CX3CR1-positive Cells Research Articles

Caspase-1 in Cx3cr1-expressing cells drives an IL-18-dependent T cell response that promotes parasite control during acute Toxoplasma gondii infection.

Inflammasome activation is a robust innate immune mechanism that promotes inflammatory responses through the release of alarmins and leaderless cytokines, including IL-1α, IL-1β, and IL-18. Various stimuli, including infectious agents and cellular stress, cause inflammasomes to assemble and activate caspase-1. Then, caspase-1 cleaves targets that lead to pore formation and leaderless cytokine activation and release. Toxoplasma gondii has been shown to promote inflammasome formation, but the cell types utilizing caspase-1 and the downstream effects on immunological outcomes during acute in vivo infection have not been explored. Here, using knockout mice, we examine the role of caspase-1 responses during acute T. gondii infection globally and in Cx3cr1-positive populations. We provide in vivo evidence that caspase-1 expression is critical for, IL-18 release, optimal interferon-γ (IFN-γ) production, monocyte and neutrophil recruitment to the site of infection, and parasite control. Specifically, we find that caspase-1 expression in Cx3cr1-positive cells drives IL-18 release, which potentiates CD4+ T cell IFN-γ production and parasite control. Notably, our Cx3cr1-Casp1 knockouts exhibited a selective T cell defect, mirroring the phenotype observed in Il18 knockouts. In further support of this finding, treatment of Cx3cr1-Casp1 knockout mice with recombinant IL-18 restored CD4+ T cell IFN-γ responses and parasite control. Additionally, we show that neutrophil recruitment is dependent on IL-1 receptor accessory protein (IL-1RAP) signaling but is dispensable for parasite control. Overall, these experiments highlight the multifaceted role of caspase-1 in multiple cell populations contributing to specific pathways that collectively contribute to caspase-1 dependent immunity to T. gondii.

An overview of the role of chemokine CX3CL1 (Fractalkine) and CX3C chemokine receptor 1 in systemic sclerosis.

Systemic sclerosis (SSc) is a complex autoimmune disease characterized by fibrosis, vascular damage, and immune dysregulation. Fractalkine or chemokine (C-X3-C motif) ligand 1 (CX3CL1), a chemokine and adhesion molecule, along with its receptor CX3CR1, have been implicated in the inflammatory processes of SSc. CX3CL1 functions as both a chemoattractant and an adhesion molecule, guiding immune cell trafficking. This systematic review examines the role of CX3CL1 and its receptor CX3CR1 in the pathogenesis of SSc, with a focus on pulmonary and vascular complications. A systematic literature search was conducted across databases including PubMed, Scopus, and Web of Science from inception to November 2020. The search focused on studies investigating the CX3CL1/CX3CR1 axis in the context of SSc. The review identified elevated CX3CL1 expression in SSc patients, particularly in the skin and lungs, where CX3CR1 is expressed on infiltrating immune cells. Higher levels of CX3CL1 were correlated with the severity of interstitial lung disease in SSc patients, indicating a potential predictive marker for disease progression. CX3CR1-positive monocytes and NK cells were recruited to inflamed tissues, contributing to fibrosis and tissue damage. Animal studies showed that inhibition of the CX3CL1/CX3CR1 axis reduced fibrosis and improved vascular function. The CX3CL1/CX3CR1 axis plays a key role in immune cell recruitment and fibrosis in SSc. Elevated CX3CL1 levels are associated with lung and vascular complications, making it a potential biomarker for disease progression and a promising therapeutic target.

Conditional Deletion of EphA4 on Cx3cr1-Expressing Microglia Fails to Influence Histopathological Outcome and Blood Brain Barrier Disruption Following Brain Injury.

