VDR, CXCR1, CXCR2, PSCA Polymorphisms and Recurrent Urinary Tract Infections in Women: Genetic Association Study.

INTRODUCTION AND HYPOTHESIS: Urinary tract infection (UTI) is one of the most common human infections. Evidence suggests that there might be a genetic predisposition to UTI. Previous small candidate gene studies have suggested that common variants in genes involved in the immune response to UTI could increase susceptibility to the development of recurrent UTI (rUTI). The objective was to conduct a gene association study to replicate previous gene association studies identifying single nucleotide polymorphisms (SNPs) putatively associated with rUTI in adult women. METHODS: Women with a history of rUTI and healthy controls were recruited (n = 1,008) from gynaecology outpatient clinics. Participants completed a signed consent form and questionnaire for phenotyping. DNA was extracted from blood or saliva samples for each participant. Putative associated SNPs were identified from a comprehensive systematic review of prior gene association studies. Primers for each selected SNP were designed, and genotyping was conducted using a competitive polymerase chain reaction (PCR) method. The Chi-squared test was used to assess the association between each variant and rUTI. Genotyping quality was assessed by checking for deviation from Hardy-Weinberg equilibrium. RESULTS: We found no association between SNPs tested in the VDR (p = 0.16, p = 0.09, p = 0.36), CXCR1 (p = 0.09), CXCR2 (p = 0.39), PSCA (p = 0.74) genes, and rUTI in adult women. CONCLUSIONS: To our knowledge, this is the largest study to date, finding no significant associations. Previously reported positive associations may have been due to type 1 error, or genotyping errors. Future studies should adjust for confounders and employ adequate sample sizes. A greater understanding of the genetic components associated with rUTI may influence future treatment guidelines and screening for susceptible patients.

Tumor-Associated Senescent Macrophages, Their Markers, and Their Role in Tumor Microenvironment.

Tumor-associated macrophages (TAMs) are an important component of the tumor microenvironment (TME) and the most abundant population of immune cells infiltrating a tumor. TAMs can largely determine direction of anti-tumor immune response by promoting it or, conversely, contribute to formation of an immunosuppressive TME that allows tumors to evade immune control. Through interactions with tumor cells or other cells in the microenvironment and, as a result of action of anti-cancer therapy, macrophages can enter senescence. In this review, we have attempted to summarize information available in the literature on the role of senescent macrophages in tumors. With the recent development of senolytic therapeutic strategies aimed at removing senescent cells from an organism, it seems important to discuss functions of the senescent macrophages and potential role of the senolytic drugs in reprogramming TAMs to enhance anti-tumor immune response and improve efficacy of cancer treatment.

Pyrrolidine dithiocarbamate in combination with L-N-monomethyl arginine alleviates Staphylococcus aureus infection via regulation of CXCL8/CXCR1 axis in peritoneal macrophages in vitro.

The CXCL8/CXCR1 axis in conjoint with the free radicals and anti-oxidants dictates the severity of inflammation caused by the bacteria, Staphylococcus aureus. S.aureus mediated inflammatory processes is regulated by NF-κB and its product, iNOS. The objective of this study was to examine the effects of inhibition of NF-κB and iNOS on CXCL8/CXCR1, alteration in M1/M2 polarization of macrophages and associated inflammatory responses during S.aureus infection in vitro. For this, the murine peritoneal macrophages were pretreated with NF-κB inhibitor, Pyrrolidine dithiocarbamate (PDTC) and iNOS inhibitor, L-N-monomethyl arginine (LNMMA), either alone or in combination, followed by time-dependent S.aureus infection. The chemotactic migrations of macrophages were determined by the agarose spot assay. The iNOS, NF-κB and CXCR1 protein expressions were evaluated. The ROS level (superoxide, H2O2, NO) and antioxidant activities (SOD, CAT, GSH, arginase) were measured. The intra-macrophage phagoctyic activity had been analyzed by confocal microscopy. S.aureus activated macrophages showed increased iNOS expression that symbolizes M1 characterization of macrophages. The results suggest that the combination treatment of LNMMA + PDTC was effective in diminution of CXCL8 production and CXCR1 expression through downregulation of NF-κB and iNOS signaling pathway. Consequently, there was decrement in macrophage migration, reduced ROS generation, elevated antioxidant enzyme activity as well as bacterial phagocytosis at 90 min post bacterial infection. The increased arginase activity further proves the switch from pro-inflammatory M1 to anti-inflammatory M2 polarization of macrophages. Concludingly, the combination of PDTC + LNMMA could resolve S.aureus mediated inflammation through mitigation of CXCL8/CXCR1 pathway switching from M1 to M2 polarization.

