Potentially probiotic bacteria induce cytokine production and suppressor of cytokine signaling 3 gene expression in human monocyte-derived macrophages

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Abstracts / Cytokine 48 (2009) 91–137

IFN-c and/or lipopolysaccharide (LPS)/IFN-c are poorly understood. In this study, we show that primary human monocytes stimulated with IFN-c alone or co-stimulated with IFN-c/LPS displayed significantly increased expression of IL-12/23p40 and IL23p19 mRNA expression as well as IL-23 protein whereas LPS alone did not affect IL-23 expression at the mRNA or protein levels in these cells. To determine the intracellular signalling pathways involved in the regulation of IFN-c-induced IL-23 expression, pharmacological inhibitors to the JAK/STAT, PI3K and MAPK pathways were employed. The results show that IFN-c and IFN-c/LPS-induced IL-23 expression is negatively regulated by the Janus activated kinase/Signal transducer and activator of transcription (JAK/STAT), phosphoinositide 3-kinase (PI3K) and the c-jun N-terminal kinase (JNK) mitogen activated protein kinase (MAPKs) pathways. In contrast, the p38 MAPKs positively regulated the expression of IL-23 and IL-12/23p40 in both IFNc and IFN-c/LPS-stimulated monocytes. In summary, the results show for the first time the differential regulation of IL-23 subunits in primary human monocytes stimulated with IFN-c and IFN-c/LPS. IFN-c and IFN-c/LPS-induced IL-12p40 and IL-23 induction was negatively regulated by the JAK/STAT, PI3K and the JNK pathway and positively regulated by the p38 MAPKs independent of the JAK/STAT signalling.

3.36 ± 0.26  107 cells, respectively) 24 h post injection of TG which returned to WT levels by 96 h. While leukocyte counts were higher in CD93 / PLF, the percentage of Gr1-positive and CD11b-positive leukocytes was similar to WT mice. In addition, WT and CD93 / mice had similar levels of apoptotic cells in the peritoneal cavity. To determine the mechanism leading to elevated leukocyte counts, proinflammatory cytokine and chemokine levels in PLF were analyzed by Luminex multiplex array. Cytokines were elevated with TG administration, however similar levels of IL-1b, IL-4, IL-5, IL-6, KC, MIP1a, RANTES, MCP1 and TNFa were detected in CD93 / and WT PLF. Since cytokine levels were comparable in CD93 / and WT mice, our current efforts are aimed at determining if sCD93 is responsible for regulating the proinflammatory phenotype in CD93 / mice. doi:10.1016/j.cyto.2009.07.420

PP2-043 Upregulation of autophagy by inhibitors of mTOR or caspases decreases splenic T cell apoptosis in sepsis

doi:10.1016/j.cyto.2009.07.418

PP2-041 Dissecting in vivo innate immune responses to TLR7/9 agonists in primates Montserrat Puig, Lucja T. Grajkowska, Joseph J. Mattapallil, Daniela Verthelyi, Poster Presentation II Dissecting in vivo innate immune responses to TLR7/9 agonists in primates

Over the last 10 years a large body of preclinical data has been generated supporting the therapeutic use of TLR agonists to treat human diseases. Underlying TLR9 species specificities made the non-human primates the preclinical model of choice for these studies. In vitro, CpG ODN types C and D (also known as A) activate TLR9 on early endosomes of pDC to induce IFNa and DC maturation. However when used in vivo to treat rhesus macaques challenged with Leishmania major, local or systemic administration of CpG ODN type D/A, but not type C (or B) reduces the severity of cutaneous lesions. In these studies we use TaqManÒ Low Density Array (TLDA) technology coupled with computational pathway analysis to gain novel insights into the systemic and local changes in gene expression underlying the difference in efficacy. We show that while in vitro stimulation of PBMCs with D or C CpG ODN induced expression of similar immune related genes, the magnitude and kinetics of type I IFNs subtypes differed significantly. We then studied the effects that CpG ODN type C and D have on the type I IFN downstream pathway by analyzing transcription factor and interferon stimulated gene (ISG) expression in order to correlate the differential regulation with the biological effect of each ODN. Lastly, we show that the 2 types of CpG ODN induce distinct responses in peripheral blood, skin and draining lymph nodes in vivo that could partly explain their distinct immunoprotective effects. These include significant differences in the local and systemic levels of type I, II & III IFNs, IL-6, and IL-10 transcripts. Our findings have implications for the therapeutic application of CpG ODN in humans.

