Yohei Maeda

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.

A point mutation in Semaphorin 4A associates with defective endosomal sorting and causes retinal degeneration

Semaphorin 4A (Sema4A) has an essential role in photoreceptor survival. In humans, mutations in Sema4A are thought to contribute to retinal degenerative diseases. Here we generate a series of knock-in mouse lines with corresponding mutations (D345H, F350C or R713Q) in the Sema4A gene and find that Sema4AF350C causes retinal degeneration phenotypes. The F350C mutation results in abnormal localization of the Sema4A protein, leading to impaired endosomal sorting of molecules indispensable for photoreceptor survival. Additionally, protein structural modelling reveals that the side chain of the 350th amino acid is critical to retain the proper protein conformation. Furthermore, Sema4A gene transfer successfully prevents photoreceptor degeneration in Sema4AF350C/F350C and Sema4A−/− mice. Thus, our findings not only indicate the importance of the Sema4A protein conformation in human and mouse retina homeostasis but also identify a novel therapeutic target for retinal degenerative diseases.

An Inhibitory Role for Sema4A in Antigen-Specific Allergic Asthma

PurposeThe class IV semaphorin Sema4A is critical for efficient TH1 differentiation and Sema4a−/− mice exhibit impaired TH1 immune responses. However, the role of Sema4A in TH2 cell-mediated allergic diseases has not been fully studied. The aim of this study was to clarify the regulatory role possessed by Sema4A in mouse models of allergic diseases, particularly allergic asthma.MethodsSema4a−/− mice on a BALB/c background were examined for the development of allergic diseases. To induce experimental asthma, mice were sensitized with ovalbumin (OVA) followed by intranasal challenges with OVA. After challenge, airway hyperreactivity (AHR) and airway inflammation were evaluated. The role of Sema4A in asthma was examined using Sema4a−/− mice and Sema4A-Fc fusion proteins. The direct effects of Sema4A-Fc on antigen-specific effector CD4+ T cells were also examined.ResultsA fraction of Sema4a−/− BALB/c mice spontaneously developed skin lesions that resembled atopic dermatitis (AD) in humans. Furthermore, AHR, airway inflammation, and TH2-type immune responses were enhanced in Sema4a−/− mice compared to wild type (WT) mice when immunized and challenged with OVA. In vivo systemic administration of Sema4A-Fc during the challenge period ameliorated AHR and lung inflammation and reduced the production of TH2-type cytokines in WT mice. The inhibitory effects of Sema4A on airway inflammation were also observed in mice deficient in Tim-2, a Sema4A receptor. Finally, we showed that Sema4A-Fc directly inhibited IL-4-producing OVA-specific CD4+ T cells.ConclusionThese results demonstrate that Sema4A plays an inhibitory role in TH2-type allergic diseases, such as allergic asthma.

mTOR Complex Signaling through the SEMA4A–Plexin B2 Axis Is Required for Optimal Activation and Differentiation of CD8+ T Cells

Mammalian target of rapamycin (mTOR) plays crucial roles in activation and differentiation of diverse types of immune cells. Although several lines of evidence have demonstrated the importance of mTOR-mediated signals in CD4+ T cell responses, the involvement of mTOR in CD8+ T cell responses is not fully understood. In this study, we show that a class IV semaphorin, SEMA4A, regulates CD8+ T cell activation and differentiation through activation of mTOR complex (mTORC) 1. SEMA4A−/− CD8+ T cells exhibited impairments in production of IFN-γ and TNF-α and induction of the effector molecules granzyme B, perforin, and FAS-L. Upon infection with OVA-expressing Listeria monocytogenes, pathogen-specific effector CD8+ T cell responses were significantly impaired in SEMA4A−/− mice. Furthermore, SEMA4A−/− CD8+ T cells exhibited reduced mTORC1 activity and elevated mTORC2 activity, suggesting that SEMA4A is required for optimal activation of mTORC1 in CD8+ T cells. IFN-γ production and mTORC1 activity in SEMA4A−/− CD8+ T cells were restored by administration of recombinant Sema4A protein. In addition, we show that plexin B2 is a functional receptor of SEMA4A in CD8+ T cells. Collectively, these results not only demonstrate the role of SEMA4A in CD8+ T cells, but also reveal a novel link between a semaphorin and mTOR signaling.

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.

Organosilica Membrane with Ionic Liquid Properties for Separation of Toluene/H2 Mixture

In this study, we present a new concept in chemically stabilized ionic liquid membranes: an ionic liquid organosilica (ILOS) membrane, which is an organosilica membrane with ionic liquid-like properties. A silylated ionic liquid was used as a precursor for synthesis. The permselectivity, permeation mechanism, and stability of the membrane in the H2/toluene binary system were then compared with a supported ionic liquid membrane. The membrane showed a superior separation factor of toluene/H2 (>17,000) in a binary mixture system based on a solution–diffusion mechanism with improved durability over the supported ionic liquid membrane.

