Toll-like receptor 4-related regulation of Ornithogalum caudatum extract on inflammatory responses in LPS activated macrophages

OBJECTIVE To investigate toll-like receptor 4(TLR4)-related the regulation of Ornithogalum caudatum extract(OCE) on inflammatory responses in lipopolysaccharide(LPS) activated macro.phages.METHODS Primary peritoneal macrophage,Raw 264.7,and THP-1 were incubated in 96-well plate for 24 h and treated with OCE of the concentration of 0-400 μg/ml for 4 h.The viability of cells was measured by MTT assay.Specific concentrations of OCE were added into the medium of primary peri.toneal macrophage,Raw 264.7,and THP-1,respectively,then following with lipopolysaccharides(LPS).Cells were harvested and the total cellular protein and nuclear protein were extracted,and the protein content was determined using BCA protein assay Kit.The expressions of TLR4,inducible nitric oxide synthase(iNOS),cyclooxygenase 2(COX-2),α-inhibitor of NF-κB(IκB-α) and nuclear factor-κB(NF-κB) were assayed by Western blot.The expressions of interleukin-1α(IL-1α),interleukin-1β(IL-1β),interleukin-18(IL-18),and tumor necrosis factor-α(TNF-α) were measured by RT-PCR.RESULTS The results of MTT showed that OCE has no cytotoxicity in Raw 264.7 cells between 1.56 μg/ml and 400 μg/ml.Compared with normal group,the expressions of TLR4,iNOS,COX-2,NF-κB and IL-1α,IL-1β,IL-18,TNF-α,the level of nitric oxide(NO) were significantly increased by LPS stimulation,while OCE pretreat.ment reduced these increase induced by LPS.However,OCE pretreatment reversed the reduction of IκB-α after LPS stimulation.CONCLUSION OCE might suppress TLR4 expression and block the inflamma.tion process of NF-κB and iNOS,further decrease the expression of COX-2 and inhibit the release of inflammatory factors.

Mechanisms of Increased Expression of Toll-Like Receptor-4 in Human Monocyte/Macrophage-derived Foam Cells

Summary： The mechanisms of increased expression of toll-like receptor 4 （TLR-4） during the formation of foam cells were explored. Low density lipoprotein （LDL） was prepared by density gradient ultracentrifugation and oxidized by incubation with CuClz. The human monocytic leukemia cell line （THP-1） was cultured in RPMI1640. The differentiation of THP-1 cells into macrophages （MPs） was induced by using myristate acetate （PMA） for 48 h. The macrophages were then incubated with oxidized LDL （ox-LDL） to generate foam cells （FCs）. The mRNA and protein expression levels of human TLR-4 were detected by immunocytochemistry, Western blotting and reverse transcription polymerase chain reaction （RT-PCR）. The results showed that the TLR-4 mRNA and the protein expression levels were significantly increased during the differentiation of monocytes into macrophages （P〈0. 05） as well as during the formation of lipid-laden foam cells （P〈0.05）. It was concluded that the upregulation of human TLR-4 gene expression during the differentiation of monocytes into macrophages and the differentiation of macrophages into foam cells could increase TLR-4 protein synthesis dramatically, which may enhance the ability of foam cells inflammation reaction in atherosclerosis.

Hepatocellular carcinoma and macrophage interaction induced tumor immunosuppressionvia Treg requires TLR4 signaling

AIM: To investigated the interaction between toll-like receptor 4 (TLR4)-activated hepatoma cells and macrophages in the induction of tumor-immune suppression mediated by CD4+CD25high family of transcription factor P3 (FOXP3) regulatory T cells (Tregs). METHODS: The proportion of FOXP3+ Tregs was identified in peripheral blood and tumor tissues of 60 hepatocellular carcinoma (HCC) patients. TLR4 expression was examined in tumor tissues and cell lines. The correlation was examined between FOXP3+ Tregs in peripheral blood and TLR4 expression of HCC tissues. Following activation of TLR4 in H22 murine hepatoma cells pre-incubated with lipopolysaccharide (LPS) and co-cultured with macrophage cell line RAW246.7, the synthesis of cytokines tumor necrosis factor-α, CCL22, and interleukin (IL)-10 by the two cell lines was detected and analyzed. RESULTS: FOXP3+ Tregs were enriched in tumor sites, and circulating FOXP3+ Tregs were increased in HCC patients in correlation with multiple tumor foci and up-regulated TLR4 expression in HCC tissues. Semi-quantitative analysis indicated that TLR4 was over-expressed in HCC compared with the matched normal tissues. Cell cultivation experiments indicated that the mRNAs of IL-10 and CCL22 were significantly up-regulated in the RAW246.7 cell line when co-cultured with LPS preincubated H22 cells. CONCLUSION: In hepatoma cell lines, TLR4 may indirectly facilitate the recruitment of Tregs to the tumor site and promote intrahepatic metastasis through its interaction with macrophages.