Ya-Ching Hsieh, Hsiang-Wei Hsueh, Chi-Hsun Hsieh, Shyng-Shiou Yuan, Poster Presentation II Upregulation of autophagy by inhibitors of MTOR or caspases decreases splenic T cell apoptosis in sepsis Ya-Ching Hsieh 1, Hsiang-Wei Hsueh 1, Chi-Hsun Hsieh 2, Shyng-Shiou Yuan 1,3, 1 Department of Medical Research, E-Da Hospital/I-Shou University Kaohsiung, Taiwan, ROC, 2 Department of Trauma and Emergency Surgery, China Medical University Hospital, Taichung, Taiwan, ROC, 3 Department of Biological Science and Technology, I-Shou University, Kaohsiung, Taiwan, ROC Apoptosis acts as an important mechanism of immunosuppression during sepsis. Autophagy, an important host mechanism for removal of intracellular bacteria, has recently emerged as an important mediator of programmed cell death pathways and may function to prevent apoptosis. We therefore hypothesized that induction of autophagy via inhibition of mTOR and caspases could reduce T cell apoptosis and inhibit the release of pro-inflammatory mediators during sepsis. To test this hypothesis, male mice were subjected to cecal ligation and puncture (CLP) or sham operation. CLP mice received either vehicle, autophagy inducer (rapamycin; mTOR inhibitor), apoptosis inhibitor (zVAD; caspase inhibitor), or rapamycin in combination with zVAD after onset of CLP. Our results show that autophagy markers, including Atg5–Atg12 complex, LC3-II, and Rab7, were decreased in the spleen at 24 h after CLP, consistent with the morphologic finding that septic mice exhibited fewer autophagic vacuoles and less digested debris within vacuoles. Administration of rapamycin, zVAD, or co-administration of rapamycin and zVAD after CLP upregulated Atg5– Atg12 complex and LC3-II as well as autophagic vacuoles in the spleen. This was accompanied by decreases in splenic caspase-3 activity and CD3+ T cell apoptosis (Fig. 1) as well as circulating levels of MCP-1 and IL-10. Moreover, administration of rapamycin alone or co-administration of rapamycin and zVAD after CLP resulted in a decrease in splenocyte capacity to release MCP-1, IL-10, and TNF-a. These results indicate that autophagy may serve as a cell survival mechanism to protect against T cell apoptosis and inflammatory mediator release during sepsis. doi:10.1016/j.cyto.2009.07.421

doi:10.1016/j.cyto.2009.07.419

PP2-042 CD93 regulates inflammation in vivo Mallary C. Greenlee, Suzanne S. Bohlson, Poster Presentation II CD93 regulates inflammation in vivo Mallary C. Greenlee 1, Suzanne S. Bohlson 1,2, 1 Department of Biological Sciences, Eck Institute for Global Health, University of Notre Dame, Notre Dame, IN, United States, 2 Department of Microbiology and Immunology, Indiana University School of Medicine – South Bend, South Bend, IN, United States Engulfment of apoptotic cells is important in the resolution of inflammation and prevention of autoimmunity. CD93 is a transmembrane glycoprotein that regulates immune cell function, including the engulfment of apoptotic cells. However, the mechanism(s) leading to CD93-dependent function has remained elusive. CD93 is shed from activated human monocytes and neutrophils, as well as mouse inflammatory macrophages, and soluble CD93 (sCD93) is elevated in mouse peritoneal lavage fluid (PLF) following thioglycollate (TG) induced peritonitis and LPS-induced septic shock. We hypothesize that sCD93 contributes to the resolution phase of inflammation, including the engulfment of apoptotic cells. Here we demonstrate that CD93-sufficient PLF from TG injected mice, containing elevated sCD93, enhanced the engulfment of apoptotic cells in vitro whereas PLF from CD93-deficient mice failed to enhance ingestion. Reconstitution of CD93 / PLF with recombinant sCD93 partially restored phagocytosis in vitro. Interestingly, CD93 / mice had elevated leukocyte counts in the peritoneal cavity compared to WT mice (6.41 ± 0.57  107 cells and

