Contrasting in vitro vs. in vivo effects of a cell membrane-specific CC-chemokine binding protein on macrophage chemotaxis

ABSTRACT: Chemokines (CK) provide directional cues that mediate the recruitment of leukocytes to sites of inflammation. Broad-spectrum blockade of the CC-CK family, using the vaccinia virus 35K protein, has been shown to cause a potent reduction of systemic inflammation in models of atherosclerosis, vein graft disease and arthritis. We have used a cell membrane-targeted form of 35K, Mem35K, to probe whether cell-associated blockade of chemokine response is sufficient to reduce cell recruitment in inflammation. In Tie2cre mice, activation of a flox-stop Mem35K transgene directed conditional expression of Mem35K in leukocytes and endothelial cells, confirmed by Western blotting, flow cytometry and immunofluorescence microscopy. This conditional Mem35K expression was sufficient to increase cell surface CCL5 binding and reduce chemotaxis in vitro to CCL5, CCL2 and CCL3 but not to non-CC-CK chemoattractants, LTB4, C5a or chemerin. However, in vivo monocyte recruitment into the peritoneum driven by zymosan or CC-chemokine injection, which was demonstrated to be CC-CK dependent using CCR2−/− mice, was not reduced by Mem35K expression, despite the expression of functional Mem35K protein. These findings highlight differing requirements for cell-associated anti-inflammatory activity in in vitro and in vivo models. KEY MESSAGE: Mem35K is a cell-associated CC-chemokine binding protein. Conditional Mem35K transgenic mice show expression Mem35K in leukocytes. Mem35K blocks in vitro primary macrophage chemotaxis specifically towards CC-chemokines. Mem35K expression is not sufficient to reduce inflammation in vivo. The requirements for anti-inflammatory activity in vitro and in vivo are different. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00109-014-1194-6) contains supplementary material, which is available to authorized users

Optimising triage of urgent referrals for suspected IBD: results from the Birmingham IBD inception study

Objective: Diagnostic delays in inflammatory bowel disease (IBD) result in adverse outcomes. We report a bespoke diagnostic pathway to assess how best to combine clinical history and faecal calprotectin (FCP) for early diagnosis and efficient resource utilisation. Methods: A rapid-access pathway was implemented for suspected IBD patients referred outside urgent ‘two-week wait’ criteria. Patients were triaged using symptoms and FCP. A 13-point symptom history was taken prediagnosis and clinical indices, including repeat FCP, collected prospectively. Results: Of 767 patients (January 2021–August 2023), 423 were diagnosed with IBD (208 Crohn’s disease (CD), 215 ulcerative colitis (UC)). Most common symptoms in CD were abdominal pain (84%), looser stools (84%) and fatigue (79%) and in UC per-rectal bleeding (94%), urgency (82%) and looser stools (81%). Strongest IBD predictors were blood mixed with stools (CD OR 4.38; 95% CI 2.40–7.98, UC OR 33.68; 15.47–73.33) and weight loss (CD OR 3.39; 2.14–5.38, UC OR 2.33; 1.37–4.00). Repeat FCP testing showed reduction from baseline in non-IBD. Both measurements >100 µg/g (area under the curve (AUC) 0.800) and >200 µg/g (AUC 0.834) collectively predicted IBD. However, a second value ≥220 µg/g considered alone, regardless of the first result, was more accurate (Youden’s index 0.735, AUC 0.923). Modelling symptoms with FCP increased AUC to 0.947. Conclusion: Serial FCP measurement prevents unnecessary colonoscopy. Two FCPs >200 µg/g could stream patients direct to colonoscopy, with two >100 µg/g prompting clinic review. A second result ≥220 µg/g was more accurate than dual-result thresholds. Coupling home FCP testing with key symptoms may form the basis of effective self-referral pathways

Absence of the non-signalling chemerin receptor CCRL2 exacerbates acute inflammatory responses in vivo

Chemerin is a chemotactic protein that induces migration of several immune cells including macrophages, immature dendritic cells, and NK cells. Chemerin binds to three G protein-coupled receptors (GPCRs), including CCRL2. The exact function of CCRL2 remains unclear. CCRL2 expression is rapidly upregulated during inflammation, but it lacks the intracellular DRYLAIV motif required for classical GPCR downstream signalling pathways, and it has not been reported to internalise chemerin upon binding. The aim of this study was to investigate what role if any CCRL2 plays during acute inflammation. Using the zymosan- and thioglycollate-induced murine models of acute inflammation, we report that mice deficient in the Ccrl2 gene display exaggerated local and systemic inflammatory responses, characterised by increased myeloid cell recruitment. This amplified myeloid cell recruitment was associated with increased chemerin and CXCL1 levels. Furthermore, we report that the inflammatory phenotype observed in these mice is dependent upon elevated levels of endogenous chemerin. Antibody neutralisation of chemerin activity in Ccrl2−/− mice abrogated the amplified inflammatory responses. Importantly, chemerin did not directly recruit myeloid cells but rather increased the production of other chemotactic proteins such as CXCL1. Administration of recombinant chemerin to wild-type mice before inflammatory challenge recapitulated the increased myeloid cell recruitment and inflammatory mediator production observed in Ccrl2−/− mice. We have demonstrated that the absence of CCRL2 results in increased levels of local and systemic chemerin levels and exacerbated inflammatory responses during acute inflammatory challenge. These results further highlight the importance of chemerin as a therapeutic target in inflammatory diseases

