Kuladip Jana

MicroRNA let-7 Modulates the Immune Response to Mycobacterium tuberculosis Infection via Control of A20, an Inhibitor of the NF-κB Pathway

The outcome of the interaction between Mycobacterium tuberculosis (Mtb) and a macrophage depends on the interplay between host defense and bacterial immune subversion mechanisms. MicroRNAs critically regulate several host defense mechanisms, but their role in the Mtb-macrophage interplay remains unclear. MicroRNA profiling of Mtb-infected macrophages revealed the downregulation of miR-let-7f in a manner dependent on the Mtb secreted effector ESAT-6. We establish that let-7f targets A20, a feedback inhibitor of the NF-κB pathway. Expression of let-7f decreases and A20 increases with progression of Mtb infection in mice. Mtb survival is attenuated in A20-deficient macrophages, and the production of TNF, IL-1β, and nitrite, which are mediators of immunity to Mtb, is correspondingly increased. Further, let-7f overexpression diminishes Mtb survival and augments the production of cytokines including TNF and IL-1β. These results uncover a role for let-7f and its target A20 in regulating immune responses to Mtb and controlling bacterial burden.

Identification of a novel role of ESAT-6-dependent miR-155 induction during infection of macrophages with mycobacterium tuberculosis

Mycobacterium tuberculosis (M.tb.) replicates in host macrophages to cause tuberculosis. We have investigated the role of miRNAs in M.tb.‐infected murine RAW264.7 cells and bone marrow‐derived macrophages (BMDMs), focusing on miR‐155, the most highly upregulated miRNA. We observed that miR‐155 upregulation is directly linked to the attenuation of expression of BTB and CNC homology 1 (Bach1) and SH2‐containing inositol 5′‐phosphatase (SHIP1). Bach1 is a transcriptional repressor of haem oxygenase‐1 (HO‐1), whereas SHIP1 inhibits the activation of the serine/threonine kinase AKT. We hypothesize that M.tb.‐induced miR‐155 induction leads to repression of Bach1, which augments the expression of HO‐1, a documented activator of the M.tb. dormancyregulon. SHIP1 repression facilitates AKT activation, which is required for M.tb. survival. In addition, M.tb.‐induced miR‐155 inhibits expression of cyclooxygenase‐2 (Cox‐2) and interleukin‐6 (Il‐6), two modulators of the innate immune response. Importantly, we observed that the virulence‐associated secreted protein ESAT‐6 plays a key role in miR‐155 induction and its subsequent effects on Bach1 and SHIP1 repression. Inhibition of miR‐155 hindered survival of M.tb. in RAW264.7 and in murine BMDMs. Thus, our results offer new insights into the role of miRNAs in modulation of the host innate immune response by M.tb. for its own benefit.

Recurrent Pregnancy Loss in Polycystic Ovary Syndrome: Role of Hyperhomocysteinemia and Insulin Resistance

Recurrent pregnancy loss (RPL) in polycystic ovary syndrome (PCOS), which occurs in ∼50% of total pregnancies is a frequent obstetric complication. Among the several hypotheses, insulin resistance (IR), obesity and hyperhomocysteinemia (HHcy) play significant role/s in RPL. This study was conducted to assess the link between elevated levels of homocysteine and IR in PCOS-associated women with RPL in Kolkata, India. A retrospective study was conducted of one hundred and twenty six PCOS women (<30 years) who experienced two or more spontaneous abortions during the first trimester presenting to Institute of Reproductive Medicine (IRM) in Kolkata during the period of March 2008 through February 2011. One hundred and seventeen non-PCOS subjects with matching age range were randomly chosen as controls. Incidence of HHcy and IR was 70.63% (n = 89) and 56.34% (n = 71), respectively, in RPL-affected PCOS population which was significantly higher (p<0.04; p<0.0001) when compared to the non-PCOS set (HHcy: 57.26%; IR: 6.83%). Rates of miscarriage were significantly higher (p<0.008; p<0.03) in hyperhomocysteinemia-induced miscarriage when compared to the normohomocysteinemic segment (PCOS: 70.63% vs.29.36% & non-PCOS: 57.26% vs. 42.73%) along with the insulin resistant (p<0.04; p<0.0001) population (PCOS: 70.63% vs. 56.34% & non-PCOS: 57.26% vs. 6.83%) in both groups. A probabilistic causal model evaluated HHcy as the strongest plausible factor for diagnosis of RPL. A probability percentage of 43.32% in the cases of HHcy- mediated RPL suggests its increased tendency when compared to IR mediated miscarriage (37.29%), further supported by ROC-AUC (HHcy: 0.778vs. IR: 0.601) values. Greater susceptibility towards HHcy may increase the incidence for miscarriage in women in India and highlights the need to combat the condition in RPL control programs in the subcontinent.

