CXCL5/CXCR2 modulates inflammation-mediated neural repair after optic nerve injury.

BACKGROUND: Previous studies reported that mild inflammation promotes retinal ganglion cell (RGC) survival and axonal regeneration after optic nerve (ON) injury with involvement of infiltrating macrophages and neutrophils. Here we aimed to evaluate the involvement and regulation of the main inflammatory chemokine pathway CXCL5/CXCR2 in the inflammation-mediated RGC survival and axonal regeneration in mice after ON injury. METHODS: The expressions and cellular locations of CXCL5 and CXCR2 were confirmed in mouse retina. Treatment effects of recombinant CXCL5 and CXCR2 antagonist SB225002 were studied in the explant culture and the ON injury model with or without lens injury. The number of RGCs, regenerating axons, and inflammatory cells were determined, and the activation of Akt andSTAT3 signaling pathways were evaluated. RESULTS: Cxcr2 and Cxcl5 expressions were increased after ON and lens injury. Addition of recombinant CXCL5 promoted RGC survival and neurite outgrowth in retinal explant culture with increase in the number of activated microglia, which was inhibited by SB225002 or clodronate liposomes. Recombinant CXCL5 also alleviated RGC death and promoted axonal regeneration in mice after ON injury, and promoted the lens injury-induced RGC protection with increase in the number of activated CD68+ cells. SB225002 inhibited lens injury-induced cell infiltration and activation, and attenuated the promotion effect on RGC survival and axonal regeneration through reduction of lens injury-induced Akt activation. CONCLUSIONS: CXCL5 promotes RGC survival and axonal regeneration after ON injury and further enhances RGC protection induced by lens injury with CD68+ cell activation, which is attenuated by CXCR2 antagonist. CXCL5/CXCR2 could be a potential therapeutic target for RGC survival promotion after ON injury.

Circ_0000215 Increases the Expression of CXCR2 and Promoted the Progression of Glioma Cells by Sponging miR-495-3p.

BACKGROUND: In recent years, accumulating studies have found that circular RNA (circRNA) exerts a great effect on tumor progression. Circ_0000215, a novel circRNA, remains largely unknown in terms of its effect and mechanism in glioma. METHOD: Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out to detect the expressions of circ_0000215, miR-495-3p and CXCR2 in human glial cell line HEB and glioma cell lines (A172, U251, U87, SHG-44, LN-18), human glioma tissues and adjacent healthy tissues. Gain- and loss-assays of circ_0000215 were conducted. Cell proliferation ability was detected via the CCK8 assay, and cell invasion ability was examined by Transwell assay. CXCR2 expression was evaluated via RT-PCR and Western blot. Moreover, bioinformatics was applied to analyze the targeting molecules of circ_0000215 and CXCR2. Verification of the relationship between these molecules were supported through the dual-luciferase reporter gene and RNA immunocoprecipitation (RIP) assay. RESULTS: Circ_0000215 and CXCR2 were remarkably upregulated in glioma tissues and cells. Overexpression of circ_0000215 notably promoted the proliferation, invasion and epithelial-mesenchymal transition (EMT) but inhibited apoptosis of glioma cells, while knocking down circ_0000215 had the opposite effects. Additionally, miR-495-3p, a sponge RNA of circ_0000215, inhibited the growth, invasion and EMT of glioma cells. Mechanistically, miR-495-3p targeted CXCR2 and negatively regulated CXCR2/PI3K/Akt pathway. However, the effects of miR-495-3p were all dampened by overexpression of circ_0000215. CONCLUSION: These data demonstrated that circ_0000215 functions as a competitive endogenous RNA by sponging miR-495-3p, thus accelerating glioma progression through CXCR2 axis.

Significant changes in circular RNA in the mouse cerebral cortex around an injury site after traumatic brain injury.

