TNF-alpha Production Research Articles

Heat shock protein 90 inhibitor radicicol attenuates matrix metalloproteinase expression in human monocytic cells (P4153)

Abstract The molecular chaperone heat shock protein 90 (HSP90) has emerged as an exciting target for anti-tumor and immunoregulatory therapy. In this study, we investigated the inhibitory effects and mechanism of radicicol, as a HSP90 inhibitor, on human monocytic matrix metalloproteinases (MMPs) and pro-inflammatory cytokine expression. Radicicol exhibited concentration-dependent inhibition of MMP-9 enzymatic activation induced by either tumor necrosis factor-alpha (TNF-alpha) or lipopolysaccharide (LPS) in human monocytic THP-1 cells. Western blot and RT-PCR analyses showed that radicicol concentration-dependently suppressed LPS-induced monocytic MMP-9 protein and mRNA expression. It was also shown that radicicol enhanced HSP70 expression. Besides, we clearly found that radicicol restore the degradation of inhibitor-kappaB-alpha and abrogate NF-kappaB reporter gene expression in activated THP-1 cells. In addition, activation of Akt and its total protein expression were reduced by radicicol. Furthermore, radicicol presented an inhibitory effect on both TNF-alpha and interleukin-6 productions by ELISA. In conclusion, we demonstrate that radicicol might involve interference with the NF-kappaB or Akt signaling pathway to attenuate production of MMP-9 and pro-inflammatory cytokines. These results provide a new opportunity for the development of anti-leukemia and cachexia-relief strategies.

A Role for Protein Phosphatase 2A in Regulating p38 Mitogen Activated Protein Kinase Activation and Tumor Necrosis Factor-Alpha Expression during Influenza Virus Infection

Influenza viruses of avian origin continue to pose pandemic threats to human health. Some of the H5N1 and H9N2 virus subtypes induce markedly elevated cytokine levels when compared with the seasonal H1N1 virus. We previously showed that H5N1/97 hyperinduces tumor necrosis factor (TNF)-alpha through p38 mitogen activated protein kinase (MAPK). However, the detailed mechanisms of p38MAPK activation and TNF-alpha hyperinduction following influenza virus infections are not known. Negative feedback regulations of cytokine expression play important roles in avoiding overwhelming production of proinflammatory cytokines. Here we hypothesize that protein phosphatases are involved in the regulation of cytokine expressions during influenza virus infection. We investigated the roles of protein phosphatases including MAPK phosphatase-1 (MKP-1) and protein phosphatase type 2A (PP2A) in modulating p38MAPK activation and downstream TNF-alpha expressions in primary human monocyte-derived macrophages (PBMac) infected with H9N2/G1 or H1N1 influenza virus. We demonstrate that H9N2/G1 virus activated p38MAPK and hyperinduced TNF-alpha production in PBMac when compared with H1N1 virus. H9N2/G1 induced PP2A activity in PBMac and, with the treatment of a PP2A inhibitor, p38MAPK phosphorylation and TNF-alpha production were further increased in the virus-infected macrophages. However, H9N2/G1 did not induce the expression of PP2A indicating that the activation of PP2A is not mediated by p38MAPK in virus-infected PBMac. On the other hand, PP2A may not be the targets of H9N2/G1 in the upstream of p38MAPK signaling pathways since H1N1 also induced PP2A activation in primary macrophages. Our results may provide new insights into the control of cytokine dysregulation.

Triggering receptor expressed on myeloid cells − 1 (Trem-1) on blood neutrophils is associated with cytokine inducibility in human E. coli sepsis

BackgroundBacterial sepsis induced immunsuppression via antigen hyporesponsibility increases the risk of nosokomial infections and mortality. Pattern recognition receptors (PRR) might have a central role in the pathophysiology of hyporesponsibility.MethodsIn this study we evaluated in a human E. coli sepsis cohort, the role of PRR including TLR’s and Trem-1. Expression of Trem-1, TLR2, TLR4, CD14 and HLA-DR on blood monozytes and neutrophils were examined using flow cytometry from 22 patients with E. coli sepsis and 6 healthy controls. LPS and LTA stimulated TNF alpha, IL-10, IL-8 and IL-6 production was studied in a 24 h whole blood assay. Free cytokine serum concentration of TNF alpha, PCT and IP-10 were evaluated.ResultsWe found a significant higher expression of Trem-1 and TLR-2 on monocytes and neutrophils in patients compared to healthy volunteers. TLR2 expression (p < 0.05) was higher and HLA-DR lower (p < 0.05) on monocytes of patients with severe sepsis compared to patients with simple sepsis. Trem-1 expression was tendentially higher (p = 0,07) on monocytes and lower on neutrophils of patients with severe sepsis. Trem-1 expression on neutrophils was associated with the IL-10 (LPS: r = 0,61, p < 0.02) and TNF-α inducibility (LPS: r = 0,78, p < 0,002). In addition Trem-1 expression on neutrophils shows a negative correlation to the serum levels of TNF alpha (r = −0,63; p < 0,005), IP-10 (r = −0,5; p < 0,035) and procalcitonin (r = −0,59; p < 0,007).ConclusionsPatients with E. coli sepsis are characterized by an association of Trem-1 expression on blood neutrophils with cytokine inducibility. The TREM-1 pathway on neutrophils might play a role in producing an adequate inflammatory and bactericidal response in bacterial sepsis.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4441869398748313

