• Journal of Plant Biotechnology
• /
• 제32권4호
• /
• pp.269-273
• /
• 2005

Methylotrophic 효모 Pichia pastoris 발현시스템을 사용하여 인간 TLR9 단백질의 세포내 TIR 도메인을 발현하였다. TIR 단백질이 P. pastoris에서 발현되어 배지 속으로 분비되는 것을 SDS-PAGE로 확인하였고, 발현된 단백질을 western-blot, MALDI-TOF 질량분석으로 동정하였다. 이를 통하여 TIR 딘백질이 P. pastoris에서 안정적으로 발현됨을 알 수 있었다. 그리고 발현된 단백질을 니켈 친화, 양이온교환수지, 겔 투과 크로마토그라피를 사용하여 순수 분리 정제하였다. P. pastoris를 이용한 단백질의 발현과 정제방법은 대장균에서 잘 발현되지 않는 단백질의 발현에 응용될 수 있을 것이다.

• Genomics & Informatics
• /
• 제10권3호
• /
• pp.153-166
• /
• 2012

A variety of ligands differ in their capacity to bind the receptor, elicit gene expression, and modulate physiological responses. Such receptors include Toll-like receptors (TLRs), which recognize various patterns of pathogens and lead to primary innate immune activation against invaders, and G-protein coupled receptors (GPCRs), whose interaction with their cognate ligands activates heterotrimeric G proteins and regulates specific downstream effectors, including immuno-stimulating molecules. Once TLRs are activated, they lead to the expression of hundreds of genes together and bridge the arm of innate and adaptive immune responses. We characterized the gene expression profile of Toll-like receptor 4 (TLR4) in RAW 264.7 cells when it bound with its ligand, 2-keto-3-deoxyoctonate (KDO), the active part of lipopolysaccharide. In addition, to determine the network communications among the TLR, Janus kinase (JAK)/signal transducer and activator of transcription (STAT), and GPCR, we tested RAW 264.7 cells with KDO, interferon-${\beta}$, or cAMP analog 8-Br. The ligands were also administered as a pair of double and triple combinations.

• 한국발생생물학회지:발생과생식
• /
• 제21권4호
• /
• pp.361-370
• /
• 2017

Koi herpesvirus (KHV), also known as Cyprinid herpes virus 3 (Cyprinid 3) is lethal disease in common carp and koi (Cyprinus carpio). Two different groups (KK and RK) were infected KHV by intraperitoneal injection. Fish for gene expression analysis were sampled at 0 h, 12 h, 24 h, 48 h and 72 h post infection (p.i). The results showed that two immune related gene, Interferons (INFs) ${\alpha}{\beta}$ and Interleukin (IL)-12 p35 induced a high response in RK. The IL-12 p35 cytokine and Toll-like receptor (TLR) 9 were significantly high expressed on 48 h post infection (p.i) in RK as compared to the KK. The histopatological examination reveals focal necrosis in liver and infiltrate of lymphocytes in spleen of KK as compared to the RK. In immunohistochemistry analysis, the KHV protein high expressed in the infected kidney cell and slenocyte of KK. Therefore, the expression of IL-12 p35, IFN ${\alpha}{\beta}$ and TLR 9 may provide a potentially genes related with KHV resistance in Koi and red common carp ${\times}$ koi.

• 생명과학회지
• /
• 제22권12호
• /
• pp.1637-1643
• /
• 2012

본 연구에서는 IL-$1{\beta}$ 발현에 PG의 영향을 조사하였고, 단핵세포에서 PG에 의한 IL-$1{\beta}$ 상향조절에 포함된 세포인자를 밝혔다. PG에 사람의 THP-1 세포를 노출시키면 IL-$1{\beta}$ 분비 증가뿐만 아니라 IL-$1{\beta}$ 유전자 전사를 유도하는 결과를 가져왔고, TLR-2/4의 억제제인 OxPAPC에 의해 저해되었다. U0126, SP6001250, Akti IV, rapamycin, DPI 같은 약리학적 저해제도 PG에 의한 IL-$1{\beta}$의 상향조절을 상당히 약화시켰다. 그러나 polymyxin B는 IL-$1{\beta}$ 발현에 영향을 미치지 않았다. 본 연구는 PG는 TLR-2, Akt, mTOR, MAPKs, ROS를 통하여 IL-$1{\beta}$의 발현을 상향시킴을 확인하였다.