BACKGROUND AND OBJECTIVE: Circular RNA (circRNA) is an important type of non-coding RNA that has not been widely researched in traumatic brain injury (TBI). The present study aimd to detect the altered circRNA expression around an injury site in the mouse cerebral cortex after TBI and explore its potential functions. METHOD: C57BL/6 mice were used to construct a controlled cortical impact (CCI) model to simulate TBI. At 24 h post-TBI, the cortex around the injury site was collected, and the total RNA was extracted to perform RNA sequencing (RNA-seq). The differentially expressed circRNAs were determined according to the following criteria: |log2(fold change)| > 1, P < .05 and FDR < 0.05. Among them, circRNA chr8_87,859,283-87,904,548 was preliminarily explored to determine its function. RESULTS: A total of 8036 altered circRNAs were discovered, and among them, 16 were significantly changed (5 up-regulated and 11 down-regulated). The circRNA chr8_87,859,283-87,904,548 significantly increased by approximately 4 times in the cerebral cortex around the injury site after TBI and promoted neuro-inflammation through increasing the CXCR2 protein by sponging mmu-let-7a-5p. As a result, the increased circRNA chr8_87,859,283-87,904,548 blocked the restoration of neurological function after TBI. CONCLUSION: Many circRNAs are significantly up-regulated or down-regulated in the traumatic cerebral penumbra cortex after TBI. Among them, the circRNA chr8_87,859,283-87,904,548 potentially plays a pro-inflammatory role, which may have a deleterious effect on neurological restoration after TBI. .

FTY720 attenuates intestinal injury and suppresses inflammation in experimental necrotizing enterocolitis via modulating CXCL5/CXCR2 axis.

Necrotizing enterocolitis (NEC) remains one of the leading causes of death in neonatal infants and new therapeutic strategies for NEC are urgently required. The immunomodulatory agent FTY720 has been shown to have protective effects in various inflammatory diseases. In this study, we hypothesized that treatment with FTY720 confers protection against experimental NEC. Experimental NEC was induced in five-day-old C57BL/6 neonatal mice by hyperosmolar formula feeding plus hypoxia and lipopolysaccharide (LPS) challenges. Induction of NEC resulted in substantial weight loss and high mortality compared to the control group, whereas FTY720 treatment significantly attenuated weight loss and improved survival in NEC-challenged neonatal mice. FTY720 treatment strongly ameliorated NEC-induced intestinal injury with reduced apoptosis and up-regulation of intestinal barrier proteins in the ileal tissues. Furthermore, FTY720 treatment abrogated NEC-initiated intestinal and systemic inflammation with markedly diminished inflammatory cytokines and chemokines. Moreover, FTY720 treatment suppressed NEC-activated CXCL5/CXCR2 axis with down-regulated expression of CXCL5 and CXCR2 at both mRNA and protein levels. Thus, we demonstrate that FTY720 protects neonatal mice against NEC-associated lethality by ameliorating intestinal injury and attenuating inflammation, possibly via its down-regulation of NEC-induced activation of intestinal CXCL5/CXCR2 axis.

Contribution of the macrophage migration inhibitory factor superfamily of cytokines in the pathogenesis of preclinical and human multiple sclerosis: In silico and in vivo evidences.

Macrophage migration inhibitory factor (MIF) is a cytokine with pleiotropic actions involved in the pathogenesis of autoimmune disorders, including Multiple Sclerosis (MS). We have first evaluated in silico the involvement of MIF, its homologue D-DT, and the receptors CD74, CD44, CXCR2 and CXCR4 in encephalitogenic T cells from a mouse model of MS, the Experimental Allergic Encephalomyelitis (EAE), as well as in circulating T helper cells from MS patients. We show an upregulation of the receptors involved in MIF signaling both in the animal model and in patients. Also, a significant increase in MIF receptors is found in the CNS lesions associated to MS. Finally, the specific inhibitor of MIF, ISO-1, improved both ex vivo and in vivo the features of EAE. Overall, our data indicate that there is a significant involvement of the MIF pathway in MS ethiopathogenesis and that interventions specifically blocking MIF receptors may represent useful therapeutic approaches in the clinical setting.

miR-940 Suppresses Tumor Cell Invasion and Migration via Regulation of CXCR2 in Hepatocellular Carcinoma.

