DataSheet_1_TLR4 activation by lysozyme induces pain without inflammation.pdf

Mostly, pain has been studied in association with inflammation, until recent studies which indicate that during bacterial infections, pain mechanisms could be independent of the inflammation. Chronic pain can sustain long after the healing from the injury, even in the absence of any visible inflammation. However, the mechanism behind this is not known. We tested inflammation in lysozyme-injected mice foot paw. Interestingly, we observed no inflammation in mice foot paw. Yet, lysozyme injections induced pain in these mice. Lysozyme induces pain in a TLR4-dependent manner and TLR4 activation by its ligands such as LPS leads to inflammatory response. We compared the intracellular signaling of MyD88 and TRIF pathways upon TLR4 activation by lysozyme and LPS to understand the underlying mechanism behind the absence of an inflammatory response upon lysozyme treatment. We observed a TLR4 induced selective TRIF and not MyD88 pathway activation upon lysozyme treatment. This is unlike any other previously known endogenous TLR4 activators. A selective activation of TRIF pathway by lysozyme induces weak inflammatory cytokine response devoid of inflammation. However, lysozyme activates glutamate oxaloacetate transaminase-2 (GOT2) in neurons in a TRIF-dependent manner, resulting in enhanced glutamate response. We propose that this enhanced glutaminergic response could lead to neuronal activation resulting in pain sensation upon lysozyme injections. Collectively we identify that TLR4 activation by lysozyme can induce pain in absence of a significant inflammation. Also, unlike other known TLR4 endogenous activators, lysozyme does not activate MyD88 signaling. These findings uncover a mechanism of selective activation of TRIF pathway by TLR4. This selective TRIF activation induces pain with negligible inflammation, constituting a chronic pain homeostatic mechanism.</p

Schematic representation of SIM (simvastatin) induced neuritogenesis pathway.

<p>SIM treatment to SH-SY5Y cells promotes retention of cell surface cholesterol (CSC) which regulates cellular function by stabilizing lipid raft signaling and maintenance of neurite outgrowth. Simultaneously, depletion of cholesterol by SIM activates an intracellular phosphatase i.e. Protein phosphatase 2A (PP2A) which inactivates AMPK by dephosphorylating at Thr 172. This is followed by activation of acetyl CoA carboxylase (ACC) through dephosphorylation at Ser-79. New fatty acids get synthesized which act as precursors for neurites during elongation process.</p

SIM promotes axon like neuritogenesis in SH-SY5Y cells.

<p>A) Morphology of SH-SY5Y under light microscope (Nikon Eclipse, 80i; 10x magnification) showing neuritogenic effect of SIM as function of concentration and time. SH-SY5Y cells were treated with SIM at concentration of 1.0 µM, 2.5 µM and 5.0 µM for a period of 24 hrs. A significant (<i>p</i> < 0.001) increase in neuritogenesis occurred at concentration of 2.5 µM SIM. Error bar graph represents the difference in neurite length (in μm) between control and SIM treated cells (mean ± SEM; N = 50 per condition from 3 separate cultures). Treatment of SH-SY5Y cells with 2.5 µM SIM for different time periods i.e. 6 hrs, 12 hrs and 24 hrs showed that 12 hrs time was the minimal time point for inducing significant (<i>p</i> < 0.001) increase in neurite length. Error bar graph represents the difference in neurite length (in μm) between control and SIM treated cells (mean ± SEM; N = 50 per condition from 3 separate cultures). Scale Bar = 100µm. <b>B</b>) Immunoblot showing SIM induced change in protein expression of neuronal differential markers like neurofilament L (NFL), β3-tubulin, GAP43 and Nestin. Note that SIM led to a significant (<i>p</i> < 0.05^*) increase in protein expression of NFL and β3-tubulin whereas GAP43 and Nestin displayed no change. phospho-Tyrosine Hydroxylase serine 31 (pTH-Ser31), a dopaminergic neuronal functional marker showed a significant (<i>p</i> < 0.05^*) decrease in protein levels at 6 hrs post SIM treatment. Error bar graph represents the fold change in protein expression of pTH, NFL and β3-tubulin normalized to loading control i.e. actin (mean ± SEM; n = 4).</p

SIM retained CSC modifies various RTK’s and MAPK’s during neuritogenesis.

