Yoshinori Yamanishi

TREM2 sustains microglial expansion during aging and response to demyelination

Microglia contribute to development, homeostasis, and immunity of the CNS. Like other tissue-resident macrophage populations, microglia express the surface receptor triggering receptor expressed on myeloid cells 2 (TREM2), which binds polyanions, such as dextran sulphate and bacterial LPS, and activates downstream signaling cascades through the adapter DAP12. Individuals homozygous for inactivating mutations in TREM2 exhibit demyelination of subcortical white matter and a lethal early onset dementia known as Nasu-Hakola disease. How TREM2 deficiency mediates demyelination and disease is unknown. Here, we addressed the basis for this genetic association using Trem2(-/-) mice. In WT mice, microglia expanded in the corpus callosum with age, whereas aged Trem2(-/-) mice had fewer microglia with an abnormal morphology. In the cuprizone model of oligodendrocyte degeneration and demyelination, Trem2(-/-) microglia failed to amplify transcripts indicative of activation, phagocytosis, and lipid catabolism in response to myelin damage. As a result, Trem2(-/-) mice exhibited impaired myelin debris clearance, axonal dystrophy, oligodendrocyte reduction, and persistent demyelination after prolonged cuprizone treatment. Moreover, myelin-associated lipids robustly triggered TREM2 signaling in vitro, suggesting that TREM2 may directly sense lipid components exposed during myelin damage. We conclude that TREM2 is required for promoting microglial expansion during aging and microglial response to insults of the white matter.

TIM1 is an endogenous ligand for LMIR5/CD300b: LMIR5 deficiency ameliorates mouse kidney ischemia/reperfusion injury

Leukocyte mono-immunoglobulin (Ig)–like receptor 5 (LMIR5)/CD300b is a DAP12-coupled activating receptor predominantly expressed in myeloid cells. The ligands for LMIR have not been reported. We have identified T cell Ig mucin 1 (TIM1) as a possible ligand for LMIR5 by retrovirus-mediated expression cloning. TIM1 interacted only with LMIR5 among the LMIR family, whereas LMIR5 interacted with TIM4 as well as TIM1. The Ig-like domain of LMIR5 bound to TIM1 in the vicinity of the phosphatidylserine (PS)-binding site within the Ig-like domain of TIM1. Unlike its binding to TIM1 or TIM4, LMIR5 failed to bind to PS. LMIR5 binding did not affect TIM1- or TIM4-mediated phagocytosis of apoptotic cells, and stimulation with TIM1 or TIM4 induced LMIR5-mediated activation of mast cells. Notably, LMIR5 deficiency suppressed TIM1-Fc–induced recruitment of neutrophils in the dorsal air pouch, and LMIR5 deficiency attenuated neutrophil accumulation in a model of ischemia/reperfusion injury in the kidneys in which TIM1 expression is up-regulated. In that model, LMIR5 deficiency resulted in ameliorated tubular necrosis and cast formation in the acute phase. Collectively, our results indicate that TIM1 is an endogenous ligand for LMIR5 and that the TIM1–LMIR5 interaction plays a physiological role in immune regulation by myeloid cells.

The Receptor LMIR3 Negatively Regulates Mast Cell Activation and Allergic Responses by Binding to Extracellular Ceramide

Mast cells (MCs) are key effector cells in allergic reactions. However, the inhibitory mechanism that prevents excessive activation of MCs remains elusive. Here we show that leukocyte mono-immunoglobulin-like receptor 3 (LMIR3; also called CD300f) is a negative regulator of MC activation in vivo. LMIR3 deficiency exacerbated MC-dependent allergic responses in mice, including anaphylaxis, airway inflammation, and dermatitis. Both physical binding and functional reporter assays via an extracellular domain of LMIR3 showed that several extracellular lipids (including ceramide) and lipoproteins were possible ligands for LMIR3. Importantly, MCs were frequently surrounded by extracellular ceramide in vivo. Upon engagement of high-affinity immunoglobulin E receptor, extracellular ceramide-LMIR3 binding inhibited MC activation via immunoreceptor tyrosine-based inhibitory and switch motifs of LMIR3. Moreover, pretreatment with LMIR3-Fc fusion protein or antibody against either ceramide or LMIR3 interfered with this binding in vivo, thereby exacerbating passive cutaneous anaphylaxis. Thus, the interaction between extracellular ceramide and LMIR3 suppressed MC-dependent allergic responses.

