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This review explores the transformative impact of omics technologies on allergy and asthma research in recent years, focusing on advancements in high-throughput technologies related to genomics and transcriptomics. In particular, the rapid spread of single-cell RNA sequencing has markedly advanced our understanding of the molecular pathology of allergic diseases. Furthermore, high-throughput genome sequencing has accelerated the discovery of monogenic disorders that were previously overlooked as ordinary intractable allergic diseases. We also introduce microbiomics, proteomics, lipidomics, and metabolomics, which are quickly growing areas of research interest, although many of their current findings remain inconclusive as solid evidence. By integrating these omics data, we will gain deeper insights into disease mechanisms, leading to the development of precision medicine approaches that promise to enhance treatment outcomes., Competing Interests: Disclosure statement Supported in part by the Japanese Agency for Medical Research and Development (AMED) (grant JP24ek0410106 [to H.M.]) and a Health, Labor and Welfare Sciences Research Grant (grant 24FE2002 [to H.M.]). Disclosure of potential conflict of interest: H. Saito reports having received a consultation fee from Teikoku Seiyaku, Co, Ltd. H. Morita reports consulting and/or advisory board agreements with Sanofi, LEO Pharma, and Otsuka Pharmaceutical Co, Ltd. The rest of the authors declare that they have no relevant conflicts of interest., (Copyright © 2024 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Pulmonary arterial hypertension (PAH) is characterized by the severe obstruction of the small pulmonary arteries and concomitant high pulmonary arterial pressure, resulting in progressive right ventricular failure. Previously, we demonstrated that long-term interleukin (IL)-33 administration in mice induces severe occlusive medial hypertrophy of pulmonary arteries (PA) in the lungs, which is mediated by group 2 innate lymphoid cells (ILC2s). In response to IL-33, ILC2s accumulate around the blood vessels and produce IL-5, leading to perivascular eosinophil recruitment. In this study, we characterized IL-33-induced medial hypertrophy of PA. We demonstrated that long-term IL-33 administration causes an increase in right ventricular pressure. In IL-33-deficient mice, medial hypertrophy of PA mediated by eggs of Schistosoma mansoni was attenuated, accompanied by a partial reduction in ILC2s, eosinophils, and CD4 + T cells. In addition, proteomic analysis revealed dramatic changes in the urine samples from mice treated with IL-33 or S. mansoni eggs. Resistin-like alpha (RELMα), a pulmonary hypertension-related molecule, was commonly detected in the urine in both treatments. Large amounts of RELMα were observed in the lungs of the IL-33-treated mice. These observations suggest that IL-33-induced medial hypertrophy of PA is a useful model for studying the mechanism underlying the development of PAH and finding biomarkers to indicate the onset of PAH., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Masashi Ikutani reports financial support was provided by Hiroshima University. Shoichi Shimizu reports financial support was provided by University of Occupational and Environmental Health, Japan. Koki Okada reports financial support was provided by Hiroshima University. Koshi Imami reports financial support was provided by RIKEN. Tadakatsu Inagaki reports financial support was provided by National Cerebral and Cardiovascular Center Research Institute. Yoshikazu Nakaoka reports financial support was provided by National Cerebral and Cardiovascular Center Research Institute. Yoshio Osada reports financial support was provided by University of Occupational and Environmental Health, Japan. Susumu Nakae reports financial support was provided by Hiroshima University., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Interleukin-7 (IL-7) is a cytokine critical for the development and maintenance of group 2 innate lymphoid cells (ILC2s). ILC2s are resident in peripheral tissues such as the intestine and lung. However, whether IL-7 produced in the lung plays a role in the maintenance and function of lung ILC2s during airway inflammation remains unknown. IL-7 was expressed in bronchoalveolar epithelial cells and lymphatic endothelial cells (LECs). To investigate the role of local IL-7 in lung ILC2s, we generated two types of IL-7 conditional knockout (IL-7cKO) mice: Sftpc-Cre (SPC-Cre) IL-7cKO mice specific for bronchial epithelial cells and type 2 alveolar epithelial cells and Lyve1-Cre IL-7cKO mice specific for LECs. In steady state, ILC2s were located near airway epithelia, although lung ILC2s were unchanged in the two lines of IL-7cKO mice. In papain-induced airway inflammation dependent on innate immunity, lung ILC2s localized near bronchia via CCR4 expression, and eosinophil infiltration and type 2 cytokine production were reduced in SPC-Cre IL-7cKO mice. In contrast, in house dust mite (HDM)-induced airway inflammation dependent on adaptive immunity, lung ILC2s localized near lymphatic vessels via their CCR2 expression 2 weeks after the last challenge. Furthermore, lung ILC2s were decreased in Lyve1-Cre IL-7cKO mice in the HDM-induced inflammation because of decreased cell survival and proliferation. Finally, administration of anti-IL-7 antibody attenuated papain-induced inflammation by suppressing the activation of ILC2s. Thus, this study demonstrates that IL-7 produced by bronchoalveolar epithelial cells and LECs differentially controls the activation and maintenance of lung ILC2s, where they are localized in airway inflammation., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Japanese Society for Immunology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Severely immunodeficient mice are useful for understanding the pathogenesis of certain tumors and for developing therapeutic agents for such tumors. In addition, engraftment of these mice with human hematopoietic cells can yield information that helps us understand the in vivo molecular mechanisms underlying actual human viral infections. In our present research, we discovered a novel, severely immunodeficient strain of mice having a mutation in exon 57 of the Prkdc gene (Prkdc Δex57/Δex57 ) in an inbred colony of B10.S/SgSlc mice. Those Prkdc Δex57/Δex57 mice showed thymic hypoplasia and lack of mature T cells and B cells in peripheral lymphoid tissues, resulting in very low levels of production of serum immunoglobulins. In addition, those mice were highly susceptible to influenza viruses due to the lack of acquired immune cells. On the other hand, since they had sufficient numbers of NK cells, they rejected tumor transplants, similarly to Prkdc +/+ mice. Next, we generated Foxn1 nu/nu Prkdc Δex57/Δex57 Il2rg -/- (NPG) mice on the BALB/cSlc background, which lack all lymphocytes such as T cells, B cells and innate lymphoid cells, including NK cells. As expected, these mice were able to undergo engraftment of human tumor cell lines. These findings suggest that Prkdc Δex57/Δex57 mice will be useful as a novel model of immunodeficiency, while NPG mice will be useful for xenografting of various malignancies., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Yumie Takagi reports financial support was provided by Japan SLC, Inc.Katsuko Sudo reports financial support was provided by Tokyo Medical University.Sachiko Yamaguchi, Masashi Ikutani and Susumu Nakae report financial support was provided by Hiroshima University.Shuzo Urata and Jiro Yasuda report financial support was provided by Nagasaki University.Tatsukuni Ohno reports financial support was provided by Tokyo Dental College.Sachiko Hirose reports financial support was provided by Toin University.Kiyoshi Matsumoto reports financial support was provided by Shinshu University.Takeshi Kuramoto reports financial support was provided by Tokyo University of Agriculture.Tadao Serikawa reports financial support was provided by Kyoto University., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Epidemiological studies demonstrated that cleaning work and frequent use of cleaning products are risk factors for asthma. Laundry detergents have been reported to have epithelial barrier-opening effects. However, whether laundry detergents directly induce airway inflammation and its mechanisms in vivo remain to be elucidated., Methods: Two commercial laundry detergents and two commonly used surfactants for cleaning and cosmetics (sodium lauryl sulfate and sodium dodecyl benzene sulfonate) were intranasally administered to mice. Lungs were analyzed using flow cytometry, histology, ELISA, and quantitative PCR. Human bronchial epithelial cells were stimulated with laundry detergents and analyzed using quantitative PCR and western blotting. Involvement of oxidative stress was assessed using an antioxidant. Dust samples from homes were analyzed to determine their detergent content by measuring their critical micelle concentration (CMC)., Results: The administered laundry detergents and surfactants-induced eosinophilic airway inflammation accompanied by increased IL-33 expression and activation of group 2 innate lymphoid cells (ILC2s). Detergent-induced eosinophilic airway inflammation was significantly attenuated in Rag2 -/- Il2rg -/- , Il33 -/- mice, and also in wild-type mice treated with NAC. Detergent-induced IL-33 expression in airways was attenuated by NAC treatment, both in vivo and in vitro. CMCs were found in all of the tested dust extracts, and they differed significantly among the homes., Conclusion: The laundry detergents and surfactants-induced eosinophilic airway inflammation in vivo through epithelial cell and ILC2 activation. They induced IL-33 expression in airway epithelial cells through oxidative stress. Furthermore, detergent residues were present in house dust and are presumably inhaled into the airway in daily life., (© 2023 European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by type 2 immune responses. Interleukin-25 (IL-25) is produced predominantly by epithelial cells. It can activate Th2 cells to produce type 2 cytokines such as IL-4, IL-5 and IL-13, contributing to host defense against nematodes. However, excessive/inappropriate production of IL-25 is considered to be involved in development of type 2 cytokine-associated allergic disorders such as asthma. On the other hand, the contribution of IL-25 to the pathogenesis of AD remains poorly understood. In the present study, we found that expression of Il25 mRNA was significantly increased in the skin of mice during oxazolone-induced chronic contact hypersensitivity (CHS), which is a mouse model of human AD. In addition, development of oxazolone-induced chronic CHS was significantly reduced in IL-25-deficient (Il25 -/- ) mice compared with wild-type mice on the C57BL/6, but not BALB/c, background, although IL-25 was not essential for IL-4 production by hapten-specific T cells. Therefore, IL-25 is crucial for development of chronic CHS, although that is partly dependent on the genetic background of the mice., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Susumu Nakae reports financial support was provided by Hiroshima University. Eri Shimura reports financial support was provided by Juntendo University. Hajime Suto reports financial support was provided by Juntendo University. Takafumi Numata reports financial support was provided by Tokyo Medical University. Sachiko Yamaguchi reports financial support was provided by Hiroshima University. Kazutoshi Harada reports financial support was provided by Tokyo Medical University. Ko Okumura reports financial support was provided by Juntendo University. Katsuko Sudo reports financial support was provided by Tokyo Medical University. Masashi Ikutani reports financial support was provided by Hiroshima University., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Group 2 innate lymphoid cells (ILC2s) are typically known for their ability to respond rapidly to parasitic infections and play a pivotal role in the development of certain allergic disorders. ILC2s produce cytokines such as Interleukin (IL)-5 and IL-13 similar to the type 2 T helper (Th2) cells. Recent findings have highlighted that ILC2s, together with IL-33 and eosinophils, participate in a considerably broad range of physiological roles such as anti-tumor immunity, metabolic regulation, and vascular disorders. Therefore, the focus of the ILC2 study has been extended from conventional Th2 responses to these unexplored areas of research. However, disease outcomes accompanied by ILC2 activities are paradoxical mostly in tumor immunity requiring further investigations. Although various environmental factors that direct the development, activation, and localization of ILC2s have been studied, IL-33/ILC2/eosinophil axis is presumably central in a multitude of inflammatory conditions and has guided the research in ILC2 biology. With a particular focus on this axis, we discuss ILC2s across different diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ikutani and Nakae.)

