IP-10 Expression Research Articles

EIAV S2 enhances pro-inflammatory cytokine and chemokine response in infected macrophages

Equine infectious anemia virus (EIAV) infection is distinctive in that it causes a rapid onset of clinical disease relative to other retroviruses. In order to understand the interaction dynamics between EIAV and the host immune response, we explored the effects of EIAV and its S2 protein in the regulation of the cytokine and chemokine response in macrophages. EIAV infection markedly altered the expression pattern of a variety of pro-inflammatory cytokines and chemokines monitored in the study. Comparative studies in the cytokine response between EIAV(17) and EIAV(17DeltaS2) infection revealed that S2 enhances the expression of IL-1alpha, IL-1beta, IL-8, MCP-2, MIP-1beta and IP-10. Moreover, S2 specifically induced the expression of the newly discovered cytokine, IL-34. Taken together, these results may help explain the effect of cytokine and chemokine dysregulation in EIAV pathogenesis and suggest a role of S2 in optimizing the host cell environment to promote viral dissemination and replication.

Imatinib Mesylate Treatment Increases Lymphoplasmocytoid Cells through SDF-1 and BMP4/7 Production in the Bone Marrow of Patients with Chronic Myelogenous Leukaemia: Relationship with Clinical/Haematological Response.

Abstract 3263Poster Board III-1▪▪This icon denotes an abstract that is clinically relevant.In the last years, tyrosine kinase inhibitors, as imatinib mesylate (Gleevec, Novartis, formerly known as STI571) are confirmed the first line treatment of Chronic Myelogenous Leukemia (CML) and of a rare form of gastroenteric stromal cancer. In the latter case, the response to the drug in vivo is mainly due to immunocompetent cells, able to produce cytokines with antineoplastic activity. In this study, 30 CML patients, prior and during treatment with imatinib mesylate, underwent bone marrow (BM) aspirates every 6 months to assess morphologic phenotypic, cytogenetic and biomolecular patterns, compared to peripheral blood. Plasma from BM and peripheral blood (PB) was also tested for cytokines able to induce B lymphocytes differentiation, such as interleukin IL-4, IL-6 (whose receptor is CD126), IL-3, IL-10 or IL-21 (by ELISPOT and real time polymerase chain reaction) togheter with the expression of MCP-1, SDF-1, BMP4, BMP7, IP-10 and IL-8 . We report that in 24 out of 30 CML patients a significant increase in the percentage of BM lymphoplasmocytoid cells was observed upon treatment with imatinib mesylate, with >10% (range 8–12%) of CD20+CD126+cells. Among this population, two third of cells coexpressed IgM and one third was IgD+, whereas a lower fraction of IgM+CD126+CD20- (3–4%) or IgD+CD126+CD20- (2–3%) cells was detected , too. In all these patients SDF1 increased in the BM plasma after imatinib (from 10–80pg/ml to 150–450pg/ml) and its receptor CXCR4 was up-regulated on CD20+CD126+cells. In some cases also IP-10 and its receptor CXCR3 were up-.regulated. No significant increase in transcription and secretion of IL-3, IL-4, IL-6, IL-10, IL-21, IL-8 or MCP1 were observed. The lasting 6 patients had<5% of CD20 +CD126+ lymphocytes (range2-4%), 2/3 coexpressing IgM and 1/3 coexpressing IgD. Every patients with increased number of CD126+ B lymphocytes achieved hematologic remission, most of them complete cytogenetic and molecular remission. On the other hand, among the patients with low or undetectable CD20+CD126+cells, only 4 obtained hemathological remission and none of them stable cytogenetyc and molecular remission. In 4 patients relapsed after six months of treatment, the fraction of BM CD20+CD126+ lymphocytes decreased from 11% and 8% to 7 and 5%, respectively, with undetectable IgM+ CD126+CD20- or IgD+ CD126+CD20- cells. The increased production of SDF-1,(partially mediated by production of BMP4 and BMP7), following imatinib administration might increase BM lymphoplasmocitoid cells, thanks to the double proliferative/chemotactic effect of the cytokine on B cells, with redistribution and in situ differentiation of CD20+ CD126+ lymphocytes. These findings shed some light on the possibility that, even in CML,immunological events may play a role in disease control ; moreover they could be useful in monitoring disease outcome . Disclosures:No relevant conflicts of interest to declare.

