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Tolerizing CTL by sustained hepatic PD-L1 expression provides a new therapy spproach in mouse sepsis
(2019)
Cytotoxic T lymphocyte (CTL) activation contributes to liver damage during sepsis, but the mechanisms involved are largely unknown. Understanding the underlying principle will permit interference with CTL activation and thus, provide a new therapeutic option.
Methods: To elucidate the mechanism leading to CTL activation we used the Hepa1-6 cell line in vitro and the mouse model of in vivo polymicrobial sepsis, following cecal-ligation and -puncture (CLP) in wildtype, myeloid specific NOX-2, global NOX2 and NOX4 knockout mice, and their survival as a final readout. In this in vivo setting, we also determined hepatic mRNA and protein expression as well as clinical parameters of liver damage - aspartate- and alanine amino-transaminases. Hepatocyte specific overexpression of PD-L1 was achieved in vivo by adenoviral infection and transposon-based gene transfer using hydrodynamic injection.
Results: We observed downregulation of PD-L1 on hepatocytes in the murine sepsis model. Adenoviral and transposon-based gene transfer to restore PD-L1 expression, significantly improved survival and reduced the release of liver damage, as PD-L1 is a co-receptor that negatively regulates T cell function. Similar protection was observed during pharmacological intervention using recombinant PD-L1-Fc. N-acetylcysteine blocked the downregulation of PD-L1 suggesting the involvement of reactive oxygen species. This was confirmed in vivo, as we observed significant upregulation of PD-L1 expression in NOX4 knockout mice, following sham operation, whereas its expression in global as well as myeloid lineage NOX2 knockout mice was comparable to that in the wild type animals. PD-L1 expression remained high following CLP only in total NOX2 knockouts, resulting in significantly reduced release of liver damage markers.
Conclusion: These results suggest that, contrary to common assumption, maintaining PD-L1 expression on hepatocytes improves liver damage and survival of mice during sepsis. We conclude that administering recombinant PD-L1 or inhibiting NOX2 activity might offer a new therapeutic option in sepsis.
Cystathionine γ lyase (CSE) is the major source of hydrogen sulfide-derived species (H2Sn) in endothelial cells and plays an important role in protecting against atherosclerosis. Here we investigated the molecular mechanisms underlying the regulation of CSE expression in endothelial cells by fluid shear stress/flow. Fluid shear stress decreased CSE expression in human and murine endothelial cells and was negatively correlated with the transcription factor Krüppel-like factor (KLF) 2. CSE was identified as a direct target of the KLF2-regulated microRNA, miR-27b and high expression of CSE in native human plaque-derived endothelial cells, was also inversely correlated with KLF2 and miR-27b levels. One consequence of decreased CSE expression was the loss of Prx6 sulfhydration (on Cys47), which resulted in Prx6 hyperoxidation, decamerization and inhibition, as well as a concomitant increase in endothelial cell reactive oxygen species and lipid membrane peroxidation. H2Sn supplementation in vitro was able to reverse the redox state of Prx6. Statin therapy, which is known to activate KLF2, also decreased CSE expression but increased CSE activity by preventing its phosphorylation on Ser377. As a result, the sulfhydration of Prx6 was partially restored in samples from plaque containing arteries from statin-treated donors. Taken together, the regulation of CSE expression by shear stress/disturbed flow is dependent on KLF2 and miR-27b. Moreover, in murine and human arteries CSE acts to maintain endothelial redox balance at least partly by targeting Prx6 to prevent its decamerization and inhibition of its peroxidase activity.
Renal cell carcinoma alters endothelial receptor expression responsible for leukocyte adhesion
(2016)
Renal cell carcinoma (RCC) escapes immune recognition. To elaborate the escape strategy the influence of RCC cells on endothelial receptor expression and endothelial leukocyte adhesion was evaluated. Human umbilical vein endothelial cells (HUVEC) were co-cultured with the RCC cell line, Caki-1, with and without tumor necrosis factor (TNF)-alpha. Intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), endothelial (E)-selectin, standard and variants (V) of CD44 were then analysed in HUVEC, using flow cytometry and Western blot analysis. To determine which components are responsible for HUVEC-Caki-1 interaction causing receptor alteration, Caki-1 membrane fragments versus cell culture supernatant were applied to HUVECS. Adhesion of peripheral blood lymphocytes (PBL) and polymorphonuclear neutrophils (PMN) to endothelium was evaluated by co-culture adhesion assays. Relevance of endothelial receptor expression for adhesion to endothelium was determined by receptor blockage. Co-culture of RCC and HUVECs resulted in a significant increase in endothelial ICAM-1, VCAM-1, E-selectin, CD44 V3 and V7 expression. Previous stimulation of HUVECs with TNF-alpha and co-cultivation with Caki-1 resulted in further elevation of endothelial CD44 V3 and V7 expression, whereas ICAM-1, VCAM-1 and E-selectin expression were significantly diminished. Since Caki-1 membrane fragments also caused these alterations, but cell culture supernatant did not, cell-cell contact may be responsible for this process. Blocking ICAM-1, VCAM-1, E-selectin or CD44 with respective antibodies led to a significant decrease in PBL and PMN adhesion to endothelium. Thus, exposing HUVEC to Caki-1 results in significant alteration of endothelial receptor expression and subsequent endothelial attachment of PBL and PMN.
