Characterization of metabolic phenotypes and distinctive genes in mice with low-weight gain.

Being overweight exacerbates various metabolic diseases, necessitating the identification of target molecules for obesity control. In the current study, we investigated common physiological features related to metabolism in mice with low weight gain: (1) G protein-coupled receptor, family C, group 5, member B-knockout; (2) gastric inhibitory polypeptide receptor-knockout; and (3) Iroquois-related homeobox 3-knockout. Moreover, we explored genes involved in metabolism by analyzing differentially expressed genes (DEGs) between low-weight gain mice and the respective wild-type control mice. The common characteristics of the low-weight gain mice were low inguinal white adipose tissue (iWAT) and liver weight despite similar food intake along with lower blood leptin levels and high energy expenditure. The DEGs of iWAT, epididymal (gonadal) WAT, brown adipose tissue, muscle, liver, hypothalamus, and hippocampus common to these low-weight gain mice were designated as candidate genes associated with metabolism. One such gene tetraspanin 7 (Tspan7) from the iWAT was validated using knockout and overexpressing mouse models. Mice with low Tspan7 expression gained more weight, while those with high Tspan7 expression gained less weight, confirming the involvement of the Tspan7 gene in weight regulation. Collectively, these findings suggest that the candidate gene list generated in this study contains potential target molecules for obesity regulation. Further validation and additional data from low-weight gain mice will aid in understanding the molecular mechanisms associated with obesity.

Activation of ILC2s through constitutive IFNγ signaling reduction leads to spontaneous pulmonary fibrosis.

Pulmonary fibrosis (PF), a condition characterized by inflammation and collagen deposition in the alveolar interstitium, causes dyspnea and fatal outcomes. Although the bleomycin-induced PF mouse model has improved our understanding of exogenous factor-induced fibrosis, the mechanism governing endogenous factor-induced fibrosis remains unknown. Here, we find that Ifngr1-/-Rag2-/- mice, which lack the critical suppression factor for group 2 innate lymphoid cells (ILC2), develop PF spontaneously. The onset phase of fibrosis includes ILC2 subpopulations with a high Il1rl1 (IL-33 receptor) expression, and fibrosis does not develop in ILC-deficient or IL-33-deficient mice. Although ILC2s are normally localized near bronchioles and blood vessels, ILC2s are increased in fibrotic areas along with IL-33 positive fibroblasts during fibrosis. Co-culture analysis shows that activated-ILC2s directly induce collagen production from fibroblasts. Furthermore, increased IL1RL1 and decreased IFNGR1 expressions are confirmed in ILC2s from individuals with idiopathic PF, highlighting the applicability of Ifngr1-/-Rag2-/- mice as a mouse model for fibrosis research.

Gut microbial carbohydrate metabolism contributes to insulin resistance.

Insulin resistance is the primary pathophysiology underlying metabolic syndrome and type 2 diabetes1,2. Previous metagenomic studies have described the characteristics of gut microbiota and their roles in metabolizing major nutrients in insulin resistance3-9. In particular, carbohydrate metabolism of commensals has been proposed to contribute up to 10% of the host's overall energy extraction10, thereby playing a role in the pathogenesis of obesity and prediabetes3,4,6. Nevertheless, the underlying mechanism remains unclear. Here we investigate this relationship using a comprehensive multi-omics strategy in humans. We combine unbiased faecal metabolomics with metagenomics, host metabolomics and transcriptomics data to profile the involvement of the microbiome in insulin resistance. These data reveal that faecal carbohydrates, particularly host-accessible monosaccharides, are increased in individuals with insulin resistance and are associated with microbial carbohydrate metabolisms and host inflammatory cytokines. We identify gut bacteria associated with insulin resistance and insulin sensitivity that show a distinct pattern of carbohydrate metabolism, and demonstrate that insulin-sensitivity-associated bacteria ameliorate host phenotypes of insulin resistance in a mouse model. Our study, which provides a comprehensive view of the host-microorganism relationships in insulin resistance, reveals the impact of carbohydrate metabolism by microbiota, suggesting a potential therapeutic target for ameliorating insulin resistance.

