Potential immune-related therapeutic mechanisms of multiple traditional Chinese medicines on type 2 diabetic nephropathy based on bioinformatics, network pharmacology and molecular docking.

BACKGROUND: The prevalence of type 2 diabetic nephropathy (T2DN) ranges from 20 % to 40 % among individuals with type 2 diabetes. Multiple immune pathways play a pivotal role in the pathogenesis of T2DN. This study aimed to investigate the immunomodulatory effects of active ingredients derived from 14 traditional Chinese medicines (TCMs) on T2DN. METHODS: By removing batch effect on the GSE30528 and GSE96804 datasets, we employed a combination of weighted gene co-expression network analysis, least absolute shrinkage and selection operator analysis, protein-protein interaction network analysis, and the CIBERSORT algorithm to identify the active ingredients of TCMs as well as potential hub biomarkers associated with immune cells. Functional analysis was conducted using Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and gene set variation analysis (GSVA). Additionally, molecular docking was employed to evaluate interactions between active ingredients and potential immunotherapy targets. RESULTS: A total of 638 differentially expressed genes (DEGs) were identified in this study, comprising 5 hub genes along with 4 potential biomarkers. Notably, CXCR1, CXCR2, and FOS exhibit significant associations with immune cells while displaying robust or favorable affinities towards the active ingredients kaempferol, quercetin, and luteolin. Furthermore, functional analysis unveiled intricate involvement of DEGs, hub genes and potential biomarkers in pathways closely linked to immunity and diabetes. CONCLUSION: The potential hub biomarkers and immunotherapy targets associated with immune cells of T2DN comprise CXCR1, CXCR2, and FOS. Furthermore, kaempferol, quercetin, and luteolin demonstrate potential immunomodulatory effects in modulating T2DN through the regulation of CXCR1, CXCR2, and FOS expression.

Circ_0026579 knockdown ameliorates lipopolysaccharide-induced human lung fibroblast cell injury by regulating CXCR1 via miR-370-3p.

Pneumonia is an inflammatory disease in lower respiratory tracts and its development involves the regulation of RNAs. Circular RNAs are a class of RNA subgroups that can mediate the progression of pneumonia. However, the molecular mechanism of circ_0026579 in regulating pneumonia occurrence remains unclear. The study is designed to reveal the role of circ_0026579 in lipopolysaccharide (LPS)-induced human lung fibroblast cell injury and the underlying mechanism. The expression levels of circ_0026579, miR-370-3p and C-X-C motif chemokine receptor 1 (CXCR1) were detected by quantitative real-time polymerase chain reaction or by western blotting. The production of tumour necrosis factor-α, interleukin (IL)-1ß and IL-6 was assessed by enzyme-linked immunosorbent assays. Malondialdehyde and superoxide dismutase levels were analysed using commercial kits. Cell viability, proliferation and apoptosis were analysed by cell counting kit-8 assay, 5-Ethynyl-2'-deoxyuridine assay and flow cytometry analysis, respectively. The binding relationship between miR-370-3p and circ_0026579 or CXCR1 was identified by dual-luciferase reporter assay, RNA immunoprecipitation assay and RNA pull-down assay. Circ_0026579 and CXCR1 expression were significantly upregulated, whereas miR-370-3p was downregulated in the serum of pneumonia patients. LPS treatment induced inflammatory response, oxidative stress and cell apoptosis and inhibited cell proliferation in MRC-5 cells; however, these effects were reversed after circ_0026579 depletion. In terms of the mechanism, circ_0026579 acted as a miR-370-3p sponge, and miR-370-3p combined with CXCR1. Additionally, circ_0026579 depletion ameliorated LPS-induced MRC-5 cell disorder by increasing miR-370-3p expression. CXCR1 overexpression also relieved the miR-370-3p-mediated effects in LPS-treated MRC-5 cells. Further, circ_0026579 induced CXCR1 expression by interacting with miR-370-3p. Circ_0026579 absence ameliorated MRC-5 cell dysfunction induced by LPS through the regulation of the miR-370-3p/CXCR1 axis.

CXCR1 drives the pathogenesis of EAE and ARDS via boosting dendritic cells-dependent inflammation.