Background: Platelets are thought to be involved in the pathophysiology of asthma, presumably through direct adhesion to inflammatory cells, including group 2 innate lymphoid cells (ILC2s). Here, we tried to elucidate the effects of platelet adhesion to ILC2s in vitro and in vivo, as well as the mechanisms involved., Methods: Alternaria-induced ILC2-dependent airway inflammation models using wild-type and c-mpl -/- mice were evaluated. Both purified CD41 + and CD41 - ILC2s were cultured with IL-2 and IL-33 to determine in vitro Type 2 (T2) cytokine production and cell proliferation. RNA-seq data of flow-cytometry-sorted CD41 + and CD41 - ILC2s were used to isolate ILC2-specific genes. Flow cytometry was performed to determine the expression of CD41 and adhesion-related molecules on ILC2s in both mouse and human tissues., Results: T2 inflammation and T2 cytokine production from ILC2s were significantly reduced in the c-mpl -/- mice compared to wild-type mice. Platelet-adherent ILC2s underwent significant proliferation and showed enhanced T2 cytokine production when exposed to IL-2 and IL-33. The functions of ILC2-specific genes were related to cell development and function. Upstream regulator analysis identified 15 molecules, that are thought to be involved in ILC2 activation. CD41 expression levels were higher in ILC2s from human PBMCs and mouse lung than in those from secondary lymphoid tissues, but they did not correlate with the P-selectin glycoprotein ligand-1 or CD24 expression level., Conclusion: Platelets spontaneously adhere to ILC2s, probably in the peripheral blood and airways, thereby potentiating ILC2s to enhance their responses to IL-33., (© 2021 European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Transforming growth factor (TGF)-β1 and prostaglandin E 2 (PGE 2 ) are humoral factors critically involved in the induction of immunosuppression in the microenvironment of various types of tumors, including melanoma. In this study, we identified a natural compound that attenuated TGF-β1- and PGE 2 -induced immunosuppression and examined its effect on B16 melanoma growth in mice. By screening 502 natural compounds for attenuating activity against TGF-β1- or PGE 2 -induced suppression of cytolysis in poly(I:C)-stimulated murine splenocytes, we found that betulin was the most potent compound. Betulin also reduced TGF-β1- and PGE 2 -induced downregulation of perforin and granzyme B mRNA expression and cell surface expression of NKG2D and CD69 in natural killer (NK) cells. Cell depletion and coculture experiments showed that NK cells, dendritic cells, B cells, and T cells were necessary for the attenuating effects of betulin. Structure-activity relationship analysis revealed that two hydroxyl groups at positions C3 and C28 of betulin, their cis-configuration, and methyl group at C30 played crucial roles in its attenuating activity. In a subcutaneous implantation model of B16 melanoma in mice, intratumor administration of betulin and LY2157299, a TGF-β1 type I receptor kinase inhibitor, significantly retarded the growth of B16 melanoma. Notably, betulin increased significantly the number of CD69 positive NK cells in tumor sites at early stages of post-tumor cell injection. Our data suggest that betulin inhibits the growth of B16 melanoma by enhancing NK cell activity through attenuating the immunosuppressive tumor microenvironment.

