Toshiyuki Takai

Microglia, the primary immune cells of the central nervous system, play a crucial role in maintaining brain homeostasis through phagocytosis of various substrates, including amyloid-β (Aβ) fibrils, a hallmark of Alzheimer disease (AD) pathology. However, the molecular mechanisms regulating microglial Aβ uptake remain poorly understood. Here, we identified GPR34, a Gi/o-coupled receptor highly expressed in microglia, as a novel regulator of fibrillar Aβ phagocytosis. Treatment with a selective GPR34 agonist, M1, specifically enhanced uptake of Aβ fibrils, but not its monomer or oligomer, in both mouse and human microglia. Mechanistically, M1 reduced intracellular cAMP levels, which inversely correlated with Aβ uptake activity. Importantly, a single intrahippocampal injection of M1 in an AD mouse model significantly increased microglial Aβ uptake in vivo. Furthermore, single-nucleus RNA-sequencing analysis of Japanese AD patient samples revealed a significant reduction of GPR34 expression in microglia from AD patients compared to controls. We also observed an age-dependent decline in microglial GPR34 expression in both human and mouse datasets, suggesting a potential contribution of GPR34 downregulation to age-related Aβ accumulation and AD risk. Collectively, our findings identify GPR34 as a promising target for modulating microglial Aβ clearance and highlight the therapeutic potential of GPR34 agonists in AD. Significance statement Alzheimer disease (AD) is characterized by amyloid-β (Aβ) accumulation in the brain. Microglia, the brain’s immune cells, play a crucial role in the metabolism of Aβ. We discovered that activating the microglial receptor GPR34 with a selective agonist enhances the phagocytosis of Aβ fibrils, a key pathogenic form of Aβ. Importantly, GPR34 expression decreases with aging and AD progression, potentially contributing to impaired Aβ clearance. Our findings highlight GPR34 as a promising therapeutic target for AD, as boosting its activity could promote Aβ clearance and slow disease progression. This study provides valuable insights into microglial function in AD and offers a novel strategy for developing disease-modifying therapies.

Loss-of-function mutations in the lysosomal nucleoside transporter SLC29A3 cause lysosomal nucleoside storage and histiocytosis: phagocyte accumulation in multiple organs. However, little is known about the mechanism by which lysosomal nucleoside storage drives histiocytosis. Herein, histiocytosis in Slc29a3−/− mice was shown to depend on Toll-like receptor 7 (TLR7), which senses a combination of nucleosides and oligoribonucleotides (ORNs). TLR7 increased phagocyte numbers by driving the proliferation of Ly6Chi immature monocytes and their maturation into Ly6Clow phagocytes in Slc29a3−/− mice. Downstream of TLR7, FcRγ and DAP10 were required for monocyte proliferation. Histiocytosis is accompanied by inflammation in SLC29A3 disorders. However, TLR7 in nucleoside-laden splenic monocytes failed to activate inflammatory responses. Enhanced production of proinflammatory cytokines was observed only after stimulation with ssRNAs, which would increase lysosomal ORNs. Patient-derived monocytes harboring the G208R SLC29A3 mutation showed enhanced survival and proliferation in a TLR8-antagonist-sensitive manner. These results demonstrated that TLR7/8 responses to lysosomal nucleoside stress drive SLC29A3 disorders.

