Correction: del Molino del Barrio et al. Breast Cancer: An Examination of the Potential of ACKR3 to Modify the Response of CXCR4 to CXCL12. Int. J. Mol. Sci. 2018, 19, 3592.

The authors and Editorial Office were made aware of an error in a figure within the original publication [...].

Dissecting the Therapeutic Mechanisms of Sphingosine-1-Phosphate Receptor Agonism during Ischaemia and Reperfusion.

Sphingosine 1-phosphate (S1P) and S1P receptors (S1PR) regulate many cellular processes, including lymphocyte migration and endothelial barrier function. As neutrophils are major mediators of inflammation, their transendothelial migration may be the target of therapeutic approaches to inflammatory conditions such as ischaemia-reperfusion injury (IRI). The aim of this project was to assess whether these therapeutic effects are mediated by S1P acting on neutrophils directly or indirectly through the endothelial cells. First, our murine model of peritoneum cell recruitment demonstrated the ability of S1P to reduce CXCL8-mediated neutrophil recruitment. Mechanistic in vitro studies revealed that S1P signals in neutrophils mainly through the S1PR1 and 4 receptors and induces phosphorylation of ERK1/2; however, this had no effect on neutrophil transmigration and adhesion. S1P treatment of endothelial cells significantly reduced TNF-α-induced neutrophil adhesion under flow (p < 0.01) and transendothelial migration towards CXCL8 during in vitro chemotaxis assays (p < 0.05). S1PR1 agonist CYM5442 treatment of endothelial cells also reduced neutrophil transmigration (p < 0.01) and endothelial permeability (p < 0.005), as shown using in vitro permeability assays. S1PR3 agonist had no effects on chemotaxis or permeability. In an in vivo mouse model of renal IRI, S1PR agonism with CYM5442 reduced endothelial permeability as shown by reduced Evan's Blue dye extravasation. Western blot was used to assess phosphorylation at different sites on vascular endothelial (VE)-cadherin and showed that CYM5442 reduced VEGF-mediated phosphorylation. Taken together, the results of this study suggest that reductions in neutrophil infiltration during IRI in response to S1P are mediated primarily by S1PR1 signalling on endothelial cells, possibly by altering phosphorylation of VE-cadherin. The results also demonstrate the therapeutic potential of S1PR1 agonist during IRI.

Hypoxia-Driven TGFß Modulation of Side Population Cells in Breast Cancer: The Potential Role of ERα.

Epithelial-to-mesenchymal transition (EMT) is known to be important in regulating the behaviour of cancer cells enabling them to acquire stem cell characteristics or by enhancing the stem cell characteristics of cancer stem cells, resulting in these cells becoming more migratory and invasive. EMT can be driven by a number of mechanisms, including the TGF-ß1 signalling pathway and/or by hypoxia. However, these drivers of EMT differ in their actions in regulating side population (SP) cell behaviour, even within SPs isolated from the same tissue. In this study we examined CoCl2 exposure and TGF-ß driven EMT on SP cells of the MDA-MB-231 and MCF7 breast cancer cell lines. Both TGF-ß1 and CoCl2 treatment led to the depletion of MDA-MB-231 SP. Whilst TGF-ß1 treatment significantly reduced the MCF7 SP cells, CoCl2 exposure led to a significant increase. Single cell analysis revealed that CoCl2 exposure of MCF7 SP leads to increased expression of ABCG2 and HES1, both associated with multi-drug resistance. We also examined the mammosphere forming efficiency in response to CoCl2 exposure in these cell lines, and saw the same effect as seen with the SP cells. We suggest that these contrasting effects are due to ERα expression and the inversely correlated expression of TGFB-RII, which is almost absent in the MCF7 cells. Understanding the EMT-mediated mechanisms of the regulation of SP cells could enable the identification of new therapeutic targets in breast cancer.

Nitration of chemokine CXCL8 acts as a natural mechanism to limit acute inflammation.

