IFN-gamma Expression Research Articles

Maternal Antibody Improves Active Neonatal Vaccination against Respiratory Syncytial Virus through Immune Complex

Respiratory syncytial virus (RSV) is a common infectious pathogen in infants. Vaccination is an important approach in the prevention of RSV infection in early age, but early vaccination is limited by low systemic and cellular immune responses. To overcome this difficulty, we in this study explored a novel vaccination strategy in a mouse model. Pregnant mother was vaccinated at day 14 of gestation and offspring were actively vaccinated at day 3 after birth. We observed a high level of maternal anti-RSV antibody in offspring born to the RSV immunized mother, but it declined 6 weeks after birth. However, anti-RSV antibody was low in offspring received active immunization alone. After offspring were nasal challenged with 2.8 × 105 i.u. RSV at age of 6 weeks old, we observed significant low levels of lung RSV load and inflammation in offspring received both passive and active vaccination as compared to the offspring received passive or active vaccination alone. The increased protection was associated with the increased endogenous production of anti-RSV antibody and Th1-biased cytokine IFN-gamma. Further in vitro study confirmed that maternal anti-RSV antibody and RSV antigen formed immune complex. The splenocytes stimulated with RSV immune complex expressed higher expression of IFN-gamma than those stimulated with RSV alone. Thus, maternal anti-RSV antibody increased efficacy of active vaccination in neonates as early as 3 days after birth and provided more protection against RSV than passive or active vaccination alone. Formation of RSV immune complex in respiratory mucusal tissues may contribute to the improved Th1-biased immune responses.

IL-17 promoted the inhibition of medulloblastoma in mice by splenocyte injection.

BackgroundInterleukin 17 (IL-17) is a proinflammatory cytokine produced by a new subset of activated CD4+ T cells, Th17 cells. We previously showed that increased Th17 cell populations were presented in human medulloblastoma-infiltrating T cells and peripheral blood. In this study, we attempted to address the possible role of Th17 cells in the biologic activity of IL-17 for tumor control.MethodsWe grafted fresh surgically obtained medulloblastoma into syngeneic athymic nude/nude mice. We intrapertonially injected splenocyte and murine IL-17 in mice on the second day. The tumor volume and the life spans of the mice were measured. Meanwhile, the IL-17, IL-6, IL-23, Ccl2, Ccl20 and IFN-gamma expression in the tumors was also examined by real-time PCR, Western blot and enzyme-linked immunosorbent assay.ResultsWe found that medulloblastoma growth in IL-17-injected mice was significantly inhibited compared to the non-IL-17 treated mice. In contrast to the IL-17 antitumor activity observed in mice injected with splenocytes, we observed that IFN-gamma, IL-6, IL-23, Ccl2, and Ccl20 proteins were significantly increased in tumor tissues of mice injected with IL-17.ConclusionsThese experiments suggest that IL-17 may promote splenocyte antitumor activity in medulloblastoma. We postulate that IL-17’s antitumor activity may be related to the increased protein levels of IFN-gamma, IL-6, IL-23, Ccl2, and Ccl20.

Colonization pattern of C. jejuni isolates of human and avian origin and differences in the induction of immune responses in chicken

Campylobacter jejuni (C. jejuni) is the most frequently reported bacterial food-borne pathogen. Poultry is regarded to be the main reservoir for human campylobacteriosis. By some authors C. jejuni is considered as a commensal of the chicken gut, but clinical signs may be observed indicating host–pathogen interaction. Little is known about C. jejuni strain dependent differences in stimulation of the immune response in chicken. Therefore we investigated the colonization pattern as well as humoral and cell-mediated immune parameters in three-week old specific pathogen-free (SPF) layer-type (LT) chicken after inoculation with different human and avian derived C. jejuni isolates between three and 21dpi. In a subsequent experiment we investigated earlier time points and additionally compared commercial broilers with SPF-LT chicken to identify possible differences after inoculation of selected C. jejuni strains from prior experiments. No clinical symptoms were observed during the experiments. The colonization pattern did not correlate with the strain origin or magnitude of circulating C. jejuni specific antibodies. Only minor changes were observed in caecal T cell populations after C. jejuni inoculation compared to non-inoculated controls. Interestingly the mRNA expression of IL-6 and IFN-gamma was down regulated at some time points after inoculation suggesting a possible immunomodulatory effect of some C. jejuni strains in the gut.Furthermore, broilers were colonized to a higher extend and the local immune cell response was different compared to the SPF –LT birds indicating an influence of genotype on C. jejuni colonization pattern.Overall, our study demonstrates that the outcome of C. jejuni infection in chicken is influenced by genotype and partially by the C. jejuni isolate, leading to differences in the early immune response and thereafter the control of colonization and infection.

