The selective vitamin D receptor agonist, elocalcitol, reduces endometriosis development in a mouse model by inhibiting peritoneal inflammation.

BACKGROUND: Endometriosis, which is characterized by the growth of endometrial tissue at ectopic locations as well as vascular development and inflammation, is still an unmet clinical need since an optimal drug that allows for both pain and infertility management does not exist. Since both the eutopic and the ectopic endometrium express the vitamin D receptor (VDR), and VDR agonists are endowed with anti-proliferative and anti-inflammatory properties, we evaluated the effect of elocalcitol, a VDR agonist with low calcaemic liability, in a mouse model of experimentally induced endometriosis. METHODS AND RESULTS: Endometriosis was induced by injection of syngeneic endometrial tissue fragments into adult Balb/c female mice. After having confirmed by immunohistochemistry that endometriotic lesions developing in mice expressed VDR, the mice were administered with elocalcitol (100 µg/kg) or vehicle orally, once a day, for various durations of time. In this model, elocalcitol was able to reduce total lesion weight up to 70% upon treatment for 1 week before and 2 weeks after disease induction. Interestingly, a therapeutic effect was also observed on already established lesions. Elocalcitol was shown to reduce the capacity of mouse endometrial cells to adhere to collagen. In addition in treated mice, a decreased state of peritoneal inflammation was demonstrated by the inhibition of macrophage recruitment and inflammatory cytokine secretion. CONCLUSIONS: The VDR agonist elocalcitol inhibits lesion development in a validated mouse model of endometriosis, and exerts a protective effect on both the implantation and organization of transferred endometrial tissue. These preliminary data in mice provide a sound rationale for further testing in primate models and eventually in humans.

Macrophages are alternatively activated in patients with endometriosis and required for growth and vascularization of lesions in a mouse model of disease.

The mechanisms that sustain endometrial tissues at ectopic sites in patients with endometriosis are poorly understood. Various leukocytes, including macrophages, infiltrate endometriotic lesions. In this study, we depleted mouse macrophages by means of either clodronate liposomes or monoclonal antibodies before the injection of syngeneic endometrial tissue. In the absence of macrophages, tissue fragments adhered and implanted into the peritoneal wall, but endometriotic lesions failed to organize and develop. When we depleted macrophages after the establishment of endometriotic lesions, blood vessels failed to reach the inner layers of the lesions, which stopped growing. Macrophages from patients with endometriosis and experimental mice, but not nonendometriotic patients who underwent surgery for uterine leiomyomas or control mice, expressed markers of alternative activation. These markers included high levels of scavenger receptors, CD163 and CD206, which are involved in both the scavenging of hemoglobin with iron transfer into macrophages and the silent clearance of inflammatory molecules. Macrophages in both inflammatory liquid and ectopic lesions were equally polarized, suggesting a critical role of environmental cues in the peritoneal cavity. Adoptively transferred, alternatively activated macrophages dramatically enhanced endometriotic lesion growth in mice. Inflammatory macrophages effectively protected mice from endometriosis. Therefore, endogenous macrophages involved in tissue remodeling appear as players in the natural history of endometriosis, required for effective vascularization and ectopic lesion growth.

Blockade of TREM-2 exacerbates experimental autoimmune encephalomyelitis.

Triggering receptor expressed on myeloid cells (TREM-2) is a membrane receptor associated with DAP12 that is expressed primarily in myeloid cells, including dendritic cells and microglia, and promotes fusion of osteoclast precursors into multinucleated cells. A rare autosomal recessive condition, Nasu-Hakola disease (NHD) is associated with loss-of-function mutations in DAP12 and TREM-2. The brain pathology observed in NHD patients suggests that disruption of the TREM-2/DAP12 pathway leads to neurodegeneration with demyelination and axonal loss. In this study, we have characterized TREM-2 protein expression on microglia using a newly produced monoclonal antibody directed against the mouse TREM-2 receptor. We report that TREM-2 expression is up-regulated in the spinal cord during both the early inflammatory and chronic phases of myelin oligodendrocyte glycoprotein (MOG)(35-55)peptide-induced experimental autoimmune encaphalomyelitis (EAE). We also demonstrate that TREM-2 is highly expressed on microglial cells in the central nervous system (CNS) during EAE and that blockade of TREM-2 during the effector phase of EAE results in disease exacerbation with more diffuse CNS inflammatory infiltrates and demyelination in the brain parenchyma. These results demonstrate a critical role for TREM-2 during inflammatory responses in the CNS.

