Fatty acid amide hydrolase deficiency enhances intraplaque neutrophil recruitment in atherosclerotic mice.

OBJECTIVE: Endocannabinoid levels are elevated in human and mouse atherosclerosis, but their causal role is not well understood. Therefore, we studied the involvement of fatty acid amide hydrolase (FAAH) deficiency, the major enzyme responsible for endocannabinoid anandamide degradation, in atherosclerotic plaque vulnerability. METHODS AND RESULTS: We assessed atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) and ApoE(-/-)FAAH(-/-) mice. Before and after 5, 10, and 15 weeks on high-cholesterol diet, we analyzed weight, serum cholesterol, and endocannabinoid levels, and atherosclerotic lesions in thoracoabdominal aortas and aortic sinuses. Serum levels of FAAH substrates anandamide, palmitoylethanolamide (PEA), and oleoylethanolamide (OEA) were 1.4- to 2-fold higher in case of FAAH deficiency. ApoE(-/-)FAAH(-/-) mice had smaller plaques with significantly lower content of smooth muscle cells, increased matrix metalloproteinase-9 expression, and neutrophil content. Circulating and bone marrow neutrophil counts were comparable between both genotypes, whereas CXC ligand1 levels were locally elevated in aortas of FAAH-deficient mice. We observed enhanced recruitment of neutrophils, but not monocytes, to large arteries of ApoE(-/-) mice treated with FAAH inhibitor URB597. Spleens of ApoE(-/-)FAAH(-/-) mice had reduced CD4+FoxP3+regulatory T-cell content, and in vitro stimulation of splenocytes revealed significantly elevated interferon-γ and tumor necrosis factor-α production in case of FAAH deficiency. CONCLUSIONS: Increased anandamide and related FAAH substrate levels are associated with the development of smaller atherosclerotic plaques with high neutrophil content, accompanied by an increased proinflammatory immune response.

NLRP12 is a neutrophil-specific, negative regulator of in vitro cell migration but does not modulate LPS- or infection-induced NF-κB or ERK signalling.

NOD-like receptors (NLR) are a family of cytosolic pattern recognition receptors that include many key drivers of innate immune responses. NLRP12 is an emerging member of the NLR family that is closely related to the well-known inflammasome scaffold, NLRP3. Since its discovery, various functions have been proposed for NLRP12, including the positive regulation of dendritic cell (DC) and neutrophil migration and the inhibition of NF-κB and ERK signalling in DC and macrophages. We show here that NLRP12 is poorly expressed in murine macrophages and DC, but is strongly expressed in neutrophils. Using myeloid cells from WT and Nlrp12(-/)(-) mice, we show that, contrary to previous reports, NLRP12 does not suppress LPS- or infection-induced NF-κB or ERK activation in myeloid cells, and is not required for DC migration in vitro. Surprisingly, we found that Nlrp12 deficiency caused increased rather than decreased neutrophil migration towards the chemokine CXCL1 and the neutrophil parasite Leishmania major, revealing NLRP12 as a negative regulator of directed neutrophil migration under these conditions.

Neuroprotection by inhibiting the c-Jun N-terminal kinase pathway after cerebral ischemia occurs independently of interleukin-6 and keratinocyte-derived chemokine (KC/CXCL1) secretion.

BACKGROUND: Cerebral ischemia is associated with the activation of glial cells, infiltration of leukocytes and an increase in inflammatory mediators in the ischemic brain and systemic circulation. How this inflammatory response influences lesion size and neurological outcome remains unclear. D-JNKI1, an inhibitor of the c-Jun N-terminal kinase pathway, is strongly neuroprotective in animal models of stroke. Intriguingly, the protection mediated by D-JNKI1 is high even with intravenous administration at very low doses with undetectable drug levels in the brain, pointing to a systemic mode of action, perhaps on inflammation. FINDINGS: We evaluated whether D-JNKI1, administered intravenously 3 h after the onset of middle cerebral artery occlusion (MCAO), modulates secretion of the inflammatory mediators interleukin-6 and keratinocyte-derived chemokine in the plasma and from the spleen and brain at several time points after MCAO. We found an early release of both mediators in the systemic circulation followed by an increase in the brain and went on to show a later systemic increase in vehicle-treated mice. Release of interleukin-6 and keratinocyte-derived chemokine from the spleen of mice with MCAO was not significantly different from sham mice. Interestingly, the secretion of these inflammatory mediators was not altered in the systemic circulation or brain after successful neuroprotection with D-JNKI1. CONCLUSIONS: We demonstrate that neuroprotection with D-JNKI1 after experimental cerebral ischemia is independent of systemic and brain release of interleukin-6 and keratinocyte-derived chemokine. Furthermore, our findings suggest that the early systemic release of interleukin-6 and keratinocyte-derived chemokine may not necessarily predict an unfavorable outcome in this model.

