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Hydroxyapatite nanoparticles drive the potency of toll-like receptor 9 agonist for amplified innate and adaptive immune response

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Nano Research 2022年第10期15卷 9286-9297页

作者： Qin Zeng Ruiqi Wang Yuchen Hua Hongfeng Wu Xuening Chen You-cai Xiao Qiang Ao Xiangdong Zhu Xingdong Zhang National Engineering Research Center for Biomaterials Sichuan UniversityChengdu 610064China NMPA Key Laboratory for Quality Research and Control of Tissue Regenerative Biomaterials&Institute of Regulatory Science for Medical Devices&NMPA Research Base of Regulatory Science for Medical Devices Sichuan UniversityChengdu 610064China College of Biomedical Engineering Sichuan UniversityChengdu 610064China Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province Sichuan UniversityChengdu 610041China

The potency of toll-like receptor 9(TLR9)agonist to drive innate immune response was limited due to immune suppression or tolerance during TLR9 signaling activation in immune cells.Herein we addressed this problem by introducing hydroxyapatite nanoparticles(HANPs)to CpG ODN(CpG),a TLR9 agonist.The study revealed that HANPs concentration and durationdependently reprogramed the immune response by enhancing the secretion of immunostimulatory cytokines(tumor necrosis factorα(TNFα)or IL-6)while reducing the production of immunosuppressive cytokine(IL-10)in macrophages in response to CpG.Next,the enhanced immune response benefited from increased intracellular Ca2+in macrophage by the addition of HANPs.Further,we found exposure to HANPs impacted the mitochondrial function of macrophages in support of the synthesis of adenosine triphosphate(ATP),the production of nicotinamide adenine dinucleotide(NAD),and reactive oxygen species(ROS)in the presence or absence of CpG.In vaccinated mice model,only one vaccination with a mixture of CpG,HANPs,and OVA,a model antigen,allowed the development of a long-lasting balanced humoral immunity in mice without any histopathological change in the local injection site.Therefore,this study revealed that HANPs could modulate the intracellular calcium level,mitochondrial function,and immune response in immune cells,and suggested a potential combination adjuvant of HANPs and TLR9 agonist for vaccine development.

关键词： hydroxyapatite nanoparticles toll-like receptor 9 intracellular calcium mitochondrial function adaptive immune response

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toll-like receptor 9 polymorphisms and Helicobacter pylori influence gene expression and risk of gastric carcinogenesis in the Brazilian population

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World Journal of Gastrointestinal Oncology 2019年第11期11卷 998-1010页

作者： Manoela Dias Susi de Matos Lourenco Caroline Lucas Trevizani Rasmussen Spencer Luis Marques Payao Ana Flavia Teixeira Rossi Ana Elizabete Silva Juliana Garcia de Oliveira-Cucolo Department of Graduate-Level Research USCSacred Heart UniversityBauru 17011-970SPBrazil Department of Genetics and Molecular Biology FAMEMA-Marilia Medical SchoolMarília 17519-030SPBrazil Department of Biology Sao Paulo State University-UNESPSao Jose do Rio Preto 15054-000SPBrazil Department of Molecular Biological and Genetics and Molecular Biology Research Unit-UPGEMFaculty of Medicine of Sao Jose do Rio Preto-FAMERPSao Jose do Rio Preto 15090-000SPBrazil

BACKGROUND toll-like receptors(TLRs)are the first line of host defense,and are involved in Helicobacter pylori(H.pylori)recognition and activation of both inflammatory and carcinogenic processes.The presence of single nucleotide polymorphisms(SNPs)in genes that activate the immune response may modulate the risk of precancerous lesions and gastric cancer(GC).Among them,toll-like receptor 9(TLR9)polymorphisms have emerged with a risk factor of infectious diseases and cancer,however the studies are still inconclusive.AIM To evaluate whether TLR9 rs5743836 and rs187084 SNPs contribute to the risk of gastric carcinogenesis,and its influence on mRNA expression.METHODS A case-control study was conducted to evaluate two TLR9 SNPs(TLR9-1237 TCrs5743836 and TLR9-1486 CT-rs187084)in chronic gastritis(CG)and GC patients.A total of 609 DNA samples of peripheral blood[248 CG,161 GC,and 200 samples from healthy individuals(C)]were genotyped by polymerase chain reaction-restriction fragment length polymorphism.All samples were tested for the H.pylori infection using Hpx1 and Hpx2 primers.Quantitative polymerase chain reaction by TaqMan?assay was used to quantify TLR9 mRNA from fresh gastric tissues(48 GC,26 CG,and 14 C).RESULTS For TLR9-1237,the TC+CC or CC genotypes were associated with a higher risk of GC than C[recessive model odds ratio(OR)=5.01,95%confidence interval(CI):2.52-9.94,P95%CI:2.44-8.79,P9-1486,an association between the CT+TT genotypes and increased risk of both GC(dominant model OR=2.72,95%CI:1.57-4.72,P9,95%CI:1.15-2.79,P=0.0094)was observed when compared to the C group.Moreover,the presence of TLR9-1237 TC/CC+TLR9-1486 CC genotypes potentiate the risk for this neoplasm(OR=18.57;95%CI:5.06-68.15,P9 mRNA level was significantly higher in the GC group(RQ=9.24,P<0.0001)in relation to the CG group(RQ=1.55,P=0.0010)and normal mucosa(RQ=1.0).When the samples were grouped according to the polymorphic