ILC2s and Adipose Tissue Homeostasis: Progress to Date and the Road Ahead.

Group 2 innate lymphoid cells (ILC2s) were initially identified as a new type of lymphocytes that produce vigorous amounts of type 2 cytokines in adipose tissue. Subsequent studies revealed that ILC2s are present not only in adipose tissue but also in various other tissues such as lung and skin. ILC2s are generally recognized as tissue-resident immune cells that regulate tissue homeostasis. ILC2s express receptors for various humoral factors and thus can change their functions or distribution depending on the environment and circumstances. In this review, we will outline our recent understanding of ILC2 biology and discuss future directions for ILC2 research, particularly in adipose tissue and metabolic homeostasis.

Cancer immunosurveillance by ILC2s.

Group 2 innate lymphoid cells (ILC2s) elicit ostensibly paradoxical responses, such as tissue repair and stimulation of tumorigenesis. Given emerging evidence that ILC2s also contribute to cancer immunosurveillance, we reassess the role of ILC2s in tumorigenesis and discuss recent insights into their tumoricidal potential.

IgM to IgG Class Switching Is a Necessary Step for Pemphigus Phenotype Induction in Desmoglein 3-Specific B Cell Receptor Knock-in Mouse.

Pemphigus vulgaris is an autoimmune blistering disease caused by IgG targeting desmoglein 3 (Dsg3), an adhesion molecule of keratinocytes. Anti-Dsg3 IgG production is prevented in healthy individuals, but it is unclear how Dsg3-specific B cells are regulated. To clarify the immunological condition regulating Dsg3-specific B cells, a pathogenic anti-Dsg3 Ig (AK23) knock-in mouse was generated. AK23 knock-in B cells developed normally without undergoing deletion or acquiring an anergic phenotype in vivo. The knock-in B cells showed Ca2+ influx upon IgM cross-linking and differentiated into AK23-IgG+ B cells after LPS and IL-4 stimulation in vitro that induced a pemphigus phenotype after adoptive transfer into Rag2 -/- mice. However, the knock-in mouse itself produced AK23-IgM but little IgG without blisters in vivo. Dsg3 immunization and skin inflammation caused AK23-IgG production and a pemphigus phenotype in vivo. Furthermore, Fcgr2b deficiency or haploinsufficiency spontaneously induced AK23-IgG production and a pemphigus phenotype with poor survival rates in AK23 knock-in mice. To assess Fcgr2b involvement in Ig class-switch efficiency, postswitch transcripts of B cells were quantified and significantly higher in Fcgr2b -/- and Fcgr2b +/- mice than wild-type mice in a gene dose-dependent manner. Finally, RNA sequencing revealed reduced expression of FCGR2B and FcγRIIB-related genes in patient B cells. These results indicated that Dsg3-specific B cells do not spontaneously perform pathogenic class switching in vivo, and pemphigus phenotype induction was prevented under normal conditions. Attenuated FcγRIIB signaling is also one of the drivers for pathogenic class switching and is consistent with immunological features identified from clinical samples. This study unveiled a characteristic immune state silencing autoreactive B cells in mice.

Innate Lymphoid Cells in Skin Homeostasis and Malignancy.

Innate lymphoid cells (ILCs) are mostly tissue resident lymphocytes that are preferentially enriched in barrier tissues such as the skin. Although they lack the expression of somatically rearranged antigen receptors present on T and B cells, ILCs partake in multiple immune pathways by regulating tissue inflammation and potentiating adaptive immunity. Emerging evidence indicates that ILCs play a critical role in the control of melanoma, a type of skin malignancy thought to trigger immunity mediated mainly by adaptive immune responses. Here, we compile our current understanding of ILCs with regard to their role as the first line of defence against melanoma development and progression. We also discuss areas that merit further investigation. We envisage that the possibility to harness therapeutic potential of ILCs might benefit patients suffering from skin malignancies such as melanoma.

An antibacterial coated polymer prevents biofilm formation and implant-associated infection.

