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

αEβ7 Integrin Identifies Subsets of Pro-Inflammatory Colonic CD4+ T Lymphocytes in Ulcerative Colitis.

Background and Aims The αEβ7 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 αEβ7-E-cadherin interactions. Methods αEβ7+ and αEβ7- 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+αEβ7+ 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+αEβ7- T lymphocytes. In UC, CD4+αEβ7+ lymphocytes expressed higher levels of IFNγ and TNFα in comparison with CD4+αEβ7- lymphocytes. Additionally the CD4+αEβ7+ 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 αEβ7 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+αEβ7+ T cells are pro-inflammatory and may play a role in UC pathobiology

Defective Leukocyte Adhesion and Chemotaxis Contributes to Combined Immunodeficiency in Humans with Autosomal Recessive MST1 Deficiency.

PURPOSE: To investigate the clinical and functional aspects of MST1 (STK4) deficiency in a profoundly CD4-lymphopenic kindred with a novel homozygous nonsense mutation in STK4. Although recent studies have described the cellular effects of murine Mst1 deficiency, the phenotype of MST1-deficient human lymphocytes has yet to be fully explored. Patient lymphocytes were therefore investigated in the context of current knowledge of murine Mst1 deficiency. METHODS: Genetic etiology was identified by whole exome sequencing of genomic DNA from two siblings, combined with linkage analysis in the wider family. MST1 protein expression was assessed by immunoblotting. The ability of patient lymphocytes to adhere to ICAM-1 under flow conditions was measured, and transwell assays were used to assess chemotaxis. Chemokine receptor expression was examined by flow cytometry and receptor signalling by immunoblotting. RESULTS: A homozygous nonsense mutation in STK4 (c.442C > T, p.Arg148Stop) was found in the patients, leading to a lack of MST1 protein expression. Patient leukocytes exhibited deficient chemotaxis after stimulation with CXCL11, despite preserved expression of CXCR3. Patient lymphocytes were also unable to bind effectively to immobilised ICAM-1 under flow conditions, in keeping with a failure to develop high affinity binding. CONCLUSION: The observed abnormalities of adhesion and migration imply a profound trafficking defect among human MST1-deficient lymphocytes. By analogy with murine Mst1 deficiency and other defects of leucocyte trafficking, this is likely to contribute to immunodeficiency by impairing key aspects of T-cell development and function such as positive selection in the thymus, thymic egress and immune synapse formation in the periphery.This is thepublished version. It first appeared at http://link.springer.com/article/10.1007%2Fs10875-016-0232-2

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio

A small molecule agonist of the chemokine receptor CXCR3 prevents experimental graft-versus-host disease

My talk was at “The Leading Edge of Leukocyte Migration”, the inaugural meeting of the British Society of Immunology Leukocyte Migration Group. It was in Glasgow on the 12-13th of September 2013

The prognostic significance and impact of the CXCR4-CXCR7-CXCL12 axis in primary cutaneous melanoma.

BACKGROUND Expression of the chemokine receptor CXCR4 is known to regulate melanoma metastasis to distant sites with high expression of the CXCL12 ligand. However, the prognostic impact of CXCR4 expression and potential for autocrine-mediated activation of prosurvival mitogen-activated protein kinase signalling remains enigmatic. Furthermore, expression of the decoy receptor CXCR7 within the local cutaneous melanoma microenvironment remains undefined. OBJECTIVES To define the contribution and prognostic impact of CXCR4-CXCR7-CXCL12 signalling in primary cutaneous melanomas and the immediate tumour microenvironment. METHODS Immunohistochemical/immunofluorescent expression of CXCR4, CXCR7 or CXC12 was analysed in human metastatic melanoma cell lines, primary cutaneous cell types and a retrospective cohort of primary melanomas/benign naevi. CXCL12 secretion by melanoma/cutaneous cells was evaluated by enzyme-linked immunosorbent assay, and autocrine CXCR4-CXCL12 signalling was investigated by addition of a CXCL12-neutralizing antibody. RESULTS CXCR4 expression was significantly higher in primary melanomas that subsequently metastasized after 7 years (P = 0·037). Stratification for American Joint Committee on Cancer (AJCC) stage II disease revealed significantly decreased disease-free survival in patients with > 50% CXCR4 expression (P = 0·036), while comparative analysis of CXCL12 expression in the adjacent epidermis of all AJCC stage melanomas revealed increased CXCL12 correlated with prolonged time to metastasis (P = 0·014). CXCR7 was expressed within the primary melanoma microenvironment but was absent on primary tumours. Addition of anti-CXCL12 to BRAF-mutant melanoma cells resulted in downregulation of phospho-CXCR4 and phospho-extracellular signal-related kinase, indicating autocrine CXCR4-CXCL12 signalling. CONCLUSIONS CXCR4 expression defines a potential prognostic biomarker for AJCC stage II melanoma. Moreover, targeting the CXCR4-CXCR7-CXCL12 axis may represent a novel therapeutic strategy to prevent early melanoma progression

Potential role of indoleamine 2,3-dioxygenase in primary biliary cirrhosis.

