A basophil-neuronal axis promotes itch.

Itch is an evolutionarily conserved sensation that facilitates expulsion of pathogens and noxious stimuli from the skin. However, in organ failure, cancer, and chronic inflammatory disorders such as atopic dermatitis (AD), itch becomes chronic, intractable, and debilitating. In addition to chronic itch, patients often experience intense acute itch exacerbations. Recent discoveries have unearthed the neuroimmune circuitry of itch, leading to the development of anti-itch treatments. However, mechanisms underlying acute itch exacerbations remain overlooked. Herein, we identify that a large proportion of patients with AD harbor allergen-specific immunoglobulin E (IgE) and exhibit a propensity for acute itch flares. In mice, while allergen-provoked acute itch is mediated by the mast cell-histamine axis in steady state, AD-associated inflammation renders this pathway dispensable. Instead, a previously unrecognized basophil-leukotriene (LT) axis emerges as critical for acute itch flares. By probing fundamental itch mechanisms, our study highlights a basophil-neuronal circuit that may underlie a variety of neuroimmune processes.

Publisher Correction: Tet2 and Tet3 in B cells are required to repress CD86 and prevent autoimmunity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Tet2 and Tet3 in B cells are required to repress CD86 and prevent autoimmunity.

A contribution of epigenetic modifications to B cell tolerance has been proposed but not directly tested. Here we report that deficiency of ten-eleven translocation (Tet) DNA demethylase family members Tet2 and Tet3 in B cells led to hyperactivation of B and T cells, autoantibody production and lupus-like disease in mice. Mechanistically, in the absence of Tet2 and Tet3, downregulation of CD86, which normally occurs following chronic exposure of self-reactive B cells to self-antigen, did not take place. The importance of dysregulated CD86 expression in Tet2- and Tet3-deficient B cells was further demonstrated by the restriction, albeit not complete, on aberrant T and B cell activation following anti-CD86 blockade. Tet2- and Tet3-deficient B cells had decreased accumulation of histone deacetylase 1 (HDAC1) and HDAC2 at the Cd86 locus. Thus, our findings suggest that Tet2- and Tet3-mediated chromatin modification participates in repression of CD86 on chronically stimulated self-reactive B cells, which contributes, at least in part, to preventing autoimmunity.

Protective neutralizing influenza antibody response in the absence of T follicular helper cells.

Virus infection induces the development of T follicular helper (TFH) and T helper 1 (TH1) cells. Although TFH cells are important in anti-viral humoral immunity, the contribution of TH1 cells to a protective antibody response remains unknown. We found that IgG2 antibodies predominated in the response to vaccination with inactivated influenza A virus (IAV) and were responsible for protective immunity to lethal challenge with pathogenic H5N1 and pandemic H1N1 IAV strains, even in mice that lacked TFH cells and germinal centers. The cytokines interleukin-21 and interferon-γ, which are secreted from TH1 cells, were essential for the observed greater persistence and higher titers of IgG2 protective antibodies. Our results suggest that TH1 induction could be a promising strategy for producing effective neutralizing antibodies against emerging influenza viruses.

Ablation of cDC2 development by triple mutations within the Zeb2 enhancer.

The divergence of the common dendritic cell progenitor1-3 (CDP) into the conventional type 1 and type 2 dendritic cell (cDC1 and cDC2, respectively) lineages4,5 is poorly understood. Some transcription factors act in the commitment of already specified progenitors-such as BATF3, which stabilizes Irf8 autoactivation at the +32 kb Irf8 enhancer4,6-but the mechanisms controlling the initial divergence of CDPs remain unknown. Here we report the transcriptional basis of CDP divergence and describe the first requirements for pre-cDC2 specification. Genetic epistasis analysis7 suggested that Nfil3 acts upstream of Id2, Batf3 and Zeb2 in cDC1 development but did not reveal its mechanism or targets. Analysis of newly generated NFIL3 reporter mice showed extremely transient NFIL3 expression during cDC1 specification. CUT&RUN and chromatin immunoprecipitation followed by sequencing identified endogenous NFIL3 binding in the -165 kb Zeb2 enhancer8 at three sites that also bind the CCAAT-enhancer-binding proteins C/EBPα and C/EBPß. In vivo mutational analysis using CRISPR-Cas9 targeting showed that these NFIL3-C/EBP sites are functionally redundant, with C/EBPs supporting and NFIL3 repressing Zeb2 expression at these sites. A triple mutation of all three NFIL3-C/EBP sites ablated Zeb2 expression in myeloid, but not lymphoid progenitors, causing the complete loss of pre-cDC2 specification and mature cDC2 development in vivo. These mice did not generate T helper 2 (TH2) cell responses against Heligmosomoides polygyrus infection, consistent with cDC2 supporting TH2 responses to helminths9-11. Thus, CDP divergence into cDC1 or cDC2 is controlled by competition between NFIL3 and C/EBPs at the -165 kb Zeb2 enhancer.

