Large Amounts Of IL-10 Research Articles

Perinatal tissue-derived exosomes ameliorate colitis in mice by regulating the Foxp3 + Treg cells and gut microbiota

BackgroundThe capacity of self-renewal and multipotent differentiation makes mesenchymal stem cells (MSC) one of the most widely investigated cell lines in preclinical studies as cell-based therapies. However, the low survival rate and poor homing efficiency of MSCs after transplantation hinder the therapeutic application. Exosomes derived from MSCs have shown promising therapeutic potential in many diseases. However, the heterogeneity of MSCs may lead to differences in the function of secreting exosomes. In this study, the therapeutic effects of hUC-Exos and hFP-Exos on the DSS-induced colitis mouse model were investigated.MethodsThe colitis mouse models were randomly divided into four groups: (1) DSS administered for 7 days and euthanasia (DSS7D), (2) DSS administered for 7 days and kept for another 7 days without any treatment (DSS14D), (3) DSS administered for 7 days and followed with hUC-EVs infusion for 7 days (hUC-EVs) and (4) DSS administered for 7 days and followed with hFP-EVs infusion for 7 days (hFP-EVs). We analyzed colon length, histopathology, Treg cells, cytokines and gut microbiota composition in each group.ResultsA large amount of IL-6, IL-17 and IFN-γ were produced along with the decrease in the number of CD4 + Foxp3 + and CD8 + Foxp3 + cells in DSS7D group, which indicated that Th17 cells were activated and Treg cells were suppressed. We found that the number of CD4 + Foxp3 + and CD8 + Foxp3 + cells increased in order to suppress inflammation, but the length of colon did not recover and the symotoms were worsened of the colonic tissue in DSS14D group. The subsequent infusion of either hUC-Exos or hFP-Exos mediated the transformation of Treg and Th17 cells in colitis mice to maintain immune balance. The infusion of hUC-Exos and hFP-Exos also both reduced the abundance of pro-inflammatory intestinal bacterial such as Verrucomicrobia and Akkermansia muciniphila to improve colitis.ConclusionsWe found that Foxp3 + Treg cells can inhibit the inflammatory response, and the over-activated Treg cells can still further damage the intestinal mucosa. hUC-Exos and hFP-Exos can control inflammation by regulating the balance between Th17 cells and Treg cells. Decreased inflammatory response improved the structure of colon wall in mice and reduced the abundance of pro-inflammatory bacteria in the intestine. The improvement of intestinal wall structure provides conditions for the reproduction of beneficial bacteria, which further contributes to the reduction of colitis.

Combinations of anti-GITR antibody and CD28 superagonist induce permanent allograft acceptance by generating type 1 regulatory T cells.

Type 1 regulatory T (Tr1) cells represent a subset of IL-10–producing CD4+Foxp3− T cells and play key roles in promoting transplant tolerance. However, no effective pharmacological approaches have been able to induce Tr1 cells in vivo. We herein report the combined use of a CD28 superagonist (D665) and anti–glucocorticoid-induced tumor necrosis factor receptor–related protein monoclonal antibody (G3c) to induce Tr1 cells in vivo. Large amounts of IL-10/interferon-γ–co-producing CD4+Foxp3− Tr1 cells were generated by D665-G3c sequential treatment in mice. Mechanistic studies suggested that D665-G3c induced Tr1 cells via transcription factors Prdm1 and Maf. G3c contributed to Tr1 cell generation via the activation of mitogen-activated protein kinase–signal transducer and activator of transcription 3 signaling. Tr1 cells suppressed dendritic cell maturation and T cell responses and mediated permanent allograft acceptance in fully major histocompatibility complex–mismatched mice in an IL-10–dependent manner. In vivo Tr1 cell induction is a promising strategy for achieving transplant tolerance.

