IL-1beta Production Research Articles

Topical thioredoxin inhibits IL-6 and IL-1beta production from keratinocytes and is effective for psoriasis-like dermatitis in mice

Topical thioredoxin inhibits IL-6 and IL-1beta production from keratinocytes and is effective for psoriasis-like dermatitis in mice

Abstract # 1820 Stimulated cytokine production is heightened among pregnant women carrying female versus male fetuses

Fetal sex plays a role in regulating maternal adaptation across pregnancy including glycemic control, blood pressure, and cortisol. However, data examining effects of fetal sex on maternal immune parameters is lacking, despite observed differences in maternal health responses and pregnancy outcomes in relation to fetal sex. In the current study, serum levels of IL-6, IL-8, and TNF-alpha as well as LPS-stimulated production of IL-6, IL-8, TNF-alpha, and IL-1 beta by PBMCs incubated for 24 h were assessed during each trimester of pregnancy in 80 women (46 with male and 34 with female fetuses). Linear mixed models showed that women carrying females versus males exhibited heightened stimulated production of IL-6 during each trimester (ps 0.85). In conclusion, in pregnant women, those carrying female versus male fetuses exhibit greater stimulated cytokine production. Heightened inflammation could affect maternal health and fetal development. Fetal sex should be considered as a factor in studies of maternal inflammation. Future investigations focused on women with health conditions (e.g., preeclampsia) and adverse pregnancy outcomes (e.g., preterm birth) would be informative.

Enhanced alpha-kinase 1 accelerates multiple early nephropathies in streptozotocin-induced hyperglycemic mice

Alpha-kinase 1 (ALPK1) is associated with chronic kidney disease (CKD), type 2 diabetes mellitus and gout. Elevated ALPK1 levels have been observed in the kidneys of patients with diabetes and the white blood cells of patients with gout. As renal injury is a common outcome of CKD, diabetes and gout, the aim of this study was to investigate the effect of ALPK1 in the development of renal injury in a hyperglycemic condition. Hyperglycemia was induced in wild-type and ALPK1 transgenic mice by an intraperitoneal injection of streptozotocin (STZ). Functional and histological examinations were performed after 3weeks. STZ-treated ALPK1 transgenic mice exclusively showed arteriolar sclerosis and fibrous thickening of the Bowman's capsule in the kidney. This was accompanied by body weight loss, severe hyperglycemia, and low serum insulin levels. Renal renin and serum renin protein levels were higher in STZ-treated ALPK1 transgenic mice, whereas cGKII protein level was decreased by ALPK1 in human embryonic kidney 293 (HEK293) cells. ALPK1 up-regulated TGF-beta1 levels and transcription of fibrosis-related genes, including MMP-9, FIBRONECTIN, and TIMP1. MSU crystals increased ALPK1 transcription in cultured kidney cells. Finally, ALPK1 enhanced production of MSU crystals-induced IL-1beta in mice. Stimulation of soluble sodium urate induced IL-1beta and Alpk1 mRNA production in mice kidney. Taken together, these data show that an increase in ALPK1 results in accelerated fibrotic nephropathies, primarily through the enhancement of renin, TGF-beta1, and IL-1beta. Renal or blood ALPK1 levels are involved in the induction of fibrotic renal injury in an experimental model of hyperglycemia.

Hyperglycemia induces mixed M1/M2 cytokine profile in primary human monocyte-derived macrophages

Hyperglycaemia is a key factor in diabetic pathology. Macrophages are essential regulators of inflammation which can be classified into two major vectors of polarisation: classically activated macrophages (M1) and alternatively activated macrophages (M2). Both types of macrophages play a role in diabetes, where M1 and M2-produced cytokines can have detrimental effects in development of diabetes-associated inflammation and diabetic vascular complications. However, the effect of hyperglycaemia on differentiation and programming of primary human macrophages was not systematically studied. We established a unique model to assess the influence of hyperglycaemia on M1 and M2 differentiation based on primary human monocyte-derived macrophages. The effects of hyperglycaemia on the gene expression and secretion of prototype M1 cytokines TNF-alpha and IL-1beta, and prototype M2 cytokines IL-1Ra and CCL18 were quantified by RT-PCR and ELISA. Hyperglycaemia stimulated production of TNF-alpha, IL-1beta and IL-1Ra during macrophage differentiation. The effect of hyperglycaemia on TNF-alpha was acute, while the stimulating effect on IL-1beta and IL-1Ra was constitutive. Expression of CCL18 was supressed in M2 macrophages by hyperglycaemia. However the secreted levels remained to be biologically significant. Our data indicate that hyperglycaemia itself, without additional metabolic factors induces mixed M1/M2 cytokine profile that can support of diabetes-associated inflammation and development of vascular complications.