Exposure to various antigens derived from house dust mites (HDM) is considered to be a risk factor for development of certain allergic diseases such as atopic asthma, atopic dermatitis, rhinitis and conjunctivitis. Chitin is an insoluble polysaccharide (β-(1-4)-poly-N-acetyl-D-glucosamine) and a major component in the outer shell of HDMs. Mice exposed to chitin develop asthma-like airway eosinophilia. On the other hand, several lines of evidence show that the effects of chitin on immune responses are highly dependent on the size of chitin particles. In the present study, we show that chitin induced production of IL-33 and TSLP by alveolar and bronchial epithelial cells, respectively, in mice. IL-25, IL-33 and TSLP were reported to be important for group 2 innate lymphoid cell (ILC2)-, but not Th2 cell-, dependent airway eosinophilia in a certain model using chitin beads. Here, we show that-in our murine models-epithelial cell-derived IL-33 and TSLP, but not IL-25, were crucial for activation of resident lung Th2 cells as well as group 2 innate lymphoid cells (ILC2s) to produce IL-5, resulting in development of chitin-induced airway eosinophilia. Our findings provide further insight into the underlying mechanisms of development of HDM-mediated allergic disorders.

Silica crystals (silica), which are a major mineral component of volcanic ash and desert dust, contribute to the pathogenesis of pulmonary disorders such as asthma and fibrosis. Although administration of silica or sand dust to rodents exacerbates development of ovalbumin-induced or house dust mite-induced asthma-like airway inflammation, the detailed mechanisms remain unclear. Here, using murine models, we found that silica can induce IL-33 expression in pulmonary epithelial cells. IL-33, but not IL-25 or TSLP, and type 2 cytokines such as IL-5 and IL-13 were critically involved in silica's exacerbation of OVA-induced airway eosinophilia in mice. Innate lymphoid cells (ILCs), but not T, B or NKT cells, were also involved in the setting. Moreover, a scavenger receptor that recognized silica was important for silica's exacerbating effect. These observations suggest that IL-33 induced in epithelial cells by silica activates ILCs to produce IL-5 and/or IL-13, contributing to silica's exacerbation of OVA-induced airway eosinophilia in mice. Our findings provide new insight into the underlying mechanisms of exacerbation of pulmonary disorders such as asthma following inhalation of silica-containing materials such as volcanic ash and desert dust., Competing Interests: Declaration of competing interest The authors declare that they have no known competing interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The function of regulatory immune cells in peripheral tissues is crucial to the onset and severity of various diseases. Interleukin-10 (IL-10)-producing regulatory B (IL-10 + B reg ) cells are known to suppress various inflammatory diseases. However, evidence for the mechanism by which IL-10 + B reg cells are generated and maintained is still very limited. Here, we found that IL-10 + B reg cells suppress the activation of IL-13-producing type 2 innate lymphoid cells (IL-13 + ILC2s) in an IL-10-dependent manner in mice with oxazolone-induced severe contact hypersensitivity (CHS). Mast cell (MC) IL-5 was important for maintaining the population of IL-10 + B reg cells in peripheral lymphoid tissues. Overall, these results uncover a previously unknown mechanism of MCs as a type of immunoregulatory cell and elucidate the cross-talk among MCs, IL-10 + B reg cells, and IL-13 + ILC2s in CHS.

