Secretion Levels Of IL-1β Research Articles

Type I Cystatin Derived from Cysticercus pisiformis-Stefins, Suppresses LPS-Mediated Inflammatory Response in RAW264.7 Cells.

Cysticercus pisiformis is a kind of tapeworm larvae of Taenia pisiformis, which parasitizes the liver envelope, omentum, mesentery, and rectum of rodents such as rabbits. Cysteine protease inhibitors derived from helminth were immunoregulatory molecules of intermediate hosts and had an immunomodulatory function that regulates the production of inflammatory factors. Thus, in the present research, the recombinant Stefin of C. pisiformis was confirmed to have the potential to fight inflammation in LPS-Mediated RAW264.7 murine macrophages. CCK8 test showed that rCpStefin below 50 μg/mL concentration did not affect cellular viability. Moreover, the NO production level determined by the Griess test was decreased. In addition, the secretion levels of IL-1β, IL-6, and TNF-α as measured by ELISA were decreased. Furthermore, it exerted anti-inflammatory activity by decreasing the production of proinflammatory cytokines and proinflammatory mediators, including IL-1β, IL-6, TNF-α, iNOS, and COX-2 at the gene transcription level, as measured by qRT-PCR. Therefore, Type I cystatin derived from C. pisiformis suppresses the LPS-Mediated inflammatory response of the intermediate host and is a potential candidate for the treatment of inflammatory diseases.

Immunoenhancement effect of cinobufagin on macrophages and the cyclophosphamide-induced immunosuppression mouse model

Cinobufagin (CBG) is a natural active substance. Although its various pharmacological activities have been explored, the immunomodulatory activity of CBG remains unexplored. Therefore, this study aimed to investigate the anti-inflammatory and immunomodulatory activities of CBG ex vivo and in vivo. The immunomodulatory activity of CBG was investigated in RAW 264.7 cells. CBG showed no significant toxicity to cells. Additionally, 0.5–8 μg/mL CBG significantly increased the phagocytosis ability of macrophages and the secretion levels of IL-1β and TNF-α. Thus, it exerted immunomodulatory effects. We established the immunosuppressive model induced by cyclophosphamide (CTX) in mice and studied the immunomodulatory activity of CBG in vivo. The experimental results showed that the intervention of CBG alleviated the CTX-induced weight loss, restored the lymphocyte nuclear cell number, and promoted the secretion and mRNA expression of cytokines IFN-γ, IL-4, IL-6, and IL-12. Moreover, CBG increased the immune organ index, protected the growth of the spleen and thymus, and improved the pathological changes in immunosuppressed mice. Western blot results showed that different concentrations of CBG upregulated the phosphorylation level of PI3K/Akt/mTOR in the spleen of CTX-induced immunosuppressed mice. This suggests that the immunomodulatory effect of CBG may be related to the regulation of PI3K/Akt/mTOR signaling pathway. This study provides a theoretical basis for developing CBG immune enhancers and opens up new ideas for the comprehensive utilization and development of CBG in factories.

Phytochemical analysis and in vitro investigation of wound healing, cytotoxicity, and inflammatory response in potentially active extracts of Anogeissus dhofarica

The traditional medicine plant Anogeissus dhofarica is only found in the Jabal Fartak region of Yemen and the Dhofar region of Oman. This plant has been used traditionally for medical purposes to treat wounds, skin tanning, and sores. People use it as an antiseptic, especially women. However, this plant has not been studied in great detail, and there is no scientific proof to back up its traditional uses of it. The goal of this study was to examine the biological properties and phytochemical analysis of various extracts made from A. dhofarica leaves. The powdered leaves of A. dhofarica were extracted using the solvents ethanol (A1), methanol (A3), acetone (A5), and ethyl acetate (A8), yielding concentrations of 3, 0.3, and 0.03 mg/ml, respectively. The total phenolic and flavonoid contents, as well as the active phytochemicals, were determined using standard methods. The proliferation potential and IC50 of human dermal fibroblasts were evaluated. Cytotoxicity, wound healing activity, and the inflammatory response to the extracts were all investigated. Our results revealed that the IC50 values for plant extracts A1, A3, A5, and A8 were 9.799 ± 0.07, 5.83 ± 0.9, 8.8 ± 0.12, and 3.98 ± 0.8 mg/ml, respectively. Moreover, the cytotoxic effect of plant extracts was observed at high concentrations (p <0.05). However, the lower concentration showed the least cytotoxic effect. For the inflammatory response, the secretion levels of IL-1β, IL-6, TNF-α, IL-8, and IL-12p70 were upregulated, and IL-10 was downregulated significantly (p < 0.05) compared to the control. For wound healing activity, 0.3 and 0.03 mg/ml of (A8) extract and 0.03 mg/ml of (A3) and (A5) extracts showed complete closure of the scratch after 72 hours of treatment. In this study, we noticed that A. dhofarica plant extracts with a lower concentration are the most effective at healing wounds and have a minimal cytotoxic effect.