<p>A) Immunoblot showing significant (<i>p</i> < 0.05^*) increase in protein levels of Flotilin-2 (a lipid raft marker) after SIM treatment from 2 hrs onwards. Error bar graph represents the fold change in protein levels with respect to untreated sample (mean ± SEM; n = 3). <b>B</b>) Confocal image at 60 X magnification showing marked increase in surface distribution of Flotilin-2 protein after SIM treatment till 6 hrs. For detection of Flotilin-2 protein, FITC-labeled secondary antibody was used which is shown as green fluorescence. Scale Bar = 10 µm. <b>C</b>) p-RTK protein array blot showing detection of various kinases represented as dots. Each kinase has been denoted by number as shown in blot and corresponding fold change in phosphorylation (ratio of SIM treated and untreated cells) at 6 hrs has been shown on right hand side of blot. The result is average of two experiments. Increased phosphorylation of various RTK’s like EGFR, ErbB4, FGFR2α, Insulin R, Dtk, MSPR, PDGF Rβ, Flt-3, c-Ret, ROR1, ROR2, Tie-2, MUSK, Eph B2, Eph A4, Eph B4 and Eph B6 was observed after SIM treatment <b>D)</b> p-MAPK protein array blot showing detection of various kinases represented as dots. Each kinase has been denoted by number as shown in blot. <b>E</b>) Morphology of SH-SY5 cells at 10 X magnification under light microscope showing significant (<i>p</i> < 0.001) reduction in neuritogenesis after addition of PI3K inhibitors i.e. wortmannin (Wort.) and LY294002 (LY.) at 12 hrs post SIM treatment. Each inhibitor was used at a concentration of 10 µM. The change in neurite length (in μm) is represented by error bar graph (mean ± SEM; N = 50 cells per condition from 3 separate cultures). Scale Bar = 100 µm.</p

Dephosphorylation of AMPK by PP2A phosphatase promotes neuritogenic effect of SIM in SH-SY5Y cells.

<p>Immunoblot in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074547#pone-0074547-g003" target="_blank">Figure <b>3 (A, B, C and D)</b></a> showing modulation of protein expression of AMPK, p-AMPK (Thr-172), Akt, pAkt (ser-473), pAkt (Thr-308), PP2Ac and PP2Ac (p-Tyr) after SIM treatment for 12 hrs. <b>A</b>) protein expression of AMPK and p-AMPK (Thr-172) at 2 hrs, 6 hrs and 12 hrs after SIM treatment. At 2 hrs a sudden increase in phosphorylation of AMPK was noted which was accompanied by rapid reversal to control. <b>B</b>) AMPK and p-AMPK (Thr-172) levels at 6 hrs in presence of SIM and PI3K inhibitors like wortmannin and LY294002, 10 µM each. No change in pAMPK (Thr-172) was observed among the samples. <b>C</b>) Akt, pAkt (ser-473) and pAkt (Thr-308) levels at 2 hrs, 6 hrs and 12 hrs after SIM addition. A significant (<i>p</i> < 0.05^*) 2.0 fold increase in pAkt (Thr-308) was observed whereas pAkt (Ser-473) displayed no change. <b>D</b>) Time dependent decrease in tyrosine phosphorylation of PP2Ac after SIM treatment till 12 hrs. Total PP2Ac protein levels remained same during SIM treatment. Simultaneously, inhibition of PP2A activity by Fostriecin showed increase in pAMPK (Thr-172) levels in presence or absence of SIM at 6 hrs <b>E</b>) Morphology of SH-SY5Y cells at 10 X magnification under light microscope showing significant (<i>p</i> < 0.001) decline in neuritogenic effect of SIM in presence of Fostriecin and an AMP analogue i.e. AICAR. Error bar graph shows the difference in neurite length between SIM, SIM + Fostriecin and SIM + AICAR treated cells for 12 hrs (mean ± SEM; N = 50 cells per condition from 3 separate cultures). Scale Bar = 100 µm.</p

Retention of CSC in plasma membrane determines SIM induced neuritogenesis in SH-SY5Y cells.

<p><b>A</b>) Confocal image of SH-SY5Y cells at 60 X magnification showing fluorescence of cholesterol binding probe i.e. Filipin III in control (a); and SIM treated cells for 2 hrs (b), 6 hrs (c) and 12 hrs (d). The fluorescence (shown as blue fluorescence) was observed not only in plasma membrane but also in neurites. The neurite has been represented by *. N = 25-30 per condition from 3 separate cultures (n = 3). Scale Bar = 10 µm. <b>B</b>) Thin layer chromatography showing total cellular cholesterol content at 2 hrs, 6 hrs and 12 hrs after SIM treatment. A significant (<i>p</i> < 0.001) decrease in cholesterol content occurred after 6 hrs. Difference in cholesterol content is represented in error bar graph calculated as percent change (mean ± SEM; n = 4). <b>C</b>) Morphology of SH-SY5Y cells under light microscope (10 X magnification) showing the neuritogenic effect of SIM in presence of agents which perturb cholesterol function. Cells were treated with SIM alone (a); or along with compounds like 200 µM MβD, cholesterol scavenger (b); 10 µM U18666a, intracellular cholesterol transport inhibitor (c); 15 µg/ml cholesterol (chol.) complexed with 200 µM MβD (d); 0.5 mM mevalonate (Meva.), a precursor for cholesterol biosynthesis (e); 10 µM GGTI-298, an inhibitor of Geranylgeranyltransferase (f); 5 µg/ml Filipin III, a cholesterol binding probe (g); and 10 µg/ml α-lysophosphatidylcholine (α-LPC), a cholesterol intercalater (h) after 1 hr post SIM treatment. Neuritogenesis induced by SIM was significantly (<i>p</i> < 0.05^*) reduced by MβD, U 18666a, mevalonate, GGTI-298, Filipin III and α-LPC whereas exogenous cholesterol supplementation did not change significantly the neuritogenic effect of SIM. Concomitantly, SIM treated SH-SY5Y cells were incubated along with sphingomyelin, SPM (i); and with inhibitors which modify endogenous sphingolipid biosynthesis like 10 µM GW4869, an inhibitor of neutral sphingomyelinase (j) and Fumonisin B1, an inhibitor for ceramide synthase (k). Application of exogenous SPM led to a significant (<i>p</i> < 0.001) decrease in SIM induced neuritogenesis whereas modulation of endogenous sphingolipid pool produced no significant effect on neuritogenic effect of SIM. The change in neurite length (in μm) is represented by error bar graph (mean ± SEM; N = 40-65 cells per condition from 3 separate cultures). Scale Bar = 100 µm.</p