Inhibition of RAW264.7 Macrophage Inflammatory Cytokines Release by Small Haparin RNAi Targeting TLR4

In order to construct an expression vector carrying small hairpin （sh） RNA （shRNA） for toll-like receptor 4 mRNA and a reporter gene of enhanced green fluorescence protein （EGFP） and study the inhibition of cytokine release by RAW264.7 cell induced by lipopolysaccharide （LPS） stimulation through transfection and expression of shRNA targeting TLR4 gene via the RNAi mecha- nism, the reporter gene plasmid pEGFP-CI （4.7 kb） and psiRNA-hHlneo （2979 bp） were used. The HI promotor and double Bbs Ⅰ restrict endoenzyme site were cloned from plasmid psiRNA-hHlneo and reconstructed them into plasmid pEGFP-CI in the Mlu Ⅰ restrict endoenzymic site, forming plasmid pEGFP-HI/siRNA, which contained Bbs site and reporter EGFP gene. Then an oligonuclear hairpin sequence targeting TLR4 gene was designed by internet tool and inserted into the plasmid pEGFP-H 1/siRNA forming plasnlid pEGFP-H 1/TLR4-siRNA. After transfection of pEGFP-H I/TLR4-siRNA into RAW264.7 cells, tumor necrosis factor-alpha （TNF-α） release by the cells after stimulation by LPS was detected. The results showed that the constructed pEGFP-HI/TLR4-siRNA carrying hairpin RNA for TLR4 gene and reporter EGFP gene were proven to be right by restriction endonuclease analysis. The expression of EGFP gene was （50.37±8.23） % and after transfection of the plasmid pEGFP-HI/ TLR4-siRNA the level of TNF-α released by RAW264.7 cell was down regulated. It was concluded that shRNA targeting TLR4 gene could inhibit the TNF-α release by RAW264.7 cells evoked by LPS.

Pharmacological Inhibition of Macrophage Toll-like Receptor 4/Nuclear Factor-kappa B Alleviates Rhabdomyolysis-induced Acute Kidney Injury

Background：Acute kidney injury （AKI） is the most common and life-threatening systemic complication ofrhabdomyolysis.Inflammation plays an important role in the development of rhabdomyolysis-induced AKI.This study aimed to investigate the kidney model of AKI caused by rhabdomyolysis to verify the role ofmacrophage Toll-like receptor 4/nuclear factor-kappa B （TLR4/NF-κB） signaling pathway.Methods：C57BL/6 mice were injected with a 50% glycerin solution at bilateral back limbs to induce rhabdomyolysis,and CLI-095 or pyrrolidine dithiocarbamate （PDTC） was intraperitoneally injected at 0.5 h before molding.Serum creatinine levels,creatine kinase,the expression of tumor necrosis factor （TNF）-c,interleukin （IL）-1β and I L-6,and hematoxylin and eosin stainings of kidney tissues were tested.The infiltration of macrophage,mRNA levels,and protein expression of TLR4 and NF-κB were investigated by immunofluorescence double-staining techniques,reverse transcriptase-quantitative polymerase chain reaction,and Western blotting,respectively.In vitro,macrophage RAW264.7 was stimulated by ferrous myoglobin;the cytokines,TLR4 and NF-κB expressions were also detected.Results：In an in vivo study,using CLI-095 or PDTC to block TLR4/NF-κB,functional and histologic results showed that the inhibition of TLR4 or NF-κB alleviated glycerol-induced renal damages （P 〈 0.0 1）.CLI-095 or PDTC administration suppressed proinflammatory cytokine （TNF-c,IL-6,and IL-1 β） production and macrophage infiltration into the kidney （P 〈 0.01）.Moreover,in an in vitro study,CLI-095 or PDTC suppressed myoglobin-induced expression ofTLR4,NF-κB,and proinflammatory cytokine levels in macrophage RAW264.7 cells （P 〈 0.01）.Conclusion：The pharmacological inhibition of TLR4/NF-κB exhibited protective effects on rhabdomyolysis-induced AKI by the regulation of proinflammatory cytokine production and macrophage infiltration.

