High expression level of CXCL1/GROα is linked to advanced stage and worse survival in uterine cervical cancer and facilitates tumor cell malignant processes.

BACKGROUND: CXCL1 belongs to a member of the ELR + CXC chemokine subgroups that also known as GRO-alpha. It has been recognized that several types of human cancers constitutively express CXCL1, which may serve as a crucial mediator involved in cancer development and metastasis via an autocrine and/or paracrine fashion. However, the expression pattern and clinical significance of CXCL1 in human uterine cervix cancer (UCC), as well as its roles and mechanisms in UCC tumor biology remains entirely unclear. METHODS: The expression and clinical significance of CXCL1 in UCC tissues was explored using immunohistochemistry and bioinformatics analyses. The expression and effects of CXCL1 in HeLa UCC cells were assessed using ELISA, CCK-8 and transwell assays. Western blotting experiments were performed to evaluate the potential mechanism of CXCL1 on malignant behaviors of HeLa UCC cells. RESULTS: The current study demonstrated that CXCL1 was expressed in HeLa UCC cells, PHM1-41 human immortalized cervical stromal cells, as well as cervical tissues, with UCC tissues having an evidently high level of CXCL1. This high level of CXCL1 in cancer tissues was notably related to poor clinical stages and worse survival probability, rather than tumor infiltration and patient age. In addition, CXCL1 expression was extremely correlated with CCL20, CXCL8 and CXCL3 cancer-associated chemokines expression. In vitro, the growth and migration abilities of HeLa cells were significantly enhanced in the presence of exogenous CXCL1. Gain-function assay revealed that CXCL1 overexpression significantly promoted growth and migration response in HeLa cells in both autocrine and paracrine manners. Finally, we found that CXCL1 overexpression in HeLa cells influenced the expression of ERK signal-related genes, and HeLa cell malignant behaviors derived from CXCL1 overexpression were further interrupted in the presence of the ERK1/2 blocker. CONCLUSION: Our findings demonstrate the potential roles of CXCL1 as a promoter and a novel understanding of the functional relationship between CXCL1 and the ERK signaling pathway in UCC.

Blocking of inflammatory heparan sulfate domains by specific antibodies is not protective in experimental glomerulonephritis.

Glomerulonephritis is an acquired serious glomerular disease, which involves the interplay of many factors such as cytokines, chemokines, inflammatory cells, and heparan sulfate (HS). We previously showed that blocking of inflammatory heparan sulfate domains on cultured glomerular endothelium by specific anti-HS single chain antibodies reduced polymorphonuclear cell (PMN) adhesion and chemokine binding. We hypothesized that injection of anti-HS antibodies in PMN-driven experimental glomerulonephritis should reduce glomerular influx of PMNs and thereby lead to a better renal outcome. In contrast to our hypothesis, co-injection of anti-HS antibodies did not alter the final outcome of anti-glomerular basement membrane (anti-GBM)-induced glomerulonephritis. Glomerular PMN influx, normally peaking 2 hours after induction of glomerulonephritis with anti-GBM IgG was not reduced by co-injection of anti-HS antibodies. Four days after induction of glomerulonephritis, albuminuria, renal function, glomerular hyalinosis and fibrin deposition were similar in mice treated and not treated with anti-HS antibodies. Interestingly, we observed transient effects in mice co-injected with anti-HS antibodies compared to mice that did not receive anti-HS antibodies: (i) a decreased renal function 2 hours and 1 day after induction of glomerulonephritis; (ii) an increased albuminuria after 2 hours and 1 day; (iii) an increased glomerular fibrin deposition after 1 day; (iv) a reduced glomerular macrophage influx after 1 day; (v) a sustained glomerular presence of PMNs at day 1 and 4, accompanied by an increased renal expression of IL-6, CXCL1, ICAM-1, L-selectin, CD11b and NF-κB. The mechanism underlying these observations induced by anti-HS antibodies remains unclear, but may be explained by a temporarily altered glycocalyx and/or altered function of PMNs due to the binding of anti-HS antibodies. Nevertheless, the evaluated anti-HS antibodies do not show therapeutic potential in anti-GBM-induced glomerulonephritis. Future research should evaluate other strategies to target HS domains involved in inflammatory processes during glomerulonephritis.

