Lysosomal cholesterol overload in macrophages promotes liver fibrosis in a mouse model of NASH.

Accumulation of lipotoxic lipids, such as free cholesterol, induces hepatocyte death and subsequent inflammation and fibrosis in the pathogenesis of nonalcoholic steatohepatitis (NASH). However, the underlying mechanisms remain unclear. We have previously reported that hepatocyte death locally induces phenotypic changes in the macrophages surrounding the corpse and remnant lipids, thereby promoting liver fibrosis in a murine model of NASH. Here, we demonstrated that lysosomal cholesterol overload triggers lysosomal dysfunction and profibrotic activation of macrophages during the development of NASH. ß-cyclodextrin polyrotaxane (ßCD-PRX), a unique supramolecule, is designed to elicit free cholesterol from lysosomes. Treatment with ßCD-PRX ameliorated cholesterol accumulation and profibrotic activation of macrophages surrounding dead hepatocytes with cholesterol crystals, thereby suppressing liver fibrosis in a NASH model, without affecting the hepatic cholesterol levels. In vitro experiments revealed that cholesterol-induced lysosomal stress triggered profibrotic activation in macrophages predisposed to the steatotic microenvironment. This study provides evidence that dysregulated cholesterol metabolism in macrophages would be a novel mechanism of NASH.

CD47 promotes peripheral T cell survival by preventing dendritic cell-mediated T cell necroptosis.

Conventional dendritic cells (cDCs) are required for peripheral T cell homeostasis in lymphoid organs, but the molecular mechanism underlying this requirement has remained unclear. We here show that T cell-specific CD47-deficient (Cd47 ΔT) mice have a markedly reduced number of T cells in peripheral tissues. Direct interaction of CD47-deficient T cells with cDCs resulted in activation of the latter cells, which in turn induced necroptosis of the former cells. The deficiency and cell death of T cells in Cd47 ΔT mice required expression of its receptor signal regulatory protein α on cDCs. The development of CD4+ T helper cell-dependent contact hypersensitivity and inhibition of tumor growth by cytotoxic CD8+ T cells were both markedly impaired in Cd47 ΔT mice. CD47 on T cells thus likely prevents their necroptotic cell death initiated by cDCs and thereby promotes T cell survival and function.

Migrants' access to COVID-19 vaccination in Japan: Progress and challenges.

Objectives: This study aimed to review the progress and challenges of COVID-19 vaccine roll-out for migrants in Japan and discuss the approaches to address the challenges and better prepare for future waves of COVID-19 and other pandemics. Methods: We conducted a literature review using an assessment framework which we developed building upon existing frameworks and tools on access to health services and COVID-19 vaccination among migrants. Results: COVID-19 vaccination coverage among foreigners might be lower than that of nationals although the data on foreigners were not widely available. A gap appeared to exist between the government's efforts to disseminate vaccine-related information through multi-lingual websites and migrant communities as recipients. A series of barriers for migrants were identified at different stages of the vaccination process. While efforts were made by different units of local governments, NGOs, migrant communities, and international exchange associations, linkages across sectors and scaling-up appeared to be an issue. No foreigners were explicitly excluded from the entitlements of COVID-19 vaccination. The national level guidance, however, allowed sub-national levels to make a decision on whether or not undocumented foreigners should be reported to the immigration office or law enforcement when providing the services. In consequence, units in charge of public health and vaccination of some municipalities did not offer vaccination to those in need. Conclusion: Migrants, especially those unregistered face various barriers in accessing COVID-19 vaccination. It is critical to assess and address challenges concerning channels of information dissemination, pathways to access services, obstacles for vulnerable migrants, and data for evidence-based actions.

