Toll-like Receptor Gene Family Research Articles

The immune function of TLR4-1 gene in Octopus sinensis revealed by RNAi and RNA-seq

Toll-like receptors (TLRs) are a class of conserved pattern recognition receptors (PRRs) that are crucial for initiating the innate immune response and aiding in the clearance of pathogenic organisms. Many studies have identified TLR4 as a distinctive member of the TLR family, capable of activating both the Myeloid differentiation factor 88-dependent signaling pathway (MyD88-dependent) and the TIR-domain-containing adaptor inducing IFN-β dependent signaling pathway (TRIF-dependent). Nevertheless, the role of TLR4 in Cephalopoda is still largely unexplored. To elucidate the immune function of the OsTLR4-1 gene in Octopus sinensis, the OsTLR4-1 gene was first validated and analyzed in this study. The cDNA comprises a 2475 bp ORF region, encoding 824 amino acids. Evolutionary tree analysis indicated a high homology and a close phylogenetic relationship between the Octopus sinensis and other mollusks. RNA interference (RNAi) experiments demonstrated that the expression level of OsTLR4-1 gene and its protein in the lymphocytes of the RNAi group treated with OsTLR4-1 dsRNA was extremely significantly lower than that of the blank control group and negative control group (P < 0.01), and the expression of downstream genes of OsTLR4-1, including ligand MyD88, IRAK4, TRAF6, MKK6, Hsp90, COX2, TRAF3, and RIP1, were significantly down-regulated compared to the blank and negative control group (P < 0.01). Additionally, OsTLR4-1 expression in lymphocytes was highly significantly up-regulated in the LPS-treated group compared to the blank control group (P < 0.01), while its expression was extremely significantly lower in the LPS-treated group after OsTLR4-1 interference than in the blank control group (P < 0.01). The expression of its downstream effector genes Big Defensin (Big-Def) and histone H2A.V (H2A.V) was highly significantly up-regulated in lymphocytes in the LPS-treated group compared to the blank control group (P < 0.01), while their expression in the LPS-treated group after OsTLR4-1 interference was extremely significantly lower than that in the blank control group (P < 0.01). Through comparative transcriptome analysis of the RNAi group and the blank control group, it was found that differentially expressed genes were enriched in the Toll-like receptor signaling pathway, PI3K-AKT signaling pathway, P53 signaling pathway, MAPK signaling pathway, and NF-κB signaling pathway. qRT-PCR results of key genes in these pathways revealed a decrease in all genes except IκB and Jun2 genes. This study enhances our understanding of the immune function of the TLR gene family in O. sinensis and provides a foundation for further research into innate immune signaling pathways in cephalopods.

Comprehensive analysis of diel rhythmic expression of the medaka toll-like receptor gene family

Several immune-related genes, including Toll-like receptors (TLR), are associated with circadian rhythms in mammals. However, information on the circadian rhythmic expression of TLRs in fish is limited. In this study, we aimed to analyze the regulation of diel oscillations in the expression of TLR genes in Japanese medaka (Oryzias latipes). The expression analysis revealed diel expression patterns of tlr1, tlr5m, tlr21, and clock genes (bmal1 and clock1) under a 12 h light:12 h dark cycle. The clock gene response element (E-box) was identified in the transcriptional regulatory regions of tlr1, tlr5m, and tlr21. Moreover, overexpressed bmal1 and clock1 enhanced expression levels of tlr1, tlr5m, and tlr21 in medaka embryo (OLHdrR-e3) cells. The expression of tlr1, tlr5m, and tlr21 was significantly decreased in OLHdrR-e3 after generating a bmal1 knockdown using a morpholino oligo. These results indicate the regulation of the diel rhythmic expression of several fish TLRs by clock genes.