Protosappanin A induces immunosuppression of rats heart transplantation targeting T cells in grafts via NF-κB pathway

Protosappanin A as one major and effective ingredient from Caesalpinia sappan L. exhibited antirejection activity obviously in heart-transplanted rat. The present study was designed to screen out the potential target genes of protosappanin A with microarray technology and reveal some molecular mechanism of immunosuppressive effect. Rats performed with ectopic peritoneal heart transplantation were randomized into three groups receiving different treatments for 7 days: protosappanin A group (25 mg kg(-1)), cyclosporine A group (10 mg kg(-1)), and control group. The differentially expressed genes responding to protosappanin A were analyzed with microarrays. Among common differentially expressed genes, the ones of interest were selected for further evaluation by real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), Western blot, immunochemistry, immunofluorescence, and ELISA. Among the 146 common differentially expressed genes, NF-kappaB and related genes like IkappaBa, IFN-r, and IP10 were selected for verification. The results of qRT-PCR, Western blot, immunochemistry, and ELISA showed that protosappanin A significantly reduced the expression of NF-kappaB, IFN-r, and IP10 (p < 0.05) and increased IkappaBa expression (p < 0.05) in graft. Moreover, the immunochemistry staining of NF-kappaB and IkappaBa was mainly observed in infiltrating mononuclear cells. Strikingly, immunofluorescent staining localized NF-kappaB to the TCR-positive T cells in graft. Furthermore, protosappanin A exhibited inhibitory effect on T cell proliferation in recipients after 7-day treatment. In conclusion, protosappanin A might act on T cells through inhibiting NF-kappaB activation and downstream gene expressions of IFN-r and IP10, meanwhile reducing T cell proliferation responding to alloantigen, so as to induce immunosuppressive effect. The results encourage a potential therapeutic evaluation of protosappanin A for clinical organ transplantation or other T cell-mediated immune disorders. Additionally, our study also verified the feasibility of microarray utilization in Chinese herb research to explore molecular mechanism and promote development of scientific theories.

α‐Fetoprotein as a modulator of the pro‐inflammatory response of human keratinocytes

The immunomodulatory effects of alpha-fetoprotein (AFP) on lymphocytes and macrophages have been described in vitro and in vivo. Recombinant forms of human AFP have been proposed as potential therapeutic entities for the treatment of autoimmune diseases. We examined the effects of embryonic and recombinant human AFP on the spontaneous, UVA- and cytokine-induced pro-inflammatory responses of human keratinocytes. Cultures of primary and immortalized human keratinocytes (HaCaT) and human blood T lymphocytes were used. The effects of AFP on cytokine expression were studied by bioplexed elisa and quantitative reverse transcriptase polymerase chain reaction assay. Kinase and nuclear factor kappa B (NFkappaB) phosphorylation were quantified by intracellular elisa. Nuclear activator protein 1 and NFkappaB DNA binding activity was measured by specific assays. Nitric oxide and H(2)O(2) production and redox status were assessed by fluorescent probe and biochemical methods. All forms of AFP enhanced baseline expression of cytokines, chemokines and growth factors. AFP dose-dependently increased tumour necrosis factor alpha-stimulated granulocyte macrophage colony stimulating factor and interleukin 8 expression and decreased tumour necrosis factor alpha-induced monocyte chemotactic protein 1 and IP-10 (interferon gamma-produced protein of 10 kDa) expression. AFP induced a marked activator protein 1 activation in human keratinocytes. AFP also increased H(2)O(2) and modulated nitrite/nitrate levels in non-stimulated keratinocytes whereas it did not affect these parameters or cytokine release from UVA-stimulated cells. Phosphorylation of extracellular signal-regulated kinase (ERK1/2) and Akt1 but not NFkappaB was activated by AFP alone or by its combination with UVA. Exogenous AFP induces activation of human keratinocytes, with de novo expression of a number of pro-inflammatory mediators and modulation of their pro-inflammatory response to cytokines or UVA. AFP may modulate inflammatory events in human skin.