To prevent infections associated with medical implants, various antimicrobial silver-coated implant materials have been developed. However, these materials do not always provide consistent antibacterial effects in vivo despite having dramatic antibacterial effects in vitro, probably because the antibacterial effects involve silver-ion-mediated reactive oxygen species generation. Additionally, the silver application process often requires extremely high temperatures, which damage non-metal implant materials. We recently developed a bacteria-resistant coating consisting of hydroxyapatite film on which ionic silver is immobilized via inositol hexaphosphate chelation, using a series of immersion and drying steps performed at low heat. Here we applied this coating to a polymer, polyetheretherketone (PEEK), and analyzed the properties and antibacterial activity of the coated polymer in vitro and in vivo. The ionic silver coating demonstrated significant bactericidal activity and prevented bacterial biofilm formation in vitro. Bio-imaging of a soft tissue infection mouse model in which a silver-coated PEEK plate was implanted revealed a dramatic absence of bacterial signals 10 days after inoculation. These animals also showed a strong reduction in histological features of infection, compared to the control animals. This innovative coating can be applied to complex structures for clinical use, and could prevent infections associated with a variety of plastic implants.

Arf1 and Arf6 Synergistically Maintain Survival of T Cells during Activation.

ADP-ribosylation factor (Arf) family consisting of six family members, Arf1-Arf6, belongs to Ras superfamily and orchestrates vesicle trafficking under the control of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins. It is well established that brefeldin A, a potent inhibitor of ArfGEFs, blocks cytokine secretion from activated T cells, suggesting that the Arf pathway plays important roles in T cell functions. In this study, because Arf1 and Arf6 are the best-characterized members among Arf family, we established T lineage-specific Arf1-deficient, Arf6-deficient, and Arf1/6 double-deficient mice to understand physiological roles of the Arf pathway in the immune system. Contrary to our expectation, Arf deficiency had little or no impact on cytokine secretion from the activated T cells. In contrast, the lack of both Arf1 and Arf6, but neither Arf1 nor Arf6 deficiency alone, rendered naive T cells susceptible to apoptosis upon TCR stimulation because of imbalanced expression of Bcl-2 family members. We further demonstrate that Arf1/6 deficiency in T cells alleviates autoimmune diseases like colitis and experimental autoimmune encephalomyelitis, whereas Ab response under Th2-polarizing conditions is seemingly normal. Our findings reveal an unexpected role for the Arf pathway in the survival of T cells during TCR-induced activation and its potential as a therapeutic target in the autoimmune diseases.

Group 2 Innate Lymphoid Cells Exacerbate Amebic Liver Abscess in Mice.

Entamoeba histolytica, a protozoan parasite in the lumen of the human large intestine, occasionally spreads to the liver and induces amebic liver abscesses (ALAs). Upon infection with E. histolytica, high levels of type 2 cytokines are induced in the liver early after infection. However, the sources and functions of these initial type 2 cytokines in ALA formation remain unclear. In this study, we examined the roles of group 2 innate lymphoid cells (ILC2s) in ALA formation. Hepatic ILC2 numbers were significantly increased and they produced robust levels of IL-5. The in vivo transfer of ILC2s into Rag2-/-common γ chain (γc)-/- KO mice aggravated ALA formation accompanied by eosinophilia and neutrophilia. Furthermore, IL-33-deficient mice and IL-5-neutralized mice had less ALA formations. These results suggest that ILC2s contribute to exacerbating the pathogenesis of ALA by producing early type 2 cytokines and promoting the accumulation of eosinophils and neutrophils in the liver.

Tumor-Derived Lactic Acid Contributes to the Paucity of Intratumoral ILC2s.