Interleukin (IL)-5 is a critical regulator of eosinophils and a therapeutic target for asthma. The administration of anti-IL-5 or anti-IL-5 receptor (IL-5R) antibodies has been shown to reduce eosinophil counts and ameliorate asthmatic symptoms in studies on animal models of allergy as well as in human clinical trials. In order to explore other potential clinical uses of IL-5R antibodies, we used an animal model of IL-33-mediated pulmonary arterial hypertrophy. We first generated chimeric monoclonal antibodies against the mouse IL-5 receptor α chain (IL-5Rα), which comprised an Fc region from human IgG1 and a Fab region from a previously established anti-mouse IL-5Rα monoclonal antibody. To investigate the role of antibody-dependent cell-mediated cytotoxicity (ADCC), chimeric antibodies that lacked ADCC were prepared. These antibodies recognized IL-5Rα to the same extent as the ADCC-sufficient antibodies. Administration of chimeric antibodies with ADCC resulted in the elimination of eosinophils from the lung and thus suppressed the development of arterial hypertrophy. This effect was attenuated in mice treated with antibodies lacking ADCC. Taken together, the results of this study provided a potential use for anti-IL-5Rα antibodies in the treatment of arterial hypertrophy, which leads to pulmonary hypertension., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Cyclosporin A (CsA) is a well-known immunosuppressant that is used against steroid-resistant asthma. Group 2 innate lymphoid cells (ILC2s) and type 2 helper T (Th2) cells produce Th2 cytokines including IL-5 and play important roles in asthma pathogenesis. Here, we studied the effects of CsA in allergen-induced lung inflammation in mice and found that CsA decreased the number of lung ILC2s and attenuated papain-induced activation of ILC2s accompanied with IL-5 expression. The ILC2 suppression mediated by CsA was not observed in culture or in lymphocyte-deficient Rag2 -/- mice. Thus, we propose a new suppressive effect of CsA, i.e., administration of CsA indirectly suppresses maintenance and activation of lung ILC2s in addition to direct suppression of T-cell activation and cytokine production., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Adipose tissue resident macrophages have important roles in the maintenance of tissue homeostasis and regulate insulin sensitivity for example by secreting pro-inflammatory or anti-inflammatory cytokines. Here, we show that M2-like macrophages in adipose tissue regulate systemic glucose homeostasis by inhibiting adipocyte progenitor proliferation via the CD206/TGFβ signaling pathway. We show that adipose tissue CD206 + cells are primarily M2-like macrophages, and ablation of CD206 + M2-like macrophages improves systemic insulin sensitivity, which was associated with an increased number of smaller adipocytes. Mice genetically engineered to have reduced numbers of CD206 + M2-like macrophages show a down-regulation of TGFβ signaling in adipose tissue, together with up-regulated proliferation and differentiation of adipocyte progenitors. Our findings indicate that CD206 + M2-like macrophages in adipose tissues create a microenvironment that inhibits growth and differentiation of adipocyte progenitors and, thereby, control adiposity and systemic insulin sensitivity.Adipose tissue contains macrophages that can influence both local and systemic metabolism via the secretion of cytokines. Here, Nawaz et al. report that M2-like macrophages, present in adipose tissue, create a microenvironment that inhibits proliferation of adipocyte progenitors due to the secretion of TGF-β1.

Pericytes are believed to originate from either mesenchymal or neural crest cells. It has recently been reported that pericytes play important roles in the central nervous system (CNS) by regulating blood-brain barrier homeostasis and blood flow at the capillary level. However, the origin of CNS microvascular pericytes and the mechanism of their recruitment remain unknown. Here, we show a new source of cerebrovascular pericytes during neurogenesis. In the CNS of embryonic day 10.5 mouse embryos, CD31 + F4/80 + hematopoietic lineage cells were observed in the avascular region around the dorsal midline of the developing midbrain. These cells expressed additional macrophage markers such as CD206 and CD11b. Moreover, the CD31 + F4/80 + cells phagocytosed apoptotic cells as functionally matured macrophages, adhered to the newly formed subventricular vascular plexus, and then divided into daughter cells. Eventually, these CD31 + F4/80 + cells transdifferentiated into NG2/PDGFRβ/desmin-expressing cerebrovascular pericytes, enwrapping and associating with vascular endothelial cells. These data indicate that a subset of cerebrovascular pericytes derive from mature macrophages in the very early phase of CNS vascular development, which in turn are recruited from sites of embryonic hematopoiesis such as the yolk sac by way of blood flow.

IL-33 is one of the critical cytokines that activates group 2 innate lymphoid cells (ILC2s) and mediates allergic reactions. Accumulating evidence suggests that IL-33 is also involved in the pathogenesis of several chronic inflammatory diseases. Previously, we generated an IL-5 reporter mouse and revealed that lung IL-5-producing ILC2s played essential roles in regulating eosinophil biology. In this study, we evaluated the consequences of IL-33 administration over a long period, and we observed significant expansion of ILC2s and eosinophils surrounding pulmonary arteries. Unexpectedly, pulmonary arteries showed severe occlusive hypertrophy that was ameliorated in IL-5- or eosinophil-deficient mice, but not in Rag2-deficient mice. This indicates that IL-5-producing ILC2s and eosinophils play pivotal roles in pulmonary arterial hypertrophy. Administration of a clinically used vasodilator was effective in reducing IL-33-induced hypertrophy and repressed the expansion of ILC2s and eosinophils. Taken together, these observations demonstrate a previously unrecognized mechanism in the development of pulmonary arterial hypertrophy and the causative roles of ILC2 in the process., Competing Interests: Conflict of interest: JF is the representative director of Pathology Institute Corp and owns its stocks.

Adipose tissue hypoxia is an important feature of pathological adipose tissue expansion. Hypoxia-inducible factor-1α (HIF-1α) in adipocytes reportedly induces oxidative stress and fibrosis, rather than neoangiogenesis via vascular endothelial growth factor (VEGF)-A. We previously reported that macrophages in crown-like structures (CLSs) are both hypoxic and inflammatory. In the current study, we examined how macrophage HIF-1α is involved in high-fat diet (HFD)-induced inflammation, neovascularization, hypoxia, and insulin resistance using mice with myeloid cell-specific HIF-1α deletion that were fed an HFD. Myeloid cell-specific HIF-1α gene deletion protected against HFD-induced inflammation, CLS formation, poor vasculature development in the adipose tissue, and systemic insulin resistance. Despite a reduced expression of Vegfa in epididymal white adipose tissue (eWAT), the preadipocytes and endothelial cells of HIF-1α-deficient mice expressed higher levels of angiogenic factors, including Vegfa, Angpt1, Fgf1, and Fgf10 in accordance with preferable eWAT remodeling. Our in vitro study revealed that lipopolysaccharide-treated bone marrow-derived macrophages directly inhibited the expression of angiogenic factors in 3T3-L1 preadipocytes. Thus, macrophage HIF-1α is involved not only in the formation of CLSs, further enhancing the inflammatory responses, but also in the inhibition of neoangiogenesis in preadipocytes. We concluded that these two pathways contribute to the obesity-related physiology of pathological adipose tissue expansion, thus causing systemic insulin resistance., (© 2016 by the American Diabetes Association.)