Identification and characterization of toll-like receptor genes in silver pomfret (Pampus argenteus) and their involvement in the host immune response to Photobacterium damselae subsp. Damselae and Nocardia seriolae infection

Toll-like receptors (TLRs) are vital pattern recognition receptors that play a critical role in the innate immune response against pathogenic attack. Among the bacteria commonly found in the culture process of silver pomfret, Photobacterium damselae subsp. Damselae (PDD, gram-negative) and Nocardia seriolae (NS, gram-positive), can cause large-scale mortality in this fish species. However, there is currently no research on the role of TLRs in mediating the immune response of silver pomfret to these two bacterial infections. Therefore, in this study, we identified nine PaTLRs family members, including several fish-specific TLRs (TLR14 and TLR21). Phylogenetic analysis revealed that these PaTLRs genes could be classified into five subfamilies, namely TLR1, TLR3, TLR5, TLR7, and TLR11, indicating their evolutionary conservation. To further explore the interactions of TLR genes with immune-related mediators, protein and protein interaction network (PPI) results were generated to explain the association of TLR genes with TNF receptor-associated factor 6 (TRAF6) and other relevant genes in the MyD88-dependent pathway and NF-κb signaling pathway. Subsequently, RT-qPCR was conducted to verify the expression patterns of the nine TLR genes in the gills, skin, kidney, liver, and spleen of healthy fish, with most of the TLRs showing high expression levels in the spleen. Following infection with PDD and NS, these PaTLRs exhibited different expression patterns in the spleen, with PaTLR2, PaTLR3, PaTLR5, PaTLR7, PaTLR9, and PaTLR14 being significantly up-regulated. Furthermore, when spleen cells were treated with bacterial compositions, the majority of PaTLRs expression was up-regulated in response to Lipopolysaccharide (LPS) and lipophosphorylcholic acid (LTA) treatment, except for PaTLR21. Finally, changes in the expression levels of TLR-interacting genes were also observed under the stimulation of bacteria and bacterial compositions. The results of this study provide a preliminary reference for further understanding the mechanism of the innate immune response of the TLR gene family in silver pomfret and offer theoretical support for addressing the disease problems encountered during large-scale fish breeding.

Comprehensive transcripts analysis based on single-molecule real-time sequencing and Illumina sequencing provides insights into the mining of Toll-like receptor family in Schizothorax lissolabiatus.

Schizothorax lissolabiatus is an economically important cold-water fish species in southwestern China. Because of water pollution and habitat destruction, the number of wild populations has dramatically decreased. In this study, we used PacBio single-molecule real-time (SMRT) sequencing and Illumina sequencing to generate the first full-length transcriptome and transcriptome, respectively. A total of 19310 polished consensus reads (PC) were obtained, with an average length of 1379 bp and an N50 length of 1485 bp. Meanwhile, 12253 transcripts were successfully annotated as known homologous genes. The pathway annotation indicated that the enrichment and expression of most genes were mainly related to membrane, signal transduction and binding, and immune response. Furthermore, we identified 16 Toll-like receptors (TLRs) by mining the data from the transcripts. Phylogeny analysis showed that S. lissolabiatus TLR genes (slTLRs) supported the classification of TLRs into six families as in other vertebrates. Selection pressure analyses showed that 16 slTLRs revealed purification selection at the overall evolutionary selection. Further, positive selection signals were still detected in eight slTLRs, and most of the positive selection sites were located in the leucine-rich repeat region (LRR domain) associated with the recognition of pathogenic microorganisms, indicating that the function of these slTLR genes may be affected. Tissue specific expression analysis showed all slTLRs are present in kidney, spleen and liver but the relative expression varied among tissues. In conclusion, this study not only provided a valuable resource of transcripts for further research on S. lissolabiatus, but also contributed to improve the current understanding of the evolutionary history of immune-related genes and the TLR gene family in S. lissolabiatus.

Genomic Structure and Molecular Characterization of Toll-like Receptors in Black Scraper Thamnaconus Modestus and Their Expression Response to Two Types of Pathogens.