Host Innate Immune Responses of Ducks Infected with Newcastle Disease Viruses of Different Pathogenicities.

Though previous studies have identified two strains of duck-origin Newcastle disease virus (NDV) with varying levels of pathogenicity, the relationship between the early-phase host innate immune response, and pathogenesis of ducks infected with these strains in the lungs and thymuses remains unclear. In this study, we compared the viral distribution and mRNA expression of immune-related genes in ducks following infection with two NDV strains, Duck/CH/GD/SS/10 (SS-10) and Duck/CH/GD/NH/10 (NH-10). Both NDV strains replicated systemically in tested tissues (i.e., small intestine, cecal tonsils, brain, lung, bursa of Fabricius, thymus, and spleen) and exhibited different biological properties in duck pathogenicity. Real-time quantitative polymerase chain reaction showed that the expression of TLR3, TLR7, RIG-I, MDA5, IL-1β, IL-2, IL-6, IL-8, IFN-alpha, IFN-beta, IFN-gamma in the lungs was significantly greater than in the respective thymus genes during the early post infection stage. However, in the lungs, the expression of TLR3, TLR7, IL-1β, IL-2, IL-8, IFN-alpha, IFN-gamma, and MHC II induced by SS-10 at 72 h post-inoculation (hpi) was less than with NH-10. Furthermore, the expression of IL-6 and IFN-beta in the lungs and thymuses following infection with SS-10 was greater than that with NH-10 at 24 and 48 hpi. These results highlight important differences in host innate immune responses, courses of infection, and pathogenesis following NDV infection. Further studies should work to expand understandings of the molecular mechanisms related to NDV infection.

The changes of serum IFN-gamma, TNF alpha and IL-10 levels in ulcerative colitis and its relevance to helicobacter pylori infection

Objective To study the changes of serum IFN-gamma, TNF alpha and IL-10 levels in ulcera-tive colitis and its relevance to helicobacter pylori infection. Methods 80 UC patients were selected as the research object, and 50 cases of 80 patients were in the activity stage of UC,30 patients were in remission. In the 50 patients with active UC：14 cases were mild,26 cases were moderate, and 10 cases were severe.30 healthy subjects were set as healthy controls. Detection of serum IFN-gamma,TNF alpha, IL-10 levels and Hp infection were made. Results The serum TNF alpha and IL-10 levels of the activity group were higher than those of the remission group and the control group, the difference was statistically significant［TNF alpha:(54.75±5.92)ng／L vs.(23.69±4.15)ng／L vs.(20.45±4.01)ng／L,F＝36.79,P＝0.000;IL-10：（35.16±6.72)ng／L vs.(25.12±5.37)ng／L vs.(23.96± 5.21)ng／L,F＝35.68,P＝0.000］，the serum IFN-gamma level of the activity group was lower than those of the remission group and the control group, the difference were statistically significant［（14.98±3.15)ng／L vs.(26.06± 4.31)ng／L vs.(27.16±4.39)ng／L,F＝35.68,P＝0.000］. As the aggravating of patients with UC in activity stage, TNF alpha and IL-10 levels showed a trend of increase(F＝32.81,24.95,all P＜0.05), while IFN-gamma showed a trend of reduce(F＝22.29,P＜0.05).In 50 cases of active UC patients, 18 patients with Hp positive, 32 cases were Hp negative. In active UC group Hp positive patients'serum TNF alpha and IL-10 levels higher than the Hp negative patients, the difference was statistically significant(t＝4.62,4.35,all P＜0.01). Serum levels of IFN-gamma of active UC patients with Hp positive group was lower than the Hp negative patients, the difference was statistically significant(t＝5.91,P＜0.01). Conclusion In active UC patients expression of TNF alpha and IL-10 are high, and IFN-gamma expression is low. The expression of IFN-gamma, TNF alpha and IL-10 are closely related to Hp infection. Key words: Colitis，Ulcerative; Interferon gamma; Tumor necrosis factor alpha; Interleukin 10; Helicobacter pylori

ID: 62: Deletion of the IFN-gamma 3′ UTR AU-rich element results in autoimmune cholangitis in female C57/BL6 mice

