High IL-10 Production Research Articles

Granulocytic and monocytic populations of tumor-infiltrating myeloid-derived suppressor cells (MDSC) suppress T cell proliferation through independent mechanisms (74.7)

Abstract Granulocytic and monocytic myeloid-derived suppressor cells (G-MDSC and M-MDSC) are hallmarks of chronic inflammatory environments and major mediators of T cell suppression in cancer. We aimed to determine the mechanisms by which tumor-associated G-MDSC and M-MDSC suppress T cell proliferation. Our results suggest that MDSC isolated from lung carcinoma-bearing mice impaired the proliferation of activated T cells thorough an arrest in the G0-G1 phase of the cell cycle. This effect was not caused by a decreased T cell activation, as an increased expression of the early activation markers CD25 and CD69, and high production of IL-2 were detected in T cells co-cultured with MDSC. The anti-proliferative effect induced by MDSC was prevented after the addition of inhibitors of arginase and inducible nitric oxide synthase (iNOS). Interestingly, arginase I was exclusively expressed in G-MDSC, whereas iNOS was expressed mainly in M-MDSC. Additional results showed that G-MDSC produced higher levels of reactive oxygen species (ROS), whereas M-MDSC produced more peroxynitrites (PNT). Expression of gp91phox in G-MDSC regulated their production of ROS. In addition, M-MDSC-induced suppression was overcome using a PNT scavenger or an inhibitor for S-nitrosylation. These results suggest that sub-populations of MDSC suppress T cell proliferation through independent mechanisms. Targeting of these pathways may enable the design of new therapeutic approaches to reverse T cell tolerance in cancer.

High IL-4 expression and IL-4 gene polymorphisms are associated with susceptibility to human paracoccidioidomycosis (164.21)

Abstract Paracoccidioidomycosis is caused by the dimorphic fungus Paracoccidioides brasiliensis. After invasion, the fungi are immediately destroyed or overcome the local defenses of the host and begin multiplication, causing initial injury. Previous studies have demonstrated that the severity of disease is associated with a Th2 immune response characterized by high IL-4 production. In the present study, we analyzed two polymorphisms in the IL-4 gene, -590 C/T and the intron-3 microsatellite, in 76 patients with paracoccidioidomycosis and in 73 control individuals from an endemic area. A significant correlation was observed between the RP2/RP2 intron-3 genotype and infection with P. brasiliensis (p=0.011), while the RP1/RP1 genotype was correlated with resistance. The IL-4 promoter polymorphism showed a significant correlation of the presence of T allele with the disease (p=0.030). Furthermore, high IL-4 expression observed on the patient group compared with control and was associated with TT genotype. This result suggests that IL-4 polymorphisms are associated with the ability of the host to control P. brasiliensis infection. The potential biological role of this polymorphism is supported by our observation that patients with disease produce high levels of IL-4 after mitogen or antigen stimulation and the association of high IL-4 levels with TT genotype. IL-4 gene is located in the cytokine cluster region of chromosome 5, where other polymorphisms have been described.

The E3 Ubiquitin Ligase Adaptor Ndfip1 Regulates Th17 Differentiation by Limiting the Production of Proinflammatory Cytokines

Ndfip1 is an adaptor for the E3 ubiquitin ligase Itch. Both Ndfip1- and Itch-deficient T cells are biased toward Th2 cytokine production. In this study, we demonstrate that lungs from Ndfip1(-/-) mice showed increased numbers of neutrophils and Th17 cells. This was not because Ndfip1(-/-) T cells are biased toward Th17 differentiation. In fact, fewer Ndfip1(-/-) T cells differentiated into Th17 cells in vitro due to high IL-4 production. Rather, Th17 differentiation was increased in Ndfip1(-/-) mice due to increased numbers of IL-6-producing eosinophils. IL-6 levels in mice that lacked both Ndfip1 and IL-4 were similar to wild-type controls, and these mice had fewer Th17 cells in their lungs. These results indicate that Th2 inflammation, such as that observed in Ndfip1(-/-) mice, can increase Th17 differentiation by recruiting IL-6-producing eosinophils into secondary lymphoid organs and tissues. This may explain why Th17 cells develop within an ongoing Th2 inflammatory response.