Immunology of Celiac Disease by Focused on Vaccine Therapy

Celiac disease (CD) is categorized by a strong genetic association with the MHC-II haplotype and about 95% of patients carrying HLA-DQ2 haplotypes, and the remaining 5% carrying HLA-DQ8 [1]. This relationship points to the principal role of the adaptive immunity of mucosal in the pathogenesis of CD. This disorder is classically mediated by TH1 because of the enhanced mucosal mRNA expression of interferon (IFN)-gamma in untreated patients with CD [2]. Evidences have shown that TH17 T cell subset (interleukin [IL]-17) are the main cells involved in the pathogenesis of autoimmune disorders and therefore it has become essential to consider the potential participation of TH17 cells in CD [2]. The result of different studies showed that the level of IL-17A is higher in patients with active CD in compare to patients on a gluten free diet (GFD). In addition, it has been confirmed that gliadin can induce the production of IL-1beta and IL-23 by TH17-polarizing in peripheral blood monocytes, and this connection providing a possible causative link between gluten exposure and TH17 cell expansion in CD [3]. On the other hands, it was shown that increased levels of adaptive immune markers such as IFN-gamma, IL-17A, IL-6 and IL-21 in the small intestinal mucosa of CD patients, are triggered by DQ2 DQ8 bounded tTG-deamidated gluten peptides and associated with TH1and TH17associated cytokine production and/or clone activation [4-9]. Among innate immune mechanisms, Toll-like receptors (TLRs) expression and function are also important in CD pathogenesis. These receptors class are able to detect microbial invasion and mediate a rapid inflammatory response. Among them TLR4 expression levels are higher on duodenal biopsies in CD cases compared to controls. Also high levels of IL-1, IL-6, IL-8, and IL-17 were detected in CD patients with high TLR4 levels [10,11]. On the other hands Kalliom aki et al. reported that, TLR2 mRNA expression was significantly lower in untreated and treated celiac patients, while TLR9 mRNA expression was higher only in untreated subjects compared to controls [12].

The MAP kinase, tumor progression locus 2 (Tpl2), activates the Akt/mTOR/S6 ribosomal protein pathway, inhibits Foxp3 expression and limits Treg immunosuppressive functions.

Abstract The serine-threonine kinase, tumor progression locus 2 (Tpl2 or Map3k8/Cot), is a potent inflammatory mediator that drives the production of TNF alpha, IL-1 beta, and IFN gamma. We previously demonstrated that Tpl2 modulates TCR signaling and T helper cell differentiation. However, little is known about how Tpl2 alters the development or function of regulatory T cells (Tregs). Tregs are a specialized subset of T cells that express Foxp3 and possess immunosuppressive properties. Because of Tpl2’s role in promoting inflammation, we hypothesized that Tpl2 inhibits Treg development and immunosuppressive functions. Tpl2−/− naïve CD4 T+ cells preferentially differentiated into Foxp3+ inducible Tregs in vitro as well as in vivo in a murine model of OVA-induced systemic tolerance. Treg biasing in Tpl2−/− T cells depended upon the TCR signal strength and correlated with reduced activation of the Akt/mTOR/S6 pathway. Importantly, freshly isolated Tpl2−/− Tregs had increased expression of Foxp3 and immunosupressive cytokines IL-10 and IL-35 and were more suppressive in a T cell transfer model of colitis, as evidenced by reduced accumulation of inflammatory T effectors, systemic inflammatory cytokines TNF, IL-6 and IFN gamma, and colonic inflammation. Therefore, Tpl2 promotes inflammation in part by limiting Foxp3 expression, iTreg differentiation and immunosuppressive functions. These findings suggest that Tpl2 inhibition may be used to preferentially drive Treg induction and limit inflammation in autoimmune diseases.

Genetic polymorphisms of cell adhesion molecules in Behcet's disease in a Chinese Han population.

