• Journal of Veterinary Science
• /
• 제23권2호
• /
• pp.27.1-27.16
• /
• 2022

Background: The role of Toll-like receptors (TLRs) in a feline infectious peritonitis virus (FIPV) infection is not completely understood. Objectives: This study examined the expression of TLR3, TLR7, TLR9, tumor necrosis factor-alpha (TNF-α), interferon (IFN)-β, and interleukin (IL)-10 upon an FIPV infection in Crandell-Reese feline kidney (CRFK) cells and feline monocytes. Methods: CRFK cells and monocytes from feline coronavirus (FCoV)-seronegative cats and FCoV-seropositive cats were infected with type II FIPV-79-1146. At four, 12, and 24 hours post-infection (hpi), the expression of TLR3, TLR7, TLR9, TNF-α, IFN-β, and IL-10, and the viral load were measured using reverse transcription quantitative polymerase chain reaction. Viral protein production was confirmed using immunofluorescence. Results: FIPV-infected CRFK showed the upregulation of TLR9, TNF-α, and IFN-β expression between 4 and 24 hpi. Uninfected monocytes from FCoV-seropositive cats showed lower TLR3 and TLR9 expression but higher TLR7 expression compared to uninfected monocytes from FCoV-seronegative cats. FIPV-infected monocytes from FCoV-seropositive cats downregulated TLR7 and TNF-α expression between 4 and 24 hpi, and 4 and 12 hpi, respectively. IFN-β was upregulated early in FIPV-infected monocytes from FCoV-seropositive cats, with a significant difference observed at 12 hpi compared to FCoV-seronegative cats. The viral load in the CRFK and FIPV-infected monocytes in both cohorts of cats was similar over time.ConclusionTLR7 may be the key TLR involved in evading the innate response against inhibiting TNF-α production. Distinct TLR expression profiles between FCoV-seronegative and FCoV-seropositive cats were observed. The associated TLR that plays a role in the induction of IFN-β needs to be explored further.

• International Journal of Oral Biology
• /
• 제46권4호
• /
• pp.190-199
• /
• 2021

Despite evidence that bacteria-sensing Toll-like receptors (TLRs) are activated in salivary gland tissues of Sjogren syndrome (SS) patients, the role of oral bacteria in SS etiopathogenesis is unclear. We previously reported that two SS-associated oral bacteria, Prevotella melaninogenica (Pm) and Rothia mucilagenosa (Rm), oppositely regulate the expression of major histocompatibility complex class I (MHC I) in human salivary gland (HSG) cells. Here, we elucidated the mechanisms underlying the differential regulation of MHC I expression by these bacteria. The ability of Pm and Rm to activate TLR2, TLR4, and TLR9 was examined using TLR reporter cells. HSG cells were stimulated by the TLR ligands, Pm, and Rm. The levels of MHC I expression, bacterial invasion, and viability of HSG cells were examined by flow cytometry. The hypoxic status of HSG cells was examined using Hypoxia Green. HSG cells upregulated MHC I expression in response to TLR2, TLR4, and TLR9 activation. Both Pm and Rm activated TLR2 and TLR9 but not TLR4. Rm-induced downregulation of MHC I strongly correlated with bacterial invasion and cell death. Rm-induced cell death was not rescued by inhibitors of the diverse cell death pathways but was associated with hypoxia. In conclusion, Pm upregulated MHC I likely through TLR2 and TLR9 activation, while Rm-induced hypoxia-associated cell death and the downregulation of MHC I, despite its ability to activate TLR2 and TLR9. These findings may provide new insight into how oral dysbiosis can contribute to salivary gland tissue damage in SS.

