Pathricia Tilstam - Academia.edu (original) (raw)

Papers by Pathricia Tilstam

Expert opinion on therapeutic targets, Jan 31, 2017

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine with chemokine-like functi... more Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine with chemokine-like functions that is being increasingly studied in different aspects of cardiovascular disease. MIF was first identified as a proinflammatory and pro-survival mediator within the immune system, and a second structurally related MIF family member, D-dopachrome tautomerase (a.k.a. MIF-2), was reported recently. Both MIF family members are released by myocardium and modulate the manifestations of cardiovascular disease, specifically in myocardial ischemia. Areas Covered: A scientific overview is provided for the involvement of MIF family cytokines in the inflammatory pathogenesis of atherosclerosis, myocardial infarction, and ischemia-reperfusion injury. We summarize findings of experimental, human genetic and clinical studies, and suggest therapeutic opportunities for modulating the activity of MIF family proteins that potentially may be applied in a MIF allele specific manner. Expert Opinion: Know...

Proceedings of the National Academy of Sciences of the United States of America, Mar 28, 2017

Constitutive photomorphogenesis 9 (COP9) signalosome 5 (CSN5), an isopeptidase that removes neura... more Constitutive photomorphogenesis 9 (COP9) signalosome 5 (CSN5), an isopeptidase that removes neural precursor cell-expressed, developmentally down-regulated 8 (NEDD8) moieties from cullins (thus termed "deNEDDylase") and a subunit of the cullin-RING E3 ligase-regulating COP9 signalosome complex, attenuates proinflammatory NF-κB signaling. We previously showed that CSN5 is up-regulated in human atherosclerotic arteries. Here, we investigated the role of CSN5 in atherogenesis in vivo by using mice with myeloid-specific Csn5 deletion. Genetic deletion of Csn5 in Apoe(-/-) mice markedly exacerbated atherosclerotic lesion formation. This was broadly observed in aortic root, arch, and total aorta of male mice, whereas the effect was less pronounced and site-specific in females. Mechanistically, Csn5 KO potentiated NF-κB signaling and proinflammatory cytokine expression in macrophages, whereas HIF-1α levels were reduced. Inversely, inhibition of NEDDylation by MLN4924 blocked proi...

PeerJ, 2017

Subcutaneous adipose tissue is a rich source of adipose tissue macrophages and adipose-derived st... more Subcutaneous adipose tissue is a rich source of adipose tissue macrophages and adipose-derived stem cells which both play a key role in wound repair. While macrophages can be divided into the classically-activated M1 and the alternatively-activated M2 phenotype, ASCs are characterized by the expression of specific stem cell markers. In the present study, we have investigated the expression of common macrophage polarization and stem cell markers in acutely inflamed adipose tissue. Subcutaneous adipose tissue adjacent to acutely inflamed wounds of 20 patients and 20 healthy subjects were harvested and underwent qPCR and flow cytometry analysis. Expression levels of the M1-specific markers CD80, iNOS, and IL-1b were significantly elevated in inflammatory adipose tissue when compared to healthy adipose tissue, whereas the M2-specific markers CD163 and TGF-β were decreased. By flow cytometry, a significant shift of adipose tissue macrophage populations towards the M1 phenotype was confir...

Nephrology Dialysis Transplantation, 2016

Proceedings of the National Academy of Sciences of the United States of America, Dec 6, 2016

Fibroblast-like synoviocytes mediate joint destruction in rheumatoid arthritis and exhibit sustai... more Fibroblast-like synoviocytes mediate joint destruction in rheumatoid arthritis and exhibit sustained proinflammatory and invasive properties. CD44 is a polymorphic transmembrane protein with defined roles in matrix interaction and tumor invasion that is also a signaling coreceptor for macrophage migration inhibitory factor (MIF), which engages cell surface CD74. High-expression MIF alleles (rs5844572) are associated with rheumatoid joint erosion, but whether MIF signaling through the CD74/CD44 receptor complex promotes upstream autoimmune responses or contributes directly to synovial joint destruction is unknown. We report here the functional regulation of CD44 by an autocrine pathway in synovial fibroblasts that is driven by high-expression MIF alleles to up-regulate an inflammatory and invasive phenotype. MIF increases CD44 expression, promotes its recruitment into a functional signal transduction complex, and stimulates alternative exon splicing, leading to expression of the CD44...

Journal of Cellular and Molecular Medicine, 2016

D-dopachrome tautomerase (D-DT/MIF-2) is a member of the macrophage migration inhibitory factor (... more D-dopachrome tautomerase (D-DT/MIF-2) is a member of the macrophage migration inhibitory factor (MIF) cytokine superfamily, and a close structural homolog of MIF. MIF and D-DT have been reported to be involved in obesity, but there is little known about the regulation of D-DT in adipose tissue inflammation and wound healing. Subcutaneous adipose tissue was collected from 54 healthy donors and 28 donors with acutely inflamed wounds undergoing wound debridement. In addition, epididymal fat pads of mice were injected with lipopolysaccharide to study receptor expression and cell migration in vivo. D-DT protein levels and mRNA expression were significantly decreased in subcutaneous adipose tissue adjacent to acutely inflamed wounds. D-DT improved fibroblast viability and increased proliferation in vitro. While D-DT alone did not have a significant effect on in vitro fibroblast wound healing, simultaneous addition of neutralizing MIF antibody resulted in a significant improvement of fibroblast wound healing. Interestingly, expression of the MIF and D-DT receptor CD74 was down-regulated while the MIF receptors CXCR2 and CXCR4 were up-regulated primarily on macrophages indicating that the MIF-CXCR2/4 axis may promote recruitment of inflammatory cells into adipose tissue. Our results describe a reciprocal role of D-DT to MIF in inflamed adipose tissue, and indicate that D-DT may be beneficial in wound repair by improving fibroblast survival and proliferation.