Cell adhesion molecules (CAMs) are involved in various immune-mediated diseases. This study was conducted to investigate the association of single nucleotide polymorphisms (SNPs) of CAMs with Behçet’s disease (BD) in a Chinese Han population. A two-stage association study was carried out in 1149 BD patients and 2107 normal controls. Genotyping of 43 SNPs was performed using MassARRAY System (Sequenom), polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and TaqMan SNP assays. The expression of CD6 and CD11c was examined by real-time PCR and cytokine production was measured by ELISA. A significantly higher frequency of the CT genotype, and a lower frequency of the CC genotype and C allele of CD6 rs11230563 were observed in BD as compared with controls. Analysis of CD11c rs2929 showed that patients with BD had a significantly higher frequency of the GG genotype and G allele, and a lower frequency of the AG genotype as compared with controls. Functional experiments showed an increased CD11c expression and increased production of TNF-α and IL-1beta by LPS stimulated PBMCs in GG carriers of CD11c rs2929 compared to AA/AG carriers. Our study provides evidence that CD6 and CD11c are involved in the susceptibility to BD in a Chinese Han population.

MP24-06 IN VIVO ROLE OF NLRP3 INFLAMMASOME IN INNATE UROTHELIAL DEFENSES AGAINST UROPATHOGENIC E. COLI

You have accessJournal of UrologyInfections/Inflammation/Cystic Disease of the Genitourinary Tract: Kidney & Bladder I1 Apr 2016MP24-06 IN VIVO ROLE OF NLRP3 INFLAMMASOME IN INNATE UROTHELIAL DEFENSES AGAINST UROPATHOGENIC E. COLI Yan Liu, Feng He, Ellen Shapiro, Herbert Lepor, and Xue-Ru Wu Yan LiuYan Liu More articles by this author , Feng HeFeng He More articles by this author , Ellen ShapiroEllen Shapiro More articles by this author , Herbert LeporHerbert Lepor More articles by this author , and Xue-Ru WuXue-Ru Wu More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.762AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The NLRP3 inflammasome is a multi-protein complex consisting of a pattern recognition receptor (NLRP3), an adaptor (ASC) and caspase 1. It is a critical integrator of innate host defenses in myeloid and non-myeloid cells by promoting the maturation of proinflammatory cytokines such as interleukin 1beta and 18. To date, no study has been reported regarding the in vivo role of NLRP3 inflammasome in urinary tract infection (UTI) caused by uropathogenic E. coli (UPEC). METHODS Experimental bladder infection was elicited in wild-type female mice through transurethral inoculation of clinical cystitis isolates, UTI89 and NU14. Formation of speck in urothelium, urine IL 1beta and cleaved caspase 1 in urothelium, hallmarks of NLRP3 inflammasome activation, were determined using immunofluorescence staining, ELISA and Western blotting, respectively. Studies were extended into NLRP3 and caspase 1 knockout (KO) mice, using UPEC strains as well as E. coli strains isolated from human asymptomatic bacteriuria (ABU; ABU83972 and ABU35) and recurrent UTI (C055 and C001). Additionally, Toll-like receptor 4 (TLR4) KO mice were analyzed to ascertain whether TLR4 status affects NLRP3 inflammasome activation. RESULTS Transurethral inoculation of UPEC strains UTI89 and NU14, but not laboratory strain AAEC191A or FimH-deletion strain NU14-1, led to urothelial speck formation, increased urine IL 1beta secretion and urothelial pro-caspase 1 cleavage. These three NLRP3 inflammasome activation markers peaked by 6 hours and subsided by 12 hours after UPEC inoculation. Such responses were significantly blunted in NLRP3 KO mice and even more so in caspase 1 KO mice. The decreased NLRP3 inflammasome response in these KO mice inversely correlated with significantly increased UPEC retention in the bladder. Whereas the two ABU strains elicited only 1/5 of urinary IL 1beta in wild-type mice, compared with the UPEC strains, the two strains from recurrent UTI elicited as much IL 1beta response as the two cystitis strains. Finally, markedly decreased IL 1beta production and secretion by urothelium was observed in TLR4 KO mice compared with wild-type mice, suggesting that lipopolysaccharide/TLR4 interaction is crucial for NLRP3 inflammasome activation in the urothelium. CONCLUSIONS Our data demonstrate for the first time that UPEC can activate inflammasomes in the urothelial cells via the NLRP3 pathway and suggest that reduced activity of this signaling pathway may predispose the urinary tract to recurrent infections by uropathogenic E. coli. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e272 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Yan Liu More articles by this author Feng He More articles by this author Ellen Shapiro More articles by this author Herbert Lepor More articles by this author Xue-Ru Wu More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Calcium pyrophosphate dihydrate crystals induce IL-1beta production by monocytes through ATP release- and ROS production-dependent pathways – involvement of potassium efflux

