CXCR2 perturbation promotes Staphylococcus aureus implant-associated infection.

Introduction. Staphylococcus aureus is the leading cause of acute medical implant infections, representing a significant modern medical concern. The success of S. aureus as a pathogen in these cases resides in its arsenal of virulence factors, resistance to multiple antimicrobials, mechanisms of immune modulation, and ability to rapidly form biofilms associated with implant surfaces. S. aureus device-associated, biofilm-mediated infections are often persistent and notoriously difficult to treat, skewing innate immune responses to promote chronic reoccurring infections. While relatively little is known of the role neutrophils play in response to acute S. aureus biofilm infections, these effector cells must be efficiently recruited to sites of infection via directed chemotaxis. Here we investigate the effects of modulating CXC chemokine receptor 2 (CXCR2) activity, predominantly expressed on neutrophils, during S. aureus implant-associated infection.Hypothesis. We hypothesize that modulation of CXCR2 expression and/or signalling activities during S. aureus infection, and thus neutrophil recruitment, extravasation and antimicrobial activity, will affect infection control and bacterial burdens in a mouse model of implant-associated infection.Aim. This investigation aims to elucidate the impact of altered CXCR2 activity during S. aureus biofilm-mediated infection that may help develop a framework for an effective novel strategy to prevent morbidity and mortality associated with implant infections.Methodology. To examine the role of CXCR2 during S. aureus implant infection, we employed a mouse model of indwelling subcutaneous catheter infection using a community-associated methicillin-resistant S. aureus (MRSA) strain. To assess the role of CXCR2 induction or inhibition during infection, treatment groups received daily intraperitoneal doses of either Lipocalin-2 (Lcn2) or AZD5069, respectively. At the end of the study, catheters and surrounding soft tissues were analysed for bacterial burdens and dissemination, and Cxcr2 transcription within the implant-associated tissues was quantified.Results. Mice treated with Lcn2 developed higher bacterial burdens within the soft tissue surrounding the implant site, which was associated with increased Cxcr2 expression. AZD5069 treatment also resulted in increased implant- and tissues-associated bacterial titres, as well as enhanced Cxcr2 expression.Conclusion. Our results demonstrate that CXCR2 plays an essential role in regulating the severity of S. aureus implant-associated infections. Interestingly, however, perturbation of CXCR2 expression or signalling both resulted in enhanced Cxcr2 transcription and elevated implant-associated bacterial burdens. Thus, CXCR2 appears finely tuned to efficiently recruit effector cells and mediate control of S. aureus biofilm-mediated infection.

Development and Initial Characterization of the First 18F-CXCR2-Targeting Radiotracer for PET Imaging of Neutrophils.

The interleukin-8 receptor beta (CXCR2) is a highly promising target for molecular imaging of inflammation and inflammatory diseases. This is due to its almost exclusive expression on neutrophils. Modified fluorinated ligands were designed based on a squaramide template, with different modification sites and synthetic strategies explored. Promising candidates were then tested for affinity to CXCR2 in a NanoBRET competition assay, resulting in tracer candidate 16b. As direct 18F-labeling using established tosyl chemistry did not yield the expected radiotracer, an indirect labeling approach was developed. The radiotracer [18F]16b was obtained with a radiochemical yield of 15% using tert-butyl (S)-3-(tosyloxy)pyrrolidine carboxylate and a pentafluorophenol ester. The subsequent time-dependent uptake of [18F]16b in CXCR2-negative and CXCR2-overexpressing human embryonic kidney cells confirmed the radiotracer's specificity. Further studies with human neutrophils revealed its diagnostic potential for functional imaging of neutrophils.

Biophysical characterization of the CXC chemokine receptor 2 ligands.