Journal of molecular and cellular cardiology, Jan 30, 2015

Phosphodiesterase 4 (PDE4) activity mediates cAMP-dependent smooth muscle cell (SMC) activation f... more Phosphodiesterase 4 (PDE4) activity mediates cAMP-dependent smooth muscle cell (SMC) activation following vascular injury. In this study we have investigated the effects of specific PDE4 inhibition with roflumilast on SMC proliferation and inflammatory activation in vitro and neointima formation following guide wire-induced injury of the femoral artery in mice in vivo. In vitro, roflumilast did not affect SMC proliferation, but diminished TNF-α induced expression of the vascular cell adhesion molecule 1 (VCAM-1). Specific activation of the cAMP effector Epac, but not PKA activation mimicked the effects of roflumilast on VCAM-1 expression. Consistently, the reduction of VCAM-1 expression was rescued following inhibition of Epac. TNF-α induced NFκB p65 translocation and VCAM-1 promoter activity were not altered by roflumilast in SMCs. However, roflumilast treatment and Epac activation repressed the induction of the activating epigenetic histone mark H3K4me2 at the VCAM-1 promoter, whi...

Thrombosis and Haemostasis, 2013

SummaryThe COP9 signalosome (CSN), a multifunctional protein complex involved in the regulation o... more SummaryThe COP9 signalosome (CSN), a multifunctional protein complex involved in the regulation of cullin-RING-E3 ubiquitin ligases (CRLs), has emerged as a regulator of NF-κB signalling. As NF-κB drives the expression of pro-inflammatory and pro-atherosclerotic genes, we probed the yet unknown role of the CSN, in particular CSN5, on NF-KB-mediated atherogenic responses in endothelial cells. Co-immunoprecipitation in human umbilical vein endothelial cells (HUVECs) revealed the presence of a super-complex between IKK and CSN, which dissociates upon TNF-α stimulation. Furthermore, CSN5 silencing enhanced TNF-α-induced IKB-α degradation and NF-κB activity in luci-ferase reporter assays. This was paralleled by an increased NF-KB-driven upregulation of atherogenic chemokines and adhesion molecules, as measured by qPCR and flow cytometry, and translated into an enhanced arrest of THP-1 monocytes on TNF-α-stimulated, CSN5-depleted HUVECs. Reverse effects on NF-κB activity and THP-1 arrest ...

Journal of Clinical Investigation

Authorship note: PVT and WS contributed equally to this work. Conflict of interest: RB and JB are... more Authorship note: PVT and WS contributed equally to this work. Conflict of interest: RB and JB are listed as co-inventors on issued patents (US20120149044A1 and US20100267714A1) for MIF antagonists.

Subject codes: [134] Pathophysiology [147] Growth factors/cytokines [145] Genetically altered mic... more Subject codes: [134] Pathophysiology [147] Growth factors/cytokines [145] Genetically altered mice Address correspondence to:

BAFF B-cell activating factor BAFFR BAFF receptor BCA Brachiocephalic artery BM Bone marrow B reg... more BAFF B-cell activating factor BAFFR BAFF receptor BCA Brachiocephalic artery BM Bone marrow B regs Regulatory B-cells BSA Bovine serum albumin CBA Cytometric bead assay CC Coiled-coil domains CCL CC chemokine ligand CD Cluster of differentiation cDC Conventional dendritic cell cDNA Complementary DNA CD40L Cd40 ligand Chuk Conserved helix-loop-helix ubiquitous kinase cIAP Calf intestinal alkaline phosphatase CLP Common lymphoid progenitor cell CMP Common myeloid progenitor cell CVD Cardiovascular disease CytD Cytochalasin D CXCR7 Chemokine (C-X-C motif) receptor 7 CXCL19 Chemokine (C-X-C motif) ligand 19 Cy3 Cyanine 3 Abbreviations iii DAPI 4',6-diamindino-2-phenylindole DC Dendritic cell DiL T regs Regulatory T-cells ULD Ubiquitin-like domain Vcam-1 Vascular cell adhesion molecule-1 VLDL Very low density lipoprotein VSMCs Vascular smooth muscle cells WHO World health organization WT Wild type ZF Zinc finger domain 18) Many immune cells, both from the innate as well as the adaptive immune system, are involved in the development and progression of atherosclerosis 14,15,19. As mentioned before, monocytes are the first cells to adhere to the endothelium and transmigrate into the intima, where they differentiate into macrophages. Macrophages are highly versatile cells, taking on many different phenotypes. M1 (classically activated macrophages) and M2 macrophages (non-classically activated macrophages) are suggested to play opposite roles during inflammation, but both are present in atherosclerotic lesions. M1 macrophages can be found in plaque 1.2.3 MicroRNAs in atherosclerosis MicroRNAs (miRs) are short, highly conserved, non-coding RNAs, which play a key regulatory role in the complex network of posttranscriptional gene expression. More than 10.000 miRs have been identified to date and 940 of them in the human organism. In mammals miRs control the expression of 50% of all protein coding genes 52. MiRs originate either from protein-independent genes being transcribed by RNA polymerase II and III or from introns of protein-coding genes. The primary transcript, so called pri-miRs, remains in the nucleus and undergoes cleavage, mediated by Drosha, an RNase III enzyme, and DGCR8 as a cofactor, to form a ~60-70 nucleotide long hairpin structure, known as pre-miR. Pre-miRs are exported through the nuclear pore complexes into the cytoplasm by binding to Exportin-5-Ran-GTP. Once in the cytoplasm, the pre-miR is further processed into mature miR by another 54) An individual miR is capable of targeting hundreds of mRNAs and a single mRNA can be regulated by multiple miRs. MiRs target mRNAs by complementary sequence in the 3´UTR regions using basic base-pairing. The gene regulation takes place by the down-regulation of the respective mRNA, either by mRNA translational repression or mRNA cleavage 54. Altered miR expression patterns have been implicated in various disease states, including cancer and cardiovascular disease 55,56. As mentioned shortly in the atherosclerosis 65. The NF-κB family has five members: RelA (p65), RelB and c-Rel and the precursor proteins NF-κB1 (p105) and NF-κB2 (p100). Both of these precursor proteins need to be processed into an active NF-κB form, being p50 and p52, respectively, whereas the other family members are already present in a mature state in the cytoplasm. The NF-κB transcription factors form dimers and bind to κB sites in promoters and enhancers of a variety of genes to induce as well as repress mediates the phosphorylation of IκB proteins on two N-terminal serine residues in an NEMO-dependent manner. This then leads to the ubiquitination and proteasomal degradation of IκB 70. Once the NF-κB dimer, mainly p65-containing heterodimers, is released from IκB, it is already in an active state and can translocate to the nucleus and start gene transcription 66,71. The transcriptional activity of NF-κB is regulated by post-translational modifications, including acetylation, phosphorylation and ubiquitination. These post-translational modifications are necessary to ensure a tight regulation of NF-κB activity, given the wide range of biological processes that are affected by NF-κB 72. Whereas the canonical NF-κB pathway mainly plays a central role in inflammatory responses, the non-canonical pathway has been implicated in B-cell The IKK kinase complex is the core element of the NF-κB cascade. It essentially consists of two kinases (IKKα and IKKβ) and a regulatory subunit, IKKγ (also known as NEMO). The complex activated by external, inflammatory stimuli, is in charge of the phosphorylation of either the inhibitory protein IκB or the NF-κB precursor p100. These two separate NF-κB activation pathways require different subunits of the IKK NEMO is a regulatory non-enzymatic scaffold protein and has been suggested to be a mainly α-helical protein containing two coiled-coil domains (CC), a leucine zipper (LZ) and a C-terminal zinc-finger (ZF) region 123,124. In addition, NEMO has a minimal oligomerization domain (MOD) and an IKKα/IKKβ binding zone located in amino acid 44-111 of NEMO (Figure 7). The MOD is an N-terminal dimerization domain that enables the formation of higher order oligomers. The ZF has been implicated in efficient IκBα binding and might direct IκBα to the ULD/SDD of IKKβ 125 .

The FASEB Journal, 2020

Sepsis is a leading cause of death worldwide and recent studies have shown white adipose tissue (... more Sepsis is a leading cause of death worldwide and recent studies have shown white adipose tissue (WAT) to be an important regulator in septic conditions. In the present study, the role of the inflammatory cytokine macrophage migration inhibitory factor (MIF) and its structural homolog D-dopachrome tautomerase (D-DT/MIF-2) were investigated in WAT in a murine endotoxemia model. Both MIF and MIF-2 levels were increased in the peritoneal fluid of LPS-challenged wildtype mice, yet in visceral WAT, the proteins were differentially regulated, with elevated MIF but down-regulated MIF-2 expression in adipocytes. Mif gene deletion polarized adipose tissue macrophages (ATM) towards an anti-inflammatory phenotype while Mif-2 gene knockout drove ATMs towards a pro-inflammatory phenotype and Mif-deficiency was found to increase fibroblast

Journal of Biological Chemistry, 2019

The cytokine macrophage migration inhibitory factor-2 (MIF-2 or D-dopachrome tautomerase) is a re... more The cytokine macrophage migration inhibitory factor-2 (MIF-2 or D-dopachrome tautomerase) is a recently characterized second member of the MIF cytokine superfamily in mammalian genomes. MIF-2 shares proinflammatory and tumorigenic properties with the clinical target MIF (MIF-1), but the precise contribution of MIF-2 to immune physiology or pathology is unclear. Like MIF-1, MIF-2 has an intrinsic keto-enol tautomerase activity and mediates biological functions by engaging the cognate, common MIF family receptor CD74. Evidence that the catalytic site of MIF family cytokines has a structural role in receptor binding has prompted the exploration of tautomerase inhibitors as potential biological antagonists and therapeutic agents, although few catalytic inhibitors inhibit receptor activation. We describe herein the discovery and biochemical characterization of a selective small-molecule inhibitor of MIF-2. An in silico screen of 1.6 million compounds targeting the MIF-2 tautomerase site yielded several hits for potential catalytic inhibitors of MIF-2 and identified 4-(3-carboxyphenyl)-2,5pyridinedicarboxylic acid (4-CPPC) as the most functionally potent compound. We found that 4-CPPC has an enzymatic IC50 of 27 µM and 17-fold selectivity for MIF-2 versus MIF-1. An in vitro binding assay for MIF-1/MIF-2 to the CD74 ectodomain (sCD74) indicated that 4-CPPC inhibits MIF-2-CD74 binding in a dose-dependent manner (0.01-10 µM) without

Expert Opinion on Therapeutic Targets, 2019

Macrophage migration inhibitory factor (MIF) as a therapeutic target for rheumatoid arthritis and... more Macrophage migration inhibitory factor (MIF) as a therapeutic target for rheumatoid arthritis and systemic lupus erythematosus, Expert Opinion on Therapeutic Targets,

FASEB journal : official publication of the Federation of American Societies for Experimental Biology, Jan 25, 2018