Chemokine CXCL8 is a key facilitator of the human host immune response, mediating neutrophil migration, and activation at the site of infection and injury. The oxidative burst is an important effector mechanism which leads to the generation of reactive nitrogen species (RNS), including peroxynitrite. The current study was performed to determine the potential for nitration to alter the biological properties of CXCL8 and its detection in human disease. Here, we show peroxynitrite nitrates CXCL8 and thereby regulates neutrophil migration and activation. The nitrated chemokine was unable to induce transendothelial neutrophil migration in vitro and failed to promote leukocyte recruitment in vivo. This reduced activity is due to impairment in both G protein-coupled receptor signaling and glycosaminoglycan binding. Using a novel antibody, nitrated CXCL8 was detected in bronchoalveolar lavage samples from patients with pneumonia. These findings were validated by mass spectrometry. Our results provide the first direct evidence of chemokine nitration in human pathophysiology and suggest a natural mechanism that limits acute inflammation.

Anti-Cholestatic Therapy with Obeticholic Acid Improves Short-Term Memory in Bile Duct-Ligated Mice.

Patients with cholestatic liver disease, including those with primary biliary cholangitis, can experience symptoms of impaired cognition or brain fog. This phenomenon remains unexplained and is currently untreatable. Bile duct ligation (BDL) is an established rodent model of cholestasis. In addition to liver changes, BDL animals develop cognitive symptoms early in the disease process (before development of cirrhosis and/or liver failure). The cellular mechanisms underpinning these cognitive symptoms are poorly understood. Herein, the study explored the neurocognitive symptom manifestations, and tested potential therapies, in BDL mice, and used human neuronal cell cultures to explore translatability to humans. BDL animals exhibited short-term memory loss and showed reduced astrocyte coverage of the blood-brain barrier, destabilized hippocampal network activity, and neuronal senescence. Ursodeoxycholic acid (first-line therapy for most human cholestatic diseases) did not reverse symptomatic or mechanistic aspects. In contrast, obeticholic acid (OCA), a farnesoid X receptor agonist and second-line anti-cholestatic agent, normalized memory function, suppressed blood-brain barrier changes, prevented hippocampal network deficits, and reversed neuronal senescence. Co-culture of human neuronal cells with either BDL or human cholestatic patient serum induced cellular senescence and increased mitochondrial respiration, changes that were limited again by OCA. These findings provide new insights into the mechanism of cognitive symptoms in BDL animals, suggesting that OCA therapy or farnesoid X receptor agonism could be used to limit cholestasis-induced neuronal senescence.

Targeting Leukocyte Trafficking in Inflammatory Bowel Disease.

In the last two decades, understanding of inflammatory bowel disease (IBD) immunopathogenesis has expanded considerably. Histopathological examination of the intestinal mucosa in IBD demonstrates the presence of a chronic inflammatory cell infiltrate. Research has focused on identifying mechanisms of immune cell trafficking to the gastrointestinal tract that may represent effective gut-selective targets for IBD therapy whilst avoiding systemic immunosuppression that may be associated with off-target adverse effects such as infection and malignancy. Integrins are cell surface receptors that can bind to cellular adhesion molecules to mediate both leukocyte homing and retention. In 2014, Vedolizumab (Entyvio®) was the first anti-integrin (anti-α4ß7 monoclonal antibody) treatment to be approved for use in IBD. Several other anti-integrin therapies are currently in advanced stages of development, including novel orally administered small-molecule drugs. Drugs targeting alternative trafficking mechanisms such as mucosal addressin cellular adhesion molecule-1 and sphingosine-1-phosphate receptors are also being evaluated. Here, we summarise key established and emerging therapies targeting leukocyte trafficking that may play an important role in realising the goal of stratified precision medicine in IBD care.

Regulation and Role of αE Integrin and Gut Homing Integrins in Migration and Retention of Intestinal Lymphocytes during Inflammatory Bowel Disease.