Aim. To investigate the expression of miR-940 in the hepatocellular carcinoma (HCC) and its impact on function and biological mechanism in the HCC cells. Methods. Quantitative RT-PCR analysis was used to quantify miR-940 expression in 46 cases of tissues and cells. Transfection of HCC cell lines was performed by miR-940 mimics; the abilities of invasion and migration were assessed through Transwell array. Western blot represents the alteration in expression of CXCR2 by miR-940 mimics. Results. miR-940 expression was decreased significantly in the HCC tissues and the relevant cell lines. miR-940 upregulation suppressed the invasion and migration of HCC cells in vitro. Furthermore, the CXCR2 was downregulated to suppress invasion and migration after miR-940 mimics. Moreover, decreased miR-940 expression was negatively correlated with Edmondson grade (P = 0.008), tumor microsatellite or multiple tumors (P = 0.04), vascular invasion (P = 0.035), and recurrence and metastasis (P = 0.038). Kaplan-Meier analysis demonstrated that decreased miR-940 expression contributed to poor overall survival (P < 0.05). Conclusions. Our findings present that miR-940 acts as a pivotal adaptor of CXCR2 and its transcription downregulated CXCR2 expression to decrease HCC invasion and migration in vitro. Our study suggests that miR-940 may be a novel poor prognostic biomarker for HCC.

Defocused low-energy shock wave activates adipose tissue-derived stem cells in vitro via multiple signaling pathways.

BACKGROUND AIMS: We found defocused low-energy shock wave (DLSW) could be applied in regenerative medicine by activating mesenchymal stromal cells. However, the possible signaling pathways that participated in this process remain unknown. In the present study, DLSW was applied in cultured rat adipose tissue-derived stem cells (ADSCs) to explore its effect on ADSCs and the activated signaling pathways. METHODS: After treating with DLSW, the cellular morphology and cytoskeleton of ADSCs were observed. The secretions of ADSCs were detected. The expressions of ADSC surface antigens were analyzed using flow cytometry. The expressions of proliferating cell nuclear antigen and Ki67 were analyzed using western blot. The expression of CXCR2 and the migrations of ADSCs in vitro and in vivo were detected. The phosphorylation of selected signaling pathways with or without inhibitors was also detected. RESULTS: DLSW did not change the morphology and phenotype of ADSCs, and could promote the secretion, proliferation and migration of ADSCs. The phosphorylation levels were significantly higher in mitogen-activated protein kinases (MAPK) pathway, phosphoinositide 3-kinase (PI-3K)/AKT pathway and nuclear factor-kappa B (NF-κB) signaling pathway but not in Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Furthermore, ADSCs were not activated by DLSW after adding the inhibitors of these pathways simultaneously. CONCLUSIONS: Our results demonstrated for the first time that DLSW could activate ADSCs through MAPK, PI-3K/AKT and NF-κB signaling pathways. Combination of DLSW and agonists targeting these pathways might improve the efficacy of ADSCs in regenerative medicine in the future.

Genetic and Pharmacologic Inhibition of the Chemokine Receptor CXCR2 Prevents Experimental Hypertension and Vascular Dysfunction.

