1,25-Dihydroxyvitamin D3 potentiates the innate immune response of peripheral blood mononuclear cells from Japanese Black cattle.

1,25-Dihydroxyvitamin D3 (1,25(OH)2 D3 ), a bioactive vitamin D, is known to regulate immune responses in mammals. However, its impact on the innate immune responses of Japanese Black cattle, which are beef cattle endemic to Japan, remains unknown. Thus, in this study, we investigated the effect of 1,25(OH)2 D3 on the immune responses of peripheral blood mononuclear cells from Japanese Black cattle. As a result, the treatment of 1,25(OH)2 D3 upregulated the expression of antibacterial peptides, bovine beta-defensin 10 (DEFB10), and lingual antimicrobial peptide (LAP), in the presence and absence of lipopolysaccharide (LPS) stimulation. Moreover, 1,25(OH)2 D3 enhanced the inflammatory responses, including C-X-C motif ligand 8 (CXCL8) and nitric oxide synthase (NOS2), while reducing the expression of anti-inflammatory cytokine IL10, leading to an inflammatory phenotype. However, in contrast to humans and mice, 1,25(OH)2 D3 did not alter the expression of tumor necrosis factor (TNF) and downregulated triggering receptor expressed on myeloid cell 1 (TREM1) with LPS treatment. These results suggest that 1,25(OH)2 D3 potentiates the innate immune responses of Japanese Black cattle, albeit with different effects and mechanisms as compared to humans and mice.

TREM2 is a receptor for non-glycosylated mycolic acids of mycobacteria that limits anti-mycobacterial macrophage activation.

Mycobacterial cell-wall glycolipids elicit an anti-mycobacterial immune response via FcRγ-associated C-type lectin receptors, including Mincle, and caspase-recruitment domain family member 9 (CARD9). Additionally, mycobacteria harbor immuno-evasive cell-wall lipids associated with virulence and latency; however, a mechanism of action is unclear. Here, we show that the DAP12-associated triggering receptor expressed on myeloid cells 2 (TREM2) recognizes mycobacterial cell-wall mycolic acid (MA)-containing lipids and suggest a mechanism by which mycobacteria control host immunity via TREM2. Macrophages respond to glycosylated MA-containing lipids in a Mincle/FcRγ/CARD9-dependent manner to produce inflammatory cytokines and recruit inducible nitric oxide synthase (iNOS)-positive mycobactericidal macrophages. Conversely, macrophages respond to non-glycosylated MAs in a TREM2/DAP12-dependent but CARD9-independent manner to recruit iNOS-negative mycobacterium-permissive macrophages. Furthermore, TREM2 deletion enhances Mincle-induced macrophage activation in vitro and inflammation in vivo and accelerates the elimination of mycobacterial infection, suggesting that TREM2-DAP12 signaling counteracts Mincle-FcRγ-CARD9-mediated anti-mycobacterial immunity. Mycobacteria, therefore, harness TREM2 for immune evasion.

Adjuvant activity of Mycobacteria-derived mycolic acids.

Successful vaccination, especially with safe vaccines such as component/subunit vaccines, requires proper activation of innate immunity and, for this purpose, adjuvant is used. For clinical use, alum is frequently used while, for experimental use, CFA, containing Mycobacterial components, was often used. In this report, we demonstrated that mycolic acids (MA), major and essential lipid components of the bacterial cell wall of the genus Mycobacterium, has adjuvant activity. MA plus model antigen-immunization induced sufficient humoral response, which was largely comparable to conventional CFA plus antigen-immunization. Importantly, while CFA plus antigen-immunization induced Th17-biased severe and destructive inflammatory responses at the injected site, MA plus antigen-immunization induced Th1-biased mild inflammation at the site. MA induced dendritic cell activation by co-stimulatory molecule induction as well as inflammatory cytokine/chemokine induction. MA plus antigen-immunization successfully protected mice from tumor progression both in prevention and in therapy models. We thus submit that MA is a promising adjuvant candidate material for clinical purposes and for experimental purposes from a perspective of animal welfare.

Essential roles of C-type lectin Mincle in induction of neuropathic pain in mice.