Targeting interactions between α4ß7 integrin and endothelial adhesion molecule MAdCAM-1 to inhibit lymphocyte migration to the gastrointestinal tract is an effective therapy in inflammatory bowel disease (IBD). Following lymphocyte entry into the mucosa, a subset of these cells expresses αEß7 integrin, which is expressed on proinflammatory lymphocytes, to increase cell retention. The factors governing lymphocyte migration into the intestinal mucosa and αE integrin expression in healthy subjects and IBD patients remain incompletely understood. We evaluated changes in factors involved in lymphocyte migration and differentiation within tissues. Both ileal and colonic tissue from active IBD patients showed upregulation of ICAM-1, VCAM-1, and MAdCAM-1 at the gene and protein levels compared with healthy subjects and/or inactive IBD patients. ß1 and ß7 integrin expression on circulating lymphocytes was similar across groups. TGF-ß1 treatment induced expression of αE on both ß7+ and ß7- T cells, suggesting that cells entering the mucosa independently of MAdCAM-1/α4ß7 can become αEß7+ ITGAE gene polymorphisms did not alter protein induction following TGF-ß1 stimulation. Increased phospho-SMAD3, which is directly downstream of TGF-ß, and increased TGF-ß-responsive gene expression were observed in the colonic mucosa of IBD patients. Finally, in vitro stimulation experiments showed that baseline ß7 expression had little effect on cytokine, chemokine, transcription factor, and effector molecule gene expression in αE+ and αE- T cells. These findings suggest cell migration to the gut mucosa may be altered in IBD and α4ß7-, and α4ß7+ T cells may upregulate αEß7 in response to TGF-ß once within the gut mucosa.

MiR-126-3p Is Dynamically Regulated in Endothelial-to-Mesenchymal Transition during Fibrosis.

In fibrotic diseases, myofibroblasts derive from a range of cell types including endothelial-to-mesenchymal transition (EndMT). Increasing evidence suggests that miRNAs are key regulators in biological processes but their profile is relatively understudied in EndMT. In human umbilical vein endothelial cells (HUVEC), EndMT was induced by treatment with TGFß2 and IL1ß. A significant decrease in endothelial markers such as VE-cadherin, CD31 and an increase in mesenchymal markers such as fibronectin were observed. In parallel, miRNA profiling showed that miR-126-3p was down-regulated in HUVECs undergoing EndMT and over-expression of miR-126-3p prevented EndMT, maintaining CD31 and repressing fibronectin expression. EndMT was investigated using lineage tracing with transgenic Cdh5-Cre-ERT2; Rosa26R-stop-YFP mice in two established models of fibrosis: cardiac ischaemic injury and kidney ureteric occlusion. In both cardiac and kidney fibrosis, lineage tracing showed a significant subpopulation of endothelial-derived cells expressed mesenchymal markers, indicating they had undergone EndMT. In addition, miR-126-3p was restricted to endothelial cells and down-regulated in murine fibrotic kidney and heart tissue. These findings were confirmed in patient kidney biopsies. MiR-126-3p expression is restricted to endothelial cells and is down-regulated during EndMT. Over-expression of miR-126-3p reduces EndMT, therefore, it could be considered for miRNA-based therapeutics in fibrotic organs.

Contribution of Heparan Sulphate Binding in CCL21-Mediated Migration of Breast Cancer Cells.

