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Objectives: There is already substantial evidence indicating that neutrophil extracellular trap (NET) formation contributes to the inflammatory cascade in ulcerative colitis (UC). However, the precise regulatory mechanisms governing this process remain elusive. This study aimed to determine the role of NET-related genes in UC and reveal possible mechanisms., Methods: Employing a two-sample MR methodology, we investigated the correlations between NET-associated genes (NRGs) and UC with summary data derived from a genome-wide association study (12,366 cases vs. 33,609 controls) and FinnGen (8,279 cases vs. 261,098 controls). The main analysis employed the inverse variance weighted method, supplemented by the MR-Egger method and weighted median method. Sensitivity analysis was conducted to rule out the interference of heterogeneity and pleiotropy among utilized instrument variables. The colocalization analysis was used to determine whether the identified NRGs and UC shared casual variants. Cross-tissue expression analysis was performed to characterize the expression patterns of target NRGs, while multi-gene correlation analysis and GSEA analysis were conducted to explore the mechanisms by which target NRGs promote UC and NET formation. Immunohistochemistry was used to validate the protein expression of target NRGs in the colon tissue of UC patients., Results: After the validation of two datasets, seven NRGs were associated with the risk of UC. The higher expression of ITGB2 was associated with increased UC risk, while the expression of CXCR1, CXCR2, IRAK4, MAPK3, SIGLEC14, and SLC22A4 were inversely associated with UC risk. Colocalization analysis supported the correlation between CXCR1/2 and UC risk. Expression analysis indicated that CXCR1/2 were down-regulated in peripheral blood, but up-regulated in colon tissue. GSEA analysis and correlation analysis indicated that CXCR1/2 promoted UC and NET formation through neutrophil chemotaxis and PAD4-mediated pathways, separately. Immunohistochemical results confirmed the high expression of CXCR1/2 in colon tissues of UC patients., Conclusions: Our study identified CXCR1/2 as candidate targets in UC among all NRGs through multi-method argumentation, providing new insights of the regulation mechanisms of NET formation in the pathogenesis of UC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Xv, Feng and Lin.)

Background: Posterior capsular opacification is a major complication following cataract surgery, marked by proliferation, migration, epithelial-mesenchymal transition, and fibrosis of residual epithelial cells. Various inflammatory cytokines are upregulated and contribute to the development of posterior capsular opacification. The effect of interleukin-8 on residual epithelial cells has not been fully determined., Methods: Aqueous humor and anterior capsules samples were collected from cataract surgery. Capsular bags from rats and pigs were cultured in DMEM media. Protein and mRNA expressions were measured using immunoblot and qPCR. Cell migration was assessed using the transwell assay., Results: Interleukin-8 is an early inflammatory factor secreted by residual lens epithelial cells. Migration of lens epithelial cells in aqueous humor positively correlates with interleukin-8 levels, and this effect is inhibited by the receptors of interleukin-8 CXCR1/2 blocker Reparaxin. The expression of tight-junction protein ZO-1 and cell-adhesion protein E-cadherin were down-regulated by administrating interleukin-8, and cell migration of both SRA01/04 cell line in vitro and capsular residual epithelial cells ex vivo were up-regulated via activating RhoA expression and RhoA/GTPase activity. The loss-of- function studies demonstrate that interleukin-8 binding to its receptor CXCR1/2 activates NF-κB/p65, which then turns on the RhoA's expression and RhoA/GTPase activity, and RhoA-modulated the downexpression of E-cadherin and ZO-1 and the increase of cell migration., Conclusions: The upregulation in interleukin-8 occurs early in posterior capsular opacification and contributes to down-regulating tight-junctions among epithelial cells and elevates cell migration via the CXCR1/2-NF-κB-RhoA signaling pathway. These demonstrated that interleukin-8 could be a potential target for preventing posterior capsular opacification., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The level of tumor and circulating CXCR1/2-expressing neutrophils and CXCR1/2 ligands correlate with poor patient outcomes, inversely correlate with tumoral lymphocyte content, and predict immune checkpoint inhibitor (ICI) treatment failure. Accordingly, CXCR2-selective and CXCR1/2 dual inhibitors exhibit activity both as single agents and in combination with ICI treatment in mouse tumor models. Based on such reports, clinical trials combining CXCR1/2 axis antagonists with ICI treatment for cancer patients are underway. It has been assumed that CXCR1/2 blockade impacts tumors by blocking neutrophil chemotaxis and reducing neutrophil content in tumors. Here, we show that while CXCR2 antagonism does slow tumor growth, it does not preclude neutrophil recruitment into tumor. Instead, CXCR1/2 inhibition alters neutrophil function by blocking the polarization of transcriptional programs toward immune suppressive phenotypes and rendering neutrophils incapable of suppressing lymphocyte proliferation. This is associated with decreased release of reactive oxygen species and Arginase-1 into the extracellular milieu. Remarkably, these therapeutics do not impact the ability of neutrophils to phagocytose and kill ingested bacteria. Taken together, these results mechanistically explain why CXCR1/2 inhibition has been active in cancer but without infectious complications., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2024 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Acute respiratory distress syndrome (ARDS) is a severe form of acute respiratory failure characterised by extensive inflammatory injury to the alveolocapillary barrier leading to alveolar oedema, impaired gas exchange and, ultimately, hypoxaemia necessitating the use of supplemental oxygen combined with some degree of positive airway pressure. Although much heterogeneity exists regarding the aetiology, localisation and endotypic characterisation of ARDS, what remains largely undisputed is the role of the innate immune system, and in particular of neutrophils, in precipitating and propagating lung injury. Activated neutrophils, recruited to the lung through chemokine gradients, promote injury by releasing oxidants, proteases and neutrophil extracellular traps, which ultimately cause platelet aggregation, microvascular thrombosis and cellular death. Among various neutrophilic chemoattractants, interleukin-8/C-X-C motif ligand 8 and related chemokines, collectively called ELR+ chemokines, acting on neutrophils through the G protein-coupled receptors CXCR1 and CXCR2, are pivotal in orchestrating the neutrophil activation status and chemotaxis in the inflamed lung. This allows efficient elimination of infectious agents while at the same time minimising collateral damage to host tissue. Therefore, understanding how CXCR1 and CXCR2 receptors are regulated is important if we hope to effectively target them for therapeutic use in ARDS. In the following narrative review, we provide an overview of the role of ELR+ chemokines in acute lung injury (ALI) and ARDS, we summarise the relevant regulatory pathways of their cognisant receptors CXCR1/2 and highlight current preclinical and clinical evidence on the therapeutic role of CXCR1 and CXCR2 inhibition in animal models of ALI, as well as in ARDS patients., Competing Interests: Conflict of interest: S. Toya, E. Gavioli, E.M. Minnella, M.C. Cesta and M. Allegretti are Dompé employees. P. Morris and L. Huerta act as Principal Investigators in studies currently conducted by Dompé and have received consulting fees for expertise in critical care clinical trials (P. Morris) as well as travel reimbursements for participating in study start-up investigators meetings (P. Morris and L. Huerta). P. Proost and S. Struyf declare no conflict of interest., (Copyright ©The authors 2024.)

Studies conducted on animal models have identified several therapeutic targets for myelofibrosis, the most severe of the myeloproliferative neoplasms. Unfortunately, many of the drugs which were effective in pre-clinical settings had modest efficacy when tested in the clinic. This discrepancy suggests that treatment for this disease requires combination therapies. To rationalize possible combinations, the efficacy in the Gata1 low model of drugs currently used for these patients (the JAK1/2 inhibitor Ruxolitinib) was compared with that of drugs targeting other abnormalities, such as p27kip1 (Aplidin), TGF-β (SB431542, inhibiting ALK5 downstream to transforming growth factor beta (TGF-β) signaling and TGF-β trap AVID200), P-selectin (RB40.34), and CXCL1 (Reparixin, inhibiting the CXCL1 receptors CXCR1/2). The comparison was carried out by expressing the endpoints, which had either already been published or had been retrospectively obtained for this study, as the fold change of the values in the corresponding vehicles. In this model, only Ruxolitinib was found to decrease spleen size, only Aplidin and SB431542/AVID200 increased platelet counts, and with the exception of AVID200, all the inhibitors reduced fibrosis and microvessel density. The greatest effects were exerted by Reparixin, which also reduced TGF-β content. None of the drugs reduced osteopetrosis. These results suggest that future therapies for myelofibrosis should consider combining JAK1/2 inhibitors with drugs targeting hematopoietic stem cells (p27Kip1) or the pro-inflammatory milieu (TGF-β or CXCL1).

