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.

The developmentally regulated fetal enterocyte gene, ZP4, mediates anti-inflammation by the symbiotic bacterial surface factor polysaccharide A on Bacteroides fragilis.

Initial colonizing bacteria play a critical role in completing the development of the immune system in the gastrointestinal tract of infants. Yet, the interaction of colonizing bacterial organisms with the developing human intestine favors inflammation over immune homeostasis. This characteristic of bacterial-intestinal interaction partially contributes to the pathogenesis of necrotizing enterocolitis (NEC), a devastating premature infant intestinal inflammatory disease. However, paradoxically some unique pioneer bacteria (initial colonizing species) have been shown to have a beneficial effect on the homeostasis of the immature intestine and the prevention of inflammation. We have reported that one such pioneer bacterium, Bacteroides fragilis (B. fragilis), and its surface component polysaccharide A (PSA) inhibit IL-1ß-induced inflammation in a human primary fetal small intestinal cell line (H4 cells). In this study, using transcription profiling of H4 cellular RNA after pretreatment with or without PSA before an inflammatory stimulation of IL-1ß, we have begun to further determine the cellular mechanism for anti-inflammation. We show that a developmentally regulated gene, zona pellucida protein 4 (ZP4), is uniquely elevated after IL-1ß stimulation and reduced with PSA exposure. ZP4 was known as a sperm receptor-mediating species-specific binding protein in the initial life of mammals. However, its intestinal epithelial function is unclear. We found that ZP4 is a developmentally regulated gene involved with immune function and regulated by both Toll-like receptor 2 and 4. Knockdown of ZP4-affected PSA inhibited IL-8 mRNA expression in response to IL-1ß. This represents an initial study of ZP4 innate immune function in immature enterocytes. This study may lead to new opportunity for efficient treatment of NEC.NEW & NOTEWORTHY This study extends previous observations to define the cellular mechanisms of polysaccharide A-induced anti-inflammation in immature enterocytes using transcription profiling of enterocyte genes after preexposure to polysaccharide A before an inflammatory stimulus with IL-1ß.

Phospholipase Cε plays a crucial role in neutrophilic inflammation accompanying acute lung injury through augmentation of CXC chemokine production from alveolar epithelial cells.

BACKGROUND: We have shown that phospholipase Cε (PLCε), an effector of Ras and Rap1 small GTPases, plays pivotal roles in inflammation and inflammation-associated carcinogenesis by augmenting proinflammatory cytokine production from epithelial cells of various organs. The purpose of this study is to analyze its role in neutrophilic alveolar inflammation accompanying acute lung injury (ALI), focusing on that in alveolar epithelial cells (AECs), which are known to make a major contribution to the pathogenesis of ALI. METHODS: We examine the effect of the PLCε genotypes on the development of ALI induced by intratracheal administration of lipopolysaccharide (LPS) to PLCε wild-type (PLCε+/+) and knockout (PLCεΔX/ΔX) mice. Pathogenesis of ALI is analyzed by histological examination of lung inflammation and measurements of the levels of various cytokines, in particular neutrophil-attracting chemokines such as Cxcl5, by quantitative reverse transcription-polymerase chain reaction and immunostaining. Primary cultures of AECs, established from PLCε+/+ and PLCεΔX/ΔX mice, are used to analyze the roles of PLCε, protein kinase D (PKD) and nuclear factor-κB (NF-κB) in augmentation of LPS-induced Cxcl5 expression. RESULTS: Compared to PLCε+/+ mice, PLCεΔX/ΔX mice exhibit marked alleviation of lung inflammation as shown by great reduction in lung wet/dry weight ratios, accumulation of inflammatory cells in the alveolar space and thickening of alveolar walls as well as the number of neutrophils and the protein concentration in bronchoalveolar lavage fluid. Also, LPS-induced expression of the CXC family of chemokines, in particular Cxcl5, is substantially diminished in the total lung and AECs of PLCεΔX/ΔX mice. Moreover, LPS-induced Cxcl5 expression in primary cultured AECs is markedly suppressed on the PLCεΔX/ΔX background (p < 0.05 versus PLCε+/+ AECs), which is accompanied by the reduction in phosphorylation of inhibitor κB (IκB), PKD and nuclear translocation of NF-κB p65. Also, it is suppressed by the treatment with inhibitors of PKD and IκB kinase, suggesting the involvement of the PLCε-PKD-IκB-NF-κB pathway. CONCLUSIONS: PLCε-mediated augmentation of the production of the CXC family of chemokines, in particular Cxcl5, in AECs plays a crucial role in neutrophilic alveolar inflammation accompanying ALI, suggesting that PLCε may be a potential molecular target for the treatment of acute respiratory distress syndrome.