The therapeutic potential of an allosteric non-competitive CXCR1/2 antagonist for diabetic nephropathy.

AIMS: Diabetic nephropathy is a major consequence of inflammation developing in type 1 diabetes, with interleukin-8 (IL-8)-CXCR1/2 axis playing a key role in kidney disease progression. In this study, we investigated the therapeutic potential of a CXCR1/2 non-competitive allosteric antagonist (Ladarixin) in preventing high glucose-mediated injury in human podocytes and epithelial cells differentiated from renal stem/progenitor cells (RSC) cultured as nephrospheres. MATERIALS AND METHODS: We used human RSCs cultured as nephrospheres through a sphere-forming functional assay to investigate hyperglycemia-mediated effects on IL-8 signalling in human podocytes and tubular epithelial cells. RESULTS: High glucose impairs RSC self-renewal, induces an increase in IL-8 transcript expression and protein secretion and induces DNA damage in RSC-differentiated podocytes, while exerting no effect on RSC-differentiated epithelial cells. Accordingly, the supernatant from epithelial cells or podocytes cultured in high glucose was able to differentially activate leucocyte-mediated secretion of pro-inflammatory cytokines, suggesting that the crosstalk between immune and non-immune cells may be involved in disease progression in vivo. CONCLUSIONS: Treatment with Ladarixin during RSC differentiation prevented high glucose-mediated effects on podocytes and modulated either podocyte or epithelial cell-dependent leucocyte secretion of pro-inflammatory cytokines, suggesting CXCR1/2 antagonists as possible pharmacological approaches for the treatment of diabetic nephropathy.

Identification of hub genes and potential biomarkers of neutrophilic asthma: evidence from a bioinformatics analysis.

OBJECTIVE: Asthma is a chronic airway inflammatory disease caused by multiple genetic and environmental factors. This study mainly sought to provide potential therapeutic targets and biomarkers for neutrophilic asthma (NA). METHODS: Three gene expression profiling datasets were obtained from the Genome Expression Omnibus (GEO) database. GSE45111 and GSE41863 were used to identify hub genes and potential biomarkers, and GSE137268 was used for data verification. We verified the repeatability of intragroup data and identified differentially expressed genes (DEGs). Then, we conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the DEGs, and a protein-protein interaction (PPI) network was constructed to identify the hub genes. Finally, receiver operating characteristic (ROC) analysis was used to verify the ability of the hub genes to differentiate between NA and eosinophilic asthma (EA). RESULTS: In this study, we identified 411 DEGs by comprehensive analysis of NA/EA patients and NA/healthy controls (HCs). Ten hub genes (CXCR1, FCGR3B, CXCR2, SELL, S100A12, CSF3R, IL6R, JAK3, CD48, and GNG2) were identified from the PPI network. Finally, based on the ROC analysis, 7 genes showed good diagnostic value for discriminating NA from EA-CXCR1, FCGR3B, CXCR2, SELL, S100A12, CSF3R, and IL6R (AUC > 0.7). CONCLUSION: We identified 7 hub genes that can distinguish NA from EA. The IL-8-mediated signaling may be the primary pathway to determine the NA phenotype in asthma. CXCR1/2 and S100A12 may be the primary genes determining the NA phenotype. CXCR1/2 and S100A12 might be biomarkers and new therapeutic targets for NA.Supplemental data for this article is available online at at.

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.

Association of CXCR1 gene polymorphism with clinical mastitis, reproductive disorders and performance traits in Hardhenu (Bos taurus × Bos indicus) cattle.