Group 2 innate lymphoid cells (ILC2s) produce a significant amount of interleukin-5 (IL-5), which supports eosinophil responses in various tissues; they also produce IL-13, which induces mucus production and contributes to tissue repair or fibrosis. The ILC2s are activated by alarmins, such as IL-33 released from epithelia, macrophages and natural killer T (NKT) cells in response to infection and allergen exposure, leading to epithelial injury. We examined gene expression in lung ILC2s and found that ILC2s expressed Ifngr1, the receptor for interferon-γ (IFN-γ). Interferon-γ severely inhibited IL-5 and IL-13 production by lung and kidney ILC2s. To evaluate the effects in vivo, we used α-galactosylceramide (α-GalCer) to induce NKT cells to produce IL-33 and IFN-γ. Intraperitoneal injection of α-GalCer in mice induced NKT cell activation resulting in IL-5 and IL-13 production by ILC2s. Administration of anti-IFN-γ together with α-GalCer significantly enhanced the production of IL-5 and IL-13 by ILC2s in lung and kidney. Conversely, cytokine production from ILC2s was markedly suppressed after injection of exogenous IL-33 in Il33(-/-) mice pre-treated with α-GalCer. Hence, IFN-γ induced or already present in tissues can impact downstream pleiotropic functions mediated by ILC2s, such as inflammation and tissue repair., (© 2015 John Wiley & Sons Ltd.)

Chronic inflammation is a pathophysiology of insulin resistance in metabolic diseases, such as obesity and type 2 diabetes. Adipose tissue macrophages (ATMs) play important roles in this inflammatory process. SIRT1 is implicated in the regulation of glucose metabolism in some metabolic tissues, such as liver or skeletal muscle. This study was performed to investigate whether SIRT1 in macrophages played any roles in the regulation of inflammation and glucose metabolism. Myeloid cell-specific SIRT1-knockout mice were originally generated and analyzed under chow-fed and high-fat-fed conditions. Myeloid cell-specific SIRT1 deletion impaired insulin sensitivity and glucose tolerance assessed by the glucose- or insulin-tolerance test, which was associated with the enhanced expression of inflammation-related genes in epididymal adipose tissue of high-fat-fed mice. Interestingly, the M1 ATMs from the SIRT1-knockout mice showed more hypoxic and inflammatory phenotypes than those from control mice. The expressions of some inflammatory genes, such as Il1b and Nos2 , which were induced by in vitro hypoxia treatment, were further enhanced by SIRT1 deletion along with the increased acetylation of HIF-1α in cultured macrophages. These results suggest that deletion of SIRT1 in myeloid cells impairs glucose metabolism by enhancing the hypoxia and inflammatory responses in ATMs, thereby possibly representing a novel therapeutic target for metabolic diseases, such as type 2 diabetes.

Syntenin-1 is an intracellular PDZ protein that binds multiple proteins and regulates protein trafficking, cancer metastasis, exosome production, synaptic formation, and IL-5 signaling. However, the functions of Syntenin-1 have not yet been clearly characterized in detail, especially in vivo. In this study, we generated a Syntenin-1 knock out (KO) mouse strain and analyzed the role(s) of Syntenin-1 in IL-5 signaling, because the direct interaction of Syntenin-1 with the cytoplasmic domain of the IL-5 receptor α subunit and the regulation of IL-5 signaling by Syntenin-1 have been reported. Unexpectedly, the number of IL-5-responding cells was normal and the levels of fecal immunoglobulins were rather higher in the Syntenin-1 KO mice. We also found that IgA and IgM production of splenic B cells stimulated in vitro was increased in Syntenin-1 KO mice. In addition, we showed that a distribution of intestinal microbial flora was influenced in Syntenin-1 KO mice. Our data indicate that Syntenin-1 negatively regulates the intestinal immunoglobulin production and has a function to maintain the intestinal homeostasis in vivo. The analysis of Syntenin-1 KO mice may provide novel information on not only mucosal immunity but also other functions of Syntenin-1 such as cancer metastasis and neural development., (Copyright © 2014 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Emerging lines of evidence have shown that extracellular vesicles (EVs) mediate cell-to-cell communication by exporting encapsulated materials, such as microRNAs (miRNAs), to target cells. Endothelial cell-derived EVs (E-EVs) are upregulated in circulating blood in different pathological conditions; however, the characteristics and the role of these E-EVs are not yet well understood. In vitro studies were conducted to determine the role of inflammation-induced E-EVs in the cell-to-cell communication between vascular endothelial cells and pericytes/vSMCs. Stimulation with inflammatory cytokines and endotoxin immediately induced release of shedding type E-EVs from the vascular endothelial cells, and flow cytometry showed that the induction was dose dependent. MiRNA array analyses revealed that group of miRNAs were specifically increased in the inflammation-induced E-EVs. E-EVs added to the culture media of cerebrovascular pericytes were incorporated into the cells. The E-EV-supplemented cells showed highly induced mRNA and protein expression of VEGF-B, which was assumed to be a downstream target of the miRNA that was increased within the E-EVs after inflammatory stimulation. The results suggest that E-EVs mediate inflammation-induced endothelial cell-pericyte/vSMC communication, and the miRNAs encapsulated within the E-EVs may play a role in regulating target cell function. E-EVs may be new therapeutic targets for the treatment of inflammatory diseases.