Increasing evidence indicates that pattern recognition receptors (PRRs) are involved in neuropathic pain after peripheral nerve injury (PNI). While a significant number of studies support an association between neuropathic pain and the innate immune response mediated through Toll-like receptors, a family of PRRs, the roles of other types of PRRs are largely unknown. In this study, we have focused on the macrophage-inducible C-type lectin (Mincle), a PRR allocated to the C-type lectin receptor family. Here, we show that Mincle is involved in neuropathic pain after PNI. Mincle-deficient mice showed impaired PNI-induced mechanical allodynia. After PNI, expression of Mincle mRNA was rapidly increased in the injured spinal nerve. Most Mincle-expressing cells were identified as infiltrating leucocytes, although the migration of leucocytes was also observed in Mincle-deficient mice. Furthermore, Mincle-deficiency affected the induction of genes, which are reported to contribute to neuropathic pain after PNI in the dorsal root ganglia and spinal dorsal horn. These results suggest that Mincle is involved in triggering sequential processes that lead to the pathogenesis of neuropathic pain.

Interleukin-27 controls basal pain threshold in physiological and pathological conditions.

Numerous studies have shown that pain sensation is affected by various immune molecules, such as cytokines, in tissues comprising the sensory pathway. Specifically, it has been shown that interleukin (IL)-17 promotes pain behaviour, but IL-10 suppresses it. IL-27 has been reported to have an anti-inflammatory effect through regulation of T cell differentiation, resulting in reduced IL-17 and induction of IL-10. Thus, we hypothesised that IL-27 would have some regulatory role in pain sensation. Here, we provide evidence that endogenous IL-27 constitutively controls thresholds for thermal and mechanical sensation in physiological and pathological conditions. Mice lacking IL-27 or its receptor WSX-1 spontaneously showed chronic pain-like hypersensitivity. Reconstitution of IL-27 in IL-27-deficient mice reversed thermal and mechanical hypersensitive behaviours. Thus, unlike many other cytokines induced by inflammatory events, IL-27 appears to be constitutively produced and to control pain sensation. Furthermore, mice lacking IL-27/WSX-1 signalling showed additional hypersensitivity when subjected to inflammatory or neuropathic pain models. Our results suggest that the mechanisms underlying hypersensitive behaviours caused by the ablation of IL-27/WSX-1 signalling are different from those underlying established chronic pain models. This novel pain control mechanism mediated by IL-27 might indicate a new mechanism for the chronic pain hypersensitivity.

Transcriptome analysis reveals key roles of AtLBR-2 in LPS-induced defense responses in plants.

BACKGROUND: Lipopolysaccharide (LPS) from Gram-negative bacteria cause innate immune responses in animals and plants. The molecules involved in LPS signaling in animals are well studied, whereas those in plants are not yet as well documented. Recently, we identified Arabidopsis AtLBR-2, which binds to LPS from Pseudomonas aeruginosa (pLPS) directly and regulates pLPS-induced defense responses, such as pathogenesis-related 1 (PR1) expression and reactive oxygen species (ROS) production. In this study, we investigated the pLPS-induced transcriptomic changes in wild-type (WT) and the atlbr-2 mutant Arabidopsis plants using RNA-Seq technology. RESULTS: RNA-Seq data analysis revealed that pLPS treatment significantly altered the expression of 2139 genes, with 605 up-regulated and 1534 down-regulated genes in WT. Gene ontology (GO) analysis on these genes showed that GO terms, "response to bacterium", "response to salicylic acid (SA) stimulus", and "response to abscisic acid (ABA) stimulus" were enriched amongst only in up-regulated genes, as compared to the genes that were down-regulated. Comparative analysis of differentially expressed genes between WT and the atlbr-2 mutant revealed that 65 genes were up-regulated in WT but not in the atlbr-2 after pLPS treatment. Furthermore, GO analysis on these 65 genes demonstrated their importance for the enrichment of several defense-related GO terms, including "response to bacterium", "response to SA stimulus", and "response to ABA stimulus". We also found reduced levels of pLPS-induced conjugated SA glucoside (SAG) accumulation in atlbr-2 mutants, and no differences were observed in the gene expression levels in SA-treated WT and the atlbr-2 mutants. CONCLUSION: These 65 AtLBR-2-dependent up-regulated genes appear to be important for the enrichment of some defense-related GO terms. Moreover, AtLBR-2 might be a key molecule that is indispensable for the up-regulation of defense-related genes and for SA signaling pathway, which is involved in defense against pathogens containing LPS.

Pertussis toxin targets the innate immunity through DAP12, FcRγ, and MyD88 adaptor proteins.

