IL-1β In Culture Supernatants Research Articles

Toxoplasma gondii infection and peripheral-blood gene expression profiling of older people reveals dysregulation of cytokines and identifies hub genes as potential therapeutic targets

Infections of humans with the protozoan parasite Toxoplasma gondii (T. gondii) can lead to the disease's development, even in an asymptomatic status. However, the mechanisms that result in these clinical outcomes after infection are poorly understood. This study aimed to explore the molecular pathogenesis of toxoplasmosis-related inflammation through next-generation sequencing, to assess RNA expression profiles in peripheral blood from 5 female patients with chronic toxoplasmosis and 5 healthy female controls. All plasma samples were analyzed for anti-Toxoplasma IgG and IgM antibody titers by using electrochemiluminescence. Detection of acute and chronic toxoplasmosis was carried out using the ELISA IgG avidity. We evaluated the levels of INF-γ, IL-2, IL-12, TNF-α, IL-10, and IL-1β in culture supernatants of Peripheral Blood Mononuclear Cells infected with Toxoplasma lysate antigen (TLA) prepared with tachyzoites of strain T. gondii RH. Differential expression analysis was performed using DESeq2, pathway and enrichment analysis of DEGs was done on WEB-based Gene SeT AnaLysis Toolkit (WebGestalt) and Protein-protein interaction was carried out using NetworkAnalyst with STRING. In older people with chronic asymptomatic infection, a significant difference in the levels of inflammatory cytokines INF-γ and IL-2 was observed compared to seronegative individuals. Our results revealed differences in the regulation of critical biological processes involved in host responses to chronic T. gondii infection. Gene ontology analysis revealed several biologically relevant inflammatory and immune-related pathways.

Bitis arietans Snake Venom and Kn-Ba, a Snake Venom Serine Protease, Induce the Production of Inflammatory Mediators in THP-1 Macrophages.

Bitis arietans is a snake of medical importance found throughout sub-Saharan Africa and in savannas and pastures of Morocco and western Arabia. The effects of its venom are characterized by local and systemic alterations, such as inflammation and cardiovascular and hemostatic disturbances, which can lead to victims’ death or permanent disability. To better characterize the inflammatory process induced by this snake’s venom, the participation of eicosanoids and PAF (platelet- activating factor) in this response were demonstrated in a previous study. In addition, edema and early increased vascular permeability followed by an accumulation of polymorphonuclear (PMN) cells in the peritoneal cavity were accompanied by the production of the eicosanoids LTB4, LTC4, TXB2, and PGE2, and local and systemic production of IL-6 and MCP-1. In this context, the present study focused on the identification of inflammatory mediators produced by human macrophages derived from THP-1 cells in response to Bitis arietans venom (BaV), and Kn-Ba, a serine protease purified from this venom. Here, we show that Kn-Ba, and even the less intensive BaV, induced the production of the cytokine TNF and the chemokines RANTES and IL-8. Only Kn-Ba was able to induce the production of IL-6, MCP-1, and IP-10, whereas PGE2 was produced only in response to BaV. Finally, the release of IL-1β in culture supernatants suggests the activation of the inflammasomes by the venom of Bitis arietans and by Kn-Ba, which will be investigated in more detail in future studies.

Maternal high-fat diet exaggerates diet-induced insulin resistance in adult offspring by enhancing inflammasome activation through noncanonical pathway of caspase-11

