Expression of 5-lipoxygenase and 15-lipoxygenase in rheumatoid arthritis synovium and effects of intraarticular glucocorticoids

The past years have witnessed tremendous progress in the treatment of rheumatoid arthritis, a chronic debilitating autoimmune disease mainly characterized by joint inflammation with progressive tissue destruction and loss of function. This condition affects 0.5-1% of the population, is associated with important co-morbidities and represents a heavy economical burden. New strategies, employing early and aggressive therapies with classical drugs or new agents, have resulted in impressive improvements in controlling disease activity. In some cases they even lead to clinical remission. Despite potent and efficient biological agents that specifically modulate distinct pathological pathways a large proportion of patients remain unresponsive to these therapies; drug-free remission is also difficult to achieve since attempting discontinuation of treatment usually results in disease flare. In rheumatoid arthritis joints there is a constant activation of complex networks of cytokines and factors mediating immune interactions and inflammation, in which prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) are important players and contributors to pathogenesis. Our research aimed to investigate the synovial expression of enzymes controlling prostaglandin E2 synthesis and degradation – cyclooxygenase (COX) 1 and 2, microsomal prostaglandin E2 synthase 1 (MPGES1) and 15-prostaglandin dihydrogenase (15-PGDH) as well as enzymes involved in leukotriene synthesis, such as 5-lipoxygenase (LO) and 15-LO. In addition, we evaluated how traditional and new therapies influence these pathways, by analyzing enzyme expression before and after systemic treatment with tumor necrosis factor (TNF) antagonists, rituximab or methotrexate, as well as before and after intra-articular treatment with glucocorticoids. We also evaluated the in vitro effects of TNF antagonists and glucocorticoids on synovial fluid cells and that of methotrexate on synovial fibroblasts. We demonstrated that synovial tissue from RA patients displayed an important expression of enzymes involved in the metabolism of PGE2, as well as 5-LO and 15-LO. MPGES1 and COX-2, the inflammation-inducible enzymes co-localized mainly in fibroblasts and macrophage-like cells and accounted for the local PGE2 production. Intra-articular glucocorticoids significantly reduced all enzymes involved in the PGE2 cascade – COX-1 and COX-2, MPGES1 and 15-PGDH, but also 5-LO, responsible for leukotriene formation. However, they did not influence the expression of 15-LO, an enzyme involved in the formation of both pro-and anti-inflammatory lipid mediators. Regarding the effects of TNF blockers, rituximab or methotrexate, they did not alter the expression profile of enzymes involved in PGE2 metabolism despite showing clinical efficiency in improving disease activity. Although anti-TNF agents reduced the in vitro expression of MPGES1 and COX-2 in synovial fluid cells, the lack of effect ex vivo in biopsies emphasized once again the differences between synovial compartments and possibly the difficulty in mimicking the micro-environment at the site of inflammation in vitro. In conclusion, this thesis demonstrates that potent anti-rheumatic drugs currently used in the clinic with good efficiency also leave inflammatory pathways un-affected, which may account for subclinical ongoing disease activity. Blocking the PGE2 pathway by using MPGES1 inhibitors as combination therapy may show benefit in dampening ongoing local inflammation

Modulation of microRNA processing by 5‐lipoxygenase

The miRNA biogenesis is tightly regulated to avoid dysfunction and consequent disease development. Here, we describe modulation of miRNA processing as a novel noncanonical function of the 5‐lipoxygenase (5‐LO) enzyme in monocytic cells. In differentiated Mono Mac 6 (MM6) cells, we found an in situ interaction of 5‐LO with Dicer, a key enzyme in miRNA biogenesis. RNA sequencing of small noncoding RNAs revealed a functional impact, knockout of 5‐LO altered the expression profile of several miRNAs. Effects of 5‐LO could be observed at two levels. qPCR analyses thus indicated that (a) 5‐LO promotes the transcription of the evolutionarily conserved miR‐99b/let‐7e/miR‐125a cluster and (b) the 5‐LO‐Dicer interaction downregulates the processing of pre‐let‐7e, resulting in an increase in miR‐125a and miR‐99b levels by 5‐LO without concomitant changes in let‐7e levels in differentiated MM6 cells. Our observations suggest that 5‐LO regulates the miRNA profile by modulating the Dicer‐mediated processing of distinct pre‐miRNAs. 5‐LO inhibits the formation of let‐7e which is a well‐known inducer of cell differentiation, but promotes the generation of miR‐99b and miR‐125a known to induce cell proliferation and the maintenance of leukemic stem cell functions

Pulmonary sarcoidosis is associated with exosomal vitamin D-binding protein and inflammatory molecules