CXCL5 as Regulator of Neutrophil Function in Cutaneous Melanoma.

Chemokines mold the tumor microenvironment by recruiting distinct immune cell populations, thereby strongly influencing disease progression. Previously, we showed that CXCL5 expression is upregulated in advanced stages of primary melanomas, which correlates with the presence of neutrophils in the tumor. The analysis of neutrophil populations in various tissues revealed a distinct phenotype of tumor-associated neutrophils. Tumor-associated neutrophils expressed PD-L1, CXCR4, CCR5, Adam17, and Nos2 and were immunosuppressive in a T-cell proliferation assay. To investigate the impact of CXCL5 and neutrophils in vivo, we established a syngeneic mouse tumor transplantation model using CXCL5-overexpressing and control melanoma cell lines. Growth behavior or vascularization of primary tumors was not affected by CXCL5 expression and neutrophils alone. However, in combination with Poly(I:C), tumor-associated neutrophils were able to attenuate induced antitumoral T-cell responses. CXCL5-overexpressing tumors had reduced lung metastasis compared with control tumors. Neutrophil depletion reversed this effect. In vitro, unstimulated lung-derived neutrophils had higher levels of reactive oxygen species compared with tumor-associated neutrophils, and CXCL5 stimulation further increased reactive oxygen species levels. In summary, in melanoma, neutrophils play a context-dependent role that is influenced by local or systemic factors, and interfere with therapies activating the acquired immune system. Actively switching neutrophils into antitumorigenic mode might be a new therapeutic strategy.

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.

Effects of extracellular orotic acid on acute contraction-induced adaptation patterns in C2C12 cells.

Dietary administration of orotic acid (OA), an intermediate in the pyrimidine biosynthetic pathway, is considered to provide a wide range of beneficial effects, including cardioprotection and exercise adaptation. Its mechanisms of action, when applied extracellularly, however, are barely understood. In this study, we evaluated potential effects of OA on skeletal muscle using an in vitro contraction model of electrically pulse-stimulated (EPS) C2C12 myotubes. By analyzing a subset of genes representing inflammatory, metabolic, and structural adaptation pathways, we could show that OA supplementation diminishes the EPS-provoked expression of inflammatory transcripts (interleukin 6, Il6; chemokine (C-X-C Motif) ligand 5, Cxcl5), and attenuated transcript levels of nuclear receptor subfamily 4 group A member 3 (Nr4A3), early growth response 1 (Egr1), activating transcription factor 3 (Atf3), and fast-oxidative MyHC-IIA isoform (Myh2). By contrast, OA had no suppressive effect on the pathogen-provoked inflammatory gene response in skeletal muscle cells, as demonstrated by stimulation of C2C12 myotubes with bacterial LPS. In addition, we observed a suppressive effect of OA on EPS-induced phosphorylation of AMP-activated protein kinase (AMPK), whereas EPS-triggered phosphorylation/activation of the mammalian target of rapamycin (mTOR) was not affected. Finally, we demonstrate that OA positively influences glycogen levels in EP-stimulated myotubes. Taken together, our results suggest that in skeletal muscle cells, OA modulates both the inflammatory and the metabolic reaction provoked by acute contraction. These results might have important clinical implications, specifically in cardiovascular and exercise medicine.

Exacerbation and Prolongation of Psoriasiform Inflammation in Diabetic Obese Mice: A Synergistic Role of CXCL5 and Endoplasmic Reticulum Stress.