The present study investigated the presence of CXCR1 gene polymorphisms and their association with clinical mastitis, reproductive disorders and performance traits in Hardhenu cattle. Genotyping of the targeted SNP rs211042414 (C>T) at the g.106216468 loci of the CXCR1 gene was performed through PCR amplification and Bsa1 restriction enzyme digestion. The genotypic frequencies revealed three genotypes: CC, CT and TT, with the C allele being the most prevalent. Significant associations were found between the targeted SNP and clinical mastitis occurrence using chi-square and logistic regression analyses. The CC genotype showed higher susceptibility to clinical mastitis with a higher odds ratio of 3.47 compared to TT (1.00) and CT (2.90) genotypes (p < .05). Furthermore, least squares analysis revealed significant associations between genotypes and performance traits such as total milk yield, 305-day milk yield and peak yield (p < .05). The CC genotype exhibited higher milk yields than CT and TT genotypes, indicating a positive association between the C allele and increased milk production. These findings have practical implications for the genetic improvement of Hardhenu cattle. Incorporating the identified CXCR1 gene polymorphisms into existing selection criteria can help enhance disease resistance and milk production traits. However, further validation with a larger sample size is necessary to strengthen the observed associations and ensure their practical applicability.

[Roles of the CXCR1/CXCL8 axis in abnormal proliferation of bile duct epithelial cells in primary biliary cholangitis].

Objective: To investigate the role of the CXC chemokine receptor 1 (CXCR1)/CXC chemokine ligand 8 (CXCL8) axis in the abnormal proliferation of bile duct epithelial cells in primary biliary cholangitis (PBC). Methods: 30 female C57BL/6 mice were randomly divided into the PBC model group (PBC group), reparixin intervention group (Rep group), and blank control group (Con group) in an in vivo experiment. PBC animal models were established after 12 weeks of intraperitoneal injection of 2-octanoic acid coupled to bovine serum albumin (2OA-BSA) combined with polyinosinic acid polycytidylic acid (polyI:C). After successful modelling, reparixin was injected subcutaneously into the Rep group (2.5 mg · kg(-1) · d(-1), 3 weeks). Hematoxylin-eosin staining was used to detect histological changes in the liver. An immunohistochemical method was used to detect the expression of cytokeratin 19 (CK-19). Tumor necrosis factor-α (TNF-α), γ-interferon (IFN-γ) and interleukin (IL)-6 mRNA expression were detected by qRT-PCR. Western blot was used to detect nuclear transcription factor-κB p65 (NF-κB p65), extracellularly regulated protein kinase 1/2 (ERK1/2), phosphorylated extracellularly regulated protein kinase 1/2 (p-ERK1/2), Bcl-2-related X protein (Bax), B lymphoma-2 (Bcl-2), and cysteine proteinase-3 (Caspase- 3) expression. Human intrahepatic bile duct epithelial cells were divided into an IL-8 intervention group (IL-8 group), an IL-8+Reparicin intervention group (Rep group), and a blank control group (Con group) in an in vitro experiment. The IL-8 group was cultured with 10 ng/ml human recombinant IL-8 protein, and the Rep group was cultured with 10 ng/ml human recombinant IL-8 protein, followed by 100 nmol/L Reparicin. Cell proliferation was detected by the EdU method. The expression of TNF-α, IFN-γ and IL-6 was detected by an enzyme-linked immunosorbent assay. The expression of CXCR1 mRNA was detected by qRT-PCR. The expression of NF-κB p65, ERK1/2 and p-ERK1/2 was detected by western blot. A one-way ANOVA was used for comparisons between data sets. Results: The results of in vivo experiments revealed that the proliferation of cholangiocytes, the expression of NF-κB and ERK pathway-related proteins, and the expression of inflammatory cytokines were increased in the Con group compared with the PBC group. However, reparixin intervention reversed the aforementioned outcomes (P<0.05). In vitro experiments showed that the proliferation of human intrahepatic cholangiocyte epithelial cells, the expression of CXCR1 mRNA, the expression of NF-κB and ERK pathway-related proteins, and the expression of inflammatory cytokines were increased in the IL-8 group compared with the Con group. Compared with the IL-8 group, the proliferation of human intrahepatic cholangiocyte epithelial cells, NF-κB and ERK pathway-related proteins, and inflammatory indicators were significantly reduced in the Rep group (P < 0.05). Conclusion: The CXCR1/CXCL8 axis can regulate the abnormal proliferation of bile duct epithelial cells in PBC, and its mechanism of action may be related to NF-κB and ERK pathways.