ObjectiveMaternal high-fat diet (HFD) has been shown to promote the development of insulin resistance (IR) in adult offspring; however, the underlying mechanisms remain unclear. MethodsEight-week-old female wild-type mice (C57BL/6) were fed either an HFD or a normal diet (ND), one week prior to mating, and the diet was continued throughout gestation and lactation. Eight-week-old male offspring of both groups were fed an HFD for 8 weeks. ResultsOffspring of HFD-fed dams (O-HFD) exhibited significantly impaired insulin sensitivity compared with the offspring of ND-fed dams (O-ND). The adipocyte size of the eWAT increased significantly in O-HFD and was accompanied by abundant crown-like structures (CLSs), as well as a higher concentration of interleukin 1β (IL-1β) in the eWAT. Treatment with an inflammasome inhibitor, MCC950, completely abrogated the enhanced IR in O-HFD. However, ex vivo caspase-1 activity in eWAT revealed no difference between the two groups. In contrast, noncanonical inflammasome activation of caspase-11 was significantly augmented in O-HFD compared with O-ND, suggesting that membrane pore formation, but not cleavage of pro-IL-1β by caspase-1, is augmented in O-HFD. To examine the membrane pore formation, we performed metabolic activation of bone marrow-derived macrophages (BMDMs). The percentage of pore formation assessed by ethidium bromide staining was significantly higher in BMDMs of O-HFD, accompanied by an enhanced active caspase-11 expression. Consistently, the concentration of IL-1β in culture supernatants was significantly higher in the BMDMs from O-HFD than those from O-ND. ConclusionsThese findings demonstrate that maternal HFD exaggerates diet-induced IR in adult offspring by enhancing noncanonical caspase-11-mediated inflammasome activation.

5220Maternal high-fat diet exaggerates the development of diet-induced insulin resistance in adult offspring by enhancing pyroptosis through augmented gasdermin D-mediated pore formation

Abstract Background Maternal high-fat diet (HFD) has been shown to promote the development of insulin resistance (IR) in adult offspring; however, the underlying mechanisms remain unclear. Approach and results Eight-week-old female wild-type mice (C57BL/6) were fed a HFD or normal diet (ND) one week prior to mating, and received during pregnancy and lactation. Eight-week-old male offspring of both groups were fed a HFD for 8 weeks. Offspring of HFD-fed dams (O-HFD) showed significantly enhanced IR compared with offspring of ND-fed dams (O-ND). There was no difference in body weight, epidydimal white adipose tissue (eWAT) weight, and cumulative caloric intake between the 2 groups. However, eWAT adipocyte size was significantly increased in O-HFD, accompanied by the abundant crown-like structures. Flow cytometric analysis revealed an increased percentage of M1, but not M2, macrophages. Serum and eWAT concentrations of IL-1β, but not TNF-α, were significantly higher in O-HFD than O-ND (3.7-fold and 2.0-fold, respectively, P<0.05). Treatment with NLRP3 inflammasome inhibitor MCC950 completely abrogated the enhanced IR in O-HFD to a similar extent of that in O-ND, although IR was modestly, but not significantly, ameliorated in O-ND even after MCC950 treatment. Consistent with in vivo findings, in vitro polarization of bone marrow-derived macrophages (BMDMs) did not show any difference in TNF-α mRNA expression after conventional stimulation. In contrast, palmitate acid (PA)-mediated metabolic activation of BMDMs following LPS priming showed a significantly higher concentration of IL-1β in culture supernatants from O-HFD (45%, P<0.05). However, protein expression levels of NLRP-3, ASC, and procaspase-1 after LPS priming were equivalent between the 2 groups. Consistently, intracellular flow cytometric analysis of caspase-1 activity after PA activation did not show any difference, which was compatible with the finding that ex vivo caspase-1 activity of eWAT assessed by fluorescent image of IVIS revealed no difference between the 2 groups. To further examine the mechanism of augmented IL-1β release in BMDM of O-HFD, we examined the cleavage of caspase substrate gasdermin D (GSDMD) and subsequent pore formation. Protein and gene expression levels of GSDM-D after LPS priming were significantly higher in O-HFD (50% and 381%, respectively, P<0.05). At 2 hrs after PA stimulation following LPS priming, cleaved GSDM-D was significantly increased in O-HFD (80%, P<0.01). Consistently, percentage of pore formation assessed by ethidium bromide staining was significantly higher in O-HFD (60%, P<0.05), while LDH release could not be observed. Conclusions Our findings demonstrate that maternal HFD exaggerates diet-induced insulin resistance in adult offspring by enhancing pyroptosis through augmented GSDM-D-mediated pore formation.