Targeting of SIRPα as a potential therapy for Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is a rare neoplastic disorder characterized by inflammatory lesions arising from the anomalous accumulation of pathogenic CD1a+ CD207+ dendritic cells (DCs). SIRPα is a transmembrane protein highly expressed in myeloid cells such as DCs and macrophages. Here we show that SIRPα is a potential therapeutic target for LCH. We found that SIRPα is expressed in CD1a+ cells of human LCH lesions as well as in CD11c+ DCs in the spleen, liver, and lung of a mouse model of LCH (BRAFV600ECD11c mouse), in which an LCH-associated active form of human BRAF is expressed in a manner dependent on the mouse Cd11c promoter. BRAFV600ECD11c mice manifested markedly increased numbers of CD4+ T cells, regulatory T cells, and macrophages as well as of CD11c+ MHCII+ DCs in the spleen. Monotherapy with a mAb to SIRPα greatly reduced the percentage of CD11c+ MHCII+ DCs in peripheral blood, LCH-like lesion size in the liver, and the number of CD11c+ MHCII+ DCs in the spleen of the mutant mice. Moreover, this mAb promoted macrophage-mediated phagocytosis of CD11c+ DCs from BRAFV600ECD11c mice, whereas it had no effects on the viability or CCL19-dependent migration of such CD11c+ DCs or on their expression of the chemokine genes Ccl5, Ccl20, Cxcl11, and Cxcl12. Our results thus suggest that anti-SIRPα monotherapy is a promising approach to the treatment of LCH that is dependent in part on the promotion of the macrophage-mediated killing of LCH cells.

Preclinical evaluation of the efficacy of an antibody to human SIRPα for cancer immunotherapy in humanized mouse models.

Tumor-associated macrophages (TAMs) are abundant in the tumor microenvironment and are considered potential targets for cancer immunotherapy. To examine the antitumor effects of agents targeting human TAMs in vivo, we here established preclinical tumor xenograft models based on immunodeficient mice that express multiple human cytokines and have been reconstituted with a human immune system by transplantation of human CD34+ hematopoietic stem and progenitor cells (HIS-MITRG mice). HIS-MITRG mice supported the growth of both human cell line (Raji)- and patient-derived B cell lymphoma as well as the infiltration of human macrophages into their tumors. We examined the potential antitumor action of an antibody to human SIRPα (SE12C3) that inhibits the interaction of CD47 on tumor cells with SIRPα on human macrophages and thereby promotes Fcγ receptor-mediated phagocytosis of the former cells by the latter. Treatment with the combination of rituximab (antibody to human CD20) and SE12C3 inhibited Raji tumor growth in HIS-MITRG mice to a markedly greater extent than did rituximab monotherapy. This enhanced antitumor effect was dependent on human macrophages and attributable to enhanced rituximab-dependent phagocytosis of lymphoma cells by human macrophages. Treatment with rituximab and SE12C3 also induced reprogramming of human TAMs toward a proinflammatory phenotype. Furthermore, the combination treatment essentially prevented the growth of patient-derived diffuse large B cell lymphoma in HIS-MITRG mice. Our findings thus support the study of HIS-MITRG mice as a model for the preclinical evaluation in vivo of potential therapeutics, such as antibodies to human SIRPα, that target human TAMs.

The Role of Type-2 Conventional Dendritic Cells in the Regulation of Tumor Immunity.

Conventional dendritic cells (cDCs) orchestrate immune responses to cancer and comprise two major subsets: type-1 cDCs (cDC1s) and type-2 cDCs (cDC2s). Compared with cDC1s, which are dedicated to the activation of CD8+ T cells, cDC2s are ontogenically and functionally heterogeneous, with their main function being the presentation of exogenous antigens to CD4+ T cells for the initiation of T helper cell differentiation. cDC1s play an important role in tumor-specific immune responses through cross-presentation of tumor-derived antigens for the priming of CD8+ T cells, whereas little is known of the role of cDC2s in tumor immunity. Recent studies have indicated that human cDC2s can be divided into at least two subsets and have implicated these cells in both anti- and pro-tumoral immune responses. Furthermore, the efficacy of cDC2-based vaccines as well as cDC2-targeted therapeutics has been demonstrated in both mouse models and human patients. Here we summarize current knowledge about the role of cDC2s in tumor immunity and address whether these cells are beneficial in the context of antitumor immune responses.

Anticancer efficacy of monotherapy with antibodies to SIRPα/SIRPß1 mediated by induction of antitumorigenic macrophages.