Indoleamine 2,3-dioxygenase (IDO)-induced immunosuppression can be clinically beneficial for autoimmune diseases. Primary biliary cirrhosis (PBC) is characterized by autoimmune lesions of intrahepatic bile duct epithelial cells that may lead to irreversible cirrhosis or hepatocellular carcinoma. The present study assessed the expression and function of IDO in a cell culture model and in PBC patients. IDO expression was monitored in a human immortalized but non-malignant biliary epithelial cell (iBEC) line. Increased expression of IDO1/2 was observed in the iBECs following stimulation with interferon-γ (IFN-γ). The induction of IDO was IFN-γ-dependent, but was independent of the transforming growth factor-β (TGF-β) pathway. IDO enzymatic activity was observed in the supernatant of iBECs following stimulation with IFN-γ using colorimetric assays. A total of 47 serum samples from PBC patients were used to examine IDO activity by high-performance liquid chromatography, with samples from 24 healthy volunteers used as controls. Patients with PBC exhibited an increased rate of tryptophan to kynurenine conversion (P>0.01). Liver sections from patients with PBC (n=5) and those of healthy controls (n=5) were used for immunohistochemical studies. IDO expression was observed in biliary epithelial cells and in hepatocytes of PBC patients. Finally, the effect of tryptophan metabolites on human cluster of differentiation (CD) 4 T cells in inducing polarization towards a regulatory T cell phenotype was examined. 3-Hydroxykynurenine significantly upregulated the fraction of CD4 cells expressing forkhead box p3 (Foxp3). The results of the present study suggest a therapeutic opportunity for the management of PBC and indicate that tryptophan catabolism could serve as a potential biomarker to monitor disease progression

Inhibition of CXCR4-CXCL12 chemotaxis in melanoma by AMD11070

BACKGROUND: Despite intensive research and novel adjuvant therapies, there is currently no cure for metastatic melanoma. The chemokine receptor CXCR4 controls metastasis to sites such as the liver; however, the therapeutic blockade with the existing agents has proven difficult. METHODS: AMD11070, a novel orally bioavailable inhibitor of CXCR4, was tested for its ability to inhibit the migration of melanoma cells compared with the commonly described antagonist AMD3100. RESULTS: AMD11070 abrogated melanoma cell migration and was significantly more effective than AMD3100. Importantly for the clinical context, the expression of B-RAF-V600E did not the affect the sensitivity of AMD11070. CONCLUSION: Liver-resident myofibroblasts excrete CXCL12, which is able to promote the migration of CXCR4-expressing tumour cells from the blood into the liver. Blockade of this axis by AMD11070 thus represents a novel therapeutic strategy for both B-RAF wild-type and mutated melanomas

Anti-inflammatory therapy by intravenous delivery of non-heparan sulfate-binding CXCL12

Interaction between chemokines and heparan sulfate (HS) is essential for leukocyte recruitment during inflammation. Previous studies have shown that a non-HS-binding mutant form of the inflammatory chemokine CCL7 can block inflammation produced by wild-type chemokines. This study examined the anti-inflammatory mechanism of a non-HS-binding mutant of the homeostatic chemokine CXCL12. Initial experiments demonstrated that mutant CXCL12 was an effective CXCR4 agonist. However, this mutant chemokine failed to promote transendothelial migration in vitro and inhibited the haptotactic response to wild-type CCL7, CXCL12, and CXCL8, and naturally occurring chemoattractants in synovial fluid from the rheumatoid synovium, including CCL2, CCL7, and CXCL8. Notably, intravenous administration of mutant CXCL12 also inhibited the recruitment of leukocytes to murine air pouches filled with wild-type CXCL12. Following intravenous administration, wild-type CXCL12 was cleared from the circulation rapidly, while the mutant chemokine persisted for >24 h. Chronic exposure to mutant CXCL12 in the circulation reduced leukocyte-surface expression of CXCR4, reduced the chemotactic response of these cells to CXCL12, and inhibited normal chemokine-mediated induction of adhesion between the α4β1 integrin, VLA-4, and VCAM-1. These data demonstrate that systemic administration of non-HS-binding variants of CXCL12 can mediate a powerful anti-inflammatory effect through chemokine receptor desensitization.—O’Boyle, G., Mellor, P., Kirby, J. A., Ali, S. Anti-inflammatory therapy by intravenous delivery of non-heparan sulfate-binding CXCL12