Inflammatory Status of Monocytes in Type 2 Diabetes Mellitus

Сhronic inflammation is considered as a key factor in the development of type 2 diabetes mellitus. Impaired tolerance of the inflammatory response of monocytes is regarded as an important mechanism in the pathogenesis of chronic inflammation. In this work, we study the inflammatory activation and tolerance of the immune response of monocytes in diabetes. In total, 40 patients with newly diagnosed diabetes and 40 control group participants were included in the study. The level of basal, LPS-stimulated and re-stimulated secretion of the TNF-α, IL-1β, and MCP-1 cytokines was assessed in a monocyte culture isolated from the blood by immunomagnetic separation of CD14+ cells. The level of basal, LPS-stimulated and re-stimulated TNF-α secretion was significantly higher in patients with diabetes; the level of IL-1β secretion did not differ significantly between the groups; basal and re-stimulated MCP-1 secretion was also significantly higher in the diabetes group. Re-stimulated secretion of TNF-α and IL-1β was reduced compared to primary-stimulated secretion in both groups, demonstrating the tolerance of the macrophage immune response to these cytokines. Re-stimulated secretion of MCP-1 in 42% of diabetes patients was higher than primary stimulated secretion, thus revealing an impaired tolerance of the immune response of macrophages. A correlation was found between TNF-α secretion and body mass index, r=0.631, p<0.001, and with glycemic level, r=0.427, p=0.037. The results obtained demonstrate inflammatory activation of monocytes with hypersecretion of TNF-α and MCP-1, impaired tolerance of the immune response of monocytes in diabetes regarding the secretion of MCP-1, as well as a correlation of TNF-α secretion with body mass index and glycemic level. This indicates an important role of TNF-α and MCP-1 in the pathogenesis of chronic inflammation in type 2 diabetes, thus allowing these cytokines to be considered as potential therapeutic targets for pathogenetic therapy of type 2 diabetes.

Identification of potential miR‑155 target genes in epidermal immune microenvironment of atopic dermatitis patients and their inflammatory effects on HaCaT cells.

Atopic dermatitis (AD) is a common inflammatory skin condition and the leading cause of morbidity associated with skin conditions worldwide. For the majority of patients, AD is a lifelong disease that cannot be cured completely. Therefore, in the present study, differentially expressed genes (DEGs) in the epidermal immune microenvironment were screened using bioinformatic techniques. Subsequently, an in vitro cellular model was constructed to investigate the role of microRNA (miR)-155 in immune infiltration during AD. In the present study, two datasets (GSE121212 and GSE157194) were downloaded from Gene Expression Omnibus, before the DEGs were screened and subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes functional enrichment analyses. miRNet was used to predict the possible target genes of miR-155 among the differentially expressed genes found. Consequently, peptidase inhibitor 3 (PI3), FOS-like 1, AP-1 transcription factor subunit (FOSL1), C-X-C motif chemokine ligand (CXCL)1 and CXCL8 were selected to be the potential target genes of miR-155 in the epidermal immune microenvironment of patients with AD. Concurrently, an inflammatory cell model using HaCaT cells was constructed by TNF-α and IFN-γ treatment. The effects of miR-155 on HaCaT cell proliferation and secretion of IL-1β, IL-6, IL-10, IL-15, PI3, FOSL1, CXCL1 and CXCL8 under inflammatory and non-inflammatory conditions were then analyzed. The results showed that after the HaCaT cells were transfected with miR-155, miR-155 inhibited HaCaT cell proliferation and decreased the mRNA expression levels of PI3 and CXCL8, increased the mRNA levels of FOSL1 and secretion levels of IL-1β, IL-6, IL-15 and CXCL1. By contrast, miR-155 decreased the secretion levels of IL-10 and CXCL8. In the inflammatory cell model of HaCaT cells, miR-155 was found to significantly inhibit the proliferation of HaCaT cells during inflammation whilst significantly increasing the secretion of IL-1β, IL-6, IL-10 and IL-15. In addition, miR-155 increased the mRNA expression and secretion levels of CXCL1 and CXCL8, whilst also increasing the mRNA expression levels of PI3. Results from the current study suggest that miR-155 can stimulate keratinocytes to produce inflammatory cytokines and proteins to enhance the inflammatory response in AD.