RGS1 regulates myeloid cell accumulation in atherosclerosis and aortic aneurysm rupture through altered chemokine signalling

Chemokine signalling drives monocyte recruitment in atherosclerosis and aortic aneurysms. The mechanisms that lead to retention and accumulation of macrophages in the vascular wall remain unclear. Regulator of G-Protein Signalling-1 (RGS1) deactivates G-protein signalling, reducing the response to sustained chemokine stimulation. Here we show that Rgs1 is upregulated in atherosclerotic plaque and aortic aneurysms. Rgs1 reduces macrophage chemotaxis and desensitizes chemokine receptor signalling. In early atherosclerotic lesions, Rgs1 regulates macrophage accumulation and is required for the formation and rupture of Angiotensin II-induced aortic aneurysms, through effects on leukocyte retention. Collectively, these data reveal a role for Rgs1 in leukocyte trafficking and vascular inflammation and identify Rgs1, and inhibition of chemokine receptor signalling as potential therapeutic targets in vascular disease

The role of chemerin and chemerin derived peptides in inflammation

Inflammation is a normal physiological response to invading pathogens or tissue injury. However, if the inciting stimulus is not efficiently cleared, the inflammatory response is not resolved and becomes chronic. Chronic inflammation plays an important role in the pathology of a variety of diseases including atherosclerosis, rheumatoid arthritis, multiple sclerosis, psoriasis, colitis and cancer. Understanding this disease process and its regulation is of paramount importance for the development of novel therapies for these diseases. Chemerin was originally isolated from inflammatory exudate fluid and found to be a ligand for the orphan G-protein coupled receptor, ChemR23. Secreted in an inactive pro- form, pro-chemerin, the carboxyl terminus can be cleaved by different proteases to generate active chemerin isoforms. Active chemerin is a chemoattractant for immature dendritic cells, natural killer cells and macrophages. The role played by chemerin during chronic inflammation remains controversial. It has been reported to be elevated in patients with a number of inflammatory and metabolic diseases but chemerin and shorter chemerin derived peptides have also been reported to exert anti-inflammatory effects in pre-clinical murine models. In this thesis, I set out to investigate what role chemerin plays during inflammation. I demonstrated that chemerin can exert both pro- and anti-inflammatory effects depending on the time of intervention. During a standard model of acute inflammation (zymosan induced peritonitis), pre-treatment of animals with recombinant murine chemerin increased neutrophil recruitment while administration of chemerin into an already inflamed peritoneum significantly reduced monocyte recruitment. These effects are likely due to further proteolytic processing of active chemerin as demonstrated by the incubation of active chemerin with neutrophil derived proteases, which produced shorter bioactive peptides. Chemerin binds to three receptors, ChemR23, GPR1 and CCRL2. Both ChemR23 and GPR1 have been demonstrated to induce downstream signalling following chemerin binding but CCRL2 does not. CCRL2 is expressed on a range of cell types and its expression is rapidly increased following inflammatory stimuli. However, its role in inflammation remains largely unknown. I have demonstrated that animals lacking the CCRL2 chemerin receptor display exaggerated monocyte and neutrophil recruitment during acute inflammatory responses. This phenotype does not seem to be due to increased levels of total chemerin either locally or systemically, nor does it seem to be due to differences in local inflammatory mediator production. I observed increased neutrophils in the blood of these animals during acute inflammatory responses, indicating CCRL2 may play a role in dampening neutrophil mobilisation from bone marrow rather then exerting effects locally. Taken together the experiments presented in this thesis suggest that targeting chemerin therapeutically may be of benefit in certain contexts but not in others. In addition, the non-signalling chemerin receptor CCRL2 plays a non-redundant role in regulating neutrophil and monocyte recruitment to local sites of inflammation as well as mobilisation of neutrophils into the blood stream.</p