BCR::ABL1 negative myeloproliferative neoplasms (MPN) form a distinct group of hematologic malignancies characterized by sustained proliferation of cells from multiple myeloid lineages. With a median survival of 16-35 months in patients with high-risk disease, primary myelofibrosis (PMF) is considered the most aggressive entity amongst all BCR::ABL1 MPN. Additionally, for a significant subset of patients, MPN evolve into secondary acute myeloid leukemia (AML), which has an even poorer prognosis compared to de novo AML. As the exact mechanisms of disease development and progression remain to be elucidated, current therapeutic approaches fail to prevent disease progression or transformation into secondary AML. As each MPN entity is characterized by sustained activation of various immune cells and raised cytokine concentrations within bone marrow (BM) and peripheral blood (PB), MPN may be considered to be typical inflammation-related malignancies. However, the exact role and consequences of increased cytokine concentrations within BM and PB plasma has still not been completely established. Up-regulated cytokines can stimulate cellular proliferation, or contribute to the development of an inflammation-related BM niche resulting in genotoxicity and thereby supporting mutagenesis. The neutrophil chemoattractant CXCL8 is of specific interest as its concentration is increased within PB and BM plasma of patients with PMF. Increased concentration of CXCL8 negatively correlates with overall survival. Furthermore, blockage of the CXCR1/2 axis appears to be able to reduce BM fibrosis and megakaryocyte dysmorphia in murine models. In this review, we summarize available evidence on the role of the CXCL8-CXCR1/2 axis within the pathogenesis of PMF, and discuss potential therapeutic modalities targeting either CXCL8 or its cognate receptors CXCR1/2.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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 C‑X‑C motif chemokine ligand 8 (CXCL8), and its receptors C‑X‑C 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 transcription‑quantitative PCR (RT‑qPCR). The quantitative analysis was carried out using the LightCycler® 480 System and GoTaq® 1‑Step RT‑qPCR System, according to the manufacturers' instructions. The concentration of CXCL8 in serum and peritoneal fluid was determined using a Human Interleukin‑8 ELISA kit, and the concentrations of CXCR1 and CXCR2 were determined using the CLOUD‑CLONE 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 CXCL8‑CXCR1 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.

CXCR1 and CXCR2 chemokine receptors, mainly activated by interleukin 8 (IL-8 or CXCL8), are expressed in a variety of cells including, leukocytes, fibroblasts, endothelial cells, and smooth muscle cells. Numerous intracellular mediators are activated by these G protein-coupled receptors based on several factors, including the nature of the ligand, its concentration, and the binding sites with the receptor, levels of the receptor, cell type, and stimulatory environment. Much focus is currently being directed towards CXCR1/2 inhibitors, as these receptors primarily induce the chemotaxis of leukocytes, especially neutrophils, during inflammation, a key process in cardiovascular disease (CVD) progression. CXCR1/2 inhibitors show beneficial effects in various animal models of CVD. These effects include reducing the atherosclerotic plaque area, improving the serum lipid profile, attenuation of the damage following ischemia-reperfusion, the regulation of blood pressure, and the restriction of cardiac remodeling. Based on these encouraging results, testing CXCR1/2 inhibitors in clinical trials could be of a great importance to limit the inflammatory complications associated with CVDs., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

In humans, Interleukin-8 (IL-8 or CXCL8) is a granulocytic chemokine with multiple roles within the tumor microenvironment (TME), such as recruiting immunosuppressive cells to the tumor, increasing tumor angiogenesis, and promoting epithelial-to-mesenchymal transition (EMT). All of these effects of CXCL8 on individual cell types can result in cascading alterations to the TME. The changes in the TME components such as the cancer-associated fibroblasts (CAFs), the immune cells, the extracellular matrix, the blood vessels, or the lymphatic vessels further influence tumor progression and therapeutic resistance. Emerging roles of the microbiome in tumorigenesis or tumor progression revealed the intricate interactions between inflammatory response, dysbiosis, metabolites, CXCL8, immune cells, and the TME. Studies have shown that CXCL8 directly contributes to TME remodeling, cancer plasticity, and the development of resistance to both chemotherapy and immunotherapy. Further, clinical data demonstrate that CXCL8 could be an easily measurable prognostic biomarker in patients receiving immune checkpoint inhibitors. The blockade of the CXCL8-CXCR1/2 axis alone or in combination with other immunotherapy will be a promising strategy to improve antitumor efficacy. Herein, we review recent advances focusing on identifying the mechanisms between TME components and the CXCL8-CXCR1/2 axis for novel immunotherapy strategies.

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., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Endothelial colony-forming cells (ECFCs) are human vasculogenic cells described as potential cell therapy product and good candidates for being a vascular liquid biopsy. Since interleukin-8 (IL-8) is a main actor in senescence, its ability to interact with ECFCs has been explored. However, expression of CXCR1 and CXCR2, the two cellular receptors for IL-8, by ECFCs remain controversial as several teams published contradictory reports. Using complementary technical approaches, we have investigated the presence of these receptors on ECFCs isolated from cord blood. First, CXCR1 and CXCR2 were not detected on several clones of cord blood- endothelial colony-forming cell using different antibodies available, in contrast to well-known positive cells. We then compared the RT-PCR primers used in different papers to search for the presence of CXCR1 and CXCR2 mRNA and found that several primer pairs used could lead to non-specific DNA amplification. Last, we confirmed those results by RNA sequencing. CXCR1 and CXCR2 were not detected in ECFCs in contrary to human-induced pluripotent stem cell-derived endothelial cells (h-iECs). In conclusion, using three different approaches, we confirmed that CXCR1 and CXCR2 were not expressed at mRNA or protein level by ECFCs. Thus, IL-8 secretion by ECFCs, its effects in angiogenesis and their involvement in senescent process need to be reanalyzed according to this absence of CXCR-1 and - 2 in ECFCs.Graphical Abstract.

Neutrophils are key inflammatory cells in the immunopathogenesis of asthma. Neutrophil migration can be initiated through activation of the CXCR1 and CXCR2 receptors by CXC chemokines, such as IL-8. Although transcription factor KLF2 has been found to maintain T cell migration patterns through repression of several chemokine receptors, whether KLF2 can regulate neutrophil migration via modulation of CXCR1 and CXCR2 is unknown. Here, we aimed to explore the functions of KLF2, CXCR1 and CXCR2 in neutrophil migration in asthma and to establish a regulatory role of KLF2 for CXCR1/2. We demonstrate that with asthma aggravation, the percentages and migration rates of peripheral blood neutrophils gradually increased in asthmatic patients and the guinea pig asthma model. Correspondingly, both the KLF2 mRNA and protein levels in neutrophils were gradually reduced. While CXCR1 and CXCR2 expression was negatively correlated with KLF2. In vitro knockdown of KLF2 dramatically increased the migration of HL-60-drived neutrophil-like cells, which was accompanied by an increase in the CXCR1 and CXCR2 mRNA and protein expression levels. Taken together, our results indicate that decreased KLF2 aggravates asthma progression by promoting neutrophil migration, which is associated with the transcriptional upregulation of CXCR1 and CXCR2. The KLF2 and/or CXCR1/2 expression levels may represent an indicator of asthma severity., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: In this study we investigate the prediction and prognostic value of CXCL8-CXCR1/2 axis for Triple-negative breast cancer (TNBC) patients underwent neoadjuvant chemotherapy (NAC) following standard radical surgery., Methods: A total of 303 TNBC patients were included in this study. The NAC regimen was weekly paclitaxel plus carboplatin (PC) for all patients. Serum CXCL8 level was measured at baseline and at surgery via Enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry was used to detect CXCR1 and CXCR2 expression in patients with residual tumors after NAC. Correlations between variables and treatment response were studied, and Cox proportional hazards regression analysis was implemented for prognostic evaluation., Results: Of the 303 patients, 103 (34.0%) patients experienced pathological complete response (pCR) after completion of NAC. CXCL8 level was significantly upgraded after NAC in CXCR1/2+ patients and downgraded after NAC in CXCR1/2- patients. Higher pCR rate was more likely observed in patients with lower CXCL8 level at surgery (P = 0.004, HR 0.939, 95% CI 0.900-0.980). In the multivariate survival model, CXCR1/2 expression was of an independent prognostic value for survival (CXCR1/2+, HR 2.149, 95% CI 0.933-4.949; CXCR1/2++, HR 3.466, 95% CI 1.569-7.655, CXCR1/2- was used as a reference; P = 0.003). Patients with higher level of CXCR1/2 expression were more likely to suffer unfavorable outcome., Conclusions: This study contributes to the clarification of the value of serum CXCL8 level to predict pCR for TNBC patients, and prognostic performance of CXCR1/2 in non-pCR responders after NAC. The CXCL8-CXCR1/2 might play an important role in tailoring and modifying the NAC strategy for advanced TNBCs; however, further confirmatory studies are needed.