Group 2 innate lymphoid cells (ILC2s) are abundant in non-lymphoid tissues and increase following infectious and inflammatory insults. In solid tumors, however, ILC2s constitute a relatively small proportion of immune cells. Here, we show, using melanoma as a model, that while the IL-33/IL C2/eosinophil axis suppresses tumor growth, tumor-derived lactate attenuates the function and survival of ILC2s. Melanomas with reduced lactate production (LDHAlow) are growth delayed and typified by an increased number of ILC2s compared with control tumors. Upon IL-33 stimulation, ILC2s accompanied by eosinophils more effectively restrain the growth of LDHAlow tumors than control melanomas. Furthermore, database analysis reveals a negative correlation between the expression of LDHA and markers associated with ILC2s and the association of high expression of IL33 and an eosinophil marker SIGLEC8 with better overall survival in human cutaneous melanoma patients. This work demonstrates that the balance between the IL-33/ILC2/eosinophil axis and lactate production by tumor cells regulates melanoma growth.

A 3D Skin Melanoma Spheroid-Based Model to Assess Tumor-Immune Cell Interactions.

Three-dimensional (3D) tumor spheroids have the potential to bridge the gap between two-dimensional (2D) monolayer tumor cell cultures and solid tumors with which they share a significant degree of similarity. However, the progression of solid tumors is often influenced by the dynamic and reciprocal interactions between tumor and immune cells. Here we present a 3D tumor spheroid-based model that might shed new light on understanding the mechanisms of tumor and immune cell interactions. The model first utilizes the hanging drop assay, which serves as one of the simplest methods for generating 3D spheroids and requires no specialized equipment. Next, pre-established spheroids can be co-cultured either directly or indirectly with an immune cell population of interest. Using skin melanoma, we provide a detailed description of the model, which might hold a significant importance for the development of successful therapeutic strategies.

Innate Lymphoid Cells in the Induction of Obesity.

Complex interactions between immune cells are an important component in the induction of obesity. Here, we show that Il2rg-/-Rag2-/- mice lacking all lymphocytes are resistant to diet-induced obesity. Transplantation of bone marrow cells from Rag2-/- mice, which lack only acquired immune cells, into Il2rg-/-Rag2-/- mice abolishes this resistance, indicating a role for innate lymphoid cells (ILCs) in this process. Mice lacking ILC2 or ILC3 cells, but not natural killer cells, are resistant to obesity. Adoptive transfer of naive ILC2s isolated from the small intestine (SI), but not ILC2s from white adipose tissue (WAT), restores the induction of diet-induced obesity in Il2rg-/-Rag2-/- mice. Analysis of transcriptional differences reveals that SI-ILC2s express higher levels of IL-2 than do WAT-ILC2s and that blockade of IL-2 signaling impairs weight gain and reduces the populations of ILC2s and ILC3s in the SI, suggesting a role for the IL-2/ILC2/3 axis in the induction of obesity.

Cancer Immunoediting by Innate Lymphoid Cells.

The immune system plays a dual role in cancer. It conveys protective immunity but also facilitates malignant progression, either by sculpting tumor immunogenicity or by creating a microenvironment that can stimulate tumor outgrowth or aid in a subsequent metastatic cascade. Innate lymphoid cells (ILCs) embody this functional heterogeneity, although the nature of their responses in cancer has only recently begun to be unveiled. We provide an overview of recent insights into the role of ILCs in cancer. We also discuss how ILCs fit into the conceptual framework of cancer immunoediting, which integrates the dual role of the immune system in carcinogenesis. A broader understanding of their relevance in cancer is essential towards the design of successful therapeutic strategies.

Hide and seek: Plasticity of innate lymphoid cells in cancer.

The advance of immunotherapies has revolutionized the treatment of cancer patients. Mostly agents modulating the adaptive immune system are currently used. More recently, attempts to stimulate the innate immune system are being promoted for clinical evaluation. Innate lymphoid cells (ILCs) are a highly plastic population of immune cells crucial for tissue homeostasis and the regulation of immune responses and maybe a promising target to improve current cancer immunotherapies. Although we have made significant progress in understanding ILC biology, their impact on tumor development, progression and therapy is controversial. In this review, we discuss the recent advances of ILC function and plasticity in the context of cancer.

The group 2 innate lymphoid cell (ILC2) regulatory network and its underlying mechanisms.