ABRO1 promotes NLRP3 inflammasome activation through regulation of NLRP3 deubiquitination.

Deubiquitination of NLRP3 has been suggested to contribute to inflammasome activation, but the roles and molecular mechanisms are still unclear. We here demonstrate that ABRO1, a subunit of the BRISC deubiquitinase complex, is necessary for optimal NLRP3-ASC complex formation, ASC oligomerization, caspase-1 activation, and IL-1β and IL-18 production upon treatment with NLRP3 ligands after the priming step, indicating that efficient NLRP3 activation requires ABRO1. Moreover, we report that ABRO1 deficiency results in a remarkable attenuation in the syndrome severity of NLRP3-associated inflammatory diseases, including MSU- and Alum-induced peritonitis and LPS-induced sepsis in mice. Mechanistic studies reveal that LPS priming induces ABRO1 binding to NLRP3 in an S194 phosphorylation-dependent manner, subsequently recruiting the BRISC to remove K63-linked ubiquitin chains of NLRP3 upon stimulation with activators. Furthermore, deficiency of BRCC3, the catalytically active component of BRISC, displays similar phenotypes to ABRO1 knockout mice. Our findings reveal an ABRO1-mediated regulatory signaling system that controls activation of the NLRP3 inflammasome and provide novel potential targets for treating NLRP3-associated inflammatory diseases.

JMJD3 is involved in neutrophil membrane proteinase 3 overexpression during the hyperinflammatory response in early sepsis

Excessive production of pro-inflammatory cytokines in early sepsis causes high early mortality rates. Membrane proteinase 3 (mPR3) expression on neutrophils plays a critical role in pro-inflammatory cytokine production. However, the mechanism underlying mPR3 overexpression in early sepsis is unknown. Here, we explored mPR3 expression in early sepsis and its regulatory mechanism. Thirty-two patients with sepsis and 20 healthy controls were prospectively enrolled. On day 1 after the onset of sepsis, mPR3 and jumonji domain-containing protein D3 (JMJD3) expression levels were measured in peripheral blood neutrophils. Lipopolysaccharide (LPS) was employed to induce JMJD3 expression in vitro, and GSK-J4 was used to inhibit JMJD3. Neutrophils were divided into four groups, control, LPS, LPS + GSK-J4, and GSK-J4, and cultured with THP-1 cells respectively. Plasma and culture supernatant cytokine levels were measured by enzyme-linked immunosorbent assays. Neutrophil mPR3 levels were significantly higher in patients with early sepsis than in healthy controls. Plasma cytokine (IL-1β and TNF-α) levels were increased in patients with sepsis exhibiting high mPR3 expression. Additionally, JMJD3 expression levels in neutrophils were increased in early sepsis. In vitro, both mPR3 on neutrophils and IL-1β in culture supernatants increased in response to LPS stimulation. Neutrophil mPR3 expression and IL-1β levels were significantly reduced by GSK-J4 in cells treated with LPS. IL-1β level was significantly higher in LPS-stimulated co-culture supernatants than in the corresponding individual cultured cells. Thus, our results suggest that JMJD3 contributes to the high expression of neutrophil mPR3, which promotes the production of proinflammatory IL-1β in early sepsis.

Matrine suppresses AGE-induced HAEC injury by inhibiting ROS-mediated NRLP3 inflammasome activation