The black scraper (Thamnaconus modestus) is an important commercial species in China. However, with the rapid expansion of aquaculture, the culture of this species faces substantial economic losses due to infectious diseases. Toll-like receptors (TLRs) recognize a wide range of pathogen-associated molecular patterns (PAMPs) and play a crucial role in disease resistance by initiating innate immune responses in the host. The genome of the black scraper comprises eight TLR members, which can be classified into five subfamilies based on evolutionary analysis. Moreover, the TmTLRs were identified on 6 out of the 20 chromosomes in the black scraper. The functional similarity within the same subfamilies is evident by conserved motifs and gene structures. The qRT-PCR experiments revealed diverse TmTLR expression patterns in the liver, intestine, spleen, head kidney, heart, and brain of black scrapers, with high expression levels observed in immune organs, suggesting that TmTLRs may participate in the regulation of immune mechanisms and other physiological functions in the black scraper. At least six TmTLRs showed significantly upregulated expression in response to poly (I: C) or lipopolysaccharide (LPS) stresses, thus indicating their potential roles in regulating abiotic stress responses. In conclusion, our findings not only provide a foundation for future research on the TLR gene family in the black scraper but also offer guidance for disease prevention and vaccine development.

Molecular characterization, adaptive evolution, and expression analysis of the Toll-like receptor gene family in Fenneropenaeus chinensis

Global warming is a challenge to animal health because of the increased environmental temperature, with subsequent induction of immune suppression and increased susceptibility to disease during summer. The Toll-like receptor (TLR) family is an essential pattern recognition receptor (PRR) that initiates the innate immune response by sensing conserved molecular patterns of pathogens. However, research on the TLR gene family in decapod crustaceans has been conducted sporadically, without systematic naming, and the relationship between pathogen immunity adaptation and adaptive evolution of immune-related genes is unclear. In this study, various TLR gene sequences in decapod crustaceans were collected, and the unified name of Fenneropenaeus chinensis was confirmed using sequence alignment. Structural characteristics and evolutionary analyses of TLR genes in decapod crustaceans were performed, and ten FcTLR genes were identified in F. chinensis. Protein domain analysis revealed that FcTLR proteins contain 4–25 LRR domains used to recognize different pathogens. Selection pressure analysis revealed that TLR1 and TLR9 were subjected to positive selection pressure in decapod crustaceans, which may be related to their resistance to environmental changes. Furthermore, the expression of ten TLR genes was detected in F. chinensis following white spot syndrome virus (WSSV) infection. The results demonstrated that FcTLR1, FcTLR7, and FcTLR9 responded positively, which was also consistent with the results of the protein domain and selection pressure analyses. This study provides new insights into the immune response and adaptive evolution of TLRs in decapod crustaceans to prevent environmental damage, such as pathogens and high temperature.

Bivalves Present the Largest and Most Diversified Repertoire of Toll-Like Receptors in the Animal Kingdom, Suggesting Broad-Spectrum Pathogen Recognition in Marine Waters.

Toll-like receptors (TLRs) are the most widespread class of membrane-bound innate immune receptors, responsible of specific pathogen recognition and production of immune effectors through the activation of intracellular signaling cascades. The repertoire of TLRs was analyzed in 85 metazoans, enriched on molluscan species, an underrepresented phylum in previous studies. Following an ancient evolutionary origin, suggested by the presence of TLR genes in Anthozoa (Cnidaria), these receptors underwent multiple independent gene family expansions, the most significant of which occurred in bivalve molluscs. Marine mussels (Mytilus spp.) had the largest TLR repertoire in the animal kingdom, with evidence of several lineage-specific expanded TLR subfamilies with different degrees of orthology conservation within bivalves. Phylogenetic analyses revealed that bivalve TLR repertoires were more diversified than their counterparts in deuterostomes or ecdysozoans. The complex evolutionary history of TLRs, characterized by lineage-specific expansions and losses, along with episodic positive selection acting on the extracellular recognition domains, suggests that functional diversification might be a leading evolutionary force. We analyzed a comprehensive transcriptomic data set from Mytilus galloprovincialis and built transcriptomic correlation clusters with the TLRs expressed in gills and in hemocytes. The implication of specific TLRs in different immune pathways was evidenced, as well as their specific modulation in response to different biotic and abiotic stimuli. We propose that, in a similar fashion to the remarkable functional specialization of vertebrate TLRs, the expansion of the TLR gene family in bivalves attends to a functional specification motivated by the biological particularities of these organisms and their living environment.

