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LKB1-MARK2 signalling mediates lipopolysaccharide-induced production of cytokines in mouse macrophages.

ABSTRACT: Lipopolysaccharide (LPS) is an endotoxin involved in a number of acute and chronic inflammatory syndromes. Although LPS-induced signalling has been extensively studied, there are still mysteries remaining to be revealed. In the current study, we used high-throughput phosphoproteomics to profile LPS-initiated signalling and aimed to find novel mediators. A total of 448 phosphoproteins with 765 phosphorylation sites were identified, and we further validated that the phosphorylation of MARK2 on T208 was important for the regulation on LPS-induced CXCL15 (human IL-8 homolog), IL-1?, IL-6 and TNF-? release, in which LKB1 had a significant contribution. In summary, induction of cytokines by LPS in mouse macrophage is regulated by LKB1-MARK2 signals. Our study provides new clues for further exploring the underlying mechanisms of LPS-induced diseases, and new therapeutic approaches concerning bacterial infection may be derived from these findings.

SUBMITTER: Deng J

PROVIDER: S-EPMC7576310 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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LKB1-MARK2 signalling mediates lipopolysaccharide-induced production of cytokines in mouse macrophages.

Deng Jie J Wen Chunmei C Ding Xiangyu X Zhang Xi X Hou Guoqing G Liu Andong A Xu Hui H Cao Xuan X Bai Yongheng Y

Journal of cellular and molecular medicine 20200825 19

Lipopolysaccharide (LPS) is an endotoxin involved in a number of acute and chronic inflammatory syndromes. Although LPS-induced signalling has been extensively studied, there are still mysteries remaining to be revealed. In the current study, we used high-throughput phosphoproteomics to profile LPS-initiated signalling and aimed to find novel mediators. A total of 448 phosphoproteins with 765 phosphorylation sites were identified, and we further validated that the phosphorylation of MARK2 on T20 ...[more]

PMID: 32841502

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Project description:Chemokine (C-C motif) receptor 8 (CCR8), the chemokine receptor for chemokine (C-C motif) ligand 1 (CCL1), is expressed in T-helper type-2 lymphocytes and peritoneal macrophages (PMφ) and is involved in various pathological conditions, including peritoneal adhesions. However, the role of CCR8 in inflammatory responses is not fully elucidated. To investigate the function of CCR8 in macrophages, we compared cytokine secretion from mouse PMφ or bone marrow-derived macrophages (BMMφ) stimulated with various Toll-like receptor (TLR) ligands in CCR8 deficient (CCR8-/-) and wild-type (WT) mice. We found that CCR8-/- PMφ demonstrated attenuated secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 when stimulated with lipopolysaccharide (LPS). In particular, LPS-induced IL-10 production absolutely required CCR8. CCR8-dependent cytokine secretion was characteristic of PMφ but not BMMφ. To further investigate this result, we selected the small molecule compound R243 from a library of compounds with CCR8-antagonistic effects on CCL1-induced Ca2+ flux and CCL1-driven PMφ aggregation. Similar to CCR8-/- PMφ, R243 attenuated secretion of TNF-α, IL-6, and most strikingly IL-10 from WT PMφ, but not BMMφ. CCR8-/- PMφ and R243-treated WT PMφ both showed suppressed c-jun N-terminal kinase activity and nuclear factor-κB signaling after LPS treatment when compared with WT PMφ. A c-Jun signaling pathway inhibitor also produced an inhibitory effect on LPS-induced cytokine secretion that was similar to that of CCR8 deficiency or R243 treatment. As seen in CCR8-/- mice, administration of R243 attenuated peritoneal adhesions in vivo. R243 also prevented hapten-induced colitis. These results are indicative of cross talk between signaling pathways downstream of CCR8 and TLR-4 that induces cytokine production by PMφ. Through use of CCR8-/- mice and the new CCR8 inhibitor, R243, we identified a novel macrophage innate immune response pathway that involves a chemokine receptor.

Project description:Previous studies have revealed that activation of the Toll‑like receptor 4 (TLR4)‑mediated proinflammatory signaling pathway plays an important role in acute inflammation, sepsis and chronic inflammatory disorders. Moreover, TLR4 significantly contributes to lipopolysaccharide (LPS)‑induced immune response. Thus, modulation of the TLR4 pathway is an important strategy to specifically target these pathologies. The aim of the present study was to develop a complete human anti‑TLR4 IgG2 antibody by screening human TLR4 Fab from a phage‑display library and integrating it with constant regions of the heavy chain of human IgG2 via antibody engineering. ELISA, a BLItz system and fluorescence‑activated cell sorting were used to assess its affinity. Furthermore, mouse‑derived peritoneal macrophages were treated with human anti‑TLR4 IgG2 and induced with LPS in vitro. Reverse transcription‑quantitative PCR and western blotting were used to determine mRNA expression levels of cytokines and phosphorylation levels of signaling pathways, respectively. It was found that human anti‑TLR4 IgG2 bound to TLR4 with a high affinity of 8.713x10‑10 M, and that preincubation with anti‑TLR4 IgG2 inhibited the LPS‑induced production of tumor necrosis factor‑α, interferon‑β and interleukin‑6 mRNA expression levels in mouse peritoneal macrophages. It was also demonstrated that human anti‑TLR4 IgG2 inhibited LPS‑induced TLR4 signaling by reducing the phosphorylation of the NF‑κB, mitogen‑activated protein kinase and interferon regulatory factor 3 signaling pathways. In addition, human anti‑TLR4 IgG2 protected mice from LPS challenge with a survival rate of 40% and also significantly increased the survival time in the cecal ligation and puncture model. Therefore, it was speculated that human anti‑TLR4 IgG2 plays a protective role against sepsis‑associated injury and is potentially applicable for the treatment of infection‑associated immune dysfunction.

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LKB1-MARK2 signalling mediates lipopolysaccharide-induced production of cytokines in mouse macrophages.

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LKB1-MARK2 signalling mediates lipopolysaccharide-induced production of cytokines in mouse macrophages.

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