Characterizations of four toll‐like receptor 4s in grass carp Ctenopharyngodon idellus and their response to grass carp reovirus infection and lipopolysaccharide stimulation

In this study, the subcellular localization, tissue distribution and response to grass carp reovirus (GCRV) infection and lipopolysaccharide (LPS) stimulation of four grass carp Ctenopharyngodon idellus toll‐like receptor 4 (tlr4) genes were investigated. All four genes were constitutively expressed in all tissues studied, but the subcellular localization and tissue exhibiting the highest expression differed for each protein. Following GCRV infection, all the four tlr4s were upregulated in all tissues examined, and stimulation of C. idellus kidney (CIK) cells with LPS resulted in downregulation of all four tlr4s. These results provide a foundation for further investigation of tlr4 genes in bony fishes.     

Characterization and expression of Megalobrama amblycephala toll‐like receptor 22 involved in the response to Aeromonas hydrophila

The toll‐like receptors (TLR) tlr22 was identified and characterized for the first time in one of the economically most important freshwater fish species in China, Megalobrama amblycephala. The full‐length cDNA (4039 bp) of M. amblycephala tlr22 contains an open reading frame of 2706 bp, encoding a 901 amino‐acid long polypeptide. The putative polypeptide contains 16 leucine‐rich repeat (LRR) motifs, an LRR C‐terminal, a transmembrane region and a cytoplasmic toll–interleukin‐1 receptor (TIR) domain. Phylogenetic analyses revealed that M. amblycephala Tlr22 shared the closest relationship with a grass carp ortholog. tlr22 was constitutively expressed in nine tissues and during 10 developmental stages studied, albeit with varying expression levels. Along with many pathological changes observed after Aeromonas hydrophila bacterium infection, tlr22 and myd88 mRNA were significantly upregulated in blood, head kidney, spleen and intestine, indicating that tlr22 is involved in the immune response. These results provide an insight into tlr22 regulation mechanisms in the innate immune response to bacterial infection.     

SNP‐based susceptibility–resistance association and mRNA expression regulation analyses of tlr7 to grass carp Ctenopharyngodon idella reovirus

Eleven single nucleotide polymorphisms (SNP) in Ctenopharyngodon idella toll‐like receptor 7 (citlr7) gene, containing two in the 5′‐flanking region, three within the single intron and six distributed in the coding sequence (CDS), were identified. A case–control study of 73 susceptible individuals and 67 resistant individuals was conducted to test the SNPs‐based susceptibility–resistance association and mRNA expression of citlr7 to grass carp reovirus (GCRV), showing that both 820 A/G and 1726 A/G were significantly correlative sites in genotype (P < 0·05). Multifactor dimensionality reduction (MDR) analysis suggested the exertion of antiviral effects of 820 A/G might rely on SNPs interactions of citlr7 and C. idella toll‐like receptor 8 (citlr8). Combining the mortality rate and citlr7 mRNA expression, it was suggested that 1726 GG‐genotyped individuals might be more resistant than 1726 A/G genotyped individuals, indicating the selection on synonymous mutations in 1726 A/G might be susceptibility–resistance‐type specific. In addition, haplotype analysis uncovered no significantly correlative haplotypes in citlr7. These findings may provide an in‐depth insight for the further functional research of citlr7. The potential genetic markers identified may contribute to the molecular and transgenic breeding of C. idella.     

The evolutionary characteristics and structural biology of Gallus toll‐like receptor 21

Toll‐like receptors (TLRs) are an important part of the innate immune system, acting as a first line of defense against many invading pathogens. The ligand known to bind Gallus toll‐like receptor 21 (gTLR21) is the unmethylated cytosine phosphate guanine dideoxy nucleotide motif; however, the evolutionary characteristics and structural biology of gTLR21 are poorly elaborated. Our results suggest that gTLR21 is phylogenetically and evolutionarily related to the TLR11 family and is perhaps a close ortholog of the Mus TLR13. Structural biology of homology modeling of the gTLR21 ectodomain structure suggests that it has no Z‐loop like that seen in Mus TLR9. The cytosolic toll‐IL‐1 receptor region of gTLR21 contains a central 4‐stranded parallel β‐sheet (βA‐βD) surrounded by 5 α‐helices (αA‐αE) on both sides, a highly conserved structure also seen in other TLRs. Molecular docking analysis reveals that the gTLR21 ectodomain has the potential to distinguish between different ligands. Homodimer analysis results also suggest that Phe842 and Pro844 of the BB loop and Cys876 of the αC helix in gTLR21 are conserved in other cytosolic toll‐IL‐1 receptor domains of other TLRs and may contribute to the docking of homodimers. Our study on the evolutionary characteristics and structural biology of gTLR21 reveals that the molecule may have a broader role to play in innate immune system; however, further experimental validation is required to confirm our findings.     

