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

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

Acupuncture alleviates inflammatory pain by activating CXCL1/CXCR2 signaling in the primary somatosensory cortex in adjuvant induced arthritis rats. / S1脑区CXCL1/CXCR2å‚ä¸Žé’ˆåˆºæ”¹å–„ä½å‰‚æ€§å ³èŠ‚ç‚Žå¤§é¼ ç‚Žæ€§ç—›çš„ä½œç”¨æœºåˆ¶.

OBJECTIVES: To observe whether acupuncture up-regulates chemokine CXC ligand 1 (CXCL1) in the brain to play an analgesic role through CXCL1/chemokine CXC receptor 2 (CXCR2) signaling in adjuvant induced arthritis (AIA) rats, so as to reveal its neuro-immunological mechanism underlying improvement of AIA. METHODS: BALB/c mice with relatively stable thermal pain reaction were subjected to planta injection of complete Freund adjuvant (CFA) for establishing AIA model, followed by dividing the AIA mice into simple AF750 (fluorochrome) and AF750+CXCL1 groups (n=2 in each group). AF750 labeled CXCL1 recombinant protein was then injected into the mouse's tail vein to induce elevation of CXCL1 level in blood for simulating the effect of acupuncture stimulation which has been demonstrated by our past study. In vivo small animal imaging technology was used to observe the AF750 and AF750+CXCL1-labelled target regions. After thermal pain screening, the Wistar rats with stable pain reaction were subjected to AIA modeling by injecting CFA into the rat's right planta, then were randomized into model and manual acupuncture groups (n=12 in each group). Other 12 rats that received planta injection of saline were used as the control group. Manual acupuncture (uniform reinforcing and reducing manipulations) was applied to bilateral "Zusanli" (ST36) for 4×2 min, with an interval of 5 min between every 2 min, once daily for 7 days. The thermal pain threshold was assessed by detecting the paw withdrawal latency (PWL) using a thermal pain detector. The contents of CXCL1 in the primary somatosensory cortex (S1), medial prefrontal cortex, nucleus accumbens, amygdala, periaqueductal gray and rostroventromedial medulla regions were assayed by using ELISA, and the expression levels of CXCL1, CXCR2 and mu-opioid receptor (MOR) mRNA in the S1 region were detected using real time-quantitative polymerase chain reaction. The immune-fluorescence positive cellular rate of CXCL1 and CXCR2 in S1 region was observed after immunofluorescence stain. The immunofluorescence double-stain of CXCR2 and astrocyte marker glial fibrillary acidic protein (GFAP) or neuron marker NeuN or MOR was used to determine whether there is a co-expression between them. RESULTS: In AIA mice, results of in vivo experiments showed no obvious enrichment signal of AF750 or AF750+CXCL1 in any organ of the body, while in vitro experiments showed that there was a stronger fluorescence signal of CXCL1 recombinant protein in the brain. In rats, compared with the control group, the PWL from day 0 to day 7 was significantly decreased (P<0.01) and the expression of CXCR2 mRNA in the S1 region significantly increased in the model group (P<0.05), while in comparison with the model group, the PWL from day 2 to day 7, CXCL1 content, CXCR2 mRNA expression and CXCR2 content, and MOR mRNA expression in the S1 region were significantly increased in the manual acupuncture group (P<0.05, P<0.01). Immunofluorescence stain showed that CXCR2 co-stained with NeuN and MOR in the S1 region, indicating that CXCR2 exists in neurons and MOR-positive neurons but not in GFAP positive astrocytes. CONCLUSIONS: Acupuncture can increase the content of CXCL1 in S1 region, up-regulate CXCR2 on neurons in the S1 region and improve MOR expression in S1 region of AIA rats, which may contribute to its effect in alleviating inflammatory pain.

Feiyanning downregulating CXCLs/CXCR2 axis to suppress TANs infiltration in the prevention of lung cancer metastasis.