The interaction of signal regulatory protein α (SIRPα) on macrophages with CD47 on cancer cells is thought to prevent antibody (Ab)-dependent cellular phagocytosis (ADCP) of the latter cells by the former. Blockade of the CD47-SIRPα interaction by Abs to CD47 or to SIRPα, in combination with tumor-targeting Abs such as rituximab, thus inhibits tumor formation by promoting macrophage-mediated ADCP of cancer cells. Here we show that monotherapy with a monoclonal Ab (mAb) to SIRPα that also recognizes SIRPß1 inhibited tumor formation by bladder and mammary cancer cells in mice, with this inhibitory effect being largely dependent on macrophages. The mAb to SIRPα promoted polarization of tumor-infiltrating macrophages toward an antitumorigenic phenotype, resulting in the killing and phagocytosis of cancer cells by the macrophages. Ablation of SIRPα in mice did not prevent the inhibitory effect of the anti-SIRPα mAb on tumor formation or its promotion of the cancer cell-killing activity of macrophages, however. Moreover, knockdown of SIRPß1 in macrophages attenuated the stimulatory effect of the anti-SIRPα mAb on the killing of cancer cells, whereas an mAb specific for SIRPß1 mimicked the effect of the anti-SIRPα mAb. Our results thus suggest that monotherapy with Abs to SIRPα/SIRPß1 induces antitumorigenic macrophages and thereby inhibits tumor growth and that SIRPß1 is a potential target for cancer immunotherapy.

Role of Ras in regulation of intestinal epithelial cell homeostasis and crosstalk with Wnt signaling.

Cross talk between different signaling pathways is thought to be important for regulation of homeostasis of, as well as oncogenesis of, the intestinal epithelium. Expression of an active form of K-Ras specifically in intestinal epithelial cells (IECs) of mice (IEC-RasDA mice) resulted in the development of hyperplasia in the small intestine and colon of mice. IEC-RasDA mice also manifested the increased proliferation of IECs. In addition, the number of goblet cells markedly increased, while that of Paneth cells decreased in IEC-RasDA mice. Development of intestinal organoids was markedly enhanced for IEC-RasDA mice compared with control mice. Whereas, the expression of Wnt target genes was significantly reduced in the in intestinal crypts from IEC-RasDA mice compared with that apparent for the control. Our results thus suggest that K-Ras promotes the proliferation of IECs as well as generation of goblet cells. By contrast, Ras counter-regulates the Wnt signaling and thereby contribute to the proper regulation of intestinal epithelial cell homeostasis.

Roles of Src family kinase, Ras, and mTOR signaling in intestinal epithelial homeostasis and tumorigenesis.

The turnover of intestinal epithelial cells (IECs) is relatively rapid (3-5 days in mouse and human), and this short existence and other aspects of the homeostasis of IECs are tightly regulated by various signaling pathways including Wnt-ß-catenin signaling. Dysregulation of IEC homeostasis likely contributes to the development of intestinal inflammation and intestinal cancer. The roles of receptor protein tyrosine kinases and their downstream signaling molecules such as Src family kinases, Ras, and mTOR in homeostatic regulation of IEC turnover have recently been evaluated. These signaling pathways have been found to promote not only the proliferation of IECs but also the differentiation of progenitor cells into secretory cell types such as goblet cells. Of note, signaling by Src family kinases, Ras, and mTOR has been shown to oppose the Wnt-ß-catenin signaling pathway and thereby to limit the number of Lgr5+ intestinal stem cells or of Paneth cells. Such cross-talk of signaling pathways is important not only for proper regulation of IEC homeostasis but for the development of intestinal tumors and potentially for anticancer therapy.

Blockade of CD47 or SIRPα: a new cancer immunotherapy.

The CD47-Signal regulatory protein α (SIRPα) singling axis acts as a crucial regulator that limits the phagocytic activity of professional phagocytes such as macrophages. Recent studies have demonstrated that the interaction between CD47 on tumor cells and SIRPα on macrophages is implicated in the ability of tumors to evade immunosurveillance. Targeting the CD47-SIRPα interaction is therefore considered to be a promising approach for cancer therapy. Herein, we review some of studies displaying the potential clinical application of antibodies and other modalities that target the CD47-SIRPα interaction. Current limitations of the CD47-SIRPα-targeted immunotherapeutic approaches are also discussed as well as other avenues for future study to improve the current strategies in targeting the CD47-SIRPα signaling axis for cancer immunotherapy.