• Journal of Microbiology and Biotechnology
• /
• 제29권5호
• /
• pp.704-712
• /
• 2019

Although nanometric dead Lactobacillus plantarum has emerged as a potentially important modulator of immune responses, its underlying mechanism of action has not been fully understood. This study aimed to identify the detailed biochemical mechanism of immune modulation by micronized and heat-treated L. plantarum LM1004 (MHT-LM1004, <$1{\mu}m$ in size). MHT-LM1004 was prepared from L. plantarum LM1004 via culture in a specifically designed membrane bioreactor and heat treatment. MHT-LM1004 was shown to effectively induce the secretion of $TNF-{\alpha}$ and IL-6 and the mRNA expression of inducible nitric oxide synthase (iNOS). MHT-LM1004 enhanced the expression of TLR-2, phosphorylation of MAPKs (ERK), and nuclear translocation of $NF-{\kappa}B$ in a dose-dependent manner. Oral administration of MHT-LM1004 ($4{\times}10^9$ or $4{\times}10^{11}cells/kg$ mouse body weight) increased the splenocyte proliferation and serum cytokine levels. These results suggested that MHT-LM1004 effectively enhances early innate immunity by activating macrophages via the TLR-2/MAPK/$NF-{\kappa}B$ signalling pathway and that this pathway is one of the major routes in immune modulation by the Lactobacillus species.

• Journal of Veterinary Science
• /
• 제24권3호
• /
• pp.37.1-37.14
• /
• 2023

Background: Toll-like receptor (TLR) agonists have been used as adjuvants to modulate immune responses in both animals and humans. Objectives: The objective of this study was to evaluate the combined effects of the TLR 4 agonist monophosphoryl lipid A (MPL) and the TLR 3 agonist polyinosinic:polycytidylic acid (Poly I:C) on equine peripheral blood mononuclear cells (PBMCs), monocyte-derived dendritic cells (MoDCs), and bone marrow-derived mesenchymal stromal cells (BM-MSCs). Methods: The PBMCs, MoDCs, and BM-MSCs collected from three mixed breed horses were treated with MPL, Poly I:C, and their combination. The mRNA expression of interferon gamma (IFN-γ), interleukin (IL)-1β, IL-4, IL-6, IL-8, IL-12p40, tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), and monocyte chemoattractant protein-1 (MCP-1) was determined using real-time polymerase chain reaction. Results: The combination of MPL and Poly I:C significantly upregulated immunomodulatory responses in equine cells/ without cytotoxicity. The combination induced greater mRNA expression of pro-inflammatory cytokines IFN-γ and IL-6 than MPL or Poly I:C stimulation alone in PBMCs. In addition, the combination induced significantly higher mRNA expression of IL-1β, IL-6, and IL-12p40 in MoDCs, and IL-8, MCP-1, and VEGF in BM-MSCs compared to stimulation with a single TLR agonist. Conclusions: The combination of MPL and Poly I:C can be used as a potential adjuvant candidate for vaccines to aid in preventing infectious diseases in horses.

• Clinical and Experimental Vaccine Research
• /
• 제12권4호
• /
• pp.328-336
• /
• 2023

Purpose: Human peripheral blood mononuclear cell (PBMC)-based in vitro systems can be of great value in the development and assessment of vaccines but require the right medium for optimal performance of the different cell types present. Here, we compare three commonly used media for their capacity to support innate and adaptive immune responses evoked in PBMCs by Toll-like receptor (TLR) ligands and whole inactivated virus (WIV) influenza vaccine. Materials and Methods: Human PBMCs were cultured for different periods of time in Roswell Park Memorial Institute (RPMI), Dulbecco's minimal essential medium (DMEM), or Iscove's modified DMEM (IMDM) supplemented with 10% fetal calf serum. The viability of the cells was monitored and their responses to TLR ligands and WIV were assessed. Results: With increasing days of incubation, the viability of PBMCs cultured in RPMI or IMDM was slightly higher than that of cells cultured in DMEM. Upon exposure of the PBMCs to TLR ligands and WIV, RPMI was superior to the other two media in terms of supporting the expression of genes related to innate immunity, such as the TLR adaptor protein gene MyD88 (myeloid differentiation factor 88), the interferon (IFN)-stimulated genes MxA (myxovirus resistance protein 1) and ISG56 (interferon-stimulated gene 56), and the leukocyte recruitment chemokine gene MCP1 (monocyte chemoattractant protein-1). RPMI also performed best with regard to the activation of antigen-presenting cells. As for adaptive immunity, when stimulated with WIV, PBMCs cultured in RPMI or IMDM contained higher numbers of IFNγ-producing T cells and secreted more immunoglobulin G than PBMCs cultured in DMEM. Conclusion: Taken together, among the different media assessed, RPMI was identified as the optimal medium for a human PBMC-based in vitro vaccine evaluation system.