Apolipoprotein E Promotes Immune Suppression in Pancreatic Cancer through NF-κB-Mediated Production of CXCL1.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with few effective therapeutic options. PDAC is characterized by an extensive fibroinflammatory stroma that includes abundant infiltrating immune cells. Tumor-associated macrophages (TAM) are prevalent within the stroma and are key drivers of immunosuppression. TAMs in human and murine PDAC are characterized by elevated expression of apolipoprotein E (ApoE), an apolipoprotein that mediates cholesterol metabolism and has known roles in cardiovascular and Alzheimer's disease but no known role in PDAC. We report here that ApoE is also elevated in peripheral blood monocytes in PDAC patients, and plasma ApoE protein levels stratify patient survival. Orthotopic implantation of mouse PDAC cells into syngeneic wild-type or in ApoE-/- mice showed reduced tumor growth in ApoE-/- mice. Histologic and mass cytometric (CyTOF) analysis of these tumors showed an increase in CD8+ T cells in tumors in ApoE-/- mice. Mechanistically, ApoE induced pancreatic tumor cell expression of Cxcl1 and Cxcl5, known immunosuppressive factors, through LDL receptor and NF-κB signaling. Taken together, this study reveals a novel immunosuppressive role of ApoE in the PDAC microenvironment. SIGNIFICANCE: This study shows that elevated apolipoprotein E in PDAC mediates immune suppression and high serum apolipoprotein E levels correlate with poor patient survival.See related commentary by Sherman, p. 4186.

MicroRNA-206 antagomiR‒enriched extracellular vesicles attenuate lung ischemia‒reperfusion injury through CXCL1 regulation in alveolar epithelial cells.

BACKGROUND: Our hypothesis is that the immunomodulatory capacities of mesenchymal stem cell‒derived extracellular vesicles (EVs) can be enhanced by specific microRNAs (miRNAs) to effectively attenuate post-transplant lung ischemia‒reperfusion (IR) injury. METHODS: The expression of miR-206 was analyzed in bronchoalveolar lavage (BAL) fluid of patients on Days 0 and 1 after lung transplantation. Lung IR injury was evaluated in C57BL/6 mice using a left lung hilar-ligation model with or without treatment with EVs or antagomiR-206‒enriched EVs. Murine lung tissue was used for miRNA microarray hybridization analysis, and cytokine expression, lung injury, and edema were evaluated. A donation after circulatory death and murine orthotopic lung transplantation model was used to evaluate the protection by enriched EVs against lung IR injury. In vitro studies analyzed type II epithelial cell activation after coculturing with EVs. RESULTS: A significant upregulation of miR-206 was observed in the BAL fluid of patients on Day 1 after lung transplantation compared with Day 0 and in murine lungs after IR injury compared with sham. Treatment with antagomiR-206‒enriched EVs attenuated lung dysfunction, injury, and edema compared with treatment with EVs alone after murine lung IR injury. Enriched EVs reduced lung injury and neutrophil infiltration as well as improved allograft oxygenation after murine orthotopic lung transplantation. Enriched EVs significantly decreased proinflammatory cytokines, especially epithelial cell‒dependent CXCL1 expression, in the in vivo and in vitro IR injury models. CONCLUSIONS: EVs can be used as biomimetic nanovehicles for protective immunomodulation by enriching them with antagomiR-206 to mitigate epithelial cell activation and neutrophil infiltration in the lungs after IR injury.

Icariin Ameliorates Lower Back Pain in Rats via Suppressing the Secretion of Cytokine-Induced Neutrophil Chemoatractant-1.

PURPOSE: To investigate whether icariin (ICA), a well-known medicine extracted from the stem and leaf of Epimedium brevicornum Maxim, had analgesic effect on lower back pain (LBP) in rats. METHODS: In a puncture-induced LBP rat model, the severity of LBP was quantified using the paw/foot withdrawal threshold method after intragastric administration of ICA at a dosage of 50 mg/kg/d or 100 mg/kg/d. The pain-related peptides of substance P (SP) and calcitonin gene-related peptide (CGRP) were also measured in intervertebral disc (IVD) tissue using RT-PCR after ICA treatment. In addition, the expression of cytokine-induced neutrophil chemoattractant-1 (CINC-1) in IVD was quantified using RT-PCR and ELISA examination. RESULTS: ICA treatment resulted in a significant amelioration of mechanical allodynia in a dose-response manner, and the analgesic effect could last for two weeks even during the washout period. More importantly, the mechanism of analgesic pharmacological effect in ICA was to suppress the upregulated CINC-1, the homolog of IL-8 in rats, which is a crucial proalgesic factor contributing to LBP, in IVDs. CONCLUSION: ICA is a novel herbal extract to relieve LBP, and it may be a promising alternative pain killer in the future.