Neonatal Plasma Polarizes TLR4-Mediated Cytokine Responses towards Low IL-12p70 and High IL-10 Production via Distinct Factors

Human neonates are highly susceptible to infection, which may be due in part to impaired innate immune function. Neonatal Toll-like receptor (TLR) responses are biased against the generation of pro-inflammatory/Th1-polarizing cytokines, yet the underlying mechanisms are incompletely defined. Here, we demonstrate that neonatal plasma polarizes TLR4-mediated cytokine production. When exposed to cord blood plasma, mononuclear cells (MCs) produced significantly lower TLR4-mediated IL-12p70 and higher IL-10 compared to MC exposed to adult plasma. Suppression by neonatal plasma of TLR4-mediated IL-12p70 production, but not induction of TLR4-mediated IL-10 production, was maintained up to the age of 1 month. Cord blood plasma conferred a similar pattern of MC cytokine responses to TLR3 and TLR8 agonists, demonstrating activity towards both MyD88-dependent and MyD88-independent agonists. The factor causing increased TLR4-mediated IL-10 production by cord blood plasma was heat-labile, lost after protein depletion and independent of lipoprotein binding protein (LBP) or soluble CD14 (sCD14). The factor causing inhibition of TLR4-mediated IL-12p70 production by cord blood plasma was resistant to heat inactivation or protein depletion and was independent of IL-10, vitamin D and prostaglandin E2. In conclusion, human neonatal plasma contains at least two distinct factors that suppress TLR4-mediated IL-12p70 production or induce IL-10 or production. Further identification of these factors will provide insight into the ontogeny of innate immune development and might identify novel targets for the prevention and treatment of neonatal infection.

IL‐10 production in non–small cell lung carcinoma patients is regulated by ERK, P38 and COX‐2

Immune dysfunction is hallmark of patients with non–small cell lung carcinoma (NSCLC). The molecular mechanism involved in COX-2– and PGE2-mediated production of immunosuppressive cytokine IL-10 is not well-understood. Our study addresses the involvement of T cell downstream signalling intermediates, cytokines (IL-10 and IFN-γ) and their transcription factors (T-bet and GATA-3) in COX-2–mediated regulation of lymphocyte functions in NSCLC patients. In comparison to healthy individual, a marked decrease in lymphocyte proliferation to anti-CD3 MAb was observed in NSCLC patients by thymidine incorporation assay. Using flow cytometry, decrease in intracellular calcium release with increase in reactive oxygen species was observed in lymphocytes of NSCLC patients. These patients showed increased IL-10 and PGE2 with reduced IFN-γ production by ELISA. Results demonstrated defect in regulation of transcription factors T-bet and GATA-3 as analysed by Western blotting (WB), immunoprecipitation and EMSA. Overexpression of p-p38, p-ERK and COX-2 were observed with diminished p-JNK by WB. IL-10/IFN-γ levels were found to be differentially regulated via p38 and ERK mitogen-activated protein kinase (MAPK) pathways in cooperation with COX-2. Inhibition of these pathways using selective inhibitors lead to increased lymphocyte proliferative response to anti-CD3 MAb and IFN-γ production with decrease in IL-10 production. Studies showed involvement of ERK, p38 and COX-2 pathways in high IL-10 production, driven by lung tumour derived PGE2. The selective COX-2 inhibitor rofecoxib showed ability to alter the cytokine balance by affecting regulation of T-bet and GATA-3 transcription factors.

Diphenyl diselenide-modulation of macrophage activation: Down-regulation of classical and alternative activation markers

Diphenyl diselenide (PhSe)(2), a simple organoselenium compound, possesses interesting pharmacological properties that are under extensive research. As macrophages respond to microenvironmental stimuli and can display activities engaged in the initiation and the resolution of inflammation, in the present report we describe the ability of (PhSe)(2) to modulate the macrophage activation. Our data indicate that (PhSe)(2) could inhibit the NO production in a dose-dependent fashion in peritoneal macrophages activated by LPS or treated with vehicle alone. We could demonstrate that this effect correlated with a reduction in the expression of the inducible NO synthase in (PhSe)(2)-treated cells. Furthermore, (PhSe)(2) suppressed the production of reactive oxygen species, diminished the activity of the arginase enzyme, and the accumulation of nitrotyrosine modified proteins in LPS-stimulated macrophages. This compound also diminished the antigen presentation capacity of classically activated macrophages, as it reduced MHCII and CD86 expression. In addition, (PhSe)(2) modulated the alternative activation phenotype of macrophages. Dexamethasone-activated macrophages presented higher production of IL-10 and CD206, which were both down-regulated by the addition of (PhSe)(2). These results suggest that (PhSe)(2) possesses antioxidant and anti-inflammatory activities in classically-activated macrophages. We could demonstrate that (PhSe)(2) can be also utilized to modulate the alternative activation phenotype of macrophages.

