Macrophages and kidney transplantation / 器官移植

Kidney transplantation is the optimal treatment for patients with end-stage renal disease, whereas long-term survival of renal allografts remains a challenging issue. Renal ischemia-reperfusion injury (IRI) and rejection of renal allografts are considered as important influencing factors of long-term survival of renal allografts, which are regulated by innate and adaptive immune cells. Macrophages are one type of innate immune cells that could assist initiating adaptive immunity and are divided into M1, M2 and regulatory macrophages. Previous studies have revealed that M1 macrophages may aggravate renal IRI and acute T cell-mediated rejection (TCMR). However, M2 macrophages may mitigate renal IRI and acute TCMR, whereas it is positively correlated with antibody-mediated rejection (AMR). Regulatory macrophages are a special subgroup of macrophages, which may induce immune tolerance in organ transplantation and have promising clinical application prospects and basic scientific research value. In this article, the relationship among macrophage typing, macrophages and renal IRI, rejection of renal allografts, regulatory macrophages and immune tolerance was reviewed, and the potential mechanism was analyzed, aiming to induce changes in macrophage subtypes or eliminate specific subtypes of macrophages, thereby improving clinical prognosis of the recipients and long-term survival of renal allografts.

Naoxintong Capsules Treat Cardiac Injury after Cerebral Ischemia via TLR2/TLR4 Signaling Pathway / 中国实验方剂学杂志

ObjectiveAfter the brain and heart injuries were simulated by myocardial injury caused by acute cerebral ischemia， this study explored the mechanism of Naoxintong capsules in treating brain and heart injuries under cerebral ischemia state with Toll-like receptor （TLR） 2/TLR4 as the breakthrough point. MethodC57BL/6 male mice were randomly assigned into the sham operation， model， Naoxintong， and Ginaton groups. The middle cerebral artery occlusion （MCAO） method was used to establish a mouse model of cerebral ischemia. The neuroethological score， cerebral infarction area， cell apoptosis， ionized calcium-binding adaptor molecule 1 （IBA-1）-positive microglia proportion， and serum levels of N-terminal pro-brain natriuretic peptide （NT-proBNP）， creatine kinase-MB （CK-MB）， and lactic dehydrogenase （LDH） were determined to evaluate the pharmacodynamic effects of Naoxintong capsules on heart and brain injuries after cerebral ischemia in mice. Western blotting was employed to determine the expression of TLR2/TLR4 protein in the brain and heart of mice. ResultCompared with the sham operation group， the model group showed increased cerebral infarction area， neuroethological score， apoptosis rate， IBA-1-positive microglia proportion， and serum levels of NT-proBNP， CK-MB， and LDH （P<0.01）. Naoxintong capsules reduced the cerebral infarction area， neuroethological score， apoptosis rate， IBA-1-positive microglia proportion （P<0.01）， and serum NT-proBNP and CK-MB levels （P<0.05） in mice compared with the model group. Western blotting results showed that Naoxintong Capsules down-regulated the expression levels of TLR2 （P<0.05） in the brain and TLR2 （P<0.01） and TLR4 （P<0.05） in the heart. ConclusionCerebral ischemia can cause myocardial damage， reflecting the pathological process of cardiac injury after cerebral ischemia. Naoxintong capsules can mitigate brain and heart injuries after cerebral ischemia and achieve the simultaneous treatment of the brain and the heart， in which TLR2/TLR4 plays a role.

Mechanism of Scutellariae Radix-Paeoniae Radix Rubra Combination of Different Proportions Against Hepatic Fibrosis in Rats / 中国实验方剂学杂志