• Asian-Australasian Journal of Animal Sciences
• /
• 제25권7호
• /
• pp.1021-1028
• /
• 2012

Peripheral blood mononuclear cells (PBMCs) discriminate microbial pathogens and induce T-cell responses of appropriate effector phenotype accordingly. Toll-like receptors (TLRs), in part, mediate this microbial recognition and differentiation while the development of T-cell effector functions critically depends on the release of Th1- or Th2- type cytokines. In the present study, buffalo PBMCs were stimulated under in vitro culture conditions by Bacillus subtilis cell wall petidoglycan, a TLR2 ligand, in a dose- and time- dependent manner. The expression of TLR2 as well as the subsequent differential induction of the Th1 and Th2 type cytokines was measured. Stimulation was analyzed across five doses of peptidoglycan ($10{\mu}g/ml$, $20{\mu}g/ml$, $30{\mu}g/ml$, $40{\mu}g/ml$ and $50{\mu}g/ml$) for 3 h, 12 h, 24 h and 36 h incubation periods. We observed the induction of TLR2 expression in a dose- and time-dependent manner and the peptidoglycan induced tolerance beyond $30{\mu}g/ml$ dose at all incubation periods. The correlation between peptidoglycan stimulation and TLR2 induction was found positive at all doses and for all incubation periods. Increased production of all the cytokines was observed at low doses for 3 h incubation, but the expression of IL-4 was relatively higher than IL-12 at the higher antigen doses, indicating tailoring towards Th2 response. At 12 h incubation, there was a pronounced decrease in IL-4 and IL-10 expression relative to IL-12 in a dose- dependent manner, indicating skewing to Th1 polarization. The expression of IL-12 was highest for all doses across all the incubation intervals at 24 h incubation, indicating Th1 polarization. The relative expression of TNF-${\alpha}$ and IFN-${\gamma}$ was also higher while that of IL-4 and IL-10 showed a decrease. For 36 h incubation, at low doses, relative increase in the expression of IL-4 and IL-10 was observed which decreased at higher doses, as did the expression of all other cytokines. The exhaustion of cytokine production at 36 h indicated that PBMCs became refractory to further stimulation. It can be concluded from this study that the cytokine response to sPGN initially was of Th2 type which skews, more pronouncedly, to Th1 type with time till the cells become refractory to further stimulation.