Chemokines secreted by dendritic cells (DCs) play a key role in the regulation of inflammation and autoimmunity through chemokine receptors. However, the role of chemokine receptor CXCR1 in inflammation-inducing experimental autoimmune encephalomyelitis (EAE) and acute respiratory distress syndrome (ARDS) remains largely enigmatic. Here we reported that compared with healthy controls, the level of CXCR1 was aberrantly increased in multiple sclerosis (MS) patients. Knockout of CXCR1 not only ameliorated disease severity in EAE mice but also suppressed the secretion of inflammatory factors (IL-6/IL-12p70) production. We observed the same results in EAE mice with DCs-specific deletion of CXCR1 and antibody neutralization of the ligand CXCL5. Mechanically, we demonstrated a positive feedback loop composed of CXCL5/CXCR1/HIF-1α direct regulating of IL-6/IL-12p70 production in DCs. Meanwhile, we found CXCR1 deficiency in DCs limited IL-6/IL-12p70 production and lung injury in LPS-induced ARDS, a disease model caused by inflammation. Overall, our study reveals CXCR1 governs DCs-mediated inflammation and autoimmune disorders and its potential as a therapeutic target for related diseases.

Structural basis of CXC chemokine receptor 1 ligand binding and activation.

Neutrophil granulocytes play key roles in innate immunity and shaping adaptive immune responses. They are attracted by chemokines to sites of infection and tissue damage, where they kill and phagocytose bacteria. The chemokine CXCL8 (also known as interleukin-8, abbreviated IL-8) and its G-protein-coupled receptors CXCR1 and CXCR2 are crucial elements in this process, and also the development of many cancers. These GPCRs have therefore been the target of many drug development campaigns and structural studies. Here, we solve the structure of CXCR1 complexed with CXCL8 and cognate G-proteins using cryo-EM, showing the detailed interactions between the receptor, the chemokine and Gαi protein. Unlike the closely related CXCR2, CXCR1 strongly prefers to bind CXCL8 in its monomeric form. The model shows that steric clashes would form between dimeric CXCL8 and extracellular loop 2 (ECL2) of CXCR1. Consistently, transplanting ECL2 of CXCR2 onto CXCR1 abolishes the selectivity for the monomeric chemokine. Our model and functional analysis of various CXCR1 mutants will assist efforts in structure-based drug design targeting specific CXC chemokine receptor subtypes.

CXCR1 participates in bone cancer pain induced by Walker 256 breast cancer cells in female rats.

Bone cancer pain (BCP) is a clinically intractable mixed pain, involving inflammation and neuropathic pain, and its mechanisms remain unclear. CXC chemokine receptor 1 (CXCR1, IL-8RA) and 2 (CXCR2, IL-8RB) are high-affinity receptors for interleukin 8 (IL8). According to previous studies, CXCR2 plays a crucial role in BCP between astrocytes and neurons, while the role of CXCR1 remains unclear. The objective of this study was to investigate the role of CXCR1 in BCP. We found that CXCR1 expression increased in the spinal dorsal horn. Intrathecal injection of CXCR1 siRNA effectively attenuated mechanical allodynia and pain-related behaviors in rats. It was found that CXCR1 was predominantly co-localized with neurons. Intrathecal injection of CXCR1-siRNA reduced phosphorylated JAK2/STAT3 protein levels and the NLRP3 inflammasome (NLRP3, caspase1, and IL-1ß) levels. Furthermore, in vitro cytological experiments confirmed this conclusion. The study results suggest that the spinal chemokine receptor CXCR1 activation mediates BCP through JAK2/STAT3 signaling pathway and NLRP3 inflammasome (NLRP3, caspase1, and IL-1ß).

Analysis of CXCL8 and its receptors CXCR1/CXCR2 at the mRNA level in neoplastic tissue, as well as in serum and peritoneal fluid in patients with ovarian cance.