Neutrophils are expanded and abundant in cancer-bearing hosts. Under the influence of CXCR1 and CXCR2 chemokine receptor agonists and other chemotactic factors produced by tumors, neutrophils, and granulocytic myeloid-derived suppressor cells (MDSCs) from cancer patients extrude their neutrophil extracellular traps (NETs). In our hands, CXCR1 and CXCR2 agonists proved to be the major mediators of cancer-promoted NETosis. NETs wrap and coat tumor cells and shield them from cytotoxicity, as mediated by CD8 + T cells and natural killer (NK) cells, by obstructing contact between immune cells and the surrounding target cells. Tumor cells protected from cytotoxicity by NETs underlie successful cancer metastases in mice and the immunotherapeutic synergy of protein arginine deiminase 4 (PAD4) inhibitors, which curtail NETosis with immune checkpoint inhibitors. Intravital microscopy provides evidence of neutrophil NETs interfering cytolytic cytotoxic T lymphocytes (CTLs) and NK cell contacts with tumor cells., Competing Interests: Declaration of Interests Ignacio Melero reports receiving commercial research grants from BMS, Bioncotech, Alligator, Pfizer, Leadartis, and Roche; has received speakers bureau honoraria from MSD; and is a consultant or advisory board member for BMS, Roche, Genmab, F-Star, Bioncotech, Bayer, Numab, Pieris, Alligator, and Merck Serono. M.A. and M.d.P. are employees at Dompé Farmaceutici S.p.A., Italy. The company has interests in the development of CXCR1 and 2 allosteric modulator for the treatment of oncologic-related diseases. J.L.P.-G. reports research grants and support from Roche, BMS, MSD, Ipsen, Eisai, Incyte, and Janssen and is a consultant or advisory board member for Roche, BMS, Ipsen, Eisai, and MSD. M.P.-S. reports receiving commercial research grants from BMS and Roche and has received speaker’s bureau honoraria from Roche. M.C. is an employee of Mavupharma. The remaining authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Proteoglycans (PGs), present in diverse environments, such as the cell membrane surface, extracellular milieu, and intracellular granules, are fundamental to life. Sulfated glycosaminoglycans (GAGs) are covalently attached to the core protein of proteoglycans. PGs are complex structures, and are diverse in terms of amino acid sequence, size, shape, and in the nature and number of attached GAG chains, and this diversity is further compounded by the phenomenal diversity in GAG structures. Chemokines play vital roles in human pathophysiology, from combating infection and cancer to leukocyte trafficking, immune surveillance, and neurobiology. Chemokines mediate their function by activating receptors that belong to the GPCR class, and receptor interactions are regulated by how, when, and where chemokines bind GAGs. GAGs fine-tune chemokine function by regulating monomer/dimer levels and chemotactic/haptotactic gradients, which are also coupled to how they are presented to their receptors. Despite their small size and similar structures, chemokines show a range of GAG-binding geometries, affinities, and specificities, indicating that chemokines have evolved to exploit the repertoire of chemical and structural features of GAGs. In this review, we summarize the current status of research on how GAG interactions regulate ELR-chemokine activation of CXCR1 and CXCR2 receptors, and discuss knowledge gaps that must be overcome to establish causal relationships governing the impact of GAG interactions on chemokine function in human health and disease., (Copyright © 2020 Rajarathnam and Desai.)

Interleukin-8 (IL-8, also named CXCL8) binds to its receptors (CXCR1 and CXCR2) with subsequent recruitment of neutrophils and enhancement of their infiltration into inflamed sites, which exaggerates inflammation in many diseases. Recent studies have proposed that metabolic disorders can be attenuated by counteracting certain inflammatory signal pathways. In this study, we examined whether intervention with G31P, an antagonist of CXCL8, could attenuate tissue inflammation and development of metabolic disorders in db/db mice. The db/m and db/db mice were subcutaneously injected with G31P or equivalent normal saline once a day for 6 wk. The physical and metabolic parameters, glucose tolerance, insulin sensitivity, hepatic lipid accumulation, and inflammation markers were measured. G31P improved hepatic insulin sensitivity by modulating expression of genes related to gluconeogenesis and phosphorylated Akt levels. The expressions of several genes encoding proteins involved in de novo lipogenesis were decreased in G31P-treated db/db mice. Meanwhile, immune cell infiltration and cytokine release were attenuated in db/db mice with G31P treatment. G31P also improved the ratio of proinflammatory M1 and anti-inflammatory M2 macrophages. Furthermore, G31P ameliorates metabolic disturbances via inhibition of CXCR1 and CXCR2 pathways in db/db mice. These data suggest that the selective inhibition of CXC chemokines may have therapeutic effects on symptoms associated with obesity and diabetes.

CXCL8 (also known as IL-8) can produce different biological effects by binding to its receptors: CXCR1, CXCR2, and the Duffy antigen receptor for chemokines (DARC). CXCL8 and its receptors are associated with the development of various tumor types, especially colorectal cancer and its liver metastases. In addition to promoting angiogenesis, proliferation, invasion, migration, and the survival of colorectal cancer (CRC) cells, CXCL8 and its receptors have also been known to induce the epithelial-mesenchymal transition (EMT) of CRC cells, to help them to escape host immunosurveillance as well as to enhance resistance to anoikis, which promotes the formation of circulating tumor cells (CTCs) and their colonization of distant organs. In this paper, we will review the established roles of CXCL8 signaling in CRC and discuss the possible strategies of targeting CXCL8 signaling for overcoming CRC drug resistance and cancer progression, including direct targeting of CXCL8/CXCR1/2 or indirect targeting through the inhibition of CXCL8-CXCR1/2 signaling., Competing Interests: The authors disclose no potential conflicts of interest., (Copyright © 2019 Yaqin Bie et al.)

Immune cells congregate at specific loci to fight infections during inflammatory responses, a process that must be transient and self-resolving. Cell dispersal promotes resolution, but it remains unclear how transition from clustering to dispersal is regulated. Here we show, using quantitative live imaging in zebrafish, that differential ligand-induced trafficking of chemokine receptors such as Cxcr1 and Cxcr2 orchestrates the state of neutrophil congregation at sites of tissue damage. Through receptor mutagenesis and biosensors, we show that Cxcr1 promotes clustering at wound sites, but is promptly desensitized and internalized, which prevents excess congregation. By contrast, Cxcr2 promotes bidirectional motility and is sustained at the plasma membrane. Persistent plasma membrane residence of Cxcr2 prolongs downstream signaling and is required for sustained exploratory motion conducive to dispersal. Thus, differential trafficking of two chemokine receptors allows coordination of antagonistic cell behaviors, promoting a self-resolving migratory response.