Chemokine receptor CCR7 is implicated in the metastasis of breast cancer to the lymph nodes. Chemokine function is dependent upon their binding to both cell-surface heparan sulphate (HS) and to their specific receptors; thus, the role of HS in CCR7-mediated lymph node metastasis was investigated by creating a non-HS binding chemokine CCL21 (mut-CCL21). Mut-CCL21 (Δ103-134) induced leukocyte chemotaxis in diffusion gradients but did not stimulate trans-endothelial migration of PBMCs (p < 0.001) and 4T1-Luc cells (p < 0.01). Furthermore, the effect of heparin and HS on the chemotactic properties of wild-type (WT) and mut-CCL21 was examined. Interestingly, heparin and HS completely inhibit the chemotaxis mediated by WT-CCL21 at 250 and 500 µg/mL, whereas minimal effect was seen with mut-CCL21. This difference could potentially be attributed to reduced HS binding, as surface plasmon resonance spectroscopy showed that mut-CCL21 did not significantly bind HS compared to WT-CCL21. A murine model was used to assess the potential of mut-CCL21 to prevent lymph node metastasis in vivo. Mice were injected with 4T1-Luc cells in the mammary fat pad and treated daily for a week with 20 µg mut-CCL21. Mice were imaged weekly with IVIS and sacrificed on day 18. Luciferase expression was significantly reduced in lymph nodes from mice that had been treated with mut-CCL21 compared to the control (p = 0.0148), suggesting the potential to target chemokine binding to HS as a therapeutic option.

A C-terminal CXCL8 peptide based on chemokine-glycosaminoglycan interactions reduces neutrophil adhesion and migration during inflammation.

Leucocyte recruitment is critical during many acute and chronic inflammatory diseases. Chemokines are key mediators of leucocyte recruitment during the inflammatory response, by signalling through specific chemokine G-protein-coupled receptors (GPCRs). In addition, chemokines interact with cell-surface glycosaminoglycans (GAGs) to generate a chemotactic gradient. The chemokine interleukin-8/CXCL8, a prototypical neutrophil chemoattractant, is characterized by a long, highly positively charged GAG-binding C-terminal region, absent in most other chemokines. To examine whether the CXCL8 C-terminal peptide has a modulatory role in GAG binding during neutrophil recruitment, we synthesized the wild-type CXCL8 C-terminal [CXCL8 (54-72)] (Peptide 1), a peptide with a substitution of glutamic acid (E) 70 with lysine (K) (Peptide 2) to increase positive charge; and also, a scrambled sequence peptide (Peptide 3). Surface plasmon resonance showed that Peptide 1, corresponding to the core CXCL8 GAG-binding region, binds to GAG but Peptide 2 binding was detected at lower concentrations. In the absence of cellular GAG, the peptides did not affect CXCL8-induced calcium signalling or neutrophil chemotaxis along a diffusion gradient, suggesting no effect on GPCR binding. All peptides equally inhibited neutrophil adhesion to endothelial cells under physiological flow conditions. Peptide 2, with its greater positive charge and binding to polyanionic GAG, inhibited CXCL8-induced neutrophil transendothelial migration. Our studies suggest that the E70K CXCL8 peptide, may serve as a lead molecule for further development of therapeutic inhibitors of neutrophil-mediated inflammation based on modulation of chemokine-GAG binding.

Bile acid receptor agonists in primary biliary cholangitis: Regulation of the cholangiocyte secretome and downstream T cell differentiation.

Primary biliary cholangitis (PBC) is a chronic autoimmune liver disease. Approximately 30% of patients do not respond to therapy with ursodeoxycholic acid (UDCA). Previous studies have implicated increased senescence of cholangiocytes in patients who do not respond to UDCA. This may increase the release of cytokines which drive pathogenic T cell polarization. As FXR agonists are beneficial in treating UDCA non-responsive patients, the current study was designed to model the interactions between cholangiocytes and CD4+ T cells to investigate potential immunomodulatory mechanisms of bile acid receptor agonists. Human cholangiocytes were co-cultured with CD4+ T cells to model the biliary stress response. Senescent cholangiocytes were able to polarize T cells toward a Th17 phenotype and suppressed expression of FoxP3 (P = 0.0043). Whilst FXR and TGR5 receptor agonists were unable directly to alter cholangiocyte cytokine expression, FGF19 was capable of significantly reducing IL-6 release (P = 0.044). Bile acid receptor expression was assessed in PBC patients with well-characterized responsiveness to UDCA therapy. A reduction in FXR staining was observed in both cholangiocytes and hepatocytes in PBC patients without adequate response to UDCA. Increased IL-6 expression by senescent cholangiocytes represents a potential mechanism by which biliary damage in PBC could contribute to excessive inflammation.