Characterization of macrophages in the liver of total body irradiated mice

Abstract Two hepatic macrophage (MΦ) populations have been described in the inflamed liver: F4/80+CD11b+ cells and F4/80+CD68+ cells. In normal conditions, hepatic F4/80+CD11b+ cells are involved in host antibacterial resistance. However, these cells in total body irradiated mice (TBI mice) are unfunctional for the elimination of bacteria translocated from intestinal tracts. In this study, the properties of hepatic MΦ populations in TBI mice were characterized. Normal mice and mice 7 days after exposure to 4 Gy of X-rays were infected i.v. with 107 CFU/mouse of Enterococcus faecalis (E. faecalis). Three day after infection, F4/80+CD11b+ cells and F4/80+CD68+ cells isolated from the liver of these mice were cultured for 24 hrs, and culture fluids were assayed for IL-12, IL-10 and CCL1 by ELISA. NSG mice were inoculated i.v. with these MΦ preparations (2 × 105 cells/mouse), then infected i.v. with 5 × 104 CFU/mouse of E. faecalis. In the results, all TBI mice died within 2 weeks, while all normal mice survived. IL-12 was produced by F4/80+CD11b+ cells from normal mice, but not by both MΦ preparations from TBI mice. IL-10 was produced by F4/80+CD68+ cells from TBI mice (529.9 ± 272.6 pg/ml) and normal mice (445.8 ± 147.7 pg/ml). Large amounts of IL-10 (912.1 ± 103.7 pg/ml) was produced by F4/80+CD11b+ cells from TBI mice, and these cells produced CCL1 (327.5 ± 9.1 pg/ml), while other MΦ preparations did not. NSG mice inoculated with F4/80+CD11b+ cells from normal mice were resistant to infection; however, NSG mice inoculated with F4/80+CD11b+ cells from TBI mice did not. These results indicate hepatic F4/80+CD11b+ cells are polarized to M2bMΦ in response to total body irradiation, and these MΦ are involved in the decreased host antibacterial resistance in TBI mice.

Intermittent Exercise Triggers Synthesis of CYP19 Aromatase as a Key Enzym for Estrogen Formation In Sprague Dawley Rat Bone Innovarectomy

Menopause is a permanent cessation of menstrual cycle due to reduced secretion of the hormone estrogen which can result in osteoporosis. Osteoporosis is characterized by the process of bone resorption faster than the process of bone formation, resulting in a decrease in bone mineral density and bone microarchitecture damage resulting in bones becoming brittle and easily fracturing. Physical exercise is a holistic intervention to prevent osteoporosis due to menopause. This study is a pure experimental study using a post-test only control group design research design. The subjects of the study were the 12-week-old Sprague Dawley rat. The number of groups is 5 with the number of rats per 6 animals. There was a significant difference between the ovarectomy group of rats and the ovarectomy group of mice and were given intermittent exercise treatment of CYP19 aromatase expression. Discussion: Muscle contractions that occur due to intermittent exercise treatment can produce large amounts of IL-6 and IL-6 mRNA which can stimulate estrogen production. Local estrogen production can reduce bone resorption and increase bone formation and bone density Intermittent exercise can trigger the process of the synthesis of the CYP19 aromatase enzyme in ovarectomy rat femur bones

Influences of interaction between dengue virus type 2-infected human umbilical vein endothelial cells and macrophages on major inflammatory cytokines

Objective To study the influences on the production of major inflammatory cytokines after co-culturing macrophages with human umbilical vein endothelial cells (HUVECs) that were infected with dengue virus type 2 (DENV-2). Methods Density gradient centrifugation was used to isolate peripheral blood mononuclear cells (PBMC) from concentrated human leukocytes. Adherent monocytes in culture flasks were obtained and stimulated with macrophage colony-stimulating factor (M-CSF) to prepare macrophages. The purity of CD14+ CD11b+ cells was measured by flow cytometry. Changes in the expression of NS1 at mRNA level in HUVECs were detected by real-time PCR following DENV-2 infection. DENV-2-infected HUVECs were co-culture with macrophages in Transwell chambers. A control group was set up by pretreating HUVECs with sphingosine-1-phosphate (S1P) type 1 (S1P1)-specific receptor agonist CYM-5442 for 24 h to remove the drug before infection and then co-culturing the infected cells with macrophages. Real-time PCR was used to detect the expression at mRNA level of IL-6 and IL-8 in HUVECs and IL-6, IL-8, TNF-α and IL-1β in macrophages. A double-antibody sandwich ELISA was used to detect the expression of above cytokines in culture supernatants. Results After HUVECs were infected with DENV-2, expression of NS1 gene at mRNA level gradually increased to the peak at 24 h (2.66±0.53, P<0.05) and then decreased. The purity of macrophages detected by flow cytometry was (89.16±2.07) %. Expression of IL-6 and IL-8 at mRNA level in DENV-2-infected HUVECs was up-regulated. The peak values reached at 24 h of IL-6 and IL-8 expression were 16.10±0.17 and 29.76±0.58, while the expression levels at 24 h in the uninfected group were 1.46±0.67 and 1.60±0.54, respectively. Expression of IL-6, IL-8, TNF-α and IL-1β at mRNA level in DENV-2-infected macrophages was increased significantly. The levels of IL-6, IL-8, TNF-α and IL-1β expression at 24 h were 45.82±3.72, 52.34±1.69 (12 h), 8.94±1.75 and 30.96±1.44 in the infected macrophages, and 1.16±0.22, 1.15±0.21, 1.11±0.09 and 1.47±0.31 in the uninfected group. Expression of these cytokines was decreased at every time points after co-culturing of DENV-2-infected HUVECs with macrophages, but still significantly higher than that in the uninfected group. In the co-culture group with DENV-2 infection, CYM-5442 pretreatment significantly decreased the expression at mRNA level of IL-6 and IL-8 in HUVECs (P<0.01) and that of IL-6, IL-8, TNF-α and IL-1β in macrophages (P<0.01). Conclusions DENV-2 could infect primary HUVECs, and then activate macrophages to promote the secretion of large amounts of IL-6, IL-8, TNF-α and IL-1β. Moreover, the activated macrophages could reduce the production of inflammatory cytokines in HUVECs to a certain extent. Key words: Dengue virus type 2 (DENV-2); Human umbilical vein endothelial cell (HUVEC); Macrophage; Cytokine; Immunoregulation

