Lipid-orchestrated paracrine circuit coordinates mast cell maturation and anaphylaxis through functional interaction with fibroblasts.

Interaction of mast cells (MCs) with fibroblasts is essential for MC maturation within tissue microenvironments, although the underlying mechanism is incompletely understood. Through a phenotypic screening of >30 mouse lines deficient in lipid-related genes, we found that deletion of the lysophosphatidic acid (LPA) receptor LPA1, like that of the phospholipase PLA2G3, the prostaglandin D2 (PGD2) synthase L-PGDS, or the PGD2 receptor DP1, impairs MC maturation and thereby anaphylaxis. Mechanistically, MC-secreted PLA2G3 acts on extracellular vesicles (EVs) to supply lysophospholipids, which are converted by fibroblast-derived autotaxin (ATX) to LPA. Fibroblast LPA1 then integrates multiple pathways required for MC maturation by facilitating integrin-mediated MC-fibroblast adhesion, IL-33-ST2 signaling, L-PGDS-driven PGD2 generation, and feedforward ATX-LPA1 amplification. Defective MC maturation resulting from PLA2G3 deficiency is restored by supplementation with LPA1 agonists or PLA2G3-modified EVs. Thus, the lipid-orchestrated paracrine circuit involving PLA2G3-driven lysophospholipid, eicosanoid, integrin, and cytokine signaling fine-tunes MC-fibroblast communication, ensuring MC maturation.

Stress-induced production of chemokines by hair follicles regulates the trafficking of dendritic cells in skin.

Langerhans cells (LCs) are epidermal dendritic cells with incompletely understood origins that associate with hair follicles for unknown reasons. Here we show that in response to external stress, mouse hair follicles recruited Gr-1(hi) monocyte-derived precursors of LCs whose epidermal entry was dependent on the chemokine receptors CCR2 and CCR6, whereas the chemokine receptor CCR8 inhibited the recruitment of LCs. Distinct hair-follicle regions had differences in their expression of ligands for CCR2 and CCR6. The isthmus expressed the chemokine CCL2; the infundibulum expressed the chemokine CCL20; and keratinocytes in the bulge produced the chemokine CCL8, which is the ligand for CCR8. Thus, distinct hair-follicle keratinocyte subpopulations promoted or inhibited repopulation with LCs via differences in chemokine production, a feature also noted in humans. Pre-LCs failed to enter hairless skin in mice or humans, which establishes hair follicles as portals for LCs.

Transcriptome analysis of the cerebral cortex of acrylamide-exposed wild-type and IL-1ß-knockout mice.

Acrylamide is an environmental electrophile that has been produced in large amounts for many years. There is concern about the adverse health effects of acrylamide exposure due to its widespread industrial use and also presence in commonly consumed foods and others. IL-1ß is a key cytokine that protects the brain from inflammatory insults, but its role in acrylamide-induced neurotoxicity remains unknown. We reported recently that deletion of IL-1ß gene exacerbates ACR-induced neurotoxicity in mice. The aim of this study was to identify genes or signaling pathway(s) involved in enhancement of ACR-induced neurotoxicity by IL-1ß gene deletion or ACR-induced neurotoxicity to generate a hypothesis mechanism explaining ACR-induced neurotoxicity. C57BL/6 J wild-type and IL-1ß KO mice were exposed to ACR at 0, 12.5, 25 mg/kg by oral gavage for 7 days/week for 4 weeks, followed by extraction of mRNA from mice cerebral cortex for RNA sequence analysis. IL-1ß deletion altered the expression of genes involved in extracellular region, including upregulation of PFN1 gene related to amyotrophic lateral sclerosis and increased the expression of the opposite strand of IL-1ß. Acrylamide exposure enhanced mitochondria oxidative phosphorylation, synapse and ribosome pathways, and activated various pathways of different neurodegenerative diseases, such as Alzheimer disease, Parkinson disease, Huntington disease, and prion disease. Protein network analysis suggested the involvement of different proteins in related to learning and cognitive function, such as Egr1, Egr2, Fos, Nr4a1, and Btg2. Our results identified possible pathways involved in IL-1ß deletion-potentiated and ACR-induced neurotoxicity in mice.