Dysregulated neutrophil extravasation contributes to the pathogenesis of many inflammatory disord... more Dysregulated neutrophil extravasation contributes to the pathogenesis of many inflammatory disorders. Pericytes (PCs) have been implicated in the regulation of neutrophil transmigration, and previous work demonstrates that endothelial cell (EC)-derived signals reduce PC barrier function; however, the signaling mechanisms are unknown. Here, we demonstrate a novel role for EC-derived macrophage migration inhibitory factor (MIF) in inhibiting PC contractility and facilitating neutrophil transmigration. With the use of micro-ELISAs, RNA sequencing, quantitative PCR, and flow cytometry, we found that ECs secrete MIF, and PCs upregulate CD74 in response to TNF-α. We demonstrate that EC-derived MIF decreases PC contractility on 2-dimensional silicone substrates via reduction of phosphorylated myosin light chain. With the use of an in vitro microvascular model of the human EC-PC barrier, we demonstrate that MIF decreases the PC barrier to human neutrophil transmigration by increasing interc...

Journal of the American College of Surgeons, 2018

Background: Acute kidney injury (AKI) refers to a broad spectrum of kidney damage and is attribut... more Background: Acute kidney injury (AKI) refers to a broad spectrum of kidney damage and is attributed a high morbidity and mortality rate at all degrees of severity. Obesity increases the risk for developing AKI. However, some studies have shown that obesity at onset of AKI is paradoxically associated with greater survival. The aim of this review is to explore the relationship between body mass index and survival in patients with AKI. Methods: An electronic search will be conducted using MEDLINE, EMBASE, CINAHL and CENTRAL using predefined search strategies. The cited and citing references of selected key studies will also be searched for relevant articles. Risk of bias will be assessed using a modified Quality in Prognosis Studies (QUIPS) tool. The primary outcome will be an exploration of the association between BMI and mortality in patients presenting with AKI. Two authors will independently select, data extract, and risk of bias assess articles. Any discrepancies will be resolved by consensus or by consulting a third author. A narrative synthesis of the findings from the included studies will be presented. Meta-analyses will be conducted where the data is available from clinically and methodologically similar studies and in the same format. Heterogeneity in such analyses, beyond that expected by chance, will be quantified using the I 2 statistic. Subgroup analyses will be performed to determine the influence of gender, AKI duration, underlying aetiology, and intervening treatments, on pooled results. Discussion: Body mass index may be an important modifiable risk factor for mortality in patients presenting with AKI. The proposed systematic review will help to elucidate the association between all categories of BMI and survival in this patient group. Systematic review registration: PROSPERO CRD42017071124.

FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2018

The inflammatory cytokine macrophage migration-inhibitory factor (MIF) promotes atherosclerosis v... more The inflammatory cytokine macrophage migration-inhibitory factor (MIF) promotes atherosclerosis via lesional monocyte and T-cell recruitment. B cells have emerged as important components in atherogenesis, but the interaction between MIF and B cells in atherogenesis is unknown. Here, we investigated the atherosclerotic phenotype of Mif-gene deletion in Apoe mice. Apoe Mif mice fed a Western diet exhibited strongly reduced atherosclerotic lesions in brachiocephalic artery (BC) and abdominal aorta compared with controls. This phenotype was accompanied by reduced circulating B cells. Flow cytometry revealed a B-cell developmental defect with increased premature and immature B-cell counts in bone marrow (BM) of Apoe Mif mice and diminished B-cell numbers in spleen. This finding was linked with a decreased expression of Baff-R and differentiation-driving transcription factors at the immature B-cell stage, whereas peritoneal B cells exhibited unchanged CD80 and CD86 expression but vastly d...

Expert opinion on therapeutic targets, Jan 31, 2017

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine with chemokine-like functi... more Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine with chemokine-like functions that is being increasingly studied in different aspects of cardiovascular disease. MIF was first identified as a proinflammatory and pro-survival mediator within the immune system, and a second structurally related MIF family member, D-dopachrome tautomerase (a.k.a. MIF-2), was reported recently. Both MIF family members are released by myocardium and modulate the manifestations of cardiovascular disease, specifically in myocardial ischemia. Areas Covered: A scientific overview is provided for the involvement of MIF family cytokines in the inflammatory pathogenesis of atherosclerosis, myocardial infarction, and ischemia-reperfusion injury. We summarize findings of experimental, human genetic and clinical studies, and suggest therapeutic opportunities for modulating the activity of MIF family proteins that potentially may be applied in a MIF allele specific manner. Expert Opinion: Know...

Proceedings of the National Academy of Sciences of the United States of America, Mar 28, 2017

Constitutive photomorphogenesis 9 (COP9) signalosome 5 (CSN5), an isopeptidase that removes neura... more Constitutive photomorphogenesis 9 (COP9) signalosome 5 (CSN5), an isopeptidase that removes neural precursor cell-expressed, developmentally down-regulated 8 (NEDD8) moieties from cullins (thus termed "deNEDDylase") and a subunit of the cullin-RING E3 ligase-regulating COP9 signalosome complex, attenuates proinflammatory NF-κB signaling. We previously showed that CSN5 is up-regulated in human atherosclerotic arteries. Here, we investigated the role of CSN5 in atherogenesis in vivo by using mice with myeloid-specific Csn5 deletion. Genetic deletion of Csn5 in Apoe(-/-) mice markedly exacerbated atherosclerotic lesion formation. This was broadly observed in aortic root, arch, and total aorta of male mice, whereas the effect was less pronounced and site-specific in females. Mechanistically, Csn5 KO potentiated NF-κB signaling and proinflammatory cytokine expression in macrophages, whereas HIF-1α levels were reduced. Inversely, inhibition of NEDDylation by MLN4924 blocked proi...