ObjectiveTo observe the effects of Scutellariae Radix （SR）-Paeoniae Radix Rubra （PRR） combination of different proportions on the expression of the Toll-like receptor 4 （TLR4）/myeloid differentiation factor 88 （MyD88）/nuclear transcription factor κB （NF-κB） and phosphatidylinositol kinase （PI3K）/protein kinase B （Akt）/mammalian target of rapamycin （mTOR） signaling pathways in liver tissues of rats with hepatic fibrosis and explore the mechanism against hepatic fibrosis. MethodSixty male SD rats of SPF grade were randomly divided into a normal group， a model group， a positive control （silymarin） group， and SR-PRR 1∶1， SR-PRR 1∶2， and SR-PRR 1∶4 groups， with 10 rats in each group. The hepatic fibrosis model was induced in rats except for those in the normal group by intraperitoneal injection of 40% tetrachloromethane （CCl4）-olive oil solution at 3 mL·kg-1， 5 mL·kg-1 for the first time， for 8 weeks， twice per week. After 4 weeks， rats were treated correspondingly at 10 mL·kg-1 by intragastric administration， and the body weight of rats in each group was weighed for 8 weeks. After administration， histopathological changes in the liver were observed. Serum alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， hyaluronic acid （HA）， laminin （LN）， albumin （ALB）， alkaline phosphatase （AKP）， and superoxide dismutase （SOD） activities， malondialdehyde （MDA）， and hydroxyproline （HYP） content in liver tissues were detected. The mRNA expression levels of TLR4， MyD88， NF-κB， PI3K， Akt， and mTOR in the liver of rats were detected by real-time fluorescence-based quantitative polymerase chain reaction （Real-time PCR）. ResultCompared with the model group， SR-PRR combination of different proportions could recover the body weight and improve the pathological injury of the liver. As revealed by enzyme linked immunosorbent assay （ELISA） results， compared with the normal group， the model group showed increased ALT， AST， HA， LN， AKP， MDA， and HYP levels to different degrees （P<0.05）. Compared with the model group， the groups with drug intervention showed decreased levels of ALT， AST， HA， LN， AKP， MDA， and HYP， potentiated SOD activity， and increased level of ALB （P<0.05）. As revealed by Real-time PCR results， compared with the normal group， the model group showed increased mRNA expression of TLR4， MyD88， NF-κB， PI3K， Akt， and mTOR （P<0.05）. Compared with the model group， the groups with drug intervention showed reduced mRNA expression of TLR4， MyD88， NF-κB， PI3K， Akt， and mTOR in the liver of rats （P<0.05）. ConclusionSR-PRR combination of different proportions can improve the histopathological injury in liver tissues caused by CCl4， with the optimal effect observed in the SR-PRR 1∶4 group. SR-PRR may inhibit the development of liver fibrosis by inhibiting the expression of TLR4/MyD88/NF-κB and PI3K/Akt/mTOR signaling pathways， thereby alleviating chemical-induced liver injury.

Regulatory Effect of Xiangshenwan on TLR/NF-κB Signal in Colonic Mucosa of Mice with Colitis / 中国实验方剂学杂志

Objective:To explore the effect and mechanism of Xiangshenwan on ulcerative colitis （UC） induced by dextran sulfate sodium （DSS） in mice based on the classic Toll-like receptor （TLR）/nuclear factor kappa B （NF-<italic>κ</italic>B） signaling pathway. Method:The experimental mice were divided into a normal group， a model group， a Xiangshenwan group， and a mesalazine group. The mice， except for those in the normal group， received 3% DSS solution for 7 days to establish the acute UC model and were treated with Xiangshenwan （5 g·kg^-1） and mesalazine （300 mg·kg^-1） continuously from the 1st day to the 10th day of modeling. The body weight， disease activity index （DAI）， colon weight， intestinal weight index， colon length， colon weight per unit length， and pathological changes of mice were evaluated respectively. The protein expression of TLR5， myeloid differentiation factor 88 （MyD88）， interleukin-1 receptor-associated kinase 4 （IRAK4）， tumor necrosis factor receptor-associated factor 6 （TRAF6）， transforming growth factor <italic>β</italic>-activated kinase 1 （TAK1）， p38 mitogen-activated protein kinase （MAPK）， NF-<italic>κ</italic>B， IRAK1， TAK1-binding protein 1 （TAB1）， TAB2， mitogen-activated protein kinase kinase 3 （MKK3）， MKK6 and cyclic adenosine monophosphate response element-binding protein （CREB） in colon tissues of mice was detected by Western blot. Result:Compared with the normal group， the model group showed decreased body weight of mice， increased DAI scores， elevated colon weight， intestinal weight index， and colon weight per unit length， shortened colon length， severe colonic mucosal injury， and up-regulated protein expression of TLR5， MyD88， IRAK4， TRAF6， TAK1， p38 MAPK， NF-<italic>κ</italic>B， IRAK1， TAB1， TAB2， MKK3， MKK6， and CREB in colon tissues （<italic>P</italic><0.05，<italic> P</italic><0.01<bold>）.</bold> Compared with the model group， the Xiangshenwan group and the mesalazine group displayed increased body weight of mice， decreased DAI scores， declining colon weight， intestinal weight index， and colon weight per unit length， increased colon length， improved colonic mucosal injury， and down-regulated protein expression of TLR5， MyD88， IRAK4， TRAF6， TAK1， p38 MAPK， NF-<italic>κ</italic>B， IRAK1， TAB1， TAB2， MKK3， MKK6， and CREB in colon tissues （<italic>P</italic><0.05，<italic> P</italic><0.01）. Conclusion:Xiangshenwan can effectively treat DSS-induced UC presumedly by the inhibition of TLR/NF-<italic>κ</italic>B signaling pathway.