Erythropoietin-producing human hepatocellular receptors play a major role in central nervous system injury. Preclinical and clinical studies revealed the upregulation of erythropoietin-producing human hepatocellular A4 (EphA4) receptors in the brain after acute traumatic brain injury. We have previously reported that Cx3cr1-expressing cells in the peri-lesion show high levels of EphA4 after the induction of controlled cortical impact (CCI) injury in mice. Cx3cr1 is a fractalkine receptor expressed on both resident microglia and peripheral-derived macrophages. The current study aimed to determine the role of microglial-specific EphA4 in CCI-induced damage. We used Cx3cr1CreER/+ knock-in/knock-out mice, which express EYFP in Cx3cr1-positive cells to establish microglia, EphA4-deficient mice following 1-month tamoxifen injection. Consistent with our previous findings, induction of CCI in wild-type (WT) Cx3cr1CreER/+EphA4+/+ mice increased EphA4 expression on EYFP-positive cells in the peri-lesion. To distinguish between peripheral-derived macrophages and resident microglia, we exploited GFP bone marrow-chimeric mice and found that CCI injury increased EphA4 expression in microglia (TMEM119+GFP–) using immunohistochemistry. Using Cx3cr1CreER/+EphA4 f/f (KO) mice, we observed that the EphA4 mRNA transcript was undetected in microglia but remained present in whole blood when compared to WT. Finally, we found no difference in lesion volume or blood-brain barrier (BBB) disruption between WT and KO mice at 3 dpi. Our data demonstrate a nonessential role of microglial EphA4 in the acute histopathological outcome in response to CCI.

Association of CX3CL1 and CX3CR1 Expression with Liver Fibrosis in a Mouse Model of Schistosomiasis.

Immunopathological mechanisms of schistosomiasis, a debilitating parasitic disease, are still unclear. In this study, we investigated the involvement of CX3C chemokine ligand 1 (CX3CL1) and its sole receptor CX3CR1 in the development of liver fibrosis in schistosomiasis. The animal model of schistosomiasis was established by infection of C57BL/6 mice with Schistosoma japonicum cercariae; mice injected with carbon tetrachloride (CCl4) were used as positive control of liver injury. After 4 and 8 weeks, the degree of liver lesions was assessed by hematoxylin and eosin staining, serum levels of hyaluronic acid (HA) were analyzed by a chemiluminescence immunoassay, liver fibrosis was evaluated by immunohistochemistry analysis of α-smooth muscle actin (α-SMA) expression, and CX3CL1 and CX3CR1 expression in the liver was measured by immunohistochemistry and real-time PCR. The results showed that at 8 weeks after Schistosoma infection, serum HA levels were increased and α-SMA-expressing cells appeared in the liver, indicating fibrogenesis. CX3CL1- and CX3CR1-positive cells were observed in the outer layer of granulomas formed around Schistosoma eggs in liver tissues, which was consistent with the significant upregulation of hepatic CX3CL1 and CX3CR1 mRNA expression at 4 and 8 weeks post-infection. Furthermore, correlation analysis revealed positive association between CX3CL1 and CX3CR1 expression and serum HA levels at 8 weeks post-infection, indicating a link between fibrogenesis and the CX3CL1/CX3CR1 axis in schistosomiasis. In conclusion, our data suggest the involvement of CX3CL1 and CX3CR1 in the progression of liver fibrosis caused by Schistosoma infection.

High Resolution Intravital Imaging of the Renal Immune Response to Injury and Infection in Mice.

We developed an experimental set up that enables longitudinal studies of immune cell behavior in situ in the challenged as well as unchallenged kidney of anesthetized mice over several hours. Using highly controlled vacuum to stabilize the kidney, the superficial renal cortex could continuously be visualized with minimal disruption of the local microenvironment. No visible changes in blood flow or neutrophils and macrophages numbers were observed after several hours of visualizing the unchallenged kidney, indicating a stable tissue preparation without apparent tissue damage. Applying this set up to monocyte/macrophage (CX3CR1GFP/+) reporter mice, we observed the extensive network of stellate-shaped CX3CR1 positive cells (previously identified as renal mononuclear phagocytes). The extended dendrites of the CX3CR1 positive cells were found to bridge multiple capillaries and tubules and were constantly moving. Light induced sterile tissue injury resulted in rapid neutrophil accumulation to the site of injury. Similarly, microinfusion of uropathogenic Escherichia coli into a single nephron induced a rapid and massive recruitment of neutrophils to the site of infection, in addition to active bacterial clearance by neutrophils. In contrast, the kidney resident mononuclear phagocytes were observed to not increase in numbers or migrate toward the site of injury or infection. In conclusion, this model allows for longitudinal imaging of responses to localized kidney challenges in the mouse.

CX3CR1 as a respiratory syncytial virus receptor in pediatric human lung.