Expert opinion on therapeutic targets, Jan 31, 2017

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine with chemokine-like functi... more Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine with chemokine-like functions that is being increasingly studied in different aspects of cardiovascular disease. MIF was first identified as a proinflammatory and pro-survival mediator within the immune system, and a second structurally related MIF family member, D-dopachrome tautomerase (a.k.a. MIF-2), was reported recently. Both MIF family members are released by myocardium and modulate the manifestations of cardiovascular disease, specifically in myocardial ischemia. Areas Covered: A scientific overview is provided for the involvement of MIF family cytokines in the inflammatory pathogenesis of atherosclerosis, myocardial infarction, and ischemia-reperfusion injury. We summarize findings of experimental, human genetic and clinical studies, and suggest therapeutic opportunities for modulating the activity of MIF family proteins that potentially may be applied in a MIF allele specific manner. Expert Opinion: Know...

Proceedings of the National Academy of Sciences of the United States of America, Mar 28, 2017

Constitutive photomorphogenesis 9 (COP9) signalosome 5 (CSN5), an isopeptidase that removes neura... more Constitutive photomorphogenesis 9 (COP9) signalosome 5 (CSN5), an isopeptidase that removes neural precursor cell-expressed, developmentally down-regulated 8 (NEDD8) moieties from cullins (thus termed "deNEDDylase") and a subunit of the cullin-RING E3 ligase-regulating COP9 signalosome complex, attenuates proinflammatory NF-κB signaling. We previously showed that CSN5 is up-regulated in human atherosclerotic arteries. Here, we investigated the role of CSN5 in atherogenesis in vivo by using mice with myeloid-specific Csn5 deletion. Genetic deletion of Csn5 in Apoe(-/-) mice markedly exacerbated atherosclerotic lesion formation. This was broadly observed in aortic root, arch, and total aorta of male mice, whereas the effect was less pronounced and site-specific in females. Mechanistically, Csn5 KO potentiated NF-κB signaling and proinflammatory cytokine expression in macrophages, whereas HIF-1α levels were reduced. Inversely, inhibition of NEDDylation by MLN4924 blocked proi...

PeerJ, 2017

Subcutaneous adipose tissue is a rich source of adipose tissue macrophages and adipose-derived st... more Subcutaneous adipose tissue is a rich source of adipose tissue macrophages and adipose-derived stem cells which both play a key role in wound repair. While macrophages can be divided into the classically-activated M1 and the alternatively-activated M2 phenotype, ASCs are characterized by the expression of specific stem cell markers. In the present study, we have investigated the expression of common macrophage polarization and stem cell markers in acutely inflamed adipose tissue. Subcutaneous adipose tissue adjacent to acutely inflamed wounds of 20 patients and 20 healthy subjects were harvested and underwent qPCR and flow cytometry analysis. Expression levels of the M1-specific markers CD80, iNOS, and IL-1b were significantly elevated in inflammatory adipose tissue when compared to healthy adipose tissue, whereas the M2-specific markers CD163 and TGF-β were decreased. By flow cytometry, a significant shift of adipose tissue macrophage populations towards the M1 phenotype was confir...

Nephrology Dialysis Transplantation, 2016

Proceedings of the National Academy of Sciences of the United States of America, Dec 6, 2016

Fibroblast-like synoviocytes mediate joint destruction in rheumatoid arthritis and exhibit sustai... more Fibroblast-like synoviocytes mediate joint destruction in rheumatoid arthritis and exhibit sustained proinflammatory and invasive properties. CD44 is a polymorphic transmembrane protein with defined roles in matrix interaction and tumor invasion that is also a signaling coreceptor for macrophage migration inhibitory factor (MIF), which engages cell surface CD74. High-expression MIF alleles (rs5844572) are associated with rheumatoid joint erosion, but whether MIF signaling through the CD74/CD44 receptor complex promotes upstream autoimmune responses or contributes directly to synovial joint destruction is unknown. We report here the functional regulation of CD44 by an autocrine pathway in synovial fibroblasts that is driven by high-expression MIF alleles to up-regulate an inflammatory and invasive phenotype. MIF increases CD44 expression, promotes its recruitment into a functional signal transduction complex, and stimulates alternative exon splicing, leading to expression of the CD44...

Journal of Cellular and Molecular Medicine, 2016

D-dopachrome tautomerase (D-DT/MIF-2) is a member of the macrophage migration inhibitory factor (... more D-dopachrome tautomerase (D-DT/MIF-2) is a member of the macrophage migration inhibitory factor (MIF) cytokine superfamily, and a close structural homolog of MIF. MIF and D-DT have been reported to be involved in obesity, but there is little known about the regulation of D-DT in adipose tissue inflammation and wound healing. Subcutaneous adipose tissue was collected from 54 healthy donors and 28 donors with acutely inflamed wounds undergoing wound debridement. In addition, epididymal fat pads of mice were injected with lipopolysaccharide to study receptor expression and cell migration in vivo. D-DT protein levels and mRNA expression were significantly decreased in subcutaneous adipose tissue adjacent to acutely inflamed wounds. D-DT improved fibroblast viability and increased proliferation in vitro. While D-DT alone did not have a significant effect on in vitro fibroblast wound healing, simultaneous addition of neutralizing MIF antibody resulted in a significant improvement of fibroblast wound healing. Interestingly, expression of the MIF and D-DT receptor CD74 was down-regulated while the MIF receptors CXCR2 and CXCR4 were up-regulated primarily on macrophages indicating that the MIF-CXCR2/4 axis may promote recruitment of inflammatory cells into adipose tissue. Our results describe a reciprocal role of D-DT to MIF in inflamed adipose tissue, and indicate that D-DT may be beneficial in wound repair by improving fibroblast survival and proliferation.