Angiopoietin-like 4 Is a Critical Regulator of Fibroblasts during Pulmonary Fibrosis Development.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and irreversible interstitial pneumonia caused by the excessive production and deposition of extracellular matrix components, including type I collagen. Activated fibroblasts, called α-SMA (α-smooth muscle actin)-expressing myofibroblasts, are the major source of type I collagen in pulmonary fibrosis (PF), but the mechanisms underlying disease progression have not been fully elucidated. Here, we obtained lung fibroblasts from patients with IPF from both nonfibrotic and fibrotic areas as determined by a lung computed tomography scan and compared gene expression between these areas by DNA microarray. We found that ANGPTL4 (angiopoietin-like 4) was highly expressed only in fibroblasts from the fibrotic area. ANGPTL4 was selectively expressed in the fibroblastic area of IPF lungs, where the myofibroblast marker α-SMA was also expressed. ANGPTL4 also regulates the gene expression of fibrosis-related markers, cell migration, and proliferation. In addition, ANGPTL4 expression in a murine model of PF induced by treatment with bleomycin was significantly induced in the lungs from the acute to the chronic phase. Single-cell transcriptome analysis during the course of bleomycin-induced PF revealed that Angptl4 was predominantly expressed in the activated fibroblasts and myofibroblasts. Moreover, the administration of recombinant ANGPTL4 to the bleomycin-induced fibrosis model significantly increased collagen deposition and exacerbated the PF. In contrast, the pathogenesis of PF in Angptl4-deficient mice was improved. These results indicate that ANGPTL4 is critical for the progression of PF and might be an early diagnostic marker and therapeutic target for IPF.

Intestinal Bacterial Translocation Contributes to Diabetic Kidney Disease.

BACKGROUND: In recent years, many studies have focused on the intestinal environment to elucidate pathogenesis of various diseases, including kidney diseases. Impairment of the intestinal barrier function, the "leaky gut," reportedly contributes to pathologic processes in some disorders. Mitochondrial antiviral signaling protein (MAVS), a component of innate immunity, maintains intestinal integrity. The effects of disrupted intestinal homeostasis associated with MAVS signaling in diabetic kidney disease remains unclear. METHODS: To evaluate the contribution of intestinal barrier impairment to kidney injury under diabetic conditions, we induced diabetic kidney disease in wild-type and MAVS knockout mice through unilateral nephrectomy and streptozotocin treatment. We then assessed effects on the kidney, intestinal injuries, and bacterial translocation. RESULTS: MAVS knockout diabetic mice showed more severe glomerular and tubular injuries compared with wild-type diabetic mice. Owing to impaired intestinal integrity, the presence of intestine-derived Klebsiella oxytoca and elevated IL-17 were detected in the circulation and kidneys of diabetic mice, especially in diabetic MAVS knockout mice. Stimulation of tubular epithelial cells with K. oxytoca activated MAVS pathways and the phosphorylation of Stat3 and ERK1/2, leading to the production of kidney injury molecule-1 (KIM-1). Nevertheless, MAVS inhibition induced inflammation in the intestinal epithelial cells and KIM-1 production in tubular epithelial cells under K. oxytoca supernatant or IL-17 stimulation. Treatment with neutralizing anti-IL-17 antibody treatment had renoprotective effects. In contrast, LPS administration accelerated kidney injury in the murine diabetic kidney disease model. CONCLUSIONS: Impaired MAVS signaling both in the kidney and intestine contributes to the disrupted homeostasis, leading to diabetic kidney disease progression. Controlling intestinal homeostasis may offer a novel therapeutic approach for this condition.

Thirty-five years since the discovery of chemotactic cytokines, interleukin-8 and MCAF: A historical overview.