LPS stimulates the TLR4/Myeloid differentiation protein-2 (MD-2) complex and promotes a variety of immune responses in B cells. TLR4 has two main signaling pathways, MyD88 and Toll/IL-1R (TIR)-domain-containing adaptor-inducing interferon-β (TRIF) pathways, but relatively few studies have examined these pathways in B cells. In this study, we investigated MyD88- or TRIF-dependent LPS responses in B cells by utilizing their knockout mice. Compared with wild-type (WT) B cells, MyD88(-/-) B cells were markedly impaired in up-regulation of CD86 and proliferation induced by lipid A moiety of LPS. TRIF(-/-) B cells were also impaired in these responses compared with WT B cells, but showed better responses than MyD88(-/-) B cells. Regarding class switch recombination (CSR) elicited by lipid A plus IL-4, MyD88(-/-) B cells showed similar patterns of CSR to WT B cells. However, TRIF(-/-) B cells showed the impaired in the CSR. Compared with WT and MyD88(-/-) B cells, TRIF(-/-) B cells exhibited reduced cell division, fewer IgG1(+) cells per division, and decreased activation-induced cytidine deaminase (Aicda) mRNA expression in response to lipid A plus IL-4. Finally, IgG1 production to trinitrophenyl (TNP)-LPS immunization was impaired in TRIF(-/-) mice, while MyD88(-/-) mice exhibited increased IgG1 production. Thus, MyD88 and TRIF pathways differently regulate TLR4-induced immune responses in B cells., (Copyright © 2014 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Inflammasome activation initiates the development of many inflammatory diseases, including obesity and type 2 diabetes. Therefore, agents that target discrete activation steps could represent very important drugs. We reported previously that ILG, a chalcone from Glycyrrhiza uralensis, inhibits LPS-induced NF-κB activation. Here, we show that ILG potently inhibits the activation of NLRP3 inflammasome, and the effect is independent of its inhibitory potency on TLR4. The inhibitory effect of ILG was stronger than that of parthenolide, a known inhibitor of the NLRP3 inflammasome. GL, a triterpenoid from G. uralensis, had similar inhibitory effects on NLRP3 activity, but high concentrations of GL were required. In contrast, activation of the AIM2 inflammasome was inhibited by GL but not by ILG. Moreover, GL inhibited NLRP3- and AIM2-activated ASC oligomerization, whereas ILG inhibited NLRP3-activated ASC oligomerization. Low concentrations of ILG were highly effective in IAPP-induced IL-1β production compared with the sulfonylurea drug glyburide. In vivo analyses revealed that ILG potently attenuated HFD-induced obesity, hypercholesterolemia, and insulin resistance. Furthermore, ILG treatment improved HFD-induced macrovesicular steatosis in the liver. Finally, ILG markedly inhibited diet-induced adipose tissue inflammation and IL-1β and caspase-1 production in white adipose tissue in ex vivo culture. These results suggest that ILG is a potential drug target for treatment of NLRP3 inflammasome-associated inflammatory diseases., (© 2014 Society for Leukocyte Biology.)

The intracellular adaptor Lnk (also known as SH2B3) regulates cytokine signals that control lymphohematopoiesis, and Lnk(-/-) mice have expanded B-cell, megakaryocyte, and hematopoietic stem-cell populations. Moreover, mutations in the LNK gene are found in patients with myeloproliferative disease, whereas LNK polymorphisms have recently been associated with inflammatory and autoimmune diseases, including celiac disease. Here, we describe a previously unrecognized function of Lnk in the control of inflammatory CD8(+) T-cell proliferation and in intestinal homeostasis. Mature T cells from newly generated Lnk-Venus reporter mice had low but substantial expression of Lnk, whereas Lnk expression was downregulated during homeostatic T-cell proliferation under lymphopenic conditions. The numbers of CD44(hi) IFN-γ(+) CD8(+) effector or memory T cells were found to be increased in Lnk(-/-) mice, which also exhibited shortening of villi in the small intestine. Lnk(-/-) CD8(+) T cells survived longer in response to stimulation with IL-15 and proliferated even in nonlymphopenic hosts. Transfer of Lnk(-/-) CD8(+) T cells together with WT CD4(+) T cells into Rag2-deficient mice recapitulated a sign of villous abnormality. Our results reveal a link between Lnk and immune cell-mediated intestinal tissue destruction., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

NAD biosynthesis is of substantial interest because of its important roles in regulating various biological processes. Nicotinamide mononucleotide adenylyltransferase 3 (Nmnat3) is considered a mitochondria-localized NAD synthesis enzyme involved in de novo and salvage pathways. Although the biochemical properties of Nmnat3 are well documented, its physiological function in vivo remains unclear. In this study, we demonstrated that Nmnat3 was localized in the cytoplasm of mature erythrocytes and critically regulated their NAD pool. Deficiency of Nmnat3 in mice caused splenomegaly and hemolytic anemia, which was associated with the findings that Nmnat3-deficient erythrocytes had markedly lower ATP levels and shortened lifespans. However, the NAD level in other tissues were not apparently affected by the deficiency of Nmnat3. LC-MS/MS-based metabolomics revealed that the glycolysis pathway in Nmnat3-deficient erythrocytes was blocked at a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) step because of the shortage of the coenzyme NAD. Stable isotope tracer analysis further demonstrated that deficiency of Nmnat3 resulted in glycolysis stall and a shift to the pentose phosphate pathway. Our findings indicate the critical roles of Nmnat3 in maintenance of the NAD pool in mature erythrocytes and the physiological impacts at its absence in mice., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Elicitation of contact hypersensitivity requires antigen-specific immunoglobulin M (IgM) antibodies that trigger recruitment of effector T cells to the local tissue. These antigen-specific IgM antibodies are produced by B-1-like 'initiator B cells'. In this study, we compared susceptibility to hapten-induced contact hypersensitivity between BALB/c and C57BL/6 mice., Methods: BALB/c and C57BL/6 mice were sensitized by painting oxazolone onto the skin and were challenged on the ears with the same hapten on day 4. Ear thickness and serum hapten-specific IgM levels were measured at 24 h post-challenge. Peritoneal cells were harvested and the numbers of B cell subpopulations were counted. Interleukin (IL)-5 was intraperitoneally injected into BALB/c and C57BL/6 mice, and the change in numbers of B cell subpopulations and serum IgM levels was monitored., Results: Oxazolone induced stronger ear swelling and specific IgM responses in BALB/c mice than in C57BL/6 mice. BALB/c mice had higher numbers of peritoneal B-1 cells than C57BL/6 mice at steady state. IL-5 injection increased the number of peritoneal B-1 cells and serum IgM levels more significantly in BALB/ mice than in C57BL/6 mice., Conclusions: BALB/c mice exhibit higher susceptibility to hapten-induced contact hypersensitivity than C57BL/6 mice, most likely because they have a higher number of B-1 cells, leading to massive production of hapten-specific IgM antibodies upon contact sensitization. The differences in the number of B-1 cells and IgM responses between the two strains of mice may be attributed to the difference in responsiveness of B-1 cells to IL-5., (Copyright © 2013 S. Karger AG, Basel.)