Abstract B035: Enhancing antitumoral activity of cell-based immunotherapies by modulating the JAK-STAT axis

Abstract Mounting a T helper 1 (Th1) type of response is required for the successful priming of antigen specific CD8 T-cells which ultimately lead to effective antitumoral responses. Several cellular components of the immune system participate of the Th1 decision-making process. They include innate cells sensing the transformed cells, mainly dendritic cells (DCs), and the further involvement of antigen specific CD4 T-cells promoting direct activation of CD8 T-cells through cross-presentation (1). The correct stimulation of DCs by type I interferons and molecular pattern sensors as STING is required to drive their differentiation to conventional DC 1 (cDC1) which through the secretion of large amounts of IL-12 and the expression of CD40 skew the CD4 T-cells differentiation to Th1 (2). However, this system is sensitive to modifications of the tumor cytokine microenvironmen,t leading to failure. Adoptive cell transfer (ACT) cancer immunotherapies are promising treatments for advanced malignancies. These therapies attempt to supply key cellular actors missing and induce proper antitumoral immunity. They include mainly the in vitro modification of autologous cells, from DCs loaded with tumor antigens to the expression of chimeric antigen receptor (CAR) on T and NK cells. Constant development of these technologies has given successful results as clinical trials have already showed up to 90 % of complete remission in relapsed/refractory B cell acute lymphoid leukemia (B-ALL) (3), treatment now approved for commercial use. However, there is still lack of consistency in the responses obtained from different individuals and in different trials. Previously our laboratory has already shown that modulation of the JAK-STAT inhibitory protein tyrosine phosphatases (PTPs), PTPN1 and PTP-N2, enhance proinflammatory type I interferon signalling while decreases the effects of immunosuppressive cytokines acting through STAT3 signaling (4, 5). Specifically, we have demonstrated that partial inhibition of these phosphatases in immune cells enhances the secretion of IL-12p70 by DCs[6] and increases the cytotoxic activity of antigen specific CD8 T-cells [Pérez-Quintero LA, Tremblay ML, unpublished results]. Moreover, the partial inhibition of these phosphatases in CD8 T-cells enhance the acquisition of a central memory (Tcm) phenotype, which has been found to be associated with better remission in CAR-T-cells therapies. Nevertheless, alternative methods to enrich this phenotype, as cytokine cocktails, have proven expensive and ineffective. Having this in mind, we propose here the use of small-molecule inhibitors specific for PTPN1 and PTPN2 as a simple and cost-effective method to enhance antitumoral responses. By treating ex vivo the cellular products in animal models for DCs and CD8 ACT therapies we show, as a proof of concept, a marked improvement on the reduction of tumor burden and remission, with the late goal of translating these findings into a clinical setup.

Abstract 127: Axl + Siglec6 + Dendritic Cells: the Role of Salt, Stretch, and Hypertension