PP2-045 Potentially probiotic bacteria induce cytokine production and suppressor of cytokine signaling 3 gene expression in human monocyte-derived macrophages Sinikka Latvala, Minja Miettinen, Riina Kekkonen, Riitta Korpela, Ilkka Julkunen, Poster Presentation II Potentially probiotic bacteria induce cytokine production and suppressor of cytokine signaling 3 gene expression in human monocyte-derived macrophages Sinikka Latvala 1, Minja Miettinen 1, Riina Kekkonen 2, Riitta Korpela 2, Ilkka Julkunen 1, 1 Department of Vaccination and Immune Protection, National Institute for Health and Welfare, Helsinki, Finland, 2 Valio Ltd., R&D, Helsinki, Finland In the present study, we have analyzed the ability of 11 potentially probiotic bacteria to activate human monocyte-derived macrophages (MO) and induce their cytokine gene expression. Our aim was to analyze whether there are significant differences in the ability of these bacteria to activate macrophage cytokine production, and whether suppressor of cytokine signaling (SOCS) 3-mediated negative feed-back systems are also operative. MOs obtained from buffy coats and differentiated in vitro with GM-CSF were stimulated with probiotic bacteria. After bacterial stimulation the cell culture supernatants were collected and cytokine levels were determined by ELISA. The kinetics of mRNA expression of cytokine genes and the involvement of SOCS3 in MO responses were analyzed by qRT-PCR. Most bacteria induced cytokine production in a dose-dependent manner. However, certain differences in the ability of these bacteria to induce MO cytokine responses were found.

Abstracts / Cytokine 48 (2009) 91–137 All bacteria induced pro-inflammatory cytokines (TNF-a, IL-1b, IL-6). In addition, some bacteria also induced anti-inflammatory cytokine (IL-10). Bifidobacterium, Streptococcus, and Lactobacillus-strains were good inducers of IL-6, IL-10, and TNF-a, while Leuconostoc mesenteroides ssp. cremoris and Propionibacteriumfreudenreichii ssp. shermanii were relatively poor inducers of cytokine gene expression. In addition to activating cytokine production all studied bacteria were also able to induce SOCS3 gene expression, which likely leads to negative feed back of extensive cytokine production in bacteria stimulated macrophages. SOCS3 gene expression was induced directly by bacterial stimulation. These results show that macrophages respond very strongly to bacterial stimulation even in the case of non-pathogenic bacteria and the responses vary between different bacterial strains.

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strating that the inflammation has a hematopoietic origin. Due to the inability of the founders to breed, bone-marrow chimeras were used to characterize the immune dysfunction in these animals. In these chimeras, we observed constitutive phosphorylation of STAT3 in lymphoid organs and high serum levels of IL-22, both of which correlated with IL-22R1 expression. Our data support a model where T-cells expressing IL-22R have a positive auto-regulatory feedback that amplifies the levels of IL-22 in the environment. These signals induce T- and B-cells to mature rapidly and migrate to sterile organs resulting in lethal inflammation. Thus, aberrant expression of the IL22R1 chain reveals a possible unexplored role for IL-22 in inflammatory conditions. doi:10.1016/j.cyto.2009.07.425