Understanding the relationship between the coexistence of inflammatory and neoplastic processes in ovarian cancer, particularly those involving chemokines and their receptors, may help to elucidate the involvement of the studied parameters in tumor pathogenesis and could lead to improved clinical applications. Therefore, the present study aimed to analyze the levels of C­X­C motif chemokine ligand 8 (CXCL8), and its receptors C­X­C chemokine receptor (CXCR)1 and CXCR2, in the serum and peritoneal fluid of women with ovarian cancer, and to evaluate the association between the expression of these parameters in tumor tissue and patient characteristics, particularly the degree of histological differentiation. The study group included women with ovarian cancer diagnosed with serous cystadenocarcinoma International Federation of Gynecology and Obstetrics stage IIIc and a control group, which consisted of women who were diagnosed with a benign lesion (serous cystadenoma). The transcript levels of CXCL8, CXCR1 and CXCR2 were evaluated using reverse transcription­quantitative PCR (RT­qPCR). The quantitative analysis was carried out using the LightCycler® 480 System and GoTaq® 1­Step RT­qPCR System, according to the manufacturers' instructions. The concentration of CXCL8 in serum and peritoneal fluid was determined using a Human Interleukin­8 ELISA kit, and the concentrations of CXCR1 and CXCR2 were determined using the CLOUD­CLONE ELISA kit. Local and systemic disturbances in immune and inflammatory responses involving the CXCL8 chemokine and its receptors indicated the involvement of these studied parameters in the pathogenesis of ovarian cancer. Immunoregulation of the CXCL8­CXCR1 system may influence the course of the inflammatory process accompanying ovarian cancer development, which may result in the identification of novel clinical applications; however, further studies are required.

The importance of CXC-receptors CXCR1-2 and CXCR4 for adaptive regulation of the stress axis in teleost fish.

In an ever-changing environment, an adaptive stress response is the pivotal regulatory mechanism to maintain allostasis. Physiologic responses to stressors enable to overcome potential threat. Glucocorticoid effects can be considered compensatory and adaptive, however prolonged or excessive glucocorticoid secretion can be also maladaptive and detrimental. Therefore, it must be tightly regulated. Apart from the essential hormonal feedback regulation, evidence accrues that cytokines, e.g., proinflammatory interleukin 1ß (IL-1ß), also play an important regulatory role in the stress axis. Here we focused on the potential role of CXC chemokines (CXCL8 and CXCL12) and their receptors (CXCR1, 2 and 4) in the regulation of the stress response in common carp. We studied changes in gene expression of CXC chemokines and CXCRs in the stress axis organs (hypothalamus-pituitary gland-head kidney) upon 11 h of restraint stress and we established how CXCR blocking affects the activation of the stress axis and the synthesis/conversion of cortisol. During restraint stress, gene expression of the majority of the proinflammatory CXCL8 and homeostatic CXCL12 chemokines and their receptors was upregulated in the stress axis organs. Inhibition of CXCR1-2 and CXCR4 differentially affected the expression of genes encoding stress-related molecules: hormones, binding proteins, receptors as well as expression of genes encoding IL-1ß and its receptor. Moreover, we observed that CXC chemokines, via interaction with their respective CXCRs, regulate gene expression of molecules involved in cortisol synthesis and conversion and consistently affect the level of cortisol released into the circulation during the stress response. We revealed that in fish, CXC chemokines and their receptors are important regulators of the stress response at multiple levels of the stress axis, with particularly pronounced effects on steroidogenesis and cortisol conversion in the head kidney.

The non-ELR CXC chemokine encoded by human cytomegalovirus UL146 genotype 5 contains a C-terminal ß-hairpin and induces neutrophil migration as a selective CXCR2 agonist.