IL-10-generated tolerogenic dendritic cells are optimal for functional regulatory T cell induction — A comparative study of human clinical-applicable DC

Tolerogenic dendritic cells (tDC) are a promising tool for specific cellular therapy to induce immunological tolerance in transplantation and autoimmunity. To date, most described tDC methods have not been converted into clinically applicable protocols and systematic comparison of required functional characteristics, i.e. migration and functional regulatory T cell (Treg) induction, is lacking. We compare clinical-grade tDC generated with vitamin D(3), IL-10, dexamethasone, TGFβ or rapamycin. For good migratory capacity and a stable phenotype, additional maturation of tDC was required. Maturation with a cocktail of TNFα, IL-1β and PGE(2) induced optimal migration. Importantly, all tDC showed a stable phenotype under pro-inflammatory conditions. Especially IL-10 DC showed most powerful tolerogenic characteristics with high IL-10 production and low T cell activation. Moreover, in a functional suppression assay only IL-10 DC induced Treg that strongly suppressed T cell reactivity. Thus, clinical-grade IL-10 DC show functional characteristics that make them best suited for tolerance-inducing therapies.

Changes of CD19 + CD5 + B cells in endometriosis

Objective To explore the changes of the frequency and cytokine production of CD19 + CD5 + B cells in Endometriosis before and after treatment.Methods Flow cytometry and quantitative PCR were used to assess the frequency,cytokine expression of CD19 + CD5 + B cells in the peripheral blood,peritoneal fluids and endometrium biopsies of 35 patients with Endometriosis and 29 patients with myoma of uterus (control subjects).Changes in the CD19+ CD5+ B cell compartment in the peripheral blood were compared before and after treatment.Results All 35 endometriosis patients had higher frequencies(all P ＜0.01 ) of CD19+ CD5 + B cells in the peripheral blood,peritoneal fluids and endometrium biopsies [ (13.1 ± 1.9)％,(12.1 ±2.0)％,(11.7 ± 1.7)％] than 29 control subjects[ (2.9 ±0.8)％,(2.6 ±0.9)％,(2.8 ±1.1 )％ ].CD19 + CD5+ B cells from endometriosis patients expressed higher IFN-γ[ (7.2 ± 1.0) × 103,(7.9±1.3) ×103,(7.4±1.1) ×103 copies] than control subjects [ (1.9±0.7) ×103,(2.2±0.8) ×103,(2.0 ±0.5) × 103 copies).The endometriosis patients also had higher IL-10 production of CD19 + CD5 + B cells [ (6.4 ±0.9) × 103,(6.8 ± 1.2) × 103,(6.1 ±0.8) × 103 copies] than control subjects [ ( 1.7 ±0.5) × 103,(2.1 ±0.9) × 103,( 1.6 ±0.4) × 103 copies].Compared with control subjects,the endometriosis patients had a clinical response to treatment demomtrated a significant decrease in CD19 + CD5 + B cells in the peripheral blood[ ( 13.1 ± 1.9)％ vs (7.3 ± 1.2)％ ],expressed lower IFN-γ [ (7.2 ± 1.0)×103 copies vs (3.3 ±0.6) × 103 copies],produced less IL-10 [(6.4 ±0.9) × 103 copies vs (3.2 ±0.7) × 103 copies].Conclusions CD19+ CD5 + B cells might play an important role in the pathogenesis of endometriosis through higher IFN-γand IL-10 expression. Key words: Endometriosis/TH/ME; Antigens, CD 19/ME; Antigens, CD5/ME; B-lymphocytes/ME; Interferon type Ⅱ/ME; Interleukin-10/ME