The NLRP3 inflammasome recognizes alpha-2 and alpha-7.3 giardins and decreases the pathogenicity of Giardia duodenalis in mice

BackgroundGiardia duodenalis is a parasitic organism that can cause giardiasis, an intestinal infection, particularly prevalent in young children, with clinical symptoms of diarrhea. We previously reported that extracellular G. duodenalis triggers intracellular nucleotide-binding oligomerization-like receptor 3 (NLRP3) inflammasome activation and regulates the host inflammatory response by secreting extracellular vesicles (EVs). However, the exact pathogen-associated molecular patterns in G. duodenalis EVs (GEVs) involved in this process and the role of the NLRP3 inflammasome in giardiasis remain to be elucidated.MethodsRecombinant eukaryotic expression plasmids of pcDNA3.1(+)-alpha-2 and alpha-7.3 giardins in GEVs were constructed, transfected into primary mouse peritoneal macrophages and screened by measuring the expression levels of the inflammasome target molecule caspase-1 p20. The preliminary identification of G. duodenalis alpha-2 and alpha-7.3 giardins was further verified by measuring the protein expression levels of key molecules of the NLRP3 inflammasome (NLRP3, pro-interleukin-1 beta [IL-1β], pro-caspase-1, and caspase-1 p20), the secretion levels of IL-1β, the level of apoptosis speck-like protein (ASC) oligomerization and the immunofluorescence localization of NLRP3 and ASC. The roles of the NLRP3 inflammasome in G. duodenalis pathogenicity were then evaluated using mice in which NLRP3 activation was blocked (NLRP3-blocked mice), and body weight, parasite burden in the duodenum and histopathological changes in the duodenum were monitored. In addition, we explored whether alpha-2 and alpha-7.3 giardins triggered IL-1β secretion in vivo through the NLRP3 inflammasome and determined the roles of these molecules in G. duodenalis pathogenicity in mice.ResultsAlpha-2 and alpha-7.3 giardins triggered NLRP3 inflammasome activation in vitro. This led to caspase-1 p20 activation, upregulation of the protein expression levels of NLRP3, pro-IL-1β and pro-caspase-1, significant enhancement of IL-1β secretion, ASC speck formation in the cytoplasm and also induction of ASC oligomerization. Deletion of the NLRP3 inflammasome aggravated G. duodenalis pathogenicity in mice. Compared to wild-type mice gavaged with cysts, mice gavaged with cysts in NLRP3-blocked mice displayed increased trophozoite loads and severe duodenal villus damage, characterized by necrotic crypts with atrophy and branching. In vivo assays revealed that alpha-2 and alpha-7.3 giardins could induce IL-1β secretion through the NLRP3 inflammasome and that immunization with alpha-2 and alpha-7.3 giardins decreased G. duodenalis pathogenicity in mice.ConclusionsOverall, the results of the present study revealed that alpha-2 and alpha-7.3 giardins trigger host NLRP3 inflammasome activation and decrease G. duodenalis infection ability in mice, which are promising targets for the prevention of giardiasis.Graphical

Type I Cystatin Derived from Fasciola gigantica Suppresses Macrophage-Mediated Inflammatory Responses

There is an inverse relationship between the high incidence of helminth infection and the low incidence of inflammatory disease. Hence, it may be that helminth molecules have anti-inflammatory effects. Helminth cystatins are being extensively studied for anti-inflammatory potential. Therefore, in this study, the recombinant type I cystatin (stefin-1) of Fasciola gigantica (rFgCyst) was verified to have LPS-activated anti-inflammatory potential, including in human THP-1-derived macrophages and RAW 264.7 murine macrophages. The results from the MTT assay suggest that rFgCyst did not alter cell viability; moreover, it exerted anti-inflammatory activity by decreasing the production of proinflammatory cytokines and mediators, including IL-1β, IL-6, IL-8, TNF-α, iNOS, and COX-2 at the gene transcription and protein expression levels, as determined by qRT-PCR and Western blot analysis, respectively. Further, the secretion levels of IL-1β, IL-6, and TNF-α determined by ELISA and the NO production level determined by the Griess test were decreased. Furthermore, in Western blot analysis, the anti-inflammatory effects involved the downregulation of pIKKα/β, pIκBα, and pNF-κB in the NF-κB signaling pathway, hence reducing the translocation from the cytosol into the nucleus of pNF-κB, which subsequently turned on the gene of proinflammatory molecules. Therefore, cystatin type 1 of F. gigantica is a potential candidate for inflammatory disease treatment.