Group 2 innate lymphoid cells (ILC2s) play critical roles in the induction of type 2 inflammation, response to parasite infection, metabolic homeostasis, and tissue repair. These multifunctional roles of ILC2s are tightly controlled by complex regulatory systems in the local microenvironment, the disruption of which may cause various health problems. This review summarizes up-to-date knowledge regarding positive and negative regulators for ILC2s based on their function and signaling pathways, including activating cytokines (IL-33, IL-25; MAPK, NF-κB pathways), co-stimulatory cytokines (IL-2, IL-7, IL-9, TSLP; STAT5, IL-4; STAT6, TNF superfamily; MAPK, NF-κB pathways), suppressive cytokines (type1 IFNs, IFN-γ, IL-27; STAT1, IL-10, TGF-ß), transdifferentiation cytokines (IL-12; STAT4, IL-1ß, IL-18), lipid mediators (LTC4, LTD4, LTE4, PGD2; Ca2+ -NFAT pathways, PGE2, PGI2; AC/cAMP/PKA pathways, LXA4, LTB4), neuropeptides (NMU; Ca2+ -NFAT, MAPK pathways, VIP, CGRP, catecholamine, acetylcholine), sex hormones (androgen, estrogen), nutrients (butyrate; HDAC inhibitors, vitamins), and cell-to-cell interactions (ICOSL-ICOS; STAT5, B7-H6-NKp30, E-cadherin-KLRG1). This comprehensive review affords a better understanding of the regulatory network system for ILC2s, providing impetus to develop new treatment strategies for ILC2-related health problems.

Innate Lymphoid Cells: 10 Years On.

Innate lymphoid cells (ILCs) are lymphocytes that do not express the type of diversified antigen receptors expressed on T cells and B cells. ILCs are largely tissue-resident cells and are deeply integrated into the fabric of tissues. The discovery and investigation of ILCs over the past decade has changed our perception of immune regulation and how the immune system contributes to the maintenance of tissue homeostasis. We now know that cytokine-producing ILCs contribute to multiple immune pathways by, for example, sustaining appropriate immune responses to commensals and pathogens at mucosal barriers, potentiating adaptive immunity, and regulating tissue inflammation. Critically, the biology of ILCs also extends beyond classical immunology to metabolic homeostasis, tissue remodeling, and dialog with the nervous system. The last 10 years have also contributed to our greater understanding of the transcriptional networks that regulate lymphocyte commitment and delineation. This, in conjunction with the recent advances in our understanding of the influence of local tissue microenvironments on the plasticity and function of ILCs, has led to a re-evaluation of their existing categorization. In this review, we distill the advances in ILC biology over the past decade to refine the nomenclature of ILCs and highlight the importance of ILCs in tissue homeostasis, morphogenesis, metabolism, repair, and regeneration.

Peripheral PDGFRα+gp38+ mesenchymal cells support the differentiation of fetal liver-derived ILC2.

Group 2 innate lymphoid cells (ILC2s) are derived from common lymphoid progenitors (CLPs) via several specific precursors, and the transcription factors essential for ILC2 differentiation have been extensively studied. However, the external factors regulating commitment to the ILC lineage as well as the sites and stromal cells that constitute the optimal microenvironment for ILC2-specific differentiation are not fully defined. In this study, we demonstrate that three key external factors, the concentration of interleukin 7 (IL-7) and strength and duration of Notch signaling, coordinately determine the fate of CLP toward the T, B, or ILC lineage. Additionally, we identified three stages of ILC2 in the fetal mesentery that require STAT5 signals for maturation: ILC progenitors, CCR9+ ILC2 progenitors, and KLRG1- immature ILC2. We further demonstrate that ILC2 development is supported by mesenteric platelet-derived growth factor receptor α (PDGFRα)+ glycoprotein 38 (gp38)+ mesenchymal cells. Collectively, our results suggest that early differentiation of ILC2 occurs in the fetal liver via IL-7 and Notch signaling, whereas final differentiation occurs in the periphery with the aid of PDGFRα+gp38+ cells.