Hepatoprotective effects of cathepsin B inhibitor on acute hepatic failure induced by lipopolysaccharide/D-galactosamine in mice

Increasing evidence suggests that the inactivation of cathepsin B attenuates hepatocyte apoptosis and liver damage. This study aimed to investigate the protective effects of a cathepsin B inhibitor (CA-074me) on lipopolysaccharide (LPS)/D-galactosamine (D-GalN)-induced acute hepatic failure (AHF) in mice. Mice were intraperitoneally injected with a combination of LPS/D-GalN to induce AHF with or without CA-074me pretreatment. The cumulative survival rates were calculated 48 hours after the induction of AHF. As well as changes in biochemical indicators and liver histology, hepatocyte apoptosis was assessed using a TUNEL method. Serum tumor necrosis factor-alpha (TNF-alpha) production, caspase-3, caspase-8, and caspase-9 activity was evaluated. Cytosolic cytochrome c and Bcl-2 expression were measured by Western blotting. The marked elevation in serum aminotransferase activity and prothrombin time found in LPS/D-GalN-treated mice was significantly improved by pretreatment with CA-074me. The efficacy of CA-074me was also confirmed by histological analysis and TUNEL assay. The survival rate significantly improved in LPS/D-GalN-induced mice given CA-074me compared with untreated mice. LPS/D-GalN-induced caspase-3 and caspase-9 activation was remarkably suppressed by CA-074me. However, the increased levels of serum TNF-alpha and elevated caspase-8 activity in AHF mice were not significantly reduced by CA-074me. Moreover, CA-074me sharply reduced the increased expression of cytosolic cytochrome c and markedly augmented Bcl-2 expression. These results suggest that CA-074me has a protective effect in acute hepatic failure induced by LPS/D-GalN.

NDP-MSH inhibits neutrophil migration through nicotinic and adrenergic receptors in experimental peritonitis

Melanocortin is a potent anti-inflammatory molecule. However, little is known about the effect of melanocortin on acute inflammatory processes such as neutrophil migration. In the present study, we investigated the ability of [Nle4, D-Phe7]-melanocyte-stimulating hormone (NDP-MSH), a semisynthetic melanocortin compound, in the inhibition of neutrophil migration in carrageenin-induced peritonitis model. Herein, subcutaneous pretreatment with NDP-MSH decreased neutrophil trafficking in the peritoneal cavity in a dose-dependent manner. NDP-MSH inhibited vascular leakage, leukocyte rolling, and adhesion and reduced peritoneal macrophage inflammatory protein 2, but not TNF-alpha, IL-1beta, IL-10, and keratinocyte-derived chemokine production. In addition, the effect on neutrophil migration was reverted by the pretreatment with both propranolol (a nonselective beta-adrenergic antagonist) and mecamylamine (a nonselective nicotinic antagonist) but not by splenectomy surgery. Moreover, NDP-MSH intracerebroventricular administration inhibited neutrophil migration, indicating participation of the central nervous system. Our results propose that the NDP-MSH effect may be due to a spleen-independent neuro-immune pathway that efficiently regulates excessive neutrophil recruitment to tissues.