Lipopolysaccharide directly stimulates aldosterone production via toll‐like receptor 2 and toll‐like receptor 4 related PI3K/Akt pathway in rat adrenal zona glomerulosa cells

The level of circulating endotoxin is related to the severity of cardiovascular disease. One of the indexes for the prognosis of cardiovascular disease is the plasma aldosterone level. Recently, the Toll‐like receptors (TLRs), lipopolysaccharide (LPS)‐regulated receptors, were found not only to mediate the inflammatory response but also to be important in the adrenal stress response. Whether LPS via TLRs induced aldosterone production in adrenal zona glomerulosa (ZG) cells was not clear. Our results suggest that LPS‐induced aldosterone secretion in a time‐ and dose‐dependent manner and via TLR2 and TLR4 signaling pathway. Administration of LPS can enhance steroidogenesis enzyme expression such as scavenger receptor‐B1 (SR‐B1), steroidogenic acute regulatory protein (StAR) and P450 side chain cleavage (P450scc) enzyme. LPS‐induced SR‐B1 and StAR protein expression are abolished by TLR2 blocker. Furthermore, we demonstrated that phosphorylation of Akt was elevated by LPS treatment and reduced by TLR2 blockers, TLR4 blockers, and LY294002 (PI₃K inhibitor). Those inhibitors of PI₃K/Akt pathways also abolish LPS‐induced aldosterone secretion and SR‐B1 protein level. In conclusion, LPS‐induced aldosterone production and SR‐B1 proteins expression are through the TLR2 and TLR4 related PI₃K/Akt pathways in adrenal ZG cells. J. Cell. Biochem. 111: 872–880, 2010. © 2010 Wiley‐Liss, Inc.     

Contrasting patterns of polymorphism and selection in bacterial‐sensing toll‐like receptor 4 in two house mouse subspecies

Detailed investigation of variation in genes involved in pathogen recognition is crucial for understanding co‐evolutionary processes between parasites and their hosts. Triggering immediate innate response to invading microbes, Toll‐like receptors (TLRs) belong presently among the best‐studied receptors of vertebrate immunity. TLRs exhibit remarkable interspecific variation and also intraspecific polymorphism is well documented. In humans and laboratory mice, several studies have recently shown that single amino acid substitution may significantly alter receptor function. Unfortunately, data concerning polymorphism in free‐living species are still surprisingly scarce. In this study, we analyzed the polymorphism of Toll‐like receptor 4 (Tlr4) over the Palearctic range of house mouse (Mus musculus). Our results reveal contrasting evolutionary patterns between the two recently (0.5 million years ago) diverged house mouse subspecies: M. m. domesticus (Mmd) and M. m. musculus (Mmm). Comparison with cytochrome b indicates strong directional selection in Mmd Tlr4. Throughout the whole Mmd western Palaearctic region, a single variant of the ligand‐binding region is spread, encoded mainly by one dominant haplotype (71% of Mmd). In contrast, Tlr4 in Mmm is much more polymorphic with several haplotypes at intermediate frequencies. Moreover, we also found clear signals of recombination between two principal haplogroups in Mmm, and we identified eight sites under positive selection in our dataset. Our results suggest that observed differences in Tlr4 diversity may be attributed to contrasting parasite‐mediated selection acting in the two subspecies.     

Endothelial toll‐like receptor 4 maintains lung integrity via epigenetic suppression of p16INK4a

We previously reported that the canonical innate immune receptor toll‐like receptor 4 (TLR4) is critical in maintaining lung integrity. However, the molecular mechanisms via which TLR4 mediates its effect remained unclear. In the present study, we identified distinct contributions of lung endothelial cells (Ec) and epithelial cells TLR4 to pulmonary homeostasis using genetic‐specific, lung‐ and cell‐targeted in vivo methods. Emphysema was significantly prevented via the reconstituting of human TLR4 expression in the lung Ec of TLR4?/? mice. Lung Ec‐silencing of TLR4 in wild‐type mice induced emphysema, highlighting the specific and distinct role of Ec‐expressed TLR4 in maintaining lung integrity. We also identified a previously unrecognized role of TLR4 in preventing expression of p16^INK4a, a senescence‐associated gene. Lung Ec‐p16^INK4a‐silencing prevented TLR4?/? induced emphysema, revealing a new functional role for p16^INK4ain lungs. TLR4 suppressed endogenous p16^INK4a expression via HDAC2‐mediated deacetylation of histone H4. These findings suggest a novel role for TLR4 in maintaining of lung homeostasis via epigenetic regulation of senescence‐related gene expression.     

