Targeting IL-8 and Its Receptors in Prostate Cancer: Inflammation, Stress Response, and Treatment Resistance.

This review delves into the intricate roles of interleukin-8 (IL-8) and its receptors, CXCR1 and CXCR2, in prostate cancer (PCa), particularly in castration-resistant (CRPC) and metastatic CRPC (mCRPC). This review emphasizes the crucial role of the tumour microenvironment (TME) and inflammatory cytokines in promoting tumour progression and response to tumour cell targeting agents. IL-8, acting through C-X-C chemokine receptor type 1 (CXCR1) and type 2 (CXCR2), modulates multiple signalling pathways, enhancing the angiogenesis, proliferation, and migration of cancer cells. This review highlights the shift in PCa research focus from solely tumour cells to the non-cancer-cell components, including vascular endothelial cells, the extracellular matrix, immune cells, and the dynamic interactions within the TME. The immunosuppressive nature of the PCa TME significantly influences tumour progression and resistance to emerging therapies. Current treatment modalities, including androgen deprivation therapy and chemotherapeutics, encounter persistent resistance and are complicated by prostate cancer's notably "immune-cold" nature, which limits immune system response to the tumour. These challenges underscore the critical need for novel approaches that both overcome resistance and enhance immune engagement within the TME. The therapeutic potential of inhibiting IL-8 signalling is explored, with studies showing enhanced sensitivity of PCa cells to treatments, including radiation and androgen receptor inhibitors. Clinical trials, such as the ACE trial, demonstrate the efficacy of combining CXCR2 inhibitors with existing treatments, offering significant benefits, especially for patients with resistant PCa. This review also addresses the challenges in targeting cytokines and chemokines, noting the complexity of the TME and the need for precision in therapeutic targeting to avoid side effects and optimize outcomes.

Interleukin-8/CXCR1 Signaling Contributes to the Progression of Pulmonary Adenocarcinoma Resulting in Malignant Pleural Effusion.

Pulmonary adenocarcinoma (PADC) treatment limited efficacy in preventing tumor progression, often resulting in malignant pleural effusion (MPE). MPE is filled with various mediators, especially interleukin-8 (IL-8). However, the role of IL-8 and its signaling mechanism within the fluid microenvironment (FME) implicated in tumor progression warrants further investigation. Primary cultured cells from samples of patients with MPE from PADC, along with a commonly utilized lung cancer cell line, were employed to examine the role of IL-8 and its receptor, CXCR1, through comparative analysis. Our study primarily assessed migration and invasion capabilities, epithelial-mesenchymal transition (EMT), and cancer stem cell (CSC) properties. Additionally, IL-8 levels in MPE fluid versus serum, along with immunohistochemical expression of IL-8/CXCR1 signaling in tumor tissue and cell blocks were analyzed. IL-8/CXCR1 overexpression enhanced EMT and CSC properties. Furthermore, the immunocytochemical examination of 17 cell blocks from patients with PADC and MPE corroborated the significant correlation between upregulated IL-8 and CXCR1 expression and the co-expression of IL-8 and CXCR1 in MPE with distant metastasis. In summary, the IL-8/ CXCR1 axis in FME is pivotal to tumor promotion via paracrine and autocrine signaling. Our study provides a therapeutic avenue for improving the prognosis of PADC patients with MPE.

Single-cell transcriptomics of blood identified IFIT1+ neutrophil subcluster expansion in NTM-PD patients.

