LRRK2 and RIPK2 variants in the NOD 2-mediated signaling pathway are associated with susceptibility to Mycobacterium leprae in Indian populations

In recent years, genome wide association studies have discovered a large number of gene loci that play a functional role in innate and adaptive immune pathways associated with leprosy susceptibility. The immunological control of intracellular bacteria M. leprae is modulated by NOD2-mediated signaling of Th1 responses. In this study, we investigated 211 clinically classified leprosy patients and 230 ethnically matched controls in Indian population by genotyping four variants in NOD2 (rs9302752A/G), LRRK2 (rs1873613A/G), RIPK2 (rs40457A/G and rs42490G/A). The LRRK2 locus is associated with leprosy outcome. The LRRK2 rs1873613A minor allele and respective rs1873613AA genotypes were significantly associated with an increased risk whereas the LRRK2 rs1873613G major allele and rs1873613GG genotypes confer protection in paucibacillary and leprosy patients. The reconstructed GA haplotypes from RIPK2 rs40457A/G and rs42490G/A variants was observed to contribute towards increased risk whereas haplotypes AA was observed to confer protective role. Our results indicate that a possible shared mechanisms underlying the development of these two clinical forms of the disease as hypothesized. Our findings confirm and validates the role of gene variants involved in NOD2-mediated signalling pathways that play a role in immunological control of intracellular bacteria M. leprae

NK-CD11c+ Cell Crosstalk in Diabetes Enhances IL-6-Mediated Inflammation during Mycobacterium tuberculosis Infection

In this study, we developed a mouse model of type 2 diabetes mellitus (T2DM) using streptozotocin and nicotinamide and identified factors that increase susceptibility of T2DM mice to infection by Mycobacterium tuberculosis (Mtb). All Mtb-infected T2DM mice and 40% of uninfected T2DM mice died within 10 months, whereas all control mice survived. In Mtb-infected mice, T2DM increased the bacterial burden and pro- and anti-inflammatory cytokine and chemokine production in the lungs relative to those in uninfected T2DM mice and infected control mice. Levels of IL-6 also increased. Anti-IL-6 monoclonal antibody treatment of Mtb-infected acute- and chronic-T2DM mice increased survival (to 100%) and reduced pro- and anti-inflammatory cytokine expression. CD11c+ cells were the major source of IL-6 in Mtb-infected T2DM mice. Pulmonary natural killer (NK) cells in Mtb-infected T2DM mice further increased IL-6 production by autologous CD11c+ cells through their activating receptors. Anti-NK1.1 antibody treatment of Mtb-infected acute-T2DM mice increased survival and reduced pro- and anti-inflammatory cytokine expression. Furthermore, IL-6 increased inflammatory cytokine production by T lymphocytes in pulmonary tuberculosis patients with T2DM. Overall, the results suggest that NK-CD11c+ cell interactions increase IL-6 production, which in turn drives the pathological immune response and mortality associated with Mtb infection in diabetic mice

Glutathione-redox balance regulates c-rel-driven IL-12 production in macrophages: possible implications in antituberculosis immunotherapy

The glutathione-redox balance, expressed as the ratio of intracellular reduced glutathione (GSH) and oxidized glutathione, plays an important role in regulating cellular immune responses. In the current study, we demonstrate that alteration of glutathione-redox balance in macrophages by GSH donors like cell-permeable glutathione ethyl ester reduced or N-acetyl-L-cysteine (NAC) can differentially regulate production of IL-12 cytokine in macrophages. A low concentration of NAC increased IL-12 p40/p70 production, whereas at high concentration, IL-12 production was inhibited due to increased calmodulin expression that binds and sequesters c-rel in the cytoplasm. Although NAC treatment increased the IκBα phosphorylation, it failed to increase TNF-α levels due to enhanced expression of suppressor of cytokine signaling 1, which specifically prevented nuclear translocation of p65 NF-κB. We demonstrate that NAC at 3 mM concentration could increase bacillus Calmette-Guerin–induced IFN-γ production by PBMCs from patients with active tuberculosis and shifts the anti–bacillus Calmette-Guerin immune response toward the protective Th1 type. Our results indicate that redox balance of glutathione plays a critical role in regulating IL-12 induction in native macrophages and NAC can be used in tailoring macrophages to induce enhanced Th1 response that may be helpful to control tuberculosis and other pathophysiological disorders. Glutathione is the most predominant intracellular low m.w. thiol and acts as a reducing agent and an antioxidant. Glutathione is implicated in many cellular functions, including synthesis and degradation of protein and DNA. The intracellular redox environment is mainly controlled by the glutathione-redox, which is defined as the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) (1) and plays critical roles in maintaining cellular homeostasis and various physiological functions (2). The cellular glutathione-redox dynamically regulates protein functions by reversible disulfide bond formation on variety of proteins, including phosphatases, kinases and transcription factors (3–5) and is known to protect against oxidative stress (6, 7). The glutathione-redox balance in macrophages is shown to be critical for mounting innate immune responses and has also been implicated in many of the pathological conditions (2, 8–10), indicating its possible potential use as a therapeutic agent. However, redox-based therapy is still in its early developmental phases. It has been observed that triggering of macrophage receptors by pathogen products could change the macrophage redox status (9) and modulate the cytokine milieu (11, 12). Various studies indicate that glutathione levels in APCs can influence the Th response (13, 14). It is well known that the Th1/Th2 lineage commitment depends to a great extent on the cytokine milieu induced during innate activation of macrophages (15, 16). For example, IL-12 and TNF-α activate the Th1 T cell, whereas IL-10 cytokine favors the Th2 T cell development. Therefore, understanding the signaling pathways modulated by the glutathione-redox in macrophages and thereby influencing induction of these cytokines is important to understand how the T cell microenvironment is controlled during various pathophysiological conditions. However, the molecular mechanisms by which glutathione-redox can directly control macrophage functions and cytokine induction profile are largely not understood, other than in a few cases like GSH-mediated regulation of NF-κB (6, 7). There are indications that Th1 immune response is downregulated in patients with active tuberculosis (TB) infection (17). Interestingly, studies also indicate that patients with TB have altered glutathione balance (18). However, it is not clear whether Mycobacterium tuberculosis modulates the T cell responses by altering the glutathione-redox balance in macrophages. In the current study, we demonstrate that IL-12 induction in native macrophages is controlled directly by the intracellular glutathione-redox through calmodulin (CaM) and c-rel and manipulation of the macrophage-glutathione-redox can influence in vitro cellular immune response of PBMCs obtained from patients with active tuberculosis to Bacillus Calmette-Guerin (BCG)