Recent progress on the roles of DAMP and NET in organ ischemia-reperfusion injury / 器官移植

Ischemia-reperfusion injury (IRI) is a common pathophysiological phenomenon, secondary to multiple pathological processes, such as organ transplantation, acute kidney injury and myocardial infarction. IRI may significantly aggravate the severity of diseases and increase the fatality of patients. Aseptic inflammation is one of the critical mechanisms of IRI. Damage-associated molecular pattern (DAMP) is a pivotal substance, which mediates aseptic inflammation. After released into extracellular space, it could effectively activate the immune system, and initiate and maintain the inflammatory responses by binding with pattern recognition receptor (PRR). Neutrophil extracellular trap (NET) is a DNA-based network structure released by neutrophils during the process of inflammatory responses, which contains histones and multiple granular proteins. Recent studies have demonstrated that DAMP and NET may aggravate IRI via aseptic inflammation. In this article, relevant studies of DAMP, NET and their relationship in IRI were reviewed, which was of great significance for understanding the pathophysiological mechanism of IRI and studying the corresponding prevention and treatment strategies.

Role of NLRP3 inflammasome and related inflammatory signaling pathways in renal ischemia-reperfusion injury / 器官移植

Renal ischemia-reperfusion injury (IRI) commonly occurs in renal transplantation, which is an important pathophysiological process that causes acute renal failure and severely affects clinical prognosis of the recipients. Inflammatory response plays a critical role in the pathogenesis and pathological process of IRI. Activated NOD-like receptor protein 3(NLRP3) inflammasome can mediate the maturation and release of various pro-inflammatory cytokines, thereby regulating the inflammatory response and relevant cell functions. In this article, the mechanism underlying NLRP3 inflammasome and its related inflammatory signaling pathway in renal IRI were reviewed, aiming to provide novel ideas for clinical prevention and treatment of renal IRI.

Regulatory Effect of Volatile Oil from Sishenwan on TLR/MyD88 Signaling Pathway in Mice with Chronic Ulcerative Colitis / 中国实验方剂学杂志