BACKGROUND: The recruitment of leukocytes to the vascular wall is a key step in hypertension development. Chemokine receptor CXCR2 mediates inflammatory cell chemotaxis in several diseases. However, the role of CXCR2 in hypertension development and the underlying mechanisms remain unknown. METHODS: Angiotensin II (490 ng·kg-1·min-1) or deoxycorticosterone acetate (DOCA) salt-induced mouse hypertensive models in genetic ablation, pharmacologic inhibition of CXCR2, and adoptive bone marrow transfer mice were used to determine the role of CXCR2 in hypertension (measured by radiotelemetry and tail-cuff system), inflammation (verified by flow cytometry and quantitative real-time polymerase chain reaction [PCR] analysis), vascular remodeling (studied by haematoxylin and eosin and Masson's trichrome staining), vascular dysfunction (assessed by aortic ring), and oxidative stress (indicated by nicotinamide adenine dinucleotide phosphate [NADPH] oxidase activity, dihydroethidium staining, and quantitative real-time PCR analysis). Moreover, the blood CXCR2+ cells in normotensive controls and hypertension patients were analyzed by flow cytometry. RESULTS: Angiotensin II significantly upregulated the expression of CXCR2 mRNA and protein and increased the number of CD45+ CXCR2+ cells in mouse aorta (n=8 per group). Selective CXCR2 knockout (CXCR2-/-) or pharmacological inhibition of CXCR2 markedly reduced angiotensin II- or DOCA-salt-induced blood pressure elevation, aortic thickness and collagen deposition, accumulation of proinflammatory cells into the vascular wall, and expression of cytokines (n=8 per group). CXCR2 inhibition also ameliorated angiotensin II-induced vascular dysfunction and reduced vascular superoxide formation, NADPH activity, and expression of NADPH oxidase subunits (n=6 per group). Bone marrow reconstitution of wild-type mice with CXCR2-/- bone marrow cells also significantly abolished angiotensin II-induced responses (n=6 per group). It is important to note that CXCR2 blockade reversed established hypertension induced by angiotensin II or DOCA-salt challenge (n=10 per group). Furthermore, we demonstrated that CXCR2+ proinflammatory cells were higher in hypertensive patients (n=30) compared with normotensive individuals (n=20). CONCLUSIONS: Infiltration of CXCR2+ cells plays a pathogenic role in arterial hypertension and vascular dysfunction. Inhibition of CXCR2 pathway may represent a novel therapeutic approach to treat hypertension.

Immunohistochemical distinction of metastases of renal cell carcinoma with molecular analysis of overexpression of the chemokines CXCR2 and CXCR3 as independent positive prognostic factors for the tumorigenesis.

Renal cell carcinoma (RCC) represents, on average, over 90% of all malignancies of the kidney that occur in adults in both sexes. Chemokine receptors expression has been found in many kinds of cancer and at tumor metastasis site. We determined CXCR2 and CXCR3 expression in RCC by immunohistochemistry method and analyzed the prognostic value of these markers. Our finding demonstrated that CXCR3 were highly overexpressed in renal cancer tissues compared with those adjacent normal kidney tissues (P < 0.001). The results showed that high expression of CXCR3 was markedly correlated with metastasis (P = 0.021) and tumor stage (P = 0.031). CXCR2 were overexpressed in renal cancer tissues compared with those adjacent normal kidney tissues (P < 0.001). Our result showed that CXCR2 expression was correlated with high grade (P = 0.024), advanced stage (P = 0.029) and metastasis (P = 0.018). The log-rank test revealed that high CXCR2 and CXCR3 expressions are related to poorer overall survival (P < 0.001; P < 0.001). In conclusion, this study indicates the correlation of CXCR3 and CXCR3 with progression of RCC. In addition, high CXCR3 andCXCR2 expressions were correlated with shorter overall survival. © 2016 IUBMB Life, 68(8):629-633, 2016.

Repertaxin, an inhibitor of the chemokine receptors CXCR1 and CXCR2, inhibits malignant behavior of human gastric cancer MKN45 cells in vitro and in vivo and enhances efficacy of 5-fluorouracil.

Chemokine-mediated activation of G protein-coupled receptors CXCR1/2 promotes tumor growth, invasion, inflammation and metastasis. Repertaxin, a CXCR1/2 small-molecule inhibitor, has been shown to attenuate many of these tumor-associated processes. The present study aimed to investigate the effects of repertaxin alone and in combination with 5-fluorouracil (5-FU) on the malignant behavior of gastric cancer and the potential mechanisms. Gastric cancer MKN45 cells were treated in vitro with repertaxin and 5-FU, either alone or in combination. MTT and colony formation assay were performed to assess proliferation. Cell cycle progression and apoptosis was completed by flow cytometry. Migration and invasion were also assessed by transwell and wound-healing assay. Western blot analysis and quantitative RT-PCR were performed to determine expression of signaling molecules. MKN45 cells were also grown as xenografts in nude mice. Mice were treated with repertaxin and 5-FU, and tumor volume and weight, angiogenesis, proliferation and apoptosis were monitored. Combination of repertaxin and 5-FU inhibited MKN45 cell proliferation and increased apoptosis better than either agent alone. Similarly, enhanced effect of the combination was also observed in migration and invasion assays. The improved effect of repertaxin and 5-FU was also observed in vivo, as xenograft models treated with both compounds exhibited significantly decreased tumor volume and increased apoptosis. In conclusion, repertaxin inhibited malignant behavior of human gastric cancer MKN45 cells in vitro and in vivo and enhances efficacy of 5-fluorouracil. These data provide rationale that targeting CXCR1/2 with small molecule inhibitors may enhance chemotherapeutic efficacy for the treatment of gastric cancer.