doi:10.1016/j.cyto.2009.07.423 PP2-048 Regulatory function of SOCS-3 in astrocytes PP2-046 Bioactivity-guided identification and cell signaling analysis to investigate the immunomodulatory effects of ginseng on U937 cells Davy Lee, Cindy Yang, Stanley Chik, James Li, Allan Lau, Poster Presentation II Bioactivity-guided identification and cell signaling analysis to investigate the immunomodulatory effects of ginseng on U937 cells Davy C.W. Lee 1, Cindy L.H. Yang 2, Stanley C.C. Chik 2, James C.B. Li 1,2, Allan S.Y. Lau 1,2, 1 Cytokine Biology Group, Department of Paediatrics and Adolescent Medicine, LKS Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China, 2 Molecular Chinese Medicine Laboratory, Department of Paediatrics and Adolescent Medicine, LKS Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China Panax ginseng (Ginseng) is one of the most commonly used medicinal herbs worldwide. It is believed to have beneficial effects against human diseases, and its active components, ginsenosides, may play critical roles in its diverse physiological actions. However, the mechanisms underlying ginseng’s effects remain to be investigated. We hypothesize that some biological effects of ginseng are due to its anti-inflammatory activities. To investigate the effects of ginseng on inflammation, human monocytic cells (U937) were treated sequentially with 70% ethanol–water extracts of Panax ginseng (PGSE) and tumor necrosis factor-alpha (TNF-a). Gene expression profiles of the cells were examined by genechip analysis (Human Genome U133 Plus 2.0 arrays, Affymetrix) and validated by Taqman quantitative RT-PCR, and protein expressions were assayed by ELISA. The composition of ginsenosides in 70% ethanol–water extracts of ginseng was analyzed by HPLC. Activation of signaling kinases was examined by Western blot analysis and the respective anti-phospho-protein kinase antibodies. PGSE significantly inhibited the transcription and secretion of CXCL-10 following TNF-a stimulation. Nine ginsenosides including Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3 and Rh1 were identified in the PGSE by HPLC. Seven out of nine ginsenosides could significantly inhibit TNF-a-induced CXCL-10 expression in U937 cells but the CXCL-10 suppressive effects of individual ginsenosides was less than that of the crude extract or the mixture of reconstituted ginsenosides. The suppressive effect of the reconstituted mixture of nine ginsenosides at the corresponding doses was comparable to the PGSE treatment in a dose-dependent manner. Furthermore, the CXCL-10 suppression can be correlated with the inactivation of ERK1/2 pathways by the PGSE. In summary, our results provide evidence that ginseng can suppress TNF-a-inducible cytokines and signaling proteins in promonocytic cells. doi:10.1016/j.cyto.2009.07.424

PP2-047 Expression of IL-22R1 on lymphocytes induces lethal inflammation Ram Savan, Della Reynolds, Adelle McFarland, Lionel Feigenbaum, Raymond P. Donnelly, Howard A. Young, Poster Presentation II Expression of IL-22R1 on lymphocytes induces lethal inflammation

Interleukin (IL)-22, a member of the IL-10 family of cytokines, has been reported to have both pro- and anti-inflammatory functions. IL-22 is known to activate cell survival and mitogenic signals through phosphorylation of STAT3. IL-22 signals through a heterodimeric receptor composed of 2 chains, IL-22R1 and IL-10R2. The private chain (IL-22R1) of this heterodimeric receptor is not expressed on normal leukocytes but aberrant IL-22 receptor expression has been found on ALK+ anaplastic large cell lymphoma. To study the effects of the receptor on lymphocytes, we generated transgenic mice that express the IL-22R1 driven by the CD2 promoter, resulting in T-cell and B-cell expression of a functional IL-22 receptor. Surprisingly, the transgenic founders succumb to inflammation within 8–12 weeks of age. Serum of these animals had high levels of inflammatory cytokines including IFN-g, IL-17, TNF-a and IL-6. As illness progresses, there is accelerated maturation of T- and B-cells with neutrophilia and increased inflammatory monocytes. Transplantation of transgenic bone-marrow into WT recipients resulted in a similar phenotype to transgenic founders demon-

Hongwei Qin, Stephanie L. Reynolds, Etty N. Benveniste, Poster Presentation II Regulatory function of SOCS-3 in astrocytes