Objective:To explore the underlying mechanism of volatile oil from Sishenwan in treating chronic ulcerative colitis through the Toll-like receptor （TLR）/myeloid differentiation factor 88 （MyD88） signaling pathway. Method:The BALB/c mice were randomly divided into a normal group （normal）， a model group ［dextran sodium sulfate （DSS）］， a Sishenwan volatile oil group， an Ershen pill volatile oil group， a Wuweizi powder volatile oil group， and a mesalazine control group. The chronic ulcerative colitis model was induced by DSS in mice. Seven days after intragastric administration， the efficacy was evaluated based on the body weight， colon weight， colon weight index， colon length， and pathological damage score under colonoscopy. The levels of interleukin （IL）-4， IL-10， IL-17A， IL-21， and interferon-<italic>γ </italic>（IFN-<italic>γ</italic>） in the supernatant of colon tissues were detected by enzyme-linked immunosorbent assay （ELISA）. Western blot was used to detect the expression levels of proteins related to the TLR/MyD88 signaling pathway in the colon mucosa of mice， including TLR2， MyD88， Ras-related C3 botulinum toxin substrate 1 （Rac1）， IL-1 receptor-associated kinase 4 （IRAK4）， IRAK1， tumor necrosis factor receptor （TNFR）-associated factor 6 （TRAF6）， transforming growth factor-<italic>β</italic>-activated kinase 1 binding protein 1 （TAB1）， TAB2， mitogen-activated protein kinase kinase 6 （MKK6）， p38 mitogen-activated protein kinase （p38 MAPK）， and cyclic adenosine monophosphate response element-binding protein （CREB）. Result:Compared with the normal group， the model group showed decreased colon length， increased colon weight， colon weight index， and pathological damage score under colonoscopy， decreased IL-10 level in the colon tissues， increased IL-4， IL-17A， IL-21， and IFN-<italic>γ</italic> levels （<italic>P<</italic>0.05， <italic>P<</italic>0.01）， and up-regulated protein expression of TLR2， MyD88， Rac1， IRAK4， IRAK1， TRAF6， TAB1， TAB2， MKK6， p38MAPK， and CREB （<italic>P<</italic>0.01）. Compared with the model group， the Sishenwan volatile oil group showed increased colon length， reduced colon weight， colon weight index， and pathological damage score under colonoscopy， elevated IL-10 level in the colon tissues， decreased IL-4， IL-17A， IL-21， and IFN-<italic>γ</italic> levels （<italic>P<</italic>0.05， <italic>P<</italic>0.01），and down-regulated protein expression of TLR2， MyD88， Rac1， IRAK4， IRAK1， TRAF6， TAB1， TAB2， MKK6， p38MAPK， and CREB （<italic>P<</italic>0.05， <italic>P<</italic>0.01）. Conclusion:The volatile oil from Sishenwan can effectively improve the inflammatory response of chronic ulcerative colitis， which may be achieved by regulating the TLR/MyD88 signaling pathway.

The role of Toll-like receptors in chemoradiotherapy-induced gastrointestinal mucositis / 口腔疾病防治

@#Mucositis is a common gastrointestinal complication in cancer patients undergoing chemoradiotherapy, including oral mucositis and gastrointestinal mucositis, with clinical manifestations of oral ulcers, vomiting, diarrhea and pain that seriously reduce the quality of life of patients and even affect anticancer therapy. Toll-like receptor (TLR) are important receptors involved in innate immunity and in the development of chemoradiation-induced mucositis by mediating the effect between microorganisms and the host. A comprehensive understanding of the role of TLR in mucositis is helpful to guide the prevention and treatment of mucositis. This paper reviews the available studies on TLR and mucositis. The results of the literature review indicate that different TLR have different roles in chemoradiation-induced mucositis: TLR2 is an important receptor in the inflammatory cascade of chemoradiation-induced mucositis; TLR4 activation can increase gastrointestinal mucosal inflammation and lead to oral epithelial ulceration; TLR5 agonists can reduce the degree of radiation-induced mucositis damage; and antagonizing or knocking out TLR9 can reduce chemoradiation-induced gastrointestinal mucositis. However, no TLR agonists or inhibitors have yet been applied in clinical practice, and additional studies are needed to explore the role of different TLR in mucositis in the future to provide a reference for the precise prevention and treatment of chemoradiation-induced mucositis.

Effect of Esculentoside on Lipopolysaccharide-induced Mice Mastitis / 中国实验方剂学杂志

Objective:To explore the effect and mechanism of esculentoside on lipopolysaccharide-induced mastitis in mice. Method:Female BALB/c mice were randomly divided into control group, model group, dexamethasone group (DEX, 5.0 mg·kg-1) and esculentoside group (50, 25, 12.5 mg·kg-1). The mastitis model of postpartum female mice (BALB/c) induced by lipopolysaccharide (LPS) was used to analyze the pathological conditions of breast tissue and the activity of recombinant myeloperoxidase（MPO）, factor content and oxidative stress level. Western blot was used to evaluate the effect of esculentoside on Toll-like receptor4 (TLR4)/nuclear transcription factor (NF-κB) signaling pathway proteins. Result:Compared with the normal group, the breast tissue of the model group had typical mastitis changes, such as hyperemia and congestion, the level of MPO increased, and the expression levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) increased significantly (P<0.01). Compared with LPS model group, esculentoside groups could significantly improve the inflammatory damage of mammary gland tissue, reduce the secretion of neutrophils and the activity of MPO, the expression levels of pro-inflammatory factors TNF-α, IL-1β and IL-6 were also significantly down-regulated, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were increased, while the level of malondialdehyde (MDA) was decreased, and the activation of TLR4/NF-κB signaling pathway was inhibited by esculentoside（P<0.05，P<0.01）. Conclusion:Esculentoside have a protective effect on lipopolysaccharide-induced mastitis in mice, which may be related to the inhibition of TLR4/NF-κB signaling pathway protein expression.

