Yoshitomo Hayama

Polarization of M2 macrophages requires Lamtor1 that integrates cytokine and amino-acid signals

Macrophages play crucial roles in host defence and tissue homoeostasis, processes in which both environmental stimuli and intracellularly generated metabolites influence activation of macrophages. Activated macrophages are classified into M1 and M2 macrophages. It remains unclear how intracellular nutrition sufficiency, especially for amino acid, influences on macrophage activation. Here we show that a lysosomal adaptor protein Lamtor1, which forms an amino-acid sensing complex with lysosomal vacuolar-type H+-ATPase (v-ATPase), and is the scaffold for amino acid-activated mTORC1 (mechanistic target of rapamycin complex 1), is critically required for M2 polarization. Lamtor1 deficiency, amino-acid starvation, or inhibition of v-ATPase and mTOR result in defective M2 polarization and enhanced M1 polarization. Furthermore, we identified liver X receptor (LXR) as the downstream target of Lamtor1 and mTORC1. Production of 25-hydroxycholesterol is dependent on Lamtor1 and mTORC1. Our findings demonstrate that Lamtor1 plays an essential role in M2 polarization, coupling immunity and metabolism.

Semaphorin 4D inhibits neutrophil activation and is involved in the pathogenesis of neutrophil-mediated autoimmune vasculitis

Objectives Inappropriate activation of neutrophils plays a pathological role in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). The aim of this study was to investigate the functions of semaphorin 4D (SEMA4D) in regulation of neutrophil activation, and its involvement in AAV pathogenesis. Methods Serum levels of soluble SEMA4D were evaluated by ELISA. Blood cell-surface expression of membrane SEMA4D was evaluated by flow cytometry. To determine the functional interactions between neutrophil membrane SEMA4D and endothelial plexin B2, wild-type and SEMA4D −/− mice neutrophils were cultured with an endothelial cell line (MS1) stained with SYTOX green, and subjected to neutrophil extracellular trap (NET) formation assays. The efficacy of treating human neutrophils with recombinant plexin B2 was assessed by measuring the kinetic oxidative burst and NET formation assays. Results Serum levels of soluble SEMA4D were elevated in patients with AAV and correlated with disease activity scores. Cell-surface expression of SEMA4D was downregulated in neutrophils from patients with AAV, a consequence of proteolytic cleavage of membrane SEMA4D. Soluble SEMA4D exerted pro-inflammatory effects on endothelial cells. Membranous SEMA4D on neutrophils bound to plexin B2 on endothelial cells, and this interaction decreased NET formation. Recombinant plexin B2 suppressed neutrophil Rac1 activation through SEMA4D’s intracellular domain, and inhibited pathogen-induced or ANCA-induced oxidative burst and NET formation. Conclusions Neutrophil surface SEMA4D functions as a negative regulator of neutrophil activation. Proteolytic cleavage of SEMA4D as observed in patients with AAV may amplify neutrophil-mediated inflammatory responses. SEMA4D is a promising biomarker and potential therapeutic target for AAV.

Development of microscopic polyangiitis-related pulmonary fibrosis in a patient with autoimmune pulmonary alveolar proteinosis

BackgroundAutoimmune pulmonary alveolar proteinosis (aPAP) is a rare lung disease caused by the autoantibody against granulocyte-macrophage colony stimulating factor (GM-CSF). The clinical course of aPAP is variable; in severe cases, patients develop lethal respiratory failure due to pulmonary fibrosis. However, the pathogenesis of pulmonary fibrosis in aPAP has never been delineated.Case presentationHere, we describe a rare case of aPAP that was subsequently complicated by microscopic polyangiitis-related pulmonary fibrosis. The patient was a 75-year-old Japanese man diagnosed with aPAP based on the crazy-paving appearance on high-resolution computed tomography (HRCT), “milky” appearance of broncho-alveolar lavage fluid (BALF), and elevated serum levels of the anti-GM-CSF antibody. The patient was followed-up without aPAP-specific treatment for 3 years. During this period, both hematuria and proteinuria appeared; in addition, serum myeloperoxidase (MPO)-anti-neutrophil cytoplasmic antibody (ANCA) turned positive and increased markedly. The second BAL performed one year after the diagnosis, showed that the “milky” appearance had resolved. The HRCT showed that fibrotic changes had developed and that the crazy-paving appearance had disappeared. These data suggest an association between pulmonary fibrosis that developed during the natural course of aPAP and ANCA-related systemic vasculitis.ConclusionThis is the first case report that suggests the existence of a pathogenetic relationship between ANCA-associated systemic vasculitis and aPAP-related pulmonary fibrosis. The link between ANCA-associated systemic vasculitis and aPAP-related pulmonary fibrosis requires further investigation.