Breast Cancer: An Examination of the Potential of ACKR3 to Modify the Response of CXCR4 to CXCL12.

Upon binding with the chemokine CXCL12, the chemokine receptor CXCR4 has been shown to promote breast cancer progression. This process, however, can be affected by the expression of the atypical chemokine receptor ACKR3. Given ACKR3's ability to form heterodimers with CXCR4, we investigated how dual expression of both receptors differed from their lone expression in terms of their signalling pathways. We created single and double CXCR4 and/or ACKR3 Chinese hamster ovary (CHO) cell transfectants. ERK and Akt phosphorylation after CXCL12 stimulation was assessed and correlated with receptor internalization. Functional consequences in cell migration and proliferation were determined through wound healing assays and calcium flux. Initial experiments showed that CXCR4 and ACKR3 were upregulated in primary breast cancer and that CXCR4 and ACKR3 could form heterodimers in transfected CHO cells. This co-expression modified CXCR4's Akt activation after CXCL12's stimulation but not ERK phosphorylation (p < 0.05). To assess this signalling disparity, receptor internalization was assessed and it was observed that ACKR3 was recycled to the surface whilst CXCR4 was degraded (p < 0.01), a process that could be partially inhibited with a proteasome inhibitor (p < 0.01). Internalization was also assessed with the ACKR3 agonist VUF11207, which caused both CXCR4 and ACKR3 to be degraded after internalization (p < 0.05 and p < 0.001), highlighting its potential as a dual targeting drug. Interestingly, we observed that CXCR4 but not ACKR3, activated calcium flux after CXCL12 stimulation (p < 0.05) and its co-expression could increase cellular migration (p < 0.01). These findings suggest that both receptors can signal through ERK and Akt pathways but co-expression can alter their kinetics and internalization pathways.

AlphaE Integrin Expression Is Increased in the Ileum Relative to the Colon and Unaffected by Inflammation.

BACKGROUND: Recent findings suggest that αE expression is enriched on effector T cells and that intestinal αE+ T cells have increased expression of inflammatory cytokines. αE integrin expression is a potential predictive biomarker for response to etrolizumab, a monoclonal antibody against ß7 integrin that targets both α4ß7 and αEß7. We evaluated the prevalence and localization of αE+ cells as well as total αE gene expression in healthy and inflammatory bowel disease patients. METHODS: αE+ cells were identified in ileal and colonic biopsies by immunohistochemistry and counted using an automated algorithm. Gene expression was assessed by quantitative reverse-transcriptase polymerase chain reaction. RESULTS: In both healthy and inflammatory bowel disease patients, significantly more αE+ cells were present in the epithelium and lamina propria of ileal compared with colonic biopsies. αE gene expression levels were also significantly higher in ileal compared with colonic biopsies. Paired biopsies from the same patient showed moderate correlation of αE expression between the ileum and colon. Inflammation did not affect αE expression, and neither endoscopy nor histology scores correlated with αE gene expression. αE expression was not different between patients based on concomitant medication use except 5-aminosalicylic acid. CONCLUSION: αE+ cells, which have been shown to have inflammatory potential, are increased in the ileum in comparison with the colon in both Crohn's disease and ulcerative colitis, as well as in healthy subjects. In inflammatory bowel disease patients, αE levels are stable, regardless of inflammatory status or most concomitant medications, which could support its use as a biomarker for etrolizumab.

Regulation of Chemokine Function: The Roles of GAG-Binding and Post-Translational Nitration.