Human cytomegalovirus (HCMV) is a major pathogen in immunocompromised patients. The UL146 gene exists as 14 diverse genotypes among clinical isolates, which encode 14 different CXC chemokines. One genotype (vCXCL1GT1) is a known agonist for CXCR1 and CXCR2, while two others (vCXCL1GT5 and vCXCL1GT6) lack the ELR motif considered crucial for CXCR1 and CXCR2 binding, thus suggesting another receptor targeting profile. To determine the receptor target for vCXCL1GT5, the chemokine was probed in a G protein signaling assay on all 18 classical human chemokine receptors, where CXCR2 was the only receptor being activated. In addition, vCXCL1GT5 recruited ß-arrestin in a BRET-based assay and induced migration in a chemotaxis assay through CXCR2, but not CXCR1. In contrast, vCXCL1GT1 stimulated G protein signaling, recruited ß-arrestin and induced migration through both CXCR1 and CXCR2. Both vCXCL1GT1 and vCXCL1GT5 induced equally potent and efficacious migration of neutrophils, and ELR vCXCL1GT4 and non-ELR vCXCL1GT6 activated only CXCR2. In contrast to most human chemokines, the 14 UL146 genotypes have remarkably long C-termini. Comparative modeling using Rosetta showed that each genotype could adopt the classic chemokine core structure, and predicted that the extended C-terminal tail of several genotypes (including vCXCL1GT1, vCXCL1GT4, vCXCL1GT5, and vCXCL1GT6) forms a novel ß-hairpin not found in human chemokines. Secondary NMR shift and TALOS+ analysis of vCXCL1GT1 supported the existence of two stable ß-strands. C-terminal deletion of vCXCL1GT1 resulted in a non-functional protein and in a shift to solvent exposure for tryptophan residues likely due to destabilization of the chemokine fold. The results demonstrate that non-ELR chemokines can activate CXCR2 and suggest that the UL146 chemokines have unique C-terminal structures that stabilize the chemokine fold. Increased knowledge of the structure and interaction partners of the chemokine variants encoded by UL146 is key to understanding why circulating HCMV strains sustain 14 stable genotypes.

Long non-coding RNA PCED1B antisense RNA 1 promotes gastric cancer progression via modulating microRNA-215-3p / C-X-C motif chemokine receptor 1 axis.

Long non-coding RNAs (lncRNAs) emerge as vital modulators and tissue-specific biomarkers of multiple cancers, including gastric cancer (GC). Instead, the expression characteristics, biological function and molecular mechanism of lncRNA PCED1B antisense RNA 1 (PCED1B-AS1) in GC await more elaboration. In this study, 48 cases of GC tissues and matched non-cancerous tissues were collected, and PCED1B-AS1, microRNA-215-3p (miR-215-3p) and C-X-C motif chemokine receptor 1 (CXCR1) expression levels were detected by qRT-PCR. Besides, CCK-8, EdU, Transwell and Western blot assays were conducted to assess the impact of PCED1B-AS1 or miR-215-3p on cell growth, migration, invasion and epithelial-mesenchymal transition (EMT). The interaction between genes was verified by bioinformatics analysis, rna immunoprecitipation (RIP) and dual-luciferase reporter gene assays. We demonstrated that, PCED1B-AS1 expression level was raised in GC tissues and cell lines, and increased expression of PCED1B-AS1 was in association with tumor size, TNM stage and lymph node metastasis in GC patients. Additionally, PCED1B-AS1 overexpression promoted GC cells proliferation, migration, invasion and EMT, and miR-215-3p overexpression counteracted the biological effects of PCED1B-AS1. Mechanistically, PCED1B-AS1 specifically inhibited miR-215-3p expressions, thus up-regulating CXCR1 expressions. In conclusion, PCED1B-AS1 accelerates GC progression via adsorbing miR-215-3p and up-regulating CXCR1, indicating that PCED1B-AS1 is a novel therapeutic target for treating GC.

Probiotics improve re-epithelialization of scratches infected by Porphyromonas gingivalis through up-regulating CXCL8-CXCR1/CXCR2 axis.

Porphyromonas gingivalis inhibits the release of CXCL8 by gingival epithelial cells and reduces their proliferation. We previously reported that Bifidocaterium sp. and Lactobacillus sp. immunomodulate gingival epithelial cells response to this periodontal pathogen, but their effects on re-epithelialization properties are still unknown. Herein we explored these activities of potential probiotics on gingival epithelial cells and clarified their mechanisms. The immortalized OBA-9 lineage was used to perform in vitro scratches. Twelve clinical isolates and commercially available strains of Bifidobacterium sp. and Lactobacillus sp. were screened. L. casei 324 m and B. pseudolongum 1191A were selected to perform mechanistic assays with P. gingivalis W83 infection and the following parameters were measured: percentage of re-epithelialization by DAPI immunofluorescence area measurement; cell number by Trypan Blue exclusion assay; CXCL8 regulation by ELISA and RT-qPCR; and expression of CXCL8 cognate receptors-CXCR1 and CXCR2 by Flow Cytometry. Complementary mechanistic assays were performed with CXCL8, in the presence or absence of the CXCR1/CXCR2 inhibitor-reparixin. L. casei 324 m and B. pseudolongum 1191A enhanced re-epithelialization/cell proliferation as well as inhibited the harmful effects of P. gingivalis W83 on these activities through an increase in the expression and release of CXCL8 and in the number of cells positive for CXCR1/CXCR2. Further, we revealed that the beneficial effects of these potential probiotics were dependent on activation of the CXCL8-CXCR1/CXCR2 axis. The current findings indicate that these potential probiotics strains may improve wound healing in the context of the periodontal tissues by a CXCL8 dependent mechanism.