Background: Club Cell protein (CC)16 correlates with lung injury and respiratory complications, which are in part triggered by polymorphonuclear leukocytes (PMNL) in severely traumatized patients (TP). CC16 exerts anti-inflammatory and immunosuppressive effects, however, its influence on PMNL functions after trauma is unknown. Here, we evaluated whether CC16 present in sera from TP could modify the biological functions of PMNL., Methods: Sera from 16 severely injured TP without pneumonia (no P, n = 8) or with pneumonia (P, n = 8) were collected at admission to emergency department (ED) and 1 day prior pneumonia and pre-incubated with or without anti-CC16 antibody for CC16 neutralization. Samples from the equal post-injury days in the corresponding no P group were used. Neutrophils were isolated from healthy volunteers (HV, n = 5) and incubated with 20% of the serum medium from TP, respectively. In PMNL, CD62L, CD11b/CD18 and CD31 expression, migratory capacity, phagocytosis rate, oxidative burst and apoptosis were investigated. In isolated PMNL, CXCR1 and CXCR2 were neutralized before stimulation with CC16, and oxidative burst, phagocytosis and apoptosis were analyzed in neutrophils and their subsets., Results: Serum from the P group enhanced significantly PMNL migration compared to no P group, while CC16-neutralization further increased the migratory rate of PMNL in both groups. CC16-neutralization increased significantly the expression of CD62L in the P group at ED. Oxidative burst was significantly increased in the P group vs. no P during the study period. CC16 seemed to have no influence on oxidative burst and phagocytosis in TP. However, in a more controlled study design, CC16 induced a significant increase of oxidative burst and a decrease of apoptosis of CD16 + granulocytes. These effects were markedly observed in mature CD16 bright CD62L bright and immune suppressive CD16 bright CD62L dim neutrophils. In mature subset, CXCR1 and CXCR2 neutralization diminished CC16-induced effects., Conclusions: CC16 in sera from multiply traumatized patients, notably of those with pneumonia, has significant effects on PMNL. The results suggest an association of CC16 with CXCR1 and CXCR2. Our data suggest that CC16 reduces the migratory capacity of PMNL and thus modulates their function in patients with respiratory complications after trauma.

One of the prominent features of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is the excessive recruitment of leukocytes to the central nervous system (CNS), which leads to an inflammatory response-with chemokines and their receptors playing the main role in this recruitment. The aim of the study was to examine the relation of CXCR1 and CXCR2, both of which are involved in the trafficking of lymphocytes into the CNS, with the outcome of HTLV-1 infection. The mRNA levels of CXCR1 and CXCR2 were examined in peripheral blood mononuclear cells (PBMCs) of HAM/TSP patients, HTLV-1 asymptomatic carriers (ACs), and healthy controls (HCs). Furthermore, the frequency of CD4 + and CD8 + T cells expressing CXCR1 and CXCR2 was evaluated in the studied groups. The results of the present study showed a substantial increase in the mean mRNA expression of CXCR2 in the HAM/TSP patients compared to the HCs and ACs (p < 0.001). A positive correlation was also found between PVL and CXCR2 mRNA expression in the total population of HTLV-1-infected subjects (R = 0.526, p < 0.001). Moreover, the percentage of CD8 + CXCR2-expressing cells was higher in HAM/TSP patients compared to ACs and HCs (p < 0.05, p < 0.01, respectively). Although the percentage of CD4 + CXCR2-expressing cells was higher in HAM/TSP patients than in ACs and HCs, a significant difference was only found between HAM/TSP patients and HCs (p < 0.05). No significant difference in the CXCR1 mRNA expression was observed in the studied groups. The frequency of the CD8 + CXCR1- and CD4 + CXCR1-expressing cells was significantly lower in HAM/TSP patients than in ACs and HCs (p < 0.001 and p < 0.01, respectively). In conclusion, the high frequency of CXCR2 CD8 + T cells and the high levels of CXCR2 mRNA expression in HAM/TSP patients are associated with disease pathogenesis, while the high frequencies of CXCR1 T cells in ACs might suggest that these cells act as effector CD8 T cells and are involved in controlling the viral spread and modulation of the immune response.

Chimeric antigen receptor (CAR) T-cell therapy targeting solid tumors has stagnated as a result of tumor heterogeneity, immunosuppressive microenvironments, and inadequate intratumoral T cell trafficking and persistence. Early (≤3 days) intratumoral presentation of CAR T cells post-treatment is a superior predictor of survival than peripheral persistence. Therefore, we have co-opted IL-8 release from tumors to enhance intratumoral T-cell trafficking through a CAR design for maximal antitumor activity in solid tumors. Here, we demonstrate that IL-8 receptor, CXCR1 or CXCR2, modified CARs markedly enhance migration and persistence of T cells in the tumor, which induce complete tumor regression and long-lasting immunologic memory in pre-clinical models of aggressive tumors such as glioblastoma, ovarian and pancreatic cancer.

Clear cell Renal Cell (RCC) and Head and Neck Squamous Cell Carcinomas (HNSCC) are characterized by a pro-angiogenic/pro-inflammatory context. Despite conventional or targeted therapies, metastatic RCC and HNSCC remain incurable. Alternative treatments to reference therapies (sunitinib, a multi tyrosine kinase inhibitor for RCC or cisplatin for HNSCC) are urgently needed on relapse. Here, we described the relevance of targeting the ELR+CXCL cytokines receptors, CXCR1/2, for the treatment of these two cancer types. Methods : The relevance to patient treatment was evaluated by correlating the ELR + CXCL/CXCR1/2 levels to survival using online available data. We report herein the synthesis of new pharmacological inhibitors of CXCR1/2 with anti-proliferation/survival activity. The latter was evaluated with the XTT assay with leukemic, breast, RCC and HNSCC cell lines. Their relevance as an alternative treatment was tested on sunitinib- and cisplatin- resistant cells. The most efficient compound was then tested in a mouse model of RCC and HNSCC. Results : RCC and HNSCC expressed the highest amounts of CXCR1/2 of all cancers. High levels of ELR + CXCL cytokines (CXCL1, 2, 3, 5, 6, 7, 8) correlated to shorter survival. Among the 33 synthesized and tested molecules, compound C29 reduced ELR + CXCL/CXCR1/2-dependent proliferation and migration of endothelial cells. C29 exerted an anti-proliferation/survival activity on a panel of cancer cells including naive and resistant RCC and HNSCC cells. C29 reduced the growth of experimental RCC and HNSCC tumors by decreasing tumor cell proliferation, angiogenesis and ELR + /CXCL-mediated inflammation. Conclusion : Our study highlights the relevance of new CXCR1/2 inhibitors for the treatment of RCC or HNSCC as first-line treatment or at relapse on reference therapies., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

CXCL8 or interleukin (IL)-8 directs neutrophil migration and activation through interaction with CXCR1 and CXCR2 that belong to the family of G protein-coupled receptors (GPCRs). Naturally occurring posttranslational modifications of the NH₂-terminal region of CXCL8 affect its biological activities, but the underlying molecular mechanisms are only partially understood. Here, we studied the implications of site-specific citrullination and truncation for the signaling potency of CXCL8. Native CXCL8(1-77), citrullinated [Cit5]CXCL8(1-77) and the major natural isoform CXCL8(6-77) were chemically synthesized and tested in internalization assays using human neutrophils. Citrullinated and truncated isoforms showed a moderately enhanced capacity to induce internalization of CXCR1 and CXCR2. Moreover, CXCL8-mediated activation of Gα i -dependent signaling through CXCR1 and CXCR2 was increased upon modification to [Cit5]CXCL8(1-77) or CXCL8(6-77). All CXCL8 variants promoted recruitment of β-arrestins 1 and 2 to CXCR1 and CXCR2. Compared to CXCL8(1-77), CXCL8(6-77) showed an enhanced potency to recruit β-arrestin 2 to both receptors, while for [Cit5]CXCL8(1-77) only the capacity to induce β-arrestin 2 recruitment to CXCR2 was increased. Both modifications had no biasing effect, i.e., did not alter the preference of CXCL8 to activate either Gα i -protein or β-arrestin-dependent signaling through its receptors. Our results support the concept that specific chemokine activities are fine-tuned by posttranslational modifications.

