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TLR signaling paralyzes monocyte chemotaxis through synergized effects of p38 MAPK and global Rap-1 activation.

ABSTRACT: Toll-like receptors (TLRs) that recognize pathogen associated molecular patterns and chemoattractant receptors (CKRs) that orchestrate leukocyte migration to infected tissue are two arms of host innate immunity. Although TLR signaling induces synthesis and secretion of proinflammatory cytokines and chemokines, which recruit leukocytes, many studies have reported the paradoxical observation that TLR stimulation inhibits leukocyte chemotaxis in vitro and impairs their recruitment to tissues during sepsis. There is consensus that physical loss of chemokine receptor (CKR) at the RNA or protein level or receptor usage switching are the mechanisms underlying this effect. We show here that a brief (<15 min) stimulation with LPS (lipopolysaccharide) at ~0.2 ng/ml inhibited chemotactic response from CCR2, CXCR4 and FPR receptors in monocytes without downmodulation of receptors. A 3 min LPS pre-treatment abolished the polarized accumulation of F-actin, integrins and PIP(3) (phosphatidylinositol-3,4,5-trisphosphate) in response to chemokines in monocytes, but not in polymorphonuclear neutrophils (PMNs). If chemoattractants were added before or simultaneously with LPS, chemotactic polarization was preserved. LPS did not alter the initial G-protein signaling, or endocytosis kinetics of agonist-occupied chemoattractant receptors (CKRs). The chemotaxis arrest did not result from downmodulation of receptors or from inordinate increase in adhesion. LPS induced rapid p38 MAPK activation, global redistribution of activated Rap1 (Ras-proximate-1 or Ras-related protein 1) GTPase and Rap1GEF (guanylate exchange factor) Epac1 (exchange proteins activated by cyclic AMP) and disruption of intracellular gradient. Co-inhibition of p38 MAPK and Rap1 GTPase reversed the LPS induced breakdown of chemotaxis suggesting that LPS effect requires the combined function of p38 MAPK and Rap1 GTPase.

SUBMITTER: Yi L

PROVIDER: S-EPMC3276499 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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TLR signaling paralyzes monocyte chemotaxis through synergized effects of p38 MAPK and global Rap-1 activation.

Yi Ling L Chandrasekaran Prabha P Venkatesan Sundararajan S

PloS one 20120209 2

Toll-like receptors (TLRs) that recognize pathogen associated molecular patterns and chemoattractant receptors (CKRs) that orchestrate leukocyte migration to infected tissue are two arms of host innate immunity. Although TLR signaling induces synthesis and secretion of proinflammatory cytokines and chemokines, which recruit leukocytes, many studies have reported the paradoxical observation that TLR stimulation inhibits leukocyte chemotaxis in vitro and impairs their recruitment to tissues during ...[more]

PMID: 22347375

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Project description:Rationale: Many viral infections are known to activate the p38 mitogen-activated protein kinase (MAPK) signaling pathway. However, the role of p38 activation in viral infection and the underlying mechanism remain unclear. The role of virus-hijacked p38 MAPK activation in viral infection was investigated in this study. Methods: The correlation of hepatitis C virus (HCV) infection and p38 activation was studied in patient tissues and primary human hepatocytes (PHHs) by immunohistochemistry and western blotting. Coimmunoprecipitation, GST pulldown and confocal microscopy were used to investigate the interaction of p38α and the HCV core protein. In vitro kinase assays and mass spectrometry were used to analyze the phosphorylation of the HCV core protein. Plaque assays, quantitative real time PCR (qRT-PCR), western blotting, siRNA and CRISPR/Cas9 were used to determine the effect of p38 activation on viral replication. Results: HCV infection was associated with p38 activation in clinical samples. HCV infection increased p38 phosphorylation by triggering the interaction of p38α and TGF-β activated kinase 1 (MAP3K7) binding protein 1 (TAB1). TAB1-mediated p38α activation facilitated HCV replication, and pharmaceutical inhibition of p38α activation by SB203580 suppressed HCV infection at the viral assembly step. Activated p38α interacted with the N-terminal region of the HCV core protein and subsequently phosphorylated the HCV core protein, which promoted HCV core protein oligomerization, an essential step for viral assembly. As expected, SB203580 or the HCV core protein N-terminal peptide (CN-peptide) disrupted the p38α-HCV core protein interaction, efficiently impaired HCV assembly and impeded normal HCV replication in both cultured cells and primary human hepatocytes. Similarly, severe fever with thrombocytopenia syndrome virus (SFTSV), herpes simplex virus type 1 (HSV-1) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection also activated p38 MAPK. Most importantly, pharmacological blockage of p38 activation by SB203580 effectively inhibited SFTSV, HSV-1 and SARS-CoV-2. Conclusion: Our study shows that virus-hijacked p38 activation is a key event for viral replication and that pharmacological blockage of p38 activation is an antiviral strategy.

Project description:The role of TIRAP (toll/interleukin-1 receptor (TIR) domain-containing adapter protein) in macrophage inflammatory signalling has been significantly evolved since its discovery in 2001 due to its dynamic nature and subcellular localization to regulate multiple signaling through several protein-protein interactions (PPIs). Structural analysis of these interactions can reveal a better understanding of their conformational dynamics and the nature of their binding. Tyrosine phosphorylation in the TIR domain of TIRAP is very critical for its function. In toll-like receptor (TLR) 4/2 signalling, Bruton's tyrosine kinase (BTK) and Protein kinase C delta (PKCδ) are known to phosphorylate the Y86, Y106, Y159, and Y187 of TIRAP which is crucial for the downstream function of MAPKs (mitogen-activated protein kinases) activation. The objective of this study is to understand the interaction of TIRAP with p38 MAPK through molecular docking and identify the importance of TIRAP tyrosine phosphorylation in p38 MAPK interaction. In this structural study, we performed an in-silico molecular docking using HADDOCK 2.4, pyDockWEB, ClusPro 2.0, and ZDOCK 3.0.2 tools to unravel the interaction between TIRAP and p38 MAPK. Further, manual in-silico phosphorylations of TIRAP tyrosines; Y86, Y106, Y159, and Y187 was created in the Discovery Studio tool to study the conformational changes in protein docking and their binding affinities with p38 MAPK in comparison to non-phosphorylated state. Our molecular docking and 500 ns of molecular dynamic (MD) simulation study demonstrates that the Y86 phosphorylation (pY86) in TIRAP is crucial in promoting the higher binding affinity (∆Gbind) with p38 MAPK. The conformational changes due to the tyrosine phosphorylation mainly at the Y86 site pull the TIRAP closer to the active site in the kinase domain of p38 MAPK and plays a significant role at the interface site which is reversed in its dephosphorylated state. The heatmap of interactions between the TIRAP and p38 MAPK after the MD simulation shows that the TIRAP pY86 structure makes the highest number of stable hydrogen bonds with p38 MAPK residues. Our findings may further be validated in an in-vitro system and would be crucial for targeting the TIRAP and p38 MAPK interaction for therapeutic purposes against the chronic inflammatory response and associated diseases.

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TLR signaling paralyzes monocyte chemotaxis through synergized effects of p38 MAPK and global Rap-1 activation.

Publications

TLR signaling paralyzes monocyte chemotaxis through synergized effects of p38 MAPK and global Rap-1 activation.

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