Effects of n-butanol extract of Pulsatilla decoction on the NLRP3 inflammasome in macrophages infected with Candida albicans

Ethnopharmacological relevancePulsatilla decoction is a traditional Chinese medicine from Shang Han Lun that has been reported to have therapeutic efficacy in vulvovaginal candidiasis (VVC), and is a growth inhibitor of Candida albicans (C. albicans) in vitro, the causative agent of VVC.Aim of the study: In previous studies, Pulsatilla decoction has shown therapeutic benefits for VVC. With further chemical extraction of the decoction, the n-butanol extract (of Pulsatilla decoction; BEPD) was most effective against C. albicans and therapeutic for VVC. The mechanism, however, has not been elucidated. The regulation of NOD-like receptor protein 3 (NLRP3) inflammasome has recently been demonstrated as a critical component of the inflammasome complex that initiates the vaginal inflammatory response. Therefore, the effect of BEPD on NLRP3 associated with VVC was investigated. Materials and methodsUltra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used for detecting the principal compounds of BEPD (Anemoside B4, Esculin, esculetin, Epiberberine, Berberine, Jatrorrhizine and Phellodendrine). BEPD-containing serum is prepared by intragastric administration of BEPD (4.6875 g/kg for seven days) in rats. PMA-induced THP-1 cells were challenged with C. albicans. The expression of CD68 to identify macrophages was examined by flow cytometry, the viability of THP-1 cells were assessed by CCK8 assay, the release of lactate dehydrogenase (LDH) was detected by LDH kit, and the secretion levels of IL-1β and IL-18 were evaluated through enzyme-linked immunosorbent assay (ELISA), the levels of NLRP3 were quantified by immunofluorescence, the levels of reactive oxygen species (ROS) were measured by ROS kit, and the expression of Dectin-1, Syk, TLR2, TLR4, MyD88, NF-κB, NLRP3, Caspase-1, and ASC proteins were detected by Western blot. ResultsAfter administration of BEPD-containing serum, the levels of IL-1β, IL-18 and LDH released from macrophages were reduced in the BEPD-containing serum group compared to the model group. In addition, BEPD-containing serum inhibited the expression of ROS in macrophages, suppressed the expression of NLRP3 and inhibited the expression of TLRs/MyD88 and Dectin-1/Syk signaling pathway-related proteins. ConclusionsBEPD suppressed the NLRP3 inflammasome and related signaling pathways in macrophages infected with C. albicans in vitro, thereby providing insight into the mechanism of BEPD action on VVC.

Oral Administration of Bacterial β Cell Expansion Factor A (BefA) Alleviates Diabetes in Mice with Type 1 and Type 2 Diabetes.

Diabetes mellitus (DM) is a group of metabolic diseases, and there is an urgent need to develop new therapeutic DM oral drugs with fewer side effects and sound therapeutic efficacy. In this study, a β cell expansion factor A (BefA) production strain of Escherichia coli (BL21-pet 28C-BefA) was constructed, and the antidiabetes effect of BefA was evaluated using type 1 DM (T1DM) and type 2 DM (T2DM) mice models. The T1DM mice results indicated that BefA significantly reduced blood glucose levels; exerted a protective effect on islet β cell morphology; downregulated the expressions of TLR-4, p-NFκB/NFκB, and Bax/Bcl-2, and the secretion levels of IL-1β and TNF-α; increased the expression of PDX-1 protein and insulin secretion in a concentration-dependent manner; and restored the disturbed microbial diversity to normal levels. Similarly with the T1DM mice, BefA obviously increased islet β cells and reduced the inflammatory reaction and apoptosis in T2DM mice, as well as improved liver lipid metabolism by downregulating the expressions of CEBP-α, ACC, and Fasn; inhibited the synthesis of triglycerides; and induced Cpt-1, Hmgcs2, and Pparα in a concentration-dependent manner. In conclusion, BefA alleviates diabetes via increasing the number of islet β cells, reducing the inflammatory reaction and apoptosis, improving liver lipid metabolism, and restoring microbial diversity to normal levels, which provides a new strategy for a DM oral drug.

Enriched Environment Attenuates Pyroptosis to Improve Functional Recovery After Cerebral Ischemia/Reperfusion Injury.