OBJECTIVE: Non-tuberculous mycobacterial pulmonary disease (NTM-PD) is caused by an imbalance between pathogens and impaired host immune responses. Mycobacterium avium complex (MAC) and Mycobacterium abscessus (MAB) are the two major pathogens that cause NTM-PD. In this study, we sought to dissect the transcriptomes of peripheral blood immune cells at the single-cell resolution in NTM-PD patients and explore potential clinical markers for NTM-PD diagnosis and treatment. METHODS: Peripheral blood samples were collected from six NTM-PD patients, including three MAB-PD patients, three MAC-PD patients, and two healthy controls. We employed single-cell RNA sequencing (scRNA-seq) to define the transcriptomic landscape at a single-cell resolution. A comprehensive scRNA-seq analysis was performed, and flow cytometry was conducted to validate the results of scRNA-seq. RESULTS: A total of 27,898 cells were analyzed. Nine T-cells, six mononuclear phagocytes (MPs), and four neutrophil subclusters were defined. During NTM infection, naïve T-cells were reduced, and effector T-cells increased. High cytotoxic activities were shown in T-cells of NTM-PD patients. The proportion of inflammatory and activated MPs subclusters was enriched in NTM-PD patients. Among neutrophil subclusters, an IFIT1+ neutrophil subcluster was expanded in NTM-PD compared to healthy controls. This suggests that IFIT1+ neutrophil subcluster might play an important role in host defense against NTM. Functional enrichment analysis of this subcluster suggested that it is related to interferon response. Cell-cell interaction analysis revealed enhanced CXCL8-CXCR1/2 interactions between the IFIT1+ neutrophil subcluster and NK cells, NKT cells, classical mononuclear phagocytes subcluster 1 (classical Mo1), classical mononuclear phagocytes subcluster 2 (classical Mo2) in NTM-PD patients compared to healthy controls. CONCLUSIONS: Our data revealed disease-specific immune cell subclusters and provided potential new targets of NTM-PD. Specific expansion of IFIT1+ neutrophil subclusters and the CXCL8-CXCR1/2 axis may be involved in the pathogenesis of NTM-PD. These insights may have implications for the diagnosis and treatment of NTM-PD.

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.

Senescent Fibroblasts Potentiate Peritoneal Metastasis of Diffuse-type Gastric Cancer Cells via IL-8-mediated Crosstalk.

BACKGROUND/AIM: Diffuse-type gastric cancer (DGC) often forms peritoneal metastases, leading to poor prognosis. However, the underlying mechanism of DGC-mediated peritoneal metastasis is poorly understood. DGC is characterized by desmoplastic stroma, in which heterogeneous cancer-associated fibroblasts (CAFs), including myofibroblastic CAFs (myCAFs) and senescent CAFs (sCAFs), play a crucial role during tumor progression. This study investigated the CAF subtypes induced by GC cells and the role of sCAFs in peritoneal metastasis of DGC cells. MATERIALS AND METHODS: Conditioned medium of human DGC cells (KATOIII, NUGC-4) and human intestinal-type GC (IGC) cells (MKN-7, N87) was used to induce CAFs. CAF subtypes were evaluated by analyzing the expression of α-smooth muscle actin (α-SMA), senescence-associated ß-galactosidase (SA-ß-gal), and p16 in human normal fibroblasts (GF, FEF-3). A cytokine array was used to explore the underlying mechanism of GC-induced CAF subtype development. The role of sCAFs in peritoneal metastasis of DGC cells was analyzed using a peritoneally metastatic DGC tumor model. The relationships between GC subtypes and CAF-related markers were evaluated using publicly available datasets. RESULTS: IGC cells significantly induced α-SMA+ myCAFs by secreting transforming growth factor-ß, whereas DGC cells induced SA-ß-gal+/p16+ sCAFs by secreting interleukin (IL)-8. sCAFs further secreted IL-8 to promote DGC cell migration. In vivo experiments demonstrated that co-inoculation of sCAFs significantly enhanced peritoneal metastasis of NUGC-4 cells, which was attenuated by administration of the IL-8 receptor antagonist navarixin. p16 and IL-8 expression was significantly associated with poor prognosis of DGC patients. CONCLUSION: sCAFs promote peritoneal metastasis of DGC via IL-8-mediated crosstalk.

Anti-CX3CL1 (fractalkine) monoclonal antibody attenuates lung and skin fibrosis in sclerodermatous graft-versus-host disease mouse model.