Neutrophils, immune cells crucial for protecting against invading pathogens, are important in sepsis. Neutrophil migration is regulated by chemokine receptors and their cognate ligands. Our previous study investigated the effect of n‑butanol extract from Folium isatidis on lipopolysaccharide (LPS)‑induced septic shock. The present study stimulated neutrophils with LPS to explore the influence of LPS on cell. Neutrophils were then pretreated with n‑butanol extract from Folium isatidis followed by LPS to examine the effect of this extract on neutrophil chemotaxis. The results showed that LPS decreased the expression levels of CXC‑chemokine receptor (CXCR)1, CXCR2 and L‑selectin (CD62L), and increased the expression of interleukin‑8 (IL‑8) by neutrophils. The addition of n‑butanol extract from Folium isatidis inhibited this LPS‑induced downregulation of CXCR1, CXCR2 and CD62L, and decreased the expression of IL‑8 on neutrophils. In addition, n‑butanol extract promoted myeloperoxidase activity in neutrophils. Taken together, LPS downregulated the expression of chemokine receptors, leading to the failure of neutrophils to migrate to sites of infection. The addition of n‑butanol extract, which promoted the ability of neutrophils to migrate, is a natural product and potential therapeutic agent with which to target neutrophil chemotaxis during LPS stimulation.

Chemokine CXCL8 plays a pivotal role in host immune response by recruiting neutrophils to the infection site. CXCL8 exists as monomers and dimers, and mediates recruitment by interacting with glycosaminoglycans (GAGs) and activating CXCR1 and CXCR2 receptors. How CXCL8 monomer and dimer interactions with both receptors and GAGs mediate trafficking is poorly understood. In particular, both haptotactic (mediated by GAG-bound chemokine) and chemotactic (mediated by soluble chemokine) gradients have been implicated, and whether it is the free or the GAG-bound CXCL8 monomer and/or dimer that activates the receptor remains unknown. Using solution NMR spectroscopy, we have now characterized the binding of heparin-bound CXCL8 monomer and dimer to CXCR1 and CXCR2 receptor N-domains. Our data provide compelling evidence that heparin-bound monomers and dimers are unable to bind either of the receptors. Cellular assays also indicate that heparin-bound CXCL8 is impaired for receptor activity. Considering dimer binds GAGs with higher affinity, dimers will exist predominantly in the GAG-bound form and the monomer in the free form. We conclude that GAG interactions determine the levels of free CXCL8, and that it is the free, and not GAG-bound, CXCL8 that activates the receptors and mediates recruitment of blood neutrophils to the infected tissue., (© 2017 The Authors.)

Studies in recent years have established that the principal effects in cardiac cell therapy are associated with paracrine/autocrine factors. We combined several complementary techniques to define human cardiac progenitor cell (CPC) secretome constituted by 914 proteins/genes; 51% of these are associated with the exosomal compartment. To define the set of proteins specifically or highly differentially secreted by CPC, we compared human mesenchymal stem cells and dermal fibroblasts; the study defined a group of growth factors, cytokines and chemokines expressed at high to medium levels by CPC. Among them, IL-1, GROa (CXCL1), CXCL6 (GCP2) and IL-8 are examples whose expression was confirmed by most techniques used. ELISA showed that CXCL6 is significantly overexpressed in CPC conditioned medium (CM) (18- to 26-fold) and western blot confirmed expression of its receptors CXCR1 and CXCR2. Addition of anti-CXCL6 completely abolished migration in CPC-CM compared with anti-CXCR2, which promoted partial inhibition, and anti-CXCR1, which was inefficient. Anti-CXCL6 also significantly inhibited CPC CM angiogenic activity. In vivo evaluation also supported a relevant role for angiogenesis. Altogether, these results suggest a notable angiogenic potential in CPC-CM and identify CXCL6 as an important paracrine factor for CPC that signals mainly through CXCR2.

Tyrosine sulfation, a post-translational modification found on many chemokine receptors, typically increases receptor affinity for the chemokine ligand. A previous bioinformatics analysis suggested that a sulfotyrosine (sY)-binding site on the surface of the chemokine CXCL12 may be conserved throughout the chemokine family. However, the extent to which receptor tyrosine sulfation contributes to chemokine binding has been examined in only a few instances. Computational solvent mapping correctly identified the conserved sulfotyrosine-binding sites on CXCL12 and CCL21 detected by nuclear magnetic resonance (NMR) spectroscopy, demonstrating its utility for hot spot analysis in the chemokine family. In this study, we analyzed five chemokines that bind to CXCR2, a subset of which also bind to CXCR1, to identify hot spots that could participate in receptor binding. A cleft containing the predicted sulfotyrosine-binding pocket was identified as a principal hot spot for ligand binding on the structures of CXCL1, CXCL2, CXCL7, and CXCL8, but not CXCL5. Sulfotyrosine titrations monitored via NMR spectroscopy showed specific binding to CXCL8, but not to CXCL5, which is consistent with the predictions from the computational solvent mapping. The lack of CXCL5-sulfotyrosine interaction and the presence of CXCL8-sulfotyrosine binding suggests a role for receptor post-translational modifications regulating ligand selectivity., Competing Interests: Brian F. Volkman and Michael B. Dwinell are co-founders and have ownership interests in Protein Foundry, LLC. The other authors declare no conflict of interest.

Background: Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis. Chemokines play important roles in the progression of many malignancies; however, the role of chemokine receptor expression in clinical cases of PDAC is unclear. Moreover, little is known about DARC, a decoy receptor of CXC chemokines, in the regulation of tumor progression., Methods: Functions of chemokine receptors were evaluated using surgical specimens collected from PDAC patients, and PDAC cell lines., Results: CXCR2 expression had no impacts on predicting prognosis, but low DARC expression in cancer cells was an independent risk factor for poor prognosis. In PDAC with low DARC expression, tumor sizes were larger and vascular invasion was increased. High CXCR2 expression was a significant predictor for poor prognosis, only in PDAC patients with low DARC expression. CXCR2 signaling induced STAT3 activation in PDAC, resulting in promoting cell cycle progression, inhibiting apoptosis, inducing angiogenesis, and enhancing invasiveness. DARC inhibited STAT3 activation by down-regulating CXCR2 signaling. These effects were confirmed by EMSA in vitro. DARC knockdown significantly increased cell proliferation in CFPAC-1 cells with high DARC expression, by activating STAT3. In contrast, CXCR2 knockdown inhibited the proliferative effects of IL-8 in MIA PaCa-2 cells with low DARC expression. Moreover, the inhibitory effect of CXCR2 antagonist on PDAC cell proliferation was more powerful in MIA PaCa-2 cells than CFPAC-1 cells., Conclusions: DARC expressing in cancer cells inhibits PDAC progression by suppressing STAT3 activation through the inhibition of CXCR2 signaling. Therefore, DARC is a novel prognostic predictor and a potential therapeutic target for PDAC., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The chemokine receptors CXCR1/2 and their ligand CXCL8 are essential for the activation and trafficking of inflammatory mediators as well as tumor progression and metastasis. The CXCL8-CXCR1/2 signaling axis is involved in the pathogenesis of several diseases including chronic obstructive pulmonary diseases (COPD), asthma, cystic fibrosis and cancer. Interaction between CXCL8 secreted by select cancer cells and CXCR1/2 in the tumor microenvironment is critical for cancer progression and metastasis. The CXCL8-CXCR1/2 axis may play an important role in tumor progression and metastasis by regulating cancer stem cell (CSC) proliferation and self-renewal. During the past two decades, several small-molecule CXCR1/2 inhibitors, CXCL8 releasing inhibitors, and neutralizing antibodies against CXCL8 and CXCR1/2 have been reported. As single agents, such inhibitors are expected to be efficacious in various inflammatory diseases. Several preclinical studies suggest that combination of CXCR1/2 inhibitors along with other targeted therapies, chemotherapies, and immunotherapy may be effective in treating select cancers. Currently, several of these inhibitors are in advanced clinical trials for COPD, asthma, and metastatic breast cancer. In this review, we provide a comprehensive analysis of the role of the CXCL8-CXCR1/2 axis and select genes co-expressed in this pathway in disease progression. We also discuss the latest progress in developing small-molecule drugs targeting this pathway., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Chemotherapy-induced peripheral neuropathy (CIPN) is a type of neuropathic pain that represents a frequent and serious consequence of chemotherapy agents. Over the last years, significant progress has been achieved in elucidating the underlying pathogenesis of CIPN. The interference of taxanes with microtubule has been proposed as a mechanism that leads to altered axonal transport and to permanent neurological damages. The inflammatory process activated by chemotherapeutic agents has been considered as a potential trigger of nociceptive process in CIPN.In this study we investigated the effect of reparixin, an inhibitor of CXCR1/CXCR2, in suppressing the development of paclitaxel-induced nociception in rats. Moreover, reparixin activity in reversing the neurotoxic effects induced by paclitaxel or GRO/KC in F11 cells was also analyzed.Reparixin administered by continuous infusion ameliorated paclitaxel-induced mechanical and cold allodynia in rats. In F11 cells, reparixin was able to inhibit the increase of acetyladed α-tubulin induced both by paclitaxel and GRO/KC. The subsequent experiments were performed in order to dissect the signal transduction pathways under GRO/KC control, eventually modulated by paclitaxel and/or reparixin. To this aim we found that reparixin significantly counteracted p-FAK, p-JAK2/p-STAT3, and PI3K-p-cortactin activation induced either by paclitaxel or GRO/KC.Overall the present results have identified IL-8/CXCR1/2 pathway as a mechanism involved in paclitaxel-induced peripheral neuropathy. In particular, the obtained data suggest that the inhibition of CXCR1/2 combined with standard taxane therapy, in addition to potentiating the taxane anti-tumor activity can reduce chemotherapy-induced neurotoxicity, thus giving some insight for the development of novel treatments.