Immunogenetic heterogeneity in a widespread ungulate: the European roe deer (Capreolus capreolus)

Understanding how immune genetic variation is shaped by selective and neutral processes in wild populations is of prime importance in both evolutionary biology and epidemiology. The European roe deer (Capreolus capreolus) has considerably expanded its distribution range these last decades, notably by colonizing agricultural landscapes. This range shift is likely to have led to bottlenecks and increased roe deer exposure to a new range of pathogens that until recently predominantly infected humans and domestic fauna. We therefore investigated the historical and contemporary forces that have shaped variability in a panel of genes involved in innate and acquired immunity in roe deer, including Mhc‐Drb and genes encoding cytokines or toll‐like receptors (TLRs). Together, our results suggest that genetic drift is the main contemporary evolutionary force shaping immunogenetic variation within populations. However, in contrast to the classical view, we found that some innate immune genes involved in micropathogen recognition (e.g. Tlrs) continue to evolve dynamically in roe deer in response to pathogen‐mediated positive selection. Most studied Tlrs (Tlr2, Tlr4 and Tlr5) had similarly high levels of amino acid diversity in the three studied populations including one recently established in southwestern France that showed a clear signature of genetic bottleneck. Tlr2 implicated in the recognition of Gram‐positive bacteria in domestic ungulates, showed strong evidence of balancing selection. The high immunogenetic variation revealed here implies that roe deer are able to cope with a wide spectrum of pathogens and to respond rapidly to emerging infectious diseases.     

Drift,not selection,shapes toll‐like receptor variation among oceanic island populations

Understanding the relative role of different evolutionary forces in shaping the level and distribution of functional genetic diversity among natural populations is a key issue in evolutionary and conservation biology. To do so accurately genetic data must be analysed in conjunction with an unambiguous understanding of the historical processes that have acted upon the populations. Here, we focused on diversity at toll‐like receptor (TLR) loci, which play a key role in the vertebrate innate immune system and, therefore, are expected to be under pathogen‐mediated selection. We assessed TLR variation within and among 13 island populations (grouped into three archipelagos) of Berthelot's pipit, Anthus berthelotii, for which detailed population history has previously been ascertained. We also compared the variation observed with that found in its widespread sister species, the tawny pipit, Anthus campestris. We found strong evidence for positive selection at specific codons in TLR1LA, TLR3 and TLR4. Despite this, we found that at the allele frequency level, demographic history has played the major role in shaping patterns of TLR variation in Berthelot's pipit. Levels of diversity and differentiation within and across archipelagos at all TLR loci corresponded very closely with neutral microsatellite variation and with the severity of the bottlenecks that occurred during colonization. Our study shows that despite the importance of TLRs in combating pathogens, demography can be the main driver of immune gene variation within and across populations, resulting in patterns of functional variation that can persist over evolutionary timescales.     

Antibacterial effect of mesenchymal stem cells against Escherichia coli is mediated by secretion of beta‐ defensin‐ 2 via toll‐ like receptor 4 signalling

Recently, we demonstrated that intratracheal transplantation of human umbilical cord blood‐ derived mesenchymal stem cells (MSCs) attenuates Escherichia (E) coli‐ induced acute lung injury primarily by down‐ modulating inflammation and enhancing bacterial clearance iQn mice. This study was performed to elucidate the mechanism underlying the antibacterial effects of MSCs. The growth of E. coli in vitro was significantly inhibited only by MSCs or their conditioned medium with bacterial preconditioning, but not by fibroblasts or their conditioned medium. Microarray analysis identified significant up‐ regulation of toll‐ like receptors (TLR)‐ 2 and TLR‐ 4, and β‐ defensin 2 (BD2) in MSCs compared with fibroblasts after E. coli exposure. The increased BD2 level and the in vitro antibacterial effects of MSCs were abolished by specific antagonist or by siRNA‐ mediated knockdown of TLR‐ 4, but not TLR‐ 2, and restored by BD2 supplementation. The in vivo down‐ modulation of the inflammatory response and enhanced bacterial clearance, increased BD2 secretion and the resultant protection against E. coli‐ induced pneumonia observed only with MSCs, but not fibroblasts, transplantation in mice, were abolished by knockdown of TLR‐ 4 with siRNA transfection. Our data indicate that BD2 secreted by the MSCs via the TLR‐ 4 signalling pathway is one of the critical paracrine factors mediating their microbicidal effects against E. coli, both in vitro and in vivo. Furthermore, TLR‐ 4 from the transplanted MSCs plays a seminal role in attenuating in vivo E. coli‐ induced pneumonia and the ensuing acute lung injury through both its anti‐ inflammatory and antibacterial effects.     