LRRK1 is critical in the regulation of B-cell responses and CARMA1-dependent NF-κB activation

B-cell receptor (BCR) signaling plays a critical role in B-cell activation and humoral immunity. In this study, we discovered a critical function of leucine-rich repeat kinase 1 (LRRK1) in BCR-mediated immune responses. Lrrk1−/− mice exhibited altered B1a-cell development and basal immunoglobulin production. In addition, these mice failed to produce IgG3 antibody in response to T cell–independent type 2 antigen due to defects in IgG3 class-switch recombination. Concomitantly, B cells lacking LRRK1 exhibited a profound defect in proliferation and survival upon BCR stimulation, which correlated with impaired BCR-mediated NF-κB activation and reduced expression of NF-κB target genes including Bcl-xL, cyclin D2, and NFATc1/αA. Furthermore, LRRK1 physically interacted and potently synergized with CARMA1 to enhance NF-κB activation. Our results reveal a critical role of LRRK1 in NF-κB signaling in B cells and the humoral immune response.

Semaphorin 6D reverse signaling controls macrophage lipid metabolism and anti-inflammatory polarization

Polarization of macrophages into pro-inflammatory or anti-inflammatory states has distinct metabolic requirements, with mechanistic target of rapamycin (mTOR) kinase signaling playing a critical role. However, it remains unclear how mTOR regulates metabolic status to promote polarization of these cells. Here we show that an mTOR–Semaphorin 6D (Sema6D)–Peroxisome proliferator receptor γ (PPARγ) axis plays critical roles in macrophage polarization. Inhibition of mTOR or loss of Sema6D blocked anti-inflammatory macrophage polarization, concomitant with severe impairments in PPARγ expression, uptake of fatty acids, and lipid metabolic reprogramming. Macrophage expression of the receptor Plexin-A4 is responsible for Sema6D-mediated anti-inflammatory polarization. We found that a tyrosine kinase, c-Abl, which associates with the cytoplasmic region of Sema6D, is required for PPARγ expression. Furthermore, Sema6D is important for generation of intestinal resident CX3CR1hi macrophages and prevents development of colitis. Collectively, these findings highlight crucial roles for Sema6D reverse signaling in macrophage polarization, coupling immunity, and metabolism via PPARγ.Semaphorins play well-known roles in axon guidance. Kumanogoh and colleagues demonstrate that Semaphorin 6D cell-intrinsically activates anti-inflammatory macrophage polarization.

Favorable response to trastuzumab plus irinotecan combination therapy in two patients with HER2-positive relapsed small-cell lung cancer

Small-cell lung cancer (SCLC) easily recurs with multidrug resistance phenotype. However, standard therapeutic strategies for relapsed-SCLC remain unestablished. Human epidermal growth factor receptor 2 (HER2) expression correlates with poor prognosis in extensive disease-SCLC. We have reported previously that HER2 expression is upregulated when HER2-positive SCLC cells acquire chemoresistance, and also demonstrated that trastuzumab exerts significant antitumor activity toward HER2-upregulated chemoresistant SCLC, mainly via antibody-dependent cell-mediated cytotoxicity mechanism. Based on these preclinical data, we treated two patients with HER2-positive SCLC by combination of trastuzumab (6 mg/kg, day 1) and irinotecan (80 mg/m(2), days 1 and 8) every 21 days as the third-line chemotherapy following two prior regimens, first-line carboplatin plus etoposide and second-line amrubicin. One patient achieved partial response after the first cycle and received 6 cycles in total without disease progression for 4.5 months. The other also received 4 cycles and kept stable disease for 3.5 months. This treatment can be continued safely at an outpatient clinic without any severe adverse event. In conclusion, trastuzumab plus irinotecan chemotherapy is promising and feasible against HER2-positive relapsed SCLC. Further clinical studies are encouraged to confirm the antitumor efficacy of trastuzumab in SCLC.

Dapsone Hypersensitivity Syndrome-related Lung Injury without Eosinophilia in the Bronchoalveolar Lavage Fluid

A 73-year-old man was admitted in respiratory failure that had subacutely progressed after five weeks of dapsone treatment for a skin rash. He also presented with fever, systemic erythroderma and liver dysfunction. Chest computed tomography showed diffuse reticular shadows with ground-glass opacity and bilateral mediastinal lymphadenopathy. Lymphocytes, but not eosinophils, were increased in the bronchoalveolar lavage fluid. Moreover, reactivation of human herpes virus-6 was confirmed on a paired serum test. Finally, we diagnosed the patient with dapsone hypersensitivity syndrome (DHS), a rare adverse event of this drug. Lung injury unaccompanied by eosinophilia in the bronchoalveolar lavage fluid is even more rare as a DHS-related lung manifestation.