ETHNOPHARMACOLOGICAL RELEVANCE: Tumor-associated neutrophils (TANs) play an important role in tumor metastasis. The Traditional Chinese medicine (TCM) Feiyanning (FYN) has been clinically proven to effectively prevent the recurrence and metastasis of lung cancer, improve immunity, and prolong the survival period of lung cancer patients. However, its anti-metastatic immune mechanism has not been fully elucidated. To this end, we studied the mechanism of FYN's regulation of neutrophils infiltration in the tumor microenvironment (TME). AIM OF THE STUDY: To explore the anti-metastatic mechanism of FYN from the perspective of anti-immunosuppressive phenotype neutrophils infiltration in the TME. MATERIALS AND METHODS: TCM network pharmacological analysis was used to predict Feiyanning effective target. Flow cytometry was used to detect the proportion of immune cell subsets in the TME. Lung metastases were investigated in C57 mice by tail vein injection. Protein expression was evaluated by immunohistochemistry and Western blot. Gene expression was evaluated by qRT-PCR. RESULTS: FYN could reshape the tumor immune microenvironment. It prevents Tregs, M2 macrophages, and neutrophils infiltration, as well as recruits T cells, NK cells, and DCs, and improves DCs activation. In addition, FYN could regulate the polarization of TANs, inhibit the infiltration of neutrophils with an immunosuppressive phenotype, downregulate CXCLs/CXCR2 axis and inhibitory factors like Arg-1 and TGF-ß, and up-regulate the immune effector molecule ICAM-1. Furthermore, FYN increases anti-tumor immune effects in the TME to prevent tumor cells from spreading to the lungs. CONCLUSION: This study clarifies the potential mechanism of FYN in regulating neutrophils infiltration and anti-metastasis. FYN may regulate neutrophils infiltration in the TME by regulating CXCLs/CXCR2 axis.

Interleukin-8: An evolving chemokine.

Early in the 1980s several laboratories mistakenly reported that partially purified interleukin-1 (IL-1) was chemotactic for neutrophils. However, further investigations by us, revealed that our purified IL-1 did not have neutrophil chemotactic activity and this activity in the LPS-stimulated human monocyte conditioned media could clearly be separated from IL-1 activity on HPLC gel filtration. This motivated Teizo Yoshimura and Kouji Matsushima to purify the monocyte-derived neutrophil chemotactic factor (MDNCF), present in LPS conditioned media and molecularly clone the cDNA for MDNCF. They found that MDNCF protein (later renamed IL-8, and finally termed CXCL8) is first translated as a precursor form consisting of 99 amino acid residues and the signal peptide is then removed, leading to the secretion and processing of biologically active IL-8 of 72 amino acid form (residues 28-99). There are four cysteine residues forming two disulfide linkage and 14 basic amino acid residues which result in a very basic property for the binding of IL-8 to heparan sulfate-proteoglycan. The IL-8 gene consists of 4 exons and 3 introns. IL-8 is produced by various types of cells in inflammation. The 5'-flanking region of IL-8 gene contains several nuclear factor binding sites, and NF-κB in combination with AP-1 or C/EBP synergistically activates IL-8 gene in response to IL-1 and TNFα. Two receptors exist for IL-8, CXCR1 and CXCR2 in humans, which belong to γ subfamily of GTP binding protein (G-protein) coupled rhodopsin-like 7 transmembrane domain receptors. Rodents express CXCR2 and do not produce IL-8, but produce numerous homologues instead. Once IL-8 binds to the receptor, ß and γ subunits of G-protein are released from Gα (Gαi2 in neutrophils) and activate PI3Kγ, PLCß2/ß3, PLA2 and PLD. Gαi2 inhibits adenyl cyclase to decrease cAMP levels. Small GTPases Ras/Rac/Rho/cdc42/Rap1, PKC and AKT (PKB) exist down-stream of ß and γ subunits and regulate cell adhesion, actin polymerization, membrane protrusion, and eventually cell migration. PLCß activation generates IP3 and induces Ca++ mobilization, DAG generation to activate protein kinase C to lead granule exocytosis and respiratory burst. MDNCF was renamed interleukin 8 (IL-8) at the International Symposium on Novel Neutrophil Chemotactic Activating Polypeptides, London, UK in 1989. The discovery of IL-8 prompted us to also purify and molecularly clone the cDNA of MCAF/MCP-1 responsible for monocyte chemotaxis, and other groups to identify a large family of chemotactic cytokines capable of attracting other types of leukocytes. In 1992, most of the investigators contributing to the discovery of this new family of chemotactic cytokines gathered in Baden, Austria and agreed to name this family "chemokines" and subsequently established the CXCL/CCL and CXCR/CCR nomenclature. The discovery of chemokines resulted in solving the long-time enigma concerning the mechanism of cell type specific leukocyte infiltration into inflamed tissues and provided a molecular basis for immune and hematopoietic cell migration and interactions under physiological as well as pathological conditions. To our surprise based on its recently identified multifunctional activities, IL-8 has evolved from a neutrophil chemoattractant to a promising therapeutic target for a wide range of inflammatory and neoplastic diseases. IL-8 was initially characterized as a chemoattractant of neutrophils engaged in acute inflammation and then discovered to also be chemotactic for endothelial cells with a major role in angiogenesis. These two activities of IL-8 foster its stimulatory effect on tumor growth. This is abetted by recent additional discoveries showing that IL-8 has stimulatory effects on stem cells and can therefore directly promote the growth of receptor expressing cancer stem cells. IL-8 by interacting with bone marrow stem/progenitor cells has also the capacity to mobilize and release hematopoietic cells into the peripheral circulation. This includes the mobilization of neutrophilic myeloid-derived suppressor cells (N-MDSC) to infiltrate into tumors and thus further promotes the immune escape of tumors. Finally, the capacity of IL-8 to induce trans-differentiation of epithelial cancer cells into mesenchymal phenotype (EMT) increases the malignancy of tumors by promoting their metastatic spread and resistance to chemotherapeutics and cytotoxic immune cells. These observations have stimulated considerable current efforts to develop receptor antagonists for IL-8 and humanized anti-IL-8 antibody for the therapy of cancer, particularly in combination with immune checkpoint inhibitors, such as anti-PD-1/PD-L1 antibodies.