Suppressive Effect on MDC and IP-10 Expression in Monocytes by Endocrine Disruptor Chemicals

The expression of chemokines is critical in leukocyte recruitment and inflammation, but the regulatory mechanisms involved remain incompletely defined. While endocrine disrupter chemicals (EDCs) are known to be ubiquitous in the environment and often associated with altered inflammatory response, their potential impact on chemokine expression in monocytes is at present unknown. To this end, the effects of EDCs on the expression of Th1- and Th2-related chemokines in a human monocytic cell line, THP-1, were investigated. THP-1 cells were pre-treated with varying concentrations of EDCs (nonylphenol and 4-octylphenol) with or without the addition of an estrogen receptor (ER) antagonist, ICI 182,780 and then stimulated by lipopolysaccharide (LPS). The levels of chemokines, CXCL10/ IFN-alpha-inducible protein 10 (IP-10, a Th1 chemokine) and monocyte-derived chemokine (MDC)/CCL22, a Th2 chemokine) were measured by ELISA. EDC-mediated signaling events and histone modifications were examined by the use of Western blotting and chromatin immunoprecipitation (ChIP) assay. Nonylphenol and 4-octylphenol were able to suppress LPS-induced MDC and IP-10 expression. This suppressive effect was not reversed by the addition of ICI 182,780. Nonylphenol and 4-octylphenol reduced LPS-induced activation of MAPK signaling pathway, MKK1/2 and ERK, concomitant with decreased levels of LPS-induced acetylated histone 4 (H4) at the IP-10 and MDC gene loci. Nonylphenol and 4-octylphenol suppressed LPS-induced MDC expression in monocytes via, at least in part, the MKK1/2-ERK MAPK pathway and histone H4 acetylation, but not the estrogen receptor.

Interferon regulatory factor-1 is a key transcription factor in murine beta cells under immune attack

IFN-gamma, together with other inflammatory cytokines such as IL-1beta and TNF-alpha, contributes to beta cell death in type 1 diabetes. We analysed the role of the transcription factor interferon regulatory factor (IRF)-1, a downstream target of IFN-gamma/signal transducer and activator of transcription (STAT)-1, in immune-mediated beta cell destruction. Islets from mice lacking Irf-1 (Irf-1 (-/-)) and control C57BL/6 mice were transplanted in overtly diabetic NOD mice. Viability and functionality of islets were evaluated in vitro. Chemokine expression by Irf-1 (-/-) islets and INS-1E cells transfected with Irf-1 short interfering RNA (siRNA) was measured by real-time PCR as well as in functional assays in vitro. IRF-1 deletion in islets was associated with higher prevalence of primary non-function (63% vs 25%, p <or= 0.05) and shorter functioning graft survival (6.0 +/- 2.6 vs 10.4 +/- 4.8 days, p <or= 0.05) in contrast to similar skin graft survival. Although Irf-1 (-/-) islets were resistant to cytokine-induced cell death, insulin secretion by them was lower than that of control C57BL/6 islets under medium and cytokine conditions. IL-1 receptor antagonist partly restored the cytokine-induced secretory defect in vitro and completely prevented primary non-function in vivo. Cytokine-exposed Irf-1 (-/-) islets and INS-1E cells transfected with Irf-1 siRNA showed increased expression of Mcp-1 (also known as Ccl2), Ip-10 (also known as Cxcl10), Mip-3alpha (also known as Ccl20) and Inos (also known as Nos2) mRNA and elevated production of monocyte chemoattractant protein-1 (MCP-1) and nitrite compared with controls. In vivo, Irf-1 (-/-) islets displayed a higher potential to attract immune cells, reflected by more aggressive immune infiltration in the grafted islets. These data indicate a key regulatory role for IRF-1 in insulin and chemokine secretion by pancreatic islets under inflammatory attack.

Varicose Veins Show Enhanced Chemokine Expression

Leucocyte infiltration in the wall of varicose veins has been reported previously. This study was designed to investigate the expression of pro-inflammatory cytokines and chemokines in control and in patients with varicose veins and to test the effect of treating varicose vein patients with acetylsalicylic acid (ASA) on cytokine expression prior to removal of varices. Sections of vein were removed during operation from both patient groups, and ribonuclease protection assays (RPAs) were performed to assess the expression of chemokines. Group I included non-varicose saphenous veins from healthy patients undergoing amputation for trauma. Varicose veins were obtained from patients with primary varicose undergoing surgical treatment who received no drug (group II) or treatment with 300 mg day(-1) of ASA for 15 days before surgery (group III). Non-varicose veins constitutively expressed low levels of monocyte-chemoattractant protein (MCP-1) and interleukin (IL)-8 mRNA. Varicose veins had a distinct chemokine expression pattern, since significant up-regulation of MCP-1 and IL-8 and a marked expression of IP-10, RANTES, MIP-1alpha and MIP-1beta mRNA were detected. Removal of the endothelium did not alter this pattern. Varicose veins obtained from patients treated with ASA showed a consistent decrease in chemokine expression, although it did not reach statistical significance. Varicose veins showed increased expression of several chemokines compared to control veins. A non-significant reduction of activation was observed following treatment with ASA for 15 days.