Apoptosis-derived membrane vesicles drive the cGAS–STING pathway and enhance type I IFN production in systemic lupus erythematosus

Objective Despite the importance of type I interferon (IFN-I) in systemic lupus erythematosus (SLE) pathogenesis, the mechanisms of IFN-I production have not been fully elucidated. Recognition of nucleic acids by DNA sensors induces IFN-I and interferon-stimulated genes (ISGs), but the involvement of cyclic guanosine monophosphate (GMP)–AMP synthase (cGAS) and stimulator of interferon genes (STING) in SLE remains unclear. We studied the role of the cGAS–STING pathway in the IFN-I-producing cascade driven by SLE serum. Methods We collected sera from patients with SLE (n=64), patients with other autoimmune diseases (n=31) and healthy controls (n=35), and assayed them using a cell-based reporter system that enables highly sensitive detection of IFN-I and ISG-inducing activity. We used Toll-like receptor-specific reporter cells and reporter cells harbouring knockouts of cGAS, STING and IFNAR2 to evaluate signalling pathway-dependent ISG induction. Results IFN-I bioactivity and ISG-inducing activities of serum were higher in patients with SLE than in patients with other autoimmune diseases or healthy controls. ISG-inducing activity of SLE sera was significantly reduced in STING-knockout reporter cells, and STING-dependent ISG-inducing activity correlated with disease activity. Double-stranded DNA levels were elevated in SLE. Apoptosis-derived membrane vesicles (AdMVs) from SLE sera had high ISG-inducing activity, which was diminished in cGAS-knockout or STING-knockout reporter cells. Conclusions AdMVs in SLE serum induce IFN-I production through activation of the cGAS–STING pathway. Thus, blockade of the cGAS–STING axis represents a promising therapeutic target for SLE. Moreover, our cell-based reporter system may be useful for stratifying patients with SLE with high ISG-inducing activity.

Lamtor1 Is Critically Required for CD4+ T Cell Proliferation and Regulatory T Cell Suppressive Function

Mechanistic target of rapamycin complex (mTORC)1 integrates intracellular sufficiency of nutrients and regulates various cellular functions. Previous studies using mice with conditional knockout of mTORC1 component proteins (i.e., mTOR, Raptor, and Rheb) gave conflicting results on the roles of mTORC1 in CD4+ T cells. Lamtor1 is the protein that is required for amino acid sensing and activation of mTORC1; however, the roles of Lamtor1 in T cells have not been investigated. In this article, we show that Lamtor1-deficient CD4+ T cells exhibited marked reductions in proliferation, IL-2 production, mTORC1 activity, and expression of purine- and lipid-synthesis genes. Polarization of Th17 cells, but not Th1 and Th2 cells, diminished following the loss of Lamtor1. Accordingly, CD4-Cre–driven Lamtor1-knockout mice exhibited reduced numbers of CD4+ and CD8+ T cells at rest, and they were completely resistant to experimental autoimmune encephalomyelitis. In contrast, genetic ablation of Lamtor1 in Foxp3+ T cells resulted in severe autoimmunity and premature death. Lamtor1-deficient regulatory T cells survived ex vivo as long as wild-type regulatory T cells; however, they exhibited a marked loss of suppressive function and expression of signature molecules, such as CTLA-4. These results indicate that Lamtor1 plays essential roles in CD4+ T cells. Our data suggest that Lamtor1 should be considered a novel therapeutic target in immune systems.

Double deletion of tetraspanins CD9 and CD81 in mice leads to a syndrome resembling accelerated aging

Chronic obstructive pulmonary disease (COPD) has been recently characterized as a disease of accelerated lung aging, but the mechanism remains unclear. Tetraspanins have emerged as key players in malignancy and inflammatory diseases. Here, we found that CD9/CD81 double knockout (DKO) mice with a COPD-like phenotype progressively developed a syndrome resembling human aging, including cataracts, hair loss, and atrophy of various organs, including thymus, muscle, and testis, resulting in shorter survival than wild-type (WT) mice. Consistent with this, DNA microarray analysis of DKO mouse lungs revealed differential expression of genes involved in cell death, inflammation, and the sirtuin-1 (SIRT1) pathway. Accordingly, expression of SIRT1 was reduced in DKO mouse lungs. Importantly, siRNA knockdown of CD9 and CD81 in lung epithelial cells additively decreased SIRT1 and Foxo3a expression, but reciprocally upregulated the expression of p21 and p53, leading to reduced cell proliferation and elevated apoptosis. Furthermore, deletion of these tetraspanins increased the expression of pro-inflammatory genes and IL-8. Hence, CD9 and CD81 might coordinately prevent senescence and inflammation, partly by maintaining SIRT1 expression. Altogether, CD9/CD81 DKO mice represent a novel model for both COPD and accelerated senescence.