Primary biliary cirrhosis (PBC, now called autoimmune cholangitis) is an autoimmune liver disease and occurs primarily in women ( > 90%), eventually leads to liver failure. IFN-gamma is elevated in patients with PBC, but the functional role of IFN-gamma on PBC is not known. Here, we characterized the distinctive pathological phenotype of PBC in a mouse model of chronic IFN-gamma expression generated by deletion of the IFN-gamma 3′ UTR AU-rich element (ARE-Del). Consistent with clinical features seen in PBC, female ARE-Del mice have moderate immune cell infiltration and bile duct destruction near hepatic portal tracts with dramatically upregulated bile acids and spontaneous production of anti-mitochondria antibodies in serum. In contrast, male ARE-Del have relatively mild histological and serological evidences of PBC compared to female ARE-Del. By focusing on genes whose expression is highly specific to female ARE-Del, type I interferon receptor and dendritic maturation are top upstream pathways for this female specific disease progression. Remarkably, deletion of the interferon a/b receptor in ARE-Del mice significantly suppressed the female-specific pathological phenotypes, specifically eliminating the sex-different phenotypes in PBC. Furthermore, female ARE-Del have notably increased plasmacytoid dendritic cells (pDC), specialized in massive secretion of type I interferon (IFN alpha and beta), suggesting that increased type I interferon via pDC is critical for the sex-difference in this disease progression. Taken together, the ARE-Del is the first mice model to present the sex differences seen in PBC and startlingly demonstrates how type II interferon coordinates with type I interferon in this disease progression.

Effects of chronic social defeat on splenic chemokine expression and myeloid cell infiltration into the brain

Dysregulation of the innate immune response has frequently been described in stress-associated psychiatric disorders such as major depressive disorder (MDD). However the kinetics and cellular origin of cytokine and chemokine expression are not well understood. Aim of this study was to elucidate stress-induced changes in the immune response. After 2, 6, or 10 daily social defeats, animals were tested for social interaction behavior, expression levels of immune-related genes were assessed in brain and spleen tissues, and splenocytes and mononuclear cells isolated from the brain were analyzed by flow cytometry. Mice that developed social avoidance behavior showed a decreased expression of genes encoding C-C chemokines accompanied by a reduction in the percentages of activated T lymphocytes and reduced IFN-gamma expression levels in the spleens of defeated mice. Also, diminished percentages of CD4+FoxP3+ regulatory T cells and lower TGF-beta mRNA levels accumulated in the spleens of these animals. In contrast, enhanced percentages of CD45hiCD11b+ cells representing peripheral myeloid cells immigrated into the brain of defeated animals. In summary, stress induced social avoidance was accompanied by an altered expression of CC chemokine genes and reduced T cell activation in the spleen, associated with an increased brain invasion of myeloid cells. Further studies will address the underlying molecular mechanisms, the cell types involved herein and in a translational approach the immune cell phenotype in patients with MDD.

PrimeFlow™ RNA assay as a method to elucidate the effect of protein transport inhibitors on cytokine gene expression in whole blood (TECH3P.941)

Abstract Intracellular antibody staining followed by flow cytometric analysis is commonly used to study cytokine expression in leukocytes. Detection of cytokines typically requires that cells be treated with protein transport inhibitors such as Brefeldin A and Monensin in order to accumulate sufficient levels of intracellular protein. However, it is not known whether the use of these inhibitors affects gene expression. PrimeFlow™ RNA is a new flow cytometric assay that allows the simultaneous interrogation of RNA and protein levels within single cells. We have used this assay to investigate the effect of protein transport inhibitors on the expression of human IL-2, TNF alpha and IFN gamma during the course of a 5 hour stimulation of whole blood with PMA and ionomycin. This represents first time that the effect of protein transport inhibitors on gene expression in whole blood has been examined in single cells.

Galectin-1 plays an essential role in intravenous tolerance in experimental autoimmune encephalomyelitis (BA4P.139)

Abstract Multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), are inflammatory demyelinating diseases of the central nervous system (CNS). EAE can be induced by immunizing mice with myelin oligodendrocyte glycoprotein-derived peptide, MOG35-55, in adjuvant. It has been previously demonstrated that intravenous (i.v.) injection of MOG35-55 post immunization induction suppresses disease development, a phenomenon called i.v. tolerance. The glycan-binding protein galectin-1, produced primarily by regulatory T cells plays an immunoregulatory role in EAE by inducing tolerogenic DCs and IL-10-producing regulatory type 1 T cells (Tr1). To examine the role of galectin-1 in i.v. tolerance, we administered MOG35-55 peptide i.v. to wild-type (WT) and galectin-1-/- (Gal1-/-) mice with ongoing EAE. MOG35-55 rapidly suppressed disease in the WT, but not in the Gal1-/- mice. Expression of pro-inflammatory cytokines IFN-gamma and IL-17A were reduced in the CNS and periphery of tolerized WT mice, while the number of Tr1 cells increased in the periphery. In contrast, i.v. MOG35-55 significantly increased IFN-gamma and IL-17A in the CNS and periphery of Gal1-/-mice and these mice had markedly fewer Tr1 cells in the periphery. Our data suggest that galectin-1 mediated i.v. tolerance induction is dependent on the downregulation of Th1 and Th17 pro-inflammatory responses, and enhanced development of Tr1 cells.