S100A8 Production in CXCR2-Expressing CD11b+Gr-1high Cells Aggravates Hepatitis in Mice Fed a High-Fat and High-Cholesterol Diet.

Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease with a spectrum of presentations. S100A8 has been suggested to play a pivotal role as an endogenous immune-activator in inflammatory diseases. In this study, we investigated the involvement of S100A8 in the development of NAFLD. We used a diet model of NAFLD, in which mice were fed either a high-fat and high-cholesterol diet (HFHCD) or a normal diet (ND) as a control. We also assessed liver tissues from patients with NAFLD, including patients with nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH). HFHCD-fed mice, but not ND-fed mice, developed steatohepatitis. S100A8 expression was significantly elevated in the livers of HFHCD-fed mice compared with the controls. S100A8 was exclusively expressed in CXCR2-expressing CD11b(+)Gr-1(high) cells, which significantly increased in the livers of HFHCD-fed mice. These cells were F4/80 negative and did not possess a suppressor function. TNF-α expression was enhanced by S100A8 in primary liver leukocytes or a hepatocyte cell line and significantly elevated in the livers of HFHCD-fed mice. TNF-α was primarily produced from CD11b(+)F4/80(+) cells in liver leukocytes in response to S100A8. TNF-α deficiency attenuated hepatitis in HFHCD-fed mice. S100A8 was significantly more expressed in the liver tissues of patients with NASH than in those of patients with NAFL. In conclusion, these results suggest that S100A8 is primarily produced from CXCR2-expressing CD11b(+)Gr-1(high) cells, and it upregulates TNF-α production in CD11b(+)F4/80(+) cells through cellular cross-talk, which is an important mechanism in the development of NAFLD.

Phospholipase D2 drives mortality in sepsis by inhibiting neutrophil extracellular trap formation and down-regulating CXCR2.

We determined the function of phospholipase D2 (PLD2) in host defense in highly lethal mouse models of sepsis using PLD2(-/-) mice and a PLD2-specific inhibitor. PLD2 deficiency not only increases survival but also decreases vital organ damage during experimental sepsis. Production of several inflammatory cytokines (TNF, IL-1ß, IL-17, and IL-23) and the chemokine CXCL1, as well as cellular apoptosis in immune tissues, kidney, and liver, are markedly decreased in PLD2(-/-) mice. Bactericidal activity is significantly increased in PLD2(-/-) mice, which is mediated by increased neutrophil extracellular trap formation and citrullination of histone 3 through peptidylarginine deiminase activation. Recruitment of neutrophils to the lung is markedly increased in PLD2(-/-) mice. Furthermore, LPS-induced induction of G protein-coupled receptor kinase 2 (GRK2) and down-regulation of CXCR2 are markedly attenuated in PLD2(-/-) mice. A CXCR2-selective antagonist abolishes the protection conferred by PLD2 deficiency during experimental sepsis, suggesting that enhanced CXCR2 expression, likely driven by GRK2 down-regulation in neutrophils, promotes survival in PLD2(-/-) mice. Furthermore, adoptively transferred PLD2(-/-) neutrophils significantly protect WT recipients against sepsis-induced death compared with transferred WT neutrophils. We suggest that PLD2 in neutrophils is essential for the pathogenesis of experimental sepsis and that pharmaceutical agents that target PLD2 may prove beneficial for septic patients.