Inflammation is a host defense response to various invading stimuli, but an excessive and persistent inflammatory response can cause tissue injury, which can lead to irreversible organ damage and dysfunction. Excessive inflammatory responses are believed to link to most human diseases. A specific type of leukocyte infiltration into invaded tissues is required for inflammation. Historically, the underlying molecular mechanisms of this process during inflammation were an enigma, compromising research in the fields of inflammation, immunology, and pathology. However, the pioneering discovery of chemotactic cytokines (chemokines), monocyte-derived neutrophil chemotactic factor (MDNCF; interleukin [IL]-8, CXCL8) and monocyte chemotactic and activating factor (MCAF; monocyte chemotactic factor 1 [MCP-1], CCL2) in the late 1980s finally enabled us to address this issue. In this review, we provide a historical overview of chemokine research over the last 35 years.

Protective effect of d-alanine against acute kidney injury.

Recent studies have revealed the connection between amino acid chirality and diseases. We have previously reported that the gut microbiota produces various d-amino acids in a murine acute kidney injury (AKI) model. Here, we further explored the pathophysiological role of d-alanine (d-Ala) in AKI. Levels of d-Ala were evaluated in a murine AKI model. We analyzed transcripts of the N-methyl-d-aspartate (NMDA) receptor, a receptor for d-Ala, in tubular epithelial cells (TECs). The therapeutic effect of d-Ala was then assessed in vivo and in vitro. Finally, the plasma level of d-Ala was evaluated in patients with AKI. The Grin genes encoding NMDA receptor subtypes were expressed in TECs. Hypoxic conditions change the gene expression of Grin1, Grin2A, and Grin2B. d-Ala protected TECs from hypoxia-related cell injury and induced proliferation after hypoxia. These protective effects are associated with the chirality of d-Ala. d-Ala inhibits reactive oxygen species (ROS) production and improves mitochondrial membrane potential, through NMDA receptor signaling. The ratio of d-Ala to l-Ala was increased in feces, plasma, and urine after the induction of ischemia-reperfusion (I/R). Moreover, Enterobacteriaceae, such as Escherichia coli and Klebsiella oxytoca, produce d-Ala. Oral administration of d-Ala ameliorated kidney injury after the induction of I/R in mice. Deficiency of NMDA subunit NR1 in tubular cells worsened kidney damage in AKI. In addition, the plasma level of d-Ala was increased and reflected the level of renal function in patients with AKI. In conclusion, d-Ala has protective effects on I/R-induced kidney injury. Moreover, the plasma level of d-Ala reflects the estimated glomerular filtration rate in patients with AKI. d-Ala could be a promising therapeutic target and potential biomarker for AKI.NEW & NOTEWORTHY d-Alanine has protective effects on I/R-induced kidney injury. d-Ala inhibits ROS production and improves mitochondrial membrane potential, resulting in reduced TEC necrosis by hypoxic stimulation. The administration of d-Ala protects the tubules from I/R injury in mice. Moreover, the plasma level of d-Ala is conversely associated with eGFR in patients with AKI. Our data suggest that d-Ala is an appealing therapeutic target and a potential biomarker for AKI.

Inhibition of the chemokine signal regulator FROUNT by disulfiram ameliorates crescentic glomerulonephritis.

Activated monocytes/macrophages promote glomerular injury, including crescent formation, in anti-glomerular basement membrane (GBM) glomerulonephritis. Disulfiram, an alcohol-aversion drug, inhibits monocyte/macrophage migration by inhibiting FROUNT, a cytosolic protein that enhances chemokine receptor signaling. Our study found that disulfiram at a human equivalent dose successfully blocked albuminuria and crescent formation with podocyte loss, and later stage kidney fibrotic lesions, in a rat model of anti-GBM glomerulonephritis. A disulfiram derivative, DSF-41, with more potent FROUNT inhibition activity, inhibited glomerulonephritis at a lower dose than disulfiram. Disulfiram markedly reduced the number of monocytes or macrophages at the early stage of glomerulonephritis and that of CD3+ and CD8+ lymphocytes at the established stage. Impaired pseudopodia formation was observed in the glomerular monocytes/macrophages of the disulfiram group; consistent with the in vitro observation that disulfiram blocked chemokine-dependent pseudopodia formation and chemotaxis of bone marrow-derived monocytes/macrophages. Furthermore, disulfiram suppressed macrophage activation as revealed by reduced expression of inflammatory cytokines and chemokines (TNF-α, CCL2, and CXCL9) and reduced CD86 and MHC class II expressions in monocytes/macrophages during glomerulonephritis. The dramatic reduction in monocyte/macrophage number might have resulted from disulfiram suppression of both the chemotactic response of monocytes/macrophages and their subsequent activation to produce cytokines and chemokines, which further recruit monocytes. Additionally, FROUNT was expressed in CD68+ monocytes/macrophages infiltrating the crescentic glomeruli in human anti-GBM glomerulonephritis. Thus, disulfiram can be a highly effective and safe drug for the treatment of glomerulonephritis by blocking the chemotactic responses of monocytes/macrophages and their activation status in the glomerulus.