Monocytes drive the induction of CD8+FOXP3+ regulatory T cells via the production of membrane TNF-alpha

Event Abstract Back to Event Monocytes drive the induction of CD8+FOXP3+ regulatory T cells via the production of membrane TNF-alpha Shawn D. P. Ellis1, Michael R. Ehrenstein1 and Clare A. Notley1* 1 University College London, United Kingdom Limiting the severity and the eventual resolution of an inflammatory response is vital for the protection of host tissues from the damaging effects of chronic inflammation. The induction of CD8+ regulatory T cells (Treg) expressing FOXP3 has been described within the context of chronic inflammation in cancer, infection and autoimmunity. For instance, we have shown that a small population of CD8+FOXP3+ Treg exists in the periphery of patients with inflammatory arthritis, which is not found in healthy individuals. The aim of our study was to dissect the mechanism of FOXP3 induction in CD8+ T cells after T cell receptor signalling, using CD3-specific antibodies. Our results found that anti-CD3 antibodies variably induce, not expand, a potent CD8+FOXP3+ Treg population. These Treg suppress CD4+ T cell proliferation, IL-17 and IFN-γ production in vitro and express TGF-β and CTLA-4. Investigation into the derivation of these cells determined that monocytes are crucial for the induction of FOXP3 expression following CD3 activation. In particular, monocytes provide co-stimulation through CD86 and membrane TNF-α. In vitro, these signals could be substituted with agonist antibodies to CD28 and TNFR2. Artificial expression of membrane TNF-α on monocytes was found to drive FOXP3 expression in a proportion of CD8+ T cells initially unresponsive to anti-CD3 stimulation. Our data identify the signalling pathways required for FOXP3 induction by CD8+ T cells and that these pathways are amenable to manipulation to augment regulatory capacity during autoimmunity or limit their effects in cancer and infection. Keywords: Treg, CD8+ T cells, TNF-α, IL-17, Rheumatoid arthritis Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Adaptive Immunity Citation: Ellis SP, Ehrenstein MR and Notley CA (2013). Monocytes drive the induction of CD8+FOXP3+ regulatory T cells via the production of membrane TNF-alpha. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00411 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 06 Apr 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Clare A Notley, University College London, London, United Kingdom, c.notley@ucl.ac.uk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Shawn D. P Ellis Michael R Ehrenstein Clare A Notley Google Shawn D. P Ellis Michael R Ehrenstein Clare A Notley Google Scholar Shawn D. P Ellis Michael R Ehrenstein Clare A Notley PubMed Shawn D. P Ellis Michael R Ehrenstein Clare A Notley Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Prone positioning attenuates TNF-alpha production in rabbits with lung injury under protective conventional mechanical ventilation (CMV).

Event Abstract Back to Event Prone positioning attenuates TNF-alpha production in rabbits with lung injury under protective conventional mechanical ventilation (CMV). Cilmery S. Kurokawa1* 1 Botucatu Medical School - São Paulo State University - UNESP, Brazil 1Cilmery Suemi Kurokawa, 1Rafaelle Batistella Pires, 1Jose Roberto Fioretto, 1Susiane O Klefens, 2João Pessoa de Araujo Júnior, 1Marcos Aurelio de Moraes, 1Ana Paula Mena Lousada, 1Rossano Cesar Bonatto, 1Mario Ferreira Carpi. 1Department of Pediatrics and Department of 2Microbiology and Immunology, Botucatu Medical School - Sao Paulo State University – UNESP. Prone positioning (PP) improves arterial oxygenation, gas exchange, distribution of ventilation toward dorsal lung areas, redirection of compressive forces on lung and facilitates secretion clearance in patients with ADRS. However, the exact mechanics of PP is still under investigation. The aim of this study aims was to measure TNF-alpha levels in rabbits with experimental ARDS under CMV and PP. Methods: Rabbits were anesthetized, intubated and assigned into three groups: 1) Control Group(CG)– animals without lung injury under CMV in supine position; 2) Prone Group (PG)– animals with lung injury under CMV in prone position; 3) Supine Group (SG)- animals with lung injury under CMV in supine position. Lung injury was induced by tracheal infusion of warm saline. The parameters evaluated were: TNF-alpha in homogenate of lung tissue, bronchoalveolar fluid (BAL) and plasma; histological and cytological analysis of lung tissue and BAL, respectively. Results: Lung injury decreased oxygenation { PaO2 - PG before[median (p25-p75)]: 451 (412- 504) > PG after: 67 (59-76), p<0,05; PaO2 - SG before: 447 (401-503) > SG after: 76 (59-87); p<0,05}. PG group had lower level of TNF-alpha in tissue than supine group {PG[median (p25-p75)]: 7,2 (3,5-11,07) > SG: 24,1 (8,1-55,55); p<0,05}. There is no difference of serum and BAL TNF-alpha levels, histopathological lung injury score and BAL neutrophil percentage between PG and SG. Conclusions: Prone positioning with CMV decreased TNF-alpha production in lung tissue in this experimental model. Acknowledgements This study was supported by FAPESP (Fundação de Amparo a Pesquisa do EStado de São Paulo), process number 2011/15144-2. We would like to thank the whole team at the PICU and Clinical and Experimental Research Center of Pediatrics Department . Keywords: lung lesion, tNF-alpha, Cytokines, mechnical ventilation, Neutrophils Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Innate immunity Citation: Kurokawa CS (2013). Prone positioning attenuates TNF-alpha production in rabbits with lung injury under protective conventional mechanical ventilation (CMV).. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00141 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 09 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Cilmery S Kurokawa, Botucatu Medical School - São Paulo State University - UNESP, Botucatu, Brazil, cskurokawa@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Cilmery S Kurokawa Google Cilmery S Kurokawa Google Scholar Cilmery S Kurokawa PubMed Cilmery S Kurokawa Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Umbilical cord blood T cells cultured with IL-7 in addition to IL-2 exhibit a higher degree of polyfunctionality and superior proliferation potential