Lymphotoxin beta receptor signaling induces the chemokine CCL20 in intestinal epithelium.

BACKGROUND & AIMS: The follicle-associated epithelium (FAE) that overlies Peyer's patches (PPs) exhibits distinct features compared with the adjacent villus epithelium. Besides the presence of antigen-sampling membranous M cells and the down-regulation of digestive functions, it constitutively expresses the chemokine CCL20. The mechanisms that induce FAE differentiation and CCL20 expression are poorly understood. The aim of this work was to test whether lymphotoxin beta receptor signaling (LTbetaR), which plays a central role in PPs' organogenesis, mediates CCL20 gene expression in intestinal epithelial cells. METHODS: CCL20, lymphotoxin beta (LTbeta) and LTbetaR expression were monitored during embryonic development by in situ hybridization of mouse intestine. The human intestinal epithelial cell line T84 was used to study CCL20 expression following LTalpha(1)/beta(2) stimulation. In vivo CCL20 expression following agonistic anti-LTbetaR antibody treatment was studied by laser microdissection and quantitative RT-PCR. RESULTS: CCL20 was expressed in the FAE before birth at the time when the first hematopoietic CD4(+)CD3(-) appeared in the PP anlage. LTbetaR was expressed in the epithelium during PP organogenesis, making it a putative target for LTalpha(1)beta(2)signals. In vitro, CCL20 was induced in T84 cells upon LTbetaR signaling, either using an agonistic ligand or anti-LTbeta receptor agonistic antibody. LTalpha(1)beta(2)-induced CCL20 expression was found to be NF-kappaB dependent. LTbetaR signaling up-regulated CCL20 expression in the small intestinal epithelium in vivo. CONCLUSIONS: Our results show that LTbetaR signaling induces CCL20 expression in intestinal epithelial cells, suggesting that this pathway triggers constitutive production of CCL20 in the FAE.

Role of the chemokine receptor CX3CR1 in the mobilization of phagocytic retinal microglial cells.

We recently showed that subretinal CX3CR1-dependent microglial cell (MC) accumulation may lead to age-related macular degeneration. The fate of MC after engulfing retinal debris is poorly understood. Severe photoreceptor degeneration was observed 40days after exposure to bright light in CX3CR1-deficient but not control mice, and more MCs accumulated in the subretinal space of the former than the latter. To study the fate of subretinal MCs in CX3CR1 competent animals, we used a dystrophic rat model in which abundant subretinal MC accumulation is observed secondary to retinal degeneration. In dystrophic rats, MCs containing rhodopsin or rod outer segment (ROS) debris were found outside the outer retina at sites suggesting choroidal and ciliary egress. In conclusion, our data indicate that MC accumulation at injury sites is independent of CX3CR1 and precedes photoreceptor degeneration. The ectopic presence of rhodopsin-positive MCs suggests that CX3CR1 participates in MC egress from the outer retina.

The chemokine CCL2 protects against methylmercury neurotoxicity.

Industrial pollution due to heavy metals such as mercury is a major concern for the environment and public health. Mercury, in particular methylmercury (MeHg), primarily affects brain development and neuronal activity, resulting in neurotoxic effects. Because chemokines can modulate brain functions and are involved in neuroinflammatory and neurodegenerative diseases, we tested the possibility that the neurotoxic effect of MeHg may interfere with the chemokine CCL2. We have used an original protocol in young mice using a MeHg-contaminated fish-based diet for 3 months relevant to human MeHg contamination. We observed that MeHg induced in the mice cortex a decrease in CCL2 concentrations, neuronal cell death, and microglial activation. Knock-out (KO) CCL2 mice fed with a vegetal control food already presented a decrease in cortical neuronal cell density in comparison with wild-type animals under similar diet conditions, suggesting that the presence of CCL2 is required for normal neuronal survival. Moreover, KO CCL2 mice showed a pronounced neuronal cell death in response to MeHg. Using in vitro experiments on pure rat cortical neurons in culture, we observed by blockade of the CCL2/CCR2 neurotransmission an increased neuronal cell death in response to MeHg neurotoxicity. Furthermore, we showed that sod genes are upregulated in brain of wild-type mice fed with MeHg in contrast to KO CCL2 mice and that CCL2 can blunt in vitro the decrease in glutathione levels induced by MeHg. These original findings demonstrate that CCL2 may act as a neuroprotective alarm system in brain deficits due to MeHg intoxication.