Enriched environment (EE) is a complex containing social, cognitive, and motor stimuli. Exposure to EE can promote functional recovery after ischemia/reperfusion (I/R) injury. However, the underlying mechanisms remained unclear. Pyroptosis has recently been identified and demonstrated a significant role in ischemic stroke. The purpose of this study was to explore the effect of EE on neuronal pyroptosis after cerebral I/R injury. In the current study, middle cerebral artery occlusion/reperfusion (MCAO/R) was applied to establish the cerebral I/R injury model. Behavior tests including the modified Neurological Severity Scores (mNSS) and the Morris Water Maze (MWM) were performed. The infarct volume was evaluated by Nissl staining. To evaluate the levels of pyroptosis-related proteins, the levels of GSDMD-N and nod-like receptor protein 1/3 (NLRP1/3) inflammasome-related proteins were examined. The mRNA levels of IL-1β and IL-18 were detected by Quantitative Real-Time PCR (qPCR). The secretion levels of IL-1β and IL-18 were analyzed by ELISA. Also, the expression of p65 and p-p65 were detected. The results showed that EE treatment improved functional recovery, reduced infarct volume, attenuated neuronal pyroptosis after cerebral I/R injury. EE treatment also suppressed the activities of NLRP1/NLRP3 inflammasomes. These may be affected by inhabiting the NF-κB p65 signaling pathway. Our findings suggested that neuronal pyroptosis was probably the neuroprotective mechanism that EE treatment rescued neurological deficits after I/R injury.

Korean Red Ginseng attenuates ultraviolet-mediated inflammasome activation in keratinocytes

BackgroundKeratinocytes form a physical barrier and act as an innate immune cell in skin. Keratinocytes secrete pro-inflammatory cytokines, such as interleukin (IL)-1β, resulting from inflammasome activation when exposed to ultraviolet (UV) irradiation. Korean Red Ginseng extracts (RGE) have been well-studied as modulators of inflammasome activation in immune cells, such as macrophages. In the study, we elucidated the role of RGE on the UV-mediated inflammasome activation in keratinocytes compared with that in macrophages. MethodsHuman skin keratinocyte cells (HaCaT), human epidermal keratinocytes (HEK), human monocyte-like cells (THP-1), and mouse macrophages were treated with RGE or a saponin fraction (SF) or non-saponin fraction (NS) of RGE before and after UV irradiation. The secretion levels of IL-1β, as an indicator of inflammasome activation, were analyzed. ResultsThe treatment of RGE or SF in macrophages after UV irradiation inhibited IL-1β secretion, but similar treatment in HaCaT cells did not. However, the treatment of RGE or SF in HaCaT cells in the presence of poly I:C, a toll-like receptor (TLR) 3 ligand, before UV exposure elicited the inhibition of the IL-1β secretion. The inhibition was caused by the disruption by RGE or SF of the TLR mediating up-regulation of the pro-IL-1β and NLRP3 genes during the priming step. ConclusionRGE and its saponins inhibit IL-1β secretion in response to UV exposure in both keratinocytes and macrophages. In particular, RGE treatment interrupted only the priming step in keratinocytes, although it did attenuate both the priming and activation steps in macrophages.

Abstract 15736: The Sphk1/s1p Signaling Axis Regulates Pulmonary Vascular Inflammation by Activating Nf-kb and Nlrp3 Inflammasome

Rationale: Pulmonary vascular inflammation is activated during the development of pulmonary arterial hypertension (PAH). Sphingosine kinase 1 (Sphk1) catalyzes the formation of sphingosine-1-phosphate (S1P), which has been shown to promote inflammation by activating nuclear factor kappa B (NF-κB) and the nucleotide oligomerization domain-like receptor 3 (NLRP3) inflammasome. However, the impact of S1P on NLRP3 inflammasome activation in PAH is unknown. Here, we tested whether Sphk1/S1P regulates pulmonary vascular inflammation via activating NF-κB and the NLRP3 inflammasome. Methods: Human pulmonary arterial smooth muscle cells (PASMCs) and endothelial cells (PAECs) were treated with S1P or were exposed to hypoxia. Sphk1 plasmid or siRNA was used to increase and decrease Sphk1/S1P levels, respectively. Activation of NF-κB signaling and the NLRP3 inflammasome (increased NLRP3, cleaved caspase-1, IL-1β and IL-18) were detected by western blots or real-time PCR. ELISA was used to measure secretion of IL-1β. Results: S1P treatment led to a biphasic phosphorylation and degradation of IκBα accompanied by increased phosphorylation and expression of NF-κB in PASMCs. S1P treatment also resulted in increased expression of inflammasome mediators, NLRP3 and cleaved caspase-1, enhanced intracellular expression of IL-1b and IL-18, and higher levels of secreted IL-1β compared to controls. Notably, over expression of Sphk1 in PASMCs led to increased NF-κB and NLRP3 expression, whereas suppression of Sphk1 expression reduced NLRP3, cleavage of IL-1β and IL-18 expression and phosphorylation of NF-κB. Similar phenomena with respect to inflammasome activation and IL-1β secretion was observed in PASMCs exposed to hypoxia. Increased NF-κB and NLRP3 activation, cleavage of caspase-1 were also observed in S1P treated PAECs, however no changes in IL-1β secretion levels were detected. Conclusion: These findings suggest that S1P induces pulmonary vascular pro-inflammatory signaling via activation of NF-κB and the NLRP3 inflammasome, and that the S1P/Sphk1 signaling axis may be involved in this context. Activation of the inflammasome may be an important mechanism underlying PAH pathogenesis, targeting of which may be exploited for the treatment of PAH.