Data on the host factors that contribute to infection of young children by respiratory syncytial virus (RSV) are limited. The human chemokine receptor, CX3CR1, has recently been implicated as an RSV receptor. Here we evaluate a role for CX3CR1 in pediatric lung RSV infections. CX3CR1 transcript levels in the upper and lower pediatric airways were assessed. Tissue localization and cell-specific expression was confirmed using in situ hybridization and immunohistochemistry. The role of CX3CR1 in RSV infection was also investigated using a novel physiological model of pediatric epithelial cells. Low levels of CX3CR1 transcript were often, but not always, expressed in both upper (62%) and lower airways (36%) of pediatric subjects. CX3CR1 transcript and protein expression was detected in epithelial cells of normal human pediatric lung tissues. CX3CR1 expression was readily detected on primary cultures of differentiated pediatric/infant human lung epithelial cells. RSV demonstrated preferential infection of CX3CR1-positive cells, and blocking CX3CR1/RSV interaction significantly decreased viral load. CX3CR1 is present in the airways of pediatric subjects where it may serve as a receptor for RSV infection. Furthermore, CX3CR1 appears to play a mechanistic role in mediating viral infection of pediatric airway epithelial cells in vitro.

Diet Modifies Colonic Microbiota and CD4+ T-Cell Repertoire to Induce Flares of Colitis in Mice With Myeloid-Cell Expression of Interleukin 23

Several studies have shown that signaling via the interleukin 23 (IL23) receptor is required for development of colitis. We studied the roles of IL23, dietary factors, alterations to the microbiota, and T cells in the development and progression of colitis in mice. All mice were maintained on laboratory diet 5053, unless otherwise noted. We generated mice that express IL23 in CX3CR1-positive myeloid cells (R23FR mice) upon cyclic administration of tamoxifen dissolved in diet 2019. Diets 2019 and 5053 have minor differences in the overall composition of protein, fat, fiber, minerals, and vitamins. CX3CR1CreER mice (FR mice) were used as controls. Some mice were given antibiotics, and others were raised in a germ-free environment. Intestinal tissues were collected and analyzed by histology and flow cytometry. Feces were collected and analyzed by 16S rDNA sequencing. Feces from C57/Bl6, R23FR, or FR mice were fed to FR and R23FR germ-free mice in microbiota transplant experiments. We also performed studies with R23FR/Rag-/-, R23FR/Mu-/-, and R23FR/Tcrd-/- mice. R23FR mice were given injections of antibodies against CD4 or CD8 to deplete T cells. Mesenteric lymph nodes and large intestine CD4+ cells from R23FR or FR mice in remission from colitis were transferred into Rag-/- mice. CD4+ cells were isolated from donor R23FR mice and recipient Rag-/- mice, and T-cell receptor sequences were determined. Expression of IL23 led to development of a relapsing-remitting colitis that was dependent on the microbiota and CD4+ T cells. The relapses were caused by switching from the conventional diet used in our facility (diet 5053) to the diet 2019 and were not dependent on tamoxifen after the first cycle. The switch in the diet modified the microbiota but did not alter levels of IL23 in intestinal tissues compared with mice that remained on the conventional diet. Mesenteric lymph nodes and large intestine CD4+ cells from R23FR mice in remission, but not from FR mice, induced colitis after transfer into Rag-/- mice, but only when these mice were placed on the diet 2019. The CD4+ T-cell receptor repertoire of Rag-/- mice with colitis (fed the 2019 diet) was less diverse than that from donor mice and Rag-/- mice without colitis (fed the 5053 diet) because of expansion of dominant T-cell clones. We developed mice that express IL23 in CX3CR1-positive myeloid cells (R23FR mice) and found that they are more susceptible to diet-induced colitis than mice that do not express IL23. The R23FR mice have a population of CD4+ T cells that becomes activated in response to dietary changes and alterations to the intestinal microbiota. The results indicate that alterations in the diet, intestinal microbiota, and IL23 signaling can contribute to pathogenesis of inflammatory bowel disease.

