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.

Ligand-dependent kinase activity of MERTK drives efferocytosis in human iPSC-derived macrophages.

Removal of apoptotic cells by phagocytes (also called efferocytosis) is a crucial process for tissue homeostasis. Professional phagocytes express a plethora of surface receptors enabling them to sense and engulf apoptotic cells, thus avoiding persistence of dead cells and cellular debris and their consequent effects. Dysregulation of efferocytosis is thought to lead to secondary necrosis and associated inflammation and immune activation. Efferocytosis in primarily murine macrophages and dendritic cells has been shown to require TAM RTKs, with MERTK and AXL being critical for clearance of apoptotic cells. The functional role of human orthologs, especially the exact contribution of each individual receptor is less well studied. Here we show that human macrophages differentiated in vitro from iPSC-derived precursor cells express both AXL and MERTK and engulf apoptotic cells. TAM RTK agonism by the natural ligand growth-arrest specific 6 (GAS6) significantly enhanced such efferocytosis. Using a newly-developed mouse model of kinase-dead MERTK, we demonstrate that MERTK kinase activity is essential for efferocytosis in peritoneal macrophages in vivo. Moreover, human iPSC-derived macrophages treated in vitro with blocking antibodies or small molecule inhibitors recapitulated this observation. Hence, our results highlight a conserved MERTK function between mice and humans, and the critical role of its kinase activity in homeostatic efferocytosis.

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.

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 in arthritis. 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 HSCs and multipotent progenitors, favoring a striking increase in myelopoiesis at the earliest hematopoietic stages. GMP accumulate in the BM in SpA and, unexpectedly, at extramedullary sites: in the inflamed joints and spleen. Furthermore, we show that GM-CSF promotes 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 are a source of GM-CSF in this model, and its pathogenic production is promoted by the alarmin IL-33.

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.

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

UNLABELLED: 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.

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.

Inhibition of late-stage autophagy synergistically enhances pyrrolo-1,5-benzoxazepine-6-induced apoptotic cell death in human colon cancer cells.

The pyrrolo-1,5-benzoxazepines (PBOXs) are a novel group of selective apoptotic agents displaying promising therapeutic potential in both ex vivo chemotherapy-refractory patient samples and in vivo murine carcinoma models. In this report, we present novel data concerning the induction of autophagy by the PBOXs in adenocarcinoma-derived colon cancer cells. Autophagy is a lysosome-dependent degradative pathway recently associated with chemotherapy. However, whether autophagy facilitates cell survival in response to chemotherapy or contributes to chemotherapy-induced cell death is highly controversial. Autophagy was identified by enhanced expression of LC3B-II, an autophagosome marker, an increase in the formation of acridine orange-stained cells, indicative of increased vesicle formation and electron microscopic confirmation of autophagic structures. The vacuolar H+ ATPase inhibitor bafilomycin-A1 (BAF-A1) inhibited vesicle formation and enhanced the apoptotic potential of PBOX-6. These findings suggest a cytoprotective role of autophagy in these cells following prolonged exposure to PBOX-6. Furthermore, BAF-A1 and PBOX-6 interactions were determined to be synergistic and caspase-dependent. Potentiation of PBOX-6-induced apoptosis by BAF-A1 was associated with a decrease in the levels of the anti-apoptotic protein, Mcl-1. The data provide evidence that autophagy functions as a survival mechanism in colon cancer cells to PBOX-6-induced apoptosis and a rationale for the use of autophagy inhibitors to further enhance PBOX­6­induced apoptosis in colon cancer.

A real time chemotaxis assay unveils unique migratory profiles amongst different primary murine macrophages.

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.