Inflammatory gene silencing in activated monocytes by a cholesterol tagged-miRNA/siRNA: a novel approach to ameliorate diabetes induced inflammation.

There is a major unmet need for the development of effective therapies for diabetes induced inflammation. Increased adenosine-uridine rich elements (AREs) containing mRNAs of inflammatory molecules are reported in inflamed monocytes. Destabilizing these inflammatory mRNAs by the miR-16 could reduce inflammation. DNA microarrays and in vitro cell studies showed that exogenous miR16 and its mimic treatment, in LPS/PMA induced monocytes, significantly downregulated several ARE containing inflammatory cytokine mRNAs similar to those seen in the normal monocytes. Ingenuity pathway analyses showed exogenous miR-16 or its synthetic mimic treatment alleviates inflammatory responses. To selectively target uptake, especially to inflamed cells, one of the CD36 substrate cholesterol was tagged to miR16/siRNA. Cholesterol tagged miR-16/ARE-siRNA showed enhanced uptake in CD36 expressing inflamed cells. In LPS or PMA, treated monocytes, candidate genes expressions levels such as IL-6, IL-8, IL-12ß, IP-10, and TNF-α mRNA were increased, as measured by RT-qPCR as seen in primary monocytes of diabetes patients. Exogenous miR16 or ARE-siRNA transfection reduced mRNAs of pro-inflammatory cytokines levels in monocyte, and its adhesion. Increased uptake of cholesterol tagged miR-16 through the CD36 receptor was observed. This destabilizes numerous inflammatory ARE containing mRNAs and alleviates inflammatory responses. Cholesterol-tagged miR-16 and its mimic are novel anti-inflammatory molecules that can be specifically targeted to, via through CD36 expressing, "inflamed" cells and thus serve as therapeutic candidates to alleviate inflammatory diseases.

HnRNPK and lysine specific histone demethylase-1 regulates IP-10 mRNA stability in monocytes.

Altered mRNA metabolism is a feature of many inflammatory diseases. Post transcriptional regulation of interferon-γ-inducible protein (IP)-10 has been uncharacterized in diabetes conditions. RNA-affinity capture method and RNA immuno-precipitation revealed S100b treatment increased the binding of heterogeneous nuclear ribonucleoprotein (hnRNP)K to the IP-10 3'UTR and increased IP-10 mRNA accumulation. Luciferase activity assay using reporter plasmids showed involvement of IP-10 3'UTR. Knocking down of hnRNPK destabilized S100b induced IP-10 mRNA accumulation. S100b promoted the translocation of hnRNPK from nucleus to the cytoplasm and this was confirmed by phosphomimetic S284/353D mutant and non-phosphatable S284/353A hnRNPK mutant. S100b treatment demethylates hnRNPK at Lys219 by Lysine Specific Demethylase (LSD)-1. HnRNPKK219I, a demethylation defective mutant increased IP-10 mRNA stability. Apparently, triple mutant hnRNPKK219I/S284D/353D promoted IP-10 mRNA stability. Interestingly, knocking down LSD-1 abolished S100b induced IP-10 mRNA accumulation. These observations show for the first time that IP-10 mRNA stability is dynamically regulated by Lysine demethylation of hnRNPK by LSD-1. These results indicate that hnRNPK plays an important role in IP-10 mRNA stability induced by S100b which could exacerbate monocyte activation, relevant to the pathogenesis of diabetic complications like atherosclerosis.

Biosynthesis of Silver Nanoparticles from Leaf Extract of Salvia coccinea and Its Effects of Anti-inflammatory Potential in Human Monocytic THP-1 Cells.

Inflammation is one of the basic pathophysiologically important components in many life-threatening diseases. Metallic nanoparticles play a crucial role in biomedical applications. The present study was aimed at investigating the ameliorative effect of biosynthesized silver nanoparticles (ScAgNPs) using Salvia coccinea leaf extracts and characterizing them using physical and chemical methods, followed by evaluation of their cytotoxic, anti-oxidant, and anti-inflammatory potentials in monocytic THP-1 cells. Luciferase reporter assays and qRT-PCRs were used for gene expression studies. As oxidative stress and inflammation are mutually induced by each other, inhibiting oxidative stress could subsequently lead to inhibition of inflammation. Spherical-shaped ScAgNPs with 24 nm average size were successfully synthesized. The DCF staining technique, in addition to DPPH and reducing power activity assays, showed that 100-400 µg/mL concentration of ScAgNPs decreased oxidative stress significantly, induced by high glucose, in THP-1 cells. Anti-inflammatory effects of ScAgNPs have corroborated inhibition of high-glucose-induced oxidative stress-sensitive transcription factor NF-κB-driven transcription of proinflammatory COX-2, MCP-1, IP-10, IL-17E, and IL-6 promoters significant in high-glucose-grown THP-1 cells, consistent with promoter inhibition, and the corresponding mRNA expression levels were also decreased, suggesting that ScAgNPs could be a potential anti-inflammatory agent, which could efficiently inhibit inflammation in THP-1 cells. Our initial in vitro studies suggested that ScAgNPs could serve as therapeutic candidates to alleviate inflammatory diseases by inhibiting oxidative stress and inflammation.

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.