Complement protein C1q activates lung fibroblasts and exacerbates silica-induced pulmonary fibrosis in mice.

Pulmonary fibrosis is a progressive fibrotic disease with a poor prognosis and has suboptimal therapeutic options. The complement protein, C1q, which has various functions, such as promoting phagocytosis and signal transduction, has been shown to exacerbate several fibrosis-related diseases such as myofibrosis. In this study, we examined the role and cellular targets of C1q in pulmonary fibrosis. Silica-induced pulmonary fibrotic C1q-deficient mice showed improvement in fibrosis, and intratracheal administration of C1q to normal mice led to the induction of fibrotic changes. Single-cell RNA sequencing analysis revealed the early activation of fibroblasts and type 2 alveolar epithelial cells after intratracheal administration of C1q, and treatment of primary lung fibroblasts with C1q induced the expression of profibrotic genes. Thus, the inhibition of C1q may be regarded as a therapeutic target for pulmonary fibrosis.

Profibrotic properties of C1q+ interstitial macrophages in silica-induced pulmonary fibrosis in mice.

Pulmonary fibrosis (PF) is a progressive fibrotic disease with poor prognosis and suboptimal therapeutic options. Although macrophages have been implicated in PF, the role of macrophage subsets, particularly interstitial macrophages (IMs), remains unknown. We performed a time-series single-cell RNA sequencing analysis of the silica-induced mouse PF model. Among the macrophage subsets in fibrotic lungs, Lyve1lo MHC IIhi IMs increased with fibrosis, and highly expressed profibrotic genes. Additionally, we identified C1q as an IM-specific marker. Experiments with C1q-diphtheria toxin receptor-GFP knock-in (C1qKI) mice revealed that IMs are distributed around fibrotic nodules. Depletion of C1q+ IMs in C1qKI mice decreased activated fibroblasts and epithelial cells; however, bodyweight loss and neutrophil infiltration were exacerbated in silica-induced PF. Collectively, these results suggest that IMs have profibrotic and anti-inflammatory properties and that the selective inhibition of the profibrotic function of IMs without compromising their anti-inflammatory effects is a potential novel therapeutic strategy for PF.

Dual blockade of MET and VEGFR2 signaling pathways as a potential therapeutic maneuver for peritoneal carcinomatosis in scirrhous gastric cancer.

Scirrhous gastric cancer frequently develops into peritoneal carcinomatosis with malignant ascites, leading to an extremely poor prognosis. We had demonstrated that paracrine hepatocyte growth factor (HGF)-induced MET activation promotes peritoneal carcinomatosis with ascites formation. The vascular endothelial growth factor (VEGF) receptor (VEGFR)/VEGF axis facilitates tumor progression and formation of malignant ascites. This study investigated the role of MET and VEGFR2 in the development of peritoneal carcinomatosis with malignant ascites. Cabozantinib is a dual inhibitor of MET and VEGFR2. We examined the effects of cabozantinib on MET- and VEGFR2-mediated progression of peritoneal carcinomatosis in human scirrhous gastric cancer in vitro and in vivo. Cabozantinib inhibited HGF-stimulated proliferation of scirrhous cancer cell lines NUGC4 and GCIY, with a high potential to generate peritoneal carcinomatosis with ascites fluid, as well as the constitutive proliferation of MKN45 cells with MET amplification. Cabozantinib also inhibited the phosphorylation of both MET and VEGFR2 in scirrhous cancer cells and HGF- or VEGF-stimulated HUVECs. It effectively reduced ascitic fluid and prolonged the survival of NUGC4-inoculated nude mice. In clinical specimens, malignant ascites fluid from patients with peritoneal carcinomatosis contained high levels of HGF and VEGF. Our results strongly suggest that MET- and VEGFR2-mediated signaling pathways play pivotal roles in the pathogenesis of peritoneal carcinomatosis in scirrhous gastric cancer. Thus, the dual blockade of MET and VEGFR2 signaling may be a potential therapeutic maneuver for peritoneal carcinomatosis in scirrhous gastric cancer.