Up-regulation of CX3CR1 on tonsillar CD8-positive cells in patients with IgA nephropathy

Although tonsillectomy are used as therapeutic options to prevent chronic renal failure in IgA nephropathy (IgAN) patients, the relationship between IgAN and tonsils is not fully proved by basic research. Recently, circulating CX3CR1-positive cells were reportedly involved in promoting hematuria in patients with IgAN. In this study, we focused on the expression of CX3CR1 in tonsillar mononuclear cells in IgAN patients. Immunohistological analysis revealed greater distribution of CX3CR1-positive cells in the inter-follicular area of tonsils in IgAN patients than in non-IgAN patients. CX3CR1-positive cells were also found in the affected renal glomerulus of IgAN patients. Flow cytometric analysis revealed the expression of CX3CR1 on tonsillar CD8-positive cells to be significantly higher in IgAN patients. CpG-oligodeoxynucleotides enhanced the expression in IgAN patients. The chemotactic response of tonsillar mononuclear cells to fractalkine was significantly higher in IgAN patients. Expression of CX3CR1 on peripheral blood CD8-positive cells in IgAN patients was significantly higher, and decreased after tonsillectomy, along with the disappearance of hematuria. These results suggest that hyper-immune response to microbial DNA enhanced the expression of CX3CR1 on tonsillar CD8-positive cells in IgAN patients, followed by the migration of the cells to renal lesions via blood circulation, resulting in the development of hematuria.

Expression and function of Fractalkine and its receptor in 5XFAD mouse

Objective: To detect the expression and the function of Fractalkine(CX3CL1) and its receptor CX3CR1 in neurodegenerative changes of the retina. Methods: Twelve 5XFAD mouse and twelve wild type mouse were enrolled. Frozen sections were prepared for histopathological tests. Immunofluorescence study for Amyloid β, CX3CL1 and CX3CR1 was carried out. Co-expression study for CX3CR1/CD11b or CX3CR1/CD68 was carried out. Total protein and total mRNA from the eyes of both 5XFAD and wild type mouse were extracted. The expression of mRNA and protein of CX3CL1 and CX3CR1 in the eyes were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blots test, respectively. Results: In wild type mouse, both CX3CL1 and its receptor CX3CR1 can be detected in the retina with low expression, however there is no Amyloid β depositions in retina. In 6 months 5XFAD mouse, an obvious up-regulated Amyloid β expression can be seen in the RPE cells, and the number of both CX3CL1 and CX3CR1 positive cells has up-regulated in the ganglion cell layer of eyes accompanied with the increased expression of their mRNA and protein. The co-expression study showed that CX3CR1 co-expressed with CD11b, but not with CD68. Conclusion: Fractalkine and its receptor play a role in Amyloid β inducing degenerative retinal inflammation in 5XFAD mouse.

The Role Of CD11b+CX3CR1+ Cells In Neovascularization

There is solid evidence that myelo-monocytic cells actively contribute to angiogeneic processes. However, the specific subpopulations of myelo-monocytic cells participating in vascularization and their precise underlying mechanisms have not been well identified. Recently it has been reported that CX3CR1, a receptor for fractalkine (CX3CL1; Fkn), is highly induced at injury sites and CX3CR1 positive cells have an active role for angiogenesis in a mouse wound healing model. Therefore, we evaluated neovascularization activities of CD11b+CX3CR1+ cells during recovery of blood perfusion in a hind-limb ischemic injury mouse model and also identified their activities during the process of angiogenic sprouting in the retina of early postnatal Sprague-Dawley (SD) rat. Ischemic injuries were induced in C57BL/6 mice by femoral artery dissections. Single-cell suspensions were made from ischemic muscles using collagenase and dispase digestion. Isolated CD11b+CX3CR1+ cells and CD11b-CX3CR1- cells from ischemic muscle (5x105 cells/mouse) were injected into ischemic muscles 2 days after femoral artery dissections. Laser Doppler Perfusion (LDP) indexes were assessed as an indicator for blood perfusion. In order to block the functions of CX3CR1 and Fkn, either anti-CX3CR1 or anti-Fkn neutralizing antibody (Ab) were injected into ischemic muscles. To determine whether CX3CR1 has a role in actively growing retinal vessels, intravitreal microinjections were done with anti-CX3CR1 Ab at postnatal 2 days (P2) SD rat. P5 retinas treated with neutralizing anti-CX3CR1 Ab at P2 were used. CD11b+CX3CR1+ cells were positive for CD45 and F4/80, monocyte/macrophage marker. CD11b+CX3CR1+ cells accumulated at the injured muscles in C57BL/6 mice. We tested for contribution of CD11b+CX3CR1+ cells to neovascularization in the injury sites by injecting ischemic muscle-derived CD11b+CX3CR1+ cells or CD11b-CX3CR1- cells into the adductor muscles of C57BL/6 mice. CD11b+CX3CR1+ cells enhanced blood perfusion when compared to CD11b-CX3CR1- cells (78.0±3.2% vs 39.0±0.3% of LDP indexes at day 28, p<0.01). Both neutralizing Abs to CX3CR1 and Fkn significantly reduced blood perfusion resulting in decreased LDP indexes of 25.9±4.9% and 29.4±4.6% compared to those of the non-ischemic limbs (p<0.01). When CX3CR1 function was blocked with anti-CX3CR1-neutralizing Ab, CD11b+CX3CR1+cells were completely eliminated and any vessel-like structures were not observed in the ischemic muscles. By staining of isolectin for P5 retina, well-organized primitive vascular plexus was observed. However, blocking the function of CX3CR1 resulted in formation of peripheral plexus that was more highly interconnected. Additionally, vessels coalesced to form a syncytium in some areas. Thus, CD11b+CX3CR1+ cells accumulate at sites of ischemic injury and enhance recovery of blood flow via new vascular structure formation. Furthermore, CX3CR1 acts to prevent overexuberant vascular growth and promotes formation of well-differentiated vascular network.In summary, CD11b+CX3CR1+ cells are a newly identified subset of myelomonocytic cells for vessel formation, capable of regulating vascular formation. CD11b+CX3CR1+ cells may be an attractive clinical tool to accelerate tissue vasculature development in various ischemic diseases. Disclosures:No relevant conflicts of interest to declare.