Flavonoids from Scutellaria barbata inhibit activation of tumor-associated macrophages by blocking the Toll-like receptor 4/myeloid differentiation factor 88/nuclear factor-κB signaling pathway

OBJECTIVE: To determine the efficacy of Scutellaria barbata flavonoids and polysaccharides on Ishikawa endometrial carcinoma cells co-cultured with U937 macrophages.METHODS: The presence of CD163 and CD206 was determined by flow cytometry. Thiazolyl Blue Tetrazolium Bromide assays were used to assess the proliferation effect of tumor-associated macrophages(TAMs) on Ishikawa cells. The secretion of interleukin(IL)-10 in the co-culture conditioned media was examined using an enzyme-linked immunosorbent assay. The protein expression levels of Toll-like receptor 4(TLR4), myeloid differentiation factor 88(MyD88) and nuclear factor(NF)-κB p65 were detected by Western blot. The mRNA expression levels of TLR4 and MyD88 were analyzed by real-time polymerase chain reaction(PCR). The expression levels of IL-12, IL-1β and tumor necrosis factor-α(TNF-α) were evaluated with real-time PCR.RESULTS: Compared with the U937 control group,the expression levels of CD163 and CD206 in the TAM group were higher(P < 0.05). TAMs co-cultured with Ishikawa cells for 24 or 48 h showed higher proliferation rates(P < 0.05). The expression levels of IL-12 decreased than compared with those in the U937 untreated group(P <0.05) and those of the Scutellaria barbata flavonoids group(P < 0.05). The expression levels of CD206, CD163, IL-10, IL-1β and TNF-α, NF-κB p65 and TLR4/MyD88 in the TAMs control group were greater than those in the U937 untreated group(P < 0.05) and those of the Scutellaria barbata flavonoids group(P < 0.05).CONCLUSION: Scutellaria barbata flavonoids may inhibit TAM activation by blocking the TLR4/MyD88/NF-κB signaling pathway.

Involvement of Cbl-b-mediated macrophage inactivation in insulin resistance

Aging and overnutrition cause obesity in rodents and humans. It is well-known that obesity causes various diseases by producing insulin resistance(IR). Macrophages infiltrate the adipose tissue(AT) of obese individuals and cause chronic low-level inflammation associated with IR. Macrophage infiltration is regulated by the chemokines that are released from hypertrophied adipocytes and the immune cells in AT. Saturated fatty acids are recognized by toll-like receptor 4(TLR4) and induce inflammatory responses in AT macrophages(ATMs). The inflammatory cytokines that are released from activated ATMs promote IR in peripheral organs, such as the liver, skeletal muscle and AT. Therefore, ATM activation is a therapeutic target for IR in obesity. The ubiquitin ligase Casitas b-lineage lymphoma-b(Cbl-b) appears to potently suppress macrophage migration and activation. Cbl-b is highly expressed in leukocytes and negatively regulates signals associated with migration and activation. Cbl-b deficiency enhances ATM accumulation and IR in aging- and diet-induced obese mice. Cbl-b inhibits migration-related signals and SFA-induced TLR4 signaling in ATMs. Thus, targeting Cbl-b may be a potential therapeutic strategy to reduce the IR induced by ATM activation. In this review, we summarize the regulatory functions of Cbl-b in ATMs.

Gastrin attenuates sepsis-induced myocardial dysfunction by down-regulation of TLR4 expression in macrophages