Identification of peptide ligands to the chemokine receptor CCR5 and their maturation by gene shuffling.

The determination of protein-protein interactions and their role in diverse pathophysiological processes is a promising approach to the identification of molecules of therapeutic potential. This paper describes the identification of peptidic CCR5 receptor ligands as potential drug leads against HIV-1 infection using in vitro evolution based on phage display. A phage-displayed peptide library was used to select for anti-CCR5 peptide. Further in vitro evolution of the peptide by exon shuffling was performed to identify peptides with optimized characteristics for CCR5 receptor. This peptide inhibited HIV coreceptor activity in a cell fusion assay with an IC50 of 5 microM. It did not exhibit either agonistic or antagonistic activity on CCR5 in the concentration range used. To our knowledge, this is a first report that describes the identification of peptide ligands specific to the CCR5 receptor from a phage-displayed library and the maturation of the selected peptide sequence by gene shuffling.

The chemokine receptor CXCR4 strongly promotes neuroblastoma primary tumour and metastatic growth, but not invasion.

Neuroblastoma (NB) is a heterogeneous, and particularly malignant childhood neoplasm in its higher stages, with a propensity to form metastasis in selected organs, in particular liver and bone marrow, and for which there is still no efficient treatment available beyond surgery. Recent evidence indicates that the CXCR4/CXCL12 chemokine/receptor axis may be involved in promoting NB invasion and metastasis. In this study, we explored the potential role of CXCR4 in the malignant behaviour of NB, using a combination of in vitro functional analyses and in vivo growth and metastasis assessment in an orthotopic NB mouse model. We show here that CXCR4 overexpression in non-metastatic CXCR4-negative NB cells IGR-NB8 and in moderately metastatic, CXCR4 expressing NB cells IGR-N91, strongly increased tumour growth of primary tumours and liver metastases, without altering the frequency or the pattern of metastasis. Moreover shRNA-mediated knock-down experiments confirmed our observations by showing that silencing CXCR4 in NB cells impairs in vitro and almost abrogates in vivo growth. High levels of CXCL12 were detected in the mouse adrenal gland (the primary tumour site), and in the liver suggesting a paracrine effect of host-derived CXCL12 on NB growth. In conclusion, this study reveals a yet unreported NB-specific predominant growth and survival-promoting role of CXCR4, which warrants a critical reconsideration of the role of CXCR4 in the malignant behaviour of NB and other cancers.

Complex interplay between LPS and IL-10 in dendritic cells: uncoupling of inflammatory chemokine receptors and positive effects on B cell chemokines

Abstract: Protective immune responses against pathogen invasion and transformed cells requires the coordinated action of distinct leukocyte subsets and soluble factors, overall termed immunological network. Among antigen-presenting cells (APC), a crucial role is played by dendritic cells (DC), which initiate, amplify and determine the outcome of the immune response. Micro-environmental conditions profoundly influence DC in such ways that the resulting immune response ranges from successful immune stimulation to abortive response or immune suppression. For instance, the presence in the milieu of anti-inflammatory cytokine interleukin-10 (IL-10) reverts most of the effects mediated on DC by even strong pro-inflammatory agents such as bacterial Lipopolysaccharide (LPS), in terms of differentiation, activation and functions. In an environment containing both LPS and IL-10, uncoupling of receptors for inflammatory chemokines already occurs after a few hours and in a reversible manner on DC, allowing scavenging of chemokines and, consequently, attenuation of the inflammatory process which could be deleterious to the organism. By studying the effects on DC of concomitant stimulation by LPS and IL-10 from the gene expression point of view, we were able to define four distinct transcriptional programs: A. the inhibition of inflammation and immunity, B. the regulation of tissue remodeling, C. the tuning of cytokine/growth factor receptors and G protein-coupled receptors, D. the stimulation of B cell function and lymphoid tissue neogenesis. Among the latter genes, we further demonstrated that IL-10 synergizes with Toll-like receptor ligands for the production of functionally active B cell attracting chemokine CXCL13. Our data provide evidence that the combined exposure of APC to LPS and IL-10, via the production of CXCL13, involves humoral immunity by attracting antibody-producing cells. It is well known that the persistent release of CXCL13 leads to the development of ectopic lymphoid tissue aggregates and production of high levels of antibodies, thus favoring the induction of auto-immunity. Our findings suggest that the IL-10 produced in chronic inflammatory conditions may promote lymphoid tissue neogenesis through increased release of CXCL13. IL-10 is an anti-inflammatory cytokine inhibiting cellular-mediated TH 1-polarized immune responses. In this study we demonstrate that IL- 10 strongly supports the development of humoral immunity. IL-10 and CXCL13 can thus be targets for specific therapies in auto-immune diseases.