Journal of molecular and cellular cardiology, Jan 30, 2015

Phosphodiesterase 4 (PDE4) activity mediates cAMP-dependent smooth muscle cell (SMC) activation f... more Phosphodiesterase 4 (PDE4) activity mediates cAMP-dependent smooth muscle cell (SMC) activation following vascular injury. In this study we have investigated the effects of specific PDE4 inhibition with roflumilast on SMC proliferation and inflammatory activation in vitro and neointima formation following guide wire-induced injury of the femoral artery in mice in vivo. In vitro, roflumilast did not affect SMC proliferation, but diminished TNF-α induced expression of the vascular cell adhesion molecule 1 (VCAM-1). Specific activation of the cAMP effector Epac, but not PKA activation mimicked the effects of roflumilast on VCAM-1 expression. Consistently, the reduction of VCAM-1 expression was rescued following inhibition of Epac. TNF-α induced NFκB p65 translocation and VCAM-1 promoter activity were not altered by roflumilast in SMCs. However, roflumilast treatment and Epac activation repressed the induction of the activating epigenetic histone mark H3K4me2 at the VCAM-1 promoter, whi...

Thrombosis and Haemostasis, 2013

SummaryThe COP9 signalosome (CSN), a multifunctional protein complex involved in the regulation o... more SummaryThe COP9 signalosome (CSN), a multifunctional protein complex involved in the regulation of cullin-RING-E3 ubiquitin ligases (CRLs), has emerged as a regulator of NF-κB signalling. As NF-κB drives the expression of pro-inflammatory and pro-atherosclerotic genes, we probed the yet unknown role of the CSN, in particular CSN5, on NF-KB-mediated atherogenic responses in endothelial cells. Co-immunoprecipitation in human umbilical vein endothelial cells (HUVECs) revealed the presence of a super-complex between IKK and CSN, which dissociates upon TNF-α stimulation. Furthermore, CSN5 silencing enhanced TNF-α-induced IKB-α degradation and NF-κB activity in luci-ferase reporter assays. This was paralleled by an increased NF-KB-driven upregulation of atherogenic chemokines and adhesion molecules, as measured by qPCR and flow cytometry, and translated into an enhanced arrest of THP-1 monocytes on TNF-α-stimulated, CSN5-depleted HUVECs. Reverse effects on NF-κB activity and THP-1 arrest ...

Journal of Clinical Investigation

Authorship note: PVT and WS contributed equally to this work. Conflict of interest: RB and JB are... more Authorship note: PVT and WS contributed equally to this work. Conflict of interest: RB and JB are listed as co-inventors on issued patents (US20120149044A1 and US20100267714A1) for MIF antagonists.

Subject codes: [134] Pathophysiology [147] Growth factors/cytokines [145] Genetically altered mic... more Subject codes: [134] Pathophysiology [147] Growth factors/cytokines [145] Genetically altered mice Address correspondence to:

BAFF B-cell activating factor BAFFR BAFF receptor BCA Brachiocephalic artery BM Bone marrow B reg... more BAFF B-cell activating factor BAFFR BAFF receptor BCA Brachiocephalic artery BM Bone marrow B regs Regulatory B-cells BSA Bovine serum albumin CBA Cytometric bead assay CC Coiled-coil domains CCL CC chemokine ligand CD Cluster of differentiation cDC Conventional dendritic cell cDNA Complementary DNA CD40L Cd40 ligand Chuk Conserved helix-loop-helix ubiquitous kinase cIAP Calf intestinal alkaline phosphatase CLP Common lymphoid progenitor cell CMP Common myeloid progenitor cell CVD Cardiovascular disease CytD Cytochalasin D CXCR7 Chemokine (C-X-C motif) receptor 7 CXCL19 Chemokine (C-X-C motif) ligand 19 Cy3 Cyanine 3 Abbreviations iii DAPI 4',6-diamindino-2-phenylindole DC Dendritic cell DiL T regs Regulatory T-cells ULD Ubiquitin-like domain Vcam-1 Vascular cell adhesion molecule-1 VLDL Very low density lipoprotein VSMCs Vascular smooth muscle cells WHO World health organization WT Wild type ZF Zinc finger domain 18) Many immune cells, both from the innate as well as the adaptive immune system, are involved in the development and progression of atherosclerosis 14,15,19. As mentioned before, monocytes are the first cells to adhere to the endothelium and transmigrate into the intima, where they differentiate into macrophages. Macrophages are highly versatile cells, taking on many different phenotypes. M1 (classically activated macrophages) and M2 macrophages (non-classically activated macrophages) are suggested to play opposite roles during inflammation, but both are present in atherosclerotic lesions. M1 macrophages can be found in plaque 1.2.3 MicroRNAs in atherosclerosis MicroRNAs (miRs) are short, highly conserved, non-coding RNAs, which play a key regulatory role in the complex network of posttranscriptional gene expression. More than 10.000 miRs have been identified to date and 940 of them in the human organism. In mammals miRs control the expression of 50% of all protein coding genes 52. MiRs originate either from protein-independent genes being transcribed by RNA polymerase II and III or from introns of protein-coding genes. The primary transcript, so called pri-miRs, remains in the nucleus and undergoes cleavage, mediated by Drosha, an RNase III enzyme, and DGCR8 as a cofactor, to form a ~60-70 nucleotide long hairpin structure, known as pre-miR. Pre-miRs are exported through the nuclear pore complexes into the cytoplasm by binding to Exportin-5-Ran-GTP. Once in the cytoplasm, the pre-miR is further processed into mature miR by another 54) An individual miR is capable of targeting hundreds of mRNAs and a single mRNA can be regulated by multiple miRs. MiRs target mRNAs by complementary sequence in the 3´UTR regions using basic base-pairing. The gene regulation takes place by the down-regulation of the respective mRNA, either by mRNA translational repression or mRNA cleavage 54. Altered miR expression patterns have been implicated in various disease states, including cancer and cardiovascular disease 55,56. As mentioned shortly in the atherosclerosis 65. The NF-κB family has five members: RelA (p65), RelB and c-Rel and the precursor proteins NF-κB1 (p105) and NF-κB2 (p100). Both of these precursor proteins need to be processed into an active NF-κB form, being p50 and p52, respectively, whereas the other family members are already present in a mature state in the cytoplasm. The NF-κB transcription factors form dimers and bind to κB sites in promoters and enhancers of a variety of genes to induce as well as repress mediates the phosphorylation of IκB proteins on two N-terminal serine residues in an NEMO-dependent manner. This then leads to the ubiquitination and proteasomal degradation of IκB 70. Once the NF-κB dimer, mainly p65-containing heterodimers, is released from IκB, it is already in an active state and can translocate to the nucleus and start gene transcription 66,71. The transcriptional activity of NF-κB is regulated by post-translational modifications, including acetylation, phosphorylation and ubiquitination. These post-translational modifications are necessary to ensure a tight regulation of NF-κB activity, given the wide range of biological processes that are affected by NF-κB 72. Whereas the canonical NF-κB pathway mainly plays a central role in inflammatory responses, the non-canonical pathway has been implicated in B-cell The IKK kinase complex is the core element of the NF-κB cascade. It essentially consists of two kinases (IKKα and IKKβ) and a regulatory subunit, IKKγ (also known as NEMO). The complex activated by external, inflammatory stimuli, is in charge of the phosphorylation of either the inhibitory protein IκB or the NF-κB precursor p100. These two separate NF-κB activation pathways require different subunits of the IKK NEMO is a regulatory non-enzymatic scaffold protein and has been suggested to be a mainly α-helical protein containing two coiled-coil domains (CC), a leucine zipper (LZ) and a C-terminal zinc-finger (ZF) region 123,124. In addition, NEMO has a minimal oligomerization domain (MOD) and an IKKα/IKKβ binding zone located in amino acid 44-111 of NEMO (Figure 7). The MOD is an N-terminal dimerization domain that enables the formation of higher order oligomers. The ZF has been implicated in efficient IκBα binding and might direct IκBα to the ULD/SDD of IKKβ 125 .