Mechanism of Anti-chronic Alcoholic Liver Injury in Rats of Tibetan Medicine Lagotis brachystachys Extracts by TLR2/MyD88/NF-κB and NALP3 Signaling Pathway / 中国实验方剂学杂志

Objective::To established the model of chronic alcoholic liver injury in rats by long-term(8 weeks) alcoholic gavage, to study the effects of Tibetan medicine Lagotis brachystachys extracts on Toll-like receptor(TLR)2/myeloid differentiation factor 88(MyD88)/nuclear factor kappa B (NF-κB)and NOD like receptor protein 3(NALP3) signaling pathways and study preliminary the mechanism of action of chronic alcoholic liver injury. Method::Sixty male Sprague-Dawley rats were randomly divided into normal group, model group, bifendate positive drug group (0.1 g·kg-1) and L. brachystachys low, medium and high-dose groups (0.5, 1, 2 g·kg-1), the corresponding drugs were given at 10 mL·kg-1 in each morning, and the 56 degree Liquor was administered by the afternoon gradient alcoholic gavage method.After 8 weeks, the levels of serum aspartate transaminase (AST), serum alanineaminotransfease(ALT), serum total cholesterol(TC), triglyceride(TG), interleukin-1β(IL-1β), and the liver levels of L-glutathione(GSH)were measured. The expression of TLR2, MyD88, NF-κB and NALP3 protein in liver were detected by Western blot.Hematoxylin-eosin (HE) staining was used to observe the pathological changes of liver tissue. Result::Compared with normal group, the serum levels of AST, ALT, TC, TG and IL-1β in model group were significantly increased (P<0.05, P<0.01). Compared with model group, the serum AST, ALT, TC, TG and IL-1β levels were decreased in the various doses of L. brachystachys, and the high dose group was particularly effective (P<0.05, P<0.01). Compared with normal group, the GSH level in the liver homogenate of model group decreased significantly, and the difference was not statistically significant. The levels of TLR2, MyD88, NF-κB and NALP3 in the liver tissue of model group were significantly increased (P<0.05, P<0.01). The GSH levels in the liver and the protein expression of TLR2, MyD88, NF-κB and NALP3 were decreased in L. brachystachys group (P<0.05, P<0.01). The liver pathological section showed that L. brachystachys can improve the pathological changes of rat liver tissue. Conclusion::L. brachystachys can protect liver from alcohol-induced chronic liver injury in rats. The mechanism was related to TLR2/MyD88/NF-κB and NALP3 signaling pathway.

Regulatory Effect of Guben Fangxiao Decoction on Expression of TLR4 and TLR7 in Mice with Asthma Remission / 中国实验方剂学杂志

Objective:To observe the regulatory effect of Guben Fangxiao decoction on Toll-like receptor (TLR)4, TLR7, nuclear factor-kappa B p65 (NF-κB p65) and NF-κB inhibitor protein alpha (IκBα) in mice with asthma remission, in order to explore the mechanism of Guben Fangxiao decoction in treating asthma remission. Method:Respiratory syncytial virus (RSV) combined with chicken ovalbumin (OVA) was used to build asthma remission model in 3-week-old BALB/c mice. Sixty mice were divided into blank group, model group, dexamethasone group (0.001 g·kg-1), low,medium and high-dose Guben Fangxiao decoction group (6.5,13,26 g·kg-1), respectively. Intervention was given once a day for 28 days. After administration, the mice were put to death. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of lung tissue and score the pulmonary inflammation. Western blot was used to detect the expressions of TLR4, TLR7, IκBα and nuclear NF-κB p65 in lung tissue of mice. Immunofluorescence was used to observe the expression of NF-κB p65 in cellnucleuses. Result:Compared with blank group, the lung tissue of model group showed obvious inflammatory cell infiltration (PκB p65 increased significantly (PκBα proteins increased significantly (PPκB p65 (PκB p65 (PκBα proteins (PConclusion:Guben Fangxiao decoction can alleviate airway inflammation, and its therapeutic effect may be achieved by regulating TLR4, TLR7, NF-κB p65 and IκBα.

