A model for the optimization of anti-inflammatory treatment with Chemerin

Routine treatment of mild to moderate pain with a combination of non-steroidal anti-inflammatory drugs such as paracetamol in combination with corticoid opioids can lead to severe complications including death from gastrointestinal injury or to drug dependence. There is a need for the development of new safer drugs. Chemerin is a mediator promoting resolution of inflammation and it is then a promising candidate for a new treatment. A pilot experimental work using the zymosan induced peritonitis model has found that injecting extra chemerin resulted in an approximately 1% reduction of the total number of inflammatory cells recruited. This paper combines and adapts existing mathematical models of inflammation to reproduce these results and to explore the therapeutic potential of chemerin through simulations. Analysis of the model predicts that the injection of chemerin with a concentration of 2,000 ng ml-1 within the first 5 minutes of inflammation onset leads to maximal inflammation inhibition. The degree of inhibition is shown to be sensitive to data used for the fit with a mean inhibition of 22±3.7% for a series of remove-one bootstrap tests whereas optimal chemerin injection parameters were not. Overall sensitivity analysis identifies parameters of the model that need to be measured more accurately or with increased sampling rate to improve model robustness and confirm chemerin’s therapeutic potential

IL-33 promotes anemia during chronic inflammation by inhibiting differentiation of erythroid progenitors

An important comorbidity of chronic inflammation is anemia, which may be related to dysregulated activity of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM). Among HSPCs, we found that the receptor for IL-33, ST2, is expressed preferentially and highly on erythroid progenitors. Induction of inflammatory spondyloarthritis in mice increased IL-33 in BM plasma, and IL-33 was required for inflammation-dependent suppression of erythropoiesis in BM. Conversely, administration of IL-33 in healthy mice suppressed erythropoiesis, decreased hemoglobin expression, and caused anemia. Using purified erythroid progenitors in vitro, we show that IL-33 directly inhibited terminal maturation. This effect was dependent on NF-κB activation and associated with altered signaling events downstream of the erythropoietin receptor. Accordingly, IL-33 also suppressed erythropoietin-accelerated erythropoiesis in vivo. These results reveal a role for IL-33 in pathogenesis of anemia during inflammatory disease and define a new target for its treatment

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

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

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

GM-CSF drives dysregulated hematopoietic stem cell activity and pathogenic extramedullary myelopoiesis in experimental spondyloarthritis

Dysregulated hematopoiesis occurs in several chronic inflammatory diseases, but it remains unclear how hematopoietic stem cells (HSCs) in the bone marrow (BM) sense peripheral inflammation and contribute to tissue damage. Here, we show the HSC gene expression program is biased toward myelopoiesis and differentiation skewed toward granulocyte-monocyte progenitors (GMP) during joint and intestinal inflammation in experimental spondyloarthritis (SpA). GM-CSF-receptor is increased on HSC and multipotent progenitors, favoring a striking increase in myelopoiesis at the earliest hematopoietic stages. GMP accumulate in the BM in SpA and, unexpectedly, at extramedulary sites: in the inflamed joints and spleen. Furthermore we show that GM-CSF promotes BM and extramedullary myelopoiesis, tissue-toxic neutrophil accumulation in target organs, and GM-CSF prophylactic or therapeutic blockade substantially decreases SpA severity. Surprisingly, besides CD4 T cells and innate lymphoid cells, mast cells (MC) are a source of GM-CSF in inflammation, and its pathogenic production is promoted by the alarmin IL-33

Human CD68 promoter GFP transgenic mice allow analysis of monocyte to macrophage differentiation in vivo.

The recruitment of monocytes and their differentiation into macrophages at sites of inflammation are key events in determining the outcome of the inflammatory response and initiating the return to tissue homeostasis. To study monocyte trafficking and macrophage differentiation in vivo, we have generated a novel transgenic reporter mouse expressing a green fluorescent protein (GFP) under the control of the human CD68 promoter. CD68-GFP mice express high levels of GFP in both monocyte and embryo-derived tissue resident macrophages in adult animals. The human CD68 promoter drives GFP expression in all CD115(+) monocytes of adult blood, spleen, and bone marrow; we took advantage of this to directly compare the trafficking of bone marrow-derived CD68-GFP monocytes to that of CX3CR1(GFP) monocytes in vivo using a sterile zymosan peritonitis model. Unlike CX3CR1(GFP) monocytes, which downregulate GFP expression on differentiation into macrophages in this model, CD68-GFP monocytes retain high-level GFP expression for 72 hours after differentiation into macrophages, allowing continued cell tracking during resolution of inflammation. In summary, this novel CD68-GFP transgenic reporter mouse line represents a powerful resource for analyzing monocyte mobilization and monocyte trafficking as well as studying the fate of recruited monocytes in models of acute and chronic inflammation

A Real Time Chemotaxis Assay Unveils Unique Migratory Profiles amongst Different Primary Murine Macrophages

<div><p>Chemotaxis assays are an invaluable tool for studying the biological activity of inflammatory mediators such as CC chemokines, which have been implicated in a wide range of chronic inflammatory diseases. Conventional chemotaxis systems such as the modified Boyden chamber are limited in terms of the data captured given that the assays are analysed at a single time-point. We report the optimisation and validation of a label-free, real-time cell migration assay based on electrical cell impedance to measure chemotaxis of different primary murine macrophage populations in response to a range of CC chemokines and other chemoattractant signalling molecules. We clearly demonstrate key differences in the migratory behavior of different murine macrophage populations and show that this dynamic system measures true macrophage chemotaxis rather than chemokinesis or fugetaxis. We highlight an absolute requirement for Gαi signaling and actin cytoskeletal rearrangement as demonstrated by Pertussis toxin and cytochalasin D inhibition. We also studied the chemotaxis of CD14⁺ human monocytes and demonstrate distinct chemotactic profiles amongst different monocyte donors to CCL2. This real-time chemotaxis assay will allow a detailed analysis of factors that regulate macrophage responses to chemoattractant cytokines and inflammatory mediators.</p> </div