Calcium pyrophosphate dihydrate crystals induce IL-1beta production by monocytes through ATP release- and ROS production-dependent pathways – involvement of potassium efflux

The Voltage-Gated Potassium Channel Kv1.3 as a Target for Inhibiting Detrimental M1 Microglia Functions

Kv1.3 was first discovered in human T cells in 1984 and has since then been pursued as a potential target for immunosuppression. It was later shown to be overexpressed in effector memory T cells, suggesting that Kv1.3 blockers would be particularly useful for the treatment of T cell mediated autoimmune diseases like multiple sclerosis, psoriasis and rheumatoid arthritis. In fact, the Kv1.3 blocking peptide ShK-186 has recently been found to be effective in Phase-1b study for psoriatic arthritis. We hypothesized that Kv1.3 blockers might also be useful for reducing microglia activation in ischemic stroke and other neurological diseases accompanied by neuroinflammation. Starting with cultured microglia, we observed that Kv1.3 expression is up-regulated in pro-inflammatory M1-like microglia and that Kv1.3 blockers preferentially reduce the production of IL-1beta and TNF-alpha without affecting IL-10 production. In organotypic hippocampal slices exposed to hypoxia/aglycemia Kv1.3 blockers significantly reduced microglia activation and increased neuronal survival. We further observed strong Kv1.3 expression on iNOS expressing inflammatory microglia in human stroke biopsies validating Kv1.3 as potential target. In both mouse and rat models of ischemic stroke the small molecule Kv1.3 blocker PAP-1 significantly reduced infarct area and improved neurological deficit 7 days after reperfusion when administered 12 hours after reperfusion. In the mouse model, PAP-1 selectively reduced brain levels of the inflammatory cytokines IL1-beta and IFN-gamma without affecting IL-10 and BDNF. Based on these findings we propose Kv1.3 inhibitors as potential therapeutic agents for preferentially inhibiting pro-inflammatory M1 microglia functions in ischemic stroke and other neurological diseases.Supported by GM076063 and AG043788.

Localized External Beam Radiation Therapy (EBRT) to the Pelvis Induces Systemic IL-1Beta and TNF-Alpha Production: Role of the TNF-Alpha Signaling in EBRT-Induced Fatigue.

Prostate cancer patients undergoing localized external beam radiation therapy (EBRT) can experience a progressive increase in fatigue, which can affect physical functioning and quality of life. The purpose of this study was to develop a mouse EBRT prostate cancer treatment model with which to determine the role of pro-inflammatory cytokines in the genesis of EBRT-related fatigue. We assessed voluntary wheel-running activity (VWRA) as a proxy for fatigue, food intake and body weight in male C57BL/6 mice undergoing EBRT to the pelvis. In the first experiment, anesthetized male C57BL/6 mice underwent fractionated EBRT to the pelvis for a total dose of 68.2 Gy, thereby mimicking a clinically relevant therapeutic dose and frequency. The day after the last treatment, levels of IL-1β and TNF-α in plasma along with mRNA levels in liver, colon and whole brain were measured. EBRT-induced fatigue resulted in reduced body weight, diminished food intake, and increased plasma and tissue levels of IL-1β and TNF-α. In a follow-up experiment, we used TNF-α-deficient mice to further delineate the role of TNF-α signaling in EBRT-induced sickness behavior. EBRT-induced changes in fatigue, food intake and body weight were no different between TNF-α deficient mice and their wild-type counterparts. Taken together our data demonstrate that a clinically relevant localized irradiation of the pelvis induces a systemic IL-1β and TNF-α response and sickness behavior in mice, but the TNF-α signaling pathway alone does not independently mediate these effects.

Total flavonoids of Bidens bipinnata L. ameliorate experimental adjuvant-induced arthritis through induction of synovial apoptosis.