The chemokines of the immune system act as first responders by operating as chemoattractants, directing immune cells to specific locations of inflamed tissues. This promiscuous network is comprised of 50 ligands and 18 receptors where the ligands may interact with the receptors in various oligomeric states i.e., monomers, homodimers, and heterodimers. Chemokine receptors are G-protein coupled receptors (GPCRs) present in the membrane of immune cells. The migration of immune cells occurs in response to a concentration gradient of the ligands. Chemotaxis of neutrophils is directed by CXC-ligand (CXCL) activation of the membrane bound CXC chemokine receptor 2 (CXCR2). CXCR2 plays an important role in human health and is linked to disorders such as autoimmune disorders, inflammation, and cancer. Yet, despite their important role, little is known about the biophysical characteristics controlling ligand:ligand and ligand:receptor interaction essential for biological activity. In this work, we study the homodimers of three of the CXCR2 cognate ligands, CXCL1, CXCL5, and CXCL8. The ligands share high structural integrity but a low sequence identity. We show that the sequence diversity has evolved different binding affinities and stabilities for the CXC-ligands resulting in diverse agonist/antagonist behavior. Furthermore, CXC-ligands fold through a three-state mechanism, populating a folded monomeric state before associating into an active dimer.

AESIS-1, a Rheumatoid Arthritis Therapeutic Peptide, Accelerates Wound Healing by Promoting Fibroblast Migration in a CXCR2-Dependent Manner.

In patients with autoimmune disorders such as rheumatoid arthritis (RA), delayed wound healing is often observed. Timely and effective wound healing is a crucial determinant of a patient's quality of life, and novel materials for skin wound repair, such as bioactive peptides, are continuously being studied and developed. One such bioactive peptide, AESIS-1, has been studied for its well-established anti-rheumatoid arthritis properties. In this study, we attempted to use the anti-RA material AESIS-1 as a therapeutic wound-healing agent based on disease-modifying antirheumatic drugs (DMARDs), which can help restore prompt wound healing. The efficacy of AESIS-1 in wound healing was assessed using a full-thickness excision model in diabetic mice; this is a well-established model for studying chronic wound repair. Initial observations revealed that mice treated with AESIS-1 exhibited significantly advanced wound repair compared with the control group. In vitro studies revealed that AESIS-1 increased the migration activity of human dermal fibroblasts (HDFs) without affecting proliferative activity. Moreover, increased HDF cell migration is mediated by upregulating chemokine receptor expression, such as that of CXC chemokine receptor 2 (CXCR2). The upregulation of CXCR2 through AESIS-1 treatment enhanced the chemotactic reactivity to CXCR2 ligands, including CXC motif ligand 8 (CXCL8). AESIS-1 directly activates the ERK and p38 mitogen-activated protein kinase (MAPK) signaling cascades, which regulate the migration and expression of CXCR2 in fibroblasts. Our results suggest that the AESIS-1 peptide is a strong wound-healing substance that increases the movement of fibroblasts and the expression of CXCR2 by turning on the ERK and p38 MAPK signaling cascades.

Endothelial CXCR2 deficiency attenuates renal inflammation and glycocalyx shedding through NF-κB signaling in diabetic kidney disease.

BACKGROUND: The incidence of diabetic kidney disease (DKD) continues to rapidly increase, with limited available treatment options. One of the hallmarks of DKD is persistent inflammation, but the underlying molecular mechanisms of early diabetic kidney injury remain poorly understood. C-X-C chemokine receptor 2 (CXCR2), plays an important role in the progression of inflammation-related vascular diseases and may bridge between glomerular endothelium and persistent inflammation in DKD. METHODS: Multiple methods were employed to assess the expression levels of CXCR2 and its ligands, as well as renal inflammatory response and endothelial glycocalyx shedding in patients with DKD. The effects of CXCR2 on glycocalyx shedding, and persistent renal inflammation was examined in a type 2 diabetic mouse model with Cxcr2 knockout specifically in endothelial cells (DKD-Cxcr2 eCKO mice), as well as in glomerular endothelial cells (GECs), cultured in high glucose conditions. RESULTS: CXCR2 was associated with early renal decline in DKD patients, and endothelial-specific knockout of CXCR2 significantly improved renal function in DKD mice, reduced inflammatory cell infiltration, and simultaneously decreased the expression of proinflammatory factors and chemokines in renal tissue. In DKD conditions, glycocalyx shedding was suppressed in endothelial Cxcr2 knockout mice compared to Cxcr2 L/L mice. Modulating CXCR2 expression also affected high glucose-induced inflammation and glycocalyx shedding in GECs. Mechanistically, CXCR2 deficiency inhibited the activation of NF-κB signaling, thereby regulating inflammation, restoring the endothelial glycocalyx, and alleviating DKD. CONCLUSIONS: Taken together, under DKD conditions, activation of CXCR2 exacerbates inflammation through regulation of the NF-κB pathway, leading to endothelial glycocalyx shedding and deteriorating renal function. Endothelial CXCR2 deficiency has a protective role in inflammation and glycocalyx dysfunction, suggesting its potential as a promising therapeutic target for DKD treatment.