Natural killer (NK) cells play pivotal roles in innate immunity as well as in anti-tumor responses via natural killing, while their activity is tightly regulated by cell-surface inhibitory receptors. Immunoglobulin (Ig)-like transcript 3/leukocyte Ig-like receptor B4 (ILT3/LILRB4, also known as gp49B in mice) is an inhibitory receptor expressed on activated NK cells as well as myeloid-lineage cells. The common physiologic ligand of human LILRB4 and gp49B is identified very recently as fibronectin (FN), particularly the N-terminal 30 kDa domain (FN30). We hypothesized that LILRB4 could bind FN on target cells in trans together with integrin, a classical FN receptor, in cis and deliver an inhibitory signal in NK cells, leading to attenuated natural killing. Flow cytometric and confocal microscopic analyses of NK cell-surface gp49B and integrin suggested that these novel and classical FN receptors, respectively, co-engage FN immobilized on a culture plate. Biochemical analyses indicated that tyrosine phosphorylation of spleen tyrosine kinase was augmented in gp49B-deficient NK cells upon binding to the immobilized FN. While surface FN-poor YAC-1 cells were evenly sensitive as to natural killing of both gp49B-positive and -negative NK cells, the killing of FN-rich Lewis lung carcinoma cells, but not the FN30-knockout cells, was augmented among gp49B-deficient NK cells. These results suggest that the natural cytotoxicity of NK cells is negatively regulated through LILRB4/gp49B sensing FN on target cells, which sheds light on the unexpected role of LILRB4 and FN as a potential attenuator of NK cell cytotoxicity in the tumor microenvironment.

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Dysregulation of osteoclasts, the multinucleated cells responsible for bone resorption, contributes to several degenerative bone disorders. Previously, we showed that blocking the leukocyte immunoglobulin (Ig)-like receptor B4 (LILRB4), a kind of inhibitory receptor that plays an important role in immune regulation, promotes osteoclast differentiation in vitro. Here, we explored whether gp49B, the murine ortholog of LILRB4, regulates osteoclastogenesis in vivo, and whether fibronectin (FN), a ligand of LILRB4/gp49B, certainly contributes to LILRB4/gp49B-mediated osteoclastogenesis. In comparison with wild-type mice, gp49B deficiency mice exhibited a loss of trabecular bone number and an increase in osteoclast formation. Gp49B knockout improved the bone resorptive capacity of osteoclasts derived from murine Raw264.7 cells by increasing osteoclast formation. We further revealed that gp49B deficiency increased the receptor activator of nuclear factor (NF)-κB ligand (RANKL)-induced signaling transduction by increasing the phosphorylation of TGF-activated kinase 1 (TAK1), NF-κB and mitogen-activated protein kinases (MAPKs). Furthermore, the N-terminal 30 kDa proteolytic fragments of FN promoted gp49B-mediated inhibition of osteoclastogenesis by increasing Src homology-2-containing tyrosine phosphatase 1 (SHP-1) phosphorylation and tumor necrosis factor receptor-associated factor 6 (TRAF6)-SHP-1 association. In summary, the FN-LILRB4/gp49B interaction negatively regulates RANKL-induced TRAF6/TAK1/NF-𝜅B/MAPK signaling in osteoclastogenesis.

LILRB4 (B4, also known as ILT3/CD85k) is an immune checkpoint of myeloid lineage cells, albeit its mode of function remains obscure. Our recent identification of a common ligand for both human B4 and its murine ortholog gp49B as the fibronectin (FN) N-terminal 30 kDa domain poses the question of how B4/gp49B regulate cellular activity upon recognition of FN in the plasma and/or the extracellular matrix. Since FN in the extracellular matrix is tethered by FN-binding integrins, we hypothesized that B4/gp49B would tether FN in cooperation with integrins on the cell surface, thus they should be in close vicinity to integrins spatially. This scenario suggests a mode of function of B4/gp49B by which the FN-induced signal is regulated. The FN pull-down complex was found to contain gp49B and integrin β ₁ in bone marrow-derived macrophages. The confocal fluorescent signals of the three molecules on the intrinsically FN-tethering macrophages were correlated to each other. When FN-poor macrophages adhered to culture plates, the gp49–integrin β ₁ signal correlation increased at the focal adhesion, supporting the notion that gp49B and integrin β ₁ become spatially closer to each other there. Adherence of RAW264.7 and THP-1 cells to immobilized FN induced phosphorylation of spleen tyrosine kinase, whose level was augmented under B4/gp49B deficiency. Thus, we concluded that B4/gp49B can co-tether FN in cooperation with integrin in the cis configuration on the same cell, forming a B4/gp49B–FN–integrin triplet as a regulatory unit of a focal adhesion-dependent pro-inflammatory signal in macrophages.