Evolutionary Impacts of Pattern Recognition Receptor Genes on Carnivora Complex Habitat Stress Adaptation.

Many mammals develop specific immune responses owing to the changes in their ecological niche and diet that are essential for animal survival. However, pattern recognition receptors (PRRs) serve as the first line of defense in innate immunity and generate immune responses in the host. However, the evolutionary impacts on PRR genes in Carnivora are not well studied. Herein, we explored the evolution of 946 PRR gene sequences in 43 Carnivora species to elucidate the molecular mechanisms of carnivore adaptation to complex habitats. We found that the PRRs were relatively conserved, and different gene families showed different evolutionary patterns. PRRs were highly purified based on their overall roles in Carnivora species but interspersed with positive-selection patterns during evolution. Different niche types may have jointly driven the evolution of PRR genes. In particular, the selection pressure of toll-like receptor (TLR) 10 was relaxed in seven species with pseudogenes, which may have emerged during recent evolutionary events. We speculated that a "functional compensation" mechanism may exist for genes with overlapping functions in the TLR gene family. Additionally, TLR2, TLR4, NLRC5, and DECTIN1 were subject to positive selection in semi-aquatic species, and the adaptive evolution of these genes may have been related to the adaptation to semi-aquatic environments. In summary, our findings offer valuable insights into the molecular and functional evolution of PRR genes, which are important for immune adaptations in Carnivora.

Genome-wide investigation and expression analysis of TLR gene family reveals its immune role in Vibrio tolerance challenge of Manila clam

Toll-like receptors (TLRs) play key roles in activating immune responses during infection. In this study, we identified TLR genes in Manila clam at the genome-wide level and characterized it into 9 types according to the Ruditapes philippinarum genome annotation, including TLR1 (1–10), TLR2 (1–10), TLR2–2 (1–5), TLR3 (1–3), TLR4 (1–9), TLR5, TLR6 (1–5), TLR7 (1–2), and TLR13 (1–4). The length of TLR proteins varied from 128 to 1257 amino acids. The molecular weights and theoretical isoelectric point (pI) values ranged from 14.63 to 143.32 kDa and 4.47 to 9.45, respectively. TLR genes showed universal expression levels in adductor muscle (AM), mantle (M), foot (F), gill (GI), pipe (P), digestive gland (DG), gonad (GO) and labial palp (L). Transcriptome analysis revealed that the expression level of TLR4, TLR5, TLR7 and TLR13 genes are significantly highly expressed in resistant individuals of Manila clam under Vibrio anguillarum challenge, indicating these TLR genes may play significant roles in response to invading pathogens. The results obtained in this work will provide valuable insights into the immune function of TLR gene in R. philippinarum.

Genome-Wide Identification, Characterization and Expression Analysis of Toll-like Receptors in Marbled Rockfish (Sebastiscus marmoratus).