BACKGROUND: Systemic sclerosis (SSc) is an autoimmune disease characterized by vascular injury and inflammation, followed by excessive fibrosis of the skin and other internal organs, including the lungs. CX3CL1 (fractalkine), a chemokine expressed on endothelial cells, supports the migration of macrophages and T cells that express its specific receptor CX3CR1 into targeted tissues. We previously reported that anti-CX3CL1 monoclonal antibody (mAb) treatment significantly inhibited transforming growth factor (TGF)-ß1-induced expression of type I collagen and fibronectin 1 in human dermal fibroblasts. Additionally, anti-mouse CX3CL1 mAb efficiently suppressed skin inflammation and fibrosis in bleomycin- and growth factor-induced SSc mouse models. However, further studies using different mouse models of the complex immunopathology of SSc are required before the initiation of a clinical trial of CX3CL1 inhibitors for human SSc. METHODS: To assess the preclinical utility and functional mechanism of anti-CX3CL1 mAb therapy in skin and lung fibrosis, a sclerodermatous chronic graft-versus-host disease (Scl-cGVHD) mouse model was analyzed with immunohistochemical staining for characteristic infiltrating cells and RNA sequencing assays. RESULTS: On day 42 after bone marrow transplantation, Scl-cGVHD mice showed increased serum CX3CL1 level. Intraperitoneal administration of anti-CX3CL1 mAb inhibited the development of fibrosis in the skin and lungs of Scl-cGVHD model, and did not result in any apparent adverse events. The therapeutic effects were correlated with the number of tissue-infiltrating inflammatory cells and α-smooth muscle actin (α-SMA)-positive myofibroblasts. RNA sequencing analysis of the fibrotic skin demonstrated that cGVHD-dependent induction of gene sets associated with macrophage-related inflammation and fibrosis was significantly downregulated by mAb treatment. In the process of fibrosis, mAb treatment reduced cGVHD-induced infiltration of macrophages and T cells in the skin and lungs, especially those expressing CX3CR1. CONCLUSIONS: Together with our previous findings in other SSc mouse models, the current results indicated that anti-CX3CL1 mAb therapy could be a rational therapeutic approach for fibrotic disorders, such as human SSc and Scl-cGVHD.

IL-8 (CXCL8) Correlations with Psychoneuroimmunological Processes and Neuropsychiatric Conditions.

Interleukin-8 (IL-8/CXCL8), an essential CXC chemokine, significantly influences psychoneuroimmunological processes and affects neurological and psychiatric health. It exerts a profound effect on immune cell activation and brain function, suggesting potential roles in both neuroprotection and neuroinflammation. IL-8 production is stimulated by several factors, including reactive oxygen species (ROS) known to promote inflammation and disease progression. Additionally, CXCL8 gene polymorphisms can alter IL-8 production, leading to potential differences in disease susceptibility, progression, and severity across populations. IL-8 levels vary among neuropsychiatric conditions, demonstrating sensitivity to psychosocial stressors and disease severity. IL-8 can be detected in blood circulation, cerebrospinal fluid (CSF), and urine, making it a promising candidate for a broad-spectrum biomarker. This review highlights the need for further research on the diverse effects of IL-8 and the associated implications for personalized medicine. A thorough understanding of its complex role could lead to the development of more effective and personalized treatment strategies for neuropsychiatric conditions.

Clinical significance of the CXCL8/CXCR1/R2 signalling axis in patients with invasive breast cancer.

The C-X-C motif chemokine ligand 8 (CXCL8)-C-X-C chemokine receptor (CXCR)1/2 signalling axis is among numerous mechanisms which stimulate the immune system to defend against tumour growth and influence the tumour microenvironment to promote tumour growth. This pathway plays an important role in the development of a number of cancers including breast cancer (BC). The aim of the present study was to analyse the levels of the chemokine CXCL8 and its receptors, CXCR1 and CXCR2, in the serum of female patients with invasive BC and to assess the expression of these parameters at the mRNA level, considering molecular subtypes and degrees of cancer malignancy. The study group consisted of 62 patients with histopathologically confirmed invasive BC. The control group consisted of 18 patients with histopathologically confirmed fibroadenoma, a benign breast tumour. The levels of CXCL8, CXCR1 and CXCR2 were determined by sandwich ELISA using the CLOUD-CLONE ELISA kit. CXCL8, CXCR1 and CXCR2 transcript levels were analysed using reverse transcription-quantitative PCR. Results showed that serum CXCL8 levels in female patients with invasive BC were significantly higher compared with those in the control group (P<0.05). In addition, significantly elevated CXCR1 levels were observed in luminal B human epidermal growth factor receptor 2+ carcinoma compared with those in the control group. Analysis of CXCL8 in the serum of female patients with BC showed a statistically significant difference between clinical stage G1 and G2 (P<0.05), G2 and G3 (P<0.01), and G1 and G3 (P<0.0001). On the other hand, the analysis of CXCR1 and CXCR2 levels in the serum of the patients revealed a statistically significant difference between G2 and G3 (P<0.05). The current study showed that abnormalities in the immune response involving the CXCL8-CXCR1/2 signalling axis in patients with invasive BC are involved in the development of these tumours. Moreover, the demonstrated severity of changes occurring at protein level may suggest the potential usefulness of their determination as potential diagnostic markers in the clinic.

Cx1Mab-1: A Novel Anti-mouse CXCR1 Monoclonal Antibody for Flow Cytometry.