Alteration in the expression of inflammatory cytokines in primary hippocampal astrocytes in response to MK-801 through ERK1/2 and PI3K signals

Our previous study showed that dizocilpine (MK-801) induced schizophrenia-like behavior in rats, enhanced GFAP expression, and activated primary cultured hippocampal astrocytes. Astrocytes play an essential role in neuroinflammation and contribute to the crosstalk that generates chronic neuro-inflammation in neurological diseases. However, the effects of MK-801 treatment on astrocytic neuroinflammatory responses and its mechanism of action have not been studied in detail. To address this issue, IL1β, IL6, TNFα and IL10 expression and secretion levels were evaluated in hippocampal astrocytes in response to MK-801 for 24 h by ELISA and real-time PCR, with and without pretreatment of either the ERK1/2 inhibitor, PD98059 or the PI3K inhibitor, LY294002. Cell apoptosis, viability, and proliferation were also examined. MK-801 treatment did not induce hippocampal astrocytes apoptosis or proliferation, however, MK-801 enhanced astrocytes viability. Additionally, the expression and secretion levels of IL1β, IL6 and TNFα were elevated, but that of IL10 was decreased, in which ERK1/2 and PI3K signals were involved. These findings suggest that hippocampal astrocytes may regulate the expressions of inflammatory cytokines through ERK1/2 and PI3K signaling pathway to participate in the pathogenesis of schizophrenia.

Interleukin-10 administration inhibits TNF-α and IL-1β, but not IL-6, secretion of LPS-stimulated peritoneal macrophages

We hypothesize that exogenous administration of anti-inflammatory cytokine interleukin (IL)-10 would affect the secretion of pro-inflammatory cytokines such as IL-1β, IL-6, or tumor necrosis factor (TNF)-α using lipopolysaccharide (LPS)-stimulated macrophages exuded from the peritoneal cavity of female BALB/c mice. The LPS-stimulated macrophages were incubated for 48 hr to investigate the secretions of pro- and anti-inflammatory cytokine. The results indicated that the secretion levels of IL-1β, IL-6, IL-10, and TNF-α by LPS-stimulated macrophages elevated in a time dependent manner during 48-hr incubation period. The amount of cytokines secretion by LPS-stimulated macrophages varied: IL-6 > TNF-α > IL-10 > IL-1β. However, the secretion of IL-10 started to decrease at 18 hr of incubation. Therefore, the macrophage cultures were extra-administrated with various concentrations (0, 75, 150, and 225 pg/ mL) of exogenous IL-10 after 18-hr incubation in order to evaluate the effect of exogenous IL-10 administration on inflammation. IL-10, 1L-1β, IL-6, and TNF-α in LPS-stimulated macrophage cultures before and after exogenous IL-10 administration were determined by ELISA. The results demonstrated that exogenous IL-10 administration inhibited IL-1β (21.3-38.6%) and TNF-α (44.7-66.8%) secretion. However, the administration did not inhibit IL-6 secretion. Low dose (75 pg/mL) of exogenous IL-10 exerted maximal effects. IL-10 levels in LPS-stimulated macrophage cultures after exogenous IL-10 administration increased from 18.4% to 35.5%. Furthermore, only low dose of exogenous IL-10 administration increased endogenous IL-10 secretion (by 10.4%), while higher doses of exogenous IL-10 inhibited endogenous IL-10 production (from 31.7% to 41.9%). The results suggest that low dose administration of exogenous IL-10 might exhibit antiinflammatory effects via inhibiting TNF-α and IL-1β, but not IL-6, secretion and increasing endogenous IL-10 production by LPS-stimulated peritoneal macrophages.

Mung Bean Protein Hydrolysate Modulates the Immune Response Through NF-κB Pathway in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages.