Accumulating evidence suggests that psoriasis is frequently accompanied by metabolic disorders, such as obesity and diabetes. However, the mechanisms underlying the association between increased psoriasis severity and concomitant metabolic syndrome have not been fully clarified. Herein, we show that imiquimod-induced psoriasiform inflammation was exacerbated and prolonged in diabetic obese mice compared to that in control mice, accompanied by remarkably increased lesional expressions of Cxcl5 and Il-1b. Notably, a large number of CXCL5+ Ly6G+ cells infiltrated the dermis and subcutaneous fat tissue of the diabetic obese mice. Most macrophages in the subcutaneous fat tissues of the diabetic obese mice were positive for expression of IL-1ß and GRP78/Bip, an endoplasmic reticulum stress marker. Depletion of Ly6G+ cells and macrophages diminished the imiquimod-induced psoriasiform inflammation. Further, CXCL5 potentiated the secretion of IL-1ß from macrophages and palmitic acid, a fatty acid released from subcutaneous adipocytes, further enhanced IL-1ß secretion via endoplasmic reticulum stress induction. Combined with the fact that the serum levels of both CXCL5 and palmitic acid are significantly elevated in patients with metabolic syndrome, our results suggest a role for CXCL5 and endoplasmic reticulum stress in the increase of psoriasis severity of patients with concomitant metabolic syndrome.

Curcumin suppresses NTHi-induced CXCL5 expression via inhibition of positive IKKß pathway and up-regulation of negative MKP-1 pathway.

Otitis media (OM) is the most common childhood bacterial infection, and leading cause of conductive hearing loss. Nontypeable Haemophilus influenzae (NTHi) is a major bacterial pathogen for OM. OM characterized by the presence of overactive inflammatory responses is due to the aberrant production of inflammatory mediators including C-X-C motif chemokine ligand 5 (CXCL5). The molecular mechanism underlying induction of CXCL5 by NTHi is unknown. Here we show that NTHi up-regulates CXCL5 expression by activating IKKß-IκBα and p38 MAPK pathways via NF-κB nuclear translocation-dependent and -independent mechanism in middle ear epithelial cells. Current therapies for OM are ineffective due to the emergence of antibiotic-resistant NTHi strains and risk of side effects with prolonged use of immunosuppressant drugs. In this study, we show that curcumin, derived from Curcuma longa plant, long known for its medicinal properties, inhibited NTHi-induced CXCL5 expression in vitro and in vivo. Curcumin suppressed CXCL5 expression by direct inhibition of IKKß phosphorylation, and inhibition of p38 MAPK via induction of negative regulator MKP-1. Thus, identification of curcumin as a potential therapeutic for treating OM is of particular translational significance due to the attractiveness of targeting overactive inflammation without significant adverse effects.

Lipopolysaccharide Upregulates the Expression of CINC-3 and LIX in Primary NG2 Cells.

Numerous NG2 cells, also called oligodendrocyte progenitor cells (OPCs), exist ubiquitously in the gray and white matter in the adult central nervous system (CNS). Although NG2 cells could become active by upregulation of NG2 expression and hypertrophy or extension of their processes under various neuropathological conditions, their actual role in the brain remains to be illustrated. In view of the fact that the synergy of cytokine and chemokine networks plays an important role in CNS inflammation and immunity, we have assumed that the NG2 cells might take part in brain inflammation and immunity by making a contribution to the pool of cytokines or chemokines. In the current study, NG2-expressing OPCs were prepared from cerebral hemispheres of postnatal day 0 or 1 Sprague-Dawley rats. Our results showed that NG2-expressing OPCs, verified by immunohistological staining of anti-NG2 antibody and anti-platelet-derived growth factor receptor alpha (PDGFRα) antibody, presented binding affinity to lipopolysaccharide (LPS), a commonly used stimulator in a neuroinflammatory model. Using cytokine antibody array, QPCR and ELISA, we have further shown that LPS could upregulate the expression of cytokine induced neutrophil chemoattractant-3 (CINC-3) and LPS induced CXC chemokine (LIX) in primary NG2-expressing OPCs, without the alteration in cell number of NG2-expressing OPCs. In addition, the cells bearing the receptor for these two cytokines included microglia and OPCs. Taken together, our results suggest that NG2-expressing OPCs could response to LPS and may take part in neuroinflammatory process, through secreting cytokines and chemokines to exert an effect on target cells (OPCs and microglia).

Cigarette smoke primes the pulmonary environment to IL-1α/CXCR-2-dependent nontypeable Haemophilus influenzae-exacerbated neutrophilia in mice.