• 한국자원식물학회지
• /
• 제36권1호
• /
• pp.15-25
• /
• 2023

이삭물수세미는 민간에서는 전초를 고름, 염증 등에 약용으로 사용하였으나, 염증에 대한 연구가 미비한 상황이다. 이에 본 연구에서는 이삭물수세미 추출물(EMS)의 항산화 효능과 항염증 효능을 분석하였다. 항산화 효능은 DPPH 라디칼 소거능과 환원력을 통해 산화적 스트레스를 통해 염증을 유발시킬 수 있는 ROS (Hong et al., 2020; Snezhkina et al., 2019)를 억제하는지 확인하였고, 항염증 효능은 염증 발현 인자인 LPS를 이용하여 RAW 264.7 대식세포에 염증을 유도한 뒤 pro-inflammatory cytokine (TNF-α, IL-1β)과 염증 매개체(NO, PGE₂)의 억제 및 TLR4/Myd88/NF-κB signaling pathway 발현 억제를 통해 확인하였다. 연구 결과, 항산화 효능에 있어서는 DPPH 라디칼 소거능과 Fe³⁺를 Fe²⁺로 환원시키는 환원력이 농도 의존적으로 증가함을 확인하였다. 무독성 상태에서 실험하기 위해 LPS와 EMS를 처리한 RAW 264.7 대식세포에서 90% 이상의 생존율을 나타내는 조건에서 실험을 진행하였다. LPS로 염증이 유도된 RAW 264.7 세포에서 EMS는 염증 매개 인자의 발현 및 생성 억제(iNOS에 의한 NO 생성 및 COX-2에 의한 PGE₂ 생성억제)와 pro-inflammatory cytokine (TNF-α 및 IL-1β)의 생성 또한 억제하였다. 특이적으로 COX-2에 의한 PGE₂ 생성 억제에서는 고농도에서 작용함을 확인하였고, IL-1β에서는 약한 억제력을 보였다. 이후 signaling pathway에서 염증 전사인자 경로를 확인하기 위하여 TLR4/MyD88의 활성을 확인하였고, EMS 처리에 따라 농도 의존적으로 억제되는 것을 확인하였다. 이에 따라 염증 초기 단계에서 NF-κB p65가 nuclear로 들어가는 것을 억제하는지 확인하기 위해 early time (LPS 처리 후 30, 60 min) 조건으로 nuclear에서 p65 인산화를 확인하였다. 그 결과, LPS 자극으로 인해 증가된 p65 인산화가 EMS에 의해 부분적으로 억제됨을 확인하였다. 이상의 결과를 통해 LPS로 염증이 유도된 RAW 264.7 대식세포에서 EMS가 COX-2에 의한 PGE₂ 생성 억제와 IL-1β의 생성에 있어 낮은 억제력을 가진 반면, iNOS에 의한 NO과 TNF-α 생성 및 TLR4/MyD88 singnaling pathway에 있어 강한 억제력을 가짐을 확인하였다. 결론적으로 EMS가 ROS를 제거하고 TLR4/MyD88/NF-κB signaling pathway를 억제함으로써 염증 인자들의 전사를 억제하고, 염증 인자 부분에서는 iNOS에 의한 NO 생성과 TNF-α 생성을 강하게 억제하여 RAW 264.7 대식세포에서 LPS로 자극된 염증을 억제하는 것으로 판단된다. 또한 TLR4/Myd88/NF-κB signaling pathway를 통한 pro-inflammatory cytokine과 염증 매개체와의 연관성에 대한 기초자료로 활용할 수 있는 근거 자료가 될 수 있을 것으로 생각된다.

• Nutrition Research and Practice
• /
• 제15권6호
• /
• pp.798-806
• /
• 2021

BACKGROUND/OBJECTIVES: Obesity is associated with chronic inflammation. The spleen is the largest organ of the lymphatic system and has an important role in immunity. Obesity-induced inflammatory responses are triggered by Toll-like receptor (TLR)-myeloid differentiation primary response 88 (MyD88) pathway signaling. Phenethyl isothiocyanate (PEITC) and 3,3'-diindolylmethane (DIM), major dietary glucosinolates present in cruciferous vegetables, have been reported to produce anti-inflammatory effects on various diseases. However, the effects of PEITC and DIM on the obesity-induced inflammatory response in the spleen are unclear. The purpose of this study was to examine the anti-inflammatory effects of PEITC and DIM on the spleen and their mechanism in high fat/cholesterol diet (HFCD)-fed C57BL/6 mice. MATERIALS/METHODS: We established an animal model of HFCD-induced obesity using C57BL/6 mice. The mice were divided into six groups: normal diet with AIN-93G diet (CON), high fat diet (60% calories from fat) with 1% cholesterol (HFCD), HFCD with PEITC 30 mg/kg/day or 75 mg/kg/day (HFCD+P30, HFCD+P75), and HFCD with DIM 1.5 mg/kg/day or 7.5 mg/kg/day (HFCD+D1.5, HFCD+D7.5). Enzyme-linked immunosorbent assay was used to evaluate pro-inflammatory cytokine secretion. Western blot and quantitative polymerase chain reaction were used to analyze protein and mRNA levels of nuclear factor kappa B (NF-κB) p65, interleukin 6 (IL-6), cyclooxygenase 2 (COX-2), TLR2, TLR4, and MyD88 in spleen tissue. RESULTS: Serum IL-6 levels were significantly higher in the HFCD group than in groups fed a HFCD with PEITC or DIM. Levels of NF-κB p65 protein and TLR2/4, MyD88, NF-κB p65, IL-6, and COX-2 mRNA were significantly higher in the HFCD group than in the CON group and were reduced by the PEITC and DIM supplements. CONCLUSIONS: PEITC- and DIM-supplemented diets improved splenic inflammation by modulating the TLR2/4-MyD88 pathway in HFCD-fed mice. We suggest that dietary glucosinolates may at least partially improve obesity-induced inflammation of the spleen.