When PLT-BMDMs and control BMDMs were stimulated with LPS for 24 h, the expression level of in PLT-BMDMs was found to be 7-fold higher than that in control BMDMs, in association with lower (~10%) expression of (Fig 4A)

When PLT-BMDMs and control BMDMs were stimulated with LPS for 24 h, the expression level of in PLT-BMDMs was found to be 7-fold higher than that in control BMDMs, in association with lower (~10%) expression of (Fig 4A). BMDMs were also cultured with Corin 0.5 U/mL thrombin, 20 M ADP, 2 g/mL collagen, or unstimulated platelet supernatant (Resting-PLT-sup). Experiments were performed in quintuplicate and repeated three times. The data are presented as the mean SEM. ***p 0.005. Representative results from the three experiments are shown.(PDF) pone.0162208.s002.pdf (383K) GUID:?AB01F9A7-3A42-4330-8533-EAAD0A12B26C S3 Fig: Effect of an arginase-1 inhibitor on LPS-induced NO production from PLT-BMDMs. BMDMs (4 105 cells) were cultured for 24 h with PLT-sup in the presence or absence of 2S-amino-4-[[(hydroxyamino)iminomethyl]amino]-butanoic acid (nor-NOHA) L-371,257 (Cayman Chemical, MI, USA) in a 24-well plate, and stimulated with complete medium made up of LPS (50 ng/mL) for 24 h. The production of NO2- was decided. Experiments were performed in quintuplicate and repeated three times. The data are presented as the mean SEM. *p 0.05, ***p 0.005 vs. controls. Representative results from the three experiments are shown.(PDF) pone.0162208.s003.pdf (379K) GUID:?E9930099-B4D5-4FDC-8B8A-904D0E892409 Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract We previously reported that mouse bone marrow-derived macrophages (BMDMs) that had been co-cultured with platelets exhibited lower susceptibility to bacterial lipopolysaccharide (LPS) and produced lower levels of nitric oxide (NO) and inflammatory cytokines including TNF- and IL-6. The suppression L-371,257 of macrophage responses was mediated, at least in part, by L-371,257 platelet supernatant. In the present study, we assessed phenotypic changes of BMDMs induced by incubation with the supernatant from thrombin-activated platelets (PLT-sup) and found that BMDMs cultured with PLT-sup (PLT-BMDMs) expressed a lower level of inducible NO synthase (iNOS) and a higher level of arginase-1, both of which are involved in the L-arginine metabolism, upon stimulation with LPS or zymosan. We also examined possible modulation of the NF-B signaling pathway and observed suppression of IB phosphorylation and a decrease of NF-B p65 expression in LPS-stimulated PLT-BMDMs. These results suggest that PLT-sup suppresses inflammatory responses of BMDMs via unfavorable regulation of NF-B signaling leading to lowered expression of iNOS and enhanced L-arginine catabolism by arginase-1. Introduction Bacterial lipopolysaccharide (LPS), a L-371,257 major component of the outer membrane of Gram-negative L-371,257 bacteria, stimulates macrophages to produce various inflammatory cytokines, including tumor necrosis factor- (TNF-), interleukin-1 (IL-1), and IL-6 [1C3]. In addition to these cytokines, nitric oxide (NO) produced by activated macrophages plays important roles in the pathogenesis of inflammation related to bacterial infection [4C7]. Excessive macrophage activation with release of these inflammatory mediators causes systemic inflammatory response syndrome (SIRS), disseminated intravascular coagulation (DIC), and multiple organ failure (MOF). We recently reported that bone marrow-derived macrophages (BMDMs) that had been co-cultured with platelets exhibited lower susceptibility to LPS and produced lower levels of NO, TNF-, and IL-6 [8]. The suppression of macrophage responses by platelets did not necessarily require the direct cellcell contact between macrophages and platelets, but appeared to be mediated by soluble factors secreted from platelets upon stimulation with thrombin or other stimulants. These observations provided a cellular basis for the results of an earlier study showing that thrombocytopenia increased mortality and aggravated organ failure in LPS-induced endotoxemia [9] and also supported the notion that platelets play critical roles in the modulation of inflammatory responses. Nitric oxide produced by activated macrophages is thought to be responsible for bacterial killing, tumor cytotoxicity and virus inactivation [10C12]. Moreover, NO interacts with reactive oxygen species (ROS) to generate peroxynitrite, which is a powerful oxidizing agent [13]. However, the excessive production of NO causes tissue damage, extensive systemic vasodilatation and hypotension, leading to organ hypoperfusion and dysfunction in association with septic shock [14C18]. The biosynthesis of NO is usually catalyzed by the enzyme nitric oxide synthase (NOS), which converts L-arginine into L-citrulline and NO. Three isoforms of NOS, neuronal NOS (nNOS, NOS1), inducible NOS (iNOS, NOS2), and endothelial NOS (eNOS, NOS3), are involved in the NO synthesis. The expression of nNOS and eNOS are constitutive in the brain and endothelium, respectively, whereas iNOS is usually expressed in response to cytokines and bacterial components, including LPS. Arginase is usually another enzyme involved in the L-arginine metabolism and converts L-arginine into L-ornithine and urea. Two isoforms of arginase, arginase-1 and arginase-2, exist in mammals and differ in their tissue distribution and physiologic functions [19, 20]. Arginase-1 is usually constitutively expressed in the liver as one of the enzymes of the urea cycle, whereas its expression in macrophages is usually regulated.

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