Toll-like receptor (TLR) is a cluster of type I transmembrane proteins that plays a role in innate immunity. Based on the marbled rockfish (Sebastiscus marmoratus) genome database, this study used bioinformatics methods to identify and analyze its TLR gene family members. The results showed that there were 11 TLR gene family members in Sebastiscus marmoratus (SmaTLR), which could be divided into five different subfamilies. The number of amino acids encoded by the Smatlr genes ranged from 637 to 1206. The physicochemical properties of the encoded proteins of different members were also computed. The results of protein structure prediction, phylogenetic relation, and motif analysis showed that the structure and function of the SmaTLRs were relatively conserved. Quantitative Real-Time PCR (qRT-PCR) analysis revealed the expression patterns of SmaTLRs in the gill, liver, spleen, head kidney, kidney, and intestine. SmaTLRs were widely detected in the tested tissues, and they tended to be expressed higher in immune-related tissues. After polyriboinosinic polyribocytidylic acid (poly(I:C)) challenge, SmaTLR14, SmaTLR3, SmaTLR5S, SmaTLR7, and SmaTLR22 were significantly upregulated in the spleen or liver. The results of this study will help to understand the status of TLR gene family members of marbled rockfish and provide a basis for further study of the functional analysis of this gene family.

Genome-Wide Scan Reveals Toll-Like Receptor Contraction Events in Oplegnathidae.

The striped knifejaw (Oplegnathus fasciatus) and spotted knifejaw (Oplegnathus punctatus) are prominent members of the Oplegnathidae family and are rocky reef-loving fishes with high ecological and economic value. However, the frequent occurrence of diseases in these fishes has severely restricted the development of their breeding industry. Toll-like receptors (TLRs) play an important role in resistance to pathogens as part of innate immunity. Genome-wide scans and cross-species comparative analysis revealed 10 TLRs in O. fasciatus (OfTLRs) and only 5 in O. punctatus (OpTLRs). In contrast to those of mammals and other fishes, the TLR family of Oplegnathidae underwent significant contraction events, especially in O. punctatus (only TLR1, TLR2, TLR14, TLR5, and TLR21 were retained). A phylogenetic tree divided the 10 OfTLRs into 5 subfamilies: TLR1, TLR3, TLR5, TLR7, and TLR11. The five OpTLR genes were divided into three different subfamilies: TLR1, TLR5, and TLR11. Quantitative real-time PCR revealed that all OpTLRs were expressed in the examined tissues, especially the immune system-related tissues, such as the spleen, gill, head kidney, and middle kidney. The expression of OpTLRs was high at the early stage of development (5 days posthatching [dph]) and decreased gradually until 30 dph. We speculated that maternal immunity or the developmental function of TLRs played an important protective role in the early stage. However, from 30 to 60 dph, TLR expression was low. At this time, juvenile fish are susceptible to viruses and begin to show TLR self-expression with weak immunity. Artificial immunity enhancement is needed to improve the environmental resistance of juvenile fish. In summary, our results not only provide valuable basic data for future studies of the TLR gene family in Oplegnathidae fish but also lay a solid foundation for Oplegnathidae fish research.

Genome-wide characterization of the Toll-like receptor gene family in Triplophysa rosa and expression profiles in response to Aeromonas hydrophila infection

Toll-like receptors (TLRs) are key pattern recognition receptors playing a pivotal role in innate immunity against pathogen invasion. Although numerous studies have clarified the characteristics and functions of TLRs in both invertebrates and vertebrates, little information is available about TLR genes in the cavefish Triplophysa rosa. Hence, 16 TLR family members, including several fish-specific TLRs (TLR18, TLR19, TLR20, and TLR21), were identified and characterized based on the T. rosa genome. Phylogenetic analysis showed that these TLR genes could be classified into six subfamilies, namely TLR1, TLR3, TLR4, TLR5, TLR7, and TLR11, revealing their evolutionary conservation together with structural analysis. By examining gene copy numbers of TLR genes from several fish species, gene duplications and losses were observed. Moreover, the expression patterns of TLR genes in the gill, skin, intestine, and liver of healthy T. rosa, as well as those in the gill and intestine, in response to Aeromonas hydrophila infection, were investigated by qPCR. Results revealed that 16 TLR genes were ubiquitously expressed in all tested tissues. Furthermore, the expression of most TLRs, including TrTLR4.1, TrTLR4.2, TrTLR5.1, TrTLR9, TrTLR18, TrTLR19, and TrTLR20, was upregulated at all time points. Most TLRs in mucosal immune organs significantly responded to A. hydrophila infection, indicating that the differential expression of TLRs plays important roles in mediating innate immune responses for host defense. Collectively, our findings might help elucidate the crucial roles of TLRs in the immune response of T. rosa, providing valuable genomic resources for future studies investigating the innate immune mechanisms underlying host defense against pathogen infection.