The C-X-C motif chemokine receptor-1 (CXCR1) is a rhodopsin-like G-protein-coupled receptor, expressed on the cell surface of immune cells and tumors. CXCR1 interacts with some C-X-C chemokines, such as CXCL6, CXCL7, and CXCL8/interleukin-8, which are produced by various cells. Since CXCR1 is involved in several diseases including tumors and diabetes mellitus, drugs targeting CXCR1 have been developed. Therefore, the development of sensitive monoclonal antibodies (mAbs) for CXCR1 has been desired for the diagnosis and treatment. This study established a novel anti-mouse CXCR1 (mCXCR1) mAb, Cx1Mab-1 (rat IgG1, kappa), using the Cell-Based Immunization and Screening method. Cx1Mab-1 reacted with mCXCR1-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR1) and mCXCR1-overexpressed LN229 glioblastoma (LN229/mCXCR1) in flow cytometry. Cx1Mab-1 demonstrated a high binding affinity for CHO/mCXCR1 and LN229/mCXCR1 with a dissociation constant of 2.6 × 10-9 M and 2.1 × 10-8 M, respectively. Furthermore, Cx1Mab-1 could detect mCXCR1 by Western blot analysis. These results indicated that Cx1Mab-1 is useful for detecting mCXCR1, and provides a possibility for targeting mCXCR1-expressing cells in vivo experiments.

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.

Investigating the impact of the genetic variant CXCR1 (rs2234671) in individuals with urinary tract infections.

BACKGROUND: Urinary tract infections (UTIs) are currently posing a worldwide health concern by affecting millions of people. The genetic variant rs2234671 in the CXCR1-interleukin-8 receptor is closely related to a raised UTI risk. OBJECTIVES: In this work, the impact of CXCR1 (rs2234671) on UTI individuals was examined. METHODS: The demographic features of 30 recurrent UTI patients and 20 controls were thoroughly investigated. Bacterial isolation and identification were performed by the implementation of cultural and biochemical methods. DNA extraction, purification of all samples from both patients and healthy people, and IL-8 rs2234671 (C/G) SNP genotyping using T-ARMS-PCR were performed. The significance of the results was evaluated by carrying out a statistical analysis. FINDINGS: The patient's average age was 34.63 ± 11.44 years, and controls averaged 30.30 ± 8.59 years (P= 0.156). No significant gender difference existed (P= 0.804). Escherichia coli (63.3%) was predominant, followed by Proteus mirabilis (26.7%), Enterococcus faecalis (23.3%), Klebsiella pneumoniae (10.0%), and Pseudomonas aeruginosa (20.0%). No significant association was found between bacterial species frequency, age, or sex. From the CXCR1 (rs2234671) frequency comparison, a higher GG genotype incidence in UTI patients than controls was extracted (26.7% vs. 15.0%), though not statistically significant. Risk analysis revealed that GG homozygous and C/G heterozygous genotypes were not UTI risk factors (OR = 2.47 and OR = 1.85, respectively). Moreover, the allele frequencies displayed no significant difference between the patients and controls (G allele: 66.7% vs. 66.7%; C allele: 33.3% vs. 33.3%). MAIN CONCLUSIONS: Although no significant association between CXCR1 (rs2234671) and UTI was found, the GG genotype may point to the increasing probability of UTI risk. Additional research is required to confirm and expand these conclusions.

Computational design of anti-cancer peptides tailored to target specific tumor markers.