Toll-like receptor 7 function is reduced in adolescents with asthma

Anti-viral innate immune responses may be impaired in asthma, although the mechanisms are not well understood. Toll-like receptors (TLRs) 7 and 3 are particularly relevant for initiating responses to common respiratory viruses, as they recognise single-stranded viral RNA and double-stranded viral RNA, respectively. The aim of the present study was to investigate TLR7 and TLR3 function in 14-yr-old adolescents with asthma. Blood mononuclear cells obtained from 17 atopic asthmatics, 29 atopic, non-asthmatics and 21 healthy, non-atopic individuals, were stimulated with the TLR7 agonist imiquimod and the TLR3 agonist poly I:C. Expression of anti-viral molecules was measured by real-time PCR. Concentrations of interferon-gamma-inducible cytokine protein (IP)-10 and interleukin (IL)-6 were measured by ELISA. TLR7-induced myxovirus resistance protein A and 2'5' oligoadenylate synthetase mRNA expression and protein levels of IP-10 were significantly lower in asthma subjects compared with healthy subjects (p = 0.041, p = 0.003 and p = 0.001 respectively). There was a significant negative correlation between total serum immunoglobulin E and IP-10 following TLR7 stimulation. However, TLR3-induced responses did not vary with asthma or atopy. IL-10 mRNA and IL-6 protein synthesis were similar in asthmatic and control subjects. In conclusion, TLR7 function is reduced in adolescents with asthma and this may contribute to susceptibility to respiratory viral infections.

Alteration of Toll-like receptor 4 activation by 4-hydroxy-2-nonenal mediated by the suppression of receptor homodimerization

Toll-like receptors (TLRs) detect invading microbial pathogens and initiate immune responses as part of host defense mechanisms. They also respond to host-derived substances released from injured cells and tissues to ensure wound healing and tissue homeostasis. Dysregulation of TLRs increases the risk of chronic inflammatory diseases and immune disorders. Inflammatory events are often accompanied by oxidative stress, which generates lipid peroxidation products such as 4-hydroxy-2-nonenal (4-HNE). Therefore, we investigated if 4-HNE affects TLR activation. We found that 4-HNE blocked LPS (a TLR4 agonist)-induced activation of NFκB and IRF3 as well as expression of IFNβ, IP-10, RANTES, and TNFα. To investigate the mechanism of inhibition by 4-HNE, we examined its effects on TLR4 dimerization, one of the initial steps in TLR4 activation. 4-HNE suppressed both ligand-induced and ligand-independent receptor dimerization. The thiol donors, DTT and NAC, prevented the inhibitory effects of 4-HNE on TLR4 dimerization, and LC–MS/MS analysis showed that 4-HNE formed adducts with cysteine residues of synthetic peptides derived from TLR4. These observations suggest that the reactivity of 4-HNE with sulfhydryl moieties is implicated in the inhibition of TLR4 activation. Furthermore, inhibition of TLR4 activation by 4-HNE resulted in down-regulation of the phagocytic activity of macrophages. Collectively, these results demonstrate that 4-HNE blocks TLR4-mediated macrophage activation, gene expression, and phagocytic functions, at least partly by suppressing receptor dimerization. They further suggest that 4-HNE influences innate immune responses at sites of infection and inflammation by inhibiting TLR4 activation.