Autocrine IL-6 Production Indicates the Level of Activated STAT3 and NF-Kb of Chronic Lymphocytic Leukemia Cells

Abstract 1784Interleukin-6 (IL-6) is a multifunctional cytokine that plays an important role in survival, apoptosis and proliferation of cancer cells. Previous studies have shown that IL-6 levels are significantly elevated in most cancer cells, while elevated serum levels of IL-6 correlated with adverse prognostic features and shorter survival in patients with chronic lymphocytic leukemia (CLL). IL-6 production is controled by transcription factors NF-kB and STAT3, which are themselves controled by tyrosine kinase phosphorylation, such as that mediated by JAK2. This creates a positive feedback loop, as JAK2 can be stimulated by IL-6.In this study we investigated the relationship between autocrine IL-6 production and activation of STAT3 and NF-kB in CLL patients. The levels of IL-6 production in cell culture medium were measured by ELISA and activation of STAT3 and NF-kB was indicated by the ratio of p-STAT3/STAT3 and p-NF-kB/NF-kB. Patients with high levels of p-STAT3/STAT3 and/or p-NF-kB/NF-kB had higher levels of IL-6 production. The level of IL-6 production significantly correlated with STAT3 activation (R=0.856, p < 0.001) and NF-kB activation (R=0.815, p < 0.001). Inhibition of constitutive STAT3 and NF-kB activation by the STAT3 phosphorylation inhibitor, DPP, and the NF-kB inhibitor, CAPE, blocked IL-6 production 40% in average (p = 0.004, p = 0.003). Analysing the level of autocrine IL-6 production and in vitro spontaneous apoptosis in CLL cells from 38 patients, we found that patients with higher levels of IL-6 production (more than median) had significantly less apoptosis compared with those below the median (23% vs. 42.2% in average, p <0.0001). In this cohort, 60% of CD38+ cases, 78% of 17p- cases and 67% of cases with a lymphocyte doubling time of less than12 months were in the high IL-6 producer group. High IL-6 production was also associated with a shorter average time to first treatment (0.5 vs. 3 years, p=0.0023).This study demonstrates that autocrine IL6 production correlates with STAT3 and NF-kB activation and apoptosis resistance in CLL. Furthermore, higher IL-6 production is associated with biological markers of poor prognosis and earlier treatment. The IL6/JAK2/STAT3 signal pathway may offer new therapeutic targets for CLL. Disclosures:No relevant conflicts of interest to declare.

Echinococcus granulosus glycoconjugates induce peritoneal B cell differentiation into antibody‐secreting cells and cytokine production

Helminth parasite infections are associated with predominant Th2-type cytokine responses, and parasite glycoconjugates have been recognized as partially responsible for such immune bias. It has been proved that Echinococcus granulosus evokes a Th2-type cytokine pattern characterized by a high production of IL-4, IL-5, IL-6 and IL-10, and no or mild IFN-γ levels in animal models and in patients with cystic echinococcosis, respectively. Here, we show that E4(+) (a glycoconjugate-enriched fraction from E. granulosus protoscolex) stimulated the secretion of a high concentration of IL-6, followed by IL-10 and TNF-α by normal peritoneal B cells. We determined that E4(+) bound to the surface of peritoneal B cells and induced their activation and, also, triggered the differentiation of peritoneal B cells into IgM-, IgG2b- and IgG3-secreting cells in a T-independent way. Interestingly, the IgM released by E4(+) -stimulated peritoneal B cells from normal mice recognized protoscolex antigens. Results showed that, after the encounter with antigens from E. granulosus protoscolex, peritoneal B cells are a source of Th2-type cytokines and polyclonal antibodies, some of which recognize parasite antigens, suggesting that peritoneal B cells can condition the outcome of the infection.