Early senescence and production of senescence-associated cytokines are major determinants of radioresistance in head-and-neck squamous cell carcinoma.

Resistance against radio(chemo)therapy-induced cell death is a major determinant of oncological treatment failure and remains a perpetual clinical challenge. The underlying mechanisms are manifold and demand for comprehensive, cancer entity- and subtype-specific examination. In the present study, resistance against radiotherapy was systematically assessed in a panel of human head-and-neck squamous cell carcinoma (HNSCC) cell lines and xenotransplants derived thereof with the overarching aim to extract master regulators and potential candidates for mechanism-based pharmacological targeting. Clonogenic survival data were integrated with molecular and functional data on DNA damage repair and different cell fate decisions. A positive correlation between radioresistance and early induction of HNSCC cell senescence accompanied by NF-κB-dependent production of distinct senescence-associated cytokines, particularly ligands of the CXCR2 chemokine receptor, was identified. Time-lapse microscopy and medium transfer experiments disclosed the non-cell autonomous, paracrine nature of these mechanisms, and pharmacological interference with senescence-associated cytokine production by the NF-κB inhibitor metformin significantly improved radiotherapeutic performance in vitro and in vivo. With regard to clinical relevance, retrospective analyses of TCGA HNSCC data and an in-house HNSCC cohort revealed that elevated expression of CXCR2 and/or its ligands are associated with impaired treatment outcome. Collectively, our study identifies radiation-induced tumor cell senescence and the NF-κB-dependent production of distinct senescence-associated cytokines as critical drivers of radioresistance in HNSCC whose therapeutic targeting in the context of multi-modality treatment approaches should be further examined and may be of particular interest for the subgroup of patients with elevated expression of the CXCR2/ligand axis.

Effects of propofol and its formulation components on macrophages and neutrophils in obese and lean animals.

We hypothesized whether propofol or active propofol component (2,6-diisopropylphenol [DIPPH] and lipid excipient [LIP-EXC]) separately may alter inflammatory mediators expressed by macrophages and neutrophils in lean and obese rats. Male Wistar rats (n = 10) were randomly assigned to receive a standard (lean) or obesity-inducing diet (obese) for 12 weeks. Animals were euthanized, and alveolar macrophages and neutrophils from lean and obese animals were exposed to propofol (50 µM), active propofol component (50 µM, 2,6-DIPPH), and lipid excipient (soybean oil, purified egg phospholipid, and glycerol) for 1 h. The primary outcome was IL-6 expression after propofol and its components exposure by alveolar macrophages extracted from bronchoalveolar lavage fluid. The secondary outcomes were the production of mediators released by macrophages from adipose tissue, and neutrophils from lung and adipose tissues, and neutrophil migration. IL-6 increased after the exposure to both propofol (median [interquartile range] 4.14[1.95-5.20]; p = .04) and its active component (2,6-DIPPH) (4.09[1.67-5.91]; p = .04) in alveolar macrophages from obese animals. However, only 2,6-DIPPH increased IL-10 expression (7.59[6.28-12.95]; p = .001) in adipose tissue-derived macrophages. Additionally, 2,6-DIPPH increased C-X-C chemokine receptor 2 and 4 (CXCR2 and CXCR4, respectively) in lung (10.08[8.23-29.01]; p = .02; 1.55[1.49-3.43]; p = .02) and adipose tissues (8.78[4.15-11.57]; p = .03; 2.86[2.17-3.71]; p = .01), as well as improved lung-derived neutrophil migration (28.00[-3.42 to 45.07]; p = .001). In obesity, the active component of propofol affected both the M1 and M2 markers as well as neutrophils in both alveolar and adipose tissue cells, suggesting that lipid excipient may hinder the effects of active propofol.

