Comparison of fertility competence in toll-like receptor 4 (TLR4)-knock out male mice fed a high-fat diet

이영주,오은영,Malavige Romesha Chandanee,Wijesooriya Mudhiyanselage Nadeema Dissanayake,이상명 충북대학교 동물의학연구소 2023 Journal of Biomedical and Translational Research Vol.24 No.2

Toll-like receptor 4 (TLR4) is known to contribute to the modulation of insulin resistance and systemic inflammation seen in obesity and the metabolic syndrome. The present study was performed to investigate the fertility competence of TLR4 knock out male mice (TLR4 mice) on a high-fat diet (HFD), compared to a normal-chow diet (NCD). The controls included wildtype (WT) mice fed on a HFD or NCD. Six-week-old male mice were fed with either a NCD or HFD for 20 weeks. Body and organ weights, serum levels of glucose, triglycerides and hepatoxicity, sperm quality and spermatogenesis were observed after the sacrifice. Also, randomly selected male mice were mated with virgin female mice after feeding of 19 weeks. The weight of the body and organs increased in WT and TLR4 mice on a HFD compared to those of mice on a NCD. The weights of the reproductive organs did not vary among the treatment groups. The motility and concentration of the epididymal spermatozoa decreased in both WT and TLR4 mice fed a HFD. The pregnancy rate and litter size declined in the HFD-fed WT mice compared to the HFD-fed TLR4 mice. In conclusion, the HFD alters energy and steroid metabolism in mice, which may lead to male reproductive disorders. However, fertility competence was somewhat restored in HFD-fed TLR4 male mice, suggesting that the TLR4 is involved in testis dysfunction due to metabolic imbalance.

Toll-like Receptor 2 Mediates Peripheral Nerve Injury-induced NADPH Oxidase 2 Expression in Spinal Cord Microglia

Lim, Hyoungsub,Kim, Donghoon,Lee, Sung Joong American Society for Biochemistry and Molecular Bi 2013 The Journal of biological chemistry Vol.288 No.11

<P>We have previously reported that NADPH oxidase 2 (Nox2) is up-regulated in spinal cord microglia after spinal nerve injury, demonstrating that it is critical for microglia activation and subsequent pain hypersensitivity. However, the mechanisms and molecules involved in Nox2 induction have not been elucidated. Previous studies have shown that Toll-like receptors (TLRs) are involved in nerve injury-induced spinal cord microglia activation. In this study, we investigated the role of TLR in Nox2 expression in spinal cord microglia after peripheral nerve injury. Studies using TLR knock-out mice have shown that nerve injury-induced microglial Nox2 up-regulation is abrogated in TLR2 but not in TLR3 or -4 knock-out mice. Intrathecal injection of lipoteichoic acid, a TLR2 agonist, induced Nox2 expression in spinal cord microglia both at the mRNA and protein levels. Similarly, lipoteichoic acid stimulation induced Nox2 expression and reactive oxygen species production in primary spinal cord glial cells <I>in vitro</I>. Studies on intracellular signaling pathways indicate that NF-κB and p38 MAP kinase activation is required for TLR2-induced Nox2 expression in glial cells. Conclusively, our data show that TLR2 mediates nerve injury-induced Nox2 gene expression in spinal cord microglia via NF-κB and p38 activation and thereby may contribute to spinal cord microglia activation.</P>

TLR10 and Its Unique Anti-Inflammatory Properties and Potential Use as a Target in Therapeutics

Faith Fore,Cut Indriputri,Janet Mamutse,Jusak Nugraha 대한면역학회 2020 Immune Network Vol.20 No.3

TLRs are pattern recognition receptors (PRRs) whose cytoplasmic signalling domain is similar to that of IL-1. The extracellular domain of TLRs serve as the binding site of pathogen associated molecular patterns. TLRs are found on both plasma and endosomal membranes and they mainly exert their function by activating genes which lead to production of inflammatory factors. The latest TLR to be discovered, TLR10 is a unique TLR which exhibit anti-inflammatory properties. TLR10 is found on the plasma membrane with other TLRs namely TLR1, TLR2, TLR4, TLR5 and TLR6. Studies have revealed that TLR10 is found on the same gene cluster with TLR1 and TLR6 and is also a coreceptor of TLR2. Up to date, TLR10 is the only TLR which exhibit anti-inflammatory property. Previously, TLR10 was thought to be an “orphan receptor” but much recent studies have identified ligands for TLR10. Currently there is no review article on TLR10 that has been published. In this narrative review, we are going to give an account of TLR10, its functions mainly as an anti-inflammatory PRR and its possible applications as a target in therapeutics.