Anti-cancer peptides (ACPs) are short peptides known for their ability to inhibit tumor cell proliferation, migration, and the formation of tumor blood vessels. In this study, we designed ACPs to target receptors often overexpressed in cancer using a systematic in silico approach. Three target receptors (CXCR1, DcR3, and OPG) were selected for their significant roles in cancer pathogenesis and tumor cell proliferation. Our peptide design strategy involved identifying interacting residues (IR) of these receptors, with their natural ligands serving as a reference for designing peptides specific to each receptor. The natural ligands of these receptors, including IL8 for CXCR1, TL1A for DcR3, and RANKL for OPG, were identified from the literature. Using the identified interacting residues (IR), we generated a peptide library through simple permutation and predicted the structure of each peptide. All peptides were analyzed using the web-based prediction server for Anticancer peptides, AntiCP. Docking simulations were then conducted to analyze the binding efficiencies of peptides with their respective target receptors, using VEGA ZZ and Chimera for interaction analysis. Our analysis identified HPKFIKELR as the interacting residues (IR) of CXCR-IL8. For DcR3, we utilized three domains from TL1A (TDSYPEP, TKEDKTF, LGLAFTK) as templates, along with two regions (SIKIPSS and PDQDATYP) from RANKL, to generate a library of peptide analogs. Subsequently, peptides for each receptor were shortlisted based on their predicted anticancer properties as determined by AntiCP and were subjected to docking analysis. After docking, peptides that exhibited the least binding energy were further analyzed for their detailed interaction with their respective receptors. Among these, peptides C9 (HPKFELY) and C7 (HPKFEWL) for CXCR1, peptides D6 (ADSYPQP) and D18 (AFSYPFP) for DcR3, and peptides P19 (PDTYPQDP) and p16 (PDQDATYP) for OPG, demonstrated the highest affinity and stronger interactions compared to the other peptides. Although in silico predictions indicated a favorable binding affinity of the designed peptides with target receptors, further experimental validation is essential to confirm their binding affinity, stability and pharmacokinetic characteristics.

CXCL17 binds efficaciously to glycosaminoglycans with the potential to modulate chemokine signaling.

Introduction: CXCL17 is a mucosally secreted protein, and the most recently identified human chemokine, an assignment based on protein fold prediction and chemotactic activity for leukocytes. However, these credentials have been the subject of much recent discussion and no experimental evidence has been presented regarding the definitive structure of CXCL17. In this study, we evaluated the structural and chemoattractant credentials of CXCL17 to better characterize this molecule, and gain deeper insights into its functional role as a glycosaminoglycan (GAG) binding protein. Methods: In the absence of structural information, in silico modeling techniques assessed the likelihood of CXCL17 adopting a chemokine fold. Recombinant CXCL17 was synthesized in mammalian and prokaryotic systems. Modified Boyden chamber and real-time chemotaxis assays assessed the ability of CXCL17 to promote chemotaxis of murine splenocytes, human neutrophils, and CXCR1 transfectants. The efficacy of CXCL17 binding to GAGs was quantified with solid-phase assays and bio-layer interferometry techniques. Results: All modeling efforts failed to support classification of CXCL17 as a chemokine based on its predicted conformation. Recombinant CXCL17 was observed to dimerize as a function of concentration, a characteristic of several chemokines. Contrary to a previous report, CXCL17 was not chemotactic for murine splenocytes, although it was a low-potency chemoattractant for human neutrophils at micromolar concentrations, several orders of magnitude higher than those required for CXCL8. As anticipated owing to its highly basic nature, CXCL17 bound to GAGs robustly, with key C-terminal motifs implicated in this process. While inactive via CXCR1, CXCL17 was found to inhibit CXCR1-mediated chemotaxis of transfectants to CXCL8 in a dose-dependent manner. Discussion: In summary, despite finding little evidence for chemokine-like structure and function, CXCL17 readily bound GAGs, and could modulate chemotactic responses to another chemokine in vitro. We postulate that such modulation is a consequence of superior GAG binding, and that C-terminal fragments of CXCL17 may serve as prototypic inhibitors of chemokine function.

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.

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.

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.

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.

Post hoc analysis of a randomized, double-blind, prospective trial evaluating a CXCR1/2 inhibitor in new-onset type 1 diabetes: endo-metabolic features at baseline identify a subgroup of responders.