Effect of Toll-like receptor nano-inhibitor P12 on pulmonary inflammation in acute lung injury mice / 上海交通大学学报（医学版）

Objective: To investigate the effect of the Toll-like receptor (TLR) nano-inhibitor P12 on THP-1 derived macrophages and acute lung injury (ALI) mouse model induced by lipopolysaccharides (LPS). Methods: In in vitro experiments, THP-1 cells were differentiated into macrophage-like cells and then treated with LPS in the absence and presence of P12. After 24 h incubation, medium was collected to quantify the secretion of pro-inflammatory cytokines using enzyme-linked immunosorbent assay (ELISA). Six- to eight-week-old C57BL/6 mice were randomly divided into three groups, i.e. PBS control, LPS challenge and P12 pretreatment plus LPS. The bronchial alveolar lavage fluid (BALF) and lung tissue of each mouse were collected, and the acute inflammatory response within lung was evaluated by total cell counts, differential cell counts and ELISA. Pathological injury scores in ALI mice were assessed with hematoxylin and eosin (H-E) staining of lung tissue sections under microscope. Results: In THP-1 derived macrophages, P12 significantly inhibited LPS-induced inflammatory cytokine production. In the LPS-induced ALI mouse model, P12 significantly attenuated the acute inflammatory response and alveolar damage in lung, including reducing the number of total cells and neutrophils in BALF, decreasing the expression of chemokine production (KC and CCL-2), and lowering lung injury scores. Conclusion: P12 exhibits potent anti-inflammatory activity in THP-1 derived macrophages and in the LPS-induced ALI mouse model, providing new concepts for the early treatment of ALI.

Vias de transdução de sinal e polimorfismo de Toll-like Receptors na carcinogenese por HPV / Toll-like Receptors signaling pathway and polymorphism on the HPV carcinogenesis

Seres humanos dependem incessantemente de um sistema de reconhecimento efetivo contra infecções para sobreviver. Dentre as diversas proteínas que compõem a resposta imune inata estão os receptores do tipo Toll (TLR Toll-like Receptors), que possuem a função de reconhecer padrões moleculares associados a patógenos e dar início a uma resposta imune adequada. O carcinoma do colo uterino é uma das principais causas de morte de mulheres por câncer mundialmente, sendo o terceiro tipo de câncer mais comum entre mulheres. Este tipo de neoplasia é vinculada etiologicamente à infecção pelo Papilomavírus humano (HPV). Dentre as principais proteínas virais, E6 e E7 são responsáveis pela manipulação dos processos celulares para promover ciclo viral, sendo essenciais no processo de transformação celular. Nesse contexto, o objetivo deste trabalho foi investigar a importância da via de sinalização de TLRs sobre a infecção por HPV. O polimorfismo rs5743836, na região promotora de TLR9, capaz de alterar a expressão deste receptor, foi estudado quanto à influência sobre a história natural da infecção por HPV em uma coorte de mulheres brasileiras; nenhuma associação relevante foi encontrada, indicando que este polimorfismo não interfere significativamente na resposta à infecção e risco de desenvolvimento de lesões no colo do útero causadas por HPV. Proteínas componentes da via de TLRs demonstraram serem alvos de interação com E6 de HPV16; dentre elas, o notável adaptador MyD88 e IKKε, enzima ativadora de importantes transfatores do sistema imune. Estas interações foram aqui estudadas. A interação de E6 com MyD88 resultou em estabilização da proteína viral, o que parece não depender do sítio LxxLL presente em MyD88, como ocorre com outros parceiros moleculares de E6. O sítio de interação de E6 com IKKε coincide com a região onde se localiza o sítio catalítico desta enzima, sugerindo a ação de E6 na ativação de proteínas alvo de IKKε. Esta interação foi observada em queratinócitos, células alvo das infecções por HPV. A produção de citocinas foi afetada por E6 de HPV16, resultando num aumento da quantidade de IL-8 e IL-6; a indução desta citocina poderia ser explicada pela ativação de IKKε. Estes resultados apontam para a capacidade do HPV16 de interferir com o sistema imune, contribuindo para o processo de carcinogênese