Evolution of toll-like receptor gene family in amphibians

The evolutionary position and lifestyle of amphibians highlights the important roles of the immune system in adaptive radiation and their adaptation to a complex pathogenic environment. Toll-like receptors (TLRs) are membrane-like sensors that recognize and bind conserved molecular motifs in pathogens to initiate downstream immune responses. To understand the evolutionary patterns of TLRs in amphibians, we analyzed TLR genes from the genomes and transcriptomes of 102 amphibian species. Phylogenetic results showed that 578 intact amphibian TLR sequences belonged to 16 TLR genes and were divided into seven subfamilies. The TLR4 subfamily was only identified in the Anura. Purification selection plays a leading role in amphibian TLR evolution and mean ω (dN/dS) values ranged from 0.252 for TLR7 to 0.381 for TLR19. Furthermore, the ω values of different domains were significantly different. We found positive selection patterns for 141 of 12,690 codons (1.1%) in all amphibian TLRs, most of which were located in leucine-rich repeats (LRRs). We also observed low to moderate levels of single-nucleotide polymorphisms (SNPs) in Pelophylax nigromaculatus and Bombina orientalis. This study provided critical primers, meaningful information regarding TLR gene family evolution in amphibians, and insights into the complex evolutionary patterns and implications of TLR polymorphisms.

Genome-wide identification and characterization of Toll-like receptor genes in black rockfish (Sebastes schlegelii) and their response mechanisms following poly (I:C) injection

Toll-like receptors (TLRs) are canonical transmembrane receptors that play an important role in defending against invading pathogens. In this study, we identified a total of 12 TLR genes in black rockfish (Sebastes schlegelii) with an analysis of their sequence characterizations. The phylogenetic analysis suggested that 12 distinct TLRs were grouped into five subfamilies (i.e., TLR1, TLR3, TLR5, TLR7, and TLR11 subfamilies), and each SsTLR gene respectively corresponded to the orthologs genes of other species. The protein domain analysis indicated that TLRs are type I transmembrane proteins, including an extracellular leucine-rich repeat (LRR), a transmembrane region (TM) domain and an intracellular Toll/IL-1 receptor (TIR) domain. The evolutionary ratios indicted that 12 SsTLRs were under purifying selection. qRT-PCR assays exhibited diverse TLRs molecular expression patterns in the heart, brain, head kidney, kidney, liver, intestine, and spleen of 3 black rockfish, and the expression levels were high in some immune tissues (e.g., head kidney, kidney, and spleen). Subsequently, 30 fish were equally divided into 2 groups i.e., poly (I:C)-treated and PBS-Control groups. After poly (I:C) injection, eight SsTLRs, i.e., SsTLR2, SsTLR2-1, SsTLR2-2, SsTLR3, SsTLR5S, SsTLR7, SsTLR8 and SsTLR22, were dramatically increased. Altogether these results contribute to understanding how SsTLRs respond to immune defense after poly (I:C) injection and provide researchers with comprehensive TLR gene family data of black rockfish.

A review of methods for detecting single-nucleotide polymorphisms in the Toll-like receptor gene family.