The objective of this study was to evaluate the immunomodulatory activity of mung bean protein hydrolysate (MBPH) in lipopolysaccharide (LPS)-induced RAW 264.7 cells and discuss the possible immune regulatory mechanism. MBPH was prepared by alcalase, trypsin, neutrase, and flavourzyme. The 3-h alcalase-hydrolyzed hydrolysate with a molecular weight less than 1,450 Da was selected for the immunological tests. Results showed that MBPH possessed strong suppressing activity to proinflammatory mediators in a dose-dependent manner. Compared to the LPS alone group, MBPH (200 µg/mL) significantly reduced nitric oxide (NO), inducible nitric oxide synthase, interleukin (IL)-6, and IL-1β secretion levels by 52.6%, 53.2%, 48.4%, and 49.7%, respectively, in LPS-induced macrophages. It also enhanced IL-10 secretion from 789 to 3,678 pg/mL. MBPH blocked nuclear factor-kappa B (NF-κB) translocation in LPS-induced macrophages through the prevention of IκBα phosphorylation, and this process further prevented p65 translocation into the nucleus. A possible mechanism of MBPH is that it regulated the expression of inflammatory factors via the NF-κB pathway, thus inhibiting inflammatory reactions. The results suggested that MBPH is of application potential in the development of immunomodulatory functional food to ameliorate immunosuppression.

Effects of ultrasonic treatment on the molecular weight and anti-inflammatory activity of oxidized konjac glucomannan

ABSTRACTThe ultrasound-degraded oxidized konjac glucomannan (U-OKGM) was manufactured and its immune activity in RAW264.7 macrophages was examined. Results showed that ultrasound treatment, which was an effective method to obtain low molecular weight OKGM, did not change the main structure of polysaccharides under the test conditions. Molecular weights (MW) of OKGM and U-OKGM are about 1.58 × 106 g/mol and 5.64 × 105 g/mol, respectively. We showed that U-OKGM could inhibit NO secretion and augment phagocytotic activity of RAW264.7 cell, and 0.8 mg/mL U-OKGM reached significant level (p < 0.05). Meanwhile, TNF-α and IL-1β secretion levels were significantly (p < 0.05) decreased by U-OKGM treatments in LPS-stimulated RAW264.7 cells. Moreover, the mRNA transcription of IL-1β, TNF-α, IL-6 and iNOS were significantly (p < 0.05) inhibited by U-OKGM in LPS-activated RAW264.7 macrophages. The results suggest that ultrasonic treatment provides a viable modification to obtain low-molecular-weight polysaccharide, and U-OKGM has strong anti-inflammatory effects on LPS-stimulated RAW264.7 macrophages.

Abstract 509: Fibronectin Containing Extra Domain a Modulates TLR4-Dependent Smooth Muscle Cell Proliferation and Neointimal Hyperplasia in Apolipoprotein E-Deficient Mice

Background: Fibronectin-splice variant containing extra domain A (Fn-EDA), an endogenous ligand of toll-like-receptor 4 (TLR4), is a characteristic feature of proliferating vascular smooth muscle cells (VSMCs) that contributes to intimal thickening and atherosclerosis. Very little is known about the role of Fn-EDA in VSMC proliferation or its clinical implication in neointima formation after vascular injury in the comorbid condition of hyperlipidemia. Methods: To explore the mechanism and therapeutic potential of targeting Fn-EDA in vascular diseases, we used carotid wire-injury model to induce neointimal hyperplasia (28 days) in hyperlipidemic Apoe -/- , Fn-EDA -/- Apoe -/- , TLR4 -/- Apoe -/- , Fn-EDA -/- TLR4 -/- Apoe -/- and SMC- specific Fn-EDA -/- mice (Fn-EDA fl/fl αSMACre + Apoe -/- ) (male and female; 10 weeks old). Furthermore, VSMCs were isolated from different genotypes and stimulated with PDGF to dissect the mechanisms. Results: Using immunostaining, we found increased expression of Fn-EDA in human atheroma, injured carotid artery and proliferating VSMCs of Apoe -/- mice (P&lt;0.05 vs. control). Irrespective of gender, genetic deletion of Fn-EDA in Apoe -/- mice reduced neointimal area after vascular injury (P&lt;0.05 vs. Apoe -/- ). Aortic VSMCs isolated from Fn-EDA -/- Apoe -/- mice exhibited reduced cell proliferation (BrdU assay, G1/S phase transition) and migration (wound healing assay) that was concomitant with elevated pAKT2 and pFOXO4 expression and reduction in pAKT1, pmTOR, pNFκB, TNF-α and IL-1β secretion levels (P&lt;0.05 vs. Apoe -/- ). Genetic ablation of TLR4 in Apoe -/- mice reduced neointimal hyperplasia concomitant with decreased VSMCs proliferation and migration (P&lt;0.05 vs. Apoe -/- ), but had no additive effect in Fn-EDA -/- Apoe -/- mice. Purified cellular Fn, which contains EDA, attenuated pAKT2 and pFOXO4 and potentiated pAKT1, pmTOR, pNFκB, TNF-α and IL-1β secretion in SMCs from Fn-EDA -/- Apoe -/- mice (P&lt;0.05 vs. plasma Fn that lacks Fn-EDA) but not from Fn-EDA -/- TLR4 -/- Apoe -/- mice. Finally, SMC-specific Fn-EDA -/- mice (Fn-EDA fl/fl αSMACre + Apoe -/- ) exhibited reduced neointimal hyperplasia. Conclusion: Fn-EDA modulates TLR4-dependent VSMCs proliferation and intimal hyperplasia after vascular injury in the comorbid condition of hyperlipidemia.