The CXCR2/CXCL2 signalling pathway - An alternative therapeutic approach in high-grade glioma.

OBJECTIVE: Besides VEGF, alternative signalling via CXCR2 and its ligands CXCL2/CXCL8 is a crucial part of angiogenesis in glioblastoma. Our aim was to understand the role of CXCR2 for glioma biology and elucidate the therapeutic potential of its specific inhibition. METHODS: GL261 glioma cells were implanted intracranially in syngeneic mice. The 14 or 7 days of local or systemic treatment with CXCR2-antagonist (SB225002) was initiated early on the day of tumour cell implantation or delayed after 14 days of tumour growth. Glioma volume was verified using MRI before and after treatment. Immunofluorescence staining was used to investigate tumour progression, angiogenesis and microglial behaviour. Furthermore, in vitro assays and gene expression analyses of glioma and endothelial cells were performed to validate inhibitor activity. RESULTS: CXCR2-blocking led to significantly reduced glioma volumes of around 50% after early and delayed local treatments. The treated tumours were comparable with controls regarding invasiveness, proliferation and apoptotic cell activity. Furthermore, no differences in CXCR2/CXCL2 expression were observed. However, immunostaining revealed reduction in vessel density and accumulation of microglia/macrophages, whereas interaction of these myeloid cells with tumour vessels was enhanced. In vitro analyses of the CXCR2-antagonist showed its direct impact on proliferation of glioma and endothelial cells if used at higher concentrations. In addition, expression of CXCR2/CXCL2 signalling genes was increased in both cell types by SB225002, but VEGF-relevant genes were unaffected. CONCLUSION: The CXCR2-antagonist inhibited glioma growth during tumour initiation and progression, whereas treatment was well-tolerated by the recipients. Thus, the CXCR2/CXCL2 signalling represents a promising therapeutic target in glioma.

CXCR2 Ligands and mTOR Activation Enhance Reprogramming of Human Somatic Cells to Pluripotent Stem Cells.

Induced pluripotent stem cell (iPSC) technology has great promise in regenerative medicine and disease modeling. In this study, we show that human placenta-derived cell conditioned medium stimulates chemokine (C-X-C motif) receptor 2 (CXCR2) in human somatic cells ectopically expressing the pluripotency-associated transcription factors Oct4, Sox2, Klf4, and cMyc (OSKM), leading to mechanistic target of rapamycin (mTOR) activation. This causes an increase in endogenous cMYC levels and a decrease in autophagy, thereby enhancing the reprogramming efficiency of human somatic cells into iPSCs. These findings were reproduced when human somatic cells after OSKM transduction were cultured in a widely used reprogramming medium (mTeSR) supplemented with CXCR2 ligands interleukin-8 and growth-related oncogene α or an mTOR activator (MHY1485). To our knowledge, this is the first report demonstrating that mTOR activation in human somatic cells with ectopic OSKM expression significantly enhances the production of iPSCs. Our results support the development of convenient protocols for iPSC generation and further our understanding of somatic cell reprogramming.

Plant-derived verbascoside and isoverbascoside regulate Toll-like receptor 2 and 4-driven neutrophils priming and activation.

BACKGROUND: Neutrophils have a short live in circulation and accelerate greatly local immune responses via increased granulopoiesis and migration at high numbers to infected or inflamed tissue. HYPOTHESIS: Since neutrophils produce a variety of factors with destructive and pro-inflammatory potential the regulation of their homeostasis and functions might be eventually beneficial in inflammation-related pathological conditions. Herein we investigated the effect of natural-derived verbascoside (Verb) and its positional isomer isoverbascoside (IsoVerb) on neutrophil functions. METHODS: We used purified murine bone marrow (BM) neutrophils to study cell responsiveness to priming or activation via Toll-like receptors (TLRs) 2 and 4. The expression of CD11b, chemokine (CXC motif) receptor 2 (CXCR2), the intracellular level of phosphorylated p38 mitogen-activated protein kinase (MAPK) and tumor necrosis factor (TNF)-α in neutrophils were determined by flow cytometry while the release of macrophage inflammatory protein (MIP)-2 in culture supernatant was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: We found that Verb appeared less powerful inhibitor of TLR2 and TLR4-mediated apoptosis than IsoVerb. However at concentrations below 16 µM and in LPS priming conditions Verb was more selective inhibitor of CD11b and CXCR2 expression than IsoVerb. Both compounds showed similar activity on integrin/chemokine receptor expression when neutrophils were stimulated with ZY or were activated with LPS. Verb sustained CXCR2 expression and turnover via regulation of the cell responsiveness to its ligand KC (CXCL1) and via the release of MIP-2 (CXCL2). Both Verb and IsoVerb increased TNF-α production and inhibited p38 phosphorylation in TNF-α+ cells. We fail to discriminate sharply between Verb's and IsoVerb's efficacy when studying p38 phosphorylation in LPS stimulated neutrophils. The multi-parametric analysis provides critical insight on the range of on-target effects of Verb and IsoVerb. CONCLUSION: The strength and selectivity of Verb and IsoVerb depended on the degree of activation and functional state of neutrophils, and both compounds are with potential to affect neutrophil-related pathologies/conditions in heterogenic populations.