关键词： toll-like receptor 9 Helicobacter pylori Gastric cancer Chronic gastritis Polymorphisms Gene expression

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Neutrophil extracellular traps contribute to myofibroblast differentiation and scar hyperplasia through the toll-like receptor 9/nuclear factor Kappa-B/interleukin-6 pathway

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Burns & Trauma 2022年第1期10卷 116-128页

作者： Yiming Shao Zaiwen Guo Yunxi Yang Lu Liu Jiamin Huang Yi Chen Linbin Li Bingwei Sun Department of Burns and Plastic Surgery Affiliated Suzhou Hospital of Nanjing Medical UniversitySuzhou 215002Jiangsu ProvinceChina

Background:Inflammation is an important factor in pathological scarring.The role of neutrophils,one of the most important inflammatory cells,in scar hyperplasia remains unclear.The purpose of this article is to study the correlation between neutrophil extracellular traps(NETs)and scar hyperplasia and identify a new target for inhibiting scar hyperplasia.Methods:Neutrophils were isolated from human peripheral blood by magnetic-bead sorting.NETs in plasma and scars were detected by enzyme-linked immunosorbent assays(ELISAs),immunofluorescence and flow cytometry.Immunohistochemistry was used to assess neutrophil(CD66B)infiltration in hypertrophic scars.To observe the entry of NETs into fibroblasts we used immunofluorescence and flow cytometry.Results:We found that peripheral blood neutrophils in patients with hypertrophic scars were more likely to form NETs(p0.05].However,inhibiting the production of NETs or degrading DNA reduced the differentiation of fibroblasts intomyofibroblasts.In vitro,NETs were found to be mediated by toll-like receptor 9(TLR-9)in fibroblasts and further phosphorylated nuclear factor Kappa-B(NF-κB).We found that IL-6,which is downstream of NF-κB,was increased in fibroblasts.Additionally,IL-6 uses autocrine and paracrine signaling to promote differentiation and secretion.Conclusions:Our experiments found that NETs activate fibroblasts through the TLR-9/NF-κB/IL-6 pathway,thereby providing a new target for regulating hypertrophic scars.

关键词： Neutrophil extracellular traps Hypertrophic scar toll-like receptor 9 Fibroblast Inflammation Differentiation Nuclear factor Kappa-B Interleukin-6

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Expression of toll-like receptor 9 in Peripheral Blood Mononuclear Cells from Patients with Different Hepatitis B and C Viral Loads

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2009年第3期29卷 313-317页

作者：周健黄元成田德英许东陈淼吴会玲 Department of Infectious Diseases Tongji Hospital Tongji Medical College Huazhong University of Science and Technology

The aim of the present study was to investigate the expression of toll-like receptors （TLR） 9 in peripheral blood mononuclear cells （PBMC） of patients with chronic hepatitis B and C with different virus copies. The study group included 90 patients （60 with chronic hepatitis B, and 30 with chronic hepatitis C）, and 20 healthy people served as control group. The protein and mRNA levels of TLR9 were detected by using flow cytometry and real-time PCR. The serum viral copies of HBV and HCV were measured in all patients, and the correlation between HBV-DNA copies or HCV-RNA copies and the TLR9 expression was analyzed. Our results demonstrated that HBV or HCV infection led to a decreased expression of TLR9 mRNA and protein compared to the control group （P〈0.05）. The TLR9 protein and mRNA levels were negatively correlated with serum viral copies of HBV and HCV （r=-0.632, r=-0.909, P〈0.01）. It was concluded that TLR9 mRNA and protein are down-regulated in PBMC of HBV-infected or HCV-infected patients, and they are negatively correlated with serum viral copies and play an important role in detecting viral replication of HBV and HCV.