MicroRNA 17-5p regulates autophagy in Mycobacterium tuberculosis-infected macrophages by targeting Mcl-1 and STAT3.

Autophagy plays a crucial role in the control of bacterial burden during Mycobacterium tuberculosis infection. MicroRNAs (miRNAs) are small non‐coding RNAs that regulate immune signalling and inflammation in response to challenge by pathogens. Appreciating the potential of host‐directed therapies designed to control autophagy during mycobacterial infection, we focused on the role of miRNAs in regulating M. tuberculosis‐induced autophagy in macrophages. Here, we demonstrate that M. tuberculosis infection leads to downregulation of miR‐17 and concomitant upregulation of its targets Mcl‐1 and STAT3, a transcriptional activator of Mcl‐1. Forced expression of miR‐17 reduces expression of Mcl‐1 and STAT3 and also the interaction between Mcl‐1 and Beclin‐1. This is directly linked to enhanced autophagy, because Mcl‐1 overexpression attenuates the effects of miR‐17. At the same time, transfection with a kinase‐inactive mutant of protein kinase C δ (PKCδ) (an activator of STAT3) augments M. tuberculosis‐induced autophagy, and miR‐17 overexpression diminishes phosphorylation of PKCδ, suggesting that an miR‐17/PKC δ/STAT3 axis regulates autophagy during M. tuberculosis infection.

Altered Trace Mineral Milieu Might Play An Aetiological Role in the Pathogenesis of Polycystic Ovary Syndrome

Insulin resistance is a very common associate of polycystic ovary syndrome (PCOS). Pathophysiology in relation with the essential elements including copper, magnesium, zinc, manganese, chromium, and calcium has been reported in women with insulin resistance. This prospective study was designed to explore whether the women with PCOS do exhibit altered serum element levels in association with/without insulin resistance. One hundred and thirty-two women with PCOS and forty-six control women were studied. Women with PCOS were further divided based on the presence of insulin resistance (insulin resistant: n = 50; non-insulin resistant: n = 82). In all women, basal levels of gonadotropins, prolactin, testosterone, insulin, glucose, and the six different elements were measured. Serum levels of testosterone (p < 0.001), luteinizing hormone (p < 0.05), and fasting insulin (p < 0.004) were significantly higher in the PCOS population compared to controls as well as PCOS women without insulin resistance. Women with PCOS exhibited a significantly high calcium (p < 0.04) and lower manganese levels (p < 0.002) when compared to controls. However, the PCOS women with insulin resistance exhibited significantly lower serum levels of magnesium and chromium (p < 0.04), in addition to higher levels of zinc and copper (p < 0.04). The differences in calcium (p < 0.03) and manganese levels (p < 0.0001) became aggravated with the presence of insulin resistance when compared to control as well as PCOS women without insulin resistance. In PCOS-associated insulin resistance, circulating serum magnesium (r = −0.31; p < 0.03) and chromium (r = −0.38; p < 0.006) status significantly correlated with fasting insulin levels. We conclude that imbalanced element status may be a key foundation for insulin resistance in PCOS. The findings in this study should be investigated with further trials in order to obtain new insights into PCOS.