• Parasites, Hosts and Diseases
• /
• 제53권4호
• /
• pp.431-438
• /
• 2015

In Trichinella spiralis infection, type 2 helper T (Th2) cell-related and regulatory T ($T_{reg}$) cell-related immune responses are the most important immune events. In order to clarify which Toll-like receptors (TLRs) are closely associated with these responses, we analyzed the expression of mouse TLR genes in the small intestine and muscle tissue during T. spiralis infection. In addition, the expression of several chemokine- and cytokine-encoding genes, which are related to Th2 and $T_{reg}$ cell mediated immune responses, were analyzed in mouse embryonic fibroblasts (MEFs) isolated from myeloid differentiation factor 88 (MyD88)/TIR-associated proteins (TIRAP) and Toll receptor-associated activator of interferons (TRIF) adapter protein deficient and wild type (WT) mice. The results showed significantly increased TLR4 and TLR9 gene expression in the small intestine after 2 weeks of T. spiralis infection. In the muscle, TLR1, TLR2, TLR5, and TLR9 gene expression significantly increased after 4 weeks of infection. Only the expression of the TLR4 and TLR9 genes was significantly elevated in WT MEF cells after treatment with excretory-secretory (ES) proteins. Gene expression for Th2 chemokine genes were highly enhanced by ES proteins in WT MEF cells, while this elevation was slightly reduced in MyD88/$TIRAP^{-/-}$ MEF cells, and quite substantially decreased in $TRIF^{-/-}$ MEF cells. In contrast, IL-10 and $TGF-{\beta}$ expression levels were not elevated in MyD88/$TIRAP^{-/-}$ MEF cells. In conclusion, we suggest that TLR4 and TLR9 might be closely linked to Th2 cell and $T_{reg}$ cell mediated immune responses, although additional data are needed to convincingly prove this observation.

• Molecules and Cells
• /
• 제38권1호
• /
• pp.26-32
• /
• 2015

Toll-like receptors (TLR) 7 and 9 transduce a cellular signal through the MyD88-dependent pathway and induce the production of inflammatory mediators against microbial nucleotide components. The repeated stimulation of TLR4 leads to endotoxin tolerance, but the molecular mechanisms of tolerance induced through the costimulation of individual TLR has not yet been established, although endosomal TLRs share signaling pathways with TLR4. In the present study, mouse macrophages were simultaneously stimulated with the TLR7 agonist, gardiquimod (GDQ), and the TLR9 agonist, CpG ODN 1826, to examine the mechanism and effector functions of macrophage tolerance. Compared with individual stimulation, the costimulation of both TLRs reduced the secretion of TNF-${\alpha}$ and IL-6 through the delayed activation of the NF-${\kappa}B$ pathway; notably, IL-10 remained unchanged in costimulated macrophages. This tolerance reflected the early induction of suppressor of cytokine signaling-1 (SOCS-1), according to the detection of elevated TNF-${\alpha}$ secretion and restored NF-${\kappa}B$ signaling in response to the siRNA-mediated abrogation of SOCS-1 signaling. In addition, the restimulation of each TLRs using the same ligand significantly reduced the expression of both TLRs in endosomes. These findings revealed that the costimulation of TLR7 and TLR9 induced macrophage tolerance via SOCS-1, and the restimulation of each receptor or both TLR7 and TLR9 downregulated TLR expression through a negative feedback mechanisms that protects the host from excessive inflammatory responses. Moreover, the insufficient and impaired immune response in chronic viral infection might also reflect the repeated and simultaneous stimulation of those endosomal TLRs.

• 한국임상수의학회지
• /
• 제26권6호
• /
• pp.520-523
• /
• 2009

본 연구는 말의 정상안과 재발성 포도막염이 있는 안구에서의 TLR-2, -4, -9 mRNA 발현의 정량적 차이를 비교하기 위해 수행되었다. 정상 및 재발성 포도막염이 있는 말 각 6두 에서 안구를 적출하여 모양체, 홍채, 망막 및 맥락막을 수집하였다. Real-time PCR assay 통해 정상안과 재발성 포도막염이 있는 안구에서의 TLR-2, -4, -9의 mRNA 발현 차이를 정량적으로 비교하였다. 말의 재발성 포도막염 시에는 모양체, 홍채에서 정상인 경우에 비해 4-12 배의 TLR-2 와 TLR-9 mRNA 발현증가를 보였으며, 맥락막 및 망막에서는 2-6 배의 TLR-2, -4, -9 mRNA 발현 증가를 보였다. 본 연구 결과는 Toll-like receptor 2, -4, -9이 말의 재발성 포도막염의 병리기전에 영향을 미치고 있음을 시사한다. 하지만 재발성 포도막염 시의 Toll-like receptor 2, -4, -9의 구체적 역할을 밝히기 위해서는 다양한 후속 연구가 요구된다.