Interleukin-8: An evolving chemokine.

Early in the 1980s several laboratories mistakenly reported that partially purified interleukin-1 (IL-1) was chemotactic for neutrophils. However, further investigations by us, revealed that our purified IL-1 did not have neutrophil chemotactic activity and this activity in the LPS-stimulated human monocyte conditioned media could clearly be separated from IL-1 activity on HPLC gel filtration. This motivated Teizo Yoshimura and Kouji Matsushima to purify the monocyte-derived neutrophil chemotactic factor (MDNCF), present in LPS conditioned media and molecularly clone the cDNA for MDNCF. They found that MDNCF protein (later renamed IL-8, and finally termed CXCL8) is first translated as a precursor form consisting of 99 amino acid residues and the signal peptide is then removed, leading to the secretion and processing of biologically active IL-8 of 72 amino acid form (residues 28-99). There are four cysteine residues forming two disulfide linkage and 14 basic amino acid residues which result in a very basic property for the binding of IL-8 to heparan sulfate-proteoglycan. The IL-8 gene consists of 4 exons and 3 introns. IL-8 is produced by various types of cells in inflammation. The 5'-flanking region of IL-8 gene contains several nuclear factor binding sites, and NF-κB in combination with AP-1 or C/EBP synergistically activates IL-8 gene in response to IL-1 and TNFα. Two receptors exist for IL-8, CXCR1 and CXCR2 in humans, which belong to γ subfamily of GTP binding protein (G-protein) coupled rhodopsin-like 7 transmembrane domain receptors. Rodents express CXCR2 and do not produce IL-8, but produce numerous homologues instead. Once IL-8 binds to the receptor, ß and γ subunits of G-protein are released from Gα (Gαi2 in neutrophils) and activate PI3Kγ, PLCß2/ß3, PLA2 and PLD. Gαi2 inhibits adenyl cyclase to decrease cAMP levels. Small GTPases Ras/Rac/Rho/cdc42/Rap1, PKC and AKT (PKB) exist down-stream of ß and γ subunits and regulate cell adhesion, actin polymerization, membrane protrusion, and eventually cell migration. PLCß activation generates IP3 and induces Ca++ mobilization, DAG generation to activate protein kinase C to lead granule exocytosis and respiratory burst. MDNCF was renamed interleukin 8 (IL-8) at the International Symposium on Novel Neutrophil Chemotactic Activating Polypeptides, London, UK in 1989. The discovery of IL-8 prompted us to also purify and molecularly clone the cDNA of MCAF/MCP-1 responsible for monocyte chemotaxis, and other groups to identify a large family of chemotactic cytokines capable of attracting other types of leukocytes. In 1992, most of the investigators contributing to the discovery of this new family of chemotactic cytokines gathered in Baden, Austria and agreed to name this family "chemokines" and subsequently established the CXCL/CCL and CXCR/CCR nomenclature. The discovery of chemokines resulted in solving the long-time enigma concerning the mechanism of cell type specific leukocyte infiltration into inflamed tissues and provided a molecular basis for immune and hematopoietic cell migration and interactions under physiological as well as pathological conditions. To our surprise based on its recently identified multifunctional activities, IL-8 has evolved from a neutrophil chemoattractant to a promising therapeutic target for a wide range of inflammatory and neoplastic diseases. IL-8 was initially characterized as a chemoattractant of neutrophils engaged in acute inflammation and then discovered to also be chemotactic for endothelial cells with a major role in angiogenesis. These two activities of IL-8 foster its stimulatory effect on tumor growth. This is abetted by recent additional discoveries showing that IL-8 has stimulatory effects on stem cells and can therefore directly promote the growth of receptor expressing cancer stem cells. IL-8 by interacting with bone marrow stem/progenitor cells has also the capacity to mobilize and release hematopoietic cells into the peripheral circulation. This includes the mobilization of neutrophilic myeloid-derived suppressor cells (N-MDSC) to infiltrate into tumors and thus further promotes the immune escape of tumors. Finally, the capacity of IL-8 to induce trans-differentiation of epithelial cancer cells into mesenchymal phenotype (EMT) increases the malignancy of tumors by promoting their metastatic spread and resistance to chemotherapeutics and cytotoxic immune cells. These observations have stimulated considerable current efforts to develop receptor antagonists for IL-8 and humanized anti-IL-8 antibody for the therapy of cancer, particularly in combination with immune checkpoint inhibitors, such as anti-PD-1/PD-L1 antibodies.