2.47 p53 Homolog p63 Promotes Apoptosis and Chemosensitivity in Chronic Lymphocytic Leukemia B-Cells

in cell culture medium were measured by enzyme-linked immunosorbent assay (ELISA) and activation of STAT3 and NFB was indicated by the ratio of p-STAT3/STAT3 and p-NFB/NFB. Patients with high levels of p-STAT3/STAT3 and/or p-NFB/ NFB had higher levels of IL-6 production. The level of IL-6 production significantly correlated with STAT3 activation (R 0.856, p 0.001) and NFB activation (R 0.815, p 0.001). Inhibition of constitutive STAT3 and NFB activation by the STAT3 phosphorylation inhibitor, diphenylporphyrin, and the NFB inhibitor, caffeic acid phenethyl ester (CAPE), blocked IL-6 production by 40% on average (p 0.004, p 0.003), active STAT3 or NFB significant increase IL-6 secretion. Analyzing the level of autocrine IL-6 production with in vitro spontaneous apoptosis in CLL cells from 38 patients, we found that patients with higher levels of IL-6 production (more than median) had significantly less apoptosis compared with those below the median (23% vs. 42.2% in average, p 0.0001). Correlation between the levels of autocrine IL-6 production and cell viability in 38 patients (CLL cells were incubated for 48 hours in vitro) was significant (R 0.72, p 0.0001). High IL-6 production was also associated with a shorter average time to first treatment (0.5 vs. 3 years, p 0.0023). This study demonstrates that autocrine IL-6 production correlates with STAT3 and NFB activation and apoptosis resistance in CLL. Furthermore, higher IL-6 production is associated with biological markers of poor prognosis and earlier treatment.

Hypertension artérielle pulmonaire liée à l’infection VIH : de la pression artérielle pulmonaire à l’interleukine-6

Vascular diseases have become the leading cause of mortality in the population treated for HIV infection. Pulmonary arterial hypertension (PAH) related to HIV (PAH-HIV), the fourth cause of PAH in France, has the same histological pattern as other PAH from the group 1 of Dana Point classification. But, conversely to idiopathic PAH in the general population, PAH-HIV is particular by its high frequency in HIV-infected population. This raises the question for the role of inflammation in the PAH-HIV pathophysiology. Its constant occurrence over the decades, despite introduction of combination antiretroviral therapy (CAT), does not preclude the hypothesis of an involvement of inflammation in the genesis of PAH-HIV. Indeed, it is well known that normalization of CD4+ by the CAT does not mean no inflammation. Especially, it persists an increased and continuous production of IL-6, a main cytokine in the genesis of PAH lesions. This inflammation mainly involves the endothelin-1 pathway, which has an action on endothelium and macrophages, leading to high production of IL-6. Moreover, plasmatic level of IL-6 has a prognostic value in PAH-HIV, independently from conventional (functional or hemodynamic) parameters. The use of endothelin receptor antagonist permits major effect on IL-6 production and dramatic effect on PAH in so-called "bosentan responders".

The suppressive effect of mesenchymal stromal cells on T cell proliferation is conserved in old age

Mesenchymal stromal cells (MSC) have become a useful tool in curing graft versus host disease (GVHD) after transplantation. No information is presently available whether the immunosuppressive properties of this cell type are maintained in old age. It was therefore the aim of our study to analyze the immunoregulatory effect of MSC on peripheral blood mononuclear cells (PBMC) in old age. We studied the proliferation, activation and cytokine production of PBMC following co-culture with MSC from young (<30years) and old (>61years) donors. Our results demonstrate that MSC from elderly donors exhibit the same suppressive effects on T cell proliferation as their young counterparts. In both age groups T cell activation was not influenced by co-culture with MSC from young and elderly donors. With the exception of IL-6, cytokine production by unstimulated or stimulated PBMC was also not affected by MSC from either age group. IL-6 production was increased during co-culture of PBMC and MSC and was higher when MSC from elderly donors were used. After PHA stimulation, however, this age-specific difference was balanced and appeared even. As high IL-6 production is a prerequisite for an effective suppression of T cell proliferation, MSC can be considered a powerful tool for immunoregulatory therapies in old age.