Regulation of colonic epithelial cell homeostasis by mTORC1.

Cell signaling important for homeostatic regulation of colonic epithelial cells (CECs) remains poorly understood. Mammalian target of rapamycin complex 1 (mTORC1), a protein complex that contains the serine-threonine kinase mTOR, mediates signaling that underlies the control of cellular functions such as proliferation and autophagy by various external stimuli. We here show that ablation of tuberous sclerosis complex 2 (Tsc2), a negative regulator of mTORC1, specifically in intestinal epithelial cells of mice resulted in increased activity of mTORC1 of, as well as increased proliferative activity of, CECs. Such Tsc2 ablation also reduced the population of Lgr5-positive colonic stem cells and the expression of Wnt target genes in CECs. The stimulatory phosphorylation of the kinase Akt and inhibitory phosphorylation of glycogen synthase kinase 3ß were both markedly decreased in the colon of the Tsc2 conditional knockout (CKO) mice. Development of colonic organoids with cryptlike structures was enhanced for Tsc2 CKO mice compared with control mice. Finally, Tsc2 CKO mice manifested increased susceptibility to dextran sulfate sodium-induced colitis. Our results thus suggest that mTORC1 activity promotes the proliferation of, as well as the expression of Wnt target genes in, CECs and thereby contributes to colonic organogenesis and homeostasis.

Macrocyclic Peptide-Mediated Blockade of the CD47-SIRPα Interaction as a Potential Cancer Immunotherapy.

Medium-sized macrocyclic peptides are an alternative to small compounds and large biomolecules as a class of pharmaceutics. The CD47-SIRPα signaling axis functions as an innate immune checkpoint that inhibits phagocytosis in phagocytes and has been implicated as a promising target for cancer immunotherapy. The potential of macrocyclic peptides that target this signaling axis as immunotherapeutic agents has remained unknown, however. Here we have developed a macrocyclic peptide consisting of 15 amino acids that binds to the ectodomain of mouse SIRPα and efficiently blocks its interaction with CD47 in an allosteric manner. The peptide markedly promoted the phagocytosis of antibody-opsonized tumor cells by macrophages in vitro as well as enhanced the inhibitory effect of anti-CD20 or anti-gp75 antibodies on tumor formation or metastasis in vivo. Our results suggest that allosteric inhibition of the CD47-SIRPα interaction by macrocyclic peptides is a potential approach to cancer immunotherapy.

SIRPα on CD11c+ cells induces Th17 cell differentiation and subsequent inflammation in the CNS in experimental autoimmune encephalomyelitis.

Signal regulatory protein α (SIRPα) is expressed predominantly on type 2 conventional dendritic cells (cDC2s) and macrophages. We previously showed that mice systemically lacking SIRPα were resistant to experimental autoimmune encephalomyelitis (EAE). Here, we showed that deletion of SIRPα in CD11c+ cells of mice (SirpaΔDC mice) also markedly ameliorated the development of EAE. The frequency of cDCs and migratory DCs (mDCs), as well as that of Th17 cells, were significantly reduced in draining lymph nodes of SirpaΔDC mice at the onset of EAE. In addition, we found the marked reduction in the number of Th17 cells and DCs in the CNS of SirpaΔDC mice at the peak of EAE. Whereas inducible systemic ablation of SIRPα before the induction of EAE prevented disease development, that after EAE onset did not ameliorate the clinical signs of disease. We also found that EAE development was partially attenuated in mice with CD11c+ cell-specific ablation of CD47, a ligand of SIRPα. Collectively, our results suggest that SIRPα expressed on CD11c+ cells, such as cDC2s and mDCs, is indispensable for the development of EAE, being required for the priming of self-reactive Th17 cells in the periphery as well as for the inflammation in the CNS.

Regulation of Small Intestinal Epithelial Homeostasis by Tsc2-mTORC1 Signaling.