Collagen-Derived N-Acetylated Proline-Glycine-Proline in Intervertebral Discs Modulates CXCR1/2 Expression and Activation in Cartilage Endplate Stem Cells to Induce Migration and Differentiation Toward a Pro-Inflammatory Phenotype.

The factors that regulate the migration and differentiation of cartilage endplate stem cells (CESCs) remain unknown. N-Acetylated proline-glycine-proline (N-Ac-PGP) is a chemokine that is involved in inflammatory diseases. The purpose of this study was to detect N-Ac-PGP in degenerative intervertebral discs (IVDs) and to determine its roles in the migration and differentiation of CESCs. Enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-mass spectrometry results indicated that the levels of the proteases that generate N-Ac-PGP as well as N-Ac-PGP levels themselves increase with the progression of IVD degeneration. Immunohistochemistry and an N-Ac-PGP generation assay demonstrated that nucleus pulposus (NP) cells generate N-Ac-PGP from collagen. The effects of N-Ac-PGP on the migration and differentiation of CESCs were determined using migration assays, RT-PCR, immunoblot analysis, and ELISA. The results showed that the expression of N-Ac-PGP receptors (CXCR1 and CXCR2) in CESCs was upregulated by N-Ac-PGP. Additionally, N-Ac-PGP induced F-actin cytoskeletal rearrangement in CESCs and increased CESC chemotaxis. Furthermore, N-Ac-PGP recruited chondrocytes and spindle-shaped cells from the cartilage endplate (CEP) into the NP in vivo. These spindle-shaped cells expressed CD105 and Stro-1 (mesenchymal stem cell markers). N-Ac-PGP induced the differentiation of CESCs toward a pro-inflammatory phenotype with increased production of inflammatory cytokines rather than toward an NP-like phenotype. Our study indicated that, in the complex microenvironment of a degenerative disc, N-Ac-PGP is generated by NP cells and induces the migration of CESCs from the CEP into the NP. N-Ac-PGP induces a pro-inflammatory phenotype in CESCs, and these cells promote the inflammatory response in degenerative discs.

CXCL7-induced macrophage infiltration in lung tumor is independent of CXCR2 expression: CXCL7-induced macrophage chemotaxis in LLC tumors.

Chemokines play diverse roles in modulating the immune response during tumor development. Levels of CXC chemokine ligand 7 (CXCL7) protein vary during tumorigenesis, and the evidence suggests that this chemokine serves as a novel biomarker of early-stage lung cancer. We investigated the effect of CXCL7 gene expression on the infiltration of myeloid cells into the tumor microenvironment in Lewis lung carcinoma (LLC). Tumors established from LLC cells overexpressing CXCL7 (CXCL7-LLC tumors) increased the infiltration of CD206(+) M2 macrophages at the early stages of tumorigenesis. This infiltration was independent of CXCR2 expression on either tumor cells or macrophages. CXCL7-LLC tumors developed faster than control-LLC tumors (IRES-LLC tumor) did. The extent of CD4(+) T cell, CD8(+) T cell, and natural killer T cell infiltration was similar between the two tumor groups. Our findings suggest that CXCL7 attracts macrophages especially at the tumor site and may accelerate lung tumor development in the early stages.

MFG-E8 inhibits neutrophil migration through αvß3-integrin-dependent MAP kinase activation.