Atopic dermatitis and alpha-chemokines.

Many studies have shown the involvement of interferon (IFN)γ-inducible protein 10 (IP-10) and T-helper 1 (Th1) cytokines in Atopic Dermatitis (AD). IFN-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-18 are potent stimulators of the expression and secretion of IP-10 in cultured keratinocytes from AD patients. Apoptosis of keratinocytes, induced mainly by T cells and mediated by IFN-γ, is the essential pathogenetic event in eczema formation. Enhanced IP-10, induced by IFN-γ, and IFN-inducible T cell alpha-chemoattractant (I-TAC) expression has been observed in lesional AD skin. It has been shown that keratinocytes undergoing apoptosis in acute eczematous lesions release chemokines that attract more T cells toward the epidermis, which may further augment the inflammation and keratinocyte apoptosis. Drugs used in the treatment of AD modulate IP-10. Antihistamines are widely used for the treatment of AD; it has been shown in human monocyte-derived dendritic cells (MoDCs) and autologous CD4+ T cells that antihistamines inhibited the production of IP-10 and�� monokine induced by IFN-gamma (MIG) expressions. It has been also shown that antimycotics and tacrolimus suppress the induced production of IP-10 in human keratinocytes.

Interferon-gamma expression and infectivity of Toxoplasma infected tissues in experimentally infected sheep in comparison with pigs

Livestock animals are a potential risk for transmission of toxoplasmosis to humans. Sheep and pigs still remain an important source because their meat is often eaten undercooked which has been regarded as a major route of infection in many countries. Moreover, porcine tissues are processed in many food products.In the current study, the IFN-gamma (T-helper 1 cells), IL-4 (Th2 cells) and IL-10 mRNA (Treg cells) expression by blood mononuclear cells, and the serum antibody response against Toxoplasma gondii total lysate antigen, recombinant T. gondii GRA1, rGRA7, rMIC3 and rEC2, a chimeric antigen composed of MIC2, MIC3 and SAG1, was studied in sheep the first two months after a T. gondii infection and compared with these responses in pigs. At the end of this period, the parasite distribution in heart, brain and two skeletal muscles in sheep was compared with this in pigs.Whereas the parasite distribution was similar in sheep and pigs, the antibody response differed considerably. In sheep, antibodies appeared against all tested T. gondii antigens, but mainly against rGRA7, rMIC3234307 and TLA whereas in pigs only rGRA7-specific antibodies could be demonstrated. Also, the cytokine response differed. Both in sheep and pigs an IFN-gamma response occurred which seemed to be a slightly more pronounced in sheep. In sheep, also IL-10 and IL-4 mRNA expression showed an increase, but later than IFN-gamma and with more variation. However, in pigs no such increase was seen.As concerning diagnosis, results indicate that serum antibodies against GRA7 in live sheep and pigs and heart tissue for bioassay and qPCR in slaughtered animals are the best targets to demonstrate presence of T. gondii infection.

P-199 Lipocalin2 Affecting Bacterial Clearance in Activated Macrophages Contributes the Onset of Intestinal Inflammation