CXCL1-CXCR1/2 signaling is induced in human temporal lobe epilepsy and contributes to seizures in a murine model of acquired epilepsy.

CXCL1, a functional murine orthologue of the human chemokine CXCL8 (IL-8), and its CXCR1 and CXCR2 receptors were investigated in a murine model of acquired epilepsy developing following status epilepticus (SE) induced by intra-amygdala kainate. CXCL8 and its receptors were also studied in human temporal lobe epilepsy (TLE). The functional involvement of the chemokine in seizure generation and neuronal cell loss was assessed in mice using reparixin (formerly referred to as repertaxin), a non-competitive allosteric inhibitor of CXCR1/2 receptors. We found a significant increase in hippocampal CXCL1 level within 24 h of SE onset that lasted for at least 1 week. No changes were measured in blood. In analogy with human TLE, immunohistochemistry in epileptic mice showed that CXCL1 and its two receptors were increased in hippocampal neuronal cells. Additional expression of these molecules was found in glia in human TLE. Mice were treated with reparixin or vehicle during SE and for additional 6 days thereafter, using subcutaneous osmotic minipumps. Drug-treated mice showed a faster SE decay, a reduced incidence of acute symptomatic seizures during 48 h post-SE, and a delayed time to spontaneous seizures onset compared to vehicle controls. Upon reparixin discontinuation, mice developed spontaneous seizures similar to vehicle mice, as shown by EEG monitoring at 14 days and 2.5 months post-SE. In the same epileptic mice, reparixin reduced neuronal cell loss in the hippocampus vs vehicle-injected mice, as assessed by Nissl staining at completion of EEG monitoring. Reparixin administration for 2 weeks in mice with established chronic seizures, reduced by 2-fold on average seizure number vs pre-treatment baseline, and this effect was reversible upon drug discontinuation. No significant changes in seizure number were measured in vehicle-injected epileptic mice that were EEG monitored in parallel. Data show that CXCL1-IL-8 signaling is activated in experimental and human epilepsy and contributes to acute and chronic seizures in mice, therefore representing a potential new target to attain anti-ictogenic effects.

An updated review on the role of the CXCL8-CXCR1/2 axis in the progression and metastasis of breast cancer.

Chronic inflammation is a major factor in tumor growth and progression. Cancer cells secrete C-X-C chemokine ligand 8 (CXCL8) along with its receptor C-X-C chemokine receptor 1 (CXCR1) and chemokine receptor 2 (CXCR2). It plays a significant role in the activation and trafficking of inflammatory mediators, tumor proliferation and interferes in breast cancer development by controlling cell adhesion, proliferation, migration, and metastasis. This axis also plays a significant role in driving different cancers and melanomas, including breast cancer progression, by controlling stem cell masses. Few small-molecule CXCR1/2 inhibitors and CXCL8 releasing inhibitors have been identified in the past two decades that bind these receptors in their inactive forms and blocks their signaling as well as the biological activities associated with inflammation. Inhibitors of certain inflammatory molecules are projected to be more efficient in different inflammatory diseases. Preclinical trials indicate that patients may be benefitted from combined treatment with targeted drugs, chemotherapies, and immunotherapies. Thus, targeting the CXCL8-CXCR1/2 signaling axis in breast cancer could be a promising approach for its therapeutics. This review examines the roles of the CXCL8-CXCR1/2 signaling axis and how it is implicated in the tumor microenvironment in breast cancer. In addition, we also discuss the potential role of the CXCL8-CXCR1/2 axis in targeted therapeutics for breast cancer.

The IL-8-CXCR1/2 axis contributes to diabetic kidney disease.