Diabetes is characterized by a high level of advanced glycation end products (AGEs), which induce damage in the arterial endothelium. Matrine has been shown to have cardioprotective effects. This study's aim was to investigate the protective effects and possible molecular mechanisms of matrine in AGE-induced human aortic endothelial cell (HAEC) injury. Cultured HAECs were treated with AGEs and/or serially diluted matrine. Cell viability was evaluated by MTT assay. Intracellular reactive oxygen species production was determined by flow cytometry. The expression levels of nucleotide-binding, leucine-rich repeat, and pyrin-domain-containing 3 (NLRP3), adaptor molecule apoptosis-associated speck-like protein, cleaved caspase-1 and interleukin-1β (IL-1β) were assessed by Western blotting; the concentration of IL-1β in culture supernatants was determined by enzyme-linked immunosorbent assay (ELISA). In a concentration-dependent manner, matrine co-treatment with AGEs substantially inhibited the reduction in cell viability and the increase in intracellular reactive oxygen species induced by AGEs. Co-treatment with matrine significantly inhibited the AGE-induced increase in NLRP3, ASC, caspase-1, p20 and IL-1β expression in HAECs in a concentration-dependent manner. Moreover, the AGE-mediated increase in IL-1β expression in cell culture supernatants was also reduced by co-treatment with matrine in a concentration-dependent manner. AGEs induced HAEC injury by inducing reactive oxygen species -mediated NLRP3 inflammasome activation. Matrine recovered HAEC viability by inhibiting reactive oxygen species -mediated NLRP3 inflammasome activation.

Effects of hydrogen sulfide on the secretion of cytokines in macrophages of deep partial-thickness burn wound in rats

To analyze the effects of exogenous hydrogen sulfide on the secretion of growth factors basic fibroblast growth factor (bFGF) and transforming growth factor β1 (TGF-β1), as well as inflammatory mediators tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) in macrophages of deep partial-thickness burn wound in rats. Seventy-eight SD rats were divided into normal control group (n=6), pure burn group, sodium hydrosulfide group, propargylglycine (PPG) group, and sodium hydrosulfide+ PPG group according to the random number table, with 18 rats in each of the latter four groups. Rats in normal control group did not receive any treatment, while rats in the other four groups were inflicted with 5% total burn surface area deep partial-thickness scald (hereinafter referred to as burn) on the back. Immediately after burn, rats in pure burn group, sodium hydrosulfide group, and group PPG were intraperitoneally injected with saline 2 mL/kg, sodium hydrosulfide 56 μmol/kg, and PPG 45 mg/kg respectively, while those in sodium hydrosulfide+ PPG group were intraperitoneally injected with sodium hydrosulfide 56 μmol/kg and PPG 45 mg/kg, once a day till the day before harvesting specimen. Six rats of normal control group fed for one week, and 6 rats from each of the rest four groups on post injury day (PID) 3, 7, 14 were collected respectively. Normal skin on the back of rats in normal control group and tissue in the base of wound of rats in the other four groups were harvested to isolate macrophages, and then the content of bFGF, TGF-β1, TNF-α, and IL-1β in culture supernatant of macrophages was detected with enzyme-linked immunosorbent assay. Data were processed with one-way analysis of variance, analysis of variance of factorial design, and LSD test. Compared with that of normal control group [(42.6±2.5) and (18±4) pg/mL], the content of bFGF and TGF-β1 in culture supernatant of macrophages of rats in pure burn group was obviously increased at each time point (with P values below 0.01), peaking on PID 14 at (141.6±7.7) and (580±16) pg/mL respectively. Compared with that of pure burn group, the content of bFGF and TGF-β1 in culture supernatant of macrophages of rats in sodium hydrosulfide group was obviously increased at each time point (with P values below 0.01), peaking on PID 14 at (193.7±10.9) and (793±12) pg/mL respectively, while the content of bFGF and TGF-β1 in culture supernatant of macrophages of rats in group PPG was obviously decreased at each time point (with P values below 0.01), reaching the nadir on PID 3 at (62.0±7.1) and (170±10) pg/mL respectively. The content of bFGF and TGF-β1 in culture supernatant of macrophages of rats in sodium hydrosulfide+ PPG group was obviously lower than that of sodium hydrosulfide group but obviously higher than that of group PPG at each time point (with P values below 0.01), peaking on PID 14 at (151.3±9.0) and (579±9) pg/mL respectively. Compared with that of normal control group [(97±6) and (31±6) pg/mL], the content of TNF-α and IL-1β in culture supernatant of macrophages of rats in pure burn group was obviously increased at each time point (with P values below 0.01), peaking on PID 3 at (924±8) and (290±10) pg/mL respectively. Compared with that of pure burn group, the content of TNF-α and IL-1β in culture supernatant of macrophages of rats in sodium hydrosulfide group was obviously decreased at each time point (with P values below 0.01), reaching the nadir on PID 14 at (346±10) and (120±5) pg/mL respectively, while the content of TNF-α and IL-1β in culture supernatant of macrophages of rats in group PPG was obviously increased at each time point (with P values below 0.01), peaking on PID 3 at (1 232±13) and (410±10) pg/mL respectively. The content of TNF-α and IL-1β in culture supernatant of macrophages of rats in sodium hydrosulfide+ PPG group was obviously higher than that of sodium hydrosulfide group but obviously lower than that of group PPG at each time point (with P values below 0.01), reaching the nadir on PID 14 at (488±16) and (144±6) pg/mL respectively. Supplementation of exogenous hydrogen sulfide in small dosage can increase the secretion of growth factors bFGF and TGF-β1 in macrophages of wound in rats with deep partial-thickness burn in the early stage and reduce the release of inflammatory mediators TNF-α and IL-1β in the meantime, thus affecting the healing of wound.