Lipocalin2 (Lcn2) is an extracellular transport protein expressed in neutrophil granules, activated macrophages, and epithelial cells of the respiratory and gastrointestinal tract in response to inflammatory signals. It is well known that Lcn2 exerts bacteriostatic effect by sequestrating iron-containing siderophores because iron is essential for the growth and virulence of most bacterial species. In this regards, Lcn2 is considered to be involved in innate immune response. However, little is known about the exact role of Lcn2 in the intestinal inflammation. The aim of this study is to investigate the role of Lcn2 in the intestinal inflammation using IL-10 knock out (KO) mice. (1) Time-dependent expression of Lcn2 in the colonic tissues of IL-10 KO mice was compared with that of C57BL/6 wild type (WT) mice by quantitative PCR (qPCR) and immunohistochemistry (IHC). (2) We compared intestinal inflammation between IL-10 KO and Lcn2/IL-10 double knock out (DKO) mice. Inflammation was quantified by histologic scores, TNF-alpha and IL-12p40 secretion from cultured colonic explants, and IFN-gamma and IL-17A secretion from unseparated mesenteric lymph node (MLN) cells stimulated with cecal bacterial lysate. (3) Thioglycollate-elicited peritoneal macrophages were obtained from IL-10 KO (IL-10 KO-derived TEPM) and Lcn2/IL-10 DKO mice (DKO-derived TEPM), and infected with Escherichia coli. We examined the bacteria-killing function of macrophages by gentamicin protection assay. Also, TNF-alpha secretion from infected macrophages was determined by enzyme-linked immune-sorbent assay (ELISA). (1) qPCR and IHC demonstrated that Lcn2 expression in the colonic tissues of IL-10 KO mice was apparently up-regulated compared to that of WT mice from early 4 weeks after birth, and further increased over time. (2) Histologic scores in DKO mice were significantly higher than those in IL-10 KO mice at 4 weeks after birth (DKO versus IL-10 KO mice, 15.0 ± 0.0 versus 0.6 ± 0.5). Gene expressions of IL-1beta, TNF-alpha, IFN-gamma, and IL-17A in the colonic tissues of DKO mice were significantly up-regulated compared to those in IL-10 KO mice. TNF-alpha and IL-12p40 secretion from colonic explants of DKO mice were significantly higher than those of IL-10 KO mice (DKO versus IL-10 KO mice, TNF-alpha; 6.5 ± 0.7 versus 3.6 ± 1.2 ng/mL, IL-12p40; 30.6 ± 9.2 versus 5.3 ± 1.9 ng/mL). IFN-gamma and IL-17A production from MLN cells of DKO mice were higher than those of IL-10 KO mice (DKO versus IL-10 KO mice, IFN-gamma; 28.6 ± 33.7 versus 21.3 ± 32.7, P = 0.29, IL-17A; 2.5 ± 3.8 versus 0.6 ± 0.8 ng/mL, P < 0.05). (3) Uptake of bacteria was significantly enhanced in DKO-derived TEPM compared to that in IL-10 KO-derived TEPM (colony forming unit (CFU) at 2 hours after infection; DKO versus IL-10 KO-derived TEPM, 1461.7 ± 201.0 versus 868.3 ± 300.0 cfu/mL). In addition, intracellular survival of phagocytosed bacteria in DKO-derived TEPM persisted compared to that in IL-10 KO-derived TEPM (CFU at 8 hours after infection; DKO versus IL-10 KO-derived TEPM, 30.0 ± 17.9 versus 8.3 ± 13.3 cfu/mL, P < 0.05). Consequently, TNF-alpha production from DKO-derived TEPM significantly increased compared to IL-10 KO-derived TEPM (DKO versus IL-10 KO-derived TEPM, 14.6 ± 1.2 versus 4.0 ± 0.9 pg/mL at 16 hours after infection). Lcn2 affecting bacterial clearance in activated macrophages contributes to the onset of intestinal inflammation.

Abstract 4084: NKG2D receptor activation of mTOR and NFκB enhances antitumor immunity in effector CD8+ T cells

Abstract To induce strong anti-tumor immune responses, naïve CD8+ T cells require stimulation through the TCR and costimulatory receptors. Each costimulatory molecule can have a unique effect on the function of T cells due to differential activation of downstream signaling pathways and gene expression. However, the effect of stimulating costimulatory receptors on activated T cells is still unclear. One costimulation receptor that is likely to be engaged in the tumor microenvironment is NKG2D since the ligands for this receptor are expressed on over 80% of tumors. In order to determine how activation of costimulatory receptors in the tumor microenvironment affects CD8+ T cell functions, this study investigated the differential activation of signaling pathways by two costimulatory receptors, CD28 and NKG2D. Specifically, this study focused on the activation of mTORC1 and mTORC2 along with NFκB, all of which have been shown to have important roles in anti-tumor functions of CD8+ T cells. Stimulation through the CD3 and NKG2D receptors differentially activated the mTOR and NFκB pathways compared to the other receptors in our study. Activation of CD8+ T cells through CD3 and NKG2D lead to increased phosphorylation of signal transduction pathway members upstream of mTOR, including AKT, p70 S6K, and GSK3β. Also, expression of genes upstream and downstream of mTORC1 and mTORC2 were differentially activated after stimulation through NKG2D, indicating that activation of mTOR pathways are altered after CD3/NKG2D stimulation as compared to stimulation through CD3 or CD3/CD28. In addition, IKKα, IκBα, and NFκB phosphorylation and IκBα degradation was increased after CD3/NKG2D stimulation. This increased activation of the NFκB pathway also lead to increased gene expression and secretion of pro-inflammatory cytokines, including IFN-alpha and IFN-gamma, and decreased gene expression and secretion of anti-inflammatory cytokines, including IL-10 and VEGF-alpha. Together, these data show that stimulation through NKG2D leads to the differential activation of signaling pathways and alters the anti-tumor functions of effector CD8+ T cells. Citation Format: Emily Whitman, Amorette E. Barber. NKG2D receptor activation of mTOR and NFκB enhances antitumor immunity in effector CD8+ T cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4084. doi:10.1158/1538-7445.AM2014-4084

In ovo carbohydrate supplementation modulates growth and immunity-related genes in broiler chickens.