AIMS/HYPOTHESIS: Inflammation has a major role in diabetic kidney disease. We thus investigated the role of the IL-8-CXCR1/2 axis in favoring kidney damage in diabetes. METHODS: Urinary IL-8 levels were measured in 1247 patients of the Joslin Kidney Study in type 2 diabetes (T2D). The expression of IL-8 and of its membrane receptors CXCR1/CXCR2 was quantified in kidney tissues in patients with T2D and in controls. The effect of CXCR1/2 blockade on diabetic kidney disease was evaluated in db/db mice. RESULTS: IL-8 urinary levels were increased in patients with T2D and diabetic kidney disease, with the highest urinary IL-8 levels found in the patients with the largest decline in glomerular filtration rate, with an increased albumin/creatine ratio and the worst renal outcome. Moreover, glomerular IL-8 renal expression was increased in patients with T2D, as compared to controls. High glucose elicits abundant IL-8 secretion in cultured human immortalized podocytes in vitro. Finally, in diabetic db/db mice and in podocytes in vitro, CXCR1/2 blockade mitigated albuminuria, reduced mesangial expansion, decreased podocyte apoptosis and reduced DNA damage. CONCLUSIONS/INTERPRETATION: The IL-8- CXCR1/2 axis may have a role in diabetic kidney disease by inducing podocyte damage. Indeed, targeting the IL-8-CXCR1/2 axis may reduce the burden of diabetic kidney disease.

MiR-144-induced KLF2 inhibition and NF-kappaB/CXCR1 activation promote neutrophil extracellular trap-induced transfusion-related acute lung injury.

Transfusion-related acute lung injury (TRALI) is a clinical syndrome which is associated with the formation of neutrophil extracellular trap (NET). Recent studies have demonstrated the roles of microRNAs (miRNAs) in the pathophysiological process of TRALI. Here, the study focused on the role of miR-144 and the molecular mechanisms in NET-induced TRALI. Up-regulated miR-144 and under-expressed KLF2 were determined in patients with TRALI. In the mouse model of a two-event TRALI induced by intraperitoneal injections with lipopolysaccharide and anti-H-2Kd mAb, we determined expression patterns of miR-144, Krüppel-like factor 2 (KLF2), chemokine (C-X-C motif) receptor 1 (CXCR1) and nuclear factor kappa-B (NF-kappaB) p65. The results confirmed that miR-144 was highly expressed, while KLF2 was poorly expressed in mice with TRALI. Dual-luciferase reporter gene assay identified that miR-144 could target KLF2. Using gain- and loss-of-function approaches, we analysed the effects of miR-144 and its interaction with KLF2 on TRALI. Enforced expression of miR-144 was found to aggravate NET-induced TRALI by down-regulating KLF2 and activating the NF-kappaB/CXCR1 signalling pathway in TRALI mice. Collectively, miR-144-targeted inhibition of KLF2 and activation of NF-kappaB/CXCR1 are possible mechanisms responsible for NET-caused TRALI. These findings aid in the development of therapeutic modalities for the treatment of TRALI.

Molecular characterization and biological function of CXCR1 in Nocardia seriolae-infected largemouth bass (Micropterus salmoides).

Interleukin-8 (IL-8, CXCL8), a pro-inflammatory chemokine secreted by a variety of cell types, plays a critical role in the development of various immune diseases. Interactions between IL-8 and its receptor CXC receptor 1/2 (CXCR1/2) are known to promote chemotaxis and phagocytosis in many immune responses. In this study, we report the molecular characteristics and pharmacological activity of CXCR1 (MsCXCR1) in largemouth bass (Micropterus salmoides) and evaluated the functional involvement of MsCXCR1 in individuals infected with the pathogen Nocardia seriolae. MsCXCR1 was cloned into the pEGFP-N1 plasmid and the subcellular localization of MsCXCR1 on the cell membrane was verified in MsCXCR1-EGFP-expressing HEK293 cells. Following observation of receptor internalization and intracellular signaling detection, we further determined the functional interaction of secreted interleukin-8 (LcIL-8, the ligand for CXCR1 in large yellow croaker) and MsCXCR1 was further determined, and the ERK phosphorylation signal activation mediated by MsCXCR1 was demonstrated. Quantitative real-time PCR assays were conducted to analyze the transcriptional distribution of MsCXCR1 in various tissues of healthy and diseased largemouth bass. These results illustrate the significant elevation of MsCXCR1 expression in the head kidney, spleen and liver of M. salmoides, suggesting that MsCXCR1 was involved in the immune response in N. seriolae-infected largemouth bass and potentially affects the digestive function of this species.