Mammalian target of rapamycin complex 1 (mTORC1), a protein complex containing the serine/threonine kinase mTOR, integrates various growth stimulating signals. mTORC1 is expressed in intestinal epithelial cells (IECs), whereas the physiological roles of this protein complex in homeostasis of IECs remain virtually unknown. We here generated mice, in which tuberous sclerosis complex 2 (Tsc2), a negative regulator of mTORC1, was specifically ablated in IECs (Tsc2 CKO mice). Ablation of Tsc2 enhanced the phosphorylation of mTORC1 downstream molecules such as ribosomal S6 protein and 4E-BP1 in IECs. Tsc2 CKO mice manifested the enhanced proliferative activity of IECs in intestinal crypts as well as the promoted migration of these cells along the crypt-villus axis. The mutant mice also manifested the increased apoptotic rate of IECs as well as the increased ectopic Paneth cells, which are one of the major differentiated IECs. In addition, in vitro study showed that ablation of Tsc2 promoted the development of intestinal organoids without epidermal growth factor, while mTORC1 inhibitor, rapamycin, diminished this phenotype. Our results thus suggest that Tsc2-mTORC1 signaling regulates the proliferation, migration, and positioning of IECs, and thereby contributes to the proper regulation of intestinal homeostasis.

SIRPα+ dendritic cells promote the development of fibroblastic reticular cells in murine peripheral lymph nodes.

Nonhematopoietic stromal cells contribute to the organization and homeostasis of secondary lymphoid organs by producing cytokines and chemokines. The development and maintenance of these stromal cells are thought to be regulated by innate immune cells. Indeed, we recently showed that signal regulatory protein α (SIRPα)-positive dendritic cells (DCs) are essential for the proliferation and survival of podoplanin (Pdpn)-positive fibroblastic reticular cells (FRCs) in mouse spleen. We have now established an in vitro culture system for lymph node stromal cells (LNSCs) isolated from mouse peripheral LNs. Activated DCs and TNF-α each promoted the proliferation of cultured LNSCs, most of which were found to be Pdpn+ FRCs. Furthermore, ablation of SIRPα in CD11c+ cells attenuated this effect of DCs on LNSC proliferation. Transplantation of activated DCs together with cultured LNSCs into the renal subcapsular space markedly increased the number of ER-TR7+ stromal cells as well as induced the accumulation of T cells and increased the expression of Ccl19 in the transplants. Ablation of SIRPα in CD11c+ cells greatly impaired the development of LN-like structure in the transplants. Our findings thus suggest that SIRPα+ DCs are important for the proliferation and differentiation of Pdpn+ FRCs in peripheral LNs.

Role of lysophosphatidic acid in proliferation and differentiation of intestinal epithelial cells.

Intestinal epithelial cells (IECs) are regenerated continuously from intestinal stem cells (ISCs) near the base of intestinal crypts in order to maintain homeostasis and structural integrity of intestinal epithelium. Epidermal growth factor (EGF) is thought to be important to drive the proliferation and differentiation of IECs from ISCs, it remains unknown whether other growth factors or lipid mediators are also important for such regulation, however. Here we show that lysophosphatidic acid (LPA), instead of EGF, robustly promoted the development of intestinal organoids prepared from the mouse small intestine. Indeed, LPA exhibited the proliferative activity of IECs as well as induction of differentiation of IECs into goblet cells, Paneth cells, and enteroendocrine cells in intestinal organoids. Inhibitors for LPA receptor 1 markedly suppressed the LPA-promoted development of intestinal organoids. LPA also promoted the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 in intestinal organoids, whereas inhibition of mitogen-activated protein kinase/ERK kinase (MEK) 1/2 significantly suppressed the development of, as well as the proliferative activity and differentiation of, intestinal organoids in response to LPA. Our results thus suggest that LPA is a key factor that drives the proliferation and differentiation of IECs.

Microglial SIRPα regulates the emergence of CD11c+ microglia and demyelination damage in white matter.

A characteristic subset of microglia expressing CD11c appears in response to brain damage. However, the functional role of CD11c+ microglia, as well as the mechanism of its induction, are poorly understood. Here we report that the genetic ablation of signal regulatory protein α (SIRPα), a membrane protein, induced the emergence of CD11c+ microglia in the brain white matter. Mice lacking CD47, a physiological ligand of SIRPα, and microglia-specific SIRPα-knockout mice exhibited the same phenotype, suggesting that an interaction between microglial SIRPα and CD47 on neighbouring cells suppressed the emergence of CD11c+ microglia. A lack of SIRPα did not cause detectable damage to the white matter, but resulted in the increased expression of genes whose expression is characteristic of the repair phase after demyelination. In addition, cuprizone-induced demyelination was alleviated by the microglia-specific ablation of SIRPα. Thus, microglial SIRPα suppresses the induction of CD11c+ microglia that have the potential to accelerate the repair of damaged white matter.