We have shown that monocyte-derived dendritic cells (DCs) are activated in hypertension to produce large amounts of cytokines and to activate T cells. DCs from hypertensive mice can convey hypertension when adoptively transferred to recipients. Single cell sequencing has recently identified a novel subset of DCs in humans that express Axl and Sigelc6 + (AS DCs). These cells have been reported to potently drive T cell proliferation and to produce large amounts of IL-8 and IL12. The role of AS DCs in hypertension remains unknown. We isolated total peripheral blood mononuclear cells (PBMCs) from normotensive (n=23) and hypertensive (n=12) patients and assessed DC populations, including AS DCs, using flow cytometry. We found a significant increase in the AS DCs in hypertensive compared to normotensive patients (297 ± 73 vs. 108 ± 26/ml; P=0.0304). In contrast, there were no differences in CD1c + DCs (3398 ± 776 vs. 5245 ± 122/ml) or CD141 + DCs (164 ± 20 vs. 218 ± 49/ml) between normotensive and hypertensive subjects. To investigate the mechanism by which AS DCs are formed in hypertension, we used two in vitro hypertensive stimuli: exposure to salt and hypertensive stretch of adjacent human endothelial cells. Human PBMCs were cultured in either normal NaCl (NS, 150 mM) or high NaCl (HS, 190 mM) for 48 hours. Flow cytometry indicated a striking increase in AS DCs by exposure to HS compared to NS (516 ± 181 vs 201 ± 57/ml) and this was prevented by co-treatment of cells with the salt-sensing Serum Glucocorticoid Kinase 1 inhibitor GSK650394. As a second approach, we co-cultured human aortic endothelial cells (HAECs) with PBMCs from normotensive donors and exposed the HAEC monolayer to either normal (5%) or hypertensive cyclical stretch (10%) for 24 hours. Co-culture of PBMCs with HAECs exposed to 10% stretch doubled AS DCs as compared to PBMCs cultured with HAECs undergoing 5% stretch (1.4 ± 0.5 vs 0.7 ± 0.3%; P=0.0217). These data show that AS DC population are increased in hypertensive patients and that known hypertensive stimuli in vitro promote formation of AS DCs. Thus, AS DCs seem to be an important immune cell subset in human hypertension and might be a novel therapeutic target for this disease.

Inhibition of SIK2 and SIK3 during differentiation enhances the anti-inflammatory phenotype of macrophages

The salt-inducible kinases (SIKs) control a novel molecular switch regulating macrophage polarization. Pharmacological inhibition of the SIKs induces a macrophage phenotype characterized by the secretion of high levels of anti-inflammatory cytokines, including interleukin (IL)-10, and the secretion of very low levels of pro-inflammatory cytokines, such as tumour necrosis factor α. The SIKs, therefore, represent attractive new drug targets for the treatment of macrophage-driven diseases, but which of the three isoforms, SIK1, SIK2 or SIK3, would be appropriate to target remains unknown. To address this question, we developed knock-in (KI) mice for SIK1, SIK2 and SIK3, in which we introduced a mutation that renders the enzymes catalytically inactive. Characterization of primary macrophages from the single and double KI mice established that all three SIK isoforms, and in particular SIK2 and SIK3, contribute to macrophage polarization. Moreover, we discovered that inhibition of SIK2 and SIK3 during macrophage differentiation greatly enhanced the production of IL-10 compared with their inhibition in mature macrophages. Interestingly, macrophages differentiated in the presence of SIK inhibitors, MRT199665 and HG-9-91-01, still produced very large amounts of IL-10, but very low levels of pro-inflammatory cytokines, even after the SIKs had been reactivated by removal of the drugs. Our data highlight an integral role for SIK2 and SIK3 in innate immunity by preventing the differentiation of macrophages into a potent and stable anti-inflammatory phenotype.

Abstract B69: Activation of the IL6/STAT3 pathway in childhood ependymoma is associated with a pro-inflammatory tumor microenvironment and a poor prognosis

