Fractalkine-CX3CR1 axis regulates tumor cell cycle and deteriorates prognosis after radical resection for hepatocellular carcinoma

BACKGROUND AND OBJECTIVES: Fractalkine is the only CX3C chemokine, and its receptor, CX3CR1, is expressed on NK cells, CD8+ T cells, monocytes, and dendritic cells (DC). Although studies have reported that fractalkine regulates the host immune response, the roles of the fractalkine-CX3CR1 axis in tumor biology and the clinical results of hepatocellular carcinoma (HCC) remain unknown. METHODS: Fractalkine and CX3CR1 expression in HCC were evaluated and compared with the clinicopathologic features, including tumor progression determined by proliferating cell nuclear antigen (PCNA) antibody and patient prognosis after surgery. RESULTS: Tumors with high expression of both fractalkine and CX3CR1 had significantly fewer intra- and extrahepatic recurrences, a low PCNA labeling index (PCNALI), and different histological grades. Patients with tumors that expressed both had a significantly better prognosis in terms of disease-free (DFS) and overall survival (OAS), and this finding was identified as one of the independent prognostic factors in the multivariate analysis. CONCLUSIONS: Our results suggest that the fractalkine-CX3CR1 axis plays a pivotal role in the prognosis of patients with HCC, which might arise from the known modulation of the host immune response, and that of the cell cycle in HCC.     

The impact of chemokine receptor CX3CR1 deficiency during respiratory infections with Mycobacterium tuberculosis or Francisella tularensis

Recruitment of immune cells to infection sites is a critical component of the host response to pathogens. This process is facilitated partly through interactions of chemokines with cognate receptors. Here, we examine the importance of fractalkine (CX3CL1) receptor, CX3CR1, which regulates function and trafficking of macrophages and dendritic cells, in the host''s ability to control respiratory infections with Mycobacterium tuberculosis or Francisella tularensis. Following low-dose aerosol challenge with M. tuberculosis, CX3CR1^−/− mice were no more susceptible to infection than wild-type C57BL/6 mice as measured by organ burden and survival time. Similarly, following inhalation of F. tularensis, CX3CR1^−/− mice displayed similar organ burdens to wild-type mice. CX3CR1^−/− mice had increased recruitment of monocytes and neutrophils in the lung; however, this did not result in increased abundance of infected monocytes or neutrophils. We conclude that CX3CR1-deficiency affects immune-cell recruitment; however, loss of CX3CR1 alone does not render the host more susceptible to M. tuberculosis or F. tularensis.     

MiR-195 inhibits proliferation and growth and induces apoptosis of endometrial stromal cells by targeting FKN

MiR-195, which exhibits a proliferation-inhibiting role in different tumors, has been reported to be down-regulated in the ectopic endometrium. The aim of this study was to determine the impact of miR-195 on the biological characteristic of the endometrial stromal cells (ESCs). MiR-195 has been presumed to target the 3’-untranslated regions (3’-UTR) of Fractalkine (FKN), which also plays important roles in endometriosis. Fluorescence reporter assays showed that miR-195 effectively binds to the 3’-UTR of FKN. The normal ESCs showed a significant higher miR-195 expression than that of eutopic and ectopic ESCs associated with endometriosis, while the FKN expression showed opposite results. MiR-195 mimics inhibited proliferation and growth and induced apoptosis of eutopic ESCs, and these effects were abolished by FKN-siRNA. miR-195 could decrease the expression of survivin, matrix metalloproteinase-9 (MMP9) and up-regulate the expression of CD82, tissue inhibitor of metalloproteinase 1 (TIMP1) and TIMP2 of eutopic ESCs by targeting FKN. Our study has demonstrated for the first time that miR-195 plays important roles in regulating the functions of ESCs through targeting FKN. The information may be useful for developing a new therapeutic strategy for endometriosis.     