We have previously demonstrated the involvement of milk fat globule-epidermal growth factor-factor 8 (MFG­E8) in reducing neutrophil infiltration in a murine model of acute lung injury (ALI). In the present study, we aimed to delineate the mechanisms through which MFG­E8 attenuates neutrophil migration. Recombinant human MFG­E8 (rhMFG­E8) was expressed and purified in our facility. The human differentiated neutrophil cell line, dHL­60, was treated with rhMFG­E8 and cell migration assay was performed in a Boyden chamber using recombinant interleukin­8 (IL­8) as the chemoattractant. Surface CXCR2 and intracellular G protein­coupled receptor kinase 2 (GRK2) levels were evaluated by flow cytometry or western blot analysis. The levels of mitogen­activated protein (MAP) kinases were determined by western blot analysis. Treatment with rhMFG­E8 resulted in a significant inhibition of dHL­60 cell migration in a dose­dependent manner. There was a 46% decrease in CXCR2 expression in the rhMFG­E8­treated dHL­60 cells, which was associated with a 32% increase in GRK2 expression. In the dHL­60 cells, treatment with rhMFG­E8 promoted the phosphorylation of p38 and extracellular signal-regulated kinase (ERK) within 10­30 min. The use of SB203580, a p38 inhibitor, and PD98059, an ERK inhibitor, resulted in the restoration of dHL­60 cell migration which was significantly inhibited treatment with rhMFG­E8. Furthermore, blocking the MFG­E8 receptors, αvß3/αvß5­integrins, by anti­αv­integrin neutralizing antibody (Ab) inhibited the activation of p38 and ERK, and reversed the rhMFG­E8­induced inhibition of dHL­60 cell migration. Finally, treatment of the dHL­60 cells with SB203580 and PD98059 neutralized the rhMFG­E8­induced downregulation of CXCR2 expression and upregulation of GRK2 expression, as well as the inhibitory effects on cell migration. Our findings reveal a novel mechanism of action of MFG­E8 through which it inhibits neutrophil migration through αvß3-integrin-dependent MAP kinase activation.

Induction of G1 Arrest by SB265610 Involves Cyclin D3 Down-regulation and Suppression of CDK2 (Thr160) Phosphorylation.

BACKGROUND/AIM: The current study investigated the mechanisms underlying the antitumor activity of SB265610, a cysteine-amino acid-cysteine (CXC) chemokines receptor 2 (CXCR2) antagonist. MATERIALS AND METHODS: Cell-cycle progression and regulatory molecules were assessed by flow cytometry, immunoblotting, real-time PCR and immunoprecipitation. Target validation was achieved via RNA interference. RESULTS: G1 arrest induced by SB265610 occurred at concentrations lacking CXCR2 selectivity, persisted upon interleukin 8 (IL8) challenge, and did not affect IL8 downstream target expression. Profiling of G1 regulators revealed cyclin-dependent kinase 2 (CDK2) (Thr160) hypophosphorylation, cyclin D3 gene down-regulation, and p21 post-translational induction. However, only cyclin D3 and CDK2 contributed towards G1 arrest. Furthermore, SB265610 induced a sustained phosphorylation of the p38MAPK. Pharmacological interference with p38MAPK significantly abrogated SB265610-induced G1 arrest and normalized the expression of cyclin D3, with restoration of its exclusive binding to CDK6, but with weak recovery of CDK2 (Thr160) hypo-phosphorylation. CONCLUSION: The present study described the mechanisms for the anti-proliferative activity of SB265610 which may be of value in IL8-rich tumor microenvironments.

Overexpression of chemokine receptor CXCR2 and ligand CXCL7 in liver metastases from colon cancer is correlated to shorter disease-free and overall survival.

Our aim was to analyze the potential role of chemokine receptors CXCR2 and CXCR4 signalling pathways in liver metastatic colorectal cancer (CRC) relapse. CXCR2, CXCR4, and their chemokine ligands were evaluated in liver metastases of colorectal cancer in order to study their correlation with overall and disease-free survival of patients having received, or not received, a neoadjuvant chemotherapy regimen. Quantitative RT-PCR and CXCR2 immunohistochemical staining were carried out using CRC liver metastasis samples. Expression levels of CXCR2, CXCR4, and their ligands were statistically analyzed according to treatment with neoadjuvant chemotherapy and patients' outcome. CXCR2 and CXCL7 overexpression are correlated to shorter overall and disease-free survival. By multivariate analysis, CXCR2 and CXCL7 expressions are independent factors of overall and disease-free survival. Neoadjuvant chemotherapy increases significantly the expression of CXCR2: treated group 1.89 (0.02-50.92) vs 0.55 (0.07-3.22), P = 0.016. CXCL7 was overexpressed close to significance, 0.40 (0.00-7.85) vs 0.15 (0.01-7.88), P = 0.12. We show the involvement of CXCL7/CXCR2 signalling pathways as a predictive factor of poor outcome in metastatic CRC. 5-Fluorouracil-based chemotherapy regimens increase the expression of these genes in liver metastasis, providing one explanation for aggressiveness of relapsed drug-resistant tumors. Selective blockage of CXCR2/CXCL7 signalling pathways could provide new potential therapeutic opportunities.