Ethanol induces mouse spermatogenic cell apoptosis in vivo through over‐expression of Fas/Fas‐L, p53, and caspase‐3 along with cytochrome c translocation and glutathione depletion

Although it has been well established that spermatogenic cells undergo apoptosis when treated with ethanol, the molecular mechanisms behind it remain to be investigated. Adult male mice were given intra‐peritoneal injection (IP) of ethanol at a dose of 3 g (15%, v/v) per kg body weight per day during the period of 14 days. Testicular androgenesis and apoptotic germ cell death, along with different interrelated proteins expression, were evaluated. Ethanol treatment induced apoptotic spermatogenic cell death with a decrease in the plasma and intra‐testicular testosterone concentration. Western blot analysis revealed that repeated ethanol treatment decreased the expression of steroidogenic acute regulatory protein (StAR), 3 beta‐hydroxysteroid dehydrogenase (3β‐HSD) and 17 beta‐hydroxysteroid dehydrogenase (17β‐HSD); increased the expression of active caspase‐3, p53, Fas and Fas‐L; and led to up‐regulation of Bax/Bcl‐2 ratio and translocation of cytochrome c from mitochondria to cytosol in testis. It has also been shown in our study that repeated ethanol treatment led to up‐regulation of caspase‐3, p53, Fas, Fas‐L transcripts; increase in caspase‐3 and caspase‐8 activities; diminution of 3β‐HSD, 17β‐HSD, and GPx activities; decrease in the mitochondrial membrane potential along with ROS generation and depletion of glutathione pool in the testicular tissue. The present study has indicated that the ethanol treatment induced apoptosis in the mouse testis through the increased expression of Fas/Fas‐L and p53, up‐regulation of Bax/Bcl‐2 ratio, cytosolic translocation of cytochrome c along with caspase‐3 activation and glutathione depletion. Mol. Reprod. Dev. 77: 820–833, 2010.

Protective effect of sodium selenite and zinc sulfate on intensive swimming-induced testicular gamatogenic and steroidogenic disorders in mature male rats

To investigate the ameliorative potential of sodium selenite and zinc sulfate on intensive-swimming-induced testicular disorders, 48 Wistar male rats (age, 4 months; mass, 146.2 +/- 3.6 g) were randomly divided into 4 groups: the unexercised-control group (n = 12); the exercised group (n = 12); the control supplemented group (n = 12); and the exercised supplemented group (n = 12). For 10 weeks, the exercised rats underwent a protocol that consisted of 4 h.d-1 swimming, for 6 d.week-1; the control rats did not exercise. For 10 weeks, both the supplemented groups received an oral daily dose of a combination of sodium selenite and zinc sulfate (6 and 3 mg.kg body mass-1, respectively). After 10 weeks, a significant reduction (p < 0.05) was seen in rats in the exercised group, compared with rats in both control groups, in paired testicular masses; in epididymal sperm count; in testicular Delta5, 3beta-hydroxysteroid dehydrogenase (HSD) and 17beta-HSD; in plasma levels of testosterone, luteinizing hormone, follicle-stimulating hormone, and prolactin; in the numbers of preleptotine spermatocytes, midpachytene spermatocytes, and stage 7 spermatids of the stage VII seminiferous epithelium cycle; and in fertility performance. As well, a significant increase (p < 0.05) was seen in the exercised group, compared with both control groups, in plasma corticosterone levels and in testicular content of malondialdehyde and catalase activity. At the same time, there was a significant reduction (p < 0.05) in the exercised group, compared with both control groups, in plasma concentrations of zinc and selenium; in the testicular content of glutathione (GSH), the glutathione and glutathione disulphide (GSSG) ratio, ascorbic acid, and alpha-tocopherol; and in testicular activities of superoxide dismutase, glutathione-peroxidase, and glutathione-S-transferase in the testes. No significant changes were seen in the number of spermatogonia-A from the stage VII seminiferous epithelium cycle or the testicular content of GSSG among the groups. Sodium selenite and zinc sulfate supplementation significantly protected against exercise-induced testicular gamatogenic and spermatogenic disorders, prevented testicular oxidative stress, and increased antioxidant status. It can be concluded that intensive-swimming-induced oxidative stress causes dysfunctions in the male reproductive system, which can be protected by the coadministration of sodium selenite and zinc sulfate.