Background: Cancer-related inflammation is recognized as a driver for tumor progression and chemokines are important players in both inflammation and the progression of many cancer types. CXC chemokines, especially CXCL8, have been implicated in melanoma growth and metastasis, while less is known for their roles in drug resistance., Methods: We generated drug-resistant cells by continuous exposure to chemotherapeutic drugs and analyzed the mechanism(s) of therapy resistance in malignant melanoma., Results: We report chemotherapies induced upregulation of a variety of chemokines in the CXCR1/CXCR2 network by an NF-κB-dependent mechanism. Notably, analysis of the drug-resistant melanoma cell line selected after prolonged exposure to chemotherapeutic drug dacarbazine revealed higher levels of CXCL8 and CXCR2 compared with parent cells as a signature of drug resistance. CXCR2 neutralization markedly improved sensitivity to dacarbazine in melanoma cells., Conclusion: These data provide insights into what drives melanoma cells to survive after chemotherapy treatment, thus pointing to strategies for developing combined drug therapies for combating the problem of chemotherapy resistance in melanoma., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Persistent infection or chronic inflammation contributes significantly to tumourigenesis and tumour progression. C-X-C motif ligand 8 (CXCL8) is a chemokine that acts as an important multifunctional cytokine to modulate tumour proliferation, invasion and migration in an autocrine or paracrine manner. Studies have suggested that CXCL8 and its cognate receptors, C-X-C chemokine receptor 1 (CXCR1) and C-X-C chemokine receptor 2 (CXCR2), mediate the initiation and development of various cancers including breast cancer, prostate cancer, lung cancer, colorectal carcinoma and melanoma. CXCL8 also integrates with multiple intracellular signalling pathways to produce coordinated effects. Neovascularisation, which provides a basis for fostering tumour growth and metastasis, is now recognised as a critical function of CXCL8 in the tumour microenvironment. In this review, we summarize the biological functions and clinical significance of the CXCL8 signalling axis in cancer. We also propose that CXCL8 may be a potential therapeutic target for cancer treatment., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

NOD1 (nucleotide-binding oligomerization domain 1) is overexpressed in head and neck squamous cell carcinoma (HNSCC) cells, as is IL-8 in cancer cells. However, the mechanism of the IL-8-mediated overexpression of NOD in HNSCC not been identified. This study determines whether IL-8 promotes tumor progression via the NOD signaling pathway in HNSCC. Higher IL-8, NOD1 and receptor-interacting protein kinase (RIP2) expressions were observed in HNSCC tissue than in non-cancerous matched tissue (NCMT), whereas NOD2 was weakly expressed. Furthermore, IL-8 stimulated the proliferation of HNSCC cells (SCC4, SCC9 and SCC25) but not dysplastic oral mucosa DOK cells. Exposure to IL-8 increased the clonogenicity of HNSCC cells. IL-8 siRNA inhibited cell proliferation and cell colony formation, suggesting that IL-8 is involved in HNSCC cancer progression. The expressions of CXCR1 and CXCR2 were higher in HNSCC tissue than in NCMT. HNSCC cells that were exposed to IL-8 exhibited higher expression of CXCR1/2 than did controls. The blocking of IL-8 by siRNA reduced CXCR1/2 expression in HNSCC cells, suggesting that the cancer progression of HNSCC cells that is induced by IL-8 depends on CXCR1/2. Additionally, IL-8 is associated with increased NOD1 and RIP2 expression and reduced NOD2 expression in three types of HNSCC cells. The blocking of IL-8 by siRNA reduces IL-8, NOD1 and RIP2 expressions in HNSCC cells, but not the level of NOD2. These results suggest that IL-8 has an important role in HNSCC progression via a CXCR1/2-meidated NOD1/RIP2 signaling pathway., Competing Interests: None.

Exosomes are small membrane vesicles released by different cell types, including hepatocytes, that play important roles in intercellular communication. We have previously demonstrated that hepatocyte-derived exosomes contain the synthetic machinery to form sphingosine-1-phosphate (S1P) in target hepatocytes resulting in proliferation and liver regeneration after ischemia/reperfusion (I/R) injury. We also demonstrated that the chemokine receptors, CXCR1 and CXCR2, regulate liver recovery and regeneration after I/R injury. In the current study, we sought to determine if the regulatory effects of CXCR1 and CXCR2 on liver recovery and regeneration might occur via altered release of hepatocyte exosomes. We found that hepatocyte release of exosomes was dependent upon CXCR1 and CXCR2. CXCR1-deficient hepatocytes produced fewer exosomes, whereas CXCR2-deficient hepatocytes produced more exosomes compared to their wild-type controls. In CXCR2-deficient hepatocytes, there was increased activity of neutral sphingomyelinase (Nsm) and intracellular ceramide. CXCR1-deficient hepatocytes had no alterations in Nsm activity or ceramide production. Interestingly, exosomes from CXCR1-deficient hepatocytes had no effect on hepatocyte proliferation, due to a lack of neutral ceramidase and sphingosine kinase. The data demonstrate that CXCR1 and CXCR2 regulate hepatocyte exosome release. The mechanism utilized by CXCR1 remains elusive, but CXCR2 appears to modulate Nsm activity and resultant production of ceramide to control exosome release. CXCR1 is required for packaging of enzymes into exosomes that mediate their hepatocyte proliferative effect., Competing Interests: The authors have declared that no competing interests exist.

Pilomatricoma is a rare benign tumor of the hair follicle matrix cells, which associates during its evolution a foreign body-like inflammatory process. We have investigated three such tumors, two of them displaying a rather poor stroma, while the third was distinctive due to its stroma and large numbers of inflammatory cells infiltrating the tumor. The analysis of IL-8 (interleukin-8), CXCR1 (IL-8RA - IL-8 receptor alpha) and CXCR2 (IL-8RB - IL-8 receptor beta) expression showed that these molecules are present not only in many different types of inflammatory and endothelial cells, but also in several tumor basaloid, transitional and even few ghost cells. Taking into consideration the roles played by IL-8 and its two receptors, CXCR1 and CXCR2, this pattern of expression offers some insights on the potential roles of these molecules in tumor survival, cell proliferation and angiogenesis. Furthermore, given the expression of IL-1 (interleukin-1) and TNF (tumor necrosis factor) receptors by the tumor cells, IL-8 production by such cells might be under the control of these pro-inflammatory cytokines.

The HIV-1 matrix protein p17 (p17) plays a crucial role in the virus life cycle. It is released in the extracellular space from HIV-1-infected cells and accumulates in the tissues of patients, even in those successfully treated with highly active antiretroviral therapy. Extracellular p17 deregulates the biological functions of many different cells that are directly or indirectly implicated in AIDS pathogenesis. All p17 actions depend on interaction between its functional epitope (AT20), located at the protein N-terminal region, and different receptors expressed on target cells. This finding corroborates the importance of impeding p17/p17 receptors interaction as a contribution to block AIDS. In this article we review the interaction of p17 with heparan sulfate proteoglycans (HSPGs) and with the chemokine (C-X-C motif) receptor 1 (CXCR1) and 2 (CXCR2). We provide details on how p17 interacts with its receptors and how these interactions are central to the p17 biological activities. Moreover, we highlight the existence of a p17 variant, named S75X, which displays opposite effects on B-cell proliferation as compared to p17. A two-site model for p17 interaction with G-coupled receptors provides a possible explanation on how mutations naturally occurring within the primary amino acid structure can lead S75X to activate the Akt signaling pathway and to promote B-cell growth and transformation. Identification of p17 interaction with HSPGs, CXCR1 and CXCR2 as a fundamental event in supporting its activity could help to find new treatment approaches aimed at blocking all p17/p17 receptors interactions and, consequently, p17 detrimental activities.