Abstract To aid development of immunotherapy for poor outcome Group A ependymoma (EPN) in children we sought to identify the molecular pathway underlying the adverse pro-inflammatory phenotype associated with this molecular subgroup. We have previously shown the importance of immunological factors in EPN outcome and that an anti-tumor immunophenotype was critical in survival whereas a pro-inflammatory phenotype was associated with a poor outcome. Moreover we showed a strong correlation between these immunophenotypes and the newly recognized molecular subgroups of posterior fossa EPN. We showed that Group A EPN have an adverse pro-inflammatory immunophenotype at presentation which adversely affects outcome even at relapse. We contrasted the gene expression profiles of patient tumor samples from Group A EPN to the more clinically favorable Group B EPN, as well as a broader panel of pediatric and adult brain tumors and normal brain, to identify potential molecular pathways that might be associated with the Group A immunophenotype. The results of these studies were validated using functional assays in short-term cultures of patient tumor samples and novel EPN cell lines. Strikingly we found a robust expression signature implicating the interleukin-6/signal transduced and activator of transcription 3 (IL6/STAT3) pathway in the Group A tumors. Comparison of transcriptomic profiles of EPN Group A (n=17) and Group B (n=20) demonstrated significant overexpression both IL6 (19.7-fold) and STAT3 (1.6 fold) in Group A. High overexpression of STAT3 driven genes IL8 (30-fold), SOCS3 (14-fold) and BIRC3 (8.5-fold) provided additional proof of IL6/STAT3 pathway activation in Group A. Furthermore, IL6 and IL8 gene expression was shown to be significantly higher (FDR&amp;lt;0.05) than any other type of pediatric or adult brain tumor or normal brain, suggesting that this phenotype is unique to Group A EPN. Western blot analysis of whole tumor samples from Groups A and B showed a significantly elevated ratio of pSTAT3 to total STAT3 in the Group A tumors compared to the Group B, denoting activation of STAT3. Measurement of IL6 secretion from primary ex-vivo tumor cells, isolated by flow sorting of disaggregated surgical tumor samples, showed a significantly higher secretion by tumors cells from Group A patients compared to Group B. To further investigate the role of the IL6/STAT3 pathway, we utilized a novel EPN cell line (811) established from a multiple recurrent EPN, with the transcriptome characteristics of a Group A EPN. This cell line secretes large amounts of IL6, considerably more than glioblastoma (GBM) cell line U87 in which the IL6/STAT3 pathway has been extensively studied. This cell line induced a contact-independent polarization of myeloid cells to a pro-inflammatory phenotype. HLA-matched donor CD14+ that we treated with 811 conditioned media showed a 9.2-fold increase in IL8 secretion (p=0.027). Demonstrating the mitogenic role of STAT3 activation in EPN, we showed 811 is more sensitive to STAT3 inhibition by S31-201 (IC50 = 81uM) than breast cancer and GBM cell lines. PhosphoSTAT3 and the downstream targets of the pathway, SOCS3 and Mcl-1 are significantly reduced in a dose dependent manner in response to S31-201. S3I-201 treatment of 811 and an EPN short-term cell line were shown to cause a significant dose-dependent increase in Caspase-3/7 cleavage demonstrating apoptosis as a result of STAT3 inhibition. To conclude: The IL6/STAT3 pathway is active in Group A EPN and is important in maintaining tumor growth in a pro-inflammatory microenvironment. Effective design of Group A-targeted immunotherapy for children with EPN may require alleviation of this potentially immunosuppressive pathway. Citation Format: Andrea Griesinger, Andrew M. Donson, Diane Birks, Vladimir Amani, Rebecca Josephson, Kathleen Dorris, Philip Reigan, Michael Handler, Nicholas Foreman. Activation of the IL6/STAT3 pathway in childhood ependymoma is associated with a pro-inflammatory tumor microenvironment and a poor prognosis. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B69.

Comparative analysis of virulence traits between a Legionella feeleii strain implicated in Pontiac fever and a strain that caused Legionnaires' disease

Legionella strains of the same species and serogroup are known to cause Legionnaires' disease (a potentially fatal atypical pneumonia) or Pontiac fever (a mild, flu-like disease), but the bacterial factors that define these dramatic differences in pathology have not been elucidated. To gain a better understanding of these factors, we compared the characteristics of Legionella feeleii strains that were isolated from either a sample of freshwater implicated in an outbreak of Pontiac fever (ATCC 35072, serogroup 1, LfPF), or a patient with Legionnaires' disease (ATCC 38549, serogroup 2, LfLD). Growth of LfPF and LfLD in BYE broth was slower than the positive control, Legionella pneumophila strain JR32. However, LfLD grew faster than LfPF at 42 °C. After in vitro infection to J774 murine or U937 human macrophage cell lines and A549 human lung epithelial cell line, LfLD showed a higher cell infection rate, stronger internalization by host cells, and greater cytotoxicity than that of LfPF. Large amounts of IL-6 and IL-8 were secreted by human host cells after infection with LfLD, but not with LfPF. LfLD possessed mono-polar flagellum while LfPF was unflagellated. When LfLD was cultured at 25, 30 and 37 °C, the bacteria had higher motility rate at lower temperatures. Based on our results, this is the first study that showed distinct characteristics between LfPF and LfLD, which may give important leads in elucidating differences in their virulence.

35. The endocannabinoid anandamide modulates adhesion, proliferation and the production of inflammatory mediators in rheumatoid arthritis synovial fibroblasts by activating CB1, TRPV1 and non-cannabinoid targets