Cigarette smoke has a broad impact on the mucosal environment with the ability to alter host defense mechanisms. Within the context of a bacterial infection, this altered host response is often accompanied by exacerbated cellular inflammation, characterized by increased neutrophilia. The current study investigated the mechanisms of neutrophil recruitment in a murine model of cigarette smoke exposure and, subsequently, a model of both cigarette smoke exposure and bacterial infection. We investigated the role of IL-1 signaling in neutrophil recruitment and found that cigarette smoke-induced neutrophilia was dependent on IL-1α produced by alveolar macrophages. In addition to being the crucial source of IL-1α, alveolar macrophages isolated from smoke-exposed mice were primed for excessive IL-1α production in response to bacterial ligands. To test the relevance of exaggerated IL-1α production in neutrophil recruitment, a model of cigarette smoke exposure and nontypeable Haemophilus influenzae infection was developed. Mice exposed to cigarette smoke elaborated an exacerbated CXCR2-dependent neutrophilia in response to nontypeable Haemophilus influenzae. Exacerbated neutrophilia was dependent on IL-1α priming of the pulmonary environment by cigarette smoke as exaggerated neutrophilia was dependent on IL-1 signaling. These data characterize a novel mechanism of cigarette smoke priming the lung mucosa toward greater IL-1-driven neutrophilic responses to bacteria, with a central role for the alveolar macrophage in this process.

An epithelial circadian clock controls pulmonary inflammation and glucocorticoid action.

The circadian system is an important regulator of immune function. Human inflammatory lung diseases frequently show time-of-day variation in symptom severity and lung function, but the mechanisms and cell types underlying these effects remain unclear. We show that pulmonary antibacterial responses are modulated by a circadian clock within epithelial club (Clara) cells. These drive circadian neutrophil recruitment to the lung via the chemokine CXCL5. Genetic ablation of the clock gene Bmal1 (also called Arntl or MOP3) in bronchiolar cells disrupts rhythmic Cxcl5 expression, resulting in exaggerated inflammatory responses to lipopolysaccharide and an impaired host response to Streptococcus pneumoniae infection. Adrenalectomy blocks rhythmic inflammatory responses and the circadian regulation of CXCL5, suggesting a key role for the adrenal axis in driving CXCL5 expression and pulmonary neutrophil recruitment. Glucocorticoid receptor occupancy at the Cxcl5 locus shows circadian oscillations, but this is disrupted in mice with bronchiole-specific ablation of Bmal1, leading to enhanced CXCL5 expression despite normal corticosteroid secretion. The therapeutic effects of the synthetic glucocorticoid dexamethasone depend on intact clock function in the airway. We now define a regulatory mechanism that links the circadian clock and glucocorticoid hormones to control both time-of-day variation and the magnitude of pulmonary inflammation and responses to bacterial infection.

CXCL5 is required for angiogenesis, but not structural adaptation after small bowel resection.

PURPOSE: Intestinal adaptation is the compensatory response to massive small bowel resection (SBR) and characterized by lengthening of villi and deepening of crypts, resulting in increased mucosal surface area. Previous studies have demonstrated increased villus capillary blood vessel density after SBR, suggesting a role for angiogenesis in the development of resection-induced adaptation. Since we have previously shown enhanced expression of the proangiogenic chemokine CXCL5 after SBR, the purpose of this study was to determine the effect of disrupted CXCL5 expression on intestinal adaptation. METHODS: CXCL5 knockout (KO) and C57BL/6 wild type (WT) mice were subjected to either a 50% proximal SBR or sham operation. Ileal tissue was harvested on postoperative day 7. To assess for adaptation, villus height and crypt depth were measured. Submucosal capillary density was measured by CD31 immunohistochemistry. RESULTS: Both CXCL5-KO and WT mice demonstrated normal structural features of adaptation. Submucosal capillary density increased in the WT but not in the KO mice following SBR. CONCLUSION: CXCL5 is required for increased intestinal angiogenesis during resection-induced adaptation. Since adaptive villus growth occurs despite impaired CXCL5 expression and enhanced angiogenesis, this suggests that the growth of new blood vessels is not needed for resection-induced mucosal surface area expansion following massive SBR.

Increased serum CXCL1 and CXCL5 are linked to obesity, hyperglycemia, and impaired islet function.