关键词： peripheral blood mononuclear cells innate immunity toll-like receptor 9

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The Recognition of CpG-oligodeoxynucleotides by toll-like receptor 9 and Cytokine Prodution are Modulated by Extracellular Acidic pH

The Recognition of CpG-oligodeoxynucleotides by Toll-like Re...

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2005全国第二届核酸疫苗研讨会

作者： HE Ying SUN Shu-Han HE Xiao-Wen LI Kai WANG Fang JIANG Lei (Department of Medical Genetics, College of Basic Medical Sciences, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, P.R.China)

Bacterial and synthetic DNAs containing CpG dinucleotides in specific sequence contexts activate the vertebrate immune system through toll-like receptor 9 (TLR9), although the mechanism of this interaction is still unclear. In the present study, we used CpG-oligodeoxynucleotides (CpG-ODNs) as immunomers, which showed immunostimulatory activity in transfected-HEK293 cells, stably expressing human TLR9, especially in acidic pH, whereas there was no significant activity in non-transfected-HEK 293 cells. When the extracellular pH is in neutral (pH 7.4), CpG-ODN induced low levels of IFN-γ, and when it shifted to acidic (pH 6.4) ,however,the CpG-ODN-induced IFN-γproduction from transfected-293 cells markedly increased. These results suggest direct involvement of TLR9 in the recognition of CpG motif and such pH change enabled CpG-ODN bind to TLR9 to trigger CpG-related signal transduction.

关键词： CpG-oligodeoxynucleotides toll-like receptor 9 Immunostimulation

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The role of MAPK in CD4(+) T cells toll-like receptor 9-mediated signaling following HHV-6 infection

The role of MAPK in CD4(+) T cells toll-like receptor 9-medi...

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第八届全国免疫学学术大会

作者： Chi J Wang F Li L Feng D Qin J Xie F Zhou F Chen Y Wang J Yao K Department of Microbiology and Immunology Nanjing Medical University

Human herpesvirus-6(HHV-6) is an important immunosuppressive and immunomodulatory virus that primarily infects immune cells(mainly CD4(+) T cells) and strongly suppresses the proliferation of infected cells.toll-like receptors are pattern-recognition receptors essential for the development of an appropriate innate immune defense against infection.To understand the role of CD4(+) T cells in the innate response to HHV-6 infection and the involvement of TLRs, we used an in vitro infection model and observed that the infection of CD4(+) T cells resulted in the activation of JNK/SAPK via up-regulation of toll-like receptor 9(TLR9).Associated with JNK activation, annexin V-PI staining indicated that HHV-6A was a strong inducer of apoptosis.Apoptotic response associated cytokines, IL-6 and TNF-α also induced by HHV-6A infection.

关键词： Mitogen-activated protein kinase toll-like receptor 9 Human herpesvirus-6

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Coexpression of TLR9 and VEGF-C is associated with lymphatic metastasis in prostate cancer

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Asian Journal of Andrology 2022年第4期24卷 380-385页

作者： Xian-Zi Zeng Zhan-Sen Huang Hong-Peng Fang Jie-Ying Wu Qun-Xiong Huang Chu-Bin Zhuang Jing Zhou Jin-Ming Di Department of Urology The Third Affiliated Hospital of Sun Yat-sen UniversityGuangzhou 510630China Department of Infertility and Sexual Medicine The Third Affiliated Hospital of Sun Yat-sen UniversityGuangzhou 510630China Department of Pathology The Third Affiliated Hospital of Sun Yat-sen UniversityGuangzhou 510630China

Prostate cancer(PCa)is one of the most frequent cancers in men,and its biomolecular targets have been extensively studied.This study aimed to analyze the expression of toll-like receptor 9(TLR9)and vascular endothelial growth factor C(VEGF-C)and the clinical value of the coexpression of TLR9 and VEGF-C in PCa.We retrospectively evaluated 55 patients with clinically localized,intermediate-risk,or high-risk PCa who underwent laparoscopic radical prostatectomy(LRP)and extended pelvic lymph node dissection(ePLND)without neoadjuvant hormonal therapy at a single institution from June 2013 to December 2016.In all 55 patients,the median number of lymph nodes(LNs)resected was 23(range:18-31),and a total of 1269 LNs were removed,of which 78 LNs were positive.Seventeen patients had positive LNs,with a positive rate of 30.9%.In addition,the immunohistochemical results in the above patients revealed that high TLR9 expression was correlated with higher Gleason score(GS)(P=0.049),increased LN metastasis(P=0.004),and more perineural invasion(PNI)(P=0.033).Moreover,VEGF-C expression was associated with GS(P=0.040),pathological stage(pT stage)(P=0.022),LN metastasis(P=0.003),and PNI(P=0.001).Furthermore,a significant positive correlation between TLR9 and VEGF-C was found(P9/VEGF-C phenotype was associated with LN metastasis(P=0.047).Collectively,we propose that TLR9 stimulation may promote LN metastasis in PCa cells through the upregulation of VEGF-C expression,thereby affecting the prognosis of PCa patients.Therefore,these markers may serve as valuable targets for the treatment of PCa.