Expression of TNF-α, TGF-β, IP-10 and IL-10 mRNA in kidneys of hamsters infected with pathogenic Leptospira

Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira. Although several components of this organism have been identified, the molecular mechanisms underlying pathogenesis of this infectious disease are still poorly understood. Besides, direct injury by microbial factors, cytokines produced in response to infection have been proposed to be involved in pathogenesis of leptospirosis. In this study, cytokine gene expression in kidneys was investigated. Hamsters were injected with pathogenic Leptospira interrogans serovar Pyrogenes and were sacrificed on days 3, 5 and 7 after infection. RNA was extracted from kidney tissues. Real-time PCR was performed to demonstrate expression of TNF-alpha, TGF-beta, IP-10 and IL-10 mRNA in kidneys. TNF-alpha, TGF-beta and IP-10 expression could be demonstrated since day 3 post-infection whereas IL-10 expression was detected later on day 5. Leptospira infection resulted in not only expression of a proinflammatory cytokine, TNF-alpha, but also a T cell chemokine, IP-10. Detection of IP-10 suggested the involvement of T cell recruitment in the immune response or pathology in infected kidneys. Expressions of anti-inflammatory cytokines, TGF-beta and IL-10 were also observed. However, the level of TGF-beta expression was prominent since day 3 post-infection whereas IL-10 expression was clearly observed on day 5. Further experiments will provide additional information whether there is a correlation between the expression of these cytokines and pathologies found in an affected organ.

WC1+γδ T Cells Indirectly Regulate Chemokine Production DuringMycobacterium bovisInfection in SCID-bo Mice

Previous studies have suggested an important role for WC1 (+)gammadelta T cells in the regulation of mycobacterial-induced inflammation in the spleen and liver of heterochimeric SCID-bovine (SCID-bo) mice. To examine the role of these cells, we investigated the levels of selected chemokines and IL12-p70 post-infection in reconstituted SCID-bo mice. Mice were treated with a monoclonal antibody specific for boWC1 to eliminate WC1-bearing cells. Isotype control treated or bovine gammadelta TCR-depleted mice were assayed in parallel. Following infection with Mycobacterium bovis, mice were examined post-infection for the expression of IL12-p70, IP-10, MIP-1alpha, lymphotactin and MIG by ELISA in plasma and from activated splenocytes. Treatment with the anti-bovine WC1 resulted in reduced serum plasma levels of IP-10, MCP-1, and IL-12p70 versus control mice. The potential of WC1 (+)gammadelta TCR-bearing cells to produce chemokines and cytokines was determined directly from peripheral blood of cattle. Our results indicate that these cells have a fairly restricted capability to produce the chemokines examined in SCID-bo mice, but may be a significant source of cytokines (IL-2, IL-10, IL-12, IL-15, and IFNgamma) and contribute to cytotoxicity through expression of FasL and perforin. In M.bovis-infected liver tissue, depletion of the WC1(+) subset was associated with increased numbers of CD3(+)T cells adjacent to venules and portal tracts. These results suggest that the WC1(+) subset in cattle may contribute to chemotaxis through indirect effects on chemokine levels. Further, activated WC1(+)gammadelta TCR(+) cells up-regulate cytokines with direct regulatory effects on T cell and macrophage function and express effector molecules with critical roles in cytotoxicity.

Cardiac troponins and autoimmunity: Their role in the pathogenesis of myocarditis and of heart failure

Despite the widespread use of cardiac troponins as biomarkers for the diagnosis and quantitation of cardiac injury, the effect of troponin release and a possible autoimmune response to the troponins is unknown. Other investigators reported that programmed cell death-1 (PD-1)-receptor deficient mice developed severe cardiomyopathy with autoantibodies to troponin I. We found that immunization of genetically susceptible mice with troponin I but not troponin T induced a robust autoimmune response leading to marked inflammation and fibrosis in the myocardium. At later times, antibodies to cardiac myosin were detected in troponin-immunized mice. The severity of inflammation correlated with expression of chemokines RANTES, MIP-2, IP-10 and MCP-1 in the myocardium. Prior immunization with troponin I increased the severity of experimental infarctions, indicating that an autoimmune response to troponin I aggravates acute cardiac damage. Cardiac inflammation, fibrosis and functional impairment were transferred from immunized to naive recipients by CD4+ T cells, and the cytokine profile suggested both Th2 and Th17 profiles in A/J mice. Finally we identified an 18-mer of troponin I containing an immuno-dominant epitope.