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The extracellular matrix (ECM) is the basis for virtually all cellular processes and is also related to tumor metastasis. Fibronectin (FN), a major ECM macromolecule expressed by different cell types and also present in plasma, consists of multiple functional modules that bind to ECM-associated, plasma, and cell-surface proteins such as integrins and FN itself, thus ensuring its cell-adhesive and modulatory role. Here we show that FN constitutes an immune checkpoint. Thus, FN was identified as a physiological ligand for a tumor/leukemia/lymphoma- as well as autoimmune-associated checkpoint, ILT3/LILRB4 (B4, CD85k). Human B4 and the murine ortholog, gp49B, bound FN with sub-micromolar affinities as assessed by bio-layer interferometry. The major B4-binding site in FN was located at the N-terminal 30-kDa module (FN30), which is apart from the major integrin-binding site present at the middle of the molecule. Blockade of B4–FN binding such as with B4 antibodies or a recombinant FN30-Fc fusion protein paradoxically ameliorated autoimmune disease in lupus-prone BXSB/Yaa mice. The unexpected nature of the B4–FN checkpoint in autoimmunity is discussed, referring to its potential role in tumor immunity.

Damaged axons in the adult mammalian central nervous system have a restricted regenerative capacity mainly because of Nogo protein, which is a major myelin‐associated axonal growth inhibitor with binding to both receptors of Nogo receptor‐1 (NgR1) and paired immunoglobulin‐like receptor (PIR)‐B. Lateral olfactory tract usher substance (LOTUS) exerts complete suppression of NgR1‐mediated axonal growth inhibition by antagonizing NgR1. However, the regulation of PIR‐B functions in neurons remains unknown. In this study, protein–protein interactions analyses found that LOTUS binds to PIR‐B and abolishes Nogo‐binding to PIR‐B completely. Reverse transcription‐polymerase chain reaction and immunocytochemistry revealed that PIR‐B is expressed in dorsal root ganglions (DRGs) from wild‐type and Ngr1‐deficient mice (male and female). In these DRG neurons, Nogo induced growth cone collapse and neurite outgrowth inhibition, but treatment with the soluble form of LOTUS completely suppressed them. Moreover, Nogo‐induced growth cone collapse and neurite outgrowth inhibition in Ngr1‐deficient DRG neurons were neutralized by PIR‐B function‐blocking antibodies, indicating that these Nogo‐induced phenomena were mediated by PIR‐B. Our data show that LOTUS negatively regulates a PIR‐B function. LOTUS thus exerts an antagonistic action on both receptors of NgR1 and PIR‐B. This may lead to an improvement in the defective regeneration of axons following injury. image

Immune homeostasis is critically regulated by the balance between activating and inhibitory receptors expressed on various immune cells such as T and B lymphocytes, and myeloid cells. The inhibitory receptors play a fundamental role in the immune checkpoint pathway, thus maintaining peripheral tolerance. We recently found that expression of leukocyte immunoglobulin-like receptor (LILR)B4, an inhibitory member of the human LILR family, is augmented in auto-antibody-producing plasmablasts/plasma cells of systemic lupus erythematosus (SLE) patients. However, the mechanism behind the ‘paradoxical’ up-regulation of this inhibitory receptor upon pathogenic antibody-secreting cells is yet to be known. To this end, in this study, we examined if glycoprotein 49B (gp49B), the murine counterpart of human LILRB4, is also elevated in auto-antibody-producing cells in several SLE mouse models, and tried to clarify the underlying mechanism. We found that gp49B is expressed on plasma cells of lupus-prone models but not of healthy C57BL/6 mice, and the level was positively correlated to the anti-double-stranded DNA IgG titer in serum. Gp49B genetic deletion, however, did not abolish the serum auto-antibodies or fully ameliorate the lethal glomerulonephritis, indicating that gp49B is not the sole regulator of lupus but a pathogenic element in the disease. We conclude that the elevated expression of this inhibitory receptor on pathogenic plasma cells was also relevant upon the murine SLE model. The mechanism of gp49B underlying the disease progression in lupus-prone mice has been discussed.