The CXCLs-CXCR2 axis modulates the cross-communication between tumor-associated neutrophils and tumor cells in cervical cancer.

OBJECTIVE: This study aimed to check the expression profile of the C-X-C motif chemokine ligands (CXCLs)-C-X-C motif chemokine receptor 2 (CXCR2) axis in cervical cancer and to explore the cross-talk between cervical cancer cells and neutrophils via CXCLs-CXCR2 axis. METHODS: Available RNA-sequencing data based on bulk tissues and single-cell/nucleus RNA-sequencing data were used for bioinformatic analysis. Cervical cancer cell lines Hela and SiHa cells were utilized for in vitro and in vivo studies. RESULTS: Except for neutrophils, CXCR2 mRNA expression is limited in other types of cells in the cervical tumor microenvironment. CXCLs bind to CXCR2 and are mainly expressed by tumor cells. CXCL1, 2, 3, 5, 6, and 8, which are consistently associated with neutrophil infiltration, are also linked to poor prognosis. SB225002 (a CXCR2 inhibitor) treatment significantly impairs SiHa cell-induced neutrophil migration. CXCL1, CXCL2, CXCL5, or CXCL8 neutralized conditioned medium from SiHa cells have weaker recruiting effects. The conditioned medium of neutrophils from healthy donors can slow cancer cell proliferation. Conditioned medium of tumor-associated neutrophils (TANs) can drastically enhance cervical cancer cell growth in vitro and in vivo. CONCLUSIONS: The CXCLs-CXCR2 axis is critical in neutrophil recruitment and tumor cell proliferation in the cervical cancer microenvironment.

Systematic assessment of chemokine ligand bias at the human chemokine receptor CXCR2 indicates G protein bias over ß-arrestin recruitment and receptor internalization.

BACKGROUND: The human CXC chemokine receptor 2 (CXCR2) is a G protein-coupled receptor (GPCR) interacting with multiple chemokines (i.e., CXC chemokine ligands CXCL1-3 and CXCL5-8). It is involved in inflammatory diseases as well as cancer. Consequently, much effort is put into the identification of CXCR2 targeting drugs. Fundamental research regarding CXCR2 signaling is mainly focused on CXCL8 (IL-8), which is the first and best described high-affinity ligand for CXCR2. Much less is known about CXCR2 activation induced by other chemokines and it remains to be determined to what extent potential ligand bias exists within this signaling system. This insight might be important to unlock new opportunities in therapeutic targeting of CXCR2. METHODS: Ligand binding was determined in a competition binding assay using labeled CXCL8. Activation of the ELR + chemokine-induced CXCR2 signaling pathways, including G protein activation, ß-arrestin1/2 recruitment, and receptor internalization, were quantified using NanoBRET-based techniques. Ligand bias within and between these pathways was subsequently investigated by ligand bias calculations, with CXCL8 as the reference CXCR2 ligand. Statistical significance was tested through a one-way ANOVA followed by Dunnett's multiple comparisons test. RESULTS: All chemokines (CXCL1-3 and CXCL5-8) were able to displace CXCL8 from CXCR2 with high affinity and activated the same panel of G protein subtypes (Gαi1, Gαi2, Gαi3, GαoA, GαoB, and Gα15) without any statistically significant ligand bias towards any one type of G protein. Compared to CXCL8, all other chemokines were less potent in ß-arrestin1 and -2 recruitment and receptor internalization while equivalently activating G proteins, indicating a G protein activation bias for CXCL1,-2,-3,-5,-6 and CXCL7. Lastly, with CXCL8 used as reference ligand, CXCL2 and CXCL6 showed ligand bias towards ß-arrestin1/2 recruitment compared to receptor internalization. CONCLUSION: This study presents an in-depth analysis of signaling bias upon CXCR2 stimulation by its chemokine ligands. Using CXCL8 as a reference ligand for bias index calculations, no ligand bias was observed between chemokines with respect to activation of separate G proteins subtypes or recruitment of ß-arrestin1/2 subtypes, respectively. However, compared to ß-arrestin recruitment and receptor internalization, CXCL1-3 and CXCL5-7 were biased towards G protein activation when CXCL8 was used as reference ligand.