Enhanced activity of NLRP3 inflammasome in peripheral blood cells of patients with active rheumatoid arthritis

IntroductionInterleukin-1β (IL-1β) is a major inflammatory cytokine, produced predominantly by innate immune cells through NLRP3-inflammasome activation. Both intrinsic and extrinsic danger signals may activate NLRP3. Genetic variations in NLRP3-inflammasome components have been reported to influence rheumatoid arthritis (RA) susceptibility and severity. We sought to assess the activity of NLRP3-inflammasome in patients with active RA compared to healthy individuals.MethodIntracellular protein expression of NLRP3, ASC, pro- and active caspase-1, pro- and active IL-1β was assessed by immunoblotting both at baseline and upon inflammasome activation. NLRP3 function (IL-1β secretion) was assessed upon priming of TLR2 (Pam(3)CysSK(4), TLR3 (poly(I:C)) or TLR4 (LPS) and ATP sequential treatment. We used caspase inhibitors (casp-1, 3/7 and 8) to assess their contribution to IL-1β maturation. All experiments were performed in whole blood cells.ResultsActive RA patients (n = 11) expressed higher basal intracellular levels of NLRP3 (p < 0.008), ASC (p < 0.003), active caspase-1 (p < 0.02) and pro-IL-1β (p < 0.001). Upon priming with TLR4 (LPS) and ATP, RA-derived cell extracts (n = 7) displayed increased expression of NLRP3 (p < 0.01) and active caspase-1 (p < 0.001). Secreted IL-1β in culture supernatants from whole blood cells activated with TLR4 (LPS) or TLR3 agonist (poly(I:C)) plus ATP was higher in RA patients (n = 20) versus controls (n = 18) (p < 0.02 for both). Caspase-1 inhibition significantly reduced IL-1β secretion induced by all stimuli, whereas caspase-8 inhibition affected only TLR4 and TLR3 cell priming.ConclusionPatients with active RA have increased expression of NLRP3 and NLRP3-mediated IL-1β secretion in whole blood cells upon stimulation via TLR3 and TLR4 but not TLR2. In these patients, IL-1β secretion seems to be predominately driven by caspase-1 and caspase-8. Targeting NLRP3 or downstream caspases may be of benefit in suppressing IL-1β production in RA.