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).

Differential Interleukin-8 thresholds for chemotaxis and netosis in human neutrophils.

In humans, IL-8 (CXCL8) is a key chemokine for chemotaxis of polymorphonuclear leukocytes and monocytes/macrophages when acting on CXCR1 and CXCR2. CXCL8 activity on neutrophils includes chemotaxis and eliciting the extrusion of neutrophil extracellular traps (NETs). In this study, we show that concentrations of IL-8 that induce NETosis surpass in at least one order of magnitude those required to elicit chemoattraction in human neutrophils. IL-8-induced NETosis was less dependent on G-proteins than migration, while extracellular Ca+2 chelation similarly inhibited both processes. Reactive oxygen species (ROS) were more important for NETosis than for chemotaxis as evidenced by neutralization with N-acetyl -cysteine. Interestingly, selective blockade with anti-CXCR1 mAb inhibited NETosis much more readily than chemotaxis, while pharmacological inhibition of both CXCR1 and CXCR2, or selective inhibition for CXCR2 alone, similarly inhibited both functions. Together, these results propose a model according to which low concentrations of IL-8 in a gradient attract neutrophils to the inflammatory foci, while high receptor-saturating concentrations of IL-8 give rise to NETosis once leukocytes reach the core of the inflammatory insult.

Blastemal progenitors modulate immune signaling during early limb regeneration.

Blastema formation, a hallmark of limb regeneration, requires proliferation and migration of progenitors to the amputation plane. Although blastema formation has been well described, the transcriptional programs that drive blastemal progenitors remain unknown. We transcriptionally profiled dividing and non-dividing cells in regenerating stump tissues, as well as the wound epidermis, during early axolotl limb regeneration. Our analysis revealed unique transcriptional signatures of early dividing cells and, unexpectedly, repression of several core developmental signaling pathways in early regenerating stump tissues. We further identify an immunomodulatory role for blastemal progenitors through interleukin 8 (IL-8), a highly expressed cytokine in subpopulations of early blastemal progenitors. Ectopic il-8 expression in non-regenerating limbs induced myeloid cell recruitment, while IL-8 knockdown resulted in defective myeloid cell retention during late wound healing, delaying regeneration. Furthermore, the il-8 receptor cxcr-1/2 was expressed in myeloid cells, and inhibition of CXCR-1/2 signaling during early stages of limb regeneration prevented regeneration. Altogether, our findings suggest that blastemal progenitors are active early mediators of immune support, and identify CXCR-1/2 signaling as an important immunomodulatory pathway during the initiation of regeneration.

Heterogenous presence of neutrophil extracellular traps in human solid tumours is partially dependent on IL-8.

Neutrophil extracellular traps (NETs) are webs of extracellular nuclear DNA extruded by dying neutrophils infiltrating tissue. NETs constitute a defence mechanism to entrap and kill fungi and bacteria. Tumours induce the formation of NETs to the advantage of the malignancy via a variety of mechanisms shown in mouse models. Here, we investigated the presence of NETs in a variety of human solid tumours and their association with IL-8 (CXCL8) protein expression and CD8+ T-cell density in the tumour microenvironment. Multiplex immunofluorescence panels were developed to identify NETs in human cancer tissues by co-staining with the granulocyte marker CD15, the neutrophil marker myeloperoxidase and citrullinated histone H3 (H3Cit), as well as IL-8 protein and CD8+ T cells. Three ELISA methods to detect and quantify circulating NETs in serum were optimised and utilised. Whole tumour sections and tissue microarrays from patients with non-small cell lung cancer (NSCLC; n = 14), bladder cancer (n = 14), melanoma (n = 11), breast cancer (n = 31), colorectal cancer (n = 20) and mesothelioma (n = 61) were studied. Also, serum samples collected retrospectively from patients with metastatic melanoma (n = 12) and NSCLC (n = 34) were ELISA assayed to quantify circulating NETs and IL-8. NETs were detected in six different human cancer types with wide individual variation in terms of tissue density and distribution. At least in NSCLC, bladder cancer and metastatic melanoma, NET density positively correlated with IL-8 protein expression and inversely correlated with CD8+ T-cell densities. In a series of serum samples from melanoma and NSCLC patients, a positive correlation between circulating NETs and IL-8 was found. In conclusion, NETs are detectable in formalin-fixed human biopsy samples from solid tumours and in the circulation of cancer patients with a considerable degree of individual variation. NETs show a positive association with IL-8 and a trend towards a negative association with CD8+ tumour-infiltrating lymphocytes. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Reduction of NETosis by targeting CXCR1/2 reduces thrombosis, lung injury, and mortality in experimental human and murine sepsis.