The Toll-like receptors play an essential role in immunity through targeting the pathogen-associated molecular patterns. Nucleotide variations in TLR genes, especially single-nucleotide polymorphisms, have been shown to alter host immune susceptibility to several infections and diseases. Since TLR genes' polymorphisms can be a promising biomarker, ongoing investigations aim to develop, optimize and validate SNP detection methods. This review discusses various TLR SNP detection methods, either used extensively or occasionally, but having a vast potential in high-throughput settings. Methods such as PCR-restriction fragment length polymorphism, TaqMan® assay, direct sequencingand matrix-assisted laser desorption ionization - time of flight mass spectroscopy MS are frequently used methods whereas Illumina GoldenGate® assay, reverse hybridization technology, PCR-confronting two-pair primers, KBiosciences KASPar® SNP assay, SNP stream®, PCR-fluorescence hybridizationand SNaPshot® are powerful but sporadically used methods. We suggest that, for individual laboratories, the detection method of choice depends on a combination of factors such as throughput volume, reproducibility, feasibility and cost-effectiveness.

Genome-wide characterization of Toll-like receptors in Japanese meagre Argyrosomus japonicus and their response to poly (I:C) injection

Japanese meagre (Argyrosomus japonicus) is well-known for its high nutritional and economic value. In recent years, with the rapid expansion and intensification of aquaculture, Japanese meagre culture has suffered serious economic losses due to infectious diseases. Toll-like receptors (TLRs) can recognize highly conserved structural motif pathogen-associated microbial patterns (PAMPs) and danger-associated molecular patterns (DAMPs), playing a key role in resisting diseases by initiating host innate immune responses. Here, a total of 12 members of the TLR gene family (TLR1, TLR2, TLR2b, TLR3, TLR5M, TLR5S, TLR7, TLR9, TLR13, TLR14, TLR22, and TLR23) were identified in the genome of Japanese meagre. The TLR genes in Japanese meagre were distributed on 10 of 24 chromosomes. The evolutionary analyses showed that 12 different TLR genes were divided into different subfamilies, including the TLR1, TLR3, TLR5, TLR7, and TLR11 subfamilies. The conserved motifs were compared with three other teleost species. The results revealed that the conserved motifs in the same subfamilies were highly consistent. The identity and similarity of the TLR-TIR domain of Japanese meagre demonstrated that the regulatory mechanisms of each of the subfamilies were similar. qRT-PCR assays showed different expression patterns of TLRs in the liver, spleen, head kidney, trunk kidney, brain, and heart of Japanese meagre, but the expression levels in immune tissues were higher. Double-stranded RNA virus (dsRNA) is a common etiological agent of diseases in aquaculture species. Therefore, a synthetic double-stranded RNA called poly (I:C) was used as an injection to understand the immune system function and disease control of Japanese meagre. Following poly (I:C) stimulation, seven of twelve ArgTLR genes, including ArgTLR2, ArgTLR2b, ArgTLR3, ArgTLR7, ArgTLR13, ArgTLR22 and ArgTLR23, were significantly upregulated. In summary, our results not only play a guiding role in disease prevention and control and vaccine development in the future, but also provide basic data for future study of the TLR gene family of Japanese meagre.

Regulation of toll-like receptors expression in muscle cells by exercise-induced stress.