Interleukin-5 (IL-5), a major hematopoietin, stimulates eosinophil proliferation, migration, and activation, which have been implicated in the pathogenesis of allergic inflammatory diseases, such as asthma. The specific IL-5 receptor (IL-5R) consists of the IL-5 receptor α subunit (IL-5RA) and the common receptor β subunit (βc). IL-5 binding to IL-5R on target cells induces rapid tyrosine phosphorylation and activation of various cellular proteins, including JAK1/JAK2 and STAT1/STAT5. Here, we report the crystal structure of dimeric IL-5 in complex with the IL-5RA extracellular domains. The structure revealed that IL-5RA sandwiches the IL-5 homodimer by three tandem domains, arranged in a "wrench-like" architecture. This association mode was confirmed for human cells expressing IL-5 and the full-length IL-5RA by applying expanded genetic code technology: protein photo-cross-linking experiments revealed that the two proteins interact with each other in vivo in the same manner as that in the crystal structure. Furthermore, a comparison with the previously reported, partial GM-CSF•GM-CSFRA•βc structure enabled us to propose complete structural models for the IL-5 and GM-CSF receptor complexes, and to identify the residues conferring the cytokine-specificities of IL-5RA and GM-CSFRA., (Copyright © 2012 The Protein Society.)

IL-5 is involved in a number of immune responses such as helminth infection and allergy. IL-5 also plays roles in innate immunity by maintaining B-1 B cells and mucosal IgA production. However, the identity of IL-5-producing cells has not been unambiguously characterized. In this report, we describe the generation of an IL-5 reporter mouse and identify IL-5-producing non-T lymphoid cells that reside in the intestine, peritoneal cavity, and lungs in naive mice. They share many characteristics with natural helper cells, nuocytes, and Ih2 cells, including surface Ags and responsiveness to cytokines. However, these phenotypes do not completely overlap with any particular one of these cell types. Innate non-T IL-5-producing cells localized most abundantly in the lung and proliferated and upregulated IL-5 production in response to IL-25 and IL-33. IL-33 was more effective than IL-25. These cells contribute to maintaining sufficient numbers of lung eosinophils and are important for eosinophil recruitment mediated by IL-25 and IL-33. Given that eosinophils are shown to possess antitumor activity, we studied lung tumor metastasis and showed that innate IL-5-producing cells were increased in response to tumor invasion, and their regulation of eosinophils is critical to suppress tumor metastasis. Genetic blockade or neutralization of IL-5 impaired eosinophil recruitment into the lung and resulted in increased tumor metastasis. Conversely, exogenous IL-5 treatment resulted in suppressed tumor metastasis and augmented eosinophil infiltration. These newly identified innate IL-5-producing cells thus play a role in tumor surveillance through lung eosinophils and may contribute to development of novel immunotherapies for cancer.

MD-1 is a secreted protein that forms a complex with radioprotective 105 (RP105) and this complex plays a crucial role in lipopolysaccharide (LPS) recognition by B cells. Disease progression is known to improve in RP105-deficient lupus-prone MRL(lpr/lpr) mice. Furthermore, a soluble form of the homologous MD-2 protein is present in the plasma of septic patients and can opsonize gram-negative bacteria in cooperation with Toll-like receptor (TLR) 4. We have now established a flow cytometry-based assay to detect the soluble form of murine MD-1 (sMD-1) and explored potential roles in autoimmunity. The assay was quantitative and validated with sera from MD-1-deficient mice. Interestingly, heat-inactivated murine serum diminished the ability of sMD-1 to bind RP105. The sMD-1 was secreted by bone marrow-derived macrophages from C57BL/6 mice. Autoimmune prone MRL(lpr/lpr) mice had higher levels of sMD-1 than control MRL(+/+) mice, and levels markedly increased with disease progression. Expression of MD-1 but not MD-2 mRNA increased with age in the liver and kidney of MRL(lpr/lpr) mice. Finally, immunohistochemical analyses revealed that MD-1 was present in infiltrated macrophages within perivascular lesions of the MRL(lpr/lpr) kidney. This correlation suggests that sMD-1 may contribute to pathogenesis in this autoimmune disease model., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Diet-induced obesity is reported to induce a phenotypic switch in adipose tissue macrophages from an antiinflammatory M2 state to a proinflammatory M1 state. Telmisartan, an angiotensin II type 1 receptor blocker and a peroxisome proliferator-activated receptor-γ agonist, reportedly has more beneficial effects on insulin sensitivity than other angiotensin II type 1 receptor blockers. In this study, we studied the effects of telmisartan on the adipose tissue macrophage phenotype in high-fat-fed mice. Telmisartan was administered for 5 wk to high-fat-fed C57BL/6 mice. Insulin sensitivity, macrophage infiltration, and the gene expressions of M1 and M2 markers in visceral adipose tissues were then examined. An insulin- or a glucose-tolerance test showed that telmisartan treatment improved insulin resistance, decreasing the body weight gain, visceral fat weight, and adipocyte size without affecting the amount of energy intake. Telmisartan reduced the mRNA expression of CD11c and TNF-α, M1 macrophage markers, and significantly increased the expressions of M2 markers, such as CD163, CD209, and macrophage galactose N-acetyl-galactosamine specific lectin (Mgl2), in a quantitative RT-PCR analysis. A flow cytometry analysis showed that telmisartan decreased the number of M1 macrophages in visceral adipose tissues. In conclusion, telmisartan improves insulin sensitivity and modulates adipose tissue macrophage polarization to an antiinflammatory M2 state in high-fat-fed mice.