Activation of the interferon‐γ signaling pathway in systemic lupus erythematosus peripheral blood mononuclear cells

To investigate interferon-gamma (IFNgamma) signaling in peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE) by analyzing IFNgamma receptor (IFNgammaR) expression, STAT-1 expression and phosphorylation, and the regulation of IFNgamma-inducible genes. Fluorocytometry was used to investigate expression of STAT-1, pSTAT-1, CD95, HLA-DR, class I major histocompatibility complex (MHC), IFNgamma-inducible 10-kd protein (IP-10), monokine induced by IFNgamma (Mig), and IFNgammaR in PBMCs from SLE patients and healthy individuals. STAT-1 phosphorylation was determined by fluorocytometry and Western blotting after stimulation with IFNalpha or IFNgamma. Quantitative polymerase chain reaction was used to assess messenger RNA (mRNA) expression of the IFNgamma-inducible genes IP-10 and Mig shortly after preparation or after stimulation with IFNgamma in monocytes. STAT-1 expression was increased in PBMCs from SLE patients and correlated significantly with disease activity and with the IFN-inducible expression of CD95 and HLA-DR. STAT-1 expression also showed a trend toward association with class I MHC expression. In addition, the expression of other IFNgamma-inducible genes, such as IP-10 or Mig, was increased in SLE monocytes. While STAT-1 phosphorylation in SLE PBMCs and PBMCs from healthy individuals was similar after IFNalpha stimulation, incubation with IFNgamma induced STAT-1 phosphorylation only in SLE lymphocytes. Moreover, SLE monocytes showed a considerably higher increase in pSTAT-1 expression upon IFNgamma stimulation than monocytes from healthy individuals. Increased responsiveness of SLE monocytes to IFNgamma was also confirmed on the mRNA level, where expression of the IFN-inducible, STAT-1-dependent genes IP-10 and Mig was more efficiently increased in SLE cells. However, IFNgammaR was similarly expressed on SLE lymphocytes and monocytes and those from healthy individuals. In addition to supporting the role of IFNs in SLE immunopathogenesis in general, the findings of the present study support a role of IFNgamma in this disease.

Role of chemokines in the enhancement of BBB permeability and inflammatory infiltration after rabies virus infection

Induction of innate immunity, particularly through the induction of interferon and chemokines, by rabies virus (RABV) infection has been reported to be inversely correlated with pathogenicity. To further investigate the association between the expression of chemokines and RABV infection, laboratory-attenuated RABV (B2C) and wild-type (wt) RABV (DRV) were administered to Balb/c mice intramuscularly. Chemokine expression, inflammatory cell infiltration, and blood–brain barrier (BBB) permeability were evaluated at various time points after infection. At day 3 post-infection (p.i.) there was very little inflammation in the central nervous system (CNS) and BBB permeability did not change in mice infected with either virus when compared with mock-infected mice. At 6 day p.i., infection with B2C induced the expression of inflammatory chemokines and infiltration of inflammatory cells into the CNS, while these changes were minimal in DRV-infected mice. Furthermore, infection with B2C significantly enhanced BBB permeability comparing to infection with DRV. Among the upregulated chemokines, the expression of IP-10 was best correlated with infiltration of inflammatory cells into the CNS and enhancement of BBB permeability. These data indicate that laboratory-attenuated RABV induces expression of chemokines and infiltration of inflammatory cells into the CNS. Upregulation of chemokines by B2C may have triggered the change in BBB permeability, which helps infiltration of inflammatory cells into the CNS, and thus attenuation of RABV.

The effect of obesity on intrahepatic cytokine and chemokine expression in chronic hepatitis C infection

BackgroundObese subjects with chronic hepatitis C virus (HCV) infection have more rapidly progressive liver disease.ObjectiveIn this study, we aimed to compare the hepatic cytokine and chemokine profiles in obese and...

CXCR3-B Expression Correlates With Tumor Necrosis Extension in Renal Cell Carcinoma