In rheumatoid arthritis (RA), synovial fibroblasts (SF) secrete large amounts of IL-6, IL-8 and matrixmetalloproteinases which are crucial for cartilage destruction. RASFs are sensitive to the action of cannabinoids and they express cannabinoid receptors type I and II (CB1 and CB2), the vanilloid receptor (TRPV1) as well as endocannabinoid degrading enzymes. Cannabinoids are regarded as antiinflammatory and since anandamide is found in RA synovial fluid we investigated how this endocannabinoid affects adhesion, proliferation and the production of inflammatory mediators of RASF. Adhesion was assessed by the XCELLigence system. Proliferation was quantified by the amount of incorporated fluorescent dye into cellular DNA. MMP-3 and cytokines were detected by ELISA. Anandamide robustly decreased the production of IL-6, IL-8 and MMP-3 by a TRPV1-mediated mechanism. The inhibitory effect was enhanced by hypoxia. Anandamide increased adhesion of SFs in a CB1-dependent manner and decreased proliferation which was blocked by inhibiting cyclooxygenase-2. Anandamide promotes an antiinflammatory phenotype in RASFs by activating TRPV1 and CB1. Additionally, cyclooxygenase-2 metabolites of anandamide exert their anti-proliferative effects independent of CB1 and CB2.

169 : Direct in vitro generation of antigen specific, memory phenotype, Cd8+ T cells using a cocktail of small molecules and gamma-chain cytokines

Adoptive T cell transfer is an emerging cancer immunotherapy that has yielded complete responses in solid tumors such as B cell lymphoma and melanoma that are incurable by standard therapies. Prior studies have demonstrated that transfer of less differentiated T cells with greater proliferative capacity is associated with better clinical responses. Conventional expansion of antigen specific CD8 T cells uses high dose IL-2 and generates effector cells with high cytolytic and little proliferative capacity. We hypothesized that altering culture conditions by adding known cytokines and small molecules that inhibit acquisition of effector function and encourage memory formation would be associated with greater efficacy of transferred cells. We have used peptide antigen pulsed dendritic cells combined with a cocktail of homeostatic and memory-inducing cytokines that bind to the common gamma chain (IL-7 and IL-21) along with small molecules that inhibit glycolysis and enhance Wnt signaling to rapidly generate tumor specific, less-differentiated CD8 T cells with memory properties. Cells grown by this method are highly antigen specific (>90%) and are ∼50% central memory (CD62L+CD44+) or naive (CD62L+CD44−) phenotype versus virtually undetectable levels of these cells in cultures grown under standard conditions (i.e. with large amounts of IL-2). Compared to highly activated effector cells grown in IL-2, these memory-like cells engraft more efficiently into lymphopenic tumor bearing mice, maintain memory (CD62L+) phenotype for several weeks, and have greater anti-tumor efficacy in vivo. This approach is particularly useful since rapid advances in bioinformatics will soon make practical the identification of CD8 T cell epitopes derived from patient specific mutations. Since this method represents a way to generate more effective T cells that target tumor specific mutations, these results have significant implications for extending the usefulness of adoptive transfer to non-immunogenic (i.e. non-TIL bearing) solid tumors that could be targeted with personalized immunotherapy.

A Review on Biology and Function of Regulatory B Cells with Special Reference to Inflammation and Autoimmune Pathogenesis

Once considered to be predominantly a positive regulator of immune function, B cells are increasingly getting attributed with new and diversified functions. In recent years a number of reports indicate that a definite regulatory function of B cell exists. A number of B cell subsets that shares various developmental markers shown to have the ability to produce the suppressive cytokine IL-10. A particularly strong candidate with distinct IL-10 production profile is CD19hiCD1dhiCD5+ population, which constitutes about 1–2 % of splenic B cells. These B cells are also called B10 cells as they produce large amount of IL-10. Increasingly it is becoming clear that a variety of regulatory B cell population exists and expands during inflammatory condition and autoimmune disease. A detailed study of development and mechanism that governs B reg generation may provide us with valuable information for therapeutic intervention.

IL-10 release by bovine epithelial cells cultured with Trichomonas vaginalis and Tritrichomonas foetus

Trichomonas vaginalis and Tritrichomonas foetus are parasitic protists of the human and bovine urogenital tracts, respectively. Several studies have described the cytotoxic effects of trichomonads on urogenital tract epithelial cells. However, little is known about the host cell response against trichomonads. The aim of this study was to determine whether T. foetus and T. vaginalis stimulated the release of the cytokine interleukin (IL)-10 from cultured bovine epithelial cells. To characterise the inflammatory response induced by these parasites, primary cultures of bovine oviduct epithelial cells were exposed to either T. vaginalis or T. foetus. Within 12 h after parasite challenge, supernatants were collected and cytokine production was analysed. Large amounts of IL-10 were detected in the supernatants of cultures that had been stimulated with T. foetus. Interestingly, T. vaginalis induced only a small increase in the release of IL-10 upon exposure to the same bovine cells. Thus, the inflammatory response of the host cell is species-specific. Only T. foetus and not T. vaginalis induced the release of IL-10 by bovine oviduct epithelial cells.