BackgroundBidens bipinnata are widely distributed in China, which have been widely used as a traditional Chinese medicine. The aim of this study was to examine the effect of total avonoids of Bidens pilosa L. (TFB) on adjuvant arthritis (AA) and its possible mechanisms.MethodsThe macroscopic scoring of paw edema, secondary paw swelling, and polyarthritis index were measured. Histological examination of the joints and the serum concentrations of IL-6, IL-1beta, and TNF-alpha were examined. Apoptosis in synovial tissue was detected. The expression of Caspase 3 cleavage, serves as a marker undergoing apoptosis, was confirmed by Western blot.ResultsTFB attenuated the severity of arthritis on paw edema, hind paw volume, and polyarthritis index of AA rats, improved the histological status in AA rats as well. TFB can inhibit the production of IL-6, IL-1beta, and TNF-alpha from serum. Clear DNA ladder formation was observed in DNA extraction of synovium from TFB treated AA rats. The number of apoptosis was increased with TFB treatment in TUNEL assay. TFB treatment on AA rats significantly increased the expression of Caspase 3 in synovium.ConclusionsOur data suggest that TFB has a signicant anti-arthritic effect in AA through the induction of apoptosis in synovial.

Manumycin A downregulates release of proinflammatory cytokines from TNF alpha stimulated human monocytes

Macrolide antibiotics such as azithromycin or clarithromycin are known to have potent anti-inflammatory and immunomodulatory effects but these properties cannot be widely used due to a risk of bacterial resistance. We studied another polyketide antibiotic, structurally related manumycin A known as a streptomycete derived farnesyltransferase inhibitor with limited antibacterial effects, with respect to its potential regulation of mRNA expression of several genes associated with proinflammatory responses. Downregulation of mRNA for IL-6, TLR-8, IL-1 beta and IL-10 was found in THP-1 cells after 4h stimulation with TNF alpha in the presence of manumycin A and downregulated TLR-8 and EGR-1 genes were observed after 8h. Among the genes upregulated in response to manumycin were HMOX-1, TNFRSF10A, IL-1R1, TICAM2, NLRP12 after 4h and only IL-1R1 after 8h.Furthermore, manumycin A was found to inhibit IL-1beta, IL-6, and IL-8 production in TNF alpha stimulated THP-1 cells and peripheral blood monocytes in a dose dependent manner (0.25–1μM of manumycin A) without affecting cell viability. Cell viability of blood monocytes decreased by about 30% at manumycin A doses of 2–5μM. Manumycin A also inhibited IL-18 release from THP-1 cells, while in cultures of blood monocytes, this cytokine was not detectable.That manumycin A mediated downregulation of proinflammatory genes in human monocytes confirmed by a measurement of cytokine levels in culture supernatants, together with a very limited effect on cell viability, might suggest potential anti-inflammatory properties of this polyketide antibiotic.