Polyinosinic-Polycytidylic Acid Induces CXCL1 Expression in Cultured hCMEC/D3 Human Cerebral Microvascular Endothelial Cells.

OBJECTIVE: Brain microvascular endothelial cells are integral components of the blood-brain barrier and play a role in protecting the brain from invading microbes. CXC motif chemokine ligand 1 (CXCL1) induces the chemotaxis of neutrophils, and neutrophils are important in host defense in the brain. However, dysregulated neutrophil infiltration leads to brain diseases. Toll-like receptor 3 (TLR3) is a pattern recognition receptor that recognizes viral double-stranded RNA (dsRNA). The aim of this study was to investigate the effect of an TLR3 agonist on the expression of CXCL1 in brain vascular endothelial cells. METHODS: hCMEC/D3 human cerebral microvascular endothelial cells were cultured and treated with polyinosinic-polycytidylic acid (poly IC), a potent synthetic dsRNA agonist for TLR3. The production of CXCL1 mRNA and protein was assessed by real-time RT-PCR and ELISA. The expression of CXCL1 was compared with that of CXCL8. The effect of pretreatment of cells with a NF-κB inhibitor (SN50), a p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580), a c-Jun N-terminal kinase (JNK) inhibitor (SP600125), an interferon (IFN) regulatory factor 3 inhibitor (MRT67307), and an anti-type I IFN-neutralizing antibody mixture was examined. Phosphorylation of p38 was examined using Western blotting. RESULTS: Treating cultured hCMEC/D3 human cells with poly IC induced the expression of CXCL1 as well as another chemokine CXCL8. Pretreatment of cells with SN50, SB203580, and SP600125 decreased the induction of CXCL1 by poly IC. However, it was not affected by MRT67307 or by an anti-type I IFN-neutralizing antibody mixture. Pretreatment of cells with SN50 decreased the poly IC-induced phosphorylation of p38. CONCLUSIONS: Poly IC induces the expression of CXCL1 in hCMEC/D3 cells. NF-κB, p38 MAPK, and JNK are involved in this reaction. There is a cross-talk between NF-κB and p38, and NF-κB partially regulates phosphorylation of p38. CXCL1 produced by brain microvascular endothelial cells may contribute to the brain's defense against viral infection and various neurological diseases associated with neutrophil accumulation.

Elevation of CXCL1 indicates poor prognosis and radioresistance by inducing mesenchymal transition in glioblastoma.

INTRODUCTION: Glioblastoma (GBM) is identified as a lethal malignant tumor derived from the nervous system. Despite the standard clinical strategy including maximum surgical resection, temozolomide (TMZ) chemotherapy, and radiotherapy, the median survival of GBM patients remains <15 months. Accumulating evidence indicates that rapid-acquired radioresistance is one of the most common reasons for GBM recurrence. Therefore, developing novel therapeutic targets for radioresistant GBM could yield long-term cures. AIMS: To investigate the functional role of CXCL1 in the acquired radioresistance and identify the molecular pathway correlated to CXCL1. RESULTS: In this study, we identified that CXCL1 is highly expressed in GBM and the elevation of CXCL1 is involved in radioresistance and poor prognosis in GBM patients. Additionally, silencing CXCL1 attenuated the proliferation and radioresistance of GBM cells. Furthermore, we demonstrated that CXCL1-overexpression induced radioresistance through mesenchymal transition of GBM via the activation of nuclear factor-kappa B (NF-κB) signaling. CONCLUSION: CXCL1 was highly enriched in GBM and positively correlated with poor prognosis in GBM patients. Additionally, elevated CXCL1 induced radioresistance in GBM through regulation of NF-κB signaling by promoting mesenchymal transition in GBM.

Interleukin-22 Ameliorates Neutrophil-Driven Nonalcoholic Steatohepatitis Through Multiple Targets.