The primary function of chemokines is to direct the migration of leukocytes to the site of injury during inflammation. The effects of chemokines are modulated by several means, including binding to G-protein coupled receptors (GPCRs), binding to glycosaminoglycans (GAGs), and through post-translational modifications (PTMs). GAGs, present on cell surfaces, bind chemokines released in response to injury. Chemokines bind leukocytes via their GPCRs, which directs migration and contributes to local inflammation. Studies have shown that GAGs or GAG-binding peptides can be used to interfere with chemokine binding and reduce leukocyte recruitment. Post-translational modifications of chemokines, such as nitration, which occurs due to the production of reactive species during oxidative stress, can also alter their biological activity. This review describes the regulation of chemokine function by GAG-binding ability and by post-translational nitration. These are both aspects of chemokine biology that could be targeted if the therapeutic potential of chemokines, like CXCL8, to modulate inflammation is to be realised.

Mechanisms Affecting the Gut of Preterm Infants in Enteral Feeding Trials.

Large randomized controlled trials (RCTs) in preterm infants offer unique opportunities for mechanistic evaluation of the risk factors leading to serious diseases, as well as the actions of interventions designed to prevent them. Necrotizing enterocolitis (NEC) a serious inflammatory gut condition and late-onset sepsis (LOS) are common feeding and nutrition-related problems that may cause death or serious long-term morbidity and are key outcomes in two current UK National Institutes for Health Research (NIHR) trials. Speed of increasing milk feeds trial (SIFT) randomized preterm infants to different rates of increases in milk feeds with a primary outcome of survival without disability at 2 years corrected age. Enteral lactoferrin in neonates (ELFIN) randomizes infants to supplemental enteral lactoferrin or placebo with a primary outcome of LOS. This is a protocol for the mechanisms affecting the gut of preterm infants in enteral feeding trials (MAGPIE) study and is funded by the UK NIHR Efficacy and Mechanistic Evaluation programme. MAGPIE will recruit ~480 preterm infants who were enrolled in SIFT or ELFIN. Participation in MAGPIE does not change the main trial protocols and uses non-invasive sampling of stool and urine, along with any residual resected gut tissue if infants required surgery. Trial interventions may involve effects on gut microbes, metabolites (e.g., short-chain fatty acids), and aspects of host immune function. Current hypotheses suggest that NEC and/or LOS are due to a dysregulated immune system in the context of gut dysbiosis, but mechanisms have not been systematically studied within large RCTs. Microbiomic analysis will use next-generation sequencing, and metabolites will be assessed by mass spectrometry to detect volatile organic and other compounds produced by microbes or the host. We will explore differences between disease cases and controls, as well as exploring the actions of trial interventions. Impacts of this research are multiple: translation of knowledge of mechanisms promoting gut health may explain outcomes or suggest alternate strategies to improve health. Results may identify new non-invasive diagnostic or monitoring techniques, preventative or treatment strategies for NEC or LOS, or provide data useful for risk stratification in future studies. Mechanistic evaluation might be especially informative where there are not clear effects on the primary outcome (ISRCTN 12554594).

αEß7 Integrin Identifies Subsets of Pro-Inflammatory Colonic CD4+ T Lymphocytes in Ulcerative Colitis.

BACKGROUND AND AIMS: The αEß7 integrin is crucial for retention of T lymphocytes at mucosal surfaces through its interaction with E-cadherin. Pathogenic or protective functions of these cells during human intestinal inflammation, such as ulcerative colitis [UC], have not previously been defined, with understanding largely derived from animal model data. Defining this phenotype in human samples is important for understanding UC pathogenesis and is of translational importance for therapeutic targeting of αEß7-E-cadherin interactions. METHODS: αEß7+ and αEß7- colonic T cell localization, inflammatory cytokine production and expression of regulatory T cell-associated markers were evaluated in cohorts of control subjects and patients with active UC by immunohistochemistry, flow cytometry and real-time PCR of FACS-purified cell populations. RESULTS: CD4+αEß7+ T lymphocytes from both healthy controls and UC patients had lower expression of regulatory T cell-associated genes, including FOXP3, IL-10, CTLA-4 and ICOS in comparison with CD4+αEß7- T lymphocytes. In UC, CD4+αEß7+ lymphocytes expressed higher levels of IFNγ and TNFα in comparison with CD4+αEß7- lymphocytes. Additionally the CD4+αEß7+ subset was enriched for Th17 cells and the recently described Th17/Th1 subset co-expressing both IL-17A and IFNγ, both of which were found at higher frequencies in UC compared to control. CONCLUSION: αEß7 integrin expression on human colonic CD4+ T cells was associated with increased production of pro-inflammatory Th1, Th17 and Th17/Th1 cytokines, with reduced expression of regulatory T cell-associated markers. These data suggest colonic CD4+αEß7+ T cells are pro-inflammatory and may play a role in UC pathobiology.