Pharmacological inhibition of the chemokine receptor CX3CR1 attenuates disease in a chronic-relapsing rat model for multiple sclerosis

One hallmark of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) is infiltration of leukocytes into the CNS, where chemokines and their receptors play a major mediatory role. CX3CR1 is a chemokine receptor involved in leukocyte adhesion and migration and hence a mediator of immune defense reactions. The role of CX3CR1 in MS and EAE pathogenesis however remains to be fully assessed. Here, we demonstrate CX3CR1 mRNA expression on inflammatory cells within active plaque areas in MS brain autopsies. To test whether blocking CNS infiltration of peripheral leukocytes expressing CX3CR1 would be a suitable treatment strategy for MS, we developed a selective, high-affinity inhibitor of CX3CR1 (AZD8797). The compound is active outside the CNS and AZD8797 treatment in Dark Agouti rats with myelin oligodendrocyte glycoprotein-induced EAE resulted in reduced paralysis, CNS pathology, and incidence of relapses. The compound is effective when starting treatment before onset, as well as after the acute phase. This treatment strategy is mechanistically similar to, but more restricted than, current very late antigen-4–directed approaches that have significant side effects. We suggest that blocking CX3CR1 on leukocytes outside the CNS could be an alternative approach to treat MS.Multiple sclerosis (MS) is a chronic inflammatory, demyelinating and degenerative disease of the central nervous system (CNS). It was already discovered in the early 1900s that a similar disease could be induced in different animal species by injection of spinal cord extracts or myelin-derived proteins (1–3). This group of animal models for MS, called experimental autoimmune encephalomyelitis (EAE), has provided an experimental platform for building an extensive understanding of the pathology of MS, as well as discovering strategies for intervention of the disease. A typical feature of the pathogenesis in both MS and EAE is the infiltration of leukocytes from the blood stream into the CNS (3). Leukocyte adhesion and extravasation includes several well defined steps and various adhesion molecules, chemokines and their receptors are important mediators for this process. In line with this, the recently developed therapeutic drugs natalizumab and fingolimod, which broadly target leukocyte migration to the brain, exhibit efficacy in EAE models (4, 5), and they are now established therapies in MS (6).Natalizumab, blocking the interaction between very late antigen-4 (VLA-4) and CD106 (VCAM-1), is an effective treatment both on clinical endpoints and MRI biomarkers (7). Fingolimod, the first oral drug for relapsing remitting MS (RRMS), acts on S1P receptors preventing lymphocytes from moving out of lymphoid tissue (8). Natalizumab is only approved as a second-line monotherapy in RRMS or in patients with very active disease, because it carries increased risk of developing the often fatal progressive multifocal leukoencephalopathy (7, 9). Treatment with fingolimod is associated with side effects such as signs of immune suppression, including increased frequency of infections (8).Considering the pronounced presence of inflammatory cells in the brain of MS patients, the significant correlation between inflammation and axonal injury (10) and the efficiency of treatments that broadly block infiltration of immune cells, a similar but more restricted therapeutic approach is appealing. Chemokines are synthesized and released at sites of inflammation, where they act on specific receptors expressed by immune cells to mediate directed cell migration in synergy with adhesion molecules, such as VLA-4, from the blood stream and into the sites of inflammation. CX3CR1 is a unique member of the chemokine receptor family (11) and binds with high affinity to its ligand CX3CL1 [fractalkine (FKN)]. FKN is produced in a membrane bound form but can also be released following proteolytic cleavage, making it important for mediating both adhesion and migration of CX3CR1-expressing cells. In contrast to VLA-4, which is broadly expressed on most leukocytes except neutrophils (7, 12), the expression of CX3CR1 is restricted to subpopulations of monocytes, T lymphocytes, and natural killer (NK) cells (13–16). We have previously demonstrated intense accumulation of CX3CR1-expressing microglia/macrophages within inflammatory foci in the spinal cord of Dark Agouti (DA) rats with EAE induced by myelin oligodendrocyte glycoprotein (MOG) (17) and formed the hypothesis that CX3CR1 would be an attractive therapeutic target for treating MS.To test the hypothesis, we have developed a selective, high-affinity small-molecule inhibitor of CX3CR1 (AZD8797) (18). This molecule has the potential to be administered as an oral drug in humans. However, because of the decrease in potency to rat CX3CR1 and a modest oral bioavailability in rats (39%), we have chosen continuous s.c. dosing for the proof-of-concept studies in rats. The MOG_1-125–induced EAE model in DA rats was used, because it exhibits pathology very similar to MS with infiltration of inflammatory cells, demyelination, and axonal degeneration in the CNS, as well as a relapsing remitting disease course (19, 20). To further mimic the human treatment situation, we have not only treated rats before the onset of paralysis but also initiated treatment during ongoing disease. We present efficacy (reduced paralysis) versus exposure data, analysis of CNS pathology, and measurements of functional inhibition of CX3CR1. These data, in combination with an analysis for CX3CR1 expression within MS brain autopsy samples, clearly demonstrate the potential of CX3CR1 inhibition as an alternative and unique approach for treating MS.     