The DNA intercalators ethidium bromide and propidium iodide also bind to core histones

Eukaryotic DNA is compacted in the form of chromatin, in a complex with histones and other non‐histone proteins. The intimate association of DNA and histones in chromatin raises the possibility that DNA‐interactive small molecules may bind to chromatin‐associated proteins such as histones. Employing biophysical and biochemical techniques we have characterized the interaction of a classical intercalator, ethidium bromide (EB) and its structural analogue propidium iodide (PI) with hierarchical genomic components: long chromatin, chromatosome, core octamer and chromosomal DNA. Our studies show that EB and PI affect both chromatin structure and function, inducing chromatin compaction and disruption of the integrity of the chromatosome. Calorimetric studies and fluorescence measurements of the ligands demonstrated and characterized the association of these ligands with core histones and the intact octamer in absence of DNA. The ligands affect acetylation of histone H3 at lysine 9 and acetylation of histone H4 at lysine 5 and lysine 8 ex vivo. PI alters the post‐translational modifications to a greater extent than EB. This is the first report showing the dual binding (chromosomal DNA and core histones) property of a classical intercalator, EB, and its longer analogue, PI, in the context of chromatin.

Clinical Evaluation of Non-surgical Sterilization of Male Cats with Single Intra-testicular Injection of Calcium Chloride

BackgroundCalcium chloride solution is an established injectable sterilant in dogs and other mammals. With cat populations a continuing problem, we sought to explore its first use in cats. Six cats per group were injected with 5%, 10% or 20% calcium chloride dihydrate in saline solution with lignocaine hydrochloride, a local anaesthetic.ResultsAt the 60th day post-injection, cat testes were collected and showed complete testicular necrosis and replacement by fibrous tissue; very low sperm counts; and reduction of serum testosterone by at least 70% in 20% dose. Androgenic enzyme activities and their expressions were also reduced in all the treated groups along with intra-testicular testosterone concentration was also low. Increased testicular lipid peroxidation, with reduced antioxidants and mitochondrial membrane potential, were evident following calcium chloride treatments. However, there were no apparent changes in serum concentrations of cortisol, fasting blood sugar level, blood urea nitrogen, packed cell volume, or total serum protein following calcium chloride injection, suggesting that this method of sterilization is not associated with any general stress response.ConclusionCalcium chloride solution demonstrates potential for androgenesis-eliminating nonsurgical sterilization of male cats in addition to its proven efficacy in dogs and other mammals.

MicroRNA 26a (miR-26a)/KLF4 and CREB-C/EBPβ regulate innate immune signaling, the polarization of macrophages and the trafficking of Mycobacterium tuberculosis to lysosomes during infection

For efficient clearance of Mycobacterium tuberculosis (Mtb), macrophages tilt towards M1 polarization leading to the activation of transcription factors associated with the production of antibacterial effector molecules such as nitric oxide (NO) and proinflammatory cytokines such as interleukin 1 β (IL-1β) and tumor necrosis factor α (TNF-α). At the same time, resolution of inflammation is associated with M2 polarization with increased production of arginase and cytokines such as IL-10. The transcriptional and post-transcriptional mechanisms that govern the balance between M1 and M2 polarization, and bacteria-containing processes such as autophagy and trafficking of Mtb to lysosomes, are incompletely understood. Here we report for the first time, that the transcription factor KLF4 is targeted by microRNA-26a (miR-26a). During Mtb infection, downregulation of miR-26a (observed both ex vivo and in vivo) facilitates upregulation of KLF4 which in turn favors increased arginase and decreased iNOS activity. We further demonstrate that KLF4 prevents trafficking of Mtb to lysosomes. The CREB-C/EBPβ signaling axis also favors M2 polarization. Downregulation of miR-26a and upregulation of C/ebpbeta were observed both in infected macrophages as well as in infected mice. Knockdown of C/ebpbeta repressed the expression of selected M2 markers such as Il10 and Irf4 in infected macrophages. The importance of these pathways is substantiated by observations that expression of miR-26a mimic or knockdown of Klf4 or Creb or C/ebpbeta, attenuated the survival of Mtb in macrophages. Taken together, our results attribute crucial roles for the miR-26a/KLF4 and CREB-C/EBPβsignaling pathways in regulating the survival of Mtb in macrophages. These studies expand our understanding of how Mtb hijacks host signaling pathways to survive in macrophages, and open up new exploratory avenues for host-targeted interventions.