Myocardial dysfunction is the most serious complication of sepsis.Sepsis-induced myocardial dysfunction(SMD)is often associated with gastrointestinal dysfunction,but its pathophysiological significance remains unclear.The present study found that patients with SMD had higher plasma gastrin concentrations than those without SMD.In mice,knockdown of the gastrin receptor,cholecystokinin B receptor(Cckbr),aggravated lipopolysaccharide(LPS)-induced cardiac dysfunction and increased inflammation in the heart,whereas the intravenous administration of gastrin ameliorated SMD and cardiac injury.Macrophage infiltration plays a significant role in SMD because depletion of macrophages by the intravenous injection of clodronate liposomes,48 h prior to LPS administration,alleviated LPSinduced cardiac injury in Cckbr-deficient mice.The intravenous injection of bone marrow macrophages(BMMs)overexpressing Cckbr reduced LPS-induced myocardial dysfunction.Furthermore,gastrin treatment inhibited toll-like receptor 4(TLR4)expression through the peroxisome proliferator-activated receptor a(PPAR-a)signaling pathway in BMMs.Thus,our findings provide insights into the mechanism of the protective role of gastrin/CCKBR in SMD,which could be used to develop new treatment modalities for SMD.

Natural anti-oxLDL IgM monoclonal antibody in the pathogenesis of atherosclerosis

Objective To explore the role and the possible molecular mechanisms of natural anti-oxLDL IgM monoclonal antibody played and involved in pathogenesis of atherosclerosis. Methods Natural anti-oxLDL IgM monoclonal antibody 3A6 was generated by using standard hybridoma production techniques. Influence of 3A6 on formation of foam cells was observed by Oil Red O staining and affinity of Na125I-conjugated oxLDL on the naive and LPS-activated macrophages. After LPS stimulation on macrophages, anti-TLR4 neutralizing mAb, p38MAPK specific inhibitor SB203580, NF-kB specific inhibitor PDTC or RNAi targeting Fcα/μ receptor (Fcamr) were applied, respectively. Results Natural anti-oxLDL IgM monoclonal antibody 3 A6 were found specifically inhibit the binding of CuoxLDL to naive macrophages but not the binding of CuoxLDL to LPS-activated macrophages. It also promoted the formation of CuoxLDL-mediated foam macrophages. 3A6 F(ab')2 or pre-incubation with un-related IgM inhibited the binding of 3A6/CuoxLDL complex to LPS-activated macrophages. LPS up-regulated the expression of Fcamr in macrophages in a dose- and time-dependent manner, which was attenuated by treatment with anti-TLR4. LPS induced the phosphorylation of p38MAPK and translocation of NF-kB p65, contributing to the up-regulated expression of Fcα/μ receptor in macrophages. Conclusions Natural anti-oxLDL IgM monoclonal antibody 3A6 specifically inhibited the binding of CuoxLDL to naive macrophages in vitro. However, LPS, through the Toll-like receptor (TLR)4 receptor, activated the p38MAPK and NF-kB pathways and up-regulated the expression of Fcα/μ receptor in macrophages, which promoted the binding of 3A6/CuoxLDL complex to macrophages through binding with Fc fragments and the formation of foam macrophages. Therefore, our findings provide a new explanation why bacterial infection deteriorates the pathogenesis of atherosclerosis.

Gut microbiota controls the development of chronic pancreatitis:A critical role of short-chain fatty acids-producing Gram-positive bacteria

Chronic pancreatitis(CP)is a progressive and irreversible fibroinflammatory disorder,accompanied by pancreatic exocrine insufficiency and dysregulated gut microbiota.Recently,accumulating evidence has supported a correlation between gut dysbiosis and CP development.However,whether gut microbiota dysbiosis contributes to CP pathogenesis remains unclear.Herein,an experimental CP was induced by repeated high-dose caerulein injections.The broad-spectrum antibiotics(ABX)and ABX targeting Gram-positive(G+)or Gram-negative bacteria(G-)were applied to explore the specific roles of these bacteria.Gut dysbiosis was observed in both mice and in CP patients,which was accompanied by a sharply reduced abundance for short-chain fatty acids(SCFAs)-producers,especially G+bacteria.Broad-spectrum ABX exacerbated the severity of CP,as evidenced by aggravated pancreatic fibrosis and gut dysbiosis,especially the depletion of SCFAs-producing G+bacteria.Additionally,depletion of SCFAs-producing G+bacteria rather than G-bacteria intensified CP progression independent of TLR4,which was attenuated by supplementation with exogenous SCFAs.Finally,SCFAs modulated pancreatic fibrosis through inhibition of macrophage infiltration and M2 phenotype switching.The study supports a critical role for SCFAs-producing G+bacteria in CP.Therefore,modulation of dietary-derived SCFAs or G+SCFAs-producing bacteria may be considered a novel interventive approach for the management of CP.