Humans constantly rely on an effective recognition system against infections in order to survive. Among various proteins that compose the innate immune response, Toll-like Receptors (TLRs) have the role to recognize pathogen associated molecular patterns and initiate a proper immune response. The cervical cancer is one of the main causes of women death worldwide, being the third most common cancer type among women. This type of neoplasia is etiologically associated with the Human papillomavirus (HPV) infection. E6 and E7, two main viral proteins, are responsible for manipulating the cellular processes to promote the virus' life-cycle, being essential to the cellular transformation process. In the context, the objective of this work was to investigate the relevance of the TLR signaling pathway on the HPV infection. The rs5743836 polymorphism, in the TLR9 promoter region, capable of altering this receptor's expression, was studied regarding its influence on the natural history of HPV infection in a Brazilian women cohort; no relevant association was found, indicating that this polymorphism does not interfere significantly in the infection response and risk of developing cervix lesions caused by HPV. Component proteins of TLR pathway were shown to be interaction targets of HPV16 E6; among them, the notable adaptor MyD88 and IKKε, enzyme that activates important immune system transfactors. These interactions were studied in this work. The interaction of E6 with MyD88 resulted in the stabilization of the viral protein, which seems independent of the LxxLL site present on MyD88, as in other E6 molecular partners. The interaction site on IKK with E6 matches with the region containing the enzyme's catalytic site, suggesting an influence of E6 in the activation of IKKε target proteins. This interaction was observed in keratinocytes, natural targets of HPV infections. The cytokines production was altered by HPV16 E6, resulting in an increase of IL-8 and IL-6 concentration; the induction of the latter could be explained by the activation of IKKε. These results point to the ability of HPV16 of interfering with the immune system, contributing to the carcinogenesis process

Toll-Like Receptor-Mediated Free Radical Generation in Clonorchis sinensis Excretory-Secretory Product-Treated Cholangiocarcinoma Cells

Clonorchiasis, caused by direct contact with Clonorchis sinensis worms and their excretory-secretory products (ESPs), is associated with chronic inflammation, malignant changes in bile ducts, and even cholangiocarcinogenesis. Our previous report revealed that intracellular free radicals enzymatically generated by C. sinensis ESPs cause NF-κB-mediated inflammation in human cholangiocarcinoma cells (HuCCT1). Therefore, the present study was conducted to examine the role of upstream Toll-like receptors (TLRs) on the initial host innate immune responses to infection. We found that treatment of HuCCT1 cells with native ESPs induced changes in TLR mRNA levels in a time-dependent manner, concomitant with the generation of free radicals. ESP-mediated free radical generation was markedly attenuated by preincubation of the cells with TLR1-4-neutralizing antibodies, indicating that at least TLR1 through 4 participate in stimulation of the host innate immune responses. These findings indicate that free radicals triggered by ESPs are critically involved in TLR signal transduction. Continuous signaling by this pathway may function in initiating C. sinensis infection-associated inflammation cascades, a detrimental event leading to progression to more severe hepatobiliary diseases.

Toll-Like Receptor-Mediated Free Radical Generation in Clonorchis sinensis Excretory-Secretory Product-Treated Cholangiocarcinoma Cells

Clonorchiasis, caused by direct contact with Clonorchis sinensis worms and their excretory-secretory products (ESPs), is associated with chronic inflammation, malignant changes in bile ducts, and even cholangiocarcinogenesis. Our previous report revealed that intracellular free radicals enzymatically generated by C. sinensis ESPs cause NF-κB-mediated inflammation in human cholangiocarcinoma cells (HuCCT1). Therefore, the present study was conducted to examine the role of upstream Toll-like receptors (TLRs) on the initial host innate immune responses to infection. We found that treatment of HuCCT1 cells with native ESPs induced changes in TLR mRNA levels in a time-dependent manner, concomitant with the generation of free radicals. ESP-mediated free radical generation was markedly attenuated by preincubation of the cells with TLR1-4-neutralizing antibodies, indicating that at least TLR1 through 4 participate in stimulation of the host innate immune responses. These findings indicate that free radicals triggered by ESPs are critically involved in TLR signal transduction. Continuous signaling by this pathway may function in initiating C. sinensis infection-associated inflammation cascades, a detrimental event leading to progression to more severe hepatobiliary diseases.