Proinflammatory cytokines are thought to play a significant role in the pathogenesis of type 2 diabetes (T2D) and are elevated in the circulation even before the onset of the disease. However, the full complement of cytokines involved in the development of T2D is not known. In this study, 32 serum cytokines were measured from diabetes-prone BKS.Cg-m+/+Lepr(db)/J (db/db) mice and heterozygous age-matched control mice at 5 weeks (non-diabetic/non-obese), 6-7 weeks (transitional-to-diabetes), or 11 weeks (hyperglycemic/obese) and then correlated with body weight, blood glucose, and fat content. Among these 32 cytokines, C-X-C motif ligand 1 (CXCL1) showed the greatest increase (+78%) in serum levels between db/db mice that were hyperglycemic (blood glucose: 519±23 mg/dl, n=6) and those that were non-hyperglycemic (193±13 mg/dl, n=8). Similarly, increased CXCL1 (+68%) and CXCL5 (+40%) were associated with increased obesity in db/db mice; note that these effects could not be entirely separated from age. We then examined whether islets could be a source of these chemokines. Exposure to cytokines mimicking low-grade systemic inflammation (10 pg/ml IL1ß+20 pg/ml IL6) for 48 h upregulated islet CXCL1 expression by 53±3-fold and CXCL5 expression by 83±10-fold (n=4, P<0.001). Finally, overnight treatment with the combination of CXCL1 and CXCL5 at serum levels was sufficient to produce a significant decrease in the peak calcium response to glucose stimulation, suggesting reduced islet function. Our findings demonstrated that CXCL1 and CXCL5 i) are increased in the circulation with the onset of T2D, ii) are produced by islets under stress, and iii) synergistically affect islet function, suggesting that these chemokines participate in the pathogenesis of T2D.

Fli1 deficiency contributes to the suppression of endothelial CXCL5 expression in systemic sclerosis.

CXCL5 is a member of CXC chemokines with neutrophilic chemoattractant and pro-angiogenic properties, which has been implicated in the pathological angiogenesis of rheumatoid arthritis and inflammatory bowel diseases. Since aberrant angiogenesis is also involved in the developmental process of systemic sclerosis (SSc), we herein measured serum CXCL5 levels in 63 SSc and 18 healthy subjects and investigated their clinical significance and the mechanism explaining altered expression of CXCL5 in SSc. Serum CXCL5 levels were significantly lower in SSc patients than in healthy subjects. In diffuse cutaneous SSc (dcSSc), serum CXCL5 levels were uniformly decreased in early stage (<1 year) and positively correlated with disease duration in patients with disease duration of <6 years. In non-early stage dcSSc (≥1 year), decreased serum CXCL5 levels were linked to the development of digital ulcers. Consistently, the expression levels of CXCL5 proteins were decreased in dermal blood vessels of early stage dcSSc. Importantly, Fli1 bound to the CXCL5 promoter and its gene silencing significantly suppressed the CXCL5 mRNA expression in human dermal microvascular endothelial cells. Furthermore, endothelial cell-specific Fli1 knockout mice, an animal model of SSc vasculopathy, exhibited decreased CXCL5 expression in dermal blood vessels. Collectively, these results indicate that CXCL5 is a member of angiogenesis-related genes, whose expression is suppressed at least partially due to Fli1 deficiency in SSc endothelial cells. Since Fli1 deficiency is deeply related to aberrant angiogenesis in SSc, it is plausible that serum CXCL5 levels inversely reflect the severity of SSc vasculopathy.

Involvement of interleukin-17A in pancreatic damage in rat experimental acute necrotizing pancreatitis.

Interleukin (IL)-17A is a proinflammatory cytokine, which has recently attracted much interest due to its pathogenic role in various inflammatory conditions such as ischemia/reperfusion injury, chronic inflammation, and autoimmune diseases, but the role of IL-17A in acute pancreatitis remains unclear. This study aimed to investigate the role of IL-17A in experimental acute necrotizing pancreatitis (ANP). We analyzed the expression of IL-17A during the pathogenesis of ANP in vivo induced by 3 % sodium taurocholate (NaTc), by microarray test, quantitative real-time PCR, Western blotting, enzyme-linked immunosorbent assay, and immunohistochemistry. The effects of IL-17A on pancreatic acinar cells and pancreatic stellate cells (PSCs) were further investigated in vitro using recombinant rat IL-17A (rIL-17A). Expression of IL-17A was significantly increased following experimental acute pancreatitis. In addition, rIL-17A induced rat pancreatic acinar cell necrosis and promoted expression of several target genes, including IL-6, IL-1ß, CXCL1, CXCL2, and CXCL5, in acinar cells and PSCs. These findings suggest that IL-17A may be involved in pancreatic damage by regulating the expression of inflammatory cytokines and chemokines during experimental acute pancreatitis.

Common and specific signatures of gene expression and protein-protein interactions in autoimmune diseases.