Event Abstract Back to Event Umbilical cord blood T cells cultured with IL-7 in addition to IL-2 exhibit a higher degree of polyfunctionality and superior proliferation potential Sofia Berglund1, 2*, Jens Gertow1, Isabelle Magalhaes1, Michael Uhlin1, 2 and Jonas Mattsson1, 2 1 Karolinska Institute, Dept of Laboratory Medicine, Sweden 2 Karolinska University Hospital Huddinge, Centre for Allogeneic Stem cell Transplantation (CAST), Sweden BACKGROUND: Umbilical cord blood transplantation (UCBT) is an increasingly used stem cell source for allogeneic stem cell transplantation. One disadvantage to UCBT is that treatment with donor lymphocyte infusion (DLI) is not possible. We wanted to explore if cultured T cells from the UCB graft could offer an alternative to DLI in this setting. METHODS: T cells were positively selected with CD3+/CD28+ magnetic beads and cultured with IL-2 at escalating concentrations. In addition, IL-7 (20 ng/ml) was added to half of the cultures. After 7 days of expansion the T cells were analyzed by flow cytometry to determine phenotype, and cytokine production. RESULTS: There was significantly greater proliferation in cultures to which IL-7 had been added. Phenotypical differeces such as a higher CD4+/CD8+, a lower percentage of central memory T cells (CD45RO+ CCR7+) and a higher percentage of effector memory T cells (CD45RO+ CCR7-) was seen with IL-7. We assessed the intracellular production of IL-2, TNF-alpha, IFN-gamma and CD107a, and found that a higher percentage of polyfunctional T cells (positive for three or four factors) was seen in cells cultured with IL-7 in CD3+ and CD3+CD8+ cells, with a trend towards the same association also in CD3+CD4+ cells. CONCLUSION: There seem to be a proliferation advantage in adding IL-7 to a lower dose of IL-2. Addition of IL-7 also seem to have an impact on the differentiation status of the cells. T cells cultured in IL-7 had more polyfunctional traits, possibly increasing the activity of a putative future DLI. Acknowledgements The authors would like to thank the maternity ward, in particular Boel Niklasson, at Karolinska University hospital Huddinge, Stockholm, Sweden, for their kind assistance in obtaining the umbilical cord blood units. This study was supported by grants from Swedish Research Council (K2012-64X-22020-01-3) and The Swedish Childhood Cancer Foundation (PROJ10/052) and Swedish society for medical research. Keywords: umbilical cord blood, IL-7, T cell expansion, DLI, Cell Culture Techniques Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Translational immunology and immune intervention Citation: Berglund S, Gertow J, Magalhaes I, Uhlin M and Mattsson J (2013). Umbilical cord blood T cells cultured with IL-7 in addition to IL-2 exhibit a higher degree of polyfunctionality and superior proliferation potential. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00697 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 12 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Sofia Berglund, Karolinska Institute, Dept of Laboratory Medicine, Stockholm, 14186, Sweden, sofia.berglund@ki.se Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Sofia Berglund Jens Gertow Isabelle Magalhaes Michael Uhlin Jonas Mattsson Google Sofia Berglund Jens Gertow Isabelle Magalhaes Michael Uhlin Jonas Mattsson Google Scholar Sofia Berglund Jens Gertow Isabelle Magalhaes Michael Uhlin Jonas Mattsson PubMed Sofia Berglund Jens Gertow Isabelle Magalhaes Michael Uhlin Jonas Mattsson Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

A New Herbal Formula, KSG-002, Suppresses Breast Cancer Growth and Metastasis by Targeting NF- κ B-Dependent TNF α Production in Macrophages.