BACKGROUND: Sarcoidosis is an inflammatory granulomatous disorder characterized by accumulation of TH1-type CD4+T cells and immune effector cells within affected organs, most frequently the lungs. Exosomes are extracellular vesicles conveying intercellular communication with possible diagnostic and therapeutic applications.OBJECTIVES: Weaimed to provide an understanding of the proinflammatory role of bronchoalveolar lavage fluid (BALF) exosomes in patients with sarcoidosis and to find candidates for disease biomarkers.METHODS: Weperformed a mass spectrometric proteomics characterization of BALF exosomes from 15 patients with sarcoidosis and 5 healthy control subjects and verified the most interesting results with flow cytometry, ELISA, and Western blot analyses in an additional 39 patients and 22 control subjects.RESULTS: Morethan 690 proteins were identified in the BALF exosomes, several of which displayed significant upregulation in patients, including inflammation-associated proteins, such as leukotriene A4 hydrolase. Most of the complement-activating factors were upregulated, whereas the complement regulator CD55 was seen less in patients comparedwith healthy control subjects. In addition, for the first time, we detected vitamin D-binding protein in BALF exosomes, which was more abundant in patients. To evaluate exosome-associated vitamin D-binding protein as a biomarker for sarcoidosis, we investigated plasma exosomes from 23 patients and 11 healthy control subjects and found significantlyhigher expression in patients.CONCLUSION: Together,these data contribute to understanding the role of exosomes in lung disease and provide suggestions for highly warranted sarcoidosis biomarkers. Furthermore, the validation of an exosome-associated biomarker in the blood of patients provides novel, and less invasive, opportunities for disease diagnosis.</h4

Arachidonic acid promotes phosphorylation of 5-lipoxygenase at Ser-271 by MAPK-activated protein kinase 2 (MK2)

We demonstrated previously that 5-lipoxygenase (5-LO), a key enzyme in leukotriene biosynthesis, can be phosphorylated by p38 MAPK-regulated MAPKAP kinases (MKs). Here we show that mutation of Ser-271 to Ala in 5-LO abolished MK2 catalyzed phosphorylation and clearly reduced phosphorylation by kinases prepared from stimulated polymorphonuclear leukocytes and Mono Mac 6 cells. Compared with heat shock protein 27 (Hsp-27), 5-LO was a weak substrate for MK2. However, the addition of unsaturated fatty acids (i.e. arachidonate 1-50 microm) up-regulated phosphorylation of 5-LO, but not of Hsp-27, by active MK2 in vitro, resulting in a similar phosphorylation as for Hsp-27. 5-LO was phosphorylated also by other serine/threonine kinases recognizing the motif Arg-Xaa-Xaa-Ser (protein kinase A, Ca(2+)/calmodulin-dependent kinase II), but these activities were not increased by fatty acids. HeLa cells expressing wild type 5-LO or S271A-5-LO, showed prominent 5-LO activity when incubated with Ca(2+)-ionophore plus arachidonate. However, when stimulated with only exogenous arachidonic acid, activity for the S271A mutant was significantly lower as compared with wild type 5-LO. It appears that phosphorylation at Ser-271 is more important for 5-LO activity induced by a stimulus that does not prominently increase intracellular Ca(2+) and that arachidonic acid stimulates leukotriene biosynthesis also by promoting this MK2-catalyzed phosphorylation

The C2-like β-barrel domain mediates the Ca2+-dependent resistance of 5-lipoxygenase activity against inhibition by glutathione peroxidase-1

Recently, we reported that in crude enzyme preparations, a monocyte-derived soluble protein (M-DSP) renders 5-lipoxygenase (5-LO) activity Ca2+-dependent. Here we provide evidence that this M-DSP is glutathione peroxidase (GPx)-1. Thus, the inhibitory effect of the M-DSP on 5-LO could be overcome by the GPx-1 inhibitor mercaptosuccinate and by the broad spectrum GPx inhibitor iodoacetate, as well as by addition of 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid (13(S)-HPODE). Also, the chromatographic characteristics and the estimated molecular mass (80-100 kDa) of the M-DSP fit to GPx-1 (87 kDa), and GPx-1, isolated from bovine erythrocytes, mimicked the effects of the M-DSP. Intriguingly, only a trace amount of thiol (10 micro M GSH) was required for reduction of 5-LO activity by GPx-1 or the M-DSP. Moreover, the requirement of Ca2+ allowing 5-LO product synthesis in various leukocytes correlated with the respective GPx-1 activities. Mutation of the Ca2+ binding sites within the C2-like domain of 5-LO resulted in strong reduction of 5-LO activity by M-DSP and GPx-1, also in the presence of Ca2+. In summary, our data suggest that interaction of Ca2+ at the C2-like domain of 5-LO protects the enzyme against the effect of GPx-1. Apparently, in the presence of Ca2+, a low lipid hydroperoxide level is sufficient for 5-LO activation