Assessment of renal function and acute rejection using Cystatin C and kidney injury Molecule-1 in renal transplant recipients

Purpose: Kidney injury molecule (KIM)-1, a transmembrane-tubular protein, is excreted in the urine within 12 h of the initial ischemic insult and before regeneration of the epithelium. Cystatin C (CysC) a low-molecular-weight nonglycosylated protein has been shown to be a good marker of kidney function. We aimed to evaluate the levels of KIM-1 and CysC immediately after transplantation as an early marker. Subjects and Methods: Urine and blood samples were collected from prospective renal transplant patients with chronic allograft dysfunction (CAD) at baseline and during the follow-up period at the interval of 12, 24 h, 7th postoperative day 15th and 6 m, and compared with healthy controls (HC) for KIM-1 CysC S.creatinine (SCr) and creatinine clearance. Results: Kidney transplant recipients showed significantly higher KIM-1and CysC values than the control group. Nonparametric receiver-operating characteristic (ROC) curve of the renal function with an area under the curve of 0.518 KIM-1, CysC was 0.841 and creatinine 0.74 indicating CysC at 12 h posttransplant (post-Tx) is a better biomarker among three. ROC of acute rejection (AR) of KIM-1 at 24 h post-Tx showed sensitivity of 0.938. ROC for distinguishing between graft survival and failure at 1 year showed a sensitivity of 0.763 for CysC, In CAD, both KIM-1and CysC were increased as compared to HC. Conclusion: Both KIM-1 and CysC are useful markers for predicting AR, renal function. Elevated urinary levels of KIM-1 independently predict AR. CysC is a valuable marker of predicting the long-term outcome

PPE17 (Rv1168c) protein of Mycobacterium tuberculosis detects individuals with latent TB infection.

Latent tuberculosis infection (LTBI) is a clinically distinct category of Mycobacterium tuberculosis (Mtb) infection that needs to be diagnosed at the initial stage. We have reported earlier that one of the Mtb proline-proline-glutamic acid (PPE) proteins, PPE17 (Rv1168c) is associated with stronger B-cell and T-cell responses and could be used to diagnose different clinical categories of active TB patients with higher specificity and sensitivity than PPD and ESAT-6. Based on these observations we further tested the potential of PPE17 for the diagnosis of LTBI. We tested 198 sera samples collected from LTBI individuals (n = 61), QFT-negative (n = 58) and active TB patients (n = 79). Individuals were defined as LTBI by QuantiFERON-TB Gold In-Tube test (QFT-GIT) positive results, while active TB patients were confirmed based on the guidelines of the Revised National TB Control Programme of India. The antibody responses against PPE17, ESAT-6:CFP-10 and PPD were compared in these subjects by enzyme-linked immunosorbent assay. We observed that LTBI individuals show a higher sero-reactivity to PPE17 as compared to currently used latent TB diagnostic antigens like ESAT-6, CFP-10 and PPD. The LTBI and active TB patients display almost similar sensitivity. Interestingly, PPE17 could discriminate LTBI positive subjects from the QFT-negative subjects (P < 0.001). Our study hints that PPE17 may be used as a novel serodiagnostic marker to screen the latently infected subjects and may also be used as a complimentary tool to the QFT-GIT

Association of Strong Immune Responses to PPE Protein Rv1168c with Active Tuberculosis ▿

Accurate diagnosis of tuberculosis (TB) infection is critical for the treatment, prevention, and control of TB. Conventional diagnostic tests based on purified protein derivative (PPD) do not achieve the required diagnostic sensitivity. Therefore, in this study, we have evaluated the immunogenic properties of Rv1168c, a member of the PPE family, in comparison with PPD, which is routinely used in the tuberculin test, and Hsp60 and ESAT-6, well-known immunodominant antigens of Mycobacterium tuberculosis. In a conventional enzyme immunoassay, the recombinant Rv1168c protein displayed stronger immunoreactivity against the sera obtained from patients with clinically active TB than did PPD, Hsp60, or ESAT-6 and could distinguish TB patients from Mycobacterium bovis BCG-vaccinated controls. Interestingly, Rv1168c antigen permits diagnosis of smear-negative pulmonary TB as well as extrapulmonary TB cases, which are often difficult to diagnose by conventional tests. The immunodominant nature of Rv1168c makes it a promising candidate to use in serodiagnosis of TB. In addition, our studies also show that Rv1168c is a potent T-cell antigen which elicits a strong gamma interferon response in sensitized peripheral blood mononuclear cells obtained from TB patients