BACKGROUND AND AIMS: Nonalcoholic fatty liver disease encompasses a spectrum of diseases ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), cirrhosis, and liver cancer. At present, how simple steatosis progresses to NASH remains obscure and effective pharmacological therapies are lacking. Hepatic expression of C-X-C motif chemokine ligand 1 (CXCL1), a key chemokine for neutrophil infiltration (a hallmark of NASH), is highly elevated in NASH patients but not in fatty livers in obese individuals or in high-fat diet (HFD)-fed mice. The aim of this study was to test whether overexpression of CXCL1 itself in the liver can induce NASH in HFD-fed mice and to test the therapeutic potential of IL-22 in this new NASH model. APPROACH AND RESULTS: Overexpression of Cxcl1 in the liver alone promotes steatosis-to-NASH progression in HFD-fed mice by inducing neutrophil infiltration, oxidative stress, and stress kinase (such as apoptosis signal-regulating kinase 1 and p38 mitogen-activated protein kinase) activation. Myeloid cell-specific deletion of the neutrophil cytosolic factor 1 (Ncf1)/p47phox gene, which encodes a component of the NADPH oxidase 2 complex that mediates neutrophil oxidative burst, markedly reduced CXCL1-induced NASH and stress kinase activation in HFD-fed mice. Treatment with interleukin (IL)-22, a cytokine with multiple targets, ameliorated CXCL1/HFD-induced NASH or methionine-choline deficient diet-induced NASH in mice. Mechanistically, IL-22 blocked hepatic oxidative stress and its associated stress kinases via the induction of metallothionein, one of the most potent antioxidant proteins. Moreover, although it does not target immune cells, IL-22 treatment attenuated the inflammatory functions of hepatocyte-derived, mitochondrial DNA-enriched extracellular vesicles, thereby suppressing liver inflammation in NASH. CONCLUSIONS: Hepatic overexpression of CXCL1 is sufficient to drive steatosis-to-NASH progression in HFD-fed mice through neutrophil-derived reactive oxygen species and activation of stress kinases, which can be reversed by IL-22 treatment via the induction of metallothionein.

Ureaplasma Urealyticum Infection Contributes to the Development of Pelvic Endometriosis Through Toll-Like Receptor 2.

Endometriosis is a chronic gynecological disorder, characterized by the presence of ectopic endometrial tissue outside the uterine cavity. Among several hypotheses, Sampson's theory of retrograde menstruation is still applicable. Recent studies have reported the importance of inflammation among endometrial tissue, the peritoneum, and immune cells. However, less is known regarding the role of bacterial infection in the pathophysiology of endometriosis. We hypothesized that Ureaplasma urealyticum infection might contribute to the development of endometriosis by inducing the production of inflammatory mediators by peritoneal mesothelial cells (PMCs), possibly through TLR2. Hence, our objective was to reveal whether PMC infection by U. urealyticum is associated with endometriosis. Moreover, we aimed to demonstrate the molecular mechanism involved in this relationship. We developed a new infection-induced mouse model of endometriosis with wild type and Tlr2-deficient mice. Based on the in vivo mouse model, U. urealyticum-infected mice showed significantly increased numbers and sizes of ectopic endometriotic lesions. U. urealyticum upregulated not only the production of IL-6, CXCL1, and CCL2, but also the expression of ICAM-1, VCAM-1, and MMP2 in murine PMCs. Similarly, endometrial stromal cells dose-dependently produced IL-6, CXCL1, and CCL2 in response to U. urealyticum infection. The series of inflammatory responses in PMCs was mediated mainly through TLR2. The phosphorylation of ERK and JNK was observed when U. urealyticum was added to PMCs and knock out of Tlr2 inhibited these MAPKs phosphorylation. Based on our co-culture study, U. urealyticum-infected PMCs exhibited significantly increased attachment to ESCs compared with uninfected PMCs. Collectively, U. urealyticum infection promotes the development of endometriosis by increasing inflammatory mediators, adhesion molecules, and MMP-2 expression in PMCs through TLR2 signaling. Through our results, we present a theory that infection-induced pelvic inflammation contributes to the initiation and progression of endometriosis. Appropriate treatment of reproductive tract infection may decrease the prevalence of endometriosis.

Mannan-binding lectin promotes keratinocyte to produce CXCL1 and enhances neutrophil infiltration at the early stages of psoriasis.