An Activating and Inhibitory Signal from an Inhibitory Receptor LMIR3/CLM-1: LMIR3 Augments Lipopolysaccharide Response through Association with FcRγ in Mast Cells

Leukocyte mono-Ig-like receptor 3 (LMIR3) is an inhibitory receptor mainly expressed in myeloid cells. Coengagement of FcεRI and LMIR3 impaired cytokine production in bone marrow-derived mast cells (BMMCs) induced by FcεRI crosslinking alone. Mouse LMIR3 possesses five cytoplasmic tyrosine residues (Y241, Y276, Y289, Y303, Y325), among which Y241 and Y289 (Y241/289) or Y325 fit the consensus sequence of ITIM or immunotyrosine-based switch motif (ITSM), respectively. The inhibitory effect was abolished by the replacement of Y325 in addition to Y241/289 with phenylalanine (Y241/189/325/F) in accordance with the potential of Y241/289/325 to cooperatively recruit Src homology region 2 domain-containing phosphatase 1 (SHP)-1 or SHP-2. Intriguingly, LMIR3 crosslinking alone induced cytokine production in BMMCs expressing LMIR3 (Y241/276/289/303/325F) mutant as well as LMIR3 (Y241/289/325F). Moreover, coimmunoprecipitation experiments revealed that LMIR3 associated with ITAM-containing FcRγ. Analysis of FcRγ-deficient BMMCs demonstrated that both Y276/303 and FcRγ played a critical role in the activating function of this inhibitory receptor. Importantly, LMIR3 crosslinking enhanced cytokine production of BMMCs stimulated by LPS, while suppressing production stimulated by other TLR agonists or stem cell factor. Thus, an inhibitory receptor LMIR3 has a unique property to associate with FcRγ and thereby functions as an activating receptor in concert with TLR4 stimulation.

Functional analysis of activating receptor LMIR4 as a counterpart of inhibitory receptor LMIR3

The leukocyte mono-Ig-like receptor (LMIR) belongs to a new family of paired immunoreceptors. In this study, we analyzed activating receptor LMIR4/CLM-5 as a counterpart of inhibitory receptor LMIR3/CLM-1. LMIR4 is expressed in myeloid cells, including granulocytes, macrophages, and mast cells, whereas LMIR3 is more broadly expressed. The association of LMIR4 with Fc receptor-γ among immunoreceptor tyrosine-based activation motif-bearing molecules was indispensable for LMIR4-mediated functions of bone marrow-derived mast cells, but dispensable for its surface expression. Cross-linking of LMIR4 led to Lyn- and Syk-dependent activation of bone marrow-derived mast cells, resulting in cytokine production and degranulation, whereas that of LMIR3 did not. The triggering of LMIR4 and TLR4 synergistically caused robust cytokine production in accordance with enhanced activation of ERK, whereas the co-ligation of LMIR4 and LMIR3 dramatically abrogated cytokine production. Notably, intraperitoneal administration of lipopolysaccharide strikingly up-regulated LMIR3 and down-regulated LMIR4, whereas that of granulocyte colony-stimulating factor up-regulated both LMIR3 and LMIR4 in granulocytes. Cross-linking of LMIR4 in bone marrow granulocytes also resulted in their activation, which was enhanced by lipopolysaccharide. Collectively, these results suggest that the innate immune system is at least in part regulated by the qualitative and quantitative balance of the paired receptors LMIR3 and LMIR4.

