CysLT1R is essential for optimal TLR4 responses and associated macrophage polarization and sepsis in response to LPS

Abstract

Background: Cysteinyl leukotrienes (cys-LTs; LTC4, LTD4 and LTE4) are arachidonic acid-derived inflammatory lipds, acting mainly via two G-protein-coupled receptors (GPCRs), cysteinyl leukotriene receptor 1 (CysLT1R) and cysteinyl leukotriene receptor 2 (CysLT2R). Cys-LTs are mainly recognized as potent modulators of asthma and allergy with their effects on mast cells. However, their role in other pathophysiological conditions are not well understood. Higher amounts of cys-LTs were reported in the lungs of mice subjected to sepsis than compared to sham mice, suggesting that CysLTs play critical role in sepsis via modulation of macrophage inflammatory responses. Objective: To investigate how cys-LT/CysLTR axis determines the inflammatory fate of macrophages in LPS/TLR4-induced sepsis. Methods: Bone marrow-derived stem cells (BMSC) from wild type (WT) and CysLT1R null ( Cysltr1-/-) mice were differentiated into macrophages and polarized to inflammatory phenotype using LPS via TLR4 pathway. Transcript and protein expression was analyzed by qPCR/RNA seq and western blotting/multiplex ELISA respectively. Results: LPS/TLR4 axis polarized WT macrophages to inflammatory phenotype by activating inflammatory intracellular signals (p38, CREB, and NFkB), upregulating surface expression of inflammatory markers (CD68, CD11b and LFA-1), enhancing phagocytosis and augmenting inflammatory cytokine secretion, in vitro. In contrast, LPS treated Cysltr1–/– macrophages exhibited attenuated TLR4 signaling observed with WT mice, suggesting that inhibition of CysLT1R prevents optimal TLR4 activation in response to LPS. Notably, reporter assays revealed a strong interaction between TLR4 and CysLT1R in HEK blue TLR4 cells overexpressing CysLT1R, further emphasizing a potential CysLT1R-TLR4 cross talk in driving TLR4-dependent inflammatory macrophage phenotype. Finally, Cysltr1–/– mice were protected from LPS-induced sepsis compared to WT mice with comcomitant reduction in the production of inflammatory cytokines. Conclusions: Our results uncover a potential crosstalk between two important innate immune receptors, TLR4-CysLT1R, in mediating macrophage inflammatory phenotype and sepsis in vivo. Understanding this interaction may offer novel potential therapeutic targets for macrophage-associated inflammatory disorders like sepsis. This work was supported by National Institutes of Health (R01AI144115 and R01HL148585) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.