Macrophages in lung fibrosis.

Pulmonary fibrosis (PF) is a disease in which excessive extracellular matrix (ECM) accumulation occurs in the lungs, which induces thickening of the alveolar walls, ultimately leading to the destruction of alveolar structures and respiratory failure. Idiopathic PF, the cause of which is unknown, has a poor prognosis with a median survival of 2-4 years after diagnosis. There is currently no known curative treatment. The mechanism underlying PF is thought to be initiated by the dysfunction of type II alveolar epithelial cells, which leads to ECM overproduction through the activation of fibroblasts. In addition, it has been suggested that a variety of cells contribute to fibrotic processes. In particular, clinical and basic research findings examining the roles of macrophages suggest that they may be pivotal regulators of PF. In this review, we discuss the characteristics, functions and origins of subsets of macrophages involved in PF, including resident alveolar, interstitial and monocyte-derived macrophages.

Suppression of liver transplant rejection by anti-donor MHC antibodies via depletion of donor immunogenic dendritic cells.

BACKGROUND: We previously found two distinct passenger dendritic cell (DC) subsets in the rat liver that played a central role in the liver transplant rejection. In addition, a tolerance-inducing protocol, donor-specific transfusion (DST), triggered systemic polytopical production of depleting alloantibodies to donor class I MHC (MHCI) antigen (DST-antibodies). METHODS: We examined the role of DST-antibodies in the trafficking of graft DC subsets and the alloresponses in a rat model. We also examined an anti-donor class II MHC (MHCII) antibody that recognizes donor DCs more selectively. RESULTS: Preoperative transfer of DST-antibodies or DST pretreatment eliminated all passenger leukocytes, including both DC subsets and depleted the sessile DCs in the graft to ~20% of control. The CD172a+CD11b/c+ immunogenic subset was almost abolished. The intrahost direct or semi-direct allorecognition pathway was successfully blocked, leading to a significant suppression of the CD8+ T-cell response in the recipient lymphoid organs and the graft with delayed graft rejection. Anti-donor MHCII antibody had similar effects without temporary graft damage. Although DST pretreatment had a priming effect on the proliferative response of recipient regulatory T cells, DST-primed sera and the anti-donor MHCII antibody did not. CONCLUSION: DST-antibodies and anti-donor MHCII antibodies could suppress the CD8+ T-cell-mediated liver transplant rejection by depleting donor immunogenic DCs, blocking the direct or semi-direct pathways of allorecognition. Donor MHCII-specific antibodies may be applicable as a selective suppressant of anti-donor immunity for clinical liver transplantation without the cellular damage of donor MHCII- graft cells and recipient cells.

A clonogenic progenitor with prominent plasmacytoid dendritic cell developmental potential.