The FASEB Journal, 2020

Sepsis is a leading cause of death worldwide and recent studies have shown white adipose tissue (... more Sepsis is a leading cause of death worldwide and recent studies have shown white adipose tissue (WAT) to be an important regulator in septic conditions. In the present study, the role of the inflammatory cytokine macrophage migration inhibitory factor (MIF) and its structural homolog D-dopachrome tautomerase (D-DT/MIF-2) were investigated in WAT in a murine endotoxemia model. Both MIF and MIF-2 levels were increased in the peritoneal fluid of LPS-challenged wildtype mice, yet in visceral WAT, the proteins were differentially regulated, with elevated MIF but down-regulated MIF-2 expression in adipocytes. Mif gene deletion polarized adipose tissue macrophages (ATM) towards an anti-inflammatory phenotype while Mif-2 gene knockout drove ATMs towards a pro-inflammatory phenotype and Mif-deficiency was found to increase fibroblast

Journal of Biological Chemistry, 2019

The cytokine macrophage migration inhibitory factor-2 (MIF-2 or D-dopachrome tautomerase) is a re... more The cytokine macrophage migration inhibitory factor-2 (MIF-2 or D-dopachrome tautomerase) is a recently characterized second member of the MIF cytokine superfamily in mammalian genomes. MIF-2 shares proinflammatory and tumorigenic properties with the clinical target MIF (MIF-1), but the precise contribution of MIF-2 to immune physiology or pathology is unclear. Like MIF-1, MIF-2 has an intrinsic keto-enol tautomerase activity and mediates biological functions by engaging the cognate, common MIF family receptor CD74. Evidence that the catalytic site of MIF family cytokines has a structural role in receptor binding has prompted the exploration of tautomerase inhibitors as potential biological antagonists and therapeutic agents, although few catalytic inhibitors inhibit receptor activation. We describe herein the discovery and biochemical characterization of a selective small-molecule inhibitor of MIF-2. An in silico screen of 1.6 million compounds targeting the MIF-2 tautomerase site yielded several hits for potential catalytic inhibitors of MIF-2 and identified 4-(3-carboxyphenyl)-2,5pyridinedicarboxylic acid (4-CPPC) as the most functionally potent compound. We found that 4-CPPC has an enzymatic IC50 of 27 µM and 17-fold selectivity for MIF-2 versus MIF-1. An in vitro binding assay for MIF-1/MIF-2 to the CD74 ectodomain (sCD74) indicated that 4-CPPC inhibits MIF-2-CD74 binding in a dose-dependent manner (0.01-10 µM) without

Expert Opinion on Therapeutic Targets, 2019

Macrophage migration inhibitory factor (MIF) as a therapeutic target for rheumatoid arthritis and... more Macrophage migration inhibitory factor (MIF) as a therapeutic target for rheumatoid arthritis and systemic lupus erythematosus, Expert Opinion on Therapeutic Targets,

FASEB journal : official publication of the Federation of American Societies for Experimental Biology, Jan 25, 2018