IL-27 regulates the differentiation of follicular helper NKT cells via metabolic adaptation of mitochondria.

Invariant natural killer T (iNKT) cells are innate-like T lymphocytes that express an invariant T cell receptor α chain and contribute to bridging innate and acquired immunity with rapid production of large amounts of cytokines after stimulation. Among effecter subsets of iNKT cells, follicular helper NKT (NKTFH) cells are specialized to help B cells. However, the mechanisms of NKTFH cell differentiation remain to be elucidated. In this report, we studied the mechanism of NKTFH cell differentiation induced by pneumococcal surface protein A and α-galactosylceramide (P/A) vaccination. We found that Gr-1+ cells helped iNKT cell proliferation and NKTFH cell differentiation in the spleen by producing interleukin-27 (IL-27) in the early phase after vaccination. The neutralization of IL-27 impaired NKTFH cell differentiation, which resulted in compromised antibody production and diminished protection against Streptococcus pneumoniae infection by the P/A vaccine. Our data indicated that Gr-1+ cell-derived IL-27 stimulated mitochondrial metabolism, meeting the energic demand required for iNKT cells to differentiate into NKTFH cells. Interestingly, Gr-1+ cell-derived IL-27 was induced by iNKT cells via interferon-γ production. Collectively, our findings suggest that optimizing the metabolism of iNKT cells was essential for acquiring specific effector functions, and they provide beneficial knowledge on iNKT cell-mediated vaccination-mediated therapeutic strategies.

Perivascular leukocyte clusters are essential for efficient activation of effector T cells in the skin.

It remains largely unclear how antigen-presenting cells (APCs) encounter effector or memory T cells efficiently in the periphery. Here we used a mouse contact hypersensitivity (CHS) model to show that upon epicutaneous antigen challenge, dendritic cells (DCs) formed clusters with effector T cells in dermal perivascular areas to promote in situ proliferation and activation of skin T cells in a manner dependent on antigen and the integrin LFA-1. We found that DCs accumulated in perivascular areas and that DC clustering was abrogated by depletion of macrophages. Treatment with interleukin 1α (IL-1α) induced production of the chemokine CXCL2 by dermal macrophages, and DC clustering was suppressed by blockade of either the receptor for IL-1 (IL-1R) or the receptor for CXCL2 (CXCR2). Our findings suggest that the dermal leukocyte cluster is an essential structure for elicitating acquired cutaneous immunity.

Role of Signal-Transducing Adaptor Protein-1 for T Cell Activation and Pathogenesis of Autoimmune Demyelination and Airway Inflammation.

Signal-transducing adaptor protein (STAP)-1 is an adaptor protein that is widely expressed in T cells. In this article, we show that STAP-1 upregulates TCR-mediated T cell activation and T cell-mediated airway inflammation. Using STAP-1 knockout mice and STAP-1-overexpressing Jurkat cells, we found that STAP-1 enhanced TCR signaling, resulting in increased calcium mobilization, NFAT activity, and IL-2 production. Upon TCR engagement, STAP-1 binding to ITK promoted formation of ITK-LCK and ITK-phospholipase Cγ1 complexes to induce downstream signaling. Consistent with the results, STAP-1 deficiency reduced the severity of symptoms in experimental autoimmune encephalomyelitis. Single-cell RNA-sequencing analysis revealed that STAP-1 is essential for accumulation of T cells and Ifng and Il17 expression in spinal cords after experimental autoimmune encephalomyelitis induction. Th1 and Th17 development was also attenuated in STAP-1 knockout naive T cells. Taken together, STAP-1 enhances TCR signaling and plays a role in T cell-mediated immune disorders.

Three-step transcriptional priming that drives the commitment of multipotent progenitors toward B cells.