Activation of the innate immunity by adjuvants, such as pertussis toxin (PTX), in the presence of autoreactive lymphocytes and antigen mimicry is sufficient to trigger autoimmunity. Toll-like, C-type lectin, and immunglobulin-like receptors play an important role in the innate immunity by sensing a variety of microbial products through several adaptor proteins, including MyD88, DAP12, and FcRγ. This study investigated the interaction between PTX and innate immune components. The direct interactions between coated PTX and receptor-fusion proteins were examined using ELISA-based binding assays. Functionally, PTX-binding receptors could be classified into two groups: inhibition (DAP12-coupled TREM2, ITIM-bearing SIRPα, SIGNR1/SIGNR3/DCSIGN) and activation (MyD88-associated TLR4, DAP12-coupled LMIR5/CD300b, FcRγ-coupled LMIR8/CD300c, CLEC9A, MGL-1). DAP12, MyD88, and FcRγ were selected for further investigation. A comprehensive analysis of gene transcription showed that PTX up-regulated the expression of various inflammatory mediators. DAP12 deficiency resulted in reduction or enhancement of inflammatory responses in a cytokine-specific manner. PTX was able to activate the TREM2-DAP12 signalling pathway. PTX induced lower expression of inflammatory mediators in the absence of FcRγ alone and substantially lost its inflammatory capacity in the absence of both FcRγ and MyD88. PTX was able to activate the MyD88-NF-κB signalling pathway in the presence of TLR2 or TLR4. The inflammatory activity of PTX was completely lost by heating. These results demonstrate that PTX targets the innate immunity through DAP12, FcRγ, and MyD88 providing new insights into the immunobiology of PTX.

Arabidopsis LBP/BPI related-1 and -2 bind to LPS directly and regulate PR1 expression.

Lipopolysaccharide (LPS) is a major constituent of the outer membrane of Gram-negative bacteria and acts as a pathogen-associated molecular pattern that triggers immune responses in both plants and animals. LPS-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI), which bind to LPS and play important roles in immunity of mammals, have been well studied. However, the molecule contributing to LPS binding in plants is mostly unknown. The Arabidopsis genome carries two genes encoding LBP/BPI-related proteins which we designated as AtLBP/BPI related-1 (AtLBR-1) and AtLBP/BPI related-2 (AtLBR-2). We found that their N-terminal domains were co-purified with cell wall-derived LPS when expressed in E. coli. Since this finding implied the direct binding of AtLBRs to LPS, we also confirmed binding by using LPS-free AtLBRs and purified LPS. AtLBRs directly bind to both rough and smooth types of LPS. We also demonstrated that LPS-treated atlbr mutant Arabidopsis exhibit a significant delay of induction of defence-related gene pathogenesis-related 1 (PR1) but no other PR genes. Furthermore, LPS-treated atlbr mutants showed defects in reactive oxygen species (ROS) generation. These results demonstrate that, as well as LBP and BPI of mammals, AtLBRs also play an important role in the LPS-induced immune response of plants.

Loss of CARD9-mediated innate activation attenuates severe influenza pneumonia without compromising host viral immunity.

Influenza virus (IFV) infection is a common cause of severe viral pneumonia associated with acute respiratory distress syndrome (ARDS), which is difficult to control with general immunosuppressive therapy including corticosteroids due to the unfavorable effect on viral replication. Studies have suggested that the excessive activation of the innate immunity by IFV is responsible for severe pathologies. In this study, we focused on CARD9, a signaling adaptor known to regulate innate immune activation through multiple innate sensor proteins, and investigated its role in anti-IFV defense and lung pathogenesis in a mouse model recapitulating severe influenza pneumonia with ARDS. We found that influenza pneumonia was dramatically attenuated in Card9-deficient mice, which showed improved mortality with reduced inflammatory cytokines and chemokines in the infected lungs. However, viral clearance, type-I interferon production, and the development of anti-viral B and T cell immunity were not compromised by CARD9 deficiency. Syk or CARD9-deficient DCs but not macrophages showed impaired cytokine but not type-I interferon production in response to IFV in vitro, indicating a possible role for the Syk-CARD9 pathway in DCs in excessive inflammation of IFV-infected lungs. Therefore, inhibition of this pathway is an ideal therapeutic target for severe influenza pneumonia without affecting viral clearance.

Evidence for TLR4 and FcRγ-CARD9 activation by cholera toxin B subunit and its direct bindings to TREM2 and LMIR5 receptors.