T93: Immune system contributes to the efficacy of cancer chemotherapy

Background Risk of metastasis formation is provided by both tumor cell biological characteristics and the microenvironment features within the primary tumor along with local and systemic conditions for metastatic niche formation. The inflammatory infiltration has been shown to strongly impact on tumor progression (Whiteside, 2013). Dronca et al. (2011) showed that immunosuppressive factors in the tumor microenvironment may impair not only local immune responses but also disturb systemic immunity. Zitvogel et al. anticipate that the comprehension of the mechanisms governing the immunogenicity of cell death will have a profound impact on the design of anticancer therapies.To study the impact of immune system on clinical response to neoadjuvant chemotherapy and metastasis-free survival in breast cancer patients. Materials and methods 350 patients with newly diagnosed invasive breast cancer treated with neoadjuvant chemotherapy (NAC) were enrolled into the study. The procedures were made in accordance with the Helsinki Declaration. Clinical response to chemotherapy, the 5-year metastasis-free survival and all major clinical and morphological parameters were determined. The original method of multidimensional data visualization was applied to present the immune system state as integral entirety in visual image for classification of patients with different risk of metastasis (NovoSpark Corporation, Canada). Copy number aberrations (CNA) of cytokine gene regions in tumor specimens were tested using high-density microarray platform CytoScanTM HD Array (Affymetrix, USA). Cytokine gene polymorphism was analyzed. Subpopulations of lymphocytes and macrophages were determined within the primary tumors by IHC. Results We found, that favorable clinical immediate response to preoperative chemotherapy was related to the high levels of IL-1beta, TNF-alpha and IL-10 production by peripheral mononuclear cells before the treatment. This correlation was further confirmed by data from the study on association between cytokine gene functional polymorphism and response to NAC. We used NovoSpark Corporation visualization approach allowing the representation the immune system state as integral unit and to discriminate breast cancer patients with high and low risk of haematogeneic metastasis. When estimated before cancer treatment, 95% of breast cancer patients had risk of metastasis. The neoadjuvant chemotherapy and surgical tumor removal reduced the risk of tumor progression to 62–71%. However, in a year after adjuvant chemo- and radiotherapy, the patient group with high risk of metastases increased to 81% again. Thus, the cancer treatment can change the primarily estimated outcome prognosis in breast cancer patients, and the monitoring of immune system is a promising approach to predict the risk of cancer progression or resistance to the therapy. We have found the connection between the profile of intra-tumor inflammatory elements and chemotherapy efficacy.Cytokine gene expression may be influenced by the chromosome anomalies (CNA – Copy Number Aberration) – deletion and amplification – of cytokine gene loci in tumor cells. We found the close relation between the clinical response to NAC and gain of function of IL-10 and CHI3L1 (YKL40) genes. In contrast, loss of TNF-alpha and IL-17 gene function due to corresponding CNA was associated with good response to NAC. Metastasis-free survival of breast cancer patients was shown to be closely related to CNA. Conclusion The parameters of the activation of systemic and intra-tumoral immune system by growing tumor and its dissemination have to be validated in order to identify the new prognostic markers for the efficiency of the neoadjuvant chemotherapy.

Monocyte Phenotype and Polyfunctionality Are Associated With Elevated Soluble Inflammatory Markers, Cytomegalovirus Infection, and Functional and Cognitive Decline in Elderly Adults.

Monocytes are mediators of the inflammatory response and include three subsets: classical, intermediate, and nonclassical. Little is known about the phenotypical and functional age-related changes in monocytes and their association with soluble inflammatory biomarkers, cytomegalovirus infection, and functional and mental decline. We assayed the activation ex vivo and the responsiveness to TLR2 and TLR4 agonists in vitro in the three subsets and assessed the intracellular production of IL1-alpha (α), IL1-beta (β), IL-6, IL-8, TNF-α, and IL-10 of elderly adults (median 83 [67-90] years old;n= 20) compared with young controls (median 35 [27-40] years old;n= 20). Ex vivo, the elderly adults showed a higher percentage of classical monocytes that expressed intracellular IL1-α (p= .001), IL1-β (p= .001), IL-6 (p= .002), and IL-8 (p= .007). Similar results were obtained both for the intermediate and nonclassical subsets and in vitro. Polyfunctionality was higher in the elderly adults. The functionality ex vivo was strongly associated with soluble inflammatory markers. The activation phenotype was independently associated with the anti-cytomegalovirus IgG levels and with functional and cognitive decline. These data demonstrate that monocytes are key cell candidates for the source of the high soluble inflammatory levels. Our findings suggest that cytomegalovirus infection might be a driving force in the activation of monocytes and is associated with the functional and cognitive decline.

Abstract LB-266: Suppression of colitis-associated colon carcinogenesis by the natural triterpenoid celastrol