Role of non-receptor tyrosine kinase Tec in the production of pro-inflammatory cytokines from macrophages induced by endotoxin/lipopolysaccharide

To investigate the role of non-receptor tyrosine kinase Tec in the production of TNF-α and IL-1β from macrophages induced by LPS and its related mechanism. RAW264.7 mononuclear-macrophages cultured in 6-well plates were divided into 4 groups according to the random number table, with 24 wells in each group. Cells in blank group were routinely cultured (cultured with DMEM medium containing 10% FBS) for 2 hours. Cells in LFM-A13 group were pretreated with 75 µmol/L Tec specific inhibitor LFM-A13 for 1 hour and then routinely cultured for 1 hour. Cells in LPS group were routinely cultured for 1 hour and then treated with 0.1 µg/mL LPS for 1 hour. Cells in LPS+LFM-A13 group were pretreated with 75 µmol/L LFM-A13 for 1 hour and then treated with 0.1 µg/mL LPS for 1 hour. The content of TNF-α and IL-1β in culture supernatant of cells was determined with ELISA. The mRNA expressions of TNF-α and IL-1β in cells were assayed with real-time fluorescent quantitative RT-PCR. The activity of intracellular Tec, p38 MAPK, and transforming growth factor activated kinase 1 (TAK1) was determined with Western blotting. Data were processed with one-way analysis of variance and LSD test. The content of TNF-α and IL-1β in culture supernatant of cells in LFM-A13 group was close to that in blank group (with P values above 0.05). The mRNA expressions of TNF-α and IL-1β in the cells of LFM-A13 group were close to those of blank group (with P values above 0.05). The content of TNF-α and IL-1β in culture supernatant of cells in LPS group was respectively (1 213 ± 154) and (636 ± 90) pg/mL, which was higher than that in blank group [(330 ± 44) and (211 ± 31) pg/mL, with P values below 0.01]. The mRNA expressions of TNF-α and IL-1β in the cells of LPS group were respectively 1.57 ± 0.22 and 1.44 ± 0.24, which were significantly higher than those of blank group (1.00 ± 0.18 and 1.00 ± 0.19, with P values below 0.01). The content of TNF-α and IL-1β in culture supernatant of cells in LPS+LFM-A13 group was respectively (787 ± 109) and (453 ± 64) pg/mL, which was significantly lower than that in LPS group (with P values below 0.05). The mRNA expressions of TNF-α and IL-1β in the cells of LPS+LFM-A13 group were respectively 1.21 ± 0.15 and 1.21 ± 0.22, and they were significantly lower than those of LPS group (with P values below 0.05). The activity of intracellular Tec, TAK1, and p38 MAPK of cells in LPS+LFM-A13 group was close to that in blank group (with P values above 0.05). The activity of intracellular Tec, TAK1, and p38 MAPK of cells in LPS group was respectively 2.69 ± 0.41, 3.99 ± 0.65, and 2.07 ± 0.31, which was significantly higher than that in blank group (1.00 ± 0.17, 1.00 ± 0.16, and 1.00 ± 0.18, with P values below 0.01) and LPS+LFM-A13 group (1.02 ± 0.17, 1.18 ± 0.20, and 1.58 ± 0.28, P < 0.05 or P < 0.01). Tec promotes the production and release of pro-inflammatory cytokines TNF-α and IL-1β from macrophages induced by LPS via TAK1-p38 MAPK signaling pathway.

3,4-Methylenedioxy-β-nitrostyrene Inhibits NLRP3 Inflammasome Activation by Blocking Assembly of the Inflammasome

The NLRP3 inflammasome is a critical component of the innate immune system. NLRP3 activation is induced by diverse stimuli associated with bacterial infection or tissue damage, but its inappropriate activation is involved in the pathogenesis of inherited and acquired inflammatory diseases. However, the mechanism by which NLRP3 is activated remains poorly understood. In this study, we explored the role of kinases in NLRP3 inflammasome activation by screening a kinase inhibitor library and identified 3,4-methylenedioxy-β-nitrostyrene (MNS) as an inhibitor for NLRP3 inflammasome activation. Notably, MNS did not affect the activation of the NLRC4 or AIM2 (absent in melanoma 2) inflammasome. Mechanistically, MNS specifically prevented NLRP3-mediated ASC speck formation and oligomerization without blocking potassium efflux induced by NLRP3 agonists. Surprisingly, Syk kinase, the reported target of MNS, did not mediate the inhibitory activity of MNS on NLRP3 inflammasome activation. We also found that the nitrovinyl group of MNS is essential for the inhibitory activity of MNS. Immunoprecipitation, mass spectrometry, and mutation studies suggest that both the nucleotide binding oligomerization domain and the leucine-rich repeat domain of NLRP3 were the intracellular targets of MNS. Administration of MNS also inhibited NLRP3 ATPase activity in vitro, suggesting that MNS blocks the NLRP3 inflammasome by directly targeting NLRP3 or NLRP3-associated complexes. These studies identified a novel chemical probe for studying the molecular mechanism of NLRP3 inflammasome activation which may advance the development of novel strategies to treat diseases associated with abnormal activation of NLRP3 inflammasome.

