pleiotropic health effects, is recognized as one of the most promising natural molecules in the prevention and treatment of chronic inflammatory disease and autoimmune disorders. In the present study, we investigated the effect of resveratrol on dextran sodium sulfate (DSS)-induced colitis in mice and found that it effectively attenuated overall clinical scores as well as various pathological markers of colitis. Resveratrol reversed the colitis-associated decrease in body weight and increased levels of serum amyloid A, tumor necrosis factor-α, interleukin (IL-6), and IL-1β. After resveratrol treatment, the percentage of CD4+ T cells in mesenteric lymph nodes (MLN) of colitis mice was restored to normal levels, and there was a decrease in these cells in the colon lamina propria (LP). Likewise, the percentages of macrophages in MLN and the LP of mice with colitis were decreased after resveratrol treatment. Resveratrol also suppressed cyclooxygenase-2 (COX-2) expression induced in DSS-exposed mice. Colitis was associated with a decrease in silent mating type information regulation-1 (SIRT1) gene expression and an increase in p-inhibitory κB expression and nuclear transcription factor-κB (NF-κB) activation. Resveratrol treatment of mice with colitis significantly reversed these changes. This study demonstrates for the first time that SIRT1 is involved in colitis, functioning as an inverse regulator of NF-κB activation and inflammation. Furthermore, our results indicate that resveratrol may protect against colitis through up-regulation of SIRT1 in immune cells in the colon.

Effect of Mycobacterium tuberculosis-Specific 10-Kilodalton Antigen on Macrophage Release of Tumor Necrosis Factor Alpha and Nitric Oxide

ABSTRACT Secreted proteins of Mycobacterium tuberculosis are major targets of the specific immunity in tuberculosis and constitute promising candidates for the development of more efficient vaccines and diagnostic tests. We show here that M. tuberculosis-specific antigen 10 (MTSA-10, originally designated CFP-10) can bind to the surface of mouse J774 macrophage-like cells and stimulate the secretion of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α). MTSA-10 also synergized with gamma interferon (IFN-γ) for the induction of the microbicidal free radical nitric oxide (NO) in J774 cells, as well as in bone marrow-derived and peritoneal macrophages. On the other hand, pretreatment of J774 cells with MTSA-10 markedly reduced NO but not TNF-α or interleukin 10 (IL-10) release upon subsequent stimulation with lipopolysaccharide or the cell lysate of M. tuberculosis. The presence of IFN-γ during stimulation with M. tuberculosis lysate antagonized the desensitizing effect of MTSA-10 pretreatment on macrophage NO production. The activation of protein tyrosine kinases (PTK) and the serine/threonine kinases p38 MAPK and ERK was apparently required for MTSA-10 induction of TNF-α and NO release, as revealed by specific kinase inhibitors. However, only p38 MAPK activity, not PTK or ERK activity, was partly responsible for MTSA-10-mediated macrophage desensitization. The modulation of macrophage function by MTSA-10 suggests a novel mechanism for its involvement in immunopathogenesis of tuberculosis and might have implications for the prevention, diagnosis, and therapy of this disease.

Mycobacterium tuberculosis Antigens Induce the Differentiation of Dendritic Cells from Bone Marrow

We show in this study that incubation of freshly isolated bone marrow cells with Mycobacterium tuberculosis (M. tb) secretory Ag (MTSA), in the absence of any growth or differentiation-inducing factor, differentiates them into dendritic cell (DC)-like APCs. These DCs expressed moderate to high levels of various markers typical of DCs. These included T cell costimulatory molecules CD80, CD86, CD40, and CD54 and high levels of surface MHC class I and II on CD11c+ cells. The levels and the kinetics of up-regulation of these molecules were comparable with those of GM-CSF-differentiated DCs. Furthermore, these DCs exhibited morphology characteristics to DCs like the presence of dendritic processes. These DCs were also potent stimulators of allogeneic T cells and preferentially induced the secretion of IFN-γ over IL-10 from the interacting T cells. Interestingly, the differentiation of bone marrow cells into DC-like APCs was obtained with many other M. tb Ags, including whole cell extract of M. tb. Further characterization of MTSA-differentiated DCs showed that they were immature in nature, as stimulation of these DCs with TNF-α, anti-CD40, or LPS further up-regulated the surface levels of various molecules together with an increase in their T cell stimulatory capacity. The Ag-specific T cell responses of MTSA-differentiated DCs were mainly contributed by the CD4+ subset, indicating that MTSA was largely MHC II restricted. Furthermore, stimulation of bone marrow cells with MTSA induced the nuclear translocation of the transcription factor NF-κB, thereby indicating its role during MTSA-induced differentiation of DCs.