Chemokine receptors in chronic obstructive pulmonary disease (COPD).

Chronic obstructive pulmonary disease (COPD) is a debilitating disease characterized by recurrent episodes of leukocyte infiltration in the lung parenchyma causing progressive pulmonary tissue damage and loss of function. Recruitment of neutrophils and CD8+ T cells is linked to disease progression and is under control of chemotactic mediators produced in the inflamed COPD lung. Recent progress in elucidation of the molecular mechanisms that regulate migration of inflammatory cells into the lung has revealed interesting novel targets for therapeutic intervention in this disease. Chemokine receptors CXCR1 and CXCR2 expressed on neutrophils and CXCR3 expressed on CD8+ T cells have been identified as potential therapeutic targets to prevent recruitment of pathogenic cells into the inflamed lung. However, the observation that chemokine receptors are also expressed and functional on various types of lung resident cells including epithelial and smooth muscle cells has raised new questions on the role played by chemokine receptors in COPD. These new findings suggest that chemokine receptor signalling could contribute to the adaptive response of lung tissue resident cells to the microenvironmental changes induced by inflammation. Thus, investigation of the role played by chemokine receptors in development of COPD remains a fertile area of research. Nevertheless, validation of chemokine receptor targets in COPD has proven a difficult challenge given the lack of predictive animal models of the disease and the still poorly defined etiology and pathogenesis.

Distribution and signaling of TREM2/DAP12, the receptor system mutated in human polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy dementia.

Together with its adaptor protein, the adaptor protein of 12 kDa also known as KARAP and TYROBP (DAP12), triggering receptor expressed in myeloid cells 2 (TREM2) is a stimulatory membrane receptor of the immunoglobulin/lectin-like superfamily, well known in myeloid cells. In humans, however, loss-of-function mutations of TREM2/DAP12 leave myeloid cells unaffected but induce an autosomal recessive disease characterized, together with bone cysts, by a spectrum of pathological lesions in the cortex, thalamus and basal ganglia with clinical symptoms of progressive dementia (polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy). Nothing was known about the role of TREM2/DAP12 in brain cell biology and physiology. By confocal immunocytochemistry we demonstrate that, in both human and mouse cerebral cortex, TREM2/DAP12, strongly expressed by microglia, is also present in a fraction of neurons but not in astrocytes and oligodendrocytes. In contrast, in the hippocampal cortex TREM2-expressing neurons are rare. Both in neurons and microglia the receptor appears to be located mostly intracellularly in a discrete compartment(s) partially coinciding with (or adjacent to) the Golgi complex/trans-Golgi network. Four nerve cell lines were identified as expressing the intracellular receptor system. In living human microglia CHME-5 and glioblastoma T98G cells, activation of TREM2 by its specific antibody induced [Ca2+]i responses, documenting its surface expression and functioning. Surface expression of TREM2, low in resting CHME-5 and T98G cells, increases significantly and transiently (60 min) when cells are stimulated by ionomycin, as revealed by both surface biotinylation and surface immunolabeling. Our results provide the first information about the expression, distribution (mostly intracellular) and functioning of TREM2/DAP12 system in nerve cells, a necessary step in the understanding of the cellular mechanisms affected in polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy.

Production of profibrotic cytokines by invariant NKT cells characterizes cirrhosis progression in chronic viral hepatitis.

Invariant (inv)NKT cells are a subset of autoreactive lymphocytes that recognize endogenous lipid ligands presented by CD1d, and are suspected to regulate the host response to cell stress and tissue damage via the prompt production of cytokines. We investigated invNKT cell response during the progression of chronic viral hepatitis caused by hepatitis B or C virus infection, a major human disease characterized by a diffused hepatic necroinflammation with scarring fibrotic reaction, which can progress toward cirrhosis and cancer. Ex vivo frequency and cytokine production were determined in circulating and intrahepatic invNKT cells from controls (healthy subjects or patients with nonviral benign or malignant focal liver damage and minimal inflammatory response) or chronic viral hepatitis patients without cirrhosis, with cirrhosis, or with cirrhosis and hepatocellular carcinoma. invNKT cells increase in chronically infected livers and undergo a substantial modification in their effector functions, consisting in the production of the type 2 profibrotic IL-4 and IL-13 cytokines, which characterizes the progression of hepatic fibrosis to cirrhosis. CD1d, nearly undetectable in noncirrhotic and control livers, is strongly expressed by APCs in cirrhotic ones. Furthermore, in vitro CD1d-dependent activation of invNKT cells from healthy donors elicits IL-4 and IL-13. Together, these findings show that invNKT cells respond to the progressive liver damage caused by chronic hepatitis virus infection, and suggest that these cells, possibly triggered by the recognition of CD1d associated with viral- or stress-induced lipid ligands, contribute to the pathogenesis of cirrhosis by expressing a set of cytokines involved in the progression of fibrosis.