Stem cell fate is orchestrated by core transcription factors (TFs) and epigenetic modifications. Although regulatory genes that control cell type specification are identified, the transcriptional circuit and the cross-talk among regulatory factors during cell fate decisions remain poorly understood. To identify the "time-lapse" TF networks during B-lineage commitment, we used multipotent progenitors harboring a tamoxifen-inducible form of Id3, an in vitro system in which virtually all cells became B cells within 6 d by simply withdrawing 4-hydroxytamoxifen (4-OHT). Transcriptome and epigenome analysis at multiple time points revealed that ∼10%-30% of differentially expressed genes were virtually controlled by the core TFs, including E2A, EBF1, and PAX5. Strikingly, we found unexpected transcriptional priming before the onset of the key TF program. Inhibition of the immediate early genes such as Nr4a2, Klf4, and Egr1 severely impaired the generation of B cells. Integration of multiple data sets, including transcriptome, protein interactome, and epigenome profiles, identified three representative transcriptional circuits. Single-cell RNA sequencing (RNA-seq) analysis of lymphoid progenitors in bone marrow strongly supported the three-step TF network model during specification of multipotent progenitors toward B-cell lineage in vivo. Thus, our findings will provide a blueprint for studying the normal and neoplastic development of B lymphocytes.

The Majority of the Serine/Threonine Phosphorylation Sites in Bcl11b Protein Are Dispensable for the Differentiation of T Cells.

Posttranslational modification, such as phosphorylation, is an important biological event that modulates and diversifies protein function. Bcl11b protein is a zinc-finger transcription factor that plays a crucial role in early T cell development and the segregation of T cell subsets. Bcl11b possesses at least 25 serine/threonine (S/T) residues that can be phosphorylated upon TCR stimulation. To understand the physiological relevance of the phosphorylation on Bcl11b protein, we replaced S/T residues with alanine (A) by targeting murine Bcl11b gene in embryonic stem cells. By combinational targeting of exons 2 and 4 in the Bcl11b gene, we generated a mouse strain, Bcl11b-phosphorylation site mutation mice, in which 23 S/T residues were replaced with A residues. Such extensive manipulation left only five putative phosphorylated residues, two of which were specific for mutant protein, and resulted in reduced amounts of Bcl11b protein. However, primary T cell development in the thymus, as well as the maintenance of peripheral T cells, remained intact even after loss of major physiological phosphorylation. In addition, in vitro differentiation of CD4+ naive T cells into effector Th cell subsets-Th1, Th2, Th17, and regulatory T-was comparable between wild-type and Bcl11b-phosphorylation site mutation mice. These findings indicate that the physiological phosphorylation on major 23 S/T residues in Bcl11b is dispensable for Bcl11b functions in early T cell development and effector Th cell differentiation.

The transcription factor NFIL3/E4BP4 regulates the developmental stage-specific acquisition of basophil function.

BACKGROUND: Basophils are rare but important effector cells in many allergic disorders. Contrary to their early progenitors, the terminal developmental processes of basophils in which they gain their unique functional properties are unknown. OBJECTIVE: We sought to identify a novel late-stage basophil precursor and a transcription factor regulating the terminal maturation of basophils. METHODS: Using flow cytometry, transcriptome analysis, and functional assays, we investigated the identification and functionality of the basophil precursors as well as basophil development. We generated mice with basophil-specific deletion of nuclear factor IL-3 (NFIL3)/E4BP4 and analyzed the functional impairment of NFIL3/E4BP4-deficient basophils in vitro and in vivo using an oxazolone-induced murine model of allergic dermatitis. RESULTS: We report a new mitotic transitional basophil precursor population (referred to as transitional basophils) that expresses the FcεRIα chain at higher levels than mature basophils. Transitional basophils are less responsive to IgE-linked degranulation but produce more cytokines in response to IL-3, IL-33, or IgE cross-linking than mature basophils. In particular, we found that the expression of NFIL3/E4BP4 gradually rises as cells mature from the basophil progenitor stage. Basophil-specific deletion of NFIL3/E4BP4 reduces the expression of genes necessary for basophil function and impairs IgE receptor signaling, cytokine secretion, and degranulation in the context of murine atopic dermatitis. CONCLUSIONS: We discovered transitional basophils, a novel late-stage mitotic basophil precursor cell population that exists between basophil progenitors and postmitotic mature basophils. We demonstrated that NFIL3/E4BP4 augments the IgE-mediated functions of basophils, pointing to a potential therapeutic regulator for allergic diseases.

Subcutaneous immunisation with zymosan generates mucosal IgA-eliciting memory and protects mice from heterologous influenza virus infection.