Down-Regulation of T Helper 1 Responses to Mycobacterial Antigens Due to Maturation of Dendritic Cells by 10-kDa Mycobacterium tuberculosis Secretory Antigen

Interactions of 10-kDa Mycobacterium tuberculosis secretory antigen (MTSA) with dendritic cells (DCs) were investigated to elucidate the role of secretory antigens in regulating immune responses to M. tuberculosis early in the course of infection. MTSA induced the maturation of different DC subsets. The cytokine profiles of these DCs were characteristic to each DC subset. Of interest, coculture of M. tuberculosis whole-cell extract (CE)-pulsed, MTSA-matured DCs with CE-specific T cells led to a marked reduction in interleukin (IL)-2 and interferon (IFN)-gamma production, thereby down-regulating proinflammatory responses to mycobacterial antigens. Attenuation of IL-2 and IFN-gamma levels of CE-specific T cells also was obtained when M. tuberculosis culture filtrate protein-activated DCs were employed as antigen-presenting cells, which suggests that MTSAs induce maturation of DCs at sites of infection, probably to down-regulate proinflammatory immune responses to mycobacteria that may subsequently be released from infected macrophages.

Cross-regulation of CD86 by CD80 differentially regulates T helper responses from Mycobacterium tuberculosis secretory antigen-activated dendritic cell subsets.

We report that stimulation of Mycobacterium tuberculosis secretory antigen‐ and tumor necrosis factor α‐matured BALB/c mouse bone marrow dendritic cells (BMDCs) with anti‐CD80 monoclonal antibody up‐regulated CD86 levels on the cell surface. Coculture of these BMDCs with naïve, allogeneic T cels now down‐regulated T helper cell type 1 (Th1) responses and up‐regulated suppressor responses. Similar results were obtained with splenic CD11c+/CD8a− DCs but not to the same extent with CD11c+/CD8a+ DCs. Following coculture with T cells, only BMDCs and CD11c+/CD8a− DCs and not CD11c+/CD8a+ DCs displayed increased levels of surface CD86, and further, coculturing these DCs with a fresh set of T cells attenuated Th1 responses and increased suppressor responses. Not only naïve but even antigen‐specific recall responses of the Th1‐committed cells were modulated by DCs expressing up‐regulated surface CD86. Further analyses showed that stimulation with anti‐CD80 increased interleukin (IL)‐10 and transforming growth factor‐β‐1 levels with a concomitant reduction in IL‐12p40 and interferon‐γ levels from BMDCs and CD11c+/CD8a− DCs and to a lesser extent, from CD11c+/CD8a+ DCs. These results suggest that cross‐talk between costimulatory molecules differentially regulates their relative surface densities leading to modulation of Th responses initiated from some DC subsets, and Th1‐committed DCs such as CD11c+/CD8a+ DCs may not allow for such modulation. Cognate antigen‐presenting cell (APC):T cell interactions then impart a level of polarization on APCs mediated via cross‐regulation of costimulatory molecules, which govern the nature of subsequent Th responses.

Intracellular expression of Mycobacterium tuberculosis-specific 10-kDa antigen down-regulates macrophage B7.1 expression and nitric oxide release.

To explore the role of the 10‐kDa Mycobacterium tuberculosis‐specific secreted antigen (MTSA‐10 or CFP‐10) in modulation of macrophage function, J774 macrophages were transfected stably with DNA encoding MTSA‐10. Compared to normal or mock‐transfected controls, MTSA‐10‐expressing macrophages had markedly lower levels of co‐stimulatory molecule B7·1 on their surface, while the expression of B7·2 and ICAM‐1 was not affected. MTSA‐transfected cells also produced significantly less microbicidal free radical nitric oxide (NO) upon stimulation with interferon (IFN)‐γ, lipopolysaccharide or M. tuberculosis cell lysate. Western blot analysis revealed the absence of tyrosine‐phosphorylated protein slightly larger than 112 kDa in MTSA‐transfected macrophages. Moreover, the treatment of control J774 cells with protein tyrosine kinase inhibitor genistein completely mimicked the effects of transfection with MTSA‐10, selectively down‐regulating NO and B7·1, but not B7·2 or ICAM‐1 expression. The observed MTSA‐10‐mediated block of B7·1 expression and NO release might contribute to the suppression of antimycobacterial response in tuberculosis.