Chemokine–Chemokine Receptor CCL2–CCR2 and CX3CL1–CX3CR1 Axis May Play a Role in the Aggravated Inflammation in Primary Biliary Cirrhosis

Senescent cells can alter local tissue environments by secretion of various senescence-associated secretory phenotypes (SASP), such as cytokines and chemokines. Given senescent biliary epithelial cells (BECs) in damaged small bile ducts in primary biliary cirrhosis (PBC) show increased expression of chemokines CCL2 and CX3CL1 as SASP, we further examined an involvement of CCL2/CCR2 and CX3CL1/CX3CR1 systems in the pathogenesis of PBC. We examined immunohistochemically the expression of CCR2, CX3CR1, CCL2 and CX3CL1 in livers taken from the patients with PBC (n=45) and control livers (n=78), such as chronic viral hepatitis (CVH; n=39). CCR2 or CX3CR1-expressing cells were characterized by double immunofluorescence with CD3, CD4, CD8, CD56 or CD68. CCR2 is expressed in round cells, epithelioid cells and dendritic cells and most CCR2-positive cells were CD68-positive. Infiltration of CCR2-positive cells in the intraepithelial layer or around small bile ducts was significantly more extensive in PBC than CVH and normal liver (p<0.05) and was significantly correlated with the expression of CCL2 in BECs (p<0.01). Most CX3CR1-expressing inflammatory cells were CD3-positive T cells (CD8>CD4). Infiltration of CX3CR1-positive cells in the intraepithelial layer and around small bile ducts was significantly more extensive in PBC than control livers (p<0.05) and was significantly correlated with the expression of CX3CL1 in BECs (p<0.05). CCL2 and CX3CL1 produced by senescent BECs may promote infiltration of corresponding CCR2 and CX3CR1-expressing cells and further aggravate inflammation in bile duct lesion in PBC.

Fractalkine receptor is expressed in mature ovarian teratomas and required for epidermal lineage differentiation

BackgroundThe goal of this study was to determine a predominant cell type expressing fractalkine receptor (CX3CR1) in mature ovarian teratomas and to establish functional significance of its expression in cell differentiation.MethodsSpecimens of ovarian teratoma and human fetal tissues were analyzed by immunohistochemistry for CX3CR1expression. Ovarian teratocarcinoma cell line PA-1 was used as a model for cell differentiation.ResultsWe found that the majority of the specimens contained CX3CR1-positive cells of epidermal lineage. Skin keratinocytes in fetal tissues were also CX3CR1- positive. PA-1 cells with downregulated CX3CR1 failed to express a skin keratinocyte marker cytokeratin 14 when cultured on Matrigel in the presence of a morphogen, bone morphogenic protein 4 (BMP-4), as compared to those expressing scrambled shRNA.ConclusionsHere we demonstrate that CX3CR1 is expressed in both normally (fetal skin) and abnormally (ovarian teratoma) differentiated keratinocytes and is required for cell differentiation into epidermal lineage.