Psoriasis is a chronic, relapsing inflammatory skin disorder. Numerous experimental evidence and therapeutic evidence have shown that the innate immune response is critical for the pathogenesis and development of psoriasis. Mannan-binding lectin (MBL), a prototypic pattern recognition molecule of the innate immune system, plays an essential role in the host defense against certain infections and also appears to be a major regulator of inflammation. In this study, we investigated the function of MBL on the course of experimental murine imiquimod (IMQ)-induced psoriasis. Our data showed that MBL-deficient (MBL-/- ) mice exhibited attenuated skin damage characterized by greatly decreased erythema compared with wild-type control mice during the early stages of IMQ-induced psoriasis-like skin inflammation. The reduced skin inflammation in MBL-/- mice was associated with the decreased infiltration of neutrophils. Furthermore, we have determined that MBL deficiency limited the chemokine CXCL1 production from skin keratinocytes upon IMQ stimulation, which might be responsible for the impaired skin recruitment of neutrophils. Additionally, we have provided the data that MBL protein promotes the IMQ-induced expression of CXCL1 and activation of MAPK/NF-κB signalling pathway in human keratinocyte HaCaT cells in vitro. In summary, our study revealed an unexpected role of MBL on keratinocyte function in skin, thus offering a new insight into the pathogenic mechanisms of psoriasis.

Cancer extracellular vesicles contribute to stromal heterogeneity by inducing chemokines in cancer-associated fibroblasts.

Cancer-associated fibroblasts (CAFs), one of the major components of a tumour microenvironment, comprise heterogeneous populations involved in tumour progression. However, it remains obscure how CAF heterogeneity is governed by cancer cells. Here, we show that cancer extracellular vesicles (EVs) induce a series of chemokines in activated fibroblasts and contribute to the formation of the heterogeneity. In a xenograft model of diffuse-type gastric cancer, we showed two distinct fibroblast subpopulations with alpha-smooth muscle actin (α-SMA) expression or chemokine expression. MicroRNAs (miRNAs) profiling of the EVs and the transfection experiment suggested that several miRNAs played a role in the induction of chemokines such as CXCL1 and CXCL8 in fibroblasts, but not for the myofibroblastic differentiation. Clinically, aberrant activation of CXCL1 and CXCL8 in CAFs correlated with poorer survival in gastric cancer patients. Thus, this link between chemokine expression in CAFs and tumour progression may provide novel targets for anticancer therapy.

4-Methylbenzenecarbothioamide, a hydrogen sulfide donor, inhibits tumor necrosis factor-α and CXCL1 production and exhibits activity in models of pain and inflammation.

The gasotransmitter hydrogen sulfide (H2S) is known to regulate many pathophysiological processes. Preclinical assays have demonstrated that H2S donors exhibit anti-inflammatory and antinociceptive activities, characterized by reduction of inflammatory mediators production, leukocytes recruitment, edema and mechanical allodynia. In the present study, the effects induced by 4-methylbenzenecarbothioamide (4-MBC) in models of pain and inflammation in mice, the mechanisms mediating such effects and the H2S-releasing property of this compound were evaluated. 4-MBC spontaneously released H2S in vitro in the absence of organic thiols. Intraperitoneal (i.p.) administration of 4-MBC (100 or 150 mg/kg) reduced the second phase of the nociceptive response induced by formaldehyde and induced a long lasting inhibitory effect on carrageenan mechanical allodynia. 4-MBC antiallodynic effect was not affected by previous administration of naltrexone or glibenclamide. 4-MBC (50, 100 or 150 mg/kg, i.p.) induced a long lasting inhibitory effect on paw edema induced by carrageenan. The highest dose (150 mg/kg, i.p.) of 4-MBC inhibited tumor necrosis factor-α and CXCL1 production and myeloperoxidase activity induced by carrageenan. Mechanical allodynia and paw edema induced by carrageenan were not inhibited by the 4-MBC oxo analogue (p-toluamide). In summary, 4-MBC, an H2S releasing thiobenzamide, exhibits antinociceptive and anti-inflammatory activities. These activities may be due to reduced cytokine and chemokine production and neutrophil recruitment. The H2S releasing property is likely essential for 4-MBC activity. Our results indicate that 4-MBC may represent a useful pharmacological tool to investigate the biological roles of H2S.

DNA Sensor IFI204 Contributes to Host Defense Against Staphylococcus aureus Infection in Mice.