Platinum based drugs are the cornerstone of chemotherapy for ovarian cancer, however the development of chemoresistance hinders its success. IL-8 is involved in regulating several pro-survival pathways in cancer. We studied the expression of IL-8 and IL-8 receptors in platinum sensitive and resistant cell lines. Using qRT-PCR and immunohistochemistry, both platinum sensitive (PEA1, PEO14) and resistant (PEA2, PEO23) show increased expression of IL-8 and IL-8 receptors. IL-8RA shows nuclear and cytoplasmic expression, whilst IL-8RB is present solely in the cytoplasm. Knockdown of IL-8 increased sensitivity to cisplatin in platinum sensitive and reversed platinum resistance in resistant cell lines, decreased the expression of anti-apoptotic Bcl-2 and decreased inhibitory phosphorylation of pro-apoptotic Bad. IL-8 receptor antagonist treatment also enhanced platinum sensitivity. Nuclear localisation of IL-8RA was only detected in platinum resistant tumours. Inhibition of IL-8 signalling can enhance response in platinum sensitive and resistant disease. Nuclear IL-8RA may have potential as a biomarker of resistant disease.

This study describes an analytical method for bioaffinity and selectivity assessment of CXCR2 antagonists and their metabolites. The method is based on liquid chromatographic separation (LC) of metabolic mixtures followed by parallel mass spectrometry (MS) identification and bioaffinity determination. The bioaffinity is assessed using radioligand binding assays in 96-well plates after at-line nanofractionation. The described method was optimized for chemokines and low-molecular weight CXCR2 ligands. The limits of detection (LODs; injected amounts) for MK-7123, a high affinity binder to both CXCR1 and CXCR2 receptors belonging to the diaminocyclobutendione chemical class, were 40pmol in CXCR1 binding and 8pmol in CXCR2 binding. For CXCL8, the LOD was 5pmol in both binding assays. A control compound was always taken along with each bioassay plate as triplicate dose-response curve. For MK-7123, the calculated IC50 values were 314±59nM (CXCR1 binding) and 38±11nM (CXCR2 binding). For CXCL8, the IC50 values were 6.9±1.4nM (CXCR1 binding) and 2.7±1.3nM (CXCR2 binding). After optimization, the method was applied to the analysis of metabolic mixtures of eight LMW CXCR2 antagonists generated by incubation with pig liver microsomes. Moreover, metabolic profiling of the MK-7123 compound was described using the developed method. Three bioactive metabolites were found, two of which were (partially) identified. This method is suitable for bioaffinity and selectivity assessment of mixtures targeting the CXCR2. In contrary to conventional LC-MS based metabolic profiling studies done at the early lead discovery stage, additional qualitative bioactivity information of drug metabolites is obtained with the method described., (Copyright © 2015 Elsevier B.V. All rights reserved.)

CXCR2 interacts with a wide range of chemokines and CXCR2 antagonists may have therapeutic value for treatment-resistant metastatic carcinomas. We aimed to explore regulation of activity of CXCR2 and its ligand, MIP-2, in metastatic breast carcinoma. We used mouse breast carcinoma cells metastasize to brain (4TBM), liver (4TLM), and heart (4THM) and explored the extra- and intracellular mechanisms effecting MIP-2 secretion using CXCR2 antagonist and inhibitors of downstream signaling molecules. 4TBM, 4TLM, and 4THM cells include cancer stem cell features and metastasize extensively. We also determined kinetics of MIP-2 secretion in 4T1 and non-metastatic 67NR mouse breast carcinoma cells. We found that there is an autocrine-inhibition of MIP-2 secretion. Specifically, metastatic cells selectively express CXCR2 only, and not CXCR1 and attenuating CXCR2 activity with SB225002 increased MIP-2 secretion. This may be due to the inhibition of protein kinase C (PKC) activity since RO318220; a specific inhibitor of PKC also increased MIP-2 secretion. Attenuating CXCR2 activity with SB225002, otherwise suppressed proliferation of 4THM and 4TBM cells. Tumor explants and cancer-associated fibroblasts obtained from 4TLM, 4THM, and 4TBM primary tumors secreted high levels of MIP-2. Surprisingly, CXCR2 expression was low in 4TLM cells demonstrating that liver metastatic cells might be resistant to the anti-tumoral effects of CXCR2 antagonists. Our results demonstrated that resistance to anti-proliferative effects of CXCR2 may also arise from feedback increases in MIP-2 secretion. Activation of PI3 K pathway augments MIP-2 secretion, hence possible resistance to the antitumor effects of CXCR2 antagonists might be prevented with inhibitors of PI3 K.

The CXCR1 and CXCR2 are the prototypical receptors and are the only known receptors for mammalian ELR+ (Glu-Leu-Arg) CXC chemokines, including CXCL8 (interleukin 8). These receptors transduce the ELR+ chemokine signals and operate the downstream signaling pathways in inflammation and innate immunity. In this study, we report the identification and characterization of CXCR1 and CXCR2 genes from rock bream fish (OfCXCR1 and OfCXCR2) at the molecular level. The cDNA and genomic DNA sequences of the OfCXCR1 and OfCXCR2 were identified from a transcriptome library and a custom-constructed BAC library, respectively. Both OfCXCR genes consisted of two exons, separated by an intron. The 5'-flanking regions of OfCXCR genes possessed multiple putative transcription factor binding sites related to immune response. The coding sequences of OfCXCR1 and OfCXCR2 encoded putative peptides of 355 and 360 amino acids (aa), respectively. The deduced aa sequences of OfCXCR1 and OfCXCR2 comprised of a G-protein coupled receptors (GPCR) family 1 profile with a GPCR signature and a DRY motif. In addition, seven conserved transmembrane regions were predicted in both OfCXCRs. While our multiple alignment study revealed the functionally significant conserved elements of the OfCXCR1 and OfCXCR2, phylogeny analyses further confirmed their position in teleost sub clade, in which they manifested an evolutionary relatedness with other fish counterparts. Based on comparative analyses, teleost CXC chemokine receptors appear to be distinct from their non-fish orthologs in terms of evolution (both CXCR1 and CXCR2) and genomic organization (CXCR2). Quantitative real-time PCR (qPCR) detected the transcripts of OfCXCR1 and OfCXCR2 in eleven examined tissues, with higher levels in head kidney, kidney and spleen highlighting their crucial importance in immunity. In vitro stimulation of peripheral blood leukocytes (PBLs) with concanavalin A (Con A) resulted in modulation of OfCXCR2 transcription, but not that of OfCXCR1. In addition, the magnitude of the OfCXCR1 and OfCXCR2 transcripts in head kidney and spleen was differentially increased after the in vivo administration of immune stimulants, LPS and poly I:C and in the infection models injected with rock bream irido virus, Edwardsiella tarda and Streptococcus iniae. These lines of evidence suggest that these receptors may play an important role(s) in immune responsive signaling during pathogenesis of rock bream., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Chemokines are small proteins that control several tissue functions, including cell recruitment and activation under homeostatic and inflammatory conditions. CXCL8 (interleukin-8) is a member of the chemokine family that acts on CXCR1 and CXCR2 receptors. CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, and CXCL7 are also ELR+ chemokine members that bind to these receptors, especially CXCR2. The majority of studies on the biology of CXCL8 and their receptors have been performed in polymorphonuclear leukocytes. However, many other cells express CXCR1/CXCR2, including epithelial, endothelial, fibroblasts and neurons, contributing to the biological effects of CXCL8. There is substantial amount of experimental data suggesting that CXCL8 and receptors contribute to elimination of pathogens, but may also contribute significantly to disease-associated processes, including tissue injury, fibrosis, angiogenesis and tumorigenesis. Here, we discuss the biology of CXCL8 family and the potential therapeutic use of antagonists or blockers of these molecules in the context of organ-specific diseases.