The endocannabinoid anandamide modulates adhesion, proliferation and the production of inflammatory mediators in rheumatoid arthritis synovial fibroblasts

In rheumatoid arthritis (RA), synovial fibroblasts (SF) secrete large amounts of IL-6, IL-8 and several matrix metalloproteinases, which are crucial for cartilage destruction. RASF are sensitive to the action of cannabinoids and they express cannabinoid receptors type I and II (CB 1 and CB 2 ) as well as endocannabinoid degrading enzymes. Cannabinoids are regarded as antiinflammatory and since anandamide is found in RA synovial fluid we investigated how this endocannabinoid affects adhesion, proliferation and the production of inflammatory mediators of RASF. Adhesion was assessed by the XCELL igence system (Roche). Proliferation was quantified by the amount of incorporated fluorescent dye into cellular DNA. MMP-3 and cytokines were detected by ELISA. Cannabinoid receptors were visualized by immunofluorescence. Anandamide slightly decreased the production of IL-6, IL-8 and MMP-3 by a non-cannabinoid receptor mediated mechanism. Anandamide increased adhesion of SFs in a CB 1 -dependent manner and decreased proliferation, which was blocked by inhibiting cyclooxygenase-2. Furthermore, proliferation was negatively correlated with CB 1 expression. In addition, GPR18 and GPR55, two recently deorphanized cannabinoid receptors, which might serve as target for anandamide, were detected in SFs. Anandamide promotes an antiinflammatory phenotype in RASFs by activating several receptors. Additionally, cyclooxygenase-2 metabolites of anandamide exert their anti-proliferative effects independent of CB 1 and CB 2 . The identification of GPR18 and GPR55 in RASFs demonstrates novel sites of action for anandamide and its metabolites and provide attractive targets for future therapeutics to be applied to patients with RA.

P044 Identification of a novel role for interleukin-27 as mediator of intestinal epithelial barrier protection acting via differential STAT signalling and induction of antibacterial and anti-inflammatory proteins

However, an inflammatory response can be induced not only by PAMPs, but also intracellular alarm signals arising from non-physiological cell death, tissue damage, or stress, the so called DAMPs. Necrotic cell death releases several endogenous pro-inflammatory DAMPs, including proteins, DNA, RNA, extracellular matrix components and lipid mediators, all of which contribute to the pathogenesis of injury and exacerbate an initial insult. Moreover, sterile inflammation secondary to ongoing tissue damage is thought to contribute to many chronic diseases. We hypothesize that DAMPs may also play a role in the intestinal inflammatory response. Methods: Necrotic cell (NC) supernatants were prepared from HT-29 cells and freshly isolated human IEC, suspended at 108 cells/ml and subjected to 5 freeze/thaw cycles. Cells were then centrifuged and supernatants collected. Human Intestinal Fibroblasts (HIF) were used as targets of DAMPderived signals by exposing them to NC supernatants for 24 h. IL-6 and IL-8 levels in the cultures were measured by ELISA. Blockade with specific antibodies to TLR2, TLR4, RAGE, IL-1RI, IL-1a and IL-1b was performed by incubating these with HIF or NC supernatants. Results: HIF secreted large amounts of IL-6 and IL-8 (up to 120,000 pg/ml) in response to NC supernatants from HT29 cells or freshly isolated IEC. Neutralization studies revealed that a functional IL-1RI, but not TLR2, TLR4 or RAGE, was critical for IL-6 and IL-8 production by HIF. Both IL-1RI agonists, IL-1a and IL-1b, could trigger IL-6 and IL-8 production but only IL-1a, and not IL-1b, was obligatory for the induction of this effect with NC. Control experiments excluded a role of histones, chromatin, mitochondria, uric acid, HMGB1, IL-33, caspase-1 and ER stress in the response to necrotic IEC. Conclusions: These results show the unexpected, highly selective and potent pro-inflammatory activity of IEC-derived IL-1a, an intracellular non-secreted cytokine that belongs to the “alarmin” group of DAMPs. Thus, IL-1a emerges as a potential major inducer of mucosal inflammation in all gastrointestinal diseases where IEC death occurs, such as IBD.