DNA methyltransferase inhibition accelerates the immunomodulation and migration of human mesenchymal stem cells.

DNA methyltransferase (DNMT) inhibitors regulate target gene expression through epigenetic modifications, and these compounds have primarily been studied for cancer therapy or reprogramming. However, the effect of DNMT inhibitors on the immunomodulatory capacity of human mesenchymal stem cells (hMSCs) has not been investigated. In the present study, we treated hMSCs with 5-azacytidine (5-aza), a DNMT inhibitor, and confirmed that the inhibitory effects on mononuclear cell proliferation and cell migration toward activated T cells were increased. To identify the immunomodulatory factors stimulated through 5-aza treatment, we investigated the changes in promoter methylation patterns using methylation arrays and observed that the promoters of immunomodulatory factors, COX2 and PTGES, and migration-related factors, CXCR2 and CXCR4, were hypomethylated after 5-aza treatment. In addition, we observed that the COX2-PGE2 pathway is one of the main pathways for the enhanced immunosuppressive activity of hMSCs through 5-aza treatment. We also determined that the migration of hMSCs toward ligands for CXCR2/CXCR4 was increased after 5-aza treatment. Moreover, using an experimental colitis model, we showed that 5-aza pre-treatment could enhance the therapeutic effect of MSCs against immune-related diseases.

The lack of type I interferon induces neutrophil-mediated pre-metastatic niche formation in the mouse lung.

Metastases are the major cause of death from cancer. Thus, understanding the regulation of metastatic processes is of utmost importance. Here we show that mice with impaired type I IFN signaling (Ifnar1(-/-)) develop more lung metastases in the 4T1 mammary and LLC lung carcinoma model, compared to control mice. In Ifnar1(-/-) mice, higher metastasis load is accompanied by massive neutrophil accumulation in lungs. Elevated G-CSF levels in serum and enhanced CXCR2 expression on neutrophils are most likely responsible for this phenomenon. Lung infiltrating neutrophils facilitate an improved pre-metastatic niche formation, supporting more efficient tumor cell extravasation and proliferation in this organ. This is due to the enhanced expression of pro-metastatic proteins, like Bv8, MMP9, S100A8 and S100A9. Development of pre-metastatic niche together with reduced neutrophil cytotoxicity against tumor cells results in enhanced metastatic processes in Ifnar1(-/-) mice. Overall, our findings describe a novel role for IFN during metastasis development and suggest that new treatment strategies should be considered for prevention of metastasis formation in patients.

Immunomodulatory oligonucleotides inhibit neutrophil migration by decreasing the surface expression of interleukin-8 and leukotriene B4 receptors.

Neutrophils play important roles in many inflammatory diseases. The migration of neutrophils to the inflammatory site is tightly regulated by specific chemokines, of which interleukin-8 (IL-8) and leukotriene B4 (LTB4 ) constitute key mediators by binding to the surface receptors CXCR1/2 and BLT1, respectively. Oligonucleotides (ODN) containing CpG motifs mediate potent immunomodulatory effects through binding to Toll-like receptor 9. So far, knowledge on how ODN can affect neutrophil migration during inflammation is lacking. This study demonstrates that several novel CpG ODN significantly down-regulate the surface expression of CXCR1/2 and BLT1. In addition, the ODN significantly blocked IL-8-induced and LTB4 -induced neutrophil migration in vitro, as well as leucocyte migration in vivo demonstrated in mice by intravital microscopy and in a model of airway inflammation. The down-regulation of CXCR1 is rapid, occurring 15 min after ODN stimulation, and can be mediated through an endosomally independent mechanism. Inhibition of the IL-8 and LTB4 pathways may provide new opportunities of therapeutic intervention using ODN to reduce neutrophil infiltration during inflammation.