Basophils have emerged as a key player in immunity.

Basophils had long been neglected in immunological studies, because of their paucity and phenotypic similarity with tissue-resident mast cells. However, recent development of analytical tools has cast new light on this neglected minority, and revealed previously unappreciated roles of basophils, distinct from those of mast cells, in various immune responses. Primary function of basophils appears to be the protection against infections with parasites, including ticks and helminths. This is why basophils are evolutionally conserved well in many animal species, albeit a small number. Nevertheless, basophils sometimes exert host-deleterious functions in immunological disorders such as allergy. Here we summarize recent advance in our understanding of basophil ontogeny and their in vivo roles under physiological and pathological conditions.

Integrin αIIbβ3 induces the adhesion and activation of mast cells through interaction with fibrinogen.

Integrin αIIb, a well-known marker of megakaryocyte-platelet lineage, has been recently recognized on hemopoietic progenitors. We now demonstrate that integrin αIIbβ3 is highly expressed on mouse and human mast cells including mouse bone marrow-derived mast cells, peritoneal mast cells, and human cord blood-derived mast cells, and that its binding to extracellular matrix proteins leads to enhancement of biological functions of mast cells in concert with various stimuli. With exposure to various stimuli, including cross-linking of FcεRI and stem cell factor, mast cells adhered to extracellular matrix proteins such as fibrinogen and von Willebrand factor in an integrin αIIbβ3-dependent manner. In addition, the binding of mast cells to fibrinogen enhanced proliferation, cytokine production, and migration and induced uptake of soluble fibrinogen in response to stem cell factor stimulation, implicating integrin αIIbβ3 in a variety of mast cell functions. In conclusion, mouse and human mast cells express functional integrin αIIbβ3.

Trogocytosis of peptide-MHC class II complexes from dendritic cells confers antigen-presenting ability on basophils

Significance Recent studies have revealed that basophils, the rarest granulocytes, have crucial roles in various immune responses. Among their properties, the MHC class II (MHC-II) expression and their function as antigen-presenting cells are matters of considerable controversy. Here we show that basophils indeed express MHC-II on the cell surface, but with little transcription of corresponding genes. This could be achieved by the acquisition of peptide–MHC-II complexes from dendritic cells via cell contact-dependent trogocytosis in vitro and in vivo. The acquired complexes enabled basophils to stimulate and differentiate T cells toward Th2 cells. Thus, the present study clarified the mechanism by which basophils display MHC-II on the cell surface and appears to reconcile some discrepancies observed in previous studies. Th2 immunity plays important roles in both protective and allergic responses. Nevertheless, the nature of antigen-presenting cells responsible for Th2 cell differentiation remains ill-defined compared with the nature of the cells responsible for Th1 and Th17 cell differentiation. Basophils have attracted attention as a producer of Th2-inducing cytokine IL-4, whereas their MHC class II (MHC-II) expression and function as antigen-presenting cells are matters of considerable controversy. Here we revisited the MHC-II expression on basophils and explored its functional relevance in Th2 cell differentiation. Basophils generated in vitro from bone marrow cells in culture with IL-3 plus GM-CSF displayed MHC-II on the cell surface, whereas those generated in culture with IL-3 alone did not. Of note, these MHC-II–expressing basophils showed little or no transcription of the corresponding MHC-II gene. The GM-CSF addition to culture expanded dendritic cells (DCs) other than basophils. Coculture of basophils and DCs revealed that basophils acquired peptide–MHC-II complexes from DCs via cell contact-dependent trogocytosis. The acquired complexes, together with CD86, enabled basophils to stimulate peptide-specific T cells, leading to their proliferation and IL-4 production, indicating that basophils can function as antigen-presenting cells for Th2 cell differentiation. Transfer of MHC-II from DCs to basophils was also detected in draining lymph nodes of mice with atopic dermatitis-like skin inflammation. Thus, the present study defined the mechanism by which basophils display MHC-II on the cell surface and appears to reconcile some discrepancies observed in previous studies.