Expression of CX3CL1 and its receptor, CX3CR1, in the development of periapical lesions

To investigate the expression of CX3CL1 and its receptor, CX3CR1, in the development of periapical lesions induced in rats and explore the possible role of these substances in the pathogenesis of periapical lesions. Periapical lesions in mandibular first molar teeth were established in 30 rats following pulp exposure to the oral environment. The animals were killed 0, 7, 14, 21, 28 and 42days after lesion induction. The development of periapical lesions was investigated by histological and enzyme histochemical examination. The distributions of CX3CL1 and CX3CR1 in the periapical tissue were examined by immunohistochemistry and immunofluorescence staining. Osteoclasts and CX3CL1-positive cells were counted in each specimen. The data were then analysed by one-way anova using the SPSS 13.0 statistical package. The lesions expanded from days 0 to 14 and stabilized thereafter. Where expansion of the periapical lesion was most evident, numerous CX3CL1-positive cells were observed around the apical foramen and adjacent periapical areas. From days 21 to 42, subjacent connective tissues presented intense CX3CL1 immunostaining in inflammatory cells with different morphologies, such as round, oval and fibroblastic. The number of CX3CL1-positive cells increased from days 7 to 28, but decreased on day 42 post-operation. Double immunofluorescence staining showed CX3CL1- and CX3CR1-positive cells around periapical lesions surrounding the apical foramen. Most CX3CL1-positive cells were mononuclear in nature, which suggests the presence of macrophages, infiltrating neutrophils and lymphocytes, and overlapped with CX3CR1-positive cells. CX3CL1 and CX3CR1 are related to the development of periapical lesions. The chemokine and its receptor may be involved in the progression of tissue destruction, including bone resorption, during periapical inflammation.

Abstract B72: CX3CR1, a fractalkine receptor, can regulate peritoneal dissemination of epithelial ovarian cancer cells

Abstract Epithelial ovarian carcinoma (EOC) is a leading cause of death from gynecologic malignancies mainly due to metastasis. EOC metastases spread by shedding the malignant cells off from the ovarian surface and seeding to organs and tissues in the peritoneal cavity. The current standard of care, a combination of chemotherapy and surgery, fails to keep patients in remission. Thus, mechanistic understanding of the biology of EOC metastasis and identification of novel targetable molecules are crucial for the development of improved therapeutic approaches. Fractalkine receptor (CX3CR1) belongs to a chemokine family of G-protein coupled receptors. CX3CR1 is expressed by EOC cells and promotes cell migration and proliferation. CX3CR1-positive EOC cells have been shown to adhere to the CX3CL1-positive peritoneal mesothelial cells in vitro. Thus, we hypothesized that CX3CL1/CX3CR1 axis could be important for peritoneal dissemination of metastatic EOC. In order to test this hypothesis, we performed short-term ex vivo and in vivo adhesion assays, as well as long-term metastasis formation assay. We found that EOC cells prefer omentum to other peritoneal tissues during the short-term in vivo and ex vivo adhesion. Importantly, reduction of CX3CR1 expression in EOC cells resulted in significantly reduced peritoneal adhesion. Furthermore, reduced adhesion correlated with the ability of EOC cells to form overt metastasis. Microenvironmental milieu has been histologically evaluated for stromal cells in the omentum expressing CX3CL1. Taken together, our data indicate that fractalkine signaling is one of the central regulators of EOC metastasis. Citation Format: Jia Xie, Hilal Gurler, Joanna Burdette, Maria Barbolina. CX3CR1, a fractalkine receptor, can regulate peritoneal dissemination of epithelial ovarian cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B72.

Fractalkine Is Expressed in Early and Advanced Atherosclerotic Lesions and Supports Monocyte Recruitment via CX3CR1

Fractalkine (CX3CL1, FKN) is expressed in the inflamed vascular wall and absence of FKN reduces atherogenesis. Whether FKN is expressed throughout all stages of atherosclerotic disease and whether it directly contributes to monocyte recruitment to atherosclerotic lesions is not known. We collected human atherosclerotic plaque material and blood samples from patients with carotid artery disease undergoing endarterectomy. Plaques were analyzed by immunohistochemistry and qPCR. We found that FKN is expressed at all stages of atherosclerotic lesion formation, and that the number of FKN-expressing cells positively correlates with the number of CX3CR1-positive cells in human carotid artery plaques. In the circulation, soluble FKN levels are significantly elevated in the presence of high-grade (sub-occlusive) stenosis. To determine the role of the FKN-CX3CR1 axis for monocyte adhesion in vivo we then performed intravital videofluorescence microscopy of the carotid artery in ApoE−/− mice. Notably, FKN-CX3CR1 interactions are critical for recruitment of circulating monocytes to the injured atherosclerotic vascular wall. Thus, this chemokine dyad could represent an attractive target for anti-atherosclerotic strategies.