Tumor-associated macrophages (TAMs) in tumor microenvironment regulate cancer progression and metastases. In breast cancer, macrophage infiltration is correlated with a poor prognosis. While metastatic breast cancer is poor prognostic with a severe mortality, therapeutic options are still limited. In this study, we demonstrate that KSG-002, a new herbal composition of radices Astragalus membranaceus and Angelica gigas, suppresses breast cancer via inhibiting TAM recruitment. KSG-002, an extract of radices Astragalus membranaceus and Angelica gigas at 3 : 1 ratio, respectively, inhibited MDA-MB-231 xenograft tumor growth and pulmonary metastasis in nude mice, while KSG-001, another composition (1 : 1 ratio, w/w), enhanced tumor growth, angiogenesis, and pulmonary metastasis, in vivo. KSG-002 further decreased the infiltrated macrophage numbers in xenograft tumor cohorts. In Raw264.7 cells, KSG-002 but not KSG-001 inhibited cell proliferation and migration and reduced TNF-alpha (TNFα) production by inhibiting NF-κB pathway. Furthermore, a combinatorial treatment of KSG-002 with TNFα inhibited a proliferation and migration of both MDA-MB-231 and Raw264.7 cells. Taken together, we conclude that KSG-002 suppresses breast cancer growth and metastasis through targeting NF-κB-mediated TNFα production in macrophages.

New Perspectives on Ancient Granulomas

EDITORIAL article Front. Immunol., 15 November 2013Sec. Inflammation Volume 4 - 2013 | https://doi.org/10.3389/fimmu.2013.00345

Differentiation and culture of dendritic cells from swine bone marrow cell

Dendritic cells (DCs) are the most potent antigen-presenting cells that play a critical roles in connecting innate and adaptive immunity. Allhough many studies about mouse and human DCs have been performed, those using other mammalian species including domestic animals are lacking. ln this study, we established an efficient experimental method for swine myeloid-origin DCs. Bone marrow cells (BMs) were cultured in vitro using an adequate cytokine for this purpose. BMs were harvested from bones of piglets and cultured in the presence of porcine recombinant granulocyte-monocyte colony stimulating factor (GM-CSF) for 6-10 days. And also, we tried to find optimal conditions for cryopreservation of BMs. After 6 day culture, semi-floating cells were harvested and characterized as DCs (BMDCs). Interestingly, BMDCs revealed the excellent production of TNF-alpha, a representative innate cytokine, upon Iipopolysaccharide exposure, but not the case of nitric oxide. In addition, we measured the expression level of immunological markers on the surface of BMDCs. Taken together, we established an efficient experimental method for swine myeloid-origin DCs. The information obtained from this study includes optimal culture conditions and thus may help researchers studying swine immunity in basic scientific and-clinicalfields.

The effects of a selective inhibitor of c-Fos/activator protein-1 on endotoxin-induced acute kidney injury in mice

BackgroundSepsis has been identified as the most common cause of acute kidney injury (AKI) in intensive care units. Lipopolysaccharide (LPS) induces the production of several proinflammatory cytokines including tumor necrosis factor (TNF)-alpha, a major pathogenetic factor in septic AKI. c-Fos/activator protein (AP)-1 controls the expression of these cytokines by binding directly to AP-1 motifs in the cytokine promoter regions. T-5224 is a new drug developed by computer-aided drug design that selectively inhibits c-Fos/AP-1 binding to DNA. In this study, we tested whether T-5224 has a potential inhibitory effect against LPS-induced AKI, by suppressing the TNF-alpha inflammatory response and other downstream effectors.MethodsTo test this hypothesis, male C57BL/6 mice at 7 weeks old were divided into three groups (control, LPS and T-5224 groups). Mice in the control group received saline intraperitoneally and polyvinylpyrrolidone solution orally. Mice in the LPS group were injected intraperitoneally with a 6 mg/kg dose of LPS and were given polyvinylpyrrolidone solution immediately after LPS injection. In the T-5224 group, mice were administered T-5224 orally at a dose of 300 mg/kg immediately after LPS injection. Serum concentrations of TNF-alpha, interleukin (IL)-1beta, IL-6 and IL-10 were measured by ELISA. Moreover, the expression of intercellular adhesion molecule (ICAM)-1 mRNA in kidney was examined by quantitative real-time RT-PCR. Finally, we evaluated renal histological changes.ResultsLPS injection induced high serum levels of TNF-alpha, IL-1beta and IL-6. However, the administration of T-5224 inhibited the LPS-induced increase in these cytokine levels. The serum levels of IL-10 in the LPS group and T-5224 group were markedly elevated compared with the control group. T-5224 also inhibited LPS-induced ICAM-1 mRNA expression. Furthermore histological studies supported an anti-inflammatory role of T-5224.ConclusionsIn endotoxin-induced AKI, T-5224 inhibited the production of TNF-alpha and other downstream effectors. In contrast, T-5224 did not inhibit IL-10, an anti-inflammatory cytokine. These data support that the use of T-5224 is a promising new treatment for septic kidney injury.