Dephosphorylation of AMPK substrate i.e. acetyl CoA carboxylase (ACC) initiates neuritogenic effect of SIM.

<p><b>A</b>) Immunoblot showing SIM induced change in phosphorylation of ACC over a period of 2 hrs, 6 hrs and 12 hrs. SIM led to a significant (<i>p</i> < 0.05^*) increase in pACC (Ser-79) levels at 2 hrs which was followed by a gradual decrease. Total ACC levels showed no change during SIM treatment. Error bar graph shows fold change (normalized to control) in pACC (Ser-79) levels at different time points (mean ± SEM; n = 4). <b>B</b>) Immunoblot showing modulation of ACC phosphorylation by SIM follows AMPK pathway. As evident, addition of AICAR (an AMPK activator) increased the phosphorylation of ACC either alone or in presence of SIM till 12 hrs. <b>C</b>) Morphology of SH-SY5Y cells under light microscope showing significant (<i>p</i> < 0.001) inhibition of SIM induced neuritogenesis in presence of acetyl CoA carboxylase (ACC) inhibitor i.e. 10 µM TOFA and fatty acid synthase (FAS) inhibitor i.e. 10 µM Cerulenin (Cerul.). Error bar graph shows the comparative difference in neurite lengths SIM, TOFA, SIM + TOFA, Cerulenin and SIM + Cerulenin treated cells for a period of 12 hrs (mean ± SEM; N = 50 cells per condition from 3 separate cultures). Morphology of PC12 cells under light microscope in presence of NGF or NGF + TOFA for a period of 3 days. Scale Bar = 100 µm.</p

SREBP-1 is dispensable but not required for neuritogenic effect of SIM.

<p>A) Immunoblot showing time dependent effect of SIM on SREBP-1 cleavage. p125 represents the immature form whereas p68 represents the mature cleaved form of SREBP-1. SIM led no change on SREBP-1 cleavage except that a significant (<i>p</i> < 0.05^*) decrease was observed at 2 hrs. Error bar graph represents the fold change (normalized to actin) in p68 protein form at different time points in presence of SIM (mean ± SEM; n = 3). <b>B</b>) Confocal image at 60 X magnification showing immunofluorescence of SREBP-1 in cytoplasm and nucleus till 6 hrs. An increase in SREBP-1 fluorescence (red) intensity is seen in cytoplasm whereas nucleus represents no change in colour. Nuclei have been stained with DAPI (blue). Error bar graph shows fold change in SREBP (p125) fluorescence before and after SIM treatment. Scale Bar = 10µm. <b>C</b>) Semi quantitative RT-PCR showing expression of ACC1, ACC2 and FAS at different time points in presence of SIM. No significant change was observed in transcripts of ACC1. FAS transcripts showed a small decrease at 12 hrs whereas ACC2 transcript showed a significant (<i>p</i> < 0.05^*) decrease at 2 hrs and 12 hrs. Error bar graph represents the comparative fold change (normalized to actin) in ACC1, ACC2 and FAS mRNA levels till 12 hrs in presence of SIM (mean ± SEM; n = 4). <b>D</b>) Morphology of SH-S5Y cells under light microscope showing abrogation of SIM induced neuritogenesis in presence of 25-hydroxy-cholesterol during 12 hrs time period. Scale Bar = 100 µm.</p

Patent intelligence of RNA viruses: Implications for combating emerging and re-emerging RNA virus based infectious diseases

The recent outbreak of one of the RNA viruses (2019-nCoV) has affected most of the population and the fatalities reported may label it as a modern-day scourge. Active research on RNA virus infections and vaccine development had more commercial impact which leads to an increase in patent filings. Patents are a goldmine of information whose mining yields crucial technological inputs for further research. In this research article, we have investigated both the patent applications and granted patents, to identify the technological trends and their impact on 2019-nCoV infection using biotechnology-related keywords such as genes, proteins, nucleic acid etc. related to the RNA virus infection. In our research, patent analysis was majorly focused on prospecting for patent data related to the RNA virus infections. Our patent analysis specifically identified spike protein (S protein) and nucleocapsid proteins (N proteins) as the most actively researched macromolecules for vaccine and/or drug development for diagnosis and treatment of RNA virus based infectious diseases. The outcomes of this patent intelligence study will be useful for the researchers who are actively working in the area of vaccine or drug development for RNA virus-based infections including 2019-nCoV and other emerging and re-emerging viral infections in the near future