Macrophage and dendritic cell (DC) progenitors (MDPs) and common DC progenitors (CDPs) are bone marrow (BM) progenitors with DC differentiation potential. However, both MDPs and CDPs give rise to large numbers of conventional DCs (cDCs) and few plasmacytoid DCs (pDCs), implying that more dedicated pDC progenitors remain to be identified. Here we have described DC progenitors with a prominent pDC differentiation potential. Although both MDPs and CDPs express the macrophage colony stimulating factor (M-CSF) receptor (M-CSFR), the progenitors were confined to a M-CSFR(-) fraction, identified as Lin(-)c-Kit(int/lo)Flt3(+)M-CSFR(-), and expressed high amounts of E2-2 (also known as Tcf4) an essential transcription factor for pDC development. Importantly, they appeared to be directly derived from either CDPs or lymphoid-primed multipotent progenitors (LMPPs). Collectively, our findings provide insight into DC differentiation pathways and may lead to progenitor-based therapeutic applications for infection and autoimmune disease.

Monocyte-derived dendritic cells perform hemophagocytosis to fine-tune excessive immune responses.

Because immune responses simultaneously defend and injure the host, the immune system must be finely regulated to ensure the host's survival. Here, we have shown that when injected with high Toll-like receptor ligand doses or infected with lymphocytic choriomeningitis virus (LCMV) clone 13, which has a high viral turnover, inflammatory monocyte-derived dendritic cells (Mo-DCs) engulfed apoptotic erythroid cells. In this process, called hemophagocytosis, phosphatidylserine (PS) served as an "eat-me" signal. Type I interferons were necessary for both PS exposure on erythroid cells and the expression of PS receptors in the Mo-DCs. Importantly, hemophagocytosis was required for interleukin-10 (IL-10) production from Mo-DCs. Blocking hemophagocytosis or Mo-DC-derived IL-10 significantly increased cytotoxic T cell lymphocyte activity, tissue damage, and mortality in virus-infected hosts, suggesting that hemophagocytosis moderates immune responses to ensure the host's survival in vivo. This sheds light on the physiological relevance of hemophagocytosis in severe inflammatory and infectious diseases.

Protective Immune Responses Elicited by Deglycosylated Live-Attenuated Simian Immunodeficiency Virus Vaccine Are Associated with IL-15 Effector Functions.

Deglycosylated, live-attenuated SIV vaccines elicited protective immune responses against heterologous SIVsmE543-3, which differs from the vaccine strain SIVmac239 to levels similar to those across HIV-1 clades. Two thirds of the vaccinees contained the chronic SIVsmE543-3 infection (controllers), whereas one third did not (noncontrollers). In this study, we investigated immune correlates of heterologous challenge control in rhesus macaques of Burmese origin. Because depletion of CD8+ cells in the controllers by administration of anti-CD8α Ab abrogated the control of viral replication, CD8+ cells were required for the protective immune response. However, classical SIV-specific CD8+ T cells did not account for the protective immune response in all controllers. Instead, IL-15-responding CD8α+ cells, including CD8+ T and NK cells, were significantly higher in the controllers than those in the noncontrollers, before and after vaccination with deglycosylated SIV. It is well established that IL-15 signal transduction occurs through "trans-presentation" in which IL-15 complexed with IL-15Rα on monocytes, macrophages, and dendritic cells binds to IL-15 Rß/γ expressed on CD8+ T and NK cells. Accordingly, levels of IL-15 stimulation were strongly affected by the depletion of monocytes from PBMCs, implying key roles of innate immune cells. These results suggest that intrinsic IL-15 responsiveness may dictate the outcome of protective responses and may lead to optimized formulations of future broadly protective HIV vaccines.

Greater extent of blood-tumor TCR repertoire overlap is associated with favorable clinical responses to PD-1 blockade.