Cholera toxin (CTX) is a virulent factor of Vibrio cholerae that causes life-threatening diarrheal disease. Its non-toxic subunit CTB has been extensively studied for vaccine delivery. In immune cells, CTB induces a number of signaling molecules related to cellular activation and cytokine production. The mechanisms by which CTB exerts its immunological effects are not understood. We report here the immunological targets of CTB. The unexpected finding that GM1 ganglioside inhibited NF-κB activation in human monocytes stimulated with CTX and agonists of Toll-like receptors (TLR) suggests the possibility of CTX-TLR interaction. Indeed, CTX-induced IL-6 production was substantially reduced in MyD88(-/-) or TLR4(-/-) macrophages. Ectopic expression of TLR4 was required for CTX-induced NF-κB activation in HEK 293 cells. Furthermore, the inflammatory capacity of CTB was lost in the absence of TLR4, adaptor protein FcRγ, or its downstream signaling molecule CARD9. Attempts have been made to identify CTB-binding targets from various C-type lectin and immunoglobulin-like receptors. CTB targeted not only GM1 and TLR4 but also TREM2 and LMIR5/CD300b. CTB-TREM2 interaction initiated signal transduction through adaptor protein DAP12. The binding of CTB inhibited LMIR5 activation induced by its endogenous ligand 3-O-sulfo-ß-d-galactosylceramide C24:1. In summary, CTB targets TLR4, FcRγ-CARD9, TREM2, and LMIR5. These findings provide new insights into the immunobiology of cholera toxin.

3-O-sulfo-ß-D-galactose moiety of endogenous sulfoglycolipids is a potential ligand for immunoglobulin-like receptor LMIR5.

Axonal Guillain-Barré syndrome (GBS) is an autoimmune neuropathy characterized by limb weakness and/or paralysis due to the presence of autoantibodies against brain glycolipids. The immune receptors that recognize these autoimmune targets have not been described. In this study, 12 C-type lectin and 10 immunoglobulin-like receptors were screened for their potential ligands from the brain glycolipids, which are the binding targets for GBS autoantibodies. These glycolipids were GM1, GM2, GD1a, GD1b, GQ1b, crude gangliosides, and 3-O-sulfo-ß-D-galactosylceramide C24:1 (designated as C24:1). A direct interaction between ligand and receptor was examined using an ELISA-based binding assay. C-type lectin (CLEC5a, SIGNR3) and immunoglobulin-like receptors (TREM2, TREM3, LMIR2, LMIR5, LMIR7, LMIR8) interacted with C24:1. In addition, TREM3 did bind to GQ1b. LMIR5 interacted with GD1a, GQ1b, and crude gangliosides. Binding with highest affinity was observed for the LMIR5-C24:1 interaction, which was selected for further verification. C24:1 was found to induce MCP-1 production, but not proinflammatory cytokines, in basophils. C24:1-induced MCP-1 production was significantly reduced in DAP12(-/-) basophils. Importantly, LMIR5 ligation by C24:1 resulted in NFAT activation through DAP12 in LMIR5-expressing reporter cells. Structural analysis showed that LMIR5 recognized the 3-O-sulfo-ß-D-galactose moiety of C24:1. The findings indicated that C24:1 is a potential ligand for DAP12-coupled LMIR5.

LMIR5 extracellular domain activates myeloid cells through toll-like receptor 4.

LMIR5/CD300b is an activating immunoglobulin-like receptor whose extracellular domain (LMIR5-Fc) is constitutively released from immune cells. The release of LMIR5-Fc is augmented upon stimulation with TLR agonists. LMIR5-Fc is reported to possess inflammatory activity and amplify LPS-induced lethal inflammation; however, its action mechanism has not been clarified. This study was aimed to identify receptors for LMIR5-Fc. Using NF-κB reporter cells in human monocytes THP1, LMIR5-Fc was solely found to trigger NF-κB activation among various signaling receptors examined. In addition, an injection of LMIR5-Fc into the mouse peritoneal resulted in a rapid production of inflammatory mediators and an amplification of LPS activity. Moreover, LMIR5-Fc-induced cytokine production was markedly reduced in TLR4-deficient mouse macrophages. Using TLR4 reporter cells, the LMIR5-Fc sample that contained a trace amount of endotoxin under the sensitivity of reporter cells triggered a potent NF-κB activation. Furthermore, the inflammatory activity of LMIR5-Fc was completely lost by heating but unchanged by polymyxin B pretreatment. Using TLR4 fusion protein, TLR4 was found to interact specifically with LMIR5-overexpressing cells. Therefore, LMIR5-Fc is new inflammatory mediator and endogenous ligand of TLR4. This study provides an insight into the positive feedback mechanism of inflammation through TLR4-LMIR5-Fc axis.

An ITAM-Syk-CARD9 signalling axis triggers contact hypersensitivity by stimulating IL-1 production in dendritic cells.