Conformational plasticity and dynamic interactions of the N-terminal domain of the chemokine receptor CXCR1.

The dynamic interactions between G protein-coupled receptors (GPCRs) and their cognate protein partners are central to several cell signaling pathways. For example, the association of CXC chemokine receptor 1 (CXCR1) with its cognate chemokine, interleukin-8 (IL8 or CXCL8) initiates pathways leading to neutrophil-mediated immune responses. The N-terminal domain of chemokine receptors confers ligand selectivity, but unfortunately the conformational dynamics of this intrinsically disordered region remains unresolved. In this work, we have explored the interaction of CXCR1 with IL8 by microsecond time scale coarse-grain simulations, complemented by atomistic models and NMR chemical shift predictions. We show that the conformational plasticity of the apo-receptor N-terminal domain is restricted upon ligand binding, driving it to an open C-shaped conformation. Importantly, we corroborated the dynamic complex sampled in our simulations against chemical shift perturbations reported by previous NMR studies and show that the trends are similar. Our results indicate that chemical shift perturbation is often not a reporter of residue contacts in such dynamic associations. We believe our results represent a step forward in devising a strategy to understand intrinsically disordered regions in GPCRs and how they acquire functionally important conformational ensembles in dynamic protein-protein interfaces.

Association of single nucleotide polymorphisms in CXCR1, CXCR2 and CXCL5 with Behçet disease: a study in the Denizli province of Turkey.

BACKGROUND: Behçet disease (BD) is associated with the immune system, especially neutrophilic activity. The CXCR1, CXCR2 and CXCL5 genes mediate the activation and migration of neutrophils. AIM: To investigate CXCR1, CXCR2 and CXCL5 single nucleotide polymorphisms (SNPs) and examine their association with BD. METHODS: We studied polymorphic sites in CXCR1 (four sites: rs16858811, rs9282752, rs16858809 and rs16858808), CXCR2 (three sites: rs2230054, rs1126579 and rs1126580) and CXCL5 (one site: rs352046) in 87 patients with BD and 111 healthy controls (HCs), using a PCR restriction-fragment length polymorphism-based approach for genotyping. RESULTS: We found that the CXCR2 rs2230054 TT genotype and the CXCL5 rs352046 polymorphism might be possible genetic factors responsible for BD. We did not find any association between the development of BD and any of the four CXCR1 polymorphisms or the other two CXCR2 SNPs. In addition, our haplotype analysis results indicated that the haplotypes of the CXCR2 and CXCR1-CXCR2 polymorphic loci were different between the BD and HC groups. CONCLUSION: Our study suggests that polymorphisms of CXCR1, CXCR2 and CXCL5 may affect susceptibility to BD and increase the risk of developing the disease. These loci need to be studied in larger groups of patients from different geographical areas around the world in order to clarify the genetic background for BD pathogenesis.

Endogenous modification of the chemoattractant CXCL5 alters receptor usage and enhances its activity toward neutrophils and monocytes.

The inflammatory human chemokine CXCL5 interacts with the G protein-coupled receptor CXCR2 to induce chemotaxis and activation of neutrophils. CXCL5 also has weak agonist activity toward CXCR1. The N-terminus of CXCL5 can be modified by proteolytic cleavage or deimination of Arg9 to citrulline (Cit), and these modifications can occur separately or together. Here, we chemically synthesized native CXCL5(1-78), truncated CXCL5 [CXCL5(9-78)], and the citrullinated (Cit9) versions and characterized their functions in vitro and in vivo. Compared with full-length CXCL5, N-terminal truncation resulted in enhanced potency to induce G protein signaling and ß-arrestin recruitment through CXCR2, increased CXCL5-initiated internalization of CXCR2, and greater Ca2+ signaling downstream of not only CXCR2 but also CXCR1. Citrullination did not affect the capacity of CXCL5 to activate classical or alternative signaling pathways. Administering the various CXCL5 forms to mice revealed that in addition to neutrophils, CXCL5 exerted chemotactic activity toward monocytes and that this activity was increased by N-terminal truncation. These findings were confirmed by in vitro chemotaxis and Ca2+ signaling assays with primary human CD14+ monocytes and human THP-1 monocytes. In vitro and in vivo analyses suggested that CXCL5 targeted monocytes through CXCR1 and CXCR2. Thus, truncation of the N-terminus makes CXCL5 a more potent chemoattractant for both neutrophils and monocytes that acts through CXCR1 and CXCR2.