Background: In endothelial cells, activation of the AMP-activated protein kinase (AMPK) has been linked with anti-inflammatory actions but the events downstream of kinase activation are not well understood. Here, we addressed the effects of AMPK activation/deletion on the activation of NFKappaB and determined whether the AMPK could contribute to the anti-inflammatory actions of nitric oxide (NO). Methodology/Principal Findings: Overexpression of a dominant negative AMPKalpha2 mutant in tumor necrosis factor-alpha-stimulated human endothelial cells resulted in increased NFKappaB activity, E-selectin expression and monocyte adhesion. In endothelial cells from AMPKalpha2-/- mice the interleukin (IL)-1beta induced expression of E-selectin was significantly increased. DETA-NO activated the AMPK and attenuated NFKappaB activation/E-selectin expression, effects not observed in human endothelial cells in the presence of the dominant negative AMPK, or in endothelial cells from AMPKalpha2-/- mice. Mechanistically, overexpression of constitutively active AMPK decreased the phosphorylation of IKappaB and p65, indicating a link between AMPK and the IKappaB kinase (IKK). Indeed, IKK (more specifically residues Ser177 and Ser181) was found to be a direct substrate of AMPKalpha2 in vitro. The hyper-phosphorylation of the IKK, which is known to result in its inhibition, was also apparent in endothelial cells from AMPKalpha2+/+ versus AMPKalpha2-/- mice. Conclusions: These results demonstrate that the IKK is a direct substrate of AMPKalpha2 and that its phosphorylation on Ser177 and Ser181 results in the inhibition of the kinase and decreased NFKappaB activation. Moreover, as NO potently activates AMPK in endothelial cells, a portion of the anti-inflammatory effects of NO are mediated by AMPK.
Myeloid-specific deletion of the AMPK2 subunit alters monocyte protein expression and atherogenesis
(2019)
The AMP-activated protein kinase (AMPK) is an energy sensing kinase that is activated by a drop in cellular ATP levels. Although several studies have addressed the role of the AMPKα1 subunit in monocytes and macrophages, little is known about the α2 subunit. The aim of this study was to assess the consequences of AMPKα2 deletion on protein expression in monocytes/macrophages, as well as on atherogenesis. A proteomics approach was applied to bone marrow derived monocytes from wild-type mice versus mice specifically lacking AMPKα2 in myeloid cells (AMPKα2∆MC mice). This revealed differentially expressed proteins, including methyltransferases. Indeed, AMPKα2 deletion in macrophages increased the ratio of S-adenosyl methionine to S-adenosyl homocysteine and increased global DNA cytosine methylation. Also, methylation of the vascular endothelial growth factor and matrix metalloproteinase-9 (MMP9) genes was increased in macrophages from AMPKα2∆MC mice, and correlated with their decreased expression. To link these findings with an in vivo phenotype, AMPKα2∆MC mice were crossed onto the ApoE-/- background and fed a western diet. ApoExAMPKα2∆MC mice developed smaller atherosclerotic plaques than their ApoExα2fl/fl littermates, that contained fewer macrophages and less MMP9 than plaques from ApoExα2fl/fl littermates. These results indicate that the AMPKα2 subunit in myeloid cells influences DNA methylation and thus protein expression and contributes to the development of atherosclerotic plaques.
AMP-activated protein kinase (AMPK) is frequently reported to phosphorylate Ser1177 of the endothelial nitric-oxide synthase (eNOS), and therefore, is linked with a relaxing effect. However, previous studies failed to consistently demonstrate a major role for AMPK on eNOS-dependent relaxation. As AMPK also phosphorylates eNOS on the inhibitory Thr495 site, this study aimed to determine the role of AMPKα1 and α2 subunits in the regulation of NO-mediated vascular relaxation. Vascular reactivity to phenylephrine and acetylcholine was assessed in aortic and carotid artery segments from mice with global (AMPKα−/−) or endothelial-specific deletion (AMPKαΔEC) of the AMPKα subunits. In control and AMPKα1-depleted human umbilical vein endothelial cells, eNOS phosphorylation on Ser1177 and Thr495 was assessed after AMPK activation with thiopental or ionomycin. Global deletion of the AMPKα1 or α2 subunit in mice did not affect vascular reactivity. The endothelial-specific deletion of the AMPKα1 subunit attenuated phenylephrine-mediated contraction in an eNOS- and endothelium-dependent manner. In in vitro studies, activation of AMPK did not alter the phosphorylation of eNOS on Ser1177, but increased its phosphorylation on Thr495. Depletion of AMPKα1 in cultured human endothelial cells decreased Thr495 phosphorylation without affecting Ser1177 phosphorylation. The results of this study indicate that AMPKα1 targets the inhibitory phosphorylation Thr495 site in the calmodulin-binding domain of eNOS to attenuate basal NO production and phenylephrine-induced vasoconstriction.