• Asian Pacific Journal of Cancer Prevention
• /
• 제15권14호
• /
• pp.5867-5872
• /
• 2014

Background: Cervical cancer is listed as one of high-incidence endemic diseases in Xinjiang. Our study aimed to evaluate the expression of TLR9 in uterine cervical tissues of Uyghur women and examine associations with clinicopathological variables. We further characterized the direct effects of TLR9 upon the selective silencing of human papillomavirus (HPV) E6 and E7 oncoprotein expression in HPV 16-positive human cervical carcinoma cells treated with siRNA in vitro. Materials and Methods: Immunohistochemistry was applied to evaluate TLR9 expression in 97 formalin-fixed paraffin-embedded cervical samples from Uyghur women; 32 diagnosed with cervical squamous cell carcinomas (CSCC), 14 with low-grade cervical intraepithelial neoplasias (CINI), 10 medium-grade (CINII), 24 high-grade (CINIII), and 17 chronic cervicitis. $BLOCK-iT^{TM}$ U6 RNAi Entry Vector $pENTR^{TM}$/U6-E6 and E7 was constructed and transfected the entry clone directly into the mammalian cell line 293FT. Then the HPV 16-positive SiHa human cervical carcinoma cell line was infected with RNAi recombinant lentivirus. RT-PCR and Western blotting were used to determine the expression of TLR9 in both SiHa and HPV 16 E6 and E7 silenced SiHa cells. Results: Immunohistochemical staining showed that TLR9 expression was undetectable (88.2%) or weak (11.8%) in chronic cervicitis tissues. However, variable staining was observed in the basal layer of all normal endocervical glands. TLR9 expression, which was mainly observed as cytoplasmic staining, gradually increased in accordance with the histopathological grade in the following order: chronic cervicitis (2/17, 11.8%)

• Clinical and Experimental Pediatrics
• /
• 제52권9호
• /
• pp.1015-1020
• /
• 2009

목 적 : 알레르기 환자와 정상인에서 형질세포양 수지상세포의 분포, TLR9 mRNA 양과 $IFN-{\alpha}$의 분비능에 차이가 있는지 알아보고자 하였다. 방 법 : 19명의 알레르기 환자와 17명의 건강한 성인을 대상으로 하였다. 말초혈액단핵세포를 채취하여 Lineage Cocktail(CD3, CD14, CD16, CD19, CD20, CD56)음성, HLA-DR 양성이면서 CD123양성을 유세포 분석기로 분석하였다. 말초혈액단핵세포에 TLR9 작용제(agonists)인 CpG-ODN 2216과 음성 대조를 위해 CpG-ODN 2206으로 자극하고 24시간 후 상청액을 추출하여 $IFN-{\alpha}$의 농도를 측정하였다. 또한 real time RT-PCR을 이용하여 TLR9 mRNA 정량분석을 시행하였다. 결 과 : 말초혈액단핵세포에서 형질세포양 수지상세포의 분포는 알레르기 환자가 평균 $0.1{\pm}0.04%$, 대조군이 평균 $0.25{\pm}0.23%$이었다. TLR9 mRNA 상대적인 양을 나타내는 ${\Delta}{\Delta}Ct$는 알레르기 환자에서 $1.29{\pm}0.41$이었고 대조군은 $1.25{\pm}0.23$이었다. TLR9 리간드인 CpG-ODN 2216 자극에 따른 $IFN-{\alpha}$의 분비능은 알레르기 환자에서 $911{\pm}829pg/mL$ 이었고 대조군에서 $1,095{\pm}888pg/mL$ 이었다. 이 세 결과에서 통계적인 차이는 없었다. 결 론 : TLR9을 통한 신호전달이 알레르기 환자의 면역반응을 대표하지는 않는 것으로 보이며, 향후 더 자세한 TLR9의 역할에 대한 연구가 필요하다.