Abstract 5323: Fractalkine receptor CX3CR1 regulates peritoneal dissemination of ovarian carcinoma

Abstract Epithelial ovarian carcinoma (EOC) is the deadliest gynecologic malignancy largely due to the metastatic disease. EOC metastases spread by shedding the malignant cells off of the ovarian surface and seeding tissues and organs of the peritoneal cavity. Currently used therapies, a combination of chemotherapy and surgery, fail to keep most patients in the remission. Mechanistic understanding of the biology of EOC metastasis will facilitate development of new therapeutic approaches. Our previous data demonstrated that fractalkine receptor (CX3CR1) is expressed by EOC cells and can support cell migration and proliferation. Moreover, our previous data suggested that CX3CR1-positive EOC cells can adhere to the CX3CL1-postive peritoneal mesothelial cells in in vitro assay. Thus, we hypothesized that CX3CL1/CX3CR1 axis could be important for peritoneal dissemination of metastatic EOC. To test this hypothesis, we performed short-term ex vivo and in vivo adhesion assay, as well as long-term metastasis formation assay. Expression of CX3CR1 in EOC cells was downregulated in SKOV-3 using shRNA. Both CX3CR1 shRNA-expressing and scrambled shRNA-expressing EOC cells were seeded atop of mouse peritoneum and omentum tissues in the ex vivo assay and allowed to adhere for up to 24 h. CX3CR1 shRNA-expressing and scrambled shRNA-expressing EOC cells were intraperitoneally injected into abdomens of athymic nude mice for 4 h following sacrifice of the animals and examination of the peritoneal organs and tissues for green fluorescence. Data analysis has demonstrated that downregulation of CX3CR1 in EOC cells led to a significant decrease of adhesion in these assays. Furthermore, parental SKOV-3 as well as CX3CR1 shRNA-, scrambled shRNA-, and vector shRNA-expressing cells were intraperitoneally injected into athymic nude mice and allowed to form metastasis for 10 weeks. As a result, we observed very small omental metastatic lesions in three out of six animals injected with CX3CR1 shRNA-expressing cells. In opposite, all of the control groups developed gross intraperitoneal metastasis in many organs and tissues. Our data suggest that CX3CR1 may be one of the key factors that determine peritoneal dissemination of EOC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5323. doi:1538-7445.AM2012-5323

Deficiency of CX3CR1 delays burn wound healing and is associated with reduced myeloid cell recruitment and decreased sub-dermal angiogenesis

The development of a good blood supply is a key step in burn wound healing and appears to be regulated in part by myeloid cells. CX3CR1 positive cells have recently been identified as myeloid cells with a potential role in angiogenesis. The role of functional CX3CR1 system in burn wound healing is not previously investigated. A 2% contact burn was induced in CX3CR1 +/gfp and CX3CR1 gfp/gfp mice. These transgenic mice facilitate the tracking of CX3CR1 cells (CX3CR1 +/gfp) and allow evaluation of the consequence of CX3CR1 functional knockout (CX3CR1 gfp/gfp) on burn wound healing. The progression of wound healing was monitored before tissue was harvested and analyzed at day 6 and day 12 for migration of CX3CR1 cells into burn wound. Deficiency of a functional CX3CR1 system resulted in decreased recruitment of CX3CR1 positive cells into the burn wound associated with decreased myeloid cell recruitment ( p < 0.001) and reduced maintenance of new vessels ( p < 0.001). Burn wound healing was prolonged ( p < 0.05). Our study is the first to establish a role for CX3CR1 in burn wound healing which is associated with sub-dermal angiogenesis. This chemokine receptor pathway may be attractive for therapeutic manipulation as it could increase sub dermal angiogenesis and thereby improve time to healing.