A variety of reactive organic compounds, called haptens, can cause allergic contact dermatitis. However, the innate immune mechanisms by which haptens stimulate dendritic cells (DCs) to sensitize T cells remain unclear. Here we show that the coupling of ITAM-Syk-CARD9 signalling to interleukin-1 (IL-1) secretion in DCs is crucial for allergic sensitization to haptens. Both MyD88 and Caspase recruitment domain-containing protein 9 (CARD9) signalling are required for contact hypersensitivity (CHS). Naïve T cells require signals received through IL-1R1-MyD88 for effector differentiation, whereas DCs require CARD9 and spleen tyrosine kinase (Syk) signalling for hapten-induced IL-1α/ß secretion and their ability to prime T cells. DC-specific deletion of CARD9, DAP12, Syk or NLRP3, but not MyD88, is sufficient to abolish CHS. All tested haptens, but not irritants, can induce Syk activation, leading to both the CARD9/BCL10-dependent pro-IL-1 synthesis (signal1) and reactive oxygen species-mediated NLRP3 inflammasome activation (signal2), required for IL-1 secretion. These data unveil an innate immune mechanism crucial for allergic contact sensitization to chemical compounds.

Direct binding of a plant LysM receptor-like kinase, LysM RLK1/CERK1, to chitin in vitro.

Plants induce immune responses against fungal pathogens by recognition of chitin, which is a component of the fungal cell wall. Recent studies have revealed that LysM receptor-like kinase 1/chitin elicitor receptor kinase 1 (LysM RLK1/CERK1) is a critical component for the immune responses to chitin in Arabidopsis thaliana. However, the molecular mechanism of the chitin recognition by LysM RLK1 still remains unknown. Here, we present the first evidence for direct binding of LysM RLK1 to chitin. We expressed LysM RLK1 fused with yeast-enhanced green fluorescent protein (LysM RLK1-yEGFP) in yeast cells. Binding studies using the solubilized LysM RLK1-yEGFP and several insoluble polysaccharides having similar structures showed that LysM RLK1-yEGFP specifically binds to chitin. Subsequently, the fluorescence microscopic observation of the solubilized LysM RLK1-yEGFP binding to chitin beads revealed that the binding was saturable and had a high affinity, with a K(d) of approximately 82 nm. This binding was competed by the addition of soluble glycol chitin or high concentration of chitin oligosaccharides having 4-8 residues of N-acetyl glucosamine. However, the competition of these chitin oligosaccharides is weaker than that of glycol chitin. These data suggest that LysM RLK1 has a higher affinity for chitin having a longer residue of N-acetyl glucosamine. We also found that LysM RLK1-yEGFP was autophosphorylated in vitro and that chitin does not affect the phosphorylation of LysM RLK1-yEGFP. Our results provide a new dimension to chitin elicitor perception in plants.

Yeast-based recombineering of DNA fragments into plant transformation vectors by one-step transformation.

T-DNA binary vectors are often used in plant transformation experiments. Because they are usually very large and have few restriction sites suitable for DNA ligation reactions, cloning DNA fragments into these vectors is difficult. We provide herein an alternative to cloning DNA fragments into very large vectors. Our yeast-based recombineering method enables DNA fragments to be cloned into certain types of T-DNA binary vectors by one-step transformation without the requirement of specific recombination sites or precisely positioned restriction ends, thus making the cloning process more flexible. Moreover, this method is inexpensive and is applicable to multifragment cloning.

Highly efficient yeast-based in vivo DNA cloning of multiple DNA fragments and the simultaneous construction of yeast/ Escherichia coli shuttle vectors.

In vivo recombinational cloning in yeast is a very efficient method. Until now, this method has been limited to experiments with yeast vectors because most animal, insect, and bacterial vectors lack yeast replication origins. We developed a new system to apply yeast-based in vivo cloning to vectors lacking yeast replication origins. Many cloning vectors are derived from the plasmid pBR322 and have a similar backbone that contains the ampicillin resistance gene and pBR322-derived replication origin for Escherichia coli. We constructed a helper plasmid pSUO that allows the in vivo conversion of a pBR322-derived vector to a yeast/E. coli shuttle vector through the use of this backbone sequence. The DNA fragment to be cloned is PCR-amplified with the addition of 40 bp of homology to a pBR322-derived vector. Cotransformation of linearized pSU0, the pBR322-derived vector, and a PCR-amplified DNA fragment, results in the conversion of the pBR322-derived vector into a yeast/E. coli shuttle vector carrying the DNA fragment of interest. Furthermore, this method is applicable to multifragment cloning, which is useful for the creation of fusion genes. Our method provides an alternative to traditional cloning methods.