Validation of Noninvasive Morphological and Diffusion Imaging in Mouse Emphysema by Micro–Computed Tomography and Hyperpolarized 129Xe Magnetic Resonance Imaging

Animal disease models are pivotal in investigating the pathogenesis of emphysema and developing novel drugs, but the modalities to evaluate murine emphysema models have been of limited validity and sensitivity. In this study, we evaluated hyperpolarized (129)Xe magnetic resonance imaging (MRI) and micro-computed tomography (micro-CT) compared with traditional methods, such as plethysmography and histology. Elastase-treated mice and adiponectin knockout mice were used as murine emphysema models to evaluate these modalities. Three weeks after elastase administration, significant and heterogeneous emphysema was evaluated according to the mean linear intercept and plethysmography parameters. Notably, the distribution of low-density areas, as examined by micro-CT, correlated with the mean linear intercept and plethysmography parameters in whole lungs. These correlations were also observed in regional areas. Furthermore, we introduced hyperpolarized (129)Xe MRI, which can evaluate gas exchange between the alveoli and blood during spontaneous breathing. Parameters of gas exchange (fD) and alveolar size (Vs/Va) were significantly decreased in elastase-treated mice, and moderately correlated with the plethysmography parameters. Of importance, we could detect a decrease of the fD value in low-density areas with micro-CT, suggesting that gas exchange decreased in emphysematous lesions. Likewise, these parameters (fD and Vs/Va) were also decreased in adiponectin knockout mice, which exhibit emphysema with a homogeneous distribution. We demonstrated the feasibility of (129)Xe MRI and micro-CT in combination with traditional modalities. These noninvasive modalities provide complementary data that can be used for repeated estimations of regional gas exchange and lung morphology.

AB0114 Semaphorin3a and semaphorin4d in rheumatoid arthritis.

Background Semaphorins are involved in a wide range of biological processes, including neuronal axon guidance, angiogenesis, immune modulation and osteogenesis (1, 2). Semaphorin 3A (Sema3A) suppress osteoclast function and increase osteoblast function (3). Semaphorin 4D (Sema4D), which is expressed by osteoclast, suppress osteoblast function (4). Sema4D and 3A might be key mediators of osteoimmunology in rheumatoid arthritis (RA), which have immune activation associated bone destruction. Objectives To determine the pathogenic role of Sema3A and Sema4D in RA, we evaluated the serum level of Sema3A and Sema4D in RA patients. Methods Concentrations of Sema3A and Sema4D in 102 RA patients and 26 normal controls were measured by ELISA (Mybiosource). Results The serum concentrations of Sema4D in RA patients and normal controls were 11.16±8.49 and 5.87±3.10ng/ml respectively (p<0.00001). The serum concentrations of Sema 3A in RA patients and normal controls were 8.97±3.71 and 6.65±3.75 ng/ml respectively (p<0.01). Serum concentrations of Sema4D in RA patients were associated with disease activity (DAS28 r=0.38, p<0.01, CRP r=0.34, p<0.01), rheumatoid factor (RF) (r=0.33, p<0.01) and marker of bone metabolism (urine deoxypyridinoline r=0.32 p<0.05). However serum Sema3A didn’t associated with disease activity and marker of bone metabolism. Image/graph Conclusions Serum concentration of Sema3A and Sema4D were elevated in RA patients. In particular Sema4D showed association with disease activity, RF, and marker of bone metabolism. Semaphorins may play an important role in RA by immune activation, angiogenesis, increasing primary afferent sensory fibers with sympathetic nerve fiber repulsion in synovium, and bone destruction in joints. References Kumanogoh A et al. Immunity. 2000;13:621-31. Takamatsu H et al. Nat Immunol. 2010;11:594-600. Negishi-Koga T et al. Nat Med. 2011;17:1473-80. Hayashi M et al. Nature. 2012;485:69-74. Disclosure of Interest None Declared

Allergic conversion of protective mucosal immunity against nasal bacteria in patients with chronic rhinosinusitis with nasal polyposis

Background Chronic rhinosinusitis with nasal polyposis (CRSwNP) is characterized by eosinophilic inflammation and polyposis at the nose and paranasal sinus and a high concentration of IgE in nasal polyps (NPs). The causative antigen and pathogenesis of CRSwNP remain unknown. Objective We aimed to identify reactive allergens of IgE antibodies produced locally in NPs of patients with CRSwNP. We also attempted to unravel the differentiation pathway of IgE‐producing B cells in NPs. Methods IgE reactivity of patients with CRSwNP was investigated by characterizing single cell–derived mAbs. T‐cell response against identified allergens was investigated in vitro. NP‐infiltrating lymphocytes were characterized by using flow cytometry. Immunoglobulins expressed in NPs were analyzed by using high‐throughput DNA sequencing for immunoglobulin. Results About 20% of isolated IgE antibodies derived from NP‐residing plasmablasts specifically recognized surface determinants of nasal bacteria, such as Staphylococcus aureus, Streptococcus pyogenes, and Haemophilus influenzae. A TH2 response against S pyogenes was observed in patients with CRSwNP. Flow cytometric analysis revealed sizable germinal center B–like cell and plasmablast subsets expressing IgE on the cell surface in NPs. High‐throughput DNA sequencing immunoglobulin analysis highlighted the clonal connectivity of IgE with IgG and IgA1. The I&egr;‐C&agr;1 circle transcript was detected in NPs. Conclusions In patients with CRSwNP, nasal bacteria–reactive B cells differentiate into IgE‐producing B cells through IgG/IgA1‐IgE class switching, suggesting that allergic conversion of the mucosal response against nasal bacteria underlies disease pathogenesis. Graphical abstract Figure. No Caption available.

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.