BACKGROUND: Neutrophil extracellular traps (NETs) facilitate bacterial clearance but also promote thrombosis and organ injury in sepsis. We quantified ex vivo NET induction in septic humans and murine models of sepsis to identify signalling pathways that may be modulated to improve outcome in human sepsis. METHODS: NET formation in human donor neutrophils was quantified after incubation with plasma obtained from patients with sepsis or systemic inflammation (double-blinded assessment of extracellular DNA using immunofluorescence microscopy). NET formation (% neutrophils forming NETs) was correlated with plasma cytokine levels (MultiPlex assay). Experimental sepsis (caecal ligation and puncture or intraperitoneal injection of Escherichia coli) was assessed in C57/BL6 male mice. The effect of pharmacological inhibition of CXCR1/2 signalling (reparixin) on NET formation, organ injury (hepatic, renal, and cardiac biomarkers), and survival in septic mice was examined. RESULTS: NET formation was higher after incubation with plasma from septic patients (median NETs=25% [10.5-46.5%]), compared with plasma obtained from patients with systemic inflammation (14% [4.0-23.3%]; P=0.02). Similar results were observed after incubation of plasma from mice with neutrophils from septic non-septic mice. Circulating CXCR1/2 ligands correlated with NETosis in patients (interleukin-8; r=0.643) and mice (macrophage inflammatory protein-2; r=0.902). In experimental sepsis, NETs were primarily observed in the lungs, correlating with fibrin deposition (r=0.702) and lung injury (r=0.692). Inhibition of CXCR1/2 using reparixin in septic mice reduced NET formation, multi-organ injury, and mortality, without impairing bacterial clearance. CONCLUSION: CXCR1/2 signalling-induced NET formation is a therapeutic target in sepsis, which may be guided by ex vivo NET assays.

Insights into the functional role of grass carp IL-8 in head kidney leukocytes: pro-inflammatory effects and signalling mechanisms.

Interleukin-8 (IL-8) is a critical chemokine regulating immune cells' chemotaxis as well as their physiological or pathological activations. In fish cells, recombinant IL-8 proteins induced transcriptions of pro-inflammatory cytokines. Nonetheless, the exact mechanisms underlying the function of fish IL-8 as a pro-inflammatory cytokine are still unclear. In this paper, the authors first prepared recombinant grass carp IL-8 (rgcIL-8) using an Escherichia coli expression system, and later confirmed rgcIL-8 increased gene expression of il8, il1ß and tumour necrosis factor alpha (tnfα) in grass carp head kidney leukocytes (HKLs). Using signalling pathway inhibitors, the authors showed that rgcIL-8 regulated transcriptions of pro-inflammatory cytokines via MAPK and/or NF-κB signalling pathways. They cloned gcIL-8-specific receptor CXCR1 and subsequently discovered that gcIL-8 could increase the activity of NF-κB and the transcription of IL-1ß via CXCR1. Simultaneously, antibody neutralization assay showed that endogenous IL-8 is partially relevant to the self-regulation of IL-1ß. Moreover, rgcIL-8 led to the expression of inducible nitric oxide synthase gene, causing an accumulation of nitric oxide in the culture medium of HKLs, suggesting the potential of gcIL-8 to mediate inflammatory response. This study not only enriched the function of IL-8 in teleost but also revealed it as a potential target for the inflammatory control in grass carp.

Preoperative blood markers for prediction of recurrence-free survival after surgical treatment of patients with stage III lung adenocarcinoma.