Lipopolysaccharide, Mediator of Sepsis Enigma: Recognition and Signaling.

The outer leaflet of Gram-negative bacterial membrane contains a great amount of lipopolysaccharides, also known as endotoxins, which play a central role in the pathogenesis of sepsis and ultimately septic shock. Lipopolysaccharide (LPS) is potent inducer of acute sepsis or chronic inflammation. Sepsis can strike anyone, but is most likely to develop from infection associated with events such as pneumonia, trauma, surgery, and burns, or serious illnesses such as cancer and AIDS. In fact, people whose deaths are ascribed to complications of cancer, AIDS, or pneumonia, often actually die as a direct result of sepsis. Sepsis involves a complex interaction between bacterial toxins and the host immune system. LPS stimulates Toll-like receptor (TLR)-4 which leads to the formation and release of range of proinflammatory mediators which are essential for the potent immune response. The massive host response to this single bacterial pattern recognition molecule is sufficient to generate diffuse endothelial injury, tissue hypoperfusion, disseminated intravascular coagulation and refractory shock. LPS recognition involves LPS binding protein (LBP), CD14 ending up in TLR4/MD-2/LPS complex. The complex leads to activation of TLR4 and subsequent signaling cascade via two pathways i. e., myeloid differentiation protein 88 (MyD88)-dependent and TRIF-dependent. Here is a brief review of TLR4 signaling and LPS recognition biology with its impact if any on downstream pathways.

Topical ALA-Photodynamic Therapy for Acne Can Induce Apoptosis of Sebocytes and Down-regulate Their TLR-2 and TLR-4 Expression

BACKGROUND: Although photodynamic therapy (PDT) is widely performed for acne, little is known about its exact therapeutic mechanism. OBJECTIVE: We aimed to estimate the efficacy and safety of PDT on acne and to discover its mode of action. METHODS: We performed PDT on 12 patients with mild to moderate acne. The clinical efficacy was assessed by counting the acne lesions and measuring the sebum secretion before and after PDT. In addition, we took biopsy samples from the peri-lesional areas before and after 3-session of PDT. To examine the degree of apoptosis of the sebaceous follicles, TUNEL assay was performed. To investigate the changes of toll-like receptor (TLR)-2 and TLR-4 expression after PDT, immunohistochemical stainings were also carried out. Finally, we performed TUNEL assay using the cultured sebocytes to confirm the apoptosis of sebocytes in vitro after PDT. RESULTS: There was a significant reduction in the number of inflammatory acne lesions after PDT, compared to baseline (p<0.05). Sebum excretion significantly decreased 2 weeks after the first PDT session except for one patient (p<0.05). The TUNEL positive cells in the peri-lesional sebaceous glands after PDT markedly increased, compared with those of before PDT. A decrease in TLR-2 and TLR-4 expression by sebaceous glands and epidermis after PDT was 50% and 30%, respectively. CONCLUSION: Our results demonstrate that apoptosis of the sebaceous glands is associated with improvement of acne by PDT. PDT has shown to down-regulate TLR-2 and TLR-4 expression in the sebaceous glands and epidermis of acne patients.

Single and Dual Ligand Effects on Gene Expression Changes in Mouse Macrophage Cells

We identified differentially expressed genes in RAW264.7 cells in response to single and double ligand treatments (LPS, IFNgamma, 2MA, LPS plus IFNgamma, and LPS plus 2MA). The majority of the regulated transcripts responded additively to dual ligand treatment. However, a significant fraction responded in a non-additive fashion. Several cytokines showing non-additive transcriptional responses to dual ligand treatment also showed non-additive protein production/secretion responses in separately performed experiments. Many of the genes with non-additive responses to LPS plus 2MA showed enhanced responses and encoded pro-inflammatory proteins. LPS plus IFNgamma appeared to induce both non-additive enhancement and non-additive attenuation of gene expression. The affected genes were associated with a variety of biological functions. These experiments reveal both dependent and independent regulatory pathways and point out the specific nature of the regulatory interactions.