Role of Csk in intestinal epithelial barrier function and protection against colitis.

Intestinal epithelial cells (IECs) play a pivotal role in the maintenance of the integrity and barrier function of the intestinal epithelium. Dysfunctions of IECs are thought to participate in the disruption of the intestinal epithelial barrier, resulting in gastrointestinal diseases, such as colitis and colorectal cancer. Here we show that IEC-specific COOH-terminal Src kinase (Csk)-deficient mice (Csk CKO mice) manifested the increased susceptibility to dextran sodium sulfate (DSS)-induced colitis, a model of inflammatory bowel disease. DSS-treated Csk CKO mice also exhibited the significantly elevated intestinal permeability. Following DSS treatment, Csk CKO mice exhibited the higher proliferative activity of colonic epithelial cells and the increased number of apoptotic cells in the colon compared with that apparent for control mice. Moreover, the abundance of the tight junction protein occludin, which regulates cell-cell adhesion as well as epithelial permeability, was markedly reduced in the colon of DSS-treated Csk CKO mice. These results thus suggest that Csk in IECs plays important roles in the regulation of the intestinal epithelial barrier function and protection against colitis.

CD47-signal regulatory protein α signaling system and its application to cancer immunotherapy.

Tumor cells evade immune surveillance through direct or indirect interactions with various types of immune cell, with much recent attention being focused on modifying immune cell responses as the basis for the development of new cancer treatments. Signal regulatory protein α (SIRPα) and CD47 are both transmembrane proteins that interact with each other and constitute a cell-cell communication system. SIRPα is particularly abundant in myeloid cells such as macrophages and dendritic cells, whereas CD47 is expressed ubiquitously and its expression level is elevated in cancer cells. Recent studies have shown that blockade of CD47-SIRPα interaction enhances the phagocytic activity of phagocytes such as macrophages toward tumor cells in vitro as well as resulting in the efficient eradication of tumor cells in a variety of xenograft or syngeneic mouse models of cancer. Moreover, CD47 blockade has been shown to promote the stimulation of tumor-specific cytotoxic T cells by macrophages or dendritic cells. Biological agents, such as Abs and recombinant proteins, that target human CD47 or SIRPα have been developed and are being tested in preclinical models of human cancer or in clinical trials with cancer patients. Preclinical studies have also suggested that CD47 or SIRPα blockade may have a synergistic antitumor effect in combination with immune checkpoint inhibitors that target the adaptive immune system. Targeting of the CD47-SIRPα signaling system is thus a promising strategy for cancer treatment based on modulation of both innate and acquired immune responses to tumor cells.

Regulation by commensal bacteria of neurogenesis in the subventricular zone of adult mouse brain.

In the mouse olfactory bulb (OB), interneurons such as granule cells and periglomerular cells are continuously replaced by adult-born neurons, which are generated in the subventricular zone (SVZ) of the brain. We have now investigated the role of commensal bacteria in regulation of such neuronal cell turnover in the adult mouse brain. Administration of mixture of antibiotics to specific pathogen-free (SPF) mice markedly attenuated the incorporation of bromodeoxyuridine (BrdU) into the SVZ cells. The treatment with antibiotics also reduced newly generated BrdU-positive neurons in the mouse OB. In addition, the incorporation of BrdU into the SVZ cells of germ-free (GF) mice was markedly reduced compared to that apparent for SPF mice. In contrast, the reduced incorporation of BrdU into the SVZ cells of GF mice was recovered by their co-housing with SPF mice, suggesting that commensal bacteria promote the incorporation of BrdU into the SVZ cells. Finally, we found that administration of ampicillin markedly attenuated the incorporation of BrdU into the SVZ cells of SPF mice. Our results thus suggest that ampicillin-sensitive commensal bacteria regulate the neurogenesis in the SVZ of adult mouse brain.