Neuroinflammatory events in the Central Nervous System (CNS) are central to the pathogenesis of Multiple Sclerosis, Alzheimer’s Disease and HIV-1 Associated Dementia. Astrocytes play a number of important physiological roles in CNS homeostasis. Suppressor Of Cytokine Signaling (SOCS) proteins, a family of proteins that negatively regulate adaptive and innate immune responses, are critical feedback inhibitors of the JAK/STAT signaling pathway and have neuroprotective effects. Herein we describe how SOCS-3 controls the secretion of inflammatory cytokines and chemokines in astrocytes. MCP-1/CCL2, MIP-1a/CCL3, RANTES/CCL5, Eotaxin/CCL11, KC/CXCL1, LIX/ CXCL5, IP-10/CXCL10, TNF-a, IL-5, IL-6, IL-12, CCL20, G-CSF and VEGF mRNA and protein expression are significant elevated in SOCS-3 deficient astrocytes compared to wild-type astrocytes after IL-6 and OSM stimulation. Furthermore, our results indicate that IL-6 and OSM activation of STAT-1, STAT-3, ERK1/2 and NF-jB p65 pathways in astrocytes is controlled by SOCS-3, which correlates with cytokine and chemokine expression in these cells. Our data suggest that properly controlled IL-6 and OSM induced JAK-STAT, MAPK and NF-jB signaling in astrocytes by SOCS-3 is essential for control of inflammatory responses in CNS, and may serve as a potential therapeutic reagent for the treatment of CNS autoimmune diseases. doi:10.1016/j.cyto.2009.07.426

PP2-049 Regulatory T cells demonstrate an injury-specific recall response Goro Tajima, Fionnuala O’Leary, Marc Hanschen, Kimiko Ikeda, Adam Delisle, Mohamed Oukka, Vijay Kuchroo, James Lederer, Poster Presentation II Regulatory T cells demonstrate an injury-specific recall response Goro Tajima 1, Fionnuala O’Leary 1, Marc Hanschen 1, Kimiko Ikeda 1, Adam Delisle 1, Mohamed Oukka 2, Vijay Kuchroo 2, James Lederer 1, 1 Department of Surgery, Brigham and Women’s Hospital/ Harvard Medical School, Boston, MA, USA, 2 Center for Neurologic Disease, Brigham and Women’s Hospital/ Harvard Medical School, Boston, MA, USA In a previous study, we demonstrated that burn injury caused rapid activation of FoxP3+ regulatory T cell (Treg) in injury-site lymph nodes in mice. Given this observation, we wished to test whether Tregs or conventional CD4 T cells might develop an injury-specific recall response. To accomplish this, we transferred CD4 T cells from sham or burn FoxP3-GFP knock-in mice into sham and burn congenic mice and tracked the expansion and activation of injury-experienced and inexperienced Tregs. FoxP3-GFP mice underwent sham or burn injury. One or 4 weeks later, CD4 T cells were purified from the lymph nodes and spleens and then transferred into CD45.1congenic mice. Recipient mice underwent sham or burn injury to represent a second injury response. After 7 days, transferred CD45.2 positive cells and FoxP3-GFP positive cells were detected by FACS to measure expansion and stained for cell-surface T cell activation and memory markers (CD62L, CD44, ICOS, and CTLA-4). When CD4 T cells from sham or burn FoxP3-GFP mice were transferred into naïve recipient sham or burn mice, we found that the combination of burn CD4 T cells into burn recipient mice caused significantly greater FoxP3+ Treg expansion and activation than other combinations. This recall response by Tregs was much higher in Tregs transferred 1 week after injury than Tregs from 4 weeks. Moreover, Tregs transferred at 1 week after the first injury showed a greater memory CD4 T cell phenotype (CD44high CD62Llow) than non-Tregs. The observation that injury-experienced Tregs respond more vigorously to the second injury suggests that Tregs develop an injury-specific recall response. Conventional CD4 T cells also showed a recall response to injury, but it was not evident until 4 weeks after injury. These findings are novel and support the concept that Tregs are injury-responsive and can develop an early, injury-specific memory-like response. doi:10.1016/j.cyto.2009.07.427