Role of CINC-1 and CXCR2 receptors on LPS-induced fever in rats.

The classic model of fever induction is based on the administration of lipopolysaccharide (LPS) from Gram-negative bacteria in experimental animals. LPS-induced fever results in the synthesis/release of many mediators that assemble an LPS-fever cascade. We have previously demonstrated that cytokine-induced neutrophil chemoattractant (CINC)-1, a Glu-Leu-Arg (ELR) + chemokine, centrally administered to rats, induces fever and increases prostaglandin E2 in the cerebrospinal fluid. We now attempt to investigate the involvement of CINC-1 and its functional receptor CXCR2 on the fever induced by exogenous and endogenous pyrogens in rats. We also investigated the effect of reparixin, an allosteric inhibitor of CXCR1/CXCR2 receptors, on fever induced by either systemic administration of LPS or intracerebroventricular injection of CINC-1, as well as TNF-α, IL-1ß, IL-6, or ET-1, known mediators of febrile response. Our results show increased CINC-1 mRNA expression in the liver, hypothalamus, CSF, and plasma following LPS injection. Moreover, reparixin administered right before CINC-1 or LPS abolished the fever induced by CINC-1 and significantly reduced the response induced by LPS. In spite of these results, reparixin does not modify the fever induced by IL-1ß, TNF-α, and IL-6, but significantly reduces ET-1-induced fever. Therefore, it is plausible to suggest that CINC-1 might contribute to LPS-induced fever in rats by activating CXCR2 receptor on the CNS. Moreover, it can be hypothesized that CINC-1 is placed upstream TNF-α, IL-1ß, and IL-6 among the prostaglandin-dependent fever-mediator cascade and amidst the prostaglandin-independent synthesis pathway of fever.

n-butanol extract from Folium isatidis inhibits the lipopolysaccharide-induced downregulation of CXCR1 and CXCR2 on human neutrophils.

Neutrophils, immune cells crucial for protecting against invading pathogens, are important in sepsis. Neutrophil migration is regulated by chemokine receptors and their cognate ligands. Our previous study investigated the effect of n­butanol extract from Folium isatidis on lipopolysaccharide (LPS)­induced septic shock. The present study stimulated neutrophils with LPS to explore the influence of LPS on cell. Neutrophils were then pretreated with n­butanol extract from Folium isatidis followed by LPS to examine the effect of this extract on neutrophil chemotaxis. The results showed that LPS decreased the expression levels of CXC­chemokine receptor (CXCR)1, CXCR2 and L­selectin (CD62L), and increased the expression of interleukin­8 (IL­8) by neutrophils. The addition of n­butanol extract from Folium isatidis inhibited this LPS­induced downregulation of CXCR1, CXCR2 and CD62L, and decreased the expression of IL­8 on neutrophils. In addition, n­butanol extract promoted myeloperoxidase activity in neutrophils. Taken together, LPS downregulated the expression of chemokine receptors, leading to the failure of neutrophils to migrate to sites of infection. The addition of n­butanol extract, which promoted the ability of neutrophils to migrate, is a natural product and potential therapeutic agent with which to target neutrophil chemotaxis during LPS stimulation.

The atypical receptor CCRL2 is required for CXCR2-dependent neutrophil recruitment and tissue damage.