Abstract Celastrol is a natural triterpenoid isolated from the medicinal herb Triptyrgium wilfordii, commonly known as the Thunder of God vine. Celastrol has been shown to have a broad range of anti-inflammatory and anti-cancer activities. Here we investigated whether celastrol administered as a dietary supplement can inhibit both colitis and colitis-associated colon cancer (CAC) in either genetic or chemically-induced CAC mouse models. As a genetic and spontaneous, inflammatory-driven intestinal neoplasia model, we generated a Smad4TKO / p27Kip1-/- (DKO) mouse in which a germ line p27Kip1 gene deletion (p27Kip1-/-) was combined with the T cell-restricted deletion of the Smad4 gene (Smad4TKO). All DKO mice developed spontaneous gastrointestinal inflammation and colon carcinoma by 3 months of age. Administration of celastrol as a dietary supplement (2 mg/day) to DKO mice increased survival and suppressed intestinal epithelial neoplasia by decreasing production of inflammatory mediators, including TNF-alpha, IL-6, IL-1beta and iNOS. We investigated the chemopreventive activity of celastrol in the chemically induced CAC mouse model using dextran sulfate sodium (DSS)/azoxymethane (AOM). Mice (C57/BL6, ages 6-8 weeks) received intraperitoneal injections with 10 mg/kg AOM, followed by exposure to 2% DSS in drinking water for 7 days. We repeated 3 cycles of 2% DSS, with 10 days of normal drinking water provided between cycles. Mice were either maintained on control diet or given celastrol (2 mg/day) diet beginning with first administration of DSS and continuing until termination of the experiment. Mice receiving celastrol diet showed less loss in body weight compared with the control diet group and marked protection from AOM/DSS-induced colon cancer. The incidence and number of colonic tumors in mice in the celastrol diet group were also significantly lower than those in control diet group. These results were accompanied by decreased expression of iNOS and lower production of pro-inflammatory cytokines. Our data demonstrate the protective effects of celastrol in the chemoprevention of CAC, was and this effect correlated with a dampening of iNOS expression and with a decrease of inflammatory cytokine production. These results highlight the potential of celastrol as an effective agent for the chemoprevention of CAC in humans. Citation Format: Sung Hee Choi, Emily Baker, Byung-Gyu Kim, Gregory Tochtrop, John J. Letterio. Suppression of colitis-associated colon carcinogenesis by the natural triterpenoid celastrol. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-266. doi:10.1158/1538-7445.AM2015-LB-266

The role of interleukin-1 beta in the pathophysiology of Schnitzler's syndrome.

IntroductionSchnitzler’s syndrome (SchS) is a disabling autoinflammatory disorder, characterized by a chronic urticarial rash, an M-protein, arthralgia, and other signs of systemic inflammation. Anti-interleukin-1 (IL-1) beta antibodies are highly effective, but the pathophysiology is still largely unknown. Here we studied the effect of in-vivo IL-1 inhibition on serum markers of inflammation and cellular immune responses.MethodsEight patients with SchS received monthly subcutaneous (s.c.) injections with 150 mg canakinumab for six months. Blood was drawn for measurement of serum markers of inflammation (12 times per patient) and for functional and phenotypic analysis of both freshly isolated and toll-like receptor (TLR)-ligand-stimulated peripheral blood mononuclear cells (PBMCs) (five times per patient). All data were compared to results of healthy controls.ResultsIL-6 levels in serum and in lysates of freshly isolated PBMCs and serum myeloid-related protein (MRP8)/14 and S100A12 levels correlated with disease activity. In vitro, LPS stimulation resulted in higher IL-6 and IL-1 beta production in PBMCs from symptomatic SchS patients compared to healthy controls, whereas patient cells were relatively hyporesponsive to poly:IC and Pam3Cys. The mRNA microarray of PBMCs showed distinct transcriptomes for controls, symptomatic patients and anti-IL-1-treated patients. Numbers of T- and B-cell subsets as well as M-protein concentrations were not affected by IL-1 inhibition. Free light chain levels were elevated in 4 out of 8 patients.ConclusionsIn conclusion, patient PBMCs are hyperresponsive to LPS, and clinical efficacy of IL-1 beta inhibition in patients with SchS is associated with in-vivo and ex-vivo suppression of inflammation. Interestingly, patient PBMCs showed divergent responses to TLR2/6, TLR3 and TLR4 ligands. Our data underscore that IL-1 beta plays a pivotal role in SchS.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0696-0) contains supplementary material, which is available to authorized users.

In Vitro infection of human dura-mater fibroblasts with Staphylococcus aureus: colonization and reactive production of IL-1beta

Objective:Post-operative meningitis, caused mainly by Staphylococcus aureus and Gram-negative rods, is a life-threatening complication after neurosurgery, and its pathogenesis is far from clear. The purpose of this work was to study the experimental infection of human dura-mater fibroblasts and whole human dura by S. aureus.Methods:In vitro cultures of human dura-mater fibroblasts and organotypic cultures of small pieces of human dura mater were inoculated with a human-derived S. aureus strain. The pattern of bacterial infection as well as cytokines secretion by the infected fibroblasts was studied.Results:Our results suggest that colonisation of human dura-mater fibroblasts in culture and whole dura-mater tissue by S. aureus includes bacterial growth on the cell surface, fibroblast intracellular invasion by bacteria and a significant synthesis of interleukin 1beta (IL-1beta) by the infected cells.Conclusion:This is the first report of human dura-mater fibroblast infection by S. aureus. Hopefully, these results can lead to a better understanding of the pathogenesis of meningitis caused by this bacterial species and to a more rational therapeutic approach.