Distinct evolution of toll‑like receptor signaling pathway genes in cetaceans

Ran Tian,Inge Seim,Zepeng Zhang,Ying Yang,Wenhua Ren,Shixia Xu,Guang Yang 한국유전학회 2019 Genes & Genomics Vol.41 No.12

Background The relatively rapid spread and diversity of marine pathogens posed an initial and ongoing challenge for cetaceans (whales, dolphins, and porpoises), descendants of terrestrial mammals that transitioned from land to sea approximately 56 million years ago. Toll-like receptors (TLRs) play important roles in regulating immunity against pathogen infections by detecting specific molecular patterns and activating a wide range of downstream signaling pathways. The ever-increasing catalogue of mammalian genomes offers unprecedented opportunities to reveal genetic changes associated with evolutionary and ecological processes. Objective This study aimed to explore the molecular evolution of TLR signaling pathway genes in cetaceans. Methods Genes involved in the TLR signaling pathway were retrieved by BLAST searches using human coding sequences as queries. We tested each gene for positive selection along the cetacean branches using PAML and Hyphy. Physicochemical property changes of amino acids at all positively selected residues were assessed by TreeSAAP and visualized with WebLogo. Bovine and dolphin TLR4 was assessed using human embryonic kidney cell line HEK293, which lacks TLR4 and its co-receptor MD-2. Results We demonstrate that eight TLR signaling pathway genes are under positive selection in cetaceans. These include key genes in the response to Gram-negative bacteria: TLR4, CD14, and LY96 (MD-2). Moreover, 41 out of 65 positively selected sites were inferred to harbor substitution that dramatically changes the physicochemical properties of amino acids, with most of them situated in or adjacent to functional regions. We also found strong evidence that positive selection occurred in the lineage of the Yangtze finless porpoise, likely reflecting relatively recent adaptions to a freshwater milieu. Species-specific differences in TLR4 response were observed between cetacean and terrestrial species. Cetacean TLR4 was significantly less responsive to lipopolysaccharides from a terrestrial E. coli strain, possibly a reflection of the arms race of host–pathogen co-evolution faced by cetaceans in an aquatic environment. Conclusion This study provides further impetus for studies on the evolution and function of the cetacean immune system.

Positive selection signatures in the TLR7 family

박승구,박동현,정유진,정은경,최선심 한국유전학회 2010 Genes & Genomics Vol.32 No.2

While adaptive immunity genes evolve rapidly under the influence of positive selection, innate immune system genes are known to evolve slowly due to strong purifying selection. Among the sensors of the innate immune system, Toll-like receptors (TLRs) are particularly important due to their ability to recognize and respond to pathogen-associated molecular patterns (PAMP), such as lipopolysaccharides, peptidoglycans,and nucleic acids from bacteria or viruses. In the present study,we examine the evolutionary process that has operated on the TLR7 family genes TLR7, TLR8, and TLR9. The results demonstrate that the average Ka/Ks (the ratio between nonsynonymous and synonymous substitution rates) of each TLR family gene is far lower than one regardless of estimating methods, supporting previous observations of strong purifying selection in this gene family. Interestingly, however, analysis of Ka/Ks ratios along the coding regions of TLR7 family genes by sliding-window analysis reveals a few narrow high peaks (Ka/Ks > 1). The most prominent peak corresponds to a specific region in the ectodomain, which exists only in the TLR7family, suggesting that this unique structure of the TLR7 fam-ily might have been a target of positive selection in a variety of lineages. Furthermore, maximum likelihood model tests suggest that positive selection is the best explanation for a certain fraction of the amino acid substitutions in the TLR9.