关键词： biochemical progression-free survival coexpression lymphatic metastasis prostate cancer toll-like receptor 9 vascular endothelial growth factor C

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TLR9 Expression and Its Role in Chemosensitivity to DDP in Human Cervical Cancer Cells in vitro

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2011年第4期31卷 550-554页

作者：翁艳洁王永军石英周文娟王鸿雁王常玉 Department of Obstetrics and Gynecology Tongji HospitalTongji Medical CollegeHuazhong University of Science and Technology Minimally Invasive Center of Gynecology Beijing Obstetrics and Gynecology HospitalCapital Medical University

Inflammation and infection play an important role in the pathogenesis of many cancers.toll-like receptors（TLRs） are a class of pattern recognition receptors that recognize conserved components of microbes and trigger the immune response against invading microorganisms.toll-like receptor 9（TLR9） recognizes non-methylated cytosine-phosphateguanosine（CpG） DNA sequences which are the surrogate for viral DNA.TLR9 may react to tumor development and progression during chronic inflammation that involves the tumor microenvironment.In order to study the role of TLR9 in cervical cancer,we analyzed the TLR9 expression in different types of HPV infection cervical cancer cells.Then we detected if CpG sequences influenced the TLR9 expression and the sensitivity to cisplatin（DDP） of these cervical cancer cells in vitro.The expression of TLR9 mRNA and protein in SiHa,Hela and C33A cells was detected by RT-PCR and Western blotting.Real-time PCR was used to examine the TLR9 expression changes induced by CpG.Chemosensitivity of the cervical cancer cells to cisplatin（DDP） was measured by MTT.It was observed that the expression of TLR9 mRNA and protein was increased gradually in SiHa（HPV16＋）,Hela（HPV18＋） and C33A（HPV-） cells.Low doses of CpG increased the TLR9 expression only in C33A（HPV-） cells,but not in SiHa（HPV16＋） and Hela（HPV18＋） cells.Furthermore,low dose of CpG significantly increased the sensitivity of C33A（HPV-） cells,but not that of SiHa（HPV16＋） and Hela（HPV18＋） cells.These results indicated that TLR9 may serve as a protective agent in HPV negative cervical cancer cells.It was concluded that TLR9 could improve the sensitivity to DDP in HPV negative cervical cancer cells and might represent a potential therapeutic option in clinical practice.

关键词： cervical cancer toll-like receptor 9 cisplatin HPV infection

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L-Arginine mediates the development of cerebral malaria by modifying the pro-inflammatory response

L-Arginine mediates the development of cerebral malaria by m...

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第八届全国免疫学学术大会

作者： Yonghui Feng Jun Liu Yaming Cao Department of Immunology Basic Medicine College of China Medical University

Background:L-Arginine(L-Arg) has been demonstrated to enhance host humoral and cellular immune function.In malaria endemic area, the use of L-Arg was found to be safe and can reverse endothelial dysfunction, with no clinically significant adverse effects.Objective:To clarify the effects of L-Arg on the development of cerebral malaria(CM).Methods:C57BL/6 mice were treated with L-Arg pre or after the P.bANKA infection.We investigated the levels of parasitaemia in different experimental groups and tested the number of Th1 cells, F4/80+CD36+ cells,DCs and Tregs in experimental and normal infection groups by flow cytometry.ELISA was used to test cytokine levels.Results:Treatment of mice with L-Arg no matter pre or after infection led to the decreased survival time and mediated the development of CM.Pro-inflammatory response related effector cells and mediators, such as CD4+T-bet+IFN-γ+ Th1 cells, F4/80+CD36+ cells, IFN-γ, TNF-α and NO, were all increased by L-Arg.CD11c+ DCs and TLR9 expressing DCs were increased in mice treated with L-Arg.However, L-Arg had no effect on Tregs and IL-10.Conclusion:We highlight that L-Arg could increase Th1 immune response which is a very important protective factor in non-CM patients but could be lethal in CM patients.Therefore the use of L-Arg in endemic area need more concern.

关键词： Malaria L-Arginine Th1 immune responses dendritic cells toll-like receptor 9 Tregs

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