We investigated the expression of the 2 spliced variants of the CXCR3 receptor (CXCR3-A and CXCR3-B) and their ligands (MIG, IP-10 and I-TAC) in patients with renal cell carcinoma according to conventional prognostic factors and the necrosis pattern. A total of 59 patients with renal cell carcinoma were selected for study. Histotype, stage, grade and tumor diameter were first analyzed. Subsequently tumor necrosis extension, stratified as low-less than 30%, intermediate-30% to 75% and high-greater than 75%, was determined while blinded to pathological data, and CXCR3-B, IP-10, MIG and I-TAC mRNA levels were assessed. The overall correlation between CXCR3-B expression with the specific ligands, and tumor histotype, stage, grade, volume, necrosis extension and ligand expression were assessed on univariate and multivariate analyses. CXCR3-B levels stratified according to necrosis pattern were analyzed with the unpaired t test. CXCR3-B correlated with tumor necrosis and I-TAC (p = 0.0005 and 0.032, respectively). We did not note any correlation between CXCR3-B and histotype, stage, grade, diameter and expression of the other ligands IP-10 and MIG. Moreover, I-TAC did not correlate with tumor necrosis (p = 0.1102). In the multiple regression model a correlation between tumor necrosis and CXCR3-B expression was noted (p = 0.0005). Significant differences in CXCR3-B expression according to the necrosis pattern were observed between low and high, and between intermediate and high patterns (p = 0.0007 and 0.0183, respectively). Results demonstrate that CXCR3-B is an independent determinant factor for the extensive tumor necrosis pattern. These data emphasize the immunoangiostatic activity of the CXCR3/CXCR3 ligand biological axis for nonmetastatic human renal cell carcinoma.

Shifts in macrophage phenotypes and macrophage competition for arginine metabolism affect the severity of muscle pathology in muscular dystrophy

Duchenne muscular dystrophy (DMD) is the most common, lethal, muscle-wasting disease of childhood. Previous investigations have shown that muscle macrophages may play an important role in promoting the pathology in the mdx mouse model of DMD. In the present study, we investigate the mechanism through which macrophages promote mdx dystrophy and assess whether the phenotype of the macrophages changes between the stage of peak muscle necrosis (4 weeks of age) and muscle regeneration (12 weeks). We find that 4-week-old mdx muscles contain a population of pro-inflammatory, classically activated M1 macrophages that lyse muscle in vitro by NO-mediated mechanisms. Genetic ablation of the iNOS gene in mdx mice also significantly reduces muscle membrane lysis in 4-week-old mdx mice in vivo. However, 4-week mdx muscles also contain a population of alternatively activated, M2a macrophages that express arginase. In vitro assays show that M2a macrophages reduce lysis of muscle cells by M1 macrophages through the competition of arginase in M2a cells with iNOS in M1 cells for their common, enzymatic substrate, arginine. During the transition from the acute peak of mdx pathology to the regenerative stage, expression of IL-4 and IL-10 increases, either of which can deactivate the M1 phenotype and promote activation of a CD163+, M2c phenotype that can increase tissue repair. Our findings further show that IL-10 stimulation of macrophages activates their ability to promote satellite cell proliferation. Deactivation of the M1 phenotype is also associated with a reduced expression of iNOS, IL-6, MCP-1 and IP-10. Thus, these results show that distinct subpopulations of macrophages can promote muscle injury or repair in muscular dystrophy, and that therapeutic interventions that affect the balance between M1 and M2 macrophage populations may influence the course of muscular dystrophy.

Synoviocyte Innate Immune Responses: I. Differential Regulation of Interferon Responses and the JNK Pathway by MAPK Kinases

JNK is a key regulator of matrix metalloproteinase production in rheumatoid arthritis. It is regulated by two upstream kinases known as MKK4 and MKK7. Previous studies demonstrated that only MKK7 is required for cytokine-mediated JNK activation and matrix metalloproteinase expression in cultured fibroblast-like synoviocytes (FLS). However, the functions of MKK4 and MKK7 in synoviocyte innate immune responses have not been determined. TNF, peptidoglycan (PGN), and LPS stimulation led to higher and more prolonged MKK7 phosphorylation compared with MKK4 in FLS. However, this pattern was reversed in poly(I-C) stimulated cells. siRNA knockdown studies showed that TNF, PGN, and LPS-induced JNK and c-Jun phosphorylation are MKK7 dependent, while poly(I-C) responses require both MKK4 and MKK7. Poly(I-C)-induced expression of IP-10, RANTES, and IFN-beta mRNA was decreased in MKK4- or MKK7-deficient FLS. However, MKK4 and MKK7 deficiency did not affect phosphorylation of IkappaB kinase-related kinases in the TLR3 signaling pathway. MKK7, but not MKK4 deficiency, significantly decreased poly(I-C)-mediated IRF3 dimerization, DNA binding, and IFN-sensitive response element-mediated gene transcription. These results were mimicked by the JNK inhibitor SP600125, indicating that JNK can directly phosphorylate IRF3. In contrast, deficiency of either MKK4 or MKK7 decreased AP-1 transcriptional activity. Therefore, JNK is differentially regulated by MKK4 and MKK7 depending on the stimulus. MKK7 is the primary activator of JNK in TNF, LPS, and PGN responses. However, TLR3 requires both MKK4 and MKK7, with the former activating c-Jun and the latter activating both c-Jun and IRF3 through JNK-dependent mechanisms.