IL-10-conditioned dendritic cells prevent autoimmune diabetes in NOD and humanized HLA-DQ8/RIP-B7.1 mice

This study was to determine whether BMDCs cultured in the presence of IL-10 (G/10-DCs) could promote T cell tolerance and prevent autoimmune diabetes in two different animal models of T1D. Our results showed that G/10-DCs suppressed both insulitis and spontaneous diabetes in NOD and HLA-DQ8/RIP-B7.1 mice. The suppression was likely to be mediated by T cells, as we found that regulatory CD4(+)CD25(+)Foxp3(+) cells were significantly increased in G/10-DC treated animals. In vivo, the G/10-DCs inhibited diabetogenic T cell proliferation; in vitro, they had reduced expression of costimulatory molecules and produced little IL-12/23 p40 or IL-6 but a large amount of IL-10 when compared with DCs matured in the presence of IL-4 (G/4-DC). We conclude that IL-10-treated DCs are tolerogenic and induce islet-directed immune tolerance, which was likely to be mediated by T regulatory cells. This non-antigen-specific DC-based approach offers potential for a new therapeutic intervention in T1D.

Susceptibility to Yersinia pseudotuberculosis Infection is Linked to the Pattern of Macrophage Activation

T helper 1 cells play a crucial role in the clearance of Yersinia pseudotuberculosis infection. By producing cytokines and presenting antigens to T cells, activated macrophages can orientate the adaptive immune response. The pathway used by macrophages to metabolize arginine has been employed as an important parameter to discriminate their activation state. In this study, the pattern of macrophage activation in Y. pseudotuberculosis-infected BALB/c (Yersinia-susceptible) and C57BL/6 (Yersinia-resistant) mice and their immunostimulatory capacity were analysed. In the early phase of infection, macrophages obtained from C57BL/6 mice produced higher levels of NO, lower arginase activity, and larger amounts of IL-12 and TNF-alpha than macrophages from BALB/c mice. On the other hand, macrophages derived from BALB/c mice produced higher levels of IL-10 and TGF-beta than C57BL/6 mice. The Y. pseudotuberculosis infection leads to a fall in the macrophage immunostimulatory capacity of both strains of mice, with T-cell proliferation significantly reduced 12 h after infection. Moreover, we observed in the supernatant of co-culture of macrophages from infected mice with T lymphocytes from heat-killed Yersinia-immunized mice lower IFN-gamma production by cells from BALB/c mice than by C57BL/6 mice, and IL-4 was produced only by BALB/c mice on the first- and third-day post-infection. These results suggest that the pattern of macrophage activation is associated with susceptibility and resistance to Y. pseudotuberculosis infection in BALB/c and C57BL/6 mice.

Induction of human CD4+ regulatory T cells by mycophenolic acid-treated dendritic cells

Depending on their degree of maturation, costimulatory molecule expression, and cytokine secretion, dendritic cells (DC) can induce immunity or tolerance. DC treated with mycophenolic acid during their maturation (MPA-DC) have a regulatory phenotype and may therefore provide a new approach to induce allograft tolerance. Purified CD4(+) T cells stimulated in a human in vitro model of mixed culture by allogeneic MPA-DC displayed much weaker proliferation than T cells activated by mature DC and were anergic. This hyporesponsiveness was alloantigen-specific. Interestingly, T cells stimulated by MPA-DC during long-term coculture in four 7-day cycles displayed potent, suppressive activity, as revealed by marked inhibition of the proliferation of naive and preactivated control T cells. These regulatory T cells (Tregs) appeared to have antigen specificity and were contact-dependent. Tregs induced by MPA-DC were CD25(+)glucocorticoid-induced TNFR(+)CTLA-4(+)CD95(+), secreted IL-5 and large amounts of IL-10 and TGF-beta, and displayed enhanced forkhead box p3 expression. These results obtained in vitro demonstrate that human MPA-DC can induce allospecific Tregs that may be exploited in cell therapy to induce allograft tolerance.

070 Reduction of IL-8 Secretion in Senescent Fibroblasts: Role in Chronic Wound Formation.

Fibroblasts in chronic wounds, and in skin in which ulcers develop, have been reported to show characteristics of senescence, presumably induced by conditions predisposing to chronic wounds or the wound environment. In monolayer culture, secretion of IL-8 declines to zero as the cells approach senescence (at about generations 50–55 in the cells tested). In three-dimensional scaffold-based tissue culture, fibroblasts, at about 30–35 population doublings, upregulate the IL-8 gene by comparison with monolayer cultures and secrete large amounts of IL-8 (30–50 ng/106 cells/day). However, it is inferred that a decline in secretion, similar to that seen in monolayer also occurs in three-dimensional culture and in the senescent fibroblasts of chronic wounds. This will result in decreased influx and activation of neutrophils. The hypothesis is proposed that chronic wounds arise because fibroblasts, following injury to ulcer-prone skin, do not recruit and activate neutrophils sufficiently to prevent bacterial colonization. As the result of the persistent presence of live bacteria, inflammatory cells or keratinocytes in a wound site may be expected to trigger mechanisms to inhibit keratinocyte migration and reepithelialization, leading to chronic ulceration.