Impact of chemokine receptor CX3CR1 in human renal allograft rejection

Chemokine receptors play pivotal roles for leukocyte recruitment in acute and chronic inflammatory processes. This study was performed to analyze the expression, distribution and cellular localization of CX3CR1 in human renal transplant biopsies and to assess its role as potential diagnostic and prognostic marker. CX3CR1 was prospectively analyzed in 174 renal graft biopsies from patients with normal morphology (n = 76), antibody-mediated acute rejection (n = 6), acute tubulointerstitial rejection (n = 27), acute vascular rejection (n = 31), and with acute tubulus necrosis (n = 34). Double immunofluorescence was additionally performed for CX3CR1 and CD4, CD8, CD20, CD68, and CD209/DC-SIGN. The number of CX3CR1 positive interstitial cells was significantly higher in the biopsies with acute tubulointerstitial and acute vascular rejection as compared to normal renal allograft biopsies. CX3CR1 positive cells were mainly CD68 positive monocytes/macrophages and CD209/DC-SIGN positive dendritic cells. The percentage of the CX3CR1 positive staining area was a predictor for steroid responsiveness and for worse clinical outcome 3 and 12 months after transplantation. CX3CR1 positive macrophages and/or dendritic cells are significantly elevated in acute renal allograft rejection. As CX3CR1 was associated with outcome parameters, it has to be further evaluated as a prognostic marker in human renal transplantation.

Angiotensin II up-regulates CX3CR1 expression in THP-1 monocytes: impact on vascular inflammation and atherogenesis

The potential regulatory effect of angiotensins on circulating mononuclear cell activation and migration has not yet been thoroughly evaluated. Using flow cytometry we assessed the possible effect of angiotensin I and II on the expression of CX3CR1 and a single representative of each major chemokine family (CCR5 and CXCR4) in THP-1 monocytes, Jurcat T lymphocytes and primary monocytes-isolated from healthy donors. Fluorescence intensity and the rate of chemokine-positive cells was measured in naïve cells and cells treated with angiotensin I and II. Neither angiotensin I nor angiotensin II exhibited any effect on fluorescence intensity and the rate of CX3CR1-, CCR5- and CXCR4-positive cells in primary peripheral blood mononuclear cells and Jurkat T cells. However, angiotensin II significantly increased the rate of CX3CR1-positive THP-1 cells. This effect was not attenuated by the pre-incubation of THP-1 cells with the AT-1 receptor blocker losartan, suggesting that this was not an AT-1-mediated effect. Angiotensin I and II had no effect on fluorescence intensity and the rate of CCR5- and CXCR4-positive THP-1 cells. In conclusion, angiotensin II increases the rate of CX3CR1-positive THP-1 cells. By extrapolating this in vitro observation to disease mechanisms, we speculate that angiotensin II induces up-regulation of CX3CR1 and promotes firm adhesion of circulation CX3CR1-positive monocytes on CX3CL1 expressing endothelial cells inducing vascular inflammation and atherogenesis.

Immunohistochemical detection of CCR2 and CX3CR1 in sepsis-induced lung injury

Sepsis is a systemic inflammatory disease with high mortality. In the present study, we immunohistochemically examined CCR2 and CX3CR1 expression in sepsis-induced lung injury, and discussed its availability for the postmortem diagnosis of sepsis. Lung samples were obtained from different lung lobes of nine sepsis and eight control cases with postmortem intervals between 12 and 48 h. Immunohistochemically, mononuclear cells recruited into the lungs expressed CCR2 and CX3CR1 in both sepsis and non-septic groups. In double-color immunofluorescence analysis, CCR2- or CX3CR1-positive cells could be identified as CD68-positive macrophages. Moreover, most of CD68-positive macrophages expressed both CCR2 and CX3CR1. Morphometrically, the average of CCR2- and CX3CR1-positive macrophages was significantly increased in sepsis group, compared with control group (sepsis vs. control: 41.6 ± 1.3% vs. 8.0 ± 0.4% in CCR2; 36.2 ± 1.3% vs. 9.2 ± 0.4% in CX3CR1). These observations implied that CCR2- or CX3CR1-positive macrophages were recruited into the lungs under several pathological conditions. In particular, their recruitment might be more evident in sepsis. Moreover, from the forensic aspects, immunohistochemical detection of CCR2 and CX3CR1 expression in the lungs can be considered as valuable diagnostic tools for the postmortem diagnosis of sepsis.