Astrocytes are very sensitive to develop innate immune responses to lipid‐carried short interfering RNA

Short interfering RNA (siRNA) inhibits the synthesis of specific proteins through RNA interference (RNAi). However, siRNA can induce innate immune responses that are mediated by toll-like receptors (TLRs) in cells of the immune system. Here, we sought to evaluate whether siRNA can induce such responses in glial cells. We examined the effects of various siRNA sequences prepared with lipids (oligofectamine). Lipid-siRNA induced variable degrees of silencing-independent nonspecific effects, e.g. increased Stat1 and Cox-2 expression and release of IL-6 and IP-10 in primary astroglia. This was prevented through chemical modification of siRNA by nucleoside 2'-O-methylation, without impairing specific gene silencing. Lipid-siRNA also induced nonspecific responses in purified astroglia, but not in microglia, or 3T3 cells. The highest TLR7 and TLR3 mRNA expression was found in microglia and purified astroglia, respectively. Accordingly, the TLR3 agonist poly(I:C) (PIC) induced higher release of IFN-beta in primary and purified astroglia than in microglia. As siRNA, PIC induced IP-10, Stat1, VCAM-1, and Cox-2 and increased TLR3 mRNA expression. The effects of lipid-siRNA in purified astrocytes were attenuated after silencing TLR3 or TLR7 expression, and by the PKR inhibitor 2-aminopurine. Furthermore, lipid-siRNA induced the expression of RIG-I. In contrast, siRNA devoid of lipids did not enter the astrocytes, did not silence gene expression, and did not induce Stat1 or Cox-2. The results show that, in astroglia, lipid-siRNA induces innate immune responses that are mediated, at least in part, by intracellular mechanism dependent on TLR7, TLR3, and helicases.

Respiratory Syncytial Virus Induces RelA Release from Cytoplasmic 100-kDa NF-κB2 Complexes via a Novel Retinoic Acid-inducible Gene-I·NF-κB-inducing Kinase Signaling Pathway

Respiratory syncytial virus (RSV) is a primary cause of severe lower respiratory tract infection in children worldwide. RSV infects airway epithelial cells, where it activates inflammatory genes via the NF-kappaB pathway. NF-kappaB is controlled by two pathways, a canonical pathway that releases sequestered RelA complexes from the IkappaBalpha inhibitor, and a second, the noncanonical pathway, that releases RelB from the 100-kDa NF-kappaB2 complex. Recently we found that the retinoic acid-inducible gene I (RIG-I) is a major intracellular RSV sensor upstream of the canonical pathway. In this study, we surprisingly found that RIG-I silencing also inhibited p100 processing to 52-kDa NF-kappaB2 ("p52"), suggesting that RIG-I was functionally upstream of the noncanonical regulatory kinase complex composed of NIK.IKKalpha subunits. Co-immunoprecipitation experiments not only demonstrated that NIK associated with RIG-I and its downstream adaptor, mitochondrial antiviral signaling (MAVS), but also showed the association between IKKalpha and MAVS. To further understand the role of the NIK.IKKalpha pathway, we compared RSV-induced NF-kappaB activation using wild type, Ikkgamma(-/-), Nik(-/-), and Ikkalpha(-/-)-deficient MEF cells. Interestingly, we found that in canonical pathway-defective Ikkgamma(-/-) cells, RSV induced RelA by liberation from p100 complexes. RSV was still able to activate IP10, Rantes, and Grobeta gene expression in Ikkgamma(-/-) cells, and this induction was inhibited by small interfering RNA-mediated RelA knockdown but not RelB silencing. These data suggest that part of the RelA activation in response to RSV infection was induced by a "cross-talk" pathway involving the noncanonical NIK.IKKalpha complex downstream of RIG-I.MAVS. This pathway may be a potential target for RSV treatment.