Interferon-inducible protein (IFI204) (p204, the murine homolog of human IFI16) is known as a cytosolic DNA sensor to recognize DNA viruses and intracellular bacteria. However, little is known about its role during extracellular bacterial infection. Here we show that IFI204 is required for host defense against the infection of Staphylococcus aureus, an extracellular bacterial pathogen. IFI204 deficiency results in decreased survival, increased bacterial loads, severe organs damage, and decreased recruitment of neutrophils and macrophages. Production of several inflammatory cytokines/chemokines including IFN-ß and KC is markedly decreased, as well as the related STING-IRF3 and NF-κB pathways are impaired. However, exogenous administration of recombinant KC or IFN-ß is unable to rescue the susceptibility of IFI204-deficient mice, suggesting that other mechanisms rather than KC and IFN-ß account for IFI204-mediated host defense. IFI204 deficiency leads to a defect in extracellular bacterial killing in macrophages and neutrophils, although bacterial engulf, and intracellular killing activity are normal. Moreover, the defect of bactericidal activity is mediated by decreased extracellular trap formation in the absence of IFI204. Adoptively transferred WT bone marrow cells significantly protect WT and IFI204-deficient recipients against Staphylococcus infection compared with transferred IFI204-deficient bone marrow cells. Hence, this study suggests that IFI204 is essential for the host defense against Staphylococcus infection.

MLN4924 protects against interleukin-17A-induced pulmonary inflammation by disrupting ACT1-mediated signaling.

An excessive inflammatory response in terminal airways, alveoli, and the lung interstitium eventually leads to pulmonary hypertension and chronic obstructive pulmonary disease. Proinflammatory cytokine interleukin-17A (IL-17A) has been implicated in the pathogenesis of pulmonary inflammatory diseases. MLN4924, an inhibitor of NEDD8-activating enzyme (NAE), is associated with the treatment of various types of cancers, but its role in the IL-17A-mediated inflammatory response has not been identified. Here, we report that MLN4924 can markedly reduce the expression of proinflammatory cytokines and chemokines such as IL-1ß, IL-6, and CXCL-1 and neutrophilia in a mouse model of IL-17A adenovirus-induced pulmonary inflammation. MLN4924 significantly inhibited IL-17A-induced stabilization of mRNA of proinflammatory cytokines and chemokines in vitro. Mechanistically, MLN4924 significantly blocked the activation of MAPK and NF-κB pathways and interfered with the interaction between ACT1 and tumor necrosis factor receptor-associated factor proteins (TRAFs), thereby inhibiting TRAF6 ubiquitination. Taken together, our data uncover a previously uncharacterized inhibitory effect of MLN4924 on the IL-17A-mediated inflammatory response; this phenomenon may facilitate the development of MLN4924 into an effective small-molecule drug for the treatment of pulmonary inflammatory diseases.

Novel Paracrine Functions of Smooth Muscle Cells in Supporting Endothelial Regeneration Following Arterial Injury.

RATIONALE: Regeneration of denuded or injured endothelium is an important component of vascular injury response. Cell-cell communication between endothelial cells and smooth muscle cells (SMCs) plays a critical role not only in vascular homeostasis but also in disease. We have previously demonstrated that PKCδ (protein kinase C-delta) regulates multiple components of vascular injury response including apoptosis of SMCs and production of chemokines, thus is an attractive candidate for a role in SMC-endothelial cells communication. OBJECTIVE: To test whether PKCδ-mediated paracrine functions of SMCs influence reendothelialization in rodent models of arterial injury. METHODS AND RESULTS: Femoral artery wire injury was performed in SMC-conditional Prkcd knockout mice, and carotid angioplasty was conducted in rats receiving transient Prkcd knockdown or overexpression. SMC-specific knockout of Prkcd impaired reendothelialization, reflected by a smaller Evans blue-excluding area in the knockout compared with the wild-type controls. A similar impediment to reendothelialization was observed in rats with SMC-specific knockdown of Prkcd. In contrast, SMC-specific gene transfer of Prkcd accelerated reendothelialization. In vitro, medium conditioned by AdPKCδ-infected SMCs increased endothelial wound closure without affecting their proliferation. A polymerase chain reaction-based array analysis identified Cxcl1 and Cxcl7 among others as PKCδ-mediated chemokines produced by SMCs. Mechanistically, we postulated that PKCδ regulates Cxcl7 expression through STAT3 (signal transducer and activator of transcription 3) as knockdown of STAT3 abolished Cxcl7 expression. The role of CXCL7 in SMC-endothelial cells communication was demonstrated by blocking CXCL7 or its receptor CXCR2, both significantly inhibited endothelial wound closure. Furthermore, insertion of a Cxcl7 cDNA in the lentiviral vector that carries a Prkcd shRNA overcame the adverse effects of Prkcd knockdown on reendothelialization. CONCLUSIONS: SMCs promote reendothelialization in a PKCδ-dependent paracrine mechanism, likely through CXCL7-mediated recruitment of endothelial cells from uninjured endothelium.