A study was undertaken to investigate the role of in ovo administrated carbohydrates on the expression pattern of growth and immune-related genes. In ovo injections (n = 400) were carried out on the 14th day of incubation into the yolk sac/amnion of the broiler chicken embryos. Expression of growth-related genes: chicken growth hormone (cGH), insulin-like growth factor-I & II (IGF-I & II) and mucin were studied in hepatic and jejunum tissues of late-term embryo and early post-hatch chicks. Expression of candidate immune genes: Interleukin-2, 6, 10 and 12 (IL-2, IL-6, IL-10 and IL-12), Tumour necrosis factor-alpha (TNF-α) and Interferon gamma (IFN-γ) were studied in peripheral blood monocyte cells of in ovo-injected and control birds following antigenic stimulation with sheep RBC (SRBC) or mitogen concanavalin A (Con-A). Glucose injection significantly increased the expression of IGF-II gene during embryonic period and both cGH and IGF-II in early post-hatch period, while ribose-injected chicks had higher expression of IGF-II gene during embryonic stage. Enhanced mucin gene expression was also observed in fructose-injected chicks during embryonic age. Glucose-injected chicks had higher expression of IL-6 or IL-10, while those injected with fructose or ribose had higher expression of IL-2, IL-12 and IFN gamma. It is concluded that in ovo supplementation of carbohydrates might help in improving the growth of late-term embryos and chicks. In ovo glucose could modulate humoral-related immunity, while fructose or ribose might help in improving the cellular immunity in broiler chickens.

The transcription factor CREMalpha regulates inflammatory T cell subsets in juvenile idiopathic arthritis (BA13P.115)

Abstract The cAMP response element (CRE) modulator (CREM)α binds to genes promoters with CREs and regulates transcription via a chromatin-dependent mechanism. CREMα is important for the T cell pathophysiology of SLE suppressing IL-2 and CD3ζ transcription, but enhances IL-17. Juvenile idiopathic arthritis (JIA) is an autoimmune disease of unknown origin. Th17 cells have a pathogenic role in arthritis and are not controlled by local FoxP3+ regulatory T cells (Tregs). Pathogenic T cells in the inflamed joints of JIA patients have enhanced expression of IL-17, IFNgamma and CD161. CD161+ CD4+ cells also contain FoxP3+ cells that produce proinflammatory cytokines. We observed enhanced CREM expression in JIA patient synovial fluid (SF) T cells and also after culture of healthy donor PBMCs with JIA SF. We furthermore found enhanced expression of CREM in CD4+CD161+ and in CD4+FoxP3+CD161+ cells, which produce inflammatory cytokines. Vice versa Helios+FoxP3+ cells, which are characteristically stable Tregs and do not express inflammatory cytokines, had lower levels of CREM. JIA SF induced IFNgamma, IL-17 and FoxP3 in T cells during incubation, which could be abrogated by CREM siRNA transfection. Within the FoxP3+ population, CREM siRNA reduced CD161+FoxP3+ cells but not Helios+FoxP3+ cells. We thus suggest CREMα overexpression in T cells contributes to T cell pathophysiology in JIA by regulating percentages of inflammatory CD4+ IL-17+ cells, as well as inflammatory CD161+FoxP3+ cells.