Rapamycin Inhibits Innate and Adaptive Immune Functions of Human Plasmacytoid Dendritic Cells

Introduction: Rapamycin is an anti-proliferative drug used to prevent organ transplant rejection and to treat cancer. Plasmacytoid dendritric cells (PDC) are important in innate immunity as they are the principal producers of IFN-α, and in adaptive immunity by presentation of antigens to T cells. PDC are critically involved in immunity to viral infections and in the pathogenesis of auto-immune disorders like Systemic Lupus Erythematosus (SLE). Here we report that innate and adaptive immune functions of human PDC are differentially regulated by Toll-Like Receptors (TLR) and CD40, and that rapamycin inhibits both innate and adaptive immune functions of PDC. Results: Human PDC activated by TLR-7 or TLR-9 ligands produced high amounts of IFN-α, but exhibited a weak T cell stimulatory capacity. Conversely, PDC activated by CD40-ligation failed to produce IFN-α, but induced robust allogeneic T cell proliferation and effector functions, among which production of pro-inflammatory cytokines IFN-gamma, TNF-alpha and IL-17. Combined stimulation via CD40 and TLR further upregulated the capacity of PDC to stimulate pro-inflammatory cytokine secretion by T cells. Rapamycin inhibited production of IFN-α by PDC, and suppressed the capacity of PDC to stimulate allogeneic T cell effector functions. Reduction of T-cell stimulatory capacity was most pronounced when rapamycin was added during activation of PDC via CD40, and was associated with inhibition of CD40 expression on PDC. Conclusions: Activation of human PDC via TLR stimulates their innate immune functions, while activation via CD40 stimulates their adaptive immune functions. Rapamycin inhibits both innate and adaptive immune functions of human PDC, and may therefore constitute a novel treatment option for SLE, but increase susceptibility to viral infections.

Nitidine chloride inhibits LPS-induced inflammatory cytokines production via MAPK and NF-kappaB pathway in RAW 264.7 cells

Ethnopharmacological relevanceZanthoxylum nitidium (Roxb.) DC. has long been used as a traditional herbal medicine for inflammatory diseases such as rheumatic arthritis and peridentitis. However, the anti-inflammatory mechanism of Nitidine chloride has not been fully elucidated. Aim of the studyTo determine the anti-inflammatory effects and mechanism of Nitidine chloride (NTD), a pentacyclic alkaloid is isolated from the root of Z. nitidium, in murine macrophages. Materials and methodsAnti-inflammatory properties of NTD were investigated using lipopolysaccharide (LPS)-stimulated Raw 264.7 macrophages as in vitro model. The pro-inflammatory cytokines were evaluated by real-time RT-PCR and ELISA. Furthermore, intracellular signaling pathways were analyzed by Western blot and Immunofluorescence staining using specific antibodies. ResultsNTD significantly reduced the production of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and IL-6 in both RNA and protein level. Moreover, transcriptional activity of NF-кB as well as the phosphorylation of mitogen-activated protein kinases (MAPKs) in LPS-treated RAW 264.7 was significantly inhibited by NTD in a dose dependent manner. These results suggested that NTD exerts an anti-inflammatory property by inhibiting TNF-α, IL-1β, and IL-6 production in association with reduced NF-κB and MAPK signaling pathways in RAW 264.7 cells. ConclusionThese results suggested that NTD exerts an anti-inflammatory property by inhibiting TNF-α, IL-1β, and IL-6 production in association with reduced NF-κB and MAPK signaling pathways in RAW 264.7 cells. Nitidine chloride inhibits LPS-induced TNF alpha, IL-1beta and IL-6 production via the suppression of phosphorylation of MAPK and the translocation of p65. In addition, these results revealed a novel role of NTD in regulation of inflammatory diseases.