Identification of a gene encoding a membrane-anchored toll-like receptor 5 (TLR5M) in <i>Oplegnathus fasciatus</i> that responds to flagellin challenge and activates NF-κB

Umasuthan, Navaneethaiyer,Bathige, S.D.N.K.,Thulasitha, William Shanthakumar,Jayasooriya, R.G.P.T.,Shin, Younhee,Lee, Jehee Elsevier 2017 Fish & shellfish immunology Vol.62 No.-

<P><B>Abstract</B></P> <P>Toll-like receptor 5 (TLR5) recognizes bacterial flagellin and induces the downstream signaling through the myeloid differentiation primary response gene 88 (MyD88) protein to produce proinflammatory cytokines. In this study, we describe a TLR5 membrane form (OfTLR5M) and its adaptor protein MyD88 (OfMyD88) in rock bream, <I>Oplegnathus fasciatus.</I> Both <I>Oftlr5m</I> (6.7 kb) and <I>Ofmyd88</I> (3.7 kb) genes displayed a quinquepartite structure with five exons and four introns. Protein structure of OfTLR5M revealed the conventional architecture of TLRs featured by an extracellular domain with 22 leucine rich repeats (LRR), a transmembrane domain and an endodomain with TIR motif. Primary OfTLR5M sequence shared a higher homology with teleost TLR5M. The evolutional analysis confirmed that TLR5 identified in the current study is a membrane receptor and the data further suggested the co-evolution of the membrane-anchored and soluble forms of TLR5 in teleosts. Inter-lineage comparison of gene structures in vertebrates indicated that the <I>tlr5m</I> gene has evolved with extensive rearrangement; whereas, the <I>myd88</I> gene has maintained a stable structure throughout the evolution. Inspection of 5′ flanking region of these genes disclosed the presence of several transcription factor binding sites including NF-κB. Quantitative real-time PCR (qPCR) detected <I>Oftlr5m</I> mRNA in eleven tissues with the highest abundance in liver. <I>In vivo</I> flagellin administration strongly induced the transcripts of both <I>Oftlr5m</I> and <I>Ofmyd88</I> in gills and head kidney tissues suggesting their ligand-mediated upregulation. In a luciferase assay, HEK293T cells transiently transfected with <I>Oftlr5m</I> and <I>Ofmyd88</I> demonstrated a higher NF-κB activity than the mock control, and the luciferase activity was intensified when cells were stimulated with flagellin. Collectively, our study represents the genomic, evolutional, expressional and functional insights into a receptor and adaptor molecules of teleost origin that are involved in flagellin sensing.</P> <P><B>Highlights</B></P> <P> <UL> <LI> <I>Oftlr5m</I> and <I>Ofmyd88</I> genes in rock bream display quinquepartite structure. </LI> <LI> While gene structure of <I>tlr5m</I> is evolved with rearrangements, <I>myd88</I> is preserved. </LI> <LI> <I>Oftlr5m</I> and <I>Ofmyd88</I> showed similar tissue mRNA profile with highest level in liver. </LI> <LI> <I>Oftlr5m</I> and <I>Ofmyd88</I> were induced by ultrapure flagellin in gill and head kidney. </LI> <LI> They individually and synergetically activated NF-κB upon flagellin-stimulation. </LI> </UL> </P>

Expression of Toll-like receptors 3, 7, 9 and cytokines in feline infectious peritonitis virus-infected CRFK cells and feline peripheral monocytes

Megat Hamzah Megat Mazhar Khair,Gayathri Thevi Selvarajah,Abdul Rahman Omar,Farina Mustaffa-Kamal 대한수의학회 2022 Journal of Veterinary Science Vol.23 No.2