Identification of Phf16 and Pnpla3 as new adipogenic factors regulated by phytochemicals.

Adipocyte differentiation is controlled by multiple signaling pathways. To identify new adipogenic factors, C3H10T1/2 adipocytes were treated with previously known antiadipogenic phytochemicals (resveratrol, butein, sulfuretin, and fisetin) for 24 hours. Commonly regulated genes were then identified by transcriptional profiling analysis. Three genes (chemokine (C-X-C motif) ligand 1 [ Cxcl1], heme oxygenase 1 [ Hmox1], and PHD (plant homeo domain) finger protein 16 [ Phf16]) were upregulated while two genes (G0/G1 switch gene 2 [ G0s2] and patatin-like phospholipase domain containing 3 [ Pnpla3]) were downregulated by these four antiadipogenic compounds. Tissue expression profiles showed that the G0s2 and Pnpla3 expressions were highly specific to adipose depots while the other three induced genes were ubiquitously expressed with significantly higher expression in adipose tissues. While Cxcl1 expression was decreased, expressions of the other four genes were significantly increased during adipogenic differentiation of C3H10T1/2 cells. Small interfering RNA-mediated knockdown including Phf16 and Pnpla3 indicated that these genes might play regulatory roles in lipid accumulation and adipocyte differentiation. Specifically, the silencing of two newly identified adipogenic genes, Phf16 or Pnpla3, suppressed lipid accumulation and expression of adipocyte markers in both 3T3-L1 and C3H10T1/2 cells. Taken together, these data showed previously uncovered roles of Phf16 and Pnpla3 in adipogenesis, highlighting the potential of using phytochemicals for further investigation of adipocyte biology.

Follicle-stimulating hormone peptide-conjugated nanoparticles for targeted shRNA delivery lead to effective gro-α silencing and antitumor activity against ovarian cancer.

The distinct hormone molecules and receptors, such as follicle-stimulating hormone receptor (FSHR) in ovarian cancer, provide opportunities for more precisely targeted therapy. We previously developed FSHR-mediated nanoparticles and found that FSH peptides on the surface of nanoparticles improved the delivery of short interfering RNA (siRNA) into ovarian cancer cells. However, the high toxicity of the nanoparticles and the transient silencing of the siRNA in vivo limited further study. Here, we developed FSH peptide-conjugated nanoparticles with an increased amount of polyethylene glycol (PEG) grafting and encapsulated short hairpin RNA (shRNA) to silence the target gene, growth-regulated oncogene α (gro-α). The nanoparticle complexes exhibited good stability over three weeks. Expression of the target gene, gro-α, was significantly down-regulated by gro-α shRNA-loaded nanoparticles conjugated with FSH peptides (FSH33-G-NP) in FSHR-positive HEY cells. Cell proliferation, migration, and invasion were also inhibited by FSH33-G-NP. Tumor growth was delayed significantly in the mice treated with FSH33-G-NP. No significant loss of body weight or severe toxic effects were observed in any groups. In conclusion, gro-α shRNA-loaded nanoparticles conjugated with FSH peptides overcame the drawbacks of the in vivo application of RNAi therapeutics and polymer-based nanocarriers and showed safe antitumor efficacy. Our study might contribute to the application of FSHR-based targeted therapy and imaging in cancer.

Anti-interleukin-6 signalling therapy rebalances the disrupted cytokine production of B cells from patients with active rheumatoid arthritis.

Rheumatoid arthritis (RA) is associated with abnormal B cell-functions implicating antibody-dependent and -independent mechanisms. B cells have emerged as important cytokine-producing cells, and cytokines are well-known drivers of RA pathogenesis. To identify novel cytokine-mediated B-cell functions in RA, we comprehensively analysed the capacity of B cells from RA patients with an inadequate response to disease modifying anti-rheumatic drugs to produce cytokines in comparison with healthy donors (HD). RA B cells displayed a constitutively higher production of the pathogenic factors interleukin (IL)-8 and Gro-α, while their production of several cytokines upon activation via the B cell receptor for antigen (BCR) was broadly suppressed, including a loss of the expression of the protective factor TRAIL, compared to HD B cells. These defects were partly erased after treatment with the IL-6-signalling inhibitor tocilizumab, indicating that abnormal IL-6 signalling contributed to these abnormalities. Noteworthy, the clinical response of individual patients to tocilizumab therapy could be predicted using the amounts of MIP-1ß and ß-NGF produced by these patients' B cells before treatment. Taken together, our study highlights hitherto unknown abnormal B-cell functions in RA patients, which are related to the unbalanced cytokine network, and are potentially relevant for RA pathogenesis and treatment.