CXCR2 chemokine receptor - a master regulator in cancer and physiology.

Recent findings have modified our understanding of the roles of chemokine receptor CXCR2 and its ligands in cancer, inflammation, and immunity. Studies in Cxcr2 tissue-specific knockout mice show that this receptor is involved in, among other things, cancer, central nervous system (CNS) function, metabolism, reproduction, COVID-19, and the response to circadian cycles. Moreover, CXCR2 involvement in neutrophil function has been revisited not only in physiology but also for its major contribution to cancers. The recent unfolding of the role of CXCR2 in numerous cancers has led to extensive evaluation of multiple CXCR2 antagonists in preclinical and clinical studies. In this review we discuss the potential of targeting CXCR2 for cancer treatment.

Cxcl1 monomer-dimer equilibrium controls neutrophil extravasation.

The chemokine Cxcl1 plays a crucial role in recruiting neutrophils in response to infection. The early events in chemokine-mediated neutrophil extravasation involve a sequence of highly orchestrated steps including rolling, adhesion, arrest, and diapedesis. Cxcl1 function is determined by its properties of reversible monomer-dimer equilibrium and binding to Cxcr2 and glycosaminoglycans. Here, we characterized how these properties orchestrate extravasation using intravital microscopy of the cremaster. Compared to WT Cxcl1, which exists as both a monomer and a dimer, the trapped dimer caused faster rolling, less adhesion, and less extravasation. Whole-mount immunofluorescence of the cremaster and arrest assays confirmed these data. Moreover, the Cxcl1 dimer showed impaired LFA-1-mediated neutrophil arrest that could be attributed to impaired Cxcr2-mediated ERK signaling. We conclude that Cxcl1 monomer-dimer equilibrium and potent Cxcr2 activity of the monomer together coordinate the early events in neutrophil recruitment.

Serum CXCL8 and CXCR2 as diagnostic biomarkers for noninvasive screening of cervical cancer.

BACKGROUND: Cervical cancer (CC) is the fourth most frequently diagnosed cancer and the fourth leading cause of cancer-related death in women. Identifying new biomarkers for the early detection of CC is an essential requirement in this field. CXCL8 was originally discovered because of its role in inflammation by binding to CXCR1 and CXCR2; however, it is now known to play an important role in cancer. In this study, we aimed to evaluate the expression levels of potential biomarkers (CXCL8, CXCR1, and CXCR2) and to explore their diagnostic potential in CC. METHODS: The expression levels of serum CXCL8, CXCR1, and CXCR2 were investigated by kit method on Immulite-1000 in 30 healthy volunteers, 30 precancerous patients and 70 CC patients. RESULTS: The results indicated that the expression of CXCL8 and CXCR2 was significantly higher in the serum of CC patients than in healthy volunteers, similar to the well-established tumor marker (squamous-cell cancerantigen [SCC]). Receiver operating characteristic analyses showed that the combination of CXCL8, CXCR2, and SCC had the highest diagnostic sensitivity and area under the curve value. Meanwhile, the positive predictive value and negative predictive value were not very low. Moreover, high concentrations of CXCL8 and CXCR2 are associated with an increased risk of CC. CONCLUSIONS: In conclusion, our data demonstrated that combined serum CXCL8, CXCR2, and SCC measurements are helpful for CC diagnosis and can be used as potential biomarkers for the early detection of CC. Cytokines, such as CXCL8 and CXCR2, can be easily measured in most university hospital laboratories and in some private laboratories with a routine test.