Toll‐like receptor signalling cross‐activates the autophagic pathway to restrict Salmonella Typhimurium growth in macrophages

It has been long recognised that activation of toll‐like receptors (TLRs) induces autophagy to restrict intracellular bacterial growth. However, the mechanisms of TLR‐induced autophagy are incompletely understood. Salmonella Typhimurium is an intracellular pathogen that causes food poisoning and gastroenteritis in humans. Whether TLR activation contributes to S. Typhimurium‐induced autophagy has not been investigated. Here, we report that S. Typhimurium and TLRs shared a common pathway to induce autophagy in macrophages. We first showed that S. Typhimurium‐induced autophagy in a RAW264.7 murine macrophage cell line was mediated by the AMP‐activated protein kinase (AMPK) through activation of the TGF‐β‐activated kinase (TAK1), a kinase activated by multiple TLRs. AMPK activation led to increased phosphorylation of Unc‐51‐like autophagy activating kinase (ULK1) at S317 and S555. ULK1 phosphorylation at these two sites in S. Typhimurium‐infected macrophages overrode the inhibitory effect of mTOR on ULK1 activity due to mTOR‐mediated ULK1 phosphorylation at S757. Lipopolysaccharide (LPS), flagellin, and CpG oligodeoxynucleotide, which activate TLR4, TLR5, and TLR9, respectively, increased TAK1 and AMPK phosphorylation and induced autophagy in RAW264.7 cells and in bone marrow‐derived macrophages. However, LPS was unable to induce TAK1 and AMPK phosphorylation and autophagy in TLR4‐deficient macrophages. TAK1 and AMPK‐specific inhibitors blocked S. Typhimurium‐induced autophagy and xenophagy and increased the bacterial growth in RAW264.7 cells. These observations collectively suggest that activation of the TAK1–AMPK axis through TLRs is essential for S. Typhimurium‐induced autophagy and that TLR signalling cross‐activates the autophagic pathway to clear intracellular bacteria.     

HMGB‐1 induces c‐kit+ cell microvascular rolling and adhesion via both toll‐like receptor‐2 and toll‐like receptor‐4 of endothelial cells

High-mobility group box 1 (HMGB-1) is a strong chemo-attractive signal for both inflammatory and stem cells. The aim of this study is to evaluate the mechanisms regulating HMGB-1–mediated adhesion and rolling of c-kit⁺ cells and assess whether toll-like receptor-2 (TLR-2) and toll-like receptor-4 (TLR-4) of endothelial cells or c-kit⁺ cells are implicated in the activation of downstream migration signals to peripheral c-kit⁺ cells. Effects of HMGB-1 on the c-kit⁺ cells/endothelial interaction were evaluated by a cremaster muscle model in wild-type (WT), TLR-2 (−/−) and Tlr4 (LPS-del) mice. The mRNA and protein expression levels of endothelial nitric oxide synthase were determined by quantitative real-time PCR and immunofluorescence staining. Induction of crucial adhesion molecules for rolling and adhesion of stem cells and leukocytes were monitored in vivo and in vitro. Following local HMGB-1 administration, a significant increase in cell rolling was detected (32.4 ± 7.1% in ‘WT’ versus 9.9 ± 3.2% in ‘control’, P < 0.05). The number of firmly adherent c-kit⁺ cells was more than 13-fold higher than that of the control group (14.6 ± 5.1 cells/mm² in ‘WT’ versus 1.1 ± 1.0 cells/mm² in ‘control’, P < 0.05). In knockout animals, the fraction of rolling cells did not differ significantly from control levels. Firm endothelial adhesion was significantly reduced in TLR-2 (−/−) and Tlr4 (LPS-del) mice compared to WT mice (1.5 ± 1.4 cells/mm² in ‘TLR-2 (−/−)’ and 2.4 ± 1.4 cells/mm² in ‘Tlr4 (LPS-del)’ versus 14.6 ± 5.1 cells/mm² in ‘WT’, P < 0.05). TLR-2 (−/−) and Tlr4 (LPS-del) stem cells in WT mice did not show significant reduction in rolling and adhesion compared to WT cells. HMGB-1 mediates c-kit⁺ cell recruitment via endothelial TLR-2 and TLR-4.     

Structures and recognition modes of toll‐like receptors

Toll‐like receptors (TLRs) recognize common structural patterns in diverse microbial molecules and play central roles in the innate immune response. The structures of extracellular domains and their ligand complexes of several TLRs have been determined by X‐ray crystallography. Here, we discuss recent advances on structures and activation mechanisms of TLRs. Despite the differences in interaction areas of ligand with TLRs, the extracellular domains of TLRs all adopt horseshoe‐shaped structures and the overall M‐shape of the TLR–ligand complexes is strikingly similar. The structural rearrangement information of TLRs sheds new light on their ligand‐recognition and ‐activation mechanisms. Proteins 2016; 85:3–9. © 2016 Wiley Periodicals, Inc.