The possibility of the preoperative level of 42 indicators characterizing the cellular composition and metabolism in blood of patients with stage III lung adenocarcinoma (AC) to predict their relapse-free survival was studied. Blood samples of 451 patients with newly diagnosed AK stage III after their surgical treatment (resection volume - R0) have been investigated. The duration of the relapse-free period (period of observation - 1 year), cellular composition of the blood, concentration of C-RP, albumin, Cyfra 21-1 antigens, SCC, TPA, chemokines CXCL5, CXCL8, pyruvate kinase TuM2 PK isoenzyme, HIF-1α and hyaluronic acid in blood serum so as the proportion of blood cells with CXCR1 and CXCR2, CD44V6 receptors in blood serum were measured. To determine the dependence of the duration of the relapse-free period after the treatment on the observation time, Kaplan-Meier graphs were built. The relationship between the determined parameters and survival was judged using single- and multi-factor Cox proportional hazard models. Comparison of groups with different risk of AK recurrence was performed using the Log Rank test and χ2. The assessment of the predictive information content of laboratory tests was carried out using ROC analysis. It was shown that the concentration of monocytes, eosinophilic leukocytes, the relative quantity of lymphocytes with CXCR1 receptor, the level of Cyfra 21-1 before surgical treatment were associated with the duration of the relapse-free period. A regression equation was compiled, which included the level of Cyfra 21-1, relative content of lymphocytes with CXCR1, and the eosinophilic leukocytes / monocytes ratio. Based on the threshold value Y=0,597, a Kaplan-Meier plot of patient survival was built and the results of it correspond to the TNM stratification. The prognostic sensitivity of the results of the equation - 85,7%, the specificity - 94,7%.

On the issue of diagnostic value of determining the level of receptors and their ligands in blood in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) occupies the first place in the structure of mortality due to oncological diseases. Late diagnosis worsens the effectiveness of its treatment. There are no informative biomarkers that allow us to judge the prevalence of the tumor process, especially in the early stages of NSCLC. To determine the level of CXCL5, CXCL8, CXCR1 and CXCR2 in the peripheral blood of patients with NSCLC to assess the possibility of their use in the diagnosis of the disease. The material was the blood of 218 patients with NSCLC, 19 patients with lung hamartoma and 42 healthy people. The concentration of CXCL5, CXCL8, and SCC in blood serum was determined by enzyme immunoassay, the CYFRA 21-1 level was determined by immunochemiluminescence analysis. The proportion of leukocytes equipped with CXCR1 and CXCR2 receptors and the fluorescence intensity of receptor complexes with antibodies (MFI) in them were measured by flow cytometry. MFI CXCR1 in granulocytes and the proportion of lymphocytes supplied CXCR2, increased in the blood already at stage I of NSCLC and showed an even more significant increase in subsequent stages. The level of these indicators was correlatively related to the stages and characteristics of NSCLC. Measuring the level of MFI CXCR1 in the blood serum makes it possible to diagnose the early stages of NSCLC with a sensitivity of 87.4% (specificity - 73.8%). Determination of the proportion of lymphocytes equipped with CXCR2 demonstrates comparable diagnostic sensitivity (87.2%) and specificity of 66.7% in the detection of stages I-II of NSCLC. MFI CXCR1 in granulocytes can also be used to differentiate stages I and II of NSCLC (diagnostic sensitivity - 75,3%, specificity - 69,6%). The sensitivity of determining for this purpose the proportion of lymphocytes equipped with CXCR2 is 75.0% with a specificity of 71.7%. In 89.7% of patients with stages III-IV NSCLC, the MFI CXCR1 in granulocytes exceeds the threshold value of 47.8 (specificity - 74.8%). Diagnostic sensitivity of determining the proportion of lymphocytes for this purpose was 90.7%.

A randomized, placebo-controlled phase 2 study of paclitaxel in combination with reparixin compared to paclitaxel alone as front-line therapy for metastatic triple-negative breast cancer (fRida).