Antiallodynic activity of leflunomide is partially inhibited by naltrexone and glibenclamide and associated with reduced production of TNF-α and CXCL-1.

Leflunomide, an immunosuppressive drug approved for the treatment of patients with rheumatoid arthritis, exhibits many mechanisms which may affect the nociceptive processing. Therefore, the present study aimed to evaluate the effect induced by leflunomide on the mechanical allodynia in models of inflammatory and neuropathic pain in mice and investigate mechanisms mediating such effects. Per os (p.o.) administration of leflunomide (25, 50 or 100mg/kg) inhibited the inflammatory edema and mechanical allodynia induced by intraplantar carrageenan. Even ongoing inflammatory edema and mechanical allodynia were reduced by leflunomide. Previous administration of naltrexone (10mg/kg, intraperitoneal) or glibenclamide (40mg/kg, p.o.) partially attenuated leflunomide antiallodynic activity. A single administration of leflunomide (50 or 100mg/kg, p.o.) also partially inhibited ongoing mechanical allodynia induced by chronic constriction injury (CCI) or repeated administrations of paclitaxel. The antiallodynic effect induced by leflunomide (50 or 100mg/kg, p.o.) in the model of neuropathic pain induced by CCI was associated with reduced production of tumor necrosis factor-α both at the injury site and ipsilateral paw. Leflunomide also reduced production of the chemokine CXCL-1 at the paw ipsilateral to the injury site. Concluding, leflunomide partially inhibited ongoing mechanical allodynia in models of inflammatory and neuropathic pain. The antiallodynic effect was associated with activation of opioidergic receptors and ATP-sensitive potassium channels and reduced production of inflammatory mediators. These data indicate leflunomide as a drug that should be further investigated aiming to identify a new analgesic pharmacotherapy and reinforces repositioning as an important strategy to identify new uses for approved drugs.

Bronchial epithelial cells produce CXCL1 in response to LPS and TNFα: A potential role in the pathogenesis of COPD.

RATIONALE: Neutrophilic airway inflammation plays a central role in chronic obstructive pulmonary disease (COPD). CXC chemokine ligand (CXCL)1 is a neutrophil chemokine involved in the pathogenesis of COPD. However, its clinical significance in COPD patients is poorly understood. AIM OF THE STUDY: To assess the production of CXCL1 by bronchial epithelial cells in response to lipopolysaccharide (LPS) and tumor necrosis factor (TNF)α. MATERIALS AND METHODS: We measured sputum CXCL1 and CXCL8 levels in patients with COPD, asthma, and asthma-COPD overlap (ACO), and compared them to those of patients with interstitial pneumonia (IP). Using primary human bronchial epithelial cells and BEAS-2B cells, CXCL1 protein release and mRNA expression were measured after LPS or TNFα stimulation. We evaluated signal transduction mechanisms for CXCL1 production using nuclear factor-κ B (NF-kB) and mitogen-activated protein kinase (MAPK) inhibitors, and examined the effects of anti-inflammatory agents on CXCL1 production in BEAS-2B cells. RESULTS: Sputum CXCL1 levels in COPD and ACO patients were higher than in IP patients, whereas sputum CXCL8 levels were not. Sputum CXCL1 levels were not affected by inhaled corticosteroid usage, whereas sputum CXCL8 levels tended to be affected. LPS and TNFα stimulated CXCL1 production and mRNA expression in bronchial epithelial cells. NF-kB and MAPK p38 were involved in LPS-induced CXCL1 production. Therapeutic anti-inflammatory agents minimally attenuated CXCL1 production and considerably inhibited CXCL8 production in BEAS-2B cells. CONCLUSIONS: Sputum CXCL1 levels is a potentially better diagnostic marker for COPD than sputum CXCL8 levels, which is explained by that CXCL1 production in bronchial epithelial cells is less affected by therapeutic anti-inflammatory agents than CXCL8 production.