Background: Interleukin 8 (IL8) has been contradictorily associated with the risk of myocardial infarction (MI)., Aim: To investigate the association of IL8 serum levels with the risk of MI and the association of the IL8 (IL8) and IL8 receptors (CXCR1 and CXCR2) genetic variants with IL8 levels and MI risk in a large case control study, the Stockholm Heart Epidemiology Program., Methods and Results: IL8 levels (pg/mL) were divided into quartiles and the MI risk was calculated by logistic regression and expressed as odds ratio (OR) and 95% CI. Two IL8 SNPs (rs4073A/T, rs2227306C/T) and three SNPs tagging CXCR1 and CXCR2 (rs4674258C/T, rs1008563C/T, rs6723449T/C) were analyzed for association with IL8 levels and with MI risk. Multivariate adjusted ORs for MI risk by IL8 levels in the highest quartiles indicated reduced point estimates in both women (OR 0.37; 95% CI 0.2-0.8) and men when compared to the lowest quartile. In female cases, IL8 levels decreased progressively in the six months after MI (p=0.03). IL8, CXCR1 and CXCR2 genetic variants were not associated with IL8 levels. In men, the T allele at the IL8 SNP rs4073 was associated with a slight increase in the MI risk under an additive and a recessive model of inheritance., Conclusions: IL8 serum levels were associated with a reduced occurrence of MI among women, whereas IL8 was associated with a slightly increased risk among men, possibly through different mechanisms. These data suggest that the biological effects of IL8 on MI risk may vary over time and warrant further cohort studies with repetitive IL8 measurements., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Mutations in the von Hippel-Lindau gene upregulate expression of the central angiogenic factor VEGF, which drives abnormal angiogenesis in clear cell renal cell carcinomas (ccRCC). However, the overexpression of VEGF in these tumors was not found to correlate with overall survival. Here, we show that the proangiogenic, proinflammatory cytokine CXCL7 is an independent prognostic factor for overall survival in this setting. CXCL7 antibodies strongly reduced the growth of ccRCC tumors in nude mice. Conversely, conditional overexpression of CXCL7 accelerated ccRCC development. CXCL7 promoted cell proliferation in vivo and in vitro, in which expression of CXCL7 was induced by the central proinflammatory cytokine interleukin (IL)-1β. ccRCC cells normally secrete low amounts of CXCL7; it was more highly expressed in tumors due to high levels of IL-1β there. We found that a pharmacological inhibitor of the CXCL7 receptors CXCR1 and CXCR2 (SB225002) was sufficient to inhibit endothelial cell proliferation and ccRCC growth. Because CXCR1 and CXCR2 are present on both endothelial and ccRCC cells, their inhibition affected both the tumor vasculature and the proliferation of tumor cells. Our results highlight the CXCL7/CXCR1/CXCR2 axis as a pertinent target for the treatment of ccRCC.

To observe the effect of acupuncture on CXCL8 receptors (CXCR1 and CXCR2) in rat endometrium experiencing embryo implantation failure, 72 pregnant rats were randomly divided into four groups: normal group (N), embryo implantation failure group (M), acupuncture treatment group (A), and progestin treatment group (W). Then the rats in each group were equally randomized into a day-6 (D6) group, a day-8 (D8) group, and a day-10 (D10) group. The rats in group M, group A, and group W were treated with mifepristone-sesame oil solution on day 1, while the rats in group N were injected with the same amount of sesame oil. Meanwhile, "Housanli" and "Sanyinjiao" were selected for acupuncture. From day 1 to the time of death, the rats in group A were fastened up and then acupuncture was administered while the rats in group N and group M were only fixed, and the rats in group W were given progestin. The number of implanted embryos was calculated. The expression of CXCR1 and CXCR2 in rat endometrium was detected by immunohistochemistry, Western blotting and real-time PCR. Compared to group N, the average number of implanted embryos, the protein and mRNA expression of CXCR1 (D6, D8 and D10), and the protein and mRNA expression of CXCR2 (D8 and D10) in rat endometrium were significantly decreased in group M. Compared to group M, there was significant elevation in the average number of implanted embryos, the protein expression (D6, D8 and D10) and mRNA expression (D8) of CXCR1 in rat endometrium of group A, and the protein expression (D8 and D10) and mRNA expression (D8) of CXCR2 in rat endometrium of group W. These findings indicated that acupuncture can increase the number of implanted embryos in rats of embryo implantation failure, which may be relevant with up-regulation the expression of CXCR1 and CXCR2 at maternal-fetal interface of rats with embryo implantation failure.

Chemokines mediate diverse functions from organogenesis to mobilizing leucocytes, and are unusual agonists for class-A GPCRs (G-protein-coupled receptors) because of their large size and multi-domain structure. The current model for receptor activation, which involves interactions between chemokine N-loop and receptor N-terminal residues (Site-I) and between chemokine N-terminal and receptor extracellular loop/transmembrane residues (Site-II), fails to describe differences in ligand/receptor selectivity and the activation of multiple signalling pathways. In the present study, we show in neutrophil-activating chemokine CXCL8 that the highly conserved GP (glycine-proline) motif located distal to both N-terminal and N-loop residues couples Site-I and Site-II interactions. GP mutants showed large differences from native-like to complete loss of function that could not be correlated with the specific mutation, receptor affinity or subtype, or a specific signalling pathway. NMR studies indicated that the GP motif does not influence Site-I interactions, but molecular dynamics simulations suggested that this motif dictates substates of the CXCL8 conformational ensemble. We conclude that the GP motif enables diverse receptor functions by controlling cross-talk between Site-I and Site-II, and further propose that the repertoire of chemokine functions is best described by a conformational ensemble model in which a network of long-range coupled indirect interactions mediate receptor activity.

The Staphylococcus aureus leukotoxin ED (LukED) is a pore-forming toxin required for the lethality associated with bacteremia in murine models. LukED targets the chemokine receptor CCR5 to kill T lymphocytes, macrophages, and dendritic cells. LukED also kills CCR5-deficient cells, like neutrophils, suggesting the existence of additional cellular receptors. Here, we identify the chemokine receptors CXCR1 and CXCR2 as the targets of LukED on neutrophils. The LukE subunit binds neutrophils in a specific and saturable manner, and this interaction is inhibited by CXCL8, the high-affinity endogenous ligand of CXCR1 and CXCR2. LukED recognition of CXCR1 and CXCR2 promotes the killing of monocytes and neutrophils in vitro. LukED-mediated targeting of CXCR1 and CXCR2(+) cells contributes to S. aureus pathogenesis and facilitates lethality in systemically infected mice. Thus, LukED is a versatile toxin that endows S. aureus with the ability to simultaneously disarm both innate and adaptive compartments of the host immune response., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Thrombosis promotes leukocyte infiltration into inflamed tissues, leading to organ injury in a broad range of diseases; however, the mechanisms by which thrombi guide leukocytes to sites of vascular injury remain ill-defined. Using mouse models of endothelial injury (traumatic or ischemia reperfusion), we demonstrate a distinct process of leukocyte recruitment, termed "directed intravascular migration," specifically mediated by platelet thrombi. Single adherent platelets and platelet aggregates stimulated leukocyte shape change at sites of endothelial injury; however, only thrombi were capable of inducing directed intravascular leukocyte migration. Leukocyte recruitment and migration induced by platelet thrombi occurred most prominently in veins but could also occur in arteries following ischemia-reperfusion injury. In vitro studies demonstrated a major role for platelet-derived NAP-2 (CXCL-7) and its CXCR1/2 receptor in regulating leukocyte polarization and motility. In vivo studies demonstrated the presence of an NAP-2 chemotactic gradient within the thrombus body. Pharmacologic blockade of CXCR1/2 as well as genetic deletion of NAP-2 markedly reduced leukocyte shape change and intrathrombus migration. These studies define a distinct process of leukocyte migration that is initiated by homotypic adhesive interactions between platelets, leading to the development of an NAP-2 chemotactic gradient within the thrombus body that guides leukocytes to sites of vascular injury.