Interstitial dendritic cell guidance by haptotactic chemokine gradients.

Directional guidance of cells via gradients of chemokines is considered crucial for embryonic development, cancer dissemination, and immune responses. Nevertheless, the concept still lacks direct experimental confirmation in vivo. Here, we identify endogenous gradients of the chemokine CCL21 within mouse skin and show that they guide dendritic cells toward lymphatic vessels. Quantitative imaging reveals depots of CCL21 within lymphatic endothelial cells and steeply decaying gradients within the perilymphatic interstitium. These gradients match the migratory patterns of the dendritic cells, which directionally approach vessels from a distance of up to 90-micrometers. Interstitial CCL21 is immobilized to heparan sulfates, and its experimental delocalization or swamping the endogenous gradients abolishes directed migration. These findings functionally establish the concept of haptotaxis, directed migration along immobilized gradients, in tissues.

Expression of Chemokine Receptors in Uveal Melanoma

Purpose:Chemokine receptors are transmembrane G coupled proteins that might be involved in the directional metastatic migration of tumor cells to specific organs. CXCR4 and CCR7 have been implicated in the selective metastasis of cutaneous melanoma cells to lung and lymph node, respectively. CCR6 is expressed in metastases from colon, ovarian and thyroid carcinomas to the liver where its ligand, CCL20, is constitutively expressed. As uveal melanomas frequently metastasize to the liver, we hypothesized that specific chemokine receptors and their respective ligands might be involved in metastasis of uveal melanoma to the liver. Methods:Tissue microarrays were constructed using 100 non irradiated primary uveal melanomas and 84 liver metastases, as well as 12 non liver metastases, collected from the files of Jules Gonin Eye Hospital and Pathology Institute, University of Lausanne. Immunohistochemistry was performed using anti-human CXCR4, SDF1, CCR7, CCL21 and CCR6 antibodies. Results:CXCR4 expression was detected in 36% of primary uveal melanomas and in 63% of liver metastases but no expression was found in metastases to other organs, except for one pancreatic metastasis. SDF1 expression was detected in 3% of primary uveal melanomas and in 26% of liver metastases, as well as in pancreas, lymph node and breast metastases. CCR6 expression was observed in the majority of primary uveal melanomas and liver metastases (73 and 88%, respectively). In addition, CCR6 was also detected in 9 metastases to other organs (pancreas, thyroid, lymph node, skin and breast). CCR7 and CCL21 were neither detected in primary uveal melanoma, nor in the metastases. Conclusions:Chemokine receptors CCR6 and CXCR4 are expressed in a large number of primary uveal melanomas and in uveal melanoma metastases to the liver. CCR6 is also expressed in a small number of metastases to other organs. These findings form the basis for further studies on the potential involvement of CXCR4 and CCR6 in the selective metastasis of uveal melanoma to the liver.

Chemokine receptor trio: CXCR3, CXCR4 and CXCR7 crosstalk via CXCL11 and CXCL12.

Although chemokines are well established to function in immunity and endothelial cell activation and proliferation, a rapidly growing literature suggests that CXC Chemokine receptors CXCR3, CXCR4 and CXCR7 are critical in the development and progression of solid tumors. The effect of these chemokine receptors in tumorigenesis is mediated via interactions with shared ligands I-TAC (CXCL11) and SDF-1 (CXCL12). Over the last decade, CXCR4 has been extensively reported to be overexpressed in most human solid tumors and has earned considerable attention toward elucidating its role in cancer metastasis. To enrich the existing armamentarium of anti-cancerous agents, many inhibitors of CXCL12-CXCR4 axis have emerged as additional or alternative agents for neo-adjuvant treatments and even many of them are in preclinical and clinical stages of their development. However, the discovery of CXCR7 as another receptor for CXCL12 with rather high binding affinity and recent reports about its involvement in cancer progression, has questioned the potential of "selective blockade" of CXCR4 as cancer chemotherapeutics. Interestingly, CXCR7 can also bind another chemokine CXCL11, which is an established ligand for CXCR3. Recent reports have documented that CXCR3 and their ligands are overexpressed in different solid tumors and regulate tumor growth and metastasis. Therefore, it is important to consider the interactions and crosstalk between these three chemokine receptors and their ligand mediated signaling cascades for the development of effective anti-cancer therapies. Emerging evidence also indicates that these receptors are differentially expressed in tumor endothelial cells as well as in cancer stem cells, suggesting their direct role in regulating tumor angiogenesis and metastasis. In this review, we will focus on the signals mediated by this receptor trio via their shared ligands and their role in tumor growth and progression.