Ceramide-CD300f binding suppresses experimental colitis by inhibiting ATP-mediated mast cell activation

Objective Extracellular ATP mediates mast cell-dependent intestinal inflammation via P2X7 purinoceptors. We have previously shown that CD300f (also called the leucocyte mono-immunoglobulin-like receptor 3 (LMIR3)) suppresses immunoglobulin E-dependent and mast cell-dependent allergic responses by binding to ceramide. The aim of the present study was to clarify the role of ceramide–LMIR3 interaction in the development of IBD. Design The dextran sodium sulfate (DSS)-induced colitis model was used in wild-type (WT), LMIR3−/−, mast cell-deficient KitW-sh/W-sh, KitW-sh/W-shLMIR3−/− or KitW-sh/W-sh mice engrafted with WT or LMIR3−/− bone marrow-derived mast cells (BMMCs). The severity of colitis was determined by clinical and histological criteria. Lamina propria cell populations were assessed by flow cytometry. Production of chemical mediators from lamina propria cells was measured by real-time reverse transcription PCR. Production of chemical mediators from ATP-stimulated BMMCs in the presence or absence of ceramide was measured by ELISA. The severity of DSS-induced colitis was assessed in mice given either an Fc fusion protein containing an extracellular domain of LMIR3, and anticeramide antibody, or ceramide liposomes. Results LMIR3 deficiency exacerbated DSS-induced colitis in mice. KitW-sh/W-sh mice harbouring LMIR3−/− mast cells exhibited more severe colitis than those harbouring WT mast cells. Ceramide–LMIR3 interaction inhibited ATP-stimulated activation of BMMCs. DSS-induced colitis was aggravated by disrupting the ceramide–LMIR3 interaction, whereas it was suppressed by treating with ceramide liposomes. Conclusions LMIR3-deficient colonic mast cells were pivotal in the exacerbation of DSS-induced colitis in LMIR3−/− mice. Ceramide liposomes attenuated DSS-induced colitis by inhibiting ATP-mediated activation of colonic mast cells through ceraimide–LMIR3 binding.

A Soluble Form of LMIR5/CD300b Amplifies Lipopolysaccharide-Induced Lethal Inflammation in Sepsis

Leukocyte mono-Ig–like receptor 5 (LMIR5, also called CD300b) is an activating receptor expressed in myeloid cells. We have previously demonstrated that T cell Ig mucin 1 works as a ligand for LMIR5 in mouse ischemia/reperfusion injury of the kidneys. In this article, we show that LMIR5 is implicated in LPS-induced sepsis in mice. Notably, neutrophils constitutively released a soluble form of LMIR5 (sLMIR5) through proteolytic cleavage of surface LMIR5. Stimulation with TLR agonists augmented the release of sLMIR5. LPS administration or peritonitis induction increased serum levels of sLMIR5 in mice, which was substantially inhibited by neutrophil depletion. Thus, neutrophils were the main source of LPS-induced sLMIR5 in vivo. On the other hand, i.p. administration of LMIR5-Fc, a surrogate of sLMIR5, bound to resident macrophages (Mϕ) and stimulated transient inflammation in mice. Consistently, LMIR5-Fc induced in vitro cytokine production of peritoneal Mϕ via its unknown ligand. Interestingly, LMIR5 deficiency profoundly reduced systemic cytokine production and septic mortality in LPS-administered mice, although it did not affect in vitro cytokine production of LPS-stimulated peritoneal Mϕ. Importantly, the resistance of LMIR5-deficient mice to LPS- or peritonitis-induced septic death was decreased by LMIR5-Fc administration, implicating sLMIR5 in LPS responses in vivo. Collectively, neutrophil-derived sLMIR5 amplifies LPS-induced lethal inflammation.