With the widespread use of programmed death receptor-1 (PD-1) blockade therapy, sensitive and specific predictive biomarkers that guide patient selection are urgently needed. T-cell receptor (TCR) repertoire, which reflects antitumor T-cell responses based on antigen specificity, is expected as a novel biomarker for PD-1 blockade therapy. In the present study, the TCR repertoire of eight patients with gastrointestinal cancer treated with anti-PD-1 antibody (nivolumab) was analyzed. To analyze the tumor-associated T-cell clones in the blood and their mobilization into the tumor, we focused on T-cell clones that presented in both blood and tumor (blood-tumor overlapping clones). Responders to PD-1 blockade tended to exhibit a higher number of overlapping clones in the tumor and a higher total frequency in the blood. Moreover, a higher total frequency of overlapping clones in blood CD8+ T cells before treatment was associated with a favorable clinical response. Collectively, these results suggest the possibility of blood-tumor TCR repertoire overlap to predict clinical response to PD-1 blockade and guide patient selection before the treatment.

D-Serine inhibits the attachment and biofilm formation of methicillin-resistant Staphylococcus aureus.

INTRODUCTION: Although therapeutic agents for methicillin-resistant Staphylococcus aureus (MRSA) are clinically available, MRSA infection is still a life-threatening disease. Bacterial attachment and biofilm formation contribute significantly to the initiation of MRSA infection. Controlling MRSA's attachment and biofilm formation might reduce the frequency of MRSA infection. According to recent data, some amino acids can reduce MRSA's attachment on plates; however, their precise inhibitory mechanisms remain unclear. Therefore, we explored the effect of the amino acids on bacterial adhesion and biofilm formation in vitro and in vivo MRSA infection models. METHODS: We tested the inhibitory effect of amino acids on MRSA and Escherichia coli (E. coli) in the attachment assay. Moreover, we evaluated the therapeutic potential of amino acids on the in vivo catheter infection model. RESULTS: Among the amino acids, D-Serine (D-Ser) was found to reduce MRSA's ability to attach on plate assay. The proliferation of MRSA was not affected by the addition of D-Ser; thus, D-Ser likely only played a role in preventing attachment and biofilm formation. Then, we analyzed the expression of genes related to attachment and biofilm formation. D-Ser was found to reduce the expressions of AgrA, SarS, IcaA, DltD, and SdrD. Moreover, the polyvinyl chloride catheters treated with D-Ser had fewer MRSA colonies. D-Ser treatment also reduced the severity of infection in the catheter-induced peritonitis model. Moreover, D-Ser reduced the attachment ability of E. coli. CONCLUSION: D-Ser inhibits the attachment and biofilm formation of MRSA by reducing the expression of the related genes. Also, the administration of D-Ser reduces the severity of catheter infection in the mouse model. Therefore, D-Ser may be a promising therapeutic option for MRSA as well as E. coli infection.

Rational Design of Monodispersed Mutants of Proteins by Identifying Aggregation Contact Sites Using Solubilizing Agents.

Suppression of protein aggregation is a subject of growing importance in the treatment of protein aggregation diseases, an urgent worldwide human health problem, and the production of therapeutic proteins, such as antibody drugs. We previously reported a method to identify compounds that suppress aggregation, based on screening using multiple terminal deletion mutants. We now present a method to determine the aggregation contact sites of proteins, using such solubilizing compounds, to design monodispersed mutants. We applied this strategy to the chemokine receptor-binding domain (CRBD) of FROUNT, which binds to the membrane-proximal C-terminal intracellular region of CCR2. Initially, the backbone NMR signals were assigned to a certain extent by available methods, and the putative locations of five α-helices were identified. Based on NMR chemical shift perturbations upon varying the protein concentrations, the first and third helices were found to contain the aggregation contact sites. The two helices are amphiphilic, and based on an NMR titration with 1,6-hexanediol, a CRBD solubilizing compound, the contact sites were identified as the hydrophobic patches located on the hydrophilic sides of the two helices. Subsequently, we designed multiple mutants targeting amino acid residues on the contact sites. Based on their NMR spectra, a doubly mutated CRBD (L538E/P612S) was selected from the designed mutants, and its monodispersed nature was confirmed by other biophysical methods. We then assessed the CCR2-binding activities of the mutants. Our method is useful for the protein structural analyses, the treatment of protein aggregation diseases, and the improvement of therapeutic proteins.