Comparison of the Interleukin-1β-Inducing Potency of Allergenic Spores from Higher Fungi (Basidiomycetes) in a Cryopreserved Human Whole Blood System

Background: Spores from basidiomycete fungi (basidiospores) are highly prevalent in the atmosphere of urban and rural settings. Studies have confirmed their potential to affect human health as allergens. Less is known about their potential to serve as stimuli of the innate immune system and induce proinflammatory reactions. Methods: In this study, we evaluated the proinflammatory potential of spores from 11 allergenic basidiomycete species (gilled: Pleurotus ostreatus,Oudemansiella radicata,Armillaria tabescens,Coprinus micaceus,Pluteus cervinus, and Chlorophyllum molybdites, and nongilled: Pisolithus arhizus,Merulius tremellosus,Calvatia cyathiformis,Lycoperdon pyriforme, andBoletus bicolor) based on their potency to induce the release of the proinflammatory cytokine interleukin (IL)-1β in a cryopreserved human whole blood system. In addition, the roles of morphological features of the spores (surface area, shape, and pigmentation) were examined for their role in the IL-1β-including potency of spores. Peripheral blood from healthy volunteers was collected, pooled, and cryopreserved. After stimulating the cryopreserved pooled blood with 10⁶ to 10³ basidiospores/ml, the concentration of IL-1β in culture supernatants was determined with ELISA. Results: Basidiospores manifested concentration-dependent IL-1β-inducing potency, which was more marked among basidiospores from gilled basidiomycetes. At higher concentrations of basidiospores, the IL-1β-inducing potency could be differentiated in the cryopreserved human whole blood system. Morphological features did not correlate with the IL-1β-inducing potency of the basidiospores, suggesting that nonmorphological properties modulate the IL-1β-inducing potency. Conclusion: Our data provide evidence of the proinflammatory potential of basidiospores, and the utility of cryopreserved human whole blood as a human-based in vitro system to study the immune reactivity of allergenic basidiospores.

THU0255 The Efficacy of Dmards Combination Therapy Using MTX and Low Dose Tacrolimus Depend on Modulation of MRP2 Expression.

BackgroundMethotrexate (MTX) is an anchor drug for the treatment of rheumatoid arthritis (RA) because of efficacy and safety. Although MTX is used in monotherapy generally, combination therapy with biologics or...

Octanoylated Ghrelin Inhibits the Activation of the Palmitic Acid-Induced TLR4/NF-κB Signaling Pathway in THP-1 Macrophages

To investigate the effect of acylated ghrelin on the activation of TLR4/NF-κB signaling pathway induced by palmitic acid in human monocyte-derived (THP-1) macrophages, THP-1 macrophages were cultured for 12 h by palmitic acid with various concentrations. The THP-1 macrophages was pretreated by acylated ghrelin at different doses for 4 h before cultivated by palmitic acid (200 μmol/L) for 12 h. We observed the level of TLR4, NF-κB p65 phosphorylation in THP-1 macrophages and TNF-α, IL-1β in culture supernatant. TLR4 mRNA was measured by real-time PCR. TLR4 protein and NF-κB p65 phosphorylation was measured by western blotting. The expression of TNF-α and IL-1β was detected by ELISA. Compared to the THP-1 macrophages without palmitic acid, the level of TLR4 mRNA protein and NF-κB p65 phosphorylation and the expression of TNF-α and IL-1β increased after treatment by palmitic acid in a dose-dependent fashion (P < 0.05). Compared to the THP-1 macrophages with palmitic acid (200 μmol/L), the level of the pervious substances decreased after preadministration by acylated ghrelin in a dose-dependent fashion. So, we make a conclusion that acylated ghrelin can regulate the activation of TLR4/NF-κB signaling pathway and inhibit the release of inflammatory cytokines in THP-1 macrophages which are stimulated by palmitic acid in a dose-dependent fashion.