47. Differing trafficking and TNF-alpha production by monocyte subsets under adrenergic activation: Blunted mobilization of “pro-inflammatory” monocytes in individuals with elevated blood pressure

Elevated blood pressure (BP) and infiltration of the vasculature by monocytes contribute to vascular pathology; but, monocyte migratory characteristics based on differing inflammatory potential under adrenergic activation remains unclear. We compared “non-classical” (CD16++CD14+), “intermediate” (CD16+CD14++), and “classical” (CD16-CD14++) monocyte trafficking and their LPS-stimulated TNF-alpha production in response to a physical stressor (20-min treadmill exercise at 65–70% VO2peak) in participants ( n = 77) with high pre-hypertension (PHT), mild PHT or normal BP (NBP). To determine adrenergic receptor sensitivity, pre-exercise cells were also treated with isoproterenol (Iso) before LPS stimulation. Monocyte subpopulations and intracellular TNF-alpha production were evaluated by flow cytometry. Epinephrine and norepinephrine levels were significantly elevated during exercise. Overall, % non-classical monocytes increased, % intermediate did not change, whereas % classical decreased post-exercise ( p < .001). However, the % increase in non-classical monocytes under adrenergic activation was blunted in PHT individuals ( p < .05). TNF-alpha production in all subsets decreased post-exercise and with in-vitro Iso treatment, irrespective of BP ( p < .001), with nonclassical and intermediate monocytes being a major source of TNF-alpha production. These findings extend our previous reports by showing that the mobilization of “pro-inflammatory” monocytes under sympathoadrenal activation is attenuated with even mild BP elevation. This may be indicative of monocytic adrenergic receptor desensitization and/or greater adhesion of “pro-inflammatory” monocytes to the vascular endothelium in hypertension with clinical implications of vascular pathology.

124. A comparative examination of the anti-inflammatory effects of SSRI and SNRI antidepressants on microglia

Selective serotonin and serotonin norepinephrine reuptake inhibitors (SSRI; SNRI) are the frontline pharmacological treatment options for major depression. While these drugs have long been assumed to exert their antidepressant effects because of their ability to alter central monoamine levels, they have also been shown to exert anti-inflammatory effects. Recently it has become apparent that these drugs can also exert anti-inflammatory effects on microglia, the principal cells within the CNS that regulate and respond to inflammatory factors. From a pharmacological standpoint, much critical information remains unknown. In particular, the relative efficacy of these drugs in modulating microglial responses is yet to be determined. To address these issues, we evaluated the ability of different SSRIs (fluoxetine, sertraline, paroxetine, fluvoxamine and citalopram) and one SNRI (venlafaxine) to suppress microglial responses to an inflammatory stimulus. Specifically, we examined their ability to alter tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) production after stimulation with lipopolysaccharide. Our results indicated that the SSRIs potently inhibited microglial TNF-alpha and NO production. We then investigated whether these effects might involve either beta-adrenoceptor or cAMP signalling. Using the protein kinase A inhibitor Rp-CAMPs, we found evidence to suggest that cAMP signalling may be partially involved in regulating the anti-inflammatory response. Findings such as these could suggest that antidepressants may owe at least some of their therapeutic effectiveness to their anti-inflammatory properties.

154. Glucocorticoid sensitivity of leukocytes predicts fatigue, depressive, and PTSD symptoms after military deployment

Posttraumatic stress disorder (PTSD), depression and severe fatigue may develop in response to severe stress and trauma. These conditions are associated with altered sensitivity of leukocytes to glucocorticoids (GCs). We investigated whether GC-sensitivity of T-cells and monocytes assessed before military deployment predicts high levels of fatigue, depressive, and/or PTSD symptoms 6 months after return from deployment. We included 523 male military personnel before deployment to Afghanistan. Logistic regression analysis was performed to predict fatigue, depressive, and PTSD symptoms 6 months after deployment based on sensitivity of LPS-induced TNF-alpha production and PHA-induced T-cell proliferation to dexamethasone (DEX)-inhibition before deployment. Severe fatigue 6 months after deployment was independently associated with low DEX-sensitivity of monocyte TNF-α production before deployment. A high level of depressive symptoms 6 months after deployment was independently associated with low DEX-sensitivity of T-cell proliferation. In contrast, a high level of PTSD symptoms after deployment was independently associated with high DEX-sensitivity of T-cell proliferation before deployment, but only in individuals who reported PTSD symptoms without depressive symptoms. DEX-sensitivity was not associated with childhood trauma, a known risk factor for development of fatigue, depressive and PTSD symptoms. GC-sensitivity of leukocytes prior to deployment predicts fatigue, depressive, and PTSD symptomatology in response to deployment. Notably, fatigue, depressive, and PTSD symptoms were differentially associated with GC-sensitivity of monocytes and T-cells and therefore may have specific biological underpinnings.