STAT3 Knockdown in B16 Melanoma by siRNA Lipopolyplexes Induces Bystander Immune Response In Vitro and In Vivo

Persistent activation of STAT3 plays a major role in cancer progression and immune escape. Therefore, targeting STAT3 in tumors is essential to enhance/reactivate antitumor immune response. In our previous studies, we demonstrated the efficacy of stearic acid-modified polyethylenimine (PEI-StA) in promoting small interfering RNA (siRNA) silencing of STAT3 in B16.F10 melanoma in vitro and in vivo. In the current study, we examine the immunologic impact of this intervention. Toward this goal, the infiltration and activation of lymphocytes and dendritic cells (DCs) in the tumor mass were assessed using flow cytometry. Moreover, the levels of IFN-γ, IL-12, and TNF-α in homogenized tumor supernatants were determined. Moreover, mixed lymphocytes reaction using splenocytes of tumor-bearing mice was used to assess DC functionality on siRNA/lipopolyplexes intervention. Our results demonstrated up to an approximately fivefold induction in the infiltration of CD3+ cells in tumor mass on STAT3 knockdown with high levels of CD4+, CD8+, and NKT cells. Consistently, DC infiltration in tumor milieu increased up to approximately fourfold. Those DCs were activated, in an otherwise suppressive microenvironment, as evidenced by a high expression of costimulatory molecules CD86 and CD40. ELISA analysis revealed a significant increase in IFN-γ, IL-12, and TNF-α. Moreover, mixed lymphocytes reaction demonstrated alloreactivity of these DCs as assessed by high T-cell proliferation and IL-2 production. Our results suggest a bystander immune response after local STAT3 silencing by siRNA. This strategy could be beneficial as an adjuvant therapy along with current cancer vaccine formulations.

TNFRp55 modulates IL-6 and nitric oxide responses following Yersinia lipopolysaccharide stimulation in peritoneal macrophages

While cytokines are major regulators of macrophage activation following host–pathogen interactions, they also act to limit inflammation to avoid tissue damage. In previous studies we reported the development of progressive Yersinia enterocolitica-induced reactive arthritis (ReA) in mice lacking the tumor necrosis factor receptor p55 (TNFRp55). In this work, we analyzed the response of TNFRp55 −/− macrophages to Y. enterocolitica antigens. We found higher concentration of nitric oxide (NO) in TNFRp55 −/− compared to wild-type macrophages in response to heat-killed Yersinia (HKY) and Yersinia outer membranes (OM). Moreover, Toll-like receptor (TLR)4 expression was increased in OM-stimulated TNFRp55 −/− versus wild-type (WT) macrophages. Accordingly, NO production was inhibited in TLR4-deficient macrophages following stimulation with OM, suggesting that LPS may function as a major OM component implicated in these responses. Thus, augmented NO production together with enhanced expression of inducible nitric oxide synthase (iNOS) and higher IL-6 production, may provide a pro-inflammatory setting in Yersinia LPS-stimulated TNFRp55 −/− macrophages. Augmented synthesis of NO and IL-6 was prevented by treatment with Polymyxin B, or by exposure to a specific NF-κB p65 oligonucleotide antisense, indicating the involvement of TLR4-mediated NF-κB activation in the unleashed pro-inflammatory response triggered by TNFRp55 deficiency. Thus, TNFRp55 modulates macrophage functions in response to Yersinia LPS stimulation through mechanisms involving NO, IL-6 and NF-κB pathways, suggesting an essential regulatory role of TNF via TNFRp55 signaling.

Functional impairment of dendritic cells in patients infected with hepatitis C virus genotype 1 who failed peginterferon plus ribavirin therapy

Although chronic hepatitis C patients have a lower frequency and functions of dendritic cells (DCs) than healthy subjects, little is known about the serial changes in frequency and functions of DCs following anti-viral treatment and the relationship with treatment outcomes. Twenty patients with hepatitis C virus genotype 1 receiving peginterferon (PEG-IFN) and ribavirin for 24 weeks were enrolled. The frequency and functions of DCs were assayed at baseline and 24 weeks post-treatment. Ten sex and age-matched healthy adults served as controls. Nineteen of the 20 chronic hepatitis C patients completed 24 weeks of combination therapy. Fifteen patients achieved rapid virologic response and 12 achieved sustained virologic response (SVR). The baseline frequency of peripheral blood myeloid DCs and plasmacytoid DCs was significantly lower in chronic hepatitis C patients than in healthy controls. In patients who achieved SVR, the frequency of DCs subsets at the end of follow-up increased to a level comparable to healthy controls. Although no functional defects of DCs was found in chronic hepatitis C patients in comparison with healthy controls, in patients without SVR had a lower CD83 expression and higher interleukin-10 production of DCs than SVR patients. The results suggest that low CD83 expression and high IL-10 production of DCs at the baseline may predict a poor virologic response to 24-week PEG-IFN plus ribavirin therapy in HCV genotype 1 patients.