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 factoralpha(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 andFCoV-seropositive cats were infected with type II FIPV-79-1146. At four, 12, and 24 hours postinfection(hpi), the expression of TLR3, TLR7, TLR9, TNF-α, IFN-β, and IL-10, and the viralload were measured using reverse transcription quantitative polymerase chain reaction. Viralprotein production was confirmed using immunofluorescence. Results: FIPV-infected CRFK showed the upregulation of TLR9, TNF-α, and IFN-βexpression between 4 and 24 hpi. Uninfected monocytes from FCoV-seropositive cats showedlower TLR3 and TLR9 expression but higher TLR7 expression compared to uninfectedmonocytes from FCoV-seronegative cats. FIPV-infected monocytes from FCoV-seropositivecats 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-seropositivecats, 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 similarover time.ConclusionTLR7 may be the key TLR involved in evading the innate responseagainst inhibiting TNF-α production. Distinct TLR expression profiles between FCoVseronegativeand FCoV-seropositive cats were observed. The associated TLR that plays a rolein the induction of IFN-β needs to be explored further.

Hypoxia Activates Toll-like Receptor 4 Signaling in Primary Mouse Hepatocytes Through the Receptor Clustering within Lipid Rafts

Dong Hee Kim,Timothy R. Billiar 대한외과학회 2011 Annals of Surgical Treatment and Research(ASRT) Vol.80 No.3

Purpose: Transient hypoxia is an initial event that accentuates ischemia/reperfusion (I/R) injury in the liver. Hepatic ischemia/reperfusion (I/R) injury is largely related to innate immunity via Toll-like receptor 4 (TLR4) signaling. However, the mechanism by which hypoxia could lead to activate TLR4 signaling remains unclear. Therefore, the aim of this experimental study investigates how TLR4 signalling is activated by hypoxia. Methods: Hepatocytes were isolated from male wild-type (C57BL/6) mice (8∼12 weeks old) by an in situ collagenase (Type Ⅳ, Sigma-Aldrich) perfusion technique. In this study, using primary mouse hepatocytes in culture to 1% oxygen, detection of TLR4 translocation to the lipid rafts on the cell membrane by immunofluorescence staining and immunoblotting was saught. Results: Hypoxia caused TLR4/MD2 and β2-Integrin (CD11b/CD18) translocation to lipid rafts associated with CD14 in hepatocytes. The cholesterol sequestering agent, Nystatin and Filipin prevented hypoxia-induced TLR4/MD2 translocation to lipid rafts. Consistent with a role for oxidative stress in this effect, in vitro H2O2 treatment of hepatocytes similarly caused TLR4/MD2 translocation to lipid rafts. In addition, translocation of hypoxia-induced TLR4 complex was inhibited by N-acetylcysteine (NAC) demonstrating that the activation of TLR4 signaling is dependent on ROS. Further, the cholesterol sequestering agent, nystatin, prevented hypoxia-induced high mobility group box 1 (HMGB1) release in hepatocytes. Conclusion: These results suggest that ROS dependent TLR4 signaling is achieved following receptor translocation to the lipid raft in hepatocytes. We hypothesized that this mechanism is required for the release of HMGB1, an early mediator of injury and inflammation in hepatic I/R injury.

Fiber Type Specific Expression of Toll-like Receptor4, IL-6, TNF-α, and Suppressor of Cytokine Signaling-3 after Acute Exercise in Rat Skeletal Muscles