Ablation of the Regulatory IE1 Protein of Murine Cytomegalovirus Alters In Vivo Pro-inflammatory TNF-alpha Production during Acute Infection

Little is known about the role of viral genes in modulating host cytokine responses. Here we report a new functional role of the viral encoded IE1 protein of the murine cytomegalovirus in sculpting the inflammatory response in an acute infection. In time course experiments of infected primary macrophages (MΦs) measuring cytokine production levels, genetic ablation of the immediate-early 1 (ie1) gene results in a significant increase in TNFα production. Intracellular staining for cytokine production and viral early gene expression shows that TNFα production is highly associated with the productively infected MΦ population of cells. The ie1- dependent phenotype of enhanced MΦ TNFα production occurs at both protein and RNA levels. Noticeably, we show in a series of in vivo infection experiments that in multiple organs the presence of ie1 potently inhibits the pro-inflammatory cytokine response. From these experiments, levels of TNFα, and to a lesser extent IFNβ, but not the anti-inflammatory cytokine IL10, are moderated in the presence of ie1. The ie1- mediated inhibition of TNFα production has a similar quantitative phenotype profile in infection of susceptible (BALB/c) and resistant (C57BL/6) mouse strains as well as in a severe immuno-ablative model of infection. In vitro experiments with infected macrophages reveal that deletion of ie1 results in increased sensitivity of viral replication to TNFα inhibition. However, in vivo infection studies show that genetic ablation of TNFα or TNFRp55 receptor is not sufficient to rescue the restricted replication phenotype of the ie1 mutant virus. These results provide, for the first time, evidence for a role of IE1 as a regulator of the pro-inflammatory response and demonstrate a specific pathogen gene capable of moderating the host production of TNFα in vivo.

In vitro comparison of the biological activity of alumina ceramic and titanium particles associated with aseptic loosening

Prosthetic wear particles are thought to play a central role in the initiation and development of periprosthetic osteolysis, leading to aseptic loosening of prostheses. This study aimed to compare the biological activity of ceramic and titanium particles that are associated with particle-induced, aseptic joint loosening. Different sizes of alumina-ceramic particles and titanium particles were prepared to stimulate murine macrophage cells RAW 264.7, of which the expressions of tumor necrosis factor alpha (TNF-alpha) and receptor activator of nuclear factor-κB ligand (RANKL) were measured by qPCR and ELISA at various time points. In the presence of all particles, the expression of TNF-alpha increased in a time-dependent manner, whereas the expression of RANKL showed no regular expression patterns. Notably, particles of smaller sizes provoked significantly higher levels of TNF-alpha and RANKL than those of larger sizes. Compared to the titanium particles, the ceramic particles provoked a significantly lower production of TNF-alpha. Thus, the bioactivities of titanium and alumina ceramic particles were inversely proportional to the sizes of the particles, and the expression of RANKL was not parallel to that of TNF-alpha. The successful outcome of ceramic-on-ceramic artificial joint prostheses may be attributed to the low biological activity of ceramic particles, as evidenced here.

Haemozoin Induces Early Cytokine-Mediated Lysozyme Release from Human Monocytes through p38 MAPK- and NF-kappaB- Dependent Mechanisms

Malarial pigment (natural haemozoin, HZ) is a ferriprotoporphyrin IX crystal produced by Plasmodium parasites after haemoglobin catabolism. HZ-fed human monocytes are functionally compromised, releasing increased amounts of pro-inflammatory molecules, including cytokines, chemokines and cytokine-related proteolytic enzyme Matrix Metalloproteinase-9 (MMP-9), whose role in complicated malaria has been recently suggested. In a previous work HZ was shown to induce through TNFalpha production the release of monocytic lysozyme, an enzyme stored in gelatinase granules with MMP-9. Here, the underlying mechanisms were investigated. Results showed that HZ lipid moiety promoted early but not late lysozyme release. HZ-dependent lysozyme induction was abrogated by anti-TNFalpha/IL-1beta/MIP-1alpha blocking antibodies and mimicked by recombinant cytokines. Moreover, HZ early activated either p38 MAPK or NF-kappaB pathways by inducing: p38 MAPK phosphorylation; cytosolic I-kappaBalpha phosphorylation and degradation; NF-kappaB nuclear translocation and DNA-binding. Inhibition of both routes through selected molecules (SB203580, quercetin, artemisinin, parthenolide) prevented HZ-dependent lysozyme release. These data suggest that HZ-triggered overproduction of TNFalpha, IL-1beta and MIP-1alpha mediates induction of lysozyme release from human monocytes through activation of p38 MAPK and NF-kappaB pathways, providing new evidence on mechanisms underlying the HZ-enhanced monocyte degranulation in falciparum malaria and the potential role for lysozyme as a new affordable marker in severe malaria.