外周血单核细胞亚群及其趋化因子在急性冠脉综合征早期的表达特点

 目的  探讨急性冠脉综合征(acute coronary syndrome, ACS)早期单核细胞亚群及其趋化因子即单核细胞趋化蛋白-1 (monocyte chemoattractant protein, MCP-1)和不规则趋化因子(fractalkine, FKN)的表达特点,并分析其相关性。方法  选取我院2016年9月至12月以胸痛症状入院拟行冠脉造影术(coronary angiography, CAG)的患者。手术当天术前抽取静脉血,采用流式细胞术检测外周血单核细胞(monocyte, Mon) 3个亚型的含量及其比例,依据分化抗原-14 (cluster differentiation-14, CD-14)和CD16表达分为3个亚型即CD14+CD16-Mon (Mon1)、CD14+CD16+Mon (Mon2)和CD14-CD16+Mon (Mon3);手术当天术前及术后一天抽取静脉血,ELISA检测Mon1的趋化因子MCP-1和Mon3的趋化因子FKN水平,比较不同组MCP-1-Mon1和FKN-Mon3水平变化,并分析其相关性。结果共入选70例患者,结合其临床症状、心肌标志物、心电图、CAG检查结果进行诊断分组:急性心肌梗死(acute myocardium infarction, AMI)组患者30例、不稳定性心绞痛(unstable angina pectoris, UAP)组患者25例、CAG完全正常者(对照组) 15例。流式细胞术结果显示AMI组Mon1所占比例高于UAP组和正常对照组(P<0.05),Mon3在各组间尚无差异。AMI组患者外周血Mon3/Mon1比值低于对照组(P<0.05)。AMI组和UAP组患者FKN、MCP-1和红细胞分布宽度均高于对照组,并且FKN和Mon3具有强相关性(P<0.05;R=0.650 2)。结论  单核细胞亚群(Mon1和Mon3)在ACS早期水平增高,并伴有其负责招募的趋化因子(MCP-1和FKN)增加,且FKN和Mon3具有强相关性,提示MCP-1 Mon1和FKN-Mon3两条通路可能参与患者ACS早期病理生理过程。     

MFG-E8 Regulates Microglial Phagocytosis of Apoptotic Neurons

Phagocytosis is an essential mechanism for clearance of pathogens, dying cells, and other unwanted debris in order to maintain tissue health in the body. Macrophages execute this process in the peripheral immune system but in the brain microglia act as resident macrophages to accomplish this function. In the peripheral immune system, macrophages secrete Milk Fat Globule Factor-E8 (MFG-E8) that recognizes phosphatidylserine “eat me” signals expressed on the surface of apoptotic cells. MFG-E8 then acts as a tether to attach the apoptotic cell to the macrophage and trigger a signaling cascade that stimulates the phagocyte development, allowing the macrophage to engulf the dying cell. When this process becomes disrupted, inflammation and autoimmunity can result. MFG-E8 resides in the brain as well as in the periphery, and microglia express MFG-E8. However, the function of MFG-E8 in the brain has not been elucidated. We measured MFG-E8 production in the BV-2 microglial cell line and the role of this protein in the recognition and engulfment of apoptotic SY5Y neuroblastoma cells. BV-2 cells produced and released MFG-E8, which apoptotic SY5Y cells and the chemokine fractalkine further stimulated. Furthermore, MFG-E8 increased phagocytosis of apoptotic SY5Y cells, and a dominant negative form of MFG-E8 inhibited phagocytosis by BV-2 cells. Finally, brain MFG-E8 levels were altered in a mouse model of Alzheimer’s disease. Our data suggest that MFG-E8 acts in the brain via microglia to aid in clearance of apoptotic neurons, and we hypothesize that a dysregulation of this process may be involved in neurodegenerative disease.