Circ_0002295 facilitated myocardial fibrosis progression through the miR-1287/CXCR2 axis.

Myocardial fibrosis (MF) is involved in hypertension, myocardial infarction and heart failure. It has been reported that circular RNA (circRNA) is a key regulatory factor of MF progression. In this study, we revealed that circ_0002295 and CXCR2 were elevated, and miR-1287 was reduced in MF patients. Knockdown of circ_0002295 effectively suppressed the proliferation, migration and MF progression. Circ_0002295 was the molecular sponge of miR-12878, and miR-1287 inhibitor reversed the biological functions of circ_0002295 on the myocardial fibrosis. CXCR2 was a target gene of miR-1287, and CXCR2 silencing relieved the impacts of miR-1287 inhibitor on cardiac myofibroblasts. Circ_0002295 promoted MF progression by regulating the miR-1287/CXCR2 axis, providing a possible circRNA-targeted therapy for MF.

Bioinformatic Analysis of the CXCR2 Ligands in Cancer Processes.

Human CXCR2 has seven ligands, i.e., CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8/IL-8-chemokines with nearly identical properties. However, no available study has compared the contribution of all CXCR2 ligands to cancer progression. That is why, in this study, we conducted a bioinformatic analysis using the GEPIA, UALCAN, and TIMER2.0 databases to investigate the role of CXCR2 ligands in 31 different types of cancer, including glioblastoma, melanoma, and colon, esophageal, gastric, kidney, liver, lung, ovarian, pancreatic, and prostate cancer. We focused on the differences in the regulation of expression (using the Tfsitescan and miRDB databases) and analyzed mutation types in CXCR2 ligand genes in cancers (using the cBioPortal). The data showed that the effect of CXCR2 ligands on prognosis depends on the type of cancer. CXCR2 ligands were associated with EMT, angiogenesis, recruiting neutrophils to the tumor microenvironment, and the count of M1 macrophages. The regulation of the expression of each CXCR2 ligand was different and, thus, each analyzed chemokine may have a different function in cancer processes. Our findings suggest that each type of cancer has a unique pattern of CXCR2 ligand involvement in cancer progression, with each ligand having a unique regulation of expression.

CXCL1 enhances COX-II expression in rheumatoid arthritis synovial fibroblasts by CXCR2, PLC, PKC, and NF-κB signal pathway.

Rheumatoid arthritis (RA) is the most common autoimmune disease, affecting the joints of the hands and feet. Several chemokines and their receptors are crucial in RA pathogenesis through immune cell recruitment. C-X-C Motif Chemokine Ligand 1 (CXCL1), a chemokine for the recruitment of various immune cells, can be upregulated in patients with RA. However, the discussion on the role of CXCL1 in RA pathogenesis is insufficient. Here, we found that CXCL1 promoted cyclooxygenase-2 (COX-II) expression in a dose- and time-dependent manner in rheumatoid arthritis synovial fibroblasts (RASFs). CXCL1 overexpression in RASFs led to a significant increase in COX-II expression, while the transfection of RASFs with the shRNA plasmid resulted in a noticeable decrease in COX-II expression. Next, we delineated the molecular mechanism underlying CXCL1-promoted COX-II expression and noted that CXC chemokine receptor 2 (CXCR2), phospholipase C (PLC), and protein kinase C (PKC) signal transduction were responsible for COX-II expression after CXCL1 incubation for RASFs. Finally, we confirmed the transcriptional activation of nuclear factor κB (NF-κB) in RASFs after incubation with CXCL1. In conclusion, the current study provided a novel insight into the role of CXCL1 in RA pathogenesis.