Objective: To characterize the phenotypic changes of adipose tissue macrophages (ATMs) under different conditions of insulin sensitivity., Research Design and Methods: The number and the expressions of marker genes for M1 and M2 macrophages from mouse epididymal fat tissue were analyzed using flow cytometry after the mice had been subjected to a high-fat diet (HFD) and pioglitazone treatment., Results: Most of the CD11c-positive M1 macrophages and the CD206-positive M2 macrophages in the epididymal fat tissue were clearly separated using flow cytometry. The M1 and M2 macrophages exhibited completely different gene expression patterns. Not only the numbers of M1 ATMs and the expression of M1 marker genes, such as tumor necrosis factor-alpha and monocyte chemoattractant protein-1, but also the M1-to-M2 ratio were increased by an HFD and decreased by subsequent pioglitazone treatment, suggesting the correlation with whole-body insulin sensitivity. We also found that the increased number of M2 ATMs after an HFD was associated with the upregulated expression of interleukin (IL)-10, an anti-inflammatory Th2 cytokine, in the adipocyte fraction as well as in adipose tissue. The systemic overexpression of IL-10 by an adenovirus vector increased the expression of M2 markers in adipose tissue., Conclusions: M1 and M2 ATMs constitute different subsets of macrophages. Insulin resistance is associated with both the number of M1 macrophages and the M1-to-M2 ratio. The increased expression of IL-10 after an HFD might be involved in the increased recruitment of M2 macrophages.

B-1 cells are a subset of B cells responsible for the production of natural antibodies. Although the amount of natural antibody is tightly regulated, how this regulation occurs remains unknown. We examined the expression of IL-5 receptor, a cytokine receptor critical for homeostatic proliferation of B-1 cells, on B-1 cell progenitors in the fetal liver. We identified B-1 progenitors expressing low levels of IL-5 receptor alpha chain (IL-5Ralpha) and eosinophil progenitors expressing higher levels of IL-5Ralpha in the fetal liver. Moreover, the number of these B-1 progenitors were significantly reduced in the fetuses of mice deficient in Bruton's tyrosine kinase (Btk), even though IL-5 and thymic stroma lymphopoietin signaling are intact in early B lineage cells in Btk-deficient mice. These data suggest that IL-5 is possibly involved in B-1 cell development and an uncharacterized, Btk-dependent regulatory signaling pathway is involved in unexpectedly early stages of B-1 cell differentiation.

Background: Elicitation of contact sensitivity (CS) depends on B-1-cell-derived antigen-specific immunoglobulin M (IgM) antibodies that recruit CS effector T cells into the local tissue, which is followed by infiltration of antigen-nonspecific mononuclear cells and polymorphonuclear cells, such as neutrophils and eosinophils. In this study, we investigated the role of interleukin (IL)-5, which has broad effects on both eosinophils and B-1 cells, in elicitation of CS., Methods: IL-5 receptor alpha-chain-deficient (IL-5Ralpha-/-) mice and IL-5Ralpha+/+ mice were contact sensitized with oxazolone hapten. Four days later, mice were challenged with the same hapten, and ear swelling responses were measured at 24 h after challenge. Eosinophil infiltration into the local tissue was determined by examination of skin histology and eosinophil peroxidase activity. To investigate the role of IL-5 in B-1 cell activation, the number of oxazolone-specific IgM-producing cells in the spleen was determined by enzyme-linked immunospot assay., Results: Ear swelling responses in IL-5Ralpha-/- mice were about half of those in IL-5Ralpha+/+ mice, and nearly no eosinophil infiltration was observed in IL-5Ralpha-/- mouse skin. Eosinophil peroxidase activity in the sensitized and challenged IL-5Ralpha-/- mice was about 11 times less than that in immunized IL-5Ralpha+/+ mice. Contact sensitization significantly increased in numbers of oxazolne-specific IgM-producing cells in IL-5Ralpha+/+ mouse spleen, but not in IL-5Ralpha-/- mouse spleen., Conclusion: We conclude that IL-5-dependent activation of eosinophils and B-1 cells is necessary for induction and elicitation of CS. These findings provide a new insight into complicated mechanisms of CS elicitation and suggest a novel role of IL-5 in the regulation of immune responses., (Copyright 2006 S. Karger AG, Basel.)

The accurate partitioning of Ig H chain V(H)DJ(H) junctions and L chain V(L)J(L) junctions is problematic. We have developed a statistical approach for the partitioning of such sequences, by analyzing the distribution of point mutations between a determined V gene segment and putative Ig regions. The establishment of objective criteria for the partitioning of sequences between V(H), D, and J(H) gene segments has allowed us to more carefully analyze intervening putative nontemplated (N) nucleotides. An analysis of 225 IgM H chain sequences, with five or fewer V mutations, led to the alignment of 199 sequences. Only 5.0% of sequences lacked N nucleotides at the V(H)D junction (N1), and 10.6% at the DJ(H) junction (N2). Long N regions (>9 nt) were seen in 20.6% of N1 regions and 17.1% of N2 regions. Using a statistical analysis based upon known features of N addition, and mutation analysis, two of these N regions aligned with D gene segments, and a third aligned with an inverted D gene segment. Nine additional sequences included possible alignments with a second D segment. Four of the remaining 40 long N1 regions included 5' sequences having six or more matches to V gene end motifs, which may be the result of V gene replacement. Such sequences were not seen in long N2 regions. The long N regions frequently seen in the expressed repertoire of human Ig gene rearrangements can therefore only partly be explained by V gene replacement and D-D fusion.