List of Primers.

<p>List of Primers.</p

Chikungunya Virus Exploits miR-146a to Regulate NF-κB Pathway in Human Synovial Fibroblasts

<div><p>Objectives</p><p>Chikungunya virus causes chronic infection with manifestations of joint pain. Human synovial fibroblasts get infected with CHIKV and could lead to pro-inflammatory responses. MicroRNAs have potentials to regulate the gene expression of various anti-viral and pro-inflammatory genes. The study aims to investigate the role of miR-146a in modulation of inflammatory responses of human synovial fibroblasts by Chikungunya virus.</p><p>Methods</p><p>To study the role of miR-146a in CHIKV pathogenesis in human synovial cells and underlying inflammatory manifestations, we performed CHIKV infection in primary human synovial fibroblasts. Western blotting, real-time PCR, luciferase reporter assay, overexpression and knockdown of cellular miR-146a strategies have been employed to validate the role of miR-146a in regulation of pro-inflammatory NF-κB pathway.</p><p>Results</p><p>CHIKV infection induced the expression of cellular miR-146a, which resulted into down-regulation of TRAF6, IRAK1, IRAK2 and increased replication of CHIKV in human synovial fibroblasts. Exogenous expression of miR-146a in human synovial fibroblasts led to decreased expression of TRAF6, IRAK1, IRAK2 and decreased replication of CHIKV. Inhibition of cellular miR-146a by anti-miR-146a restored the expression levels of TRAF6, IRAK1 and IRAK2. Downregulation of TRAF6, IRAK1 and IRAK2 led to downstream decreased NF-κB activation through negative feedback loop.</p><p>Conclusion</p><p>This study demonstrated the mechanism of exploitation of cellular miR-146a by CHIKV in modulating the host antiviral immune response in primary human synovial fibroblasts.</p></div

CHIKV suppresses <b>NF-κB</b> activity through miR-146a.

<p>Increase in miR-146a expression levels suppresses the activation of NF-κB. 2 µg of NF-κB -FLuc plasmid was co-transfected with 700 ng of pCMV-β-gal plasmid in HEK 293 T cells for reporter assay. 6–8 hours post transfection, cells were infected with CHIKV (MOI 2) and harvested 32 hours post infection for Luciferase assay. (<b>A</b>) The graph bars are representing relative luciferase activity of NF-κB in control and CHIKV infected primary human synovial fibroblasts after normalization with β-galactosidase expression units showing decreased NF-κB activity. (<b>B</b>) The graph bars representing relative luciferase units in miR-146a overexpression. Transfection of miR-146a decreases NF-κB activity compared to controls and scramble miR-146a. (<b>C</b>) HEK cells were infected with CHIKV after the 24 hours of anti-miR-146a transfection. Anti-miR-146a transfection rescues the activity of NF-κB even in cells infected with CHIKV. The graph bars are showing elevated luciferase activity in anti-miR-146a and anti-miR-146a+CHIKV infection. All experiments were performed in biological triplicates and data represented as mean ± SEM. (*for p value≤0.05, **for p value≤0.005).</p

miR-146a overexpression decreases IRAK1 and IRAK2 protein expression levels in primary human synovial fibroblasts.

<p>(A) Western blot analysis showing down regulation of IRAK1 at the protein expression level in miR-146a over expressed primary human synovial fibroblasts. (B) Densitometry analysis of IRAK1 expression levels normalized with β-tubulin. (C). Western blot analysis showing decreased expression of IRAK2 at the protein levels in miR-146a overexpressed primary human synovial fibroblasts. (D) Densitometry analysis of IRAK2 protein expression levels upon normalization with β-tubulin. Experiments were repeated three times and results shown as mean ± SEM. (* for p value≤0.05, ** for p value≤0.005).</p

Induced expression of miR-146a decreases NF-κB activation.

<p>CHIKV infection decreases activation of NF-κB in negative feedback loop. (<b>A</b>). Western blot image showing decreased phosphorylation of NF-κB (p65), phospho NF-κB (p65) and total NF-κB (p65) after CHIKV infection in primary human synovial fibroblasts. (<b>B</b>) Densitometry analysis shown as graph bars for protein expression levels of p- NF-κB (p65) and total NF-κB (p65) normalized with β-tubulin. (<b>C</b>) Western blot analysis of p- NF-κB (p65) and total NF-κB (p65) protein expression levels in primary human synovial fibroblasts transfected with 100 pm scrambled and 100 pm miR-146a mimics. Increase in miR-146a significantly decreases p- NF-κB (p65) levels compared to controls and scramble miR-146a. (<b>D</b>) The graph bars representing densitometry of protein expression levels of p- NF-κB (p65) and total NF-κB (p65) after normalization with β-tubulin. Three independent experiments were performed and data is presented as mean ± SEM. *p<0.05.</p

Anti-miR-146a rescues the target gene expression levels.

<p>Anti-miR-146a suppresses the miR-146a levels and rescues target gene expression levels. (<b>A</b>) The graph bars are showing a significant decrease in the expression of cellular miR-146a after transfection with anti-miR-146a (miR-146a inhibitors) compared to CHIKV infection (*≤0.05). Cy3 labelled controls anti-miR was used to check the transfection efficiency. RNU24 expression levels have been used as normalizer and fold change in miR-146a has been calculated by ΔΔct methods. (<b>B</b>) Western blot images showing the rescued expression of TRAF6, IRAK1 and IRAK2 due to knockdown of cellular miR-146a, 48 hours post anti-miR-146a transfection. (<b>C</b>) The graph bars representing densitometry analysis of western blot images for TRAF6, IRAK1 and IRAK2 normalized with β-tubulin. All the experiments were repeated in three biological replicates and represented as mean ± SEM. * above bars are representing the p value≤0.05 as level of significance, n = 3. (* for p value≤0.05, ** for p value≤0.005, *** for p value≤0.001).</p

CHIKV mediated regulation of NF-κB by miR-146a modulation in primary human synovial fibroblasts.

<p>CHIKV infection induces the expression of cellular miR-146a in primary human synovial fibroblasts, which in turn downregulates the expression of TRAF6, IRAK1 and IRAK2. Decreased expression of these immune modulators results into reduced NF-κB phosphorylation and activation in primary human synovial fibroblasts.</p

MicroRNA sequences.

<p>MicroRNA sequences.</p

Neglected tropical diseases in China and India.

<p>Neglected tropical diseases in China and India.</p

Socioeconomic aspects of China and India.

<p>Socioeconomic aspects of China and India.</p