Safety and efficacy of the immunosuppressive agent 6-tioguanine in murine model of acute and chronic colitis

BackgroundOral thiopurines are effective and widely used in treatment of inflammatory bowel disease (IBD) in humans, although their use is limited due the development of adverse events. Here, we examine the efficacy and toxicity of oral treatment with 6-tioguanine (6-TG) and azathioprine (AZA) in a murine model of IBD.MethodsWe induced acute or chronic colitis in BALB/c mice by one or four cycles of 3% dextran sulphate sodium (DSS), respectively. Mice were treated by daily gavages of various dosages of 6-tioguanine, azathioprine, or by phosphate buffered saline (PBS) starting the first day of DSS or after two cycles of DSS, respectively. We monitored the efficacy and toxicity by measuring the weight change and serum alanine aminotransferase (ALT) activity and by disease severity and histology, at the end of the experiment. Moreover, we measured cytokine production after colon fragment cultivation by enzyme-linked immunoabsorbent assay and numbers of apoptotic cells in the spleen by flow cytometry.Results6-TG is effective in the treatment of acute DSS-induced colitis in a dose-dependent manner and 40 μg of 6-TG is significantly more effective in the treatment of acute colitis than both AZA and PBS. This effect is accompanied by decrease of IL-6 and IFN-γ production in colon. We did not observe histological abnormalities in liver samples from control (PBS) or 6-TG treated mice. However, liver samples from most mice treated with AZA showed mild, yet distinct signs of hepatotoxicity. In chronic colitis, all thiopurine derivatives improved colitis, 20 μg of 6-TG per dose was superior. High doses of 6-TG led to significant weight loss at the end of the therapy, but none of the thiopurine derivatives increased levels of serum ALT. Both thiopurine derivatives reduced the proportion of apoptotic T helper cells, but a high production of both IL-6 and TGF-β was observed only in colon of AZA-treated mice.ConclusionsUse of 6-TG in the treatment of experimental colitis in mice appears superior to AZA administration and placebo. In contrast to 6-TG, the use of AZA resulted in histological liver abnormalities.

Abstract 3653: Enrichment of Foxp3+ CD4 Tregs in migrated T-cells to IL-6- and IL-8-expressing tumors through predominant induction of CXCR1 by IL-6

Abstract Background: Analysis of cytokine and chemokine production by tumor cell lines including 5 lung cancers, a malignant mesothelioma and a malignant melanoma recently established in our laboratory showed rather high production of IL-8 in all tumors and IL-6 in one lung cancer, the malignant mesothelioma and the malignant melanoma. We investigated migration of PBMCs to these tumor cells using Transwell plates and showed enrichment of Foxp3+ CD4 Tregs in migrated T-cells to both IL-6- and IL-8-producing tumors. Marked induction of CXCR1 expression on Foxp3+ CD4 Tregs by IL-6 followed by IL-8-mediated migration appeared to be the mechanisms for enriched migration. Frequent production of IL-8 by the tumors and Treg migration to those tumors through induction of IL-8 receptor expression by IL-6 is one of the mechanisms for tumor escape. Purpose: In this study, we investigated cytokine and chemokine production by tumor cell lines including 5 lung cancers, a malignant mesothelioma and a malignant melanoma recently established in our laboratory. We observed IL-8 production in all tumors and IL-6 production in one lung cancer, a malignant mesothelioma and a malignant melanoma. We investigated migration of Foxp3+ CD4 Tregs in PBMCs to those tumor cells using Transwell plates. Experimental Design: Cytokine and chemokine production was analyzed in tumor cell lines including 5 lung cancers, a malignant mesothelioma and a malignant melanoma recently established in our laboratory. IL-8 production was detected in all tumors and IL-6 production in 3 of them. We then investigated the role of IL-6 and IL-8 on migration of Foxp3+ CD4 Tregs to those tumor cells using Transwell plates. Induction of IL-8 receptor on Foxp3+ CD4 T-cells by IL-6 was investigated by flow cytometry and its role on their migration was examined by anti-IL-8R or anti-IL-6R blocking. Furthermore, to investigate relationships between the effect of IL-6 and STAT3 signaling in the induction of IL-8R expression, we examined whether IL-8R expression would inhibit by blocking of STAT3 signaling. Results: We showed enrichment of Foxp3+ CD4 Tregs in migrated T-cells to both IL-6 and IL-8-producing tumors. Marked induction of CXCR1 (IL-8RA) expression was observed on Foxp3+ CD4 Tregs by the treatment with IL-6. Migration of IL-6-treated Foxp3+ CD4 T-cells to the tumors was blocked by the addition of anti-CXCR1 or anti-IL-6R. Furthermore, high level of IL-6 was detected in pleural effusion in most of lung cancer patients.Conclusions: Frequent production of IL-8 by the tumors and Treg migration to those tumors through induction of IL-8 receptor expression by IL-6 is one of the mechanisms for tumor escape. The findings indicate that the blocking of Treg migration by inhibiting IL-6-mediated IL-8 receptor expression could be a new strategy for tumor immunotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3653. doi:10.1158/1538-7445.AM2011-3653