Macrophages are plastic and heterogeneous immune cells that adapt pro- or anti-inflammatory phenotypes upon exposure to different stimuli. Even though there has been evidence supporting a crosstalk between coagulation and innate immunity, the way in which protein components of the hemostasis pathway influence macrophages remains unclear. We investigated the effect of thrombin on macrophage polarization. On the basis of gene expression and cytokine secretion, our results suggest that polarization with thrombin induces an anti-inflammatory, M2-like phenotype. In functional studies, thrombin polarization promoted oxLDL phagocytosis by macrophages, and conditioned medium from the same cells increased endothelial cell proliferation. There were, however, clear differences between the classical M2a polarization and the effects of thrombin on gene expression. Finally, the deletion and inactivation of secreted modular Ca2+-binding protein 1 (SMOC1) attenuated phagocytosis by thrombin-stimulated macrophages, a phenomenon revered by the addition of recombinant SMOC1. Manipulation of SMOC1 levels also had a pronounced impact on the expression of TGF-β-signaling-related genes. Taken together, our results show that thrombin induces an anti-inflammatory macrophage phenotype with similarities as well as differences to the classical alternatively activated M2 polarization states, highlighting the importance of tissue levels of SMOC1 in modifying thrombin-induced macrophage polarization.
Cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) are important modulators of endothelial cell homeostasis. We investigated the signaling pathway linking the activation of CYP 2C9 to enhanced endothelial cell proliferation. Overexpression of CYP 2C9 in cultured human endothelial cells markedly increased proliferation. This effect was paralleled by an up-regulation of the G1 phase regulatory protein, cyclin D1. The specific CYP 2C9 inhibitor, sulfaphenazole, prevented both the enhanced cell proliferation and up-regulation of cyclin D1. CYP 2C9 overexpression also decreased the activity of the c-Jun N-terminal kinase (JNK). Coexpression of wild type JNK with CYP 2C9 attenuated the CYP 2C9-induced increase in cyclin D1 expression and abolished the CYP 2C9-induced proliferation response. In contrast, cotransfecting dominant negative JNK with CYP 2C9 restored the CYP 2C9-mediated up-regulation of cyclin D1 and proliferation. The inactivation of JNK is linked to its dephosphorylation by dual specificity mitogen-activated protein (MAP) kinase phosphatases (MKPs). Overexpression of CYP 2C9 significantly increased the expression of MKP-1, as did incubation with 11,12-EET. These data demonstrate that the mitogenic effect of CYP 2C9 is due to the generation of EETs, which promote the MKP-1-mediated dephosphorylation and inactivation of JNK, effects ultimately culminating in the expression of cyclin D1 and endothelial cell proliferation.
Cytochrome P450 (CYP) signalling pathway has been shown to play a vital role in the vasoreactivity of wild type mouse ophthalmic artery. In this study, we determined the expression, vascular responses and potential mechanisms of the CYP-derived arachidonic acid metabolites. The expression of murine CYP (Cyp2c44) and soluble epoxide hydrolase (sEH) in the wild type ophthalmic artery was determined with immunofluorescence, which showed predominant expression of Cyp2c44 in the vascular smooth muscle cells (VSMC), while sEH was found mainly in the endothelium of the wild type ophthalmic artery. Artery of Cyp2c44−/− and sEH−/− mice were used as negative controls. Targeted mass spectrometry-based lipidomics analysis of endogenous epoxide and diols of the wild type artery detected only 14, 15-EET. Vasorelaxant responses of isolated vessels in response to selective pharmacological blockers and agonist were analysed ex vivo. Direct antagonism of epoxyeicosatrienoic acids (EETs) with a selective inhibitor caused partial vasodilation, suggesting that EETs may behave as vasoconstrictors. Exogenous administration of synthetic EET regioisomers significantly constricted the vessels in a concentration-dependent manner, with the strongest responses elicited by 11, 12- and 14, 15-EETs. Our results provide the first experimental evidence that Cyp2c44-derived EETs in the VSMC mediate vasoconstriction of the ophthalmic artery.