PURPOSE: CXCR1, one of the receptors for CXCL8, has been identified as a druggable target on breast cancer cancer stem cells (CSC). Reparixin (R), an investigational oral inhibitor of CXCR1, was safely administered to metastatic breast cancer patients in combination with paclitaxel (P) and appeared to reduce CSC in a window-of-opportunity trial in operable breast cancer. The fRida trial (NCT02370238) evaluated the addition of R to weekly as first-line therapy for metastatic (m) TNBC. SUBJECTS AND METHODS: Subjects with untreated mTNBC were randomized 1:1 to R or placebo days 1-21 in combination with weekly P 80 mg/m2 on days 1, 8, 15 of 28-day cycles. The primary endpoint was PFS by central review. RESULTS: 123 subjects were randomized (62 to R + P and 61 to placebo + P). PFS was not different between the 2 groups (median 5.5 and 5.6 months for R + P and placebo + P, respectively; HR 1.13, p = 0.5996). ALDH+ and CD24-/CD44+ CSC centrally evaluated by IHC were found in 16 and 34 of the 54 subjects who provided a metastatic tissue biopsy at study entry. Serious adverse events (21.3 and 20% of subjects) and grade ≥ 3 adverse reactions (ADR) (9.1 and 6.3% of all ADRs) occurred at similar frequency in both groups. CONCLUSION: fRida is the first randomized, double-blind clinical trial of a CSC-targeting agent in combination with chemotherapy in breast cancer. The primary endpoint of prolonged PFS was not met. CLINICAL TRIAL REGISTRATION/DATE OF REGISTRATION: NCT01861054/February 24, 2015.

MiR-126, IL-7, CXCR1/2 receptors, inflammation and circulating endothelial progenitor cells: The study on targets for treatment pathways in a model of subclinical cardiovascular disease (type 1 diabetes mellitus).

BACKGROUND: Type 1 diabetes (T1DM) is associated with premature cardiovascular disease (CVD) and a pro-inflammatory state whilst the proangiogenic miR-126-3p/-5p may play a role in CVD. Animal studies established miR-126 to be pro-angiogenic. We hypothesised miR-126-3p/-5p are reduced in T1DM whilst pro-inflammatory cytokines are increased. METHODS: 29 well controlled, T1DM patients without CVD and 20 healthy controls (HCs) were studied. MiR-126-3p/-5p were assayed in plasma and peripheral blood mononuclear cells (PBMCs) whilst Chemokine C-X-C Receptor 1/2 (CXCR1/2) mRNA in PBMCs by real-time quantitative PCR. Cytokines were assayed by the Mesoscale Discovery. Ingenuity Pathway Analysis (IPA) was used to predict target genes, cellular functions and pathological states regulated by miR-126-3p/-5p. IPA generated both direct and indirect causations between different targets and analysed whether these effects would be inhibitory or stimulatory based on the published evidence. RESULTS: T1DM patients had a relatively good diabetic control (HbA1c = 7.4 ± 0.7% or 57.3 ± 7.6 mmol/mol). Homeostatic cytokine IL-7, pro-inflammatory cytokines IL-8 and TNF-α, and vascular endothelial growth factor-C (VEGF-C) were increased in T1DM, versus HCs; p = 0.008, p = 0.003, p = 0.041 and p = 0.013 respectively. MiR-126-5p was significantly upregulated in PBMCs in T1DM versus HCs; p = 0.01, but not in plasma. MiR-126-3p was unchanged. CXCR1/2 were elevated in T1DM versus HCs; p = 0.009 and p < 0.001 respectively. MiR-126-5p was positively correlated with CXCR1/2, and with HbA1c whilst negatively correlated with circulating endothelial progenitor cells (CD34+CD133+CD45dim) and fibronectin adhesion assay in a combined group of T1DM patients and HCs; p = 0.028 p = 0.049 p = 0.035 p = 0.047 and p = 0.004 respectively. IPA predicted miR-126-5p to be anti-inflammatory through the inhibition of chemokine C-C motif ligand 27, chymotrypsin-like elastase 2A and IL-7, whilst miR-126-3p had no direct anti-inflammatory effect. Simultaneously IPA predicted IL-7 as the most upstream cytokine target. CONCLUSIONS: T1DM without apparent CVD or diabetic complications is an inflammatory state characterised not only by raised pro-inflammatory cytokines but also by increased receptor CXCR1/2 and miR-126-5p. MiR-126-5p upregulation may represent a compensatory response. Pro-miR-126-5p therapies or anti-IL-7 therapies may be a new option to reduce both inflammation and CVD risk in T1DM. Further research is required in a large prospective study in patients with T1DM.