Exercise training inhibits macrophage-derived IL-17A-CXCL5-CXCR2 inflammatory axis to attenuate pulmonary fibrosis in mice exposed to silica.

Exposure to crystalline silica leads to health effects beyond occupational silicosis. Exercise training's potential benefits on pulmonary diseases yield inconsistent outcomes. In this study, we utilized experimental silicotic mice subjected to exercise training and pharmacological interventions, including interleukin-17A (IL-17A) neutralizing antibody or clodronate liposome for macrophage depletion. Findings reveal exercise training's ability to mitigate silicosis progression in mice by suppressing scavenger receptor B (SRB)/NOD-like receptor thermal protein domain associated protein 3 (NLRP3) and Toll-like receptor 4 (TLR4) pathways. Macrophage-derived IL-17A emerges as primary source and trigger for silica-induced pulmonary inflammation and fibrosis. Exercise training effectively inhibits IL-17A-CXC motif chemokine ligand 5 (CXCL5)-Chemokine (C-X-C motif) Receptor 2 (CXCR2) axis in silicotic mice. Our study evidences exercise training's potential to reduce collagen deposition, preserve elastic fibers, slow pulmonary fibrosis advancement, and enhance pulmonary function post silica exposure by impeding macrophage-derived IL-17A-CXCL5-CXCR2 axis.

Chemokine Cxcl1-Cxcl2 heterodimer is a potent neutrophil chemoattractant.

Microbial infection is characterized by release of multiple proinflammatory chemokines that direct neutrophils to the insult site. How collective function of these chemokines orchestrates neutrophil recruitment is not known. Here, we characterized the role for heterodimer and show that the Cxcl1-Cxcl2 heterodimer is a potent neutrophil chemoattractant in mice and can recruit more neutrophils than the individual chemokines. Chemokine-mediated neutrophil recruitment is determined by Cxcr2 receptor signaling, Cxcr2 endocytosis, and binding to glycosaminoglycans. We have now determined heterodimer's Cxcr2 activity using cellular assays and Cxcr2 density in blood and recruited neutrophils in heterodimer-treated mice. We have shown that the heterodimer binds glycosaminoglycans with higher affinity and more efficiently than Cxcl1 or Cxcl2. These data collectively indicate that optimal glycosaminoglycan interactions and dampened receptor activity acting in concert in a dynamic fashion promote heterodimer-mediated robust neutrophil recruitment. We propose that this could play a critical role in combating infection.

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

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

Fluorescent Ligands Enable Target Engagement Studies for the Intracellular Allosteric Binding Site of the Chemokine Receptor CXCR2.

Herein, we report the structure-based development of fluorescent ligands targeting the intracellular allosteric binding site (IABS) of CXC chemokine receptor 2 (CXCR2), a G protein-coupled receptor (GPCR) that has been pursued as a drug target in oncology and inflammation. Starting from the cocrystallized intracellular CXCR2 antagonist 00767013 (1), tetramethylrhodamine (TAMRA)-labeled CXCR2 ligands were designed, synthesized, and tested for their suitability as fluorescent reporters to probe binding to the IABS of CXCR2. By means of these studies, we developed Mz438 (9a) as a high-affinity and selective fluorescent CXCR2 ligand, enabling cell-free as well as cellular NanoBRET-based binding studies in a nonisotopic and high-throughput manner. Further, we show that 9a can be used as a tool to visualize intracellular target engagement for CXCR2 via fluorescence microscopy. Thus, our small-molecule-based fluorescent CXCR2 ligand 9a represents a promising tool for future studies of CXCR2 pharmacology.

Design, Synthesis, and Application of Fluorescent Ligands Targeting the Intracellular Allosteric Binding Site of the CXC Chemokine Receptor 2.