The human cytomegalovirus-encoded chemokine receptor US28 induces caspase-dependent apoptosis.

Viral subversion of apoptosis regulation plays an important role in the outcome of host/virus interactions. Although human cytomegalovirus (HCMV) encodes several immediate early (IE) antiapoptotic proteins (IE1, IE2, vMIA and vICA), no proapoptotic HCMV protein has yet been identified. Here we show that US28, a functional IE HCMV-encoded chemokine receptor, which may be involved in both viral dissemination and immune evasion, constitutively induces apoptosis in several cell types. In contrast, none of nine human cellular chemokine receptors, belonging to three different subfamilies, induced any significant level of apoptosis. US28-induced cell death involves caspase 10 and caspase 8 activation, but does not depend on the engagement of cell-surface death receptors of the tumour necrosis factor receptor/CD95 family. US28 cell-death induction is prevented by coexpression of C-FLIP, a protein that inhibits Fas-associated death domain protein (FADD)-mediated activation of caspase 10 and caspase 8, and by coexpression of the HCMV antiapoptotic protein IE1. The use of US28 mutants indicated that the DRY sequence of its third transmenbrane domain, required for constitutive G-protein signalling, and the US28 intracellular terminal domain required for constitutive US28 endocytosis, are each partially required for cell-death induction. Thus, in HCMV-infected cells, US28 may function either as a chemokine receptor, a phospholipase C activator, or a proapoptotic factor, depending on expression levels of HCMV and/or cellular antiapoptotic proteins.

Critical role for the chemokine receptor CXCR6 in NK cell-mediated antigen-specific memory of haptens and viruses.

Hepatic natural killer (NK) cells mediate antigen-specific contact hypersensitivity (CHS) in mice deficient in T cells and B cells. We report here that hepatic NK cells, but not splenic or naive NK cells, also developed specific memory of vaccines containing antigens from influenza, vesicular stomatitis virus (VSV) or human immunodeficiency virus type 1 (HIV-1). Adoptive transfer of virus-sensitized NK cells into naive recipient mice enhanced the survival of the mice after lethal challenge with the sensitizing virus but not after lethal challenge with a different virus. NK cell memory of haptens and viruses depended on CXCR6, a chemokine receptor on hepatic NK cells that was required for the persistence of memory NK cells but not for antigen recognition. Thus, hepatic NK cells can develop adaptive immunity to structurally diverse antigens, an activity that requires NK cell-expressed CXCR6.

The Ovarian Cancer Chemokine Landscape Is Conducive to Homing of Vaccine-Primed and CD3/CD28-Costimulated T Cells Prepared for Adoptive Therapy.

PURPOSE: Chemokines are implicated in T-cell trafficking. We mapped the chemokine landscape in advanced stage ovarian cancer and characterized the expression of cognate receptors in autologous dendritic cell (DC)-vaccine primed T cells in the context of cell-based immunotherapy. EXPERIMENTAL DESIGN: The expression of all known human chemokines in patients with primary ovarian cancer was analyzed on two independent microarray datasets and validated on tissue microarray. Peripheral blood T cells from five HLA-A2 patients with recurrent ovarian cancer, who previously received autologous tumor DC vaccine, underwent CD3/CD28 costimulation and expansion ex vivo. Tumor-specific T cells were identified by HER2/neu pentamer staining and were evaluated for the expression and functionality of chemokine receptors important for homing to ovarian cancer. RESULTS: The chemokine landscape of ovarian cancer is heterogeneous with high expression of known lymphocyte-recruiting chemokines (CCL2, CCL4, and CCL5) in tumors with intraepithelial T cells, whereas CXCL10, CXCL12, and CXCL16 are expressed quasi-universally, including in tumors lacking tumor-infiltrating T cells. DC-vaccine primed T cells were found to express the cognate receptors for the above chemokines. Ex vivo CD3/CD28 costimulation and expansion of vaccine-primed Tcells upregulated CXCR3 and CXCR4, and enhanced their migration toward universally expressed chemokines in ovarian cancer. CONCLUSIONS: DC-primed tumor-specific T cells are armed with the appropriate receptors to migrate toward universal ovarian cancer chemokines, and these receptors are further upregulated by ex vivo CD3/CD28 costimulation, which render T cells more fit for migrating toward these chemokines. Clin Cancer Res; 21(12); 2840-50. ©2015 AACR.