The role of chemerin and chemerin derived peptides in inflammation

Inflammation is a normal physiological response to invading pathogens or tissue injury. However, if the inciting stimulus is not efficiently cleared, the inflammatory response is not resolved and becomes chronic. Chronic inflammation plays an important role in the pathology of a variety of diseases including atherosclerosis, rheumatoid arthritis, multiple sclerosis, psoriasis, colitis and cancer. Understanding this disease process and its regulation is of paramount importance for the development of novel therapies for these diseases. Chemerin was originally isolated from inflammatory exudate fluid and found to be a ligand for the orphan G-protein coupled receptor, ChemR23. Secreted in an inactive pro- form, pro-chemerin, the carboxyl terminus can be cleaved by different proteases to generate active chemerin isoforms. Active chemerin is a chemoattractant for immature dendritic cells, natural killer cells and macrophages. The role played by chemerin during chronic inflammation remains controversial. It has been reported to be elevated in patients with a number of inflammatory and metabolic diseases but chemerin and shorter chemerin derived peptides have also been reported to exert anti-inflammatory effects in pre-clinical murine models. In this thesis, I set out to investigate what role chemerin plays during inflammation. I demonstrated that chemerin can exert both pro- and anti-inflammatory effects depending on the time of intervention. During a standard model of acute inflammation (zymosan induced peritonitis), pre-treatment of animals with recombinant murine chemerin increased neutrophil recruitment while administration of chemerin into an already inflamed peritoneum significantly reduced monocyte recruitment. These effects are likely due to further proteolytic processing of active chemerin as demonstrated by the incubation of active chemerin with neutrophil derived proteases, which produced shorter bioactive peptides. Chemerin binds to three receptors, ChemR23, GPR1 and CCRL2. Both ChemR23 and GPR1 have been demonstrated to induce downstream signalling following chemerin binding but CCRL2 does not. CCRL2 is expressed on a range of cell types and its expression is rapidly increased following inflammatory stimuli. However, its role in inflammation remains largely unknown. I have demonstrated that animals lacking the CCRL2 chemerin receptor display exaggerated monocyte and neutrophil recruitment during acute inflammatory responses. This phenotype does not seem to be due to increased levels of total chemerin either locally or systemically, nor does it seem to be due to differences in local inflammatory mediator production. I observed increased neutrophils in the blood of these animals during acute inflammatory responses, indicating CCRL2 may play a role in dampening neutrophil mobilisation from bone marrow rather then exerting effects locally. Taken together the experiments presented in this thesis suggest that targeting chemerin therapeutically may be of benefit in certain contexts but not in others. In addition, the non-signalling chemerin receptor CCRL2 plays a non-redundant role in regulating neutrophil and monocyte recruitment to local sites of inflammation as well as mobilisation of neutrophils into the blood stream.</p

Mucins Dynamics in Physiological and Pathological Conditions

Maintaining intestinal health requires clear segregation between epithelial cells and luminal microbes. The intestinal mucus layer, produced by goblet cells (GCs), is a key element in maintaining the functional protection of the epithelium. The importance of the gut mucus barrier is highlighted in mice lacking Muc2, the major form of secreted mucins. These mice show closer bacterial residence to epithelial cells, develop spontaneous colitis and became moribund when infected with the attaching and effacing pathogen, Citrobacter rodentium. Furthermore, numerous observations have associated GCs and mucus layer dysfunction to the pathogenesis of inflammatory bowel disease (IBD). However, the molecular mechanisms that regulate the physiology of GCs and the mucus layer remain obscured. In this review, we consider novel findings describing divergent functionality and expression profiles of GCs subtypes within intestinal crypts. We also discuss internal (host) and external (diets and bacteria) factors that modulate different aspects of the mucus layer as well as the contribution of an altered mucus barrier to the onset of IBD

Identifying Spatial Co-occurrence in Healthy and InflAmed tissues (ISCHIA)

Spatial transcriptomics techniques are able to chart the distribution and localization of cell types and RNA molecules across a tissue. Here, we generated matched sequencing-based (Visium) and hybridization-based (Molecular Cartography) spatial transcriptomics data of human IBD samples. We then developed ISCHIA (Identifying Spatial Co-occurrence in Healthy and InflAmed tissues), a computational framework to analyze the spatial co-occurrence of cell types and transcript species in the tissue environment. ISCHIA revealed tightly associated cellular networks, ligand-receptor interactions enriched in the inflamed human colon, and their associated gene signatures, highlighting the hypothesis-generating power of co-occurrence analysis on spatial transcriptomics data

Table S1 from A model for the optimization of anti-inflammatory treatment with chemerin

Measurement of the mean and standard deviation of the concentration of CCL2 CXXL1 and chemerin concentrations at different times during the zymosan induced peritonitis experiment

Matlab code from A model for the optimization of anti-inflammatory treatment with chemerin

Matlab code developed for analysing the anti-inflammatory potential of chemeri