CCRL2 is a 7-transmembrane domain receptor that shares structural and functional similarities with the family of atypical chemokine receptors (ACKRs). CCRL2 is upregulated by inflammatory signals and, unlike other ACKRs, it is not a chemoattractant-scavenging receptor, does not activate ß-arrestins, and is widely expressed by many leukocyte subsets. Therefore, the biological role of CCRL2 in immunity is still unclear. We report that CCRL2-deficient mice have a defect in neutrophil recruitment and are protected in 2 models of inflammatory arthritis. In vitro, CCRL2 was found to constitutively form homodimers and heterodimers with CXCR2, a main neutrophil chemotactic receptor. By heterodimerization, CCRL2 could regulate membrane expression and promote CXCR2 functions, including the activation of ß2-integrins. Therefore, upregulation of CCRL2 observed under inflammatory conditions is functional to finely tune CXCR2-mediated neutrophil recruitment at sites of inflammation.

Astragaloside IV alleviates E. coli-caused peritonitis via upregulation of neutrophil influx to the site of infection.

Astragaloside IV (AS-IV), an active saponin purified from Astragali Radix, has been identified with broad biological and pharmacological activities. In the present study, we continue to explore the potential effect of AS-IV on antibacterial response using an acute E. coli peritoneal infection model. Our findings implied that administration of AS-IV decreases mortality in mice challenged by lethal E. coli infection. The protection of AS-IV was related to promotion of neutrophil extravasation into the peritoneum and bacterial clearance. Toll-like receptor (TLR) activation in neutrophils has been reported to reduce CXCR2 expression and subsequent neutrophil migration. Our data indicated that AS-IV prevented the reduction of CXCR2 expression and neutrophil migration induced by LPS, the activator for TLR4. Moreover, we found that AS-IV blocks LPS-induced suppression of CXCR2 on neutrophils by inhibiting the expression of G protein-coupled receptor kinase-2 (GRK2), an agonist that regulates desensitization and internalization of chemokine receptors. Taken together, these data propose that AS-IV, through modulating GRK2-CXCR2 signal in neutrophils, offers an essential efficacy on host antibacterial immunity.

Facilitation of Allergic Sensitization and Allergic Airway Inflammation by Pollen-Induced Innate Neutrophil Recruitment.

Neutrophil recruitment is a hallmark of rapid innate immune responses. Exposure of airways of naive mice to pollens rapidly induces neutrophil recruitment. The innate mechanisms that regulate pollen-induced neutrophil recruitment and the contribution of this neutrophilic response to subsequent induction of allergic sensitization and inflammation need to be elucidated. Here we show that ragweed pollen extract (RWPE) challenge in naive mice induces C-X-C motif ligand (CXCL) chemokine synthesis, which stimulates chemokine (C-X-C motif) receptor 2 (CXCR2)-dependent recruitment of neutrophils into the airways. Deletion of Toll-like receptor 4 (TLR4) abolishes CXCL chemokine secretion and neutrophil recruitment induced by a single RWPE challenge and inhibits induction of allergic sensitization and airway inflammation after repeated exposures to RWPE. Forced induction of CXCL chemokine secretion and neutrophil recruitment in mice lacking TLR4 also reconstitutes the ability of multiple challenges of RWPE to induce allergic airway inflammation. Blocking RWPE-induced neutrophil recruitment in wild-type mice by administration of a CXCR2 inhibitor inhibits the ability of repeated exposures to RWPE to stimulate allergic sensitization and airway inflammation. Administration of neutrophils derived from naive donor mice into the airways of Tlr4 knockout recipient mice after each repeated RWPE challenge reconstitutes allergic sensitization and inflammation in these mice. Together these observations indicate that pollen-induced recruitment of neutrophils is TLR4 and CXCR2 dependent and that recruitment of neutrophils is a critical rate-limiting event that stimulates induction of allergic sensitization and airway inflammation. Inhibiting pollen-induced recruitment of neutrophils, such as by administration of CXCR2 antagonists, may be a novel strategy to prevent initiation of pollen-induced allergic airway inflammation.

CXCR2 Inhibition Combined with Sorafenib Improved Antitumor and Antiangiogenic Response in Preclinical Models of Ovarian Cancer.