The aim of this study is to understand intracellular regulatory mechanisms in peripheral blood mononuclear cells (PBMCs), which are either common to many autoimmune diseases or specific to some of them. We incorporated large-scale data such as protein-protein interactions, gene expression and demographical information of hundreds of patients and healthy subjects, related to six autoimmune diseases with available large-scale gene expression measurements: multiple sclerosis (MS), systemic lupus erythematosus (SLE), juvenile rheumatoid arthritis (JRA), Crohn's disease (CD), ulcerative colitis (UC) and type 1 diabetes (T1D). These data were analyzed concurrently by statistical and systems biology approaches tailored for this purpose. We found that chemokines such as CXCL1-3, 5, 6 and the interleukin (IL) IL8 tend to be differentially expressed in PBMCs of patients with the analyzed autoimmune diseases. In addition, the anti-apoptotic gene BCL3, interferon-γ (IFNG), and the vitamin D receptor (VDR) gene physically interact with significantly many genes that tend to be differentially expressed in PBMCs of patients with the analyzed autoimmune diseases. In general, similar cellular processes tend to be differentially expressed in PBMC in the analyzed autoimmune diseases. Specifically, the cellular processes related to cell proliferation (for example, epidermal growth factor, platelet-derived growth factor, nuclear factor-κB, Wnt/ß-catenin signaling, stress-activated protein kinase c-Jun NH2-terminal kinase), inflammatory response (for example, interleukins IL2 and IL6, the cytokine granulocyte-macrophage colony-stimulating factor and the B-cell receptor), general signaling cascades (for example, mitogen-activated protein kinase, extracellular signal-regulated kinase, p38 and TRK) and apoptosis are activated in most of the analyzed autoimmune diseases. However, our results suggest that in each of the analyzed diseases, apoptosis and chemotaxis are activated via different subsignaling pathways. Analyses of the expression levels of dozens of genes and the protein-protein interactions among them demonstrated that CD and UC have relatively similar gene expression signatures, whereas the gene expression signatures of T1D and JRA relatively differ from the signatures of the other autoimmune diseases. These diseases are the only ones activated via the Fcɛ pathway. The relevant genes and pathways reported in this study are discussed at length, and may be helpful in the diagnoses and understanding of autoimmunity and/or specific autoimmune diseases.

Modulatory effect of fenofibrate on endothelial production of neutrophil chemokines IL-8 and ENA-78.

PURPOSE: The PPAR-alpha agonists (fibrates) are commonly used in the treatment of dyslipidemia. It has been hypothesized that the cardio-protective effects of fibrates are partially due to immunomodulatory effects. However, there is a paucity of data regarding the effect of fibrates on neutrophilic chemokines such as epithelial neutrophil activating protein (ENA-78) and interleukin (IL)-8. We investigated the influence of fenofibrate on IL-1ß-stimulated production of ENA-78 and IL-8 from human endothelial cells (HUVECs). METHODS: HUVECs were cultured in the presence or absence of IL-1ß and fenofibrate ranging from 1-50 uM. ENA-78 and IL-8 were measured and normalized to total protein content in cell culture supernates by multiplex immunofluorescence detection. Experimental samples were measured in triplicate. Significance was set at P < 0.05 by ANOVA with correction for multiple comparisons. RESULTS: Endothelial production of both ENA-78 and IL-8 was induced by the proinflammatory cytokine IL-1ß. ENA-78 concentrations increased by more than 160-fold over constitutively produced ENA-78 upon IL-1ß stimulation (mean ± SEM: 10,129 ± 1591 pg/mg vs. 61 ± 9.5 mg/mg; P < 0.0001). IL-8 concentrations increased by slightly over 5-fold (6145 ± 860 pg/mg vs. 1160 ± 201 pg/mg; P = 0.0003). ENA-78 protein and mRNA were significantly reduced by fenofibrate while no drug effects were observed on IL-8 production. CONCLUSIONS: Fenofibrate blunts IL-1ß-mediated ENA-78 production with no effect on IL-8. This represents a novel mechanism by which fenofibrate exerts anti-inflammatory effects and should be further explored.

Mesenchymal stem cells promote mammary cancer cell migration in vitro via the CXCR2 receptor.