Rat resident testicular macrophages have an alternatively activated phenotype and constitutively produce interleukin-10 in vitro

The ability of the rodent testis to tolerate graft alloantigens and spermatogenic cell autoantigens is well known. The mechanisms underlying this "immune privilege" are poorly understood, but the numerous resident TMs have been implicated. Although it has been assumed that TMs display a phenotype consistent with immune privilege, this has not been formally established. Consequently, TMs were isolated from adult rats and cultured under basal conditions and following stimulation with LPS and IFN-γ (classical activation) or IL-4 (alternative activation). BMMs matured in vitro were used as control. Expression of the classical (proinflammatory) activation markers TNF-α, IL-1β, iNOS, IL-6, RANTES, IL-12p40, and SOCS3 and alternative (immunoregulatory) activation markers IL-10, TGF-β1, CXCL2, and SOCS1 was measured by QPCR or ELISA. In culture, TMs were characterized by poor expression of classical activation genes and TGF-β1 but constitutively high IL-10 production and reduced costimulatory activity in a polyclonal T cell activation assay. This pattern of gene expression was associated with TMs expressing the scavenger receptor CD163, which is characteristic of tissue resident macrophages and alternative activation. By contrast, CD163-negative TMs displayed reduced inflammatory gene expression but did not constitutively produce IL-10. These data indicate that under the influence of the testicular environment, macrophages adopt an alternatively activated phenotype, involving reduced capacity for proinflammatory gene expression, constitutive IL-10 production, and impaired ability to support T cell activation, consistent with a role in maintaining testicular immune privilege.

Novel method for the development of regulatory T cells (166.22)

Abstract Background: Regulatory T cells (Treg) are important in the inhibition of inflammatory disorders, like asthma. Unfortunately, current protocols used to develop them in vitro for study rely on complicated cloning methods that may not represent biologically relevant Treg. LPS-free ovalbumin (OVA) given intranasal (i.n.) can induce mucosal tolerance in mice. Therefore, we hypothesized that Dendritic cells (DCs) taken from the draining lymph nodes (LNs) of mice given LPS-free OVA would induce Treg in vitro. Methods: Mice were given i.n. LPS-free OVA. 24 h later cervical and bronchial LNs were excised and DCs were isolated (~98% purity). DCs were cultured in vitro with transgenic OVA-specific T cells for four days. The development of Treg was assessed by flowcytometry (Foxp3 expression), ELISA (IL-10 production), and inhibition of T cell proliferation (CFSE staining/BrdU uptake). Finally, we studied the effectiveness of these cells to inhibit the development of AHR in a mouse model of asthma. Results: T cells from mice treated with LPS-free OVA showed high levels of Foxp3 expression, high IL-10 production, and the ability to inhibit OVA specific T cells in vitro. compared to control mice. Furthermore, T cells from LPS-free OVA treated mice prevented the development of AHR when given to mice before OVA challenge (i.n.). Conclusion: We believe our protocol could represent an alternative method to develop Treg cells for study.