Naloxone Inhibition of Lipopolysaccharide-Induced Activation of Retinal Microglia is Partly Mediated via the p38 Mitogen Activated Protein Kinase Signalling Pathway

To investigate the effects and underlying mechanism of action of naloxone on lipopolysaccharide (LPS)-induced activation of retinal microglia in vitro. Rat retinal microglia primary cultures were divided into four treatment groups: untreated; 1 μg/ml LPS for 12 h; 0.5, 1.0 or 2.0 μM naloxone for 30 min before LPS; 2.5 or 5.0 μM SB203580 for 12 h before LPS and naloxone. Levels of tumour necrosis factor (TNF)-α and interleukin (IL)-1β were determined by enzyme-linked immuno sorbent assay. Western blot analysis and double immunofluorescence were used to examine activation of the mitogen activated protein kinase (MAPK) signalling pathway. LPS induced an increase in TNF-α and IL-1β in culture supernatants, which was dose-dependently inhibited by naloxone. Naloxone also dose-dependently inhibited LPS-induced increases in phosphorylated p38 MAPK. Pretreatment of cells with SB203580 attenuated the inhibitory effect of naloxone on TNF-α and IL-1β production. Naloxone suppressed LPS-induced activation of cultured retinal microglia and this suppression appeared to occur partly through the p38 MAPK signalling pathway. Naloxone may have therapeutic potential in neuro degenerative diseases characterized by the activation of microglia.

Carica papaya increases regulatory T cells and reduces IFN‐γ+CD4+ T cells in healthy human subjects

Fruit and vegetables have therapeutic potential as they dampen inflammation, have no known side-effects and as whole foods have prospective additive and synergistic benefits. Th1 (IFN-γ(+) CD4(+))/Th2 (IL-4(+)CD4(+)) T cells play a vital role in mediating inflammatory responses and may be regulated by regulatory T cells (Tregs). Effects of Carica papaya on cells of healthy individuals were determined using flow cytometry methods. Significant down-regulation of IFN-γ(+) CD4(+) (p=0.03, n=13), up-regulation of IL-4(+) CD4(+) (p=0.04, n=13) T cells and up-regulation of CD3(+) CD4(+) CD25(+) CD127(-) (p=0.001, n=15) Tregs were observed after papaya consumption. In vitro cultures showed up-regulation of Tregs in male subjects and was significantly associated with levels of IL-1β in culture supernatants (R(2) =0.608, p=0.04, n=12). Other inflammatory cytokines were significantly suppressed. Papaya consumption may exert an anti-inflammatory response mediated through Tregs and have potential in alleviating inflammatory conditions.

Characterisation of a monoclonal antibody to carp IL-1β and the development of a sensitive capture ELISA

A carp IL-1β gene was identified from a subtraction hybridisation technology based cDNA library from activated carp leucocytes. This gene was cloned into pQE vector carrying 6xHis tag and the protein was expressed. Recombinant IL-1β was used to produce hybridomas specific for carp IL-1β. Monoclonal antibodies were purified by affinity column and a sandwich ELISA for IL-1β was developed with a detection limit of 10ng of the recombinant protein. Using the capture ELISA, the presence of native IL-1β in culture supernatant of PHA-stimulated leucocytes from carp was identified, which was confirmed by SDS-PAGE and Western blot. Since IL-1β is known to stimulate proliferation of T&B cells and macrophages, its ability to stimulate proliferation of carp leucocytes was studied using tritiated thymidine. The recombinant protein was found to significantly stimulate proliferation of head kidney and spleen cells from carp.