ObjectiveThis study investigates the expression patterns of toll-like receptors (TLRs) and intracellular mediators in horse muscle cells after exercise, and the relationship between TLRS expression in stressed horse muscle cells and immune cell migration toward them.MethodsThe expression patterns of the TLRs (TLR2, TLR4, and TLR8) and downstream signaling pathway-related genes (myeloid differentiation primary response 88 [MYD88]; activating transcription factor 3 [ATF3]) are examined in horse tissues, and horse peripheral blood mononuclear cells (PBMCs), polymorphonuclear cells (PMNs) and muscles in response to exercise, using the quantitative reverse transcription-polymerase chain reaction (qPCR). Expressions of chemokine receptor genes, i.e., C-X-C motif chemokine receptor 2 (CXCR2) and C-C motif chemokine receptor 5 (CCR5), are studied in PBMCs and PMNs. A horse muscle cell line is developed by transfecting SV-T antigen into fetal muscle cells, followed by examination of muscle-specific genes. Horse muscle cells are treated with stressors, i.e., cortisol, hydrogen peroxide (H2O2), and heat, to mimic stress conditions in vitro, and the expression of TLR4 and TLR8 are examined in stressed muscle cells, in addition to migration activity of PBMCs toward stressed muscle cells.ResultsThe qPCR revealed that TLR4 message was expressed in cerebrum, cerebellum, thymus, lung, liver, kidney, and muscle, whereas TLR8 expressed in thymus, lung, and kidney, while TLR2 expressed in thymus, lung, and kidney. Expressions of TLRs, i.e., TLR4 and TLR8, and mediators, i.e., MYD88 and ATF3, were upregulated in muscle, PBMCs and PMNs in response to exercise. Expressions of CXCR2 and CCR5 were also upregulated in PBMCs and PMNs after exercise. In the muscle cell line, TLR4 and TLR8 expressions were upregulated when cells were treated with stressors such as cortisol, H2O2, and heat. Migration of PBMCs toward stressed muscle cells was increased by exercise and oxidative stresses, and combinations of these. Treatment with methylsulfonylmethane (MSM), an antioxidant on stressed muscle cells, reduced migration of PBMCs toward stressed muscle cells.ConclusionIn this study, we have successfully cultured horse skeletal muscle cells, isolated horse PBMCs, and established an in vitro system for studying stress-related gene expressions and function. Expression of TLR4, TLR8, CXCR2, and CCR5 in horse muscle cells was higher in response to stressors such as cortisol, H2O2, and heat, or combinations of these. In addition, migration of PBMCs toward muscle cells was increased when muscle cells were under stress, but inhibition of reactive oxygen species by MSM modulated migratory activity of PBMCs to stressed muscle cells. Further study is necessary to investigate the biological function(s) of the TLR gene family in horse muscle cells.

Prediction of Toll-Like Receptor Gene Family in Asian Sea Bass

Prediction of Toll-Like Receptor Gene Family in Asian Sea Bass

Structural Characterization and Evolutionary Analysis of Toll-Like Receptor Gene Family in African Hunting Dog

Structural Characterization and Evolutionary Analysis of Toll-Like Receptor Gene Family in African Hunting Dog

Evolutionary History of the Toll-Like Receptor Gene Family across Vertebrates.

Adaptation to a wide range of pathogenic environments is a major aspect of the ecological adaptations of vertebrates during evolution. Toll-like receptors (TLRs) are ancient membrane-bound sensors in animals and are best known for their roles in detecting and defense against invading pathogenic microorganisms. To understand the evolutionary history of the vertebrate TLR gene family, we first traced the origin of single-cysteine cluster TLRs that share the same protein architecture with vertebrate TLRs in early-branching animals and then analyzed all members of the TLR family in over 200 species covering all major vertebrate clades. Our results indicate that although the emergence of single-cysteine cluster TLRs predates the separation of bilaterians and cnidarians, most vertebrate TLR members originated shortly after vertebrate emergence. Phylogenetic analyses divided 1,726 vertebrate TLRs into 8 subfamilies, and TLR3 may represent the most ancient subfamily that emerged before the branching of deuterostomes. Our analysis reveals that purifying selection predominated in the evolution of all vertebrate TLRs, with mean dN/dS (ω) values ranging from 0.082 for TLR21 in birds to 0.434 for TLR11 in mammals. However, we did observe patterns of positive selection acting on specific codons (527 of 60,294 codons across all vertebrate TLRs, 8.7‰), which are significantly concentrated in ligand-binding extracellular domains and suggest host–pathogen coevolutionary interactions. Additionally, we found stronger positive selection acting on nonviral compared with viral TLRs, indicating the more essential nonredundant function of viral TLRs in host immunity. Taken together, our findings provide comprehensive insight into the complex evolutionary processes of the vertebrate TLR gene family, involving gene duplication, pseudogenization, purification, and positive selection.