Aim: In a recent randomized, multicenter trial (NCT02814838) a short-term anti-inflammatory treatment with ladarixin (LDX; an inhibitor of the CXCR1/2 chemokine receptors) did not show benefit on preserving residual beta cell function in new-onset type 1 diabetes. We present a post hoc analysis of trial patients in the predefined subgroup analysis developed according to baseline daily insulin requirement (DIR) tertiles. Method: A double-blind, randomized (2:1), placebo-controlled study was conducted in 45 men and 31 women (aged 18-46 years) within 100 days of the first insulin administration. Patients received LDX (400 mg twice daily) for three cycles of 14 days on/14 days off, or placebo. The primary endpoint was the area under the curve for C-peptide [AUC (0-120 min)] in response to a 2-h mixed meal tolerance test (MMTT) at week 13 ± 1. Seventy-five patients completed the week 13 MMTT and were divided into three groups according to the DIR tertiles: lower, ≤ 0.23U/kg/die (n = 25); middle, 0.24-0.40 U/kg/die (n = 24); upper, ≥ 0.41 U/kg/die (n = 26). Results: When considering the patients in the upper tertile (HIGH-DIR), C-peptide AUC (0-120 min) at 13 weeks was higher in the LDX group (n = 16) than in the placebo (n = 10) group [difference: 0.72 nmol/L (95% CI 0.9-1.34), p = 0.027]. This difference reduced over time (0.71 nmol/L at 26 weeks, p = 0.04; 0.42 nmol/L at 52 weeks, p = 0.29), while it has never been significant at any time in patients in the lower and/or middle tertile (LOW-DIR). We characterized at baseline the HIGH-DIR and found that endo-metabolic (HOMA-B, adiponectin, and glucagon-to-C-peptide ratio) and immunologic (chemokine (C-C motif) ligand 2 (CCL2)/monocyte chemoattractant protein 1 (MCP1) and Vascular Endothelial Growth Factor (VEGF)) features distinguished this group from LOW-DIR. Conclusion: While LDX did not prevent the progressive loss of beta-cell function in the majority of treated subjects, the post hoc analysis suggests that it could work in subjects with HIGH-DIR at baseline. As we found differences in endo-metabolic and immunologic parameters within this subgroup, this generates the hypothesis that the interactions between host factors and drug action can contribute to its efficacy. Further research is needed to evaluate this hypothesis.

CXCL8 and the peritoneal metastasis of ovarian and gastric cancer.

CXCL8 is the most representative chemokine produced autocrine or paracrine by tumor cells, endothelial cells and lymphocytes. It can play a key role in normal tissues and tumors by activating PI3K-Akt, PLC, JAK-STAT, and other signaling pathways after combining with CXCR1/2. The incidence of peritoneal metastasis in ovarian and gastric cancer is extremely high. The structure of the peritoneum and various peritoneal-related cells supports the peritoneal metastasis of cancers, which readily produces a poor prognosis, low 5-year survival rate, and the death of patients. Studies show that CXCL8 is excessively secreted in a variety of cancers. Thus, this paper will further elaborate on the mechanism of CXCL8 and the peritoneal metastasis of ovarian and gastric cancer to provide a theoretical basis for the proposal of new methods for the prevention, diagnosis, and treatment of cancer peritoneal metastasis.

IL-10 in combination with IL-12 and TNF-α attenuates CXCL8/CXCR1 axis in peritoneal macrophages of mice infected with Staphylococcus aureus through the TNFR1-IL-1R-NF-κB pathway.

Overexpression of Staphylococcus aureus mediated CXCL8/CXCR1 axis is a major cause of sepsis and severe inflammatory diseases. This chemokine acts conjointly with various pro-inflammatory and anti-inflammatory cytokines that govern the severity of inflammation. The effects of different combinations of exogenous cytokines on CXCR1 expression in macrophages remain undetermined. Exogenous cytokine and anti-inflammatory cytokine therapy had been used to modulate CXCL8 and CXCR1 expression in peritoneal macrophages. Male Swiss albino mice were inoculated with live S. aureus (106 cells/ mouse) for the development of infection. Exogenous cytokines (TNF-α, IL-12, IFN-γ and IL-10) were administered intraperitoneally (single or combination) 24 h post S. aureus infection. The mice were sacrificed and peritoneal macrophages were isolated three days post infection. CXCL8, IL-12, IL-10 secretion, ROS generation and the bacterial phagocytic process had been evaluated. Western blot was used to study the expressions of TNFR1, IL-1R, CXCR1 and NF-κB. TNF-α, IL-12 and IFN-γ treatments aggravated CXCL8 and CXCR1 expression in the macrophages of infected mice. TNF-α + IFN-γ treatment was a major inducer of nitric oxide release and mediated maximum bacterial killing. IL-12 + TNF-α treatment was most potent in increasing ROS, CXCL8/CXCR1 expression through increased levels of TNFR1, IL-1R and NF-κB activation. IL-10 reversed the effects of exogenous cytokines but also impaired the bacterial clearance phenomenon in peritoneal lavage. Treatment with IL-12 + TNF-α + IL-10 was most effective in ameliorating oxidative stress, reduced CXCL8 release and expression levels of TNFR1, IL-1R, and NF-κB. Concludingly, IL-12 + TNF-α + IL-10 treatment mitigated CXCL8/CXCR1 expression and inflammatory signalling via downregulation of TNFR1-IL-1R-NF-κB pathway in peritoneal macrophages and inflammatory sequelae during S. aureus infection.