CXCR1 correlates to poor outcomes of EGFR-TKI against advanced non-small cell lung cancer by activating chemokine and JAK/STAT pathway.

OBJECTIVE: CXCR1, a member of the seven-transmembrane chemokine receptor family, promotes cell proliferation and metastasis in many tumors. The present study was undertaken to explore the interrelation between CXCR1 expression and the prognosis of advanced non-small cell lung cancer (NSCLC) in addition to the efficacy of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in lung adenocarcinoma (LUAD). METHODS: The expression of CXCR1 in NSCLC tissues was assessed by immunohistochemistry. The relationships between CXCR1 expression and clinical-pathological factors were investigated. Concomitantly, the relationship between CXCR1 expression and EGFR-TKI treatment efficacy was investigated. Gene set enrichment analysis (GSEA) was employed for the exploration of pathway enrichment, tumor immune estimation resource (TIMER) and gene expression profiling interactive analysis (GEPIA) for the inspection of the interrelationship between infiltration immune cells and CXCR1. After gain-and loss-of-function of CXCR1 in NSCLC cells, qRT-PCR and Western blot were applied to measure the levels of proteins associated with the chemokine pathway (CCL3 and CXCL2) and the JAK/STAT pathway (IL9R, PIAS4 and STAT5A). RESULTS: CXCR1 significantly correlated with poor prognosis of NSCLC patients. Additionally, CXCR1 limited the clinical efficacy of EGFR-TKIs in advanced LUAD (P = 0.029). In the tumor microenvironment, CXCR1 was positively associated with infiltration levels of immune markers in lung squamous cell carcinoma (LUSC) and LUAD. High expression of CXCR1 was implicated in the NOD-like receptor (NLR), cytokine/cytokine receptor, JAK/STAT and chemokine signaling pathways in LUAD and LUSC. Overexpression of CXCR1 in NSCLC cell lines enhanced expressions of CCL3, CXCL2, IL9R, PIAS4 and STAT5A, while knockdown of CXCR1 repressed expressions of CCL3, CXCL2, IL9R, PIAS4 and STAT5A. CONCLUSION: CXCR1 is correlated with poor prognosis of NSCLC and affects the efficacy of EGFR-TKIs in LUAD.

Neutralization of TNF-α and IL-1ß Regulates CXCL8 Production through CXCL8/CXCR1 Axis in Macrophages during Staphylococcus aureus Infection.

Anti-cytokine therapy is widely acknowledged as an anti-inflammatory technique to treat varied infectious diseases. TNF-α and IL-1ß are major cytokines that regulate every aspect of the inflammatory process. However, the effects of single or dual cytokine neutralization on S. aureus mediated CXCL8 secretion and CXCR1 expression in murine peritoneal macrophages remained noninvestigated. Thus we aimed to explore the effects of kinetic-dose dependent neutralization of TNF-α and IL-1ß using specific anti-cytokine antibodies and its influential impact on the CXCL8/CXCR1 axis at different stages of S. aureus (30, 60, and 90 min) infection. The murine peritoneal macrophages were isolated and infected with viable S. aureus followed by subsequent addition of anti-TNF-α and anti-IL-1ß into the medium. The treated cells were centrifuged and lysate and supernatant collected for various experiments. The ROS generation was measured and cytokine production was estimated by ELISA. The expression of TNFR1, IL-1R, CXCR1, signaling molecules (NF-κB and JNK) were evaluated by Western blot. The role of single or dual cytokine neutralization on intracellular bacterial phagocytosis had also been analyzed by confocal microscopy. Dual cytokine neutralization significantly suppressed ROS, cytokines, CXCL8 secretion, and intracellular bacterial count compared to single cytokine neutralization and it was more apparent at 90 min post S. aureus infection. There was a drastic reduction in TNFR1, IL-1R, and CXCR1 expression on macrophage surface due to reduced expression of downstream signaling molecules, NF-κB and JNK. Hence dual cytokine neutralization was more effectual compared to single cytokine neutralization in the downregulation of S. aureus induced CXCR1 expression.