Evaluation of tetravalent and conserved synthetic peptides vaccines derived from Dengue virus Envelope domain I and II

Dengue is a major worldwide public health problem, especially in the tropical and subtropical regions of the world. Primary infection with a single Dengue virus (DENV) serotype causes a mild, self-limiting febrile illness called dengue fever. However, a subset of patients experiencing a secondary infection with a different serotype progress to the severe form of the disease, called dengue hemorrhagic fever. In this study, the vaccine potential of three tetravalent and conserved synthetic peptides derived from DENV envelope domain I (named Pep01) and II (named Pep02 and Pep03) was evaluated. Human dengue IgM/IgG positive serum (n=16) showed reactivity against Pep01, Pep02 and Pep03 in different degrees. Mice immunization experiments showed that these peptides were able to induce a humoral response characterized by antibodies with low neutralizing activity. The spleen cells derived from mice immunized with the peptides showed a significant cytotoxic activity (only for Pep02 and Pep03), a high expression of IL-10 (P<0.01) and a reduced expression of TNF-α and IFN-gamma (P<0.001) compared to DENV-1 infected splenocytes. Thus these peptides, and specially the Pep03, can induce a humoral response characterized by antibodies with low neutralizing activities and probably a T cell response that could be beneficial to induce an effective immune response against all DENV serotypes and do not contributed to the immunopathogenesis. However, further studies in peptide sequence will be required to induce the production of neutralizing antibodies against all four DENV serotypes and also to improve immunogenicity of these peptides.

Experimental study on anti-tumor effect of Xihuang pill and its immune clearance function

To discuss the anti-tumor effect of Xihuang pill on tumor-bearing rats and its effect on the immune clearance function of tumor-bearing organisms. Walker256 tumor cells were adopted to establish the tumor-bearing rat model. The rats were randomly divided into five groups: the normal control group, the model control group, the lentinan group and Xihuang pill low dose, middle dose and high dose groups, with 10 rats in each group, and continuously treated and given drugs for 14 d after modeling. Blood and tumors were collected from abdominal aorta to calculate the tumor inhibition rate. The content of CD3+, CD4+, CD8+ T cells and adhesion molecule B7-1 (CD80) in peripheral blood were detected by flow cytometry (FCM). The expressions of IL-2 and IFN-gamma in were determined by ELISA. The tumor inhibition rate of the Xihuang pill high dose group was 33. 1 percent. Compared with the model group, the Xihuang pill large dose group showed significantly low IL-2, IFN-gamma, CD3+, CD4+, B7-1 in peripheral blood, with statistical significance in their differences (P < 0.05). Xihuang pill could show its anti-tumor effect by enhancing the immune clearance function and increasing IL-2, IFN-gamma, CD3+ T, CD4+ T, B7-1 in peripheral blood.

OC.12.4 INNATE LYMPHOID CELLS ARE INCREASED IN CELIAC DISEASE INTESTINAL MUCOSA AND HAVE A PATHOGENIC ROLE IN A MOUSE MODEL OF SMALL INTESTINE ATROPHY

Background and aim: Innate lymphoid cells (ILCs) are an emerging family of innate hematopoietic cells producing inflammatory cytokines and involved in the pathogenesis of several immune-mediated diseases. The aim of our study was to characterize the tissue distribution of ILCs in celiac disease (CD) and analyze their role in gut tissue damage. Materials and methods: ILC subpopulations were analyzed in lamina propria mononuclear cells (LPMCs), isolated from duodenal biopsies of patients with active CD, patients with inactive CD on a gluten-free diet and healthy controls (HC) and jejunal specimens of patients undergoing gastro-intestinal bypass by flow cytometry. Cytokines, transcription factors and receptors for interleukin (IL)-15, interferon (IFN)-alpha and Toll-like receptors (TLR) were assessed in ILCs either freshly isolated or following incubation of control LPMC with IL-15, IFNalpha and poly I:C, a TLR agonist, by flow cytometry. The role of ILCs in gut tissue damage was evaluated in a mouse model of poly I:C-driven small intestine atrophy. Results: The percentage of total ILCs did not differ between CD (both active and inactive) patients and HC. However, the fractions of ILCs type 1 expressing T-bet and ILCs type 3 expressing ROR-(g)t were significantly increased in active CD compared to controls, while there was no difference in term of ILCs type 2 among groups. ILCs expressed TLR3, IL-15R, little TLR2 but not IFN-alphaR and TLR4. IL-15 and poly I:C, but not IFN-alpha increased IFNgamma expression in ILCs. In vivo in mice, depletion of ILCs significantly reduced TLR3driven small intestinal damage. Conclusions: Active CD is characterized by preferential accumulation of type 1 and type 3 ILC subpopulations and these cells respond to IL-15, a cytokine over-produced in CD, by enhancing IFN-gamma production. The demonstration that ILCs express TLR3, are functionally able to respond to poly I:C with increased synthesis of inflammatory cytokines, and contribute to TLR3-driven small intestinal atrophy delineates a novel mechanism underlying the CD-associated tissue damage.