Abstract O.21: Inositol 1,4,5 triphosphate 3- kinase C regulates NLRP3 Inflammasome activation in Kawasaki disease

Background: ITPKC controls calcium homeostasis and was identified in genome-wide studies to be associated with susceptibility and severity of KD. NLRP3 is activated by danger signals, leading to inflammasome activation and release of IL-1beta and IL-18. Ca 2+ mobilization mediates NLRP3 activation. We studied the contribution of ITPKC and NLRP3 inflammasome activation in KD. Methods: Cytokine levels were measured using ELISA. Gene expression was assayed using Illumina HumanHT-12v4. EBV-transformed B-cell lines from KD patients with different ITPKC genotypes were used to study the gene and protein expression and Ca 2+ flux using qRT-PCR, Western blot analysis, and FACS respectively. In vivo and in vitro analyses from ITPKC-deficient and wildtype mice were performed using ELISA, spinning disc confocal and Westerns, to determine cytokine release, Ca 2+ flux and protein expression responses respectively. Results: Children with KD show increased circulating IL-1beta, IL-18, IL-1RA and IL18BP protein during acute phase of KD (KD n=48, febrile controls n=41 p<0.001). NLRP3 and its associated inflammasome complex, and the IL-1[[Unsupported Character - Symbol Font ]] and IL18 receptor genes were up-regulated nearly two fold during the acute KD compared to matched convalescent controls (n=171, p<0.001). Pathway analysis showed specific up-regulation of NLRP3 related genes. The KD-associated genetic polymorphism in ITPKC (rs28493229) is functional, directly modulating [Ca 2+ ] i mobilization resulting in NLRP3 activation and increased production of IL-1beta and IL-18, as demonstrated by gene expression, circulating protein levels and functional assays of PBMCs and immortalized cell lines from affected children. These biologic data are partnered with evidence showing resistance to IVIG therapy in those with the ITPKC polymorphism associated with the highest basal and stimulated [Ca 2+ ] i levels. Studies using ITPKC-deficient mice in an animal model of KD support the molecular, cellular and clinical findings in affected children. Significance: ITPKC regulates NLRP3 activation via control of [Ca 2+ ] i mobilization, directing production of IL-1beta and IL-18, pointing to the key role of calcium mobilization in the immunopathogenesis of KD.

190-POS

Objectives Preeclampsia (PE) is a specific disorder of pregnancy associated with hyperuricemia, higher levels of pro-inflammatory cytokines, enhanced leukocyte activation and oxidative stress. Adenosine deaminase (ADA) is an enzyme present in cells of the immune system, and is considered an indicator of cellular inflammation. This study aimed to compare serum levels of ADA with TNF-alpha and IL-1beta produced by peripheral blood mononuclear cells (PBMC) in pregnant women with PE, in normotensive pregnant (NT) and in non-pregnant (NP) women. Methods Sixty women with PE and 30 NT paired by gestational age were evaluated. Twenty healthy NP women matched for age with pregnant women were included as a control group. PBMC were obtained from the three groups studied and cultured with or without lipopolysaccharide (LPS) for 18h at 37 °C, and TNF-alpha and IL-1beta production was assessed in the supernatant of cultures by enzyme-linked immunosorbent assay. ADA and uric acid serum concentrations were determined by colorimetric methods. Differences between groups were analyzed by non-parametric tests with a significance level of 5%. Results Serum levels of ADA were significantly higher in PE group compared with NT and NP groups. A positive correlation between ADA and uric acid levels was detected in preeclamptic women ( r = 0.4082; p = 0.0024). There was no significant difference in relation to ADA levels when PE patients were classified in early and late-onset PE. The endogenous production of IL-1beta and TNF-alpha by PBMC was significantly higher in PE group than in NT and NP women, showing the activation state of these cells in PE. Conclusions The increased levels of ADA associated with hyperuricemia and proinflammatory cytokines suggest that this enzyme plays an important role in the systemic inflammation observed in PE. Disclosures V.S. Giorgi: None. V.T. Borges: None. S.S. Witkin: None. C.F. Bannwart-Castro: None. J.C. Peracoli: None. M.T. Peracoli: None.