CCL2 nitration is a negative regulator of chemokine-mediated inflammation.

Chemokines promote leukocyte recruitment during inflammation. The oxidative burst is an important effector mechanism, this leads to the generation of reactive nitrogen species (RNS), including peroxynitrite (ONOO). The current study was performed to determine the potential for nitration to alter the chemical and biological properties of the prototypical CC chemokine, CCL2. Immunofluorescence was performed to assess the presence of RNS in kidney biopsies. Co-localisation was observed between RNS-modified tyrosine residues and the chemokine CCL2 in diseased kidneys. Nitration reduced the potential of CCL2 to stimulate monocyte migration in diffusion gradient chemotaxis assays (p < 0.05). This was consistent with a trend towards reduced affinity of the nitrated chemokine for its cognate receptor CCR2b. The nitrated chemokine was unable to induce transendothelial monocyte migration in vitro and failed to promote leukocyte recruitment when added to murine air pouches (p < 0.05). This could potentially be attributed to reduced glycosaminoglycan binding ability, as surface plasmon resonance spectroscopy showed that nitration reduced heparan sulphate binding by CCL2. Importantly, intravenous administration of nitrated CCL2 also inhibited the normal recruitment of leukocytes to murine air pouches filled with unmodified CCL2. Together these data suggest that nitration of CCL2 during inflammation provides a mechanism to limit and resolve acute inflammation.

Effect of cobalt-mediated Toll-like receptor 4 activation on inflammatory responses in endothelial cells.

Cobalt-containing metal-on-metal hip replacements are associated with adverse reactions to metal debris (ARMD), including inflammatory pseudotumours, osteolysis, and aseptic implant loosening. The exact cellular and molecular mechanisms leading to these responses are unknown. Cobaltions (Co2+) activate human Toll-like receptor 4 (TLR4), an innate immune receptor responsible for inflammatory responses to Gram negative bacterial lipopolysaccharide (LPS).We investigated the effect of Co2+-mediated TLR4 activation on human microvascular endothelial cells (HMEC-1), focusing on the secretion of key inflammatory cytokines and expression of adhesion molecules. We also studied the role of TLR4 in Co2+-mediated adhesion molecule expression in MonoMac 6 macrophages.We show that Co2+ increases secretion of inflammatory cytokines, including IL-6 and IL-8, in HMEC-1. The effects are TLR4-dependent as they can be prevented with a small molecule TLR4 antagonist. Increased TLR4-dependent expression of intercellular adhesion molecule 1 (ICAM1) was also observed in endothelial cells and macrophages. Furthermore, we demonstrate for the first time that Co2+ activation of TLR4 upregulates secretion of a soluble adhesion molecule, sICAM-1, in both endothelial cells and macrophages. Although sICAM-1 can be generated through activity of matrix metalloproteinase-9 (MMP-9), we did not find any changes in MMP9 expression following Co2+ stimulation.In summary we show that Co2+ can induce endothelial inflammation via activation of TLR4. We also identify a role for TLR4 in Co2+-mediated changes in adhesion molecule expression. Finally, sICAM-1 is a novel target for further investigation in ARMD studies.