Dominance of CCL22 over CCL17 in induction of chemokine receptor CCR4 desensitization and internalization on human Th2 cells.

Chemokines and their receptors play a pivotal role in controlling T cell trafficking in immunity and inflammation. Two chemokines, CCL17 and CCL22, activate the chemokine receptor CCR4, expressed on functionally distinct subsets of T cells: cutaneous leukocyte-associated antigen (CLA)+ skin-homing, T helper (Th) 2, and CD25+ T suppressor cells. Here, we compared the ability of CCL17 and CCL22 to promote CCR4 internalization as a mechanism of regulation of receptor function on human Th2 cells. We report that CCL22 is a potent and rapid inducer of CCR4 internalization, while CCL17 is not. CCR4 internalization does not require G protein coupling, while being dependent on lipid rafts integrity and clathrin-coated pits functionality. Cell surface disappearance of CCR4 is rapidly reversed upon removal of exogenous ligand by virtue of receptor recycling. CCR4 internalization leads to a loss of functional responsiveness, while recovery of surface expression leads to re-acquisition of chemotactic sensitivity of Th2 cells. The differential CCR4 desensitization and internalization reported here and the distinct expression patterns of CCL17 and CCL22 observed in vivo suggest that while CCL17 may act first on CCR4 at the endothelial surface to promote vascular recognition, CCL22 could subsequently engage the receptor within the tissue microenvironment to guide cellular localization.

Analysis of homing receptor expression on infiltrating leukocytes in disease states.

Chemokines represent a large family of polypeptides that signal through G-protein-coupled receptors and have a role in chemotaxis, leukocyte homing, inflammation, hematopoiesis, angiogenesis and tumor growth. The chemokine/chemokine receptor system acts in coordination with a complex cytokine network to elicit and direct leukocyte infiltration into the inflamed tissue. In addition to promoting movement into the inflamed tissue, the chemokine/chemokine receptor system may also activate infiltrating cells, such as neutrophils and eosinophils, and induce local damage. In recent years, the elucidation of intricate chemokine networks has led to the identification of potential target molecules for therapeutic intervention. Of considerable interest has been the role of chemokine/chemokine receptors in regulating allergic lung inflammation. In this review, techniques to study in situ expression of chemokine receptors in inflamed tissues are presented and discussed.

Increased expression of the chemokine receptor CXCR3 and its ligand CXCL10 in peripheral airways of smokers with chronic obstructive pulmonary disease.

CXCR3 is a chemokine receptor preferentially expressed on lymphocytes, particularly on type-1 T-lymphocytes. Smokers who develop chronic obstructive pulmonary disease (COPD) have a chronic bronchopulmonary inflammation that is characterized by an increased infiltration of T-lymphocytes, particularly CD8(+), in the airways and lung parenchyma. To investigate the expression of CXCR3 and its ligand interferon-induced protein 10/CXCL10 in COPD, we counted the number of CXCR3(+) cells and analyzed the expression of CXCL10 in the peripheral airways of 19 patients undergoing lung resection for localized pulmonary lesions. We examined lung specimens from seven smokers with fixed airflow limitation (COPD), five smokers with normal lung function, and seven nonsmoking subjects with normal lung function. The number of CXCR3(+) cells was immunohistochemically quantified in the epithelium, in the submucosa, and in the adventitia of peripheral airways. The number of CXCR3(+) cells in the epithelium and submucosa was increased in smokers with COPD as compared with nonsmoking subjects, but not as compared with smokers with normal lung function. Immunoreactivity for the CXCR3-ligand CXCL10 was present in the bronchiolar epithelium of smokers with COPD but not in the bronchiolar epithelium of smoking and nonsmoking control subjects. Most CXCR3(+) cells coexpressed CD8 and produced interferon gamma. These findings suggest that the CXCR3/CXCL10 axis may be involved in the T cell recruitment that occurs in peripheral airways of smokers with COPD and that these T cells may have a type-1 profile.