Antiangiogenic therapy is important for the treatment of gynecological cancer. However, the therapeutic benefit derived from these treatments is transient, predominantly due to the selective activation of compensatory proangiogenic pathways that lead to rapid development of resistance. We aimed to identify and target potential alternative signaling to anti-vascular endothelial growth factor (VEGF) therapy, with a view toward developing a combination of antiangiogenic agents to provide extended therapeutic benefits. We developed a preclinical in vivo phenotypic resistance model of ovarian cancer resistant to antiangiogenic therapy. We measured dynamic changes in secreted chemokines and angiogenic signaling in tumors and plasma in response to anti-VEGF treatment, as tumors advanced from the initial responsive phase to progressive disease. In tumors that progressed following sorafenib treatment, gene and protein expression levels of proangiogenic CXC chemokines and their receptors were significantly elevated, compared with responsive tumors. The chemokine (C-X-C motif) ligand 8 (CXCL8), also known as interleukin-8 (IL-8) increase was time-dependent and coincided with the dynamics of tumor progression. We used SB225002, a pharmacological inhibitor of chemokine (C-X-C motif) receptor 2 (CXCR2), to disrupt the CXC chemokine-mediated functions of ovarian cancer cells in in vitro assays of cell growth inhibition, spheroid formation, and cell migration. The combination of CXCR2 inhibitor with sorafenib led to a synergistic inhibition of cell growth in vitro, and further stabilized tumor progression following sorafenib in vivo. Our results suggest that CXCR2-mediated chemokines may represent an important compensatory pathway that promotes resistance to antiangiogenic therapy in ovarian cancer. Thus, simultaneous blockage of this proangiogenic cytokine pathway using CXCR2 inhibitors and the VEGF receptor (VEGFR) pathway could improve the outcomes of antiangiogenic therapy.

Cordyceps Militaris Alleviates Severity of Murine Acute Lung Injury Through miRNAs-Mediated CXCR2 Inhibition.

BACKGROUND/AIMS: Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are lethal diseases in humans, and the current treatments have limited therapeutic effects. Cordyceps militaris (CM) is a caterpillar-grown traditional medicinal mushroom, and has been used as a natural invigorant for longevity, endurance, and vitality in China. Recently, purified extracts from CM have been shown to have beneficial effects on various diseases including cancer. Nevertheless, a role of CM in ALI has not been examined previously. METHODS: Here, we used a bleomycin-induced ALI model to study the effects of CM on the severity of ALI in mice. The levels of CXCR2, a receptor for Interleukin 8 (IL-8) in pulmonary microvascular endothelial cells, were examined in different experimental groups. The levels of microRNA (miR)-1321 and miR-3188 were also examined in lung samples and in CM. Adeno-associated viruses carrying miR-1321 and miR-3188 were injected into bleomycin-treated mice for evaluation their effects on the severity of ALI. RESULTS: CM treatment significantly alleviated the severity of bleomycin-induced ALI in mice. The increases in lung CXCR2 by bleomycin were significantly reduced by CM at protein level, but not at mRNA level. CM contained high levels of 2 miRNAs (miR-1321 and miR-3188) that target 3'-UTR of CXCR2 mRNA to inhibit its expression. Overexpression of miR-1321 and miR-3188 in mouse lung through AAV-mediated gene therapy mimicked the effects of CM. CONCLUSION: CM may alleviate severity of murine ALI through miRNAs-mediated CXCR2 inhibition.

Effect of acupuncture on CXCL8 receptors in rats suffering from embryo implantation failure.

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

[Effects of Xiaotan Sanjie Decoction on expressions of interleukin-8 and its receptors in gastric tumor xenografts and gastric tissue adjacent to the tumor in mice].

OBJECTIVE: To explore the mechanisms of Xiaotan Sanjie Decoction (XTSJD), a compound traditional Chinese herbal medicine, in inhibiting the tumor growth and preventing recurrence by testing the protein expressions of interleukin-8 (IL-8) and its receptors chemokine receptor 1 (CXCR1) and chemokine receptor 2 (CXCR2) in gastric tumor xenografts and gastric tissue adjacent to the tumor in mice. METHODS: Fifty Kunming mice were randomly divided into normal group, normal saline (NS) group, Heat-clearing and Detoxicating Decoction (HCDD) group, tegafur (FT-207) group and XTSJD group. Except for mice in the normal group, S180 tumor block was transplanted into the gastric walls of the mice, and the mice were administered with corresponding medicine for 3 weeks. Weight of tumor xenografts was measured and tumor inhibition rate was calculated. IL-8 protein expression was tested by enzyme-linked immunosorbent assay. Expressions of CXCR1 and CXCR2 were tested by immunohistochemical method. RESULTS: The protein expressions of IL-8 and its receptors in tumor xenografts and gastric tissue adjacent to the tumor were markedly higher than those in the gastric tissue in normal mice (P<0.01); compared with HCDD and FT-207, XTSJD could significantly decrease the IL-8 protein expression in tumor xenografts and gastric tissue adjacent to the tumor (P<0.05); compared with FT-207, XTSJD could significantly decrease the CXCR1 protein expression in tumor xenografts (P<0.01), and XTSJD could also significantly decrease the CXCR1 protein expression in gastric tissue adjacent to the tumor as compared with HCDD and FT-207 (P<0.01); compared with HCDD and FT-207, XTSJD could significantly decrease the CXCR2 protein expression in tumor xenografts (P<0.01), and there was no significant difference among the three drug-treated groups in CXCR2 protein expression in gastric tissue adjacent to the tumor (P>0.05). CONCLUSION: XTSJD can decrease the protein expressions of IL-8 and its receptors in tumor xenografts and gastric tissue adjacent to the tumor. It may be one of the mechanisms of XTSJD in inhibiting the tumor growth and preventing recurrence.