Dysregulated neutrophil extravasation contributes to the pathogenesis of many inflammatory disord... more Dysregulated neutrophil extravasation contributes to the pathogenesis of many inflammatory disorders. Pericytes (PCs) have been implicated in the regulation of neutrophil transmigration, and previous work demonstrates that endothelial cell (EC)-derived signals reduce PC barrier function; however, the signaling mechanisms are unknown. Here, we demonstrate a novel role for EC-derived macrophage migration inhibitory factor (MIF) in inhibiting PC contractility and facilitating neutrophil transmigration. With the use of micro-ELISAs, RNA sequencing, quantitative PCR, and flow cytometry, we found that ECs secrete MIF, and PCs upregulate CD74 in response to TNF-α. We demonstrate that EC-derived MIF decreases PC contractility on 2-dimensional silicone substrates via reduction of phosphorylated myosin light chain. With the use of an in vitro microvascular model of the human EC-PC barrier, we demonstrate that MIF decreases the PC barrier to human neutrophil transmigration by increasing interc...

Journal of the American College of Surgeons, 2018

Background: Acute kidney injury (AKI) refers to a broad spectrum of kidney damage and is attribut... more Background: Acute kidney injury (AKI) refers to a broad spectrum of kidney damage and is attributed a high morbidity and mortality rate at all degrees of severity. Obesity increases the risk for developing AKI. However, some studies have shown that obesity at onset of AKI is paradoxically associated with greater survival. The aim of this review is to explore the relationship between body mass index and survival in patients with AKI. Methods: An electronic search will be conducted using MEDLINE, EMBASE, CINAHL and CENTRAL using predefined search strategies. The cited and citing references of selected key studies will also be searched for relevant articles. Risk of bias will be assessed using a modified Quality in Prognosis Studies (QUIPS) tool. The primary outcome will be an exploration of the association between BMI and mortality in patients presenting with AKI. Two authors will independently select, data extract, and risk of bias assess articles. Any discrepancies will be resolved by consensus or by consulting a third author. A narrative synthesis of the findings from the included studies will be presented. Meta-analyses will be conducted where the data is available from clinically and methodologically similar studies and in the same format. Heterogeneity in such analyses, beyond that expected by chance, will be quantified using the I 2 statistic. Subgroup analyses will be performed to determine the influence of gender, AKI duration, underlying aetiology, and intervening treatments, on pooled results. Discussion: Body mass index may be an important modifiable risk factor for mortality in patients presenting with AKI. The proposed systematic review will help to elucidate the association between all categories of BMI and survival in this patient group. Systematic review registration: PROSPERO CRD42017071124.

FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2018

The inflammatory cytokine macrophage migration-inhibitory factor (MIF) promotes atherosclerosis v... more The inflammatory cytokine macrophage migration-inhibitory factor (MIF) promotes atherosclerosis via lesional monocyte and T-cell recruitment. B cells have emerged as important components in atherogenesis, but the interaction between MIF and B cells in atherogenesis is unknown. Here, we investigated the atherosclerotic phenotype of Mif-gene deletion in Apoe mice. Apoe Mif mice fed a Western diet exhibited strongly reduced atherosclerotic lesions in brachiocephalic artery (BC) and abdominal aorta compared with controls. This phenotype was accompanied by reduced circulating B cells. Flow cytometry revealed a B-cell developmental defect with increased premature and immature B-cell counts in bone marrow (BM) of Apoe Mif mice and diminished B-cell numbers in spleen. This finding was linked with a decreased expression of Baff-R and differentiation-driving transcription factors at the immature B-cell stage, whereas peritoneal B cells exhibited unchanged CD80 and CD86 expression but vastly d...

Expert opinion on therapeutic targets, Jan 31, 2017

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine with chemokine-like functi... more Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine with chemokine-like functions that is being increasingly studied in different aspects of cardiovascular disease. MIF was first identified as a proinflammatory and pro-survival mediator within the immune system, and a second structurally related MIF family member, D-dopachrome tautomerase (a.k.a. MIF-2), was reported recently. Both MIF family members are released by myocardium and modulate the manifestations of cardiovascular disease, specifically in myocardial ischemia. Areas Covered: A scientific overview is provided for the involvement of MIF family cytokines in the inflammatory pathogenesis of atherosclerosis, myocardial infarction, and ischemia-reperfusion injury. We summarize findings of experimental, human genetic and clinical studies, and suggest therapeutic opportunities for modulating the activity of MIF family proteins that potentially may be applied in a MIF allele specific manner. Expert Opinion: Know...

Proceedings of the National Academy of Sciences of the United States of America, Mar 28, 2017

Constitutive photomorphogenesis 9 (COP9) signalosome 5 (CSN5), an isopeptidase that removes neura... more Constitutive photomorphogenesis 9 (COP9) signalosome 5 (CSN5), an isopeptidase that removes neural precursor cell-expressed, developmentally down-regulated 8 (NEDD8) moieties from cullins (thus termed "deNEDDylase") and a subunit of the cullin-RING E3 ligase-regulating COP9 signalosome complex, attenuates proinflammatory NF-κB signaling. We previously showed that CSN5 is up-regulated in human atherosclerotic arteries. Here, we investigated the role of CSN5 in atherogenesis in vivo by using mice with myeloid-specific Csn5 deletion. Genetic deletion of Csn5 in Apoe(-/-) mice markedly exacerbated atherosclerotic lesion formation. This was broadly observed in aortic root, arch, and total aorta of male mice, whereas the effect was less pronounced and site-specific in females. Mechanistically, Csn5 KO potentiated NF-κB signaling and proinflammatory cytokine expression in macrophages, whereas HIF-1α levels were reduced. Inversely, inhibition of NEDDylation by MLN4924 blocked proi...