Won Jun Lee(이원준) 한국생명과학회 2011 생명과학회지 Vol.21 No.9

본 연구의 목적은 일회성 유산소 운동이 TLR4, IL-6, TNF-a, SOCS-3 유전자 발현에 미치는 영향을 쥐의 골격근에서 살펴보는데 있었다. 또한 이러한 일회성 운동의 영향이 근섬유 형태 특이적으로 나타나는 지에 대한 연구도 수행되었다. 실험은 Balb/c 수컷 쥐(♂:7주령, 몸무게 22.78±0.27 g) 13마리 대상으로 하였으며, 대조군과 운동군으로 무선배정되었다. 운동은 일회성으로 지칠 때까지 트레드밀 운동(경사도 10°, speed 17 cm/sec 10 min, 33 cm/sec 10 min, 50 cm/sec)을 실시하였으며, 운동 후 24시간이 지난 시점에서 가자미근과 족저근을 적출하였다. 가자미근과 족저근의 TLR4, IL-6, TNF-a, SOCS-3 mRNA 수준 변화는 real-time PCR을 이용하여 측정하였다. 일회성 유산소 트레드밀 운동은 가자미근에서 TLR4 mRNA 발현을 유의하게 증가시켰지만, 족저근의 TLR4 mRNA 발현에는 유의한 영향을 미치지 않았다. 또한 IL-6, TNF-a, SOCS-3 mRNA 발현은 가자미근에서 일회성 트레드밀 운동에 의해 유의하게 증가되었다. 하지만 족저근에서 이들 유전자의 mRNA 발현은 일회성 운동에 의해 영향을 받지 않았다. 결론적으로 TLR4, IL-6, TNF-a, SOCS-3와 같은 면역관련 유전자의 발현 수준은 일회성 트레드밀 운동에 의해 근섬유 형태 특이적으로 조절됨을 알 수 있었다. The purpose of this study was to determine whether a single bout of aerobic exercise affects the expression level of toll-like receptor4 (TLR4), IL-6, TNF-α, and suppressor of cytokine signaling-3 (SOCS-3) expression in rat hindlimb muscles depending on fiber types. To accomplish this, thirteen 7-wk Balb/c male mice were randomly assigned to an experimental group or a control group. The exercise protocol consisted of a single bout of treadmill exercise (inclination 10˚, speed 17 cm/sec 10 min, 33 cm/sec 10 min, 50 cm/sec) and the animals were killed 24 hr after the exhaustion protocol. The level of TLR4, IL-6, TNF-α, and SOCS-3 mRNA expression was measured by quantitative real- time PCR in soleus and plantaris muscles. A single bout of aerobic treadmill exercise increased TLR4 mRNA expression in the soleus muscle (p<0.05), whereas plantaris TLR4 mRNA expression did not change. Additionally, acute exercise led to a significant increase in IL-6, TNF-α, and SOCS-33 mRNA in the soleus muscle, while transcripts of these genes were not affected by exercise in the plantaris muscle. In conclusion, expression level of several immune-related genes such as TLR4, cytokines, and SOCS-3 is regulated by acute exercise in a fiber type specific manner.

FCP 17 : Effect of three sea salt type hot spring (HS) water on cytokine expression in primary human keratinocyte (KC) under Toll-like receptors (TLRs) stimulation

( Hyun Jee Kim ),( So Min Kim ),( Kyung Ho Lee ),( Su Jin Kim ),( So Youn Woo ),( Hyung Jin Hahn ),( Young Bok Lee ),( Dong Soo Yu ),( Jin Wou Kim ) 대한피부과학회 2014 대한피부과학회 학술발표대회집 Vol.66 No.2

Background: Balneotherapy is widely used for the management of inflammatory skin diseases, but the mechanism is partly understood. Korea has abundant resources of hot spring(HS) and among them are sea salt type of Na(Ca)-Cl. We previously reported that different HS water affected the secretion of proinflammatory cytokines from HaCaT cells. Objectives: We evaluated effect on proinflammatory cytokine secretion in primary keratinocytes(KCs) of three different sea salt type HS water, i.e., Haeundae, Magumsan, and Seokmodo HS area, selected by salt concentration gradient. Methods: To know whether primary KCs express Toll-like receptors(TLRs), we isolated mRNA from KCs and evaluated the expression of TLR1 to 10 by RT-PCR. Next, we investigated the effect of three different HS waters on theexpression of cytokines(IL-8, TNF-α, IL-1β, IL-6, and GM-CSF) in the primary KCs under TLR1, 2, 3, 5, and TLR6 stimulation by ELISA. Results: The treatment of HS waters induced suppression of proinflammatory cytokines under TLR stimulation in the primary KCs. Cytokine secretion from primary human KCs to TLR3 ligand was the most prominent. Decrease of IL-8, TNF-α, IL-1β, IL-6, or GM-CSF secretion under TLR stimulation were observed to all HS waters. Conclusion: Three different sea salt type HS water treatment affected the secretion of proinflammatory cytokine production from KCs under TLR stimulation. These parameters and protocols can be used to evaluate anti-inflammatory effect of sea salt type HS water.