The anti-inflammatory effects of baicalin through suppression of NLRP3 inflammasome pathway in LPS-challenged piglet mononuclear phagocytes.

In this study, the anti-inflammatory effects and mechanisms of baicalin on LPS-induced NLRP3 inflammatory pathway were investigated in piglet mononuclear phagocytes (control, LPS stimulation, LPS stimulation + 12.5 µg/ml baicalin, LPS stimulation + 25 µg/ml baicalin, LPS stimulation + 50 µg/ml baicalin and LPS stimulation + 100 µg/ml baicalin). The levels of reactive oxygen species (ROS), the secretion levels of IL-1β, IL-18 and TNF-α, mRNA expression levels of IL-1β, IL-18, TNF-α and NLRP3, as well as the protein levels of cleaved caspase-1 p20 were significantly increased after LPS-challengein vitro However, LPS stimulation did not influence apoptosis-associated speck-like protein and caspase-1 mRNA levels, which are also components of the NLRP3 inflammasome. Baicalin at 50 µg/ml and 100 µg/ml could inhibit the production of ROS, TNF-α, IL-1β and IL-18, and down-regulate mRNA expression of IL-1β, IL-18, TNF-α and NLRP3, as well as expression of cleaved caspase-1 p20. These results showed that the anti-inflammatory effects of baicalin occurred via the regulation of the release of ROS and mRNA expression of NLRP3. The anti-inflammatory activity of baicalin could be related to the suppression of NLRP3 inflammasome pathway under LPS stimulation.

Capsular Polysaccharide Is Involved in NLRP3 Inflammasome Activation by Klebsiella pneumoniae Serotype K1.

Klebsiella pneumoniae (strain 43816, K2 serotype) induces interleukin-1β (IL-1β) secretion, but neither the bacterial factor triggering the activation of these inflammasome-dependent responses nor whether they are mediated by NLRP3 or NLRC4 is known. In this study, we identified a capsular polysaccharide (K1-CPS) in K. pneumoniae (NTUH-K2044, K1 serotype), isolated from a primary pyogenic liver abscess (PLA K. pneumoniae), as the Klebsiella factor that induces IL-1β secretion in an NLRP3-, ASC-, and caspase-1-dependent manner in macrophages. K1-CPS induced NLRP3 inflammasome activation through reactive oxygen species (ROS) generation, mitogen-activated protein kinase phosphorylation, and NF-κB activation. Inhibition of both the mitochondrial membrane permeability transition and mitochondrial ROS generation inhibited K1-CPS-mediated NLRP3 inflammasome activation. Furthermore, IL-1β secretion in macrophages infected with PLA K. pneumoniae was shown to depend on NLRP3 but also on NLRC4 and TLR4. In macrophages infected with a K1-CPS deficiency mutant, an lipopolysaccharide (LPS) deficiency mutant, or K1-CPS and LPS double mutants, IL-1β secretion levels were lower than those in cells infected with wild-type PLA K. pneumoniae. Our findings indicate that K1-CPS is one of the Klebsiella factors of PLA K. pneumoniae that induce IL-1β secretion through the NLRP3 inflammasome.

Factors of Th17 and Treg Lymphocyte Differentiation in Pulmonary Tuberculosis.

In patients with infiltrative pulmonary tuberculosis, the increase in IL-6 secretion, the decrease in TGFβ production (in case of drug resistance of the causative agent), and unchanged level of IL-1β secretion by mononuclear leukocytes in vitro were associated with increased number of CD4(+)CD161(+)IL-17A(+) Th17 lymphocytes in the peripheral blood. In patients with disseminated drug-resistant pulmonary tuberculosis, TGFβ hyperproduction promoted differentiation of CD4(+)CD25(+)FoxP3(+) Treg lymphocytes with immunosuppressive activity.