• Journal of Microbiology and Biotechnology
• /
• 제33권9호
• /
• pp.1213-1227
• /
• 2023

Fetal growth restriction (FGR) is a prevalent obstetric condition. This study aimed to investigate the role of Toll-like receptor 9 (TLR9) in regulating the inflammatory response and gut microbiota structure in FGR. An FGR animal model was established in rats, and ODN1668 and hydroxychloroquine (HCQ) were administered. Changes in gut microbiota structure were assessed using 16S rRNA sequencing, and fecal microbiota transplantation (FMT) was conducted. HTR-8/Svneo cells were treated with ODN1668 and HCQ to evaluate cell growth. Histopathological analysis was performed, and relative factor levels were measured. The results showed that FGR rats exhibited elevated levels of TLR9 and myeloid differentiating primary response gene 88 (MyD88). In vitro experiments demonstrated that TLR9 inhibited trophoblast cell proliferation and invasion. TLR9 upregulated lipopolysaccharide (LPS), LPS-binding protein (LBP), interleukin (IL)-1β and tumor necrosis factor (TNF)-α while downregulating IL-10. TLR9 activated the TARF3-TBK1-IRF3 signaling pathway. In vivo experiments showed HCQ reduced inflammation in FGR rats, and the relative cytokine expression followed a similar trend to that observed in vitro. TLR9 stimulated neutrophil activation. HCQ in FGR rats resulted in changes in the abundance of Eubacterium_coprostanoligenes_group at the family level and the abundance of Eubacterium_coprostanoligenes_group and Bacteroides at the genus level. TLR9 and associated inflammatory factors were correlated with Bacteroides, Prevotella, Streptococcus, and Prevotellaceae_Ga6A1_group. FMT from FGR rats interfered with the therapeutic effects of HCQ. In conclusion, our findings suggest that TLR9 regulates the inflammatory response and gut microbiota structure in FGR, providing new insights into the pathogenesis of FGR and suggesting potential therapeutic interventions.

• Clinical and Experimental Reproductive Medicine
• /
• 제45권1호
• /
• pp.1-9
• /
• 2018

Objective: To determine the localization, expression, and function of Toll-like receptors (TLRs) in fallopian tube epithelial cells. Methods: The localization of TLRs in fallopian tube epithelial cells was investigated by immunostaining. Surprisingly, the intensity of staining was not equal in the secretory and ciliated cells. After primary cell culture of fallopian tube epithelial cells, ring cloning was used to isolate colonies of ciliated epithelial cells, distinct from non-ciliated epithelial cells. The expression of TLRs 1-10 was examined by quantitative real-time polymerase chain reaction, and protein localization was confirmed by immunostaining. The function of the TLRs was determined by interleukin (IL)-6 and IL-8 production in response to TLR2, TLR3, TLR5, TLR7, and TLR9 ligands. Results: Fallopian tube epithelial cells expressed TLRs 1-10 in a cell-type-specific manner. Exposing fallopian tube epithelial cells to TLR2, TLR3, TLR5, TLR7, and TLR9 agonists induced the secretion of proinflammatory cytokines such as IL-6 and IL-8. Conclusion: Our findings suggest that TLR expression in the fallopian tubes is cell-type-specific. According to our results, ciliated cells may play more effective role than non-ciliated cells in the innate immune defense of the fallopian tubes, and in interactions with gametes and embryos.

• Journal of Ginseng Research
• /
• 제31권4호
• /
• pp.181-190
• /
• 2007

본 연구에서는 고려홍삼으로부터 새로 분리한 CK 함유분획을 이용하여 마우스 대식세포에 대한 선천면역반응 조절에 미치는 영향을 조사하였다. 본 연구에서 사용된 농도의 CK 함유분획에서는 세포독성 효과가 관찰되지 않았으며 CK함유 분획의 전처리에 의하여 그람음성세균의 LPS, 또는 CpG-ODN에 의해 유도되는 NF-${\kappa}B$와 MAPK 활성 및 전염증성 사이토카인, NO의 분비가 TLR4 및 TLR9 특이적으로 억제되었다. 이와 같은 결과는 CK 함유분획이 TLR4을 매개로하는 염증반응뿐만 아니라 TLR9을 통한 염증반응에도 영향을 미치는 것으로 해석된다. 따라서 앞으로 CK 함유 분획에 포함된 개별 사포닌 등 시료 성분에 대한 면밀한 분석, 그리고 이들 개별 물질이 각각의 신호전달 체계에 미치는 영향과 그 기작에 대한 연구가 더욱 필요할 것으로 사료되며 염증억제제로서의 개발 가능성을 탐구하기 위한 생체 내에서의 효능 및 작용기전 분석이 요구된다.