Hepatitis B-Induced IL8 Promotes Hepatocellular Carcinoma Venous Metastasis and Intrahepatic Treg Accumulation.

Hepatitis B-associated hepatocellular carcinoma (HCC) is often accompanied by severe vascular invasion and portal vein tumor thrombus, leading to a poor prognosis. However, the underlying mechanism of this disease remains obscure. In this study, we demonstrate that the hepatitis B virus (HBV)-encoded gene HBx induces high IL8 production through MEK-ERK signal activation, leading to enhanced endothelial permeability to facilitate tumor vascular invasion. In a vascular metastatic model using a tail vein injection in a transgenic mouse with selective expression of human CXCR1 in the endothelium, activation of the IL8-CXCR1 cascade by overexpression of IL8 in tumor cells dramatically enhanced liver metastasis. Mechanistically, IL8 selectively induced GARP-latent-TGFß in liver sinusoidal endothelial cells and subsequently provoked preferential regulatory T-cell polarization to suppress antitumor immunity. Collectively, these findings reveal a hepatitis B-associated IL8-CXCR1 signaling axis that mediates vascular invasion and local microenvironmental immune escape of HCC to induce intrahepatic metastasis, which may serve as potential therapeutic targets for HBV-associated HCC. SIGNIFICANCE: This study identifies a hepatitis B-induced IL8/CXCR1/TGFß signaling cascade that suppresses antitumor immunity and enhances metastasis in hepatocellular carcinoma, providing new potential targets for therapeutic intervention.

Molecular characterization and expression analysis of Japanese flounder (Paralichthys olivaceus) chemokine receptor CXCR2 in comparison with CXCR1.

Chemokines are categorized into five families; one of the families is the CXC chemokines, which are critical in the pro-inflammatory process. CXC chemokines transmit signals and mediate a cell's biological activities by binding to cell surface receptors known as chemokine receptors (CXCRs). In this study, the CXCR2 from Japanese flounder (Paralichthys olivaceus) (JfCXCR2) was identified and characterized at the molecular level. The JfCXCR2 gene has a 1077 bp open reading frame that encodes a protein of 359 amino acid residues with seven transmembrane domains. Phylogenetic analysis of JfCXCR2 revealed that it belonged to the fish CXCR2 subfamily. Furthermore, JfCXCR2 was compared with the previously identified Japanese flounder CXCR1 (JfCXCR1). The expression analysis of uninfected Japanese flounder showed that JfCXCR1 and JfCXCR2 were expressed in all the tissues and organs tested but mainly in immune-related organs, including the kidney and spleen. Infection by Streptococcus iniae significantly increased the level of JfCXCR1 and JfCXCR2 mRNA in the kidney at days 1 and 3 post-infection. On the other hand, VHSV (viral hemorrhagic septicemia virus) and Edwardsiella tarda infection significantly increased JfCXCR2 mRNA levels in the kidney at days 3 and 6 post-infection, respectively. Conversely, JfCXCR1 expression was not significantly changed by either E. tarda or VHSV infection. Additionally, the peripheral blood leukocytes (PBLs) stimulated by recombinant proteins rCXCL8_L1a and rCXCL8_L1b were found to have significantly increased levels of JfCXCR1 and JfCXCR2 mRNA. Interestingly, even higher levels of JfCXCR1 and JfCXCR2 expression were observed in PBLs stimulated with rCXCL8_L1a and rCXCL8_L1b than in PBLs stimulated with either recombinant protein. These data suggest that bacterial infections may activate JfCXCR1. By contrast, JfCXCR2 may be activated by both bacterial and viral infection to mediate the immune response. These data can contribute to further understanding the functions of CXCR1 and CXCR2 in the fish immune system.