The inhibition of CXC chemokine receptor 2 (CXCR2), a key inflammatory mediator, is a potential strategy in the treatment of several pulmonary diseases and cancers. The complexity of endogenous chemokine interaction with the orthosteric binding site has led to the development of CXCR2 negative allosteric modulators (NAMs) targeting an intracellular pocket near the G protein binding site. Our understanding of NAM binding and mode of action has been limited by the availability of suitable tracer ligands for competition studies, allowing direct ligand binding measurements. Here, we report the rational design, synthesis, and pharmacological evaluation of a series of fluorescent NAMs, based on navarixin (2), which display high affinity and preferential binding for CXCR2 over CXCR1. We demonstrate their application in fluorescence imaging and NanoBRET binding assays, in whole cells or membranes, capable of kinetic and equilibrium analysis of NAM binding, providing a platform to screen for alternative chemophores targeting these receptors.

CXCR2 antagonism attenuates neuroinflammation after subarachnoid hemorrhage.

OBJECTIVES: Overactivation of neuroinflammation can worsen the prognosis of subarachnoid hemorrhage (SAH) patients. CXCR2 is a widely expressed G protein-coupled receptor that participates in the regulation of inflammation, indicating a potential role of CXCR2 in SAH. MATERIALS AND METHODS: Herein, we examined the expression pattern of CXCR2 in the ipsilateral brain tissue of SAH mice. Then, we evaluated the effects of CXCR2 antagonist on neuroinflammation and neurological function after SAH. RESULTS: Western blotting and immunohistochemistry revealed that CXCR2 expression was upregulated following SAH. Our results demonstrated that treatment with SB225002 inhibited inflammatory cytokine (IL-1ß, IL-6, TNF-α, MCP-1) production in the brain and cerebrospinal fluid (CSF) following SAH. Our further findings confirmed that treatment with SB225002 ameliorated astrocytosis and microgliosis after SAH. Interestingly, SB225002 significantly improved neurological impairment after SAH. CONCLUSIONS: Altogether, these results suggest that pharmacologically targeting CXCR2 may be an effective disease-modifying treatment for SAH.

Gastric cancer mesenchymal stem cells via the CXCR2/HK2/PD-L1 pathway mediate immunosuppression.

BACKGROUND: Anti-PD-1 immunotherapy has emerged as an important therapeutic modality in advanced gastric cancer (GC). However, drug resistance frequently develops, limiting its effectiveness. METHODS: The role of gastric cancer mesenchymal stem cells (GCMSCs) in anti-PD-1 resistance was evaluated in vivo in NPGCD34+ or NCGPBMC xenograft mouse model. In addition, we investigated CD8+T cell infiltration and effector function by spectral cytometry and IHC. The effects of GCMSCs conditional medium (GCMSC-CM) on GC cell lines were characterized at the level of the proteome, secretome using western blot, and ELISA assays. RESULTS: We reported that GCMSCs mediated tolerance mechanisms contribute to tumor immunotherapy tolerance. GCMSC-CM attenuated the antitumor activity of PD-1 antibody and inhibited immune response in humanized mouse model. In GC cells under serum deprivation and hypoxia, GCMSC-CM promoted GC cells proliferation via upregulating PD-L1 expression. Mechanistically, GCMSC-derived IL-8 and AKT-mediated phosphorylation facilitated HK2 nuclear localization. Phosphorylated-HK2 promoted PD-L1 transcription by binding to HIF-1α. What is more, GCMSC-CM also induced lactate overproduction in GC cells in vitro and xenograft tumors in vivo, leading to impaired function of CD8+ T cells. Furthermore, CXCR1/2 receptor depletion, CXCR2 receptor antagonist AZD5069 and IL-8 neutralizing antibody application also significantly reversed GCMSCs mediated immunosuppression, restoring the antitumor capacity of PD-1 antibody. CONCLUSIONS: Our findings reveal that blocking GCMSCs-derived IL-8/CXCR2 pathway decreasing PD-L1 expression and lactate production, improving antitumor efficacy of anti-PD-1 immunotherapy, may be of value for the treatment of advanced gastric carcinoma.