Hydrogen sulfide augments neutrophil migration through enhancement of adhesion molecule expression and prevention of CXCR2 internalization: role of ATP-sensitive potassium channels.

In this study, we have addressed the role of H(2)S in modulating neutrophil migration in either innate (LPS-challenged naive mice) or adaptive (methylated BSA (mBSA)-challenged immunized mice) immune responses. Treatment of mice with H(2)S synthesis inhibitors, dl-propargylglycine (PAG) or beta-cyanoalanine, reduced neutrophil migration induced by LPS or methylated BSA (mBSA) into the peritoneal cavity and by mBSA into the femur/tibial joint of immunized mice. This effect was associated with decreased leukocyte rolling, adhesion, and P-selectin and ICAM-1 expression on endothelium. Predictably, treatment of animals with the H(2)S donors, NaHS or Lawesson's reagent, enhanced these parameters. Moreover, the NaHS enhancement of neutrophil migration was not observed in ICAM-1-deficient mice. Neither PAG nor NaHS treatment changed LPS-induced CD18 expression on neutrophils, nor did the LPS- and mBSA-induced release of neutrophil chemoattractant mediators TNF-alpha, keratinocyte-derived chemokine, and LTB(4). Furthermore, in vitro MIP-2-induced neutrophil chemotaxis was inhibited by PAG and enhanced by NaHS treatments. Accordingly, MIP-2-induced CXCR2 internalization was enhanced by PAG and inhibited by NaHS treatments. Moreover, NaHS prevented MIP-2-induced CXCR2 desensitization. The PAG and NaHS effects correlated, respectively, with the enhancement and inhibition of MIP-2-induced G protein-coupled receptor kinase 2 expression. The effects of NaHS on neutrophil migration both in vivo and in vitro, together with CXCR2 internalization and G protein-coupled receptor kinase 2 expression were prevented by the ATP-sensitive potassium (K(ATP)(+)) channel blocker, glybenclamide. Conversely, diazoxide, a K(ATP)(+) channel opener, increased neutrophil migration in vivo. Together, our data suggest that during the inflammatory response, H(2)S augments neutrophil adhesion and locomotion, by a mechanism dependent on K(ATP)(+) channels.

A potent and selective nonpeptide antagonist of CXCR2 inhibits acute and chronic models of arthritis in the rabbit.

Much evidence implicates IL-8 as a major mediator of inflammation and joint destruction in rheumatoid arthritis. The effects of IL-8 and its related ligands are mediated via two receptors, CXCR1 and CXCR2. In the present study, we demonstrate that a potent and selective nonpeptide antagonist of human CXCR2 potently inhibits (125)I-labeled human IL-8 binding to, and human IL-8-induced calcium mobilization mediated by, rabbit CXCR2 (IC(50) = 40.5 and 7.7 nM, respectively), but not rabbit CXCR1 (IC(50) = >1000 and 2200 nM, respectively). These data suggest that the rabbit is an appropriate species in which to examine the anti-inflammatory effects of a human CXCR2-selective antagonist. In two acute models of arthritis in the rabbit induced by knee joint injection of human IL-8 or LPS, and a chronic Ag (OVA)-induced arthritis model, administration of the antagonist at 25 mg/kg by mouth twice a day significantly reduced synovial fluid neutrophils, monocytes, and lymphocytes. In addition, in the more robust LPS- and OVA-induced arthritis models, which were characterized by increased levels of proinflammatory mediators in the synovial fluid, TNF-alpha, IL-8, PGE(2), leukotriene B(4), and leukotriene C(4) levels were significantly reduced, as was erythrocyte sedimentation rate, possibly as a result of the observed decreases in serum TNF-alpha and IL-8 levels. In vitro, the antagonist potently inhibited human IL-8-induced chemotaxis of rabbit neutrophils (IC(50) = 0.75 nM), suggesting that inhibition of leukocyte migration into the knee joint is a likely mechanism by which the CXCR2 antagonist modulates disease.