IFN-gamma AU-rich element removal promotes chronic IFN-gamma expression and autoimmunity in mice

We generated a mouse model with a 162 nt AU-rich element (ARE) region deletion in the 3′ untranslated region (3′UTR) of the interferon-gamma (IFN-γ) gene that results in chronic circulating serum IFN-γ levels. Mice homozygous for the ARE deletion (ARE-Del) −/− present both serologic and cellular abnormalities typical of patients with systemic lupus erythematosus (SLE). ARE-Del−/− mice display increased numbers of pDCs in bone marrow and spleen. Addition of IFN-γ to Flt3-ligand (Flt3L) treated in vitro bone marrow cultures results in a 2-fold increase in pDCs with concurrent increases in IRF8 expression. Marginal zone B (MZB) cells and marginal zone macrophages (MZMs) are absent in ARE-Del−/− mice. ARE-Del+/− mice retain both MZB cells and MZMs and develop no or mild autoimmunity. However, low dose clodronate treatment in ARE-Del+/− mice specifically eliminates MZMs and promotes anti-DNA antibody development and glomerulonephritis. Our findings demonstrate the consequences of a chronic IFN-γ milieu on B220+ cell types and in particular the impact of MZB cell loss on MZM function in autoimmunity. Furthermore, similarities between disease states in ARE-Del−/− mice and SLE patients suggest that IFN-γ may not only be a product of SLE but may be critical for disease onset and progression.

OP012 Epigenetic control of colonic inflammation via the methyl-binding protein Mbd2

Background: Methyl-CpG binding protein domain protein2 (Mbd2) is a transcriptional co-repressor that binds to methylated DNA. Mbd2 can recruit a nucleosome remodelling complex which contains chromatin remodelling and histone deacetylase properties. Mbd2 deficient mice are viable and fertile but display a dysregulated T cell cytokine response. The impact of Mbd2 deficiency on innate immune cell function and the immunological phenotype in the GI tract is unknown. Methods: Mbd2 / , CD11cCre and littermate Mbd2+/+ or Cre negative control (WT) mice were given 2% Dextran Sulphate Sodium (DSS) in drinking water for 7 10 d. 0.5×106 Mbd2 / or WT CD4+ CD25 CD45RbHi T cells were adoptively transferred by intra-peritoneal injection into Rag / in a T cell model of chronic enterocolitis. Both models were assessed for weight loss, symptom score, colon histology score and mRNA expression of selected cytokines. Single cell suspension of colon lamina propria (LP) leucocytes were isolated and assessed by FACS for CD4+, FoxP3, CD8, CD25, CD11b, CD11c, CD103, CD64, MertK, F4/80, Ly6C, MHC-II, SiglecF, Ly6G. Intracellular cytokines (IL1b, TNF-alpha, IL-10, IL-17 and IFN-gamma) were assessed by FACS post 4hrs ex vivo Golgistop or PMA/ionomycin incubation. Results: Mbd2 / and CD11cCre mice had equivalent proportions of all myeloid subsets in the LP compared to WT in the steady state. Mbd2 / displayed significantly worse colitis post-DSS compared to WT, with greater weight loss, histology score, and mRNA expression of IFN-gamma and IL-17. CD11cCre mice similarly displayed significantly greater weight loss with increased proportion of LP neutrophils and Ly6C+ MHC-II +/ inflammatory monocytes after 8 days of DSS compared to WT. CD11cCre DSS treated LP inflammatory monocytes also had significantly greater TNF and IL-1b producing ability than WT as assessed by FACS suggesting for the first time epigenetic processes can regulate this cell type, which is previously shown to be the dominant pathogenic lineage in this model. Mbd2 / adoptively transferred T cells produced a significantly greater colitis with increased weight loss, histology score and mRNA expression of IL-17 and IFN-gamma. Mbd2 / CD4+ LP T cells also had significantly greater proportion of double positive IL-17 and IFN-gamma producing cells demonstrating epigenetic regulation of T cells is critical in this established Th17 model of colitis. Conclusions: These data reveal for the first time that Mbd2 is of critical importance in the orchestration of a T cell dependent and independent model of murine colitis in vivo. They also identify methyl-binding proteins and/or genes that they regulate as exciting new targets for therapeutic modulation of GI inflammation. OP013 Status of ER stress and autophagy in Crohn’s disease: From genetics to functional read-outs K. Nys1 *, W. Vanhove1, I. Cleynen1, M. Ferrante1, G. Van Assche1, P. Rutgeerts1, P. Agostinis2, S. Vermeire1. 1Department of Clinical and Experimental Medicine, KU Leuven, Translational Research in GastroIntestinal Disorders, Leuven, Belgium, 2KU Leuven, Cellular & Molecular Medicine, Leuven, Belgium