Bone metastasis is a common event during breast cancer progression. Recently, mesenchymal stem cells (MSCs) have been implicated in the metastasis of primary mammary cancer. Given that bone is the native environment for MSCs, we hypothesized MSCs facilitate the homing of circulating mammary cancer cells to the bone. To test this hypothesis, we examined in vitro whether bone derived MSCs from FVB mice could influence the migration of syngeneic murine mammary cancer cell lines derived from the polyoma virus middle-T (PyMT) model of mammary gland tumorigenesis. Our data show that conditioned media derived from MSCs significantly enhanced the migration of PyMT mammary cancer cell lines. Analysis of conditioned media using a cytokine array revealed the presence of numerous cytokines in the MSC conditioned media, most notably, the murine orthologs of CXCL1 and CXCL5 that are cognate ligands of the CXCR2 receptor. Further investigation identified that: (1) CXCL1, CXCL5 and CXCR2 mRNA and protein were expressed by the MSCs and PyMT cell lines and; (2) neutralizing antibodies to CXCL1, CXCL5 and CXCR2 or a CXCR2 small molecule inhibitor (SB265610) significantly abrogated the migratory effect of the MSC conditioned media on the PyMT cells. Therefore, in vitro evidence demonstrates that bone derived MSCs play a role in the migration of mammary cancer cells, a conclusion that has potential implications for breast to bone metastasis in vivo.

IL-17A and TNF-α exert synergistic effects on expression of CXCL5 by alveolar type II cells in vivo and in vitro.

CXCL5, a member of the CXC family of chemokines, contributes to neutrophil recruitment during lung inflammation, but its regulation is poorly understood. Because the T cell-derived cytokine IL-17A enhances host defense by triggering production of chemokines, particularly in combination with TNF-α, we hypothesized that IL-17A would enhance TNF-α-induced expression of CXCL5. Intratracheal coadministration of IL-17A and TNF-α in mice induced production of CXCL1, CXCL2, and CXCL5, which was associated with increased neutrophil influx in the lung at 8 and 24 h. The synergistic effects of TNF-α and IL17A were greatly attenuated in Cxcl5(-/-) mice at 24 h, but not 8 h, after exposure, a time when CXCL5 expression was at its peak in wild-type mice. Bone marrow chimeras produced using Cxcl5(-/-) donors and recipients demonstrated that lung-resident cells were the source of CXCL5. Using differentiated alveolar epithelial type II (ATII) cells derived from human fetal lung, we found that IL-17A enhanced TNF-α-induced CXCL5 transcription and stabilized TNF-α-induced CXCL5 transcripts. Whereas expression of CXCL5 required activation of NF-κB, IL-17A did not increase TNF-α-induced NF-κB activation. Apical costimulation of IL-17A and TNF-α provoked apical secretion of CXCL5 by human ATII cells in a transwell system, whereas basolateral costimulation led to both apical and basolateral secretion of CXCL5. The observation that human ATII cells secrete CXCL5 in a polarized fashion may represent a mechanism to recruit neutrophils in host defense in a fashion that discriminates the site of initial injury.

The BH3 mimetic ABT-737 induces cancer cell senescence.

ABT-737, a small molecule cell-permeable Bcl-2 antagonist that acts by mimicking BH3 proteins, induces apoptotic cell death in multiple cancer types. However, when incubated with this agent many solid tumor cell lines do not undergo apoptosis. The current study reveals a novel mechanism whereby ABT-737 when added to apoptosis-resistant cancer cells has profound biologic effects. In PV-10 cells, a renal cell carcinoma that does not die after ABT-737 treatment, this agent induces a two-fold change in the transcription of nearly 430 genes. Many of these induced mRNA changes are in secreted proteins, IL-6, IL-8, and IL-11 and chemokines CXCL2 and CXCL5, or genes associated with an "inflammatory" phenotype. Strikingly, these gene changes are highly similar to those changes previously identified in cellular senescence. Brief exposure of apoptosis-resistant renal, lung and prostate cancer cell lines to ABT-737, although not capable of inducing cell death, causes the induction of senescence-associated ß-galactosidase and inhibition of cell growth consistent with the induction of cellular senescence. Evidence indicates that the induction of senescence occurs as a result of reactive oxygen species elevation followed by low-level activation of the caspase cascade, insufficient to induce apoptosis, but sufficient to lead to minor DNA damage and increases in p53, p21, IL-6 and 8 proteins. By overexpression of a dominant-negative p53 protein, we show that ABT-737-induced cellular senescence is p53-dependent. Thus, in multiple cancer types in which ABT-737 is incapable of causing cell death, ABT-737 may have additional cellular activities that make its use as an anticancer agent highly attractive.