Immunoglobulin A (IgA) is the most abundant isotype of antibodies and provides a first line of defense at the mucosa against pathogens invading the host. It has been widely accepted that the mucosal IgA response provided by vaccination requires mucosal inoculation, and intranasal inoculation has been proposed for vaccines against influenza virus. Considering the difficulty of intranasal vaccination in infants or elderly people, however, parenteral vaccination that provides the mucosal IgA response is desirable. Here, we demonstrate that subcutaneous immunisation with zymosan, a yeast cell wall constituent known to be recognised by Dectin-1 and TLR2, potentiates the production of antigen-specific IgA antibodies in the sera and airway mucosa upon intranasal antigen challenge. We confirmed that the antigen-specific IgA-secreting cells accumulated in the lung and nasal-associated lymphoid tissues after the antigen challenge. Such an adjuvant effect of zymosan in the primary immunisation for the IgA response depended on Dectin-1 signalling, but not on TLR2. The IgA response to the antigen challenge required both antigen-specific memory B and T cells, and the generation of memory T cells, but not memory B cells, depended on zymosan as an adjuvant. Finally, we demonstrated that subcutaneous inoculation of inactivated influenza virus with zymosan, but not with alum, mostly protected the mice from infection with a lethal dose of a heterologous virus strain. These data suggest that zymosan is a possible adjuvant for parenteral immunisation that generates memory IgA responses to respiratory viruses such as influenza virus.

An Interleukin-33-Mast Cell-Interleukin-2 Axis Suppresses Papain-Induced Allergic Inflammation by Promoting Regulatory T Cell Numbers.

House dust mite-derived proteases contribute to allergic disorders in part by disrupting epithelial barrier function. Interleukin-33 (IL-33), produced by lung cells after exposure to protease allergens, can induce innate-type airway eosinophilia by activating natural helper (NH) cells, a member of group 2 innate lymphoid cells (ILC2), to secrete Th2 type-cytokines. Because IL-33 also can induce mast cells (MCs) to secrete Th2 type-cytokines, MCs are thought to cooperate with NH cells in enhancing protease or IL-33-mediated innate-type airway eosinophilia. However, we found that MC-deficient Kit(W-sh/W-sh) mice exhibited exacerbated protease-induced lung inflammation associated with reduced numbers of regulatory T (Treg) cells. Moreover, IL-2 produced by IL-33-stimulated MCs promoted expansion of numbers of Treg cells, thereby suppressing development of papain- or IL-33-induced airway eosinophilia. We have thus identified a unique anti-inflammatory pathway that can limit induction of innate-type allergic airway inflammation mediated by NH cells.

C-peptide index at 2 h post-meal is a useful predictor of endogenous insulin secretory capacity and withdrawal from insulin therapy in subjects with type 2 diabetes.

AIMS: To evaluate the correlation between C-peptide index (CPI) at 2 h post-meal and endogenous insulin secretory capacity and to develop clinical models to predict the possibility of withdrawal from insulin therapy in patients with type 2 diabetes. METHOD: This was a single-centre retrospective study of patients with type 2 diabetes admitted to our hospital. Patients were divided into a withdrawal group (n = 72) and a non-withdrawal group (n = 75) based on whether they were able to withdraw from insulin therapy at discharge, and the correlation between CPI at 2 h after meal and diabetes-related parameters was evaluated. In addition, we created two clinical models to predict the possibility of withdrawal from insulin therapy using machine learning. RESULTS: The glycated haemoglobin values of the study participants were 87.8 ± 22.6 mmol/mo. The CPI at 2 h post-meal was 1.93 ± 1.28 in the non-withdrawal group and 2.97 ± 2.07 in the withdrawal group (p < 0.001). CPI at 2 h post-meal was an independent predictor of withdrawal from insulin therapy. In addition, CPI at 2 h post-meal was a better predictor than fasting CPI. Six factors associated with insulin therapy withdrawal (age, duration of diabetes, creatinine, alanine aminotransferase, insulin therapy until hospitalization, and CPI at 2 h post-meal) were used to generate two clinical models by machine learning. The accuracy of the generated clinical models ranged from 78.3% to 82.6%. CONCLUSION: The CPI at 2 h post-meal is a clinically useful measure of endogenous insulin secretory capacity under non-fasting conditions.

The enhancer HS2 critically regulates GATA-3-mediated Il4 transcription in T(H)2 cells.

GATA-3 is a master regulator of T helper type 2 (T(H)2) differentiation. However, the molecular basis of GATA-3-mediated T(H)2 lineage commitment is poorly understood. Here we identify the DNase I-hypersensitive site 2 (HS2) element located in the second intron of the interleukin 4 locus (Il4) as a critical enhancer strictly controlled by GATA-3 binding. Mice lacking HS2 showed substantial impairment in their asthmatic responses and their production of IL-4 but not of other T(H)2 cytokines. Overexpression of Gata3 in HS2-deficient T cells failed to restore Il4 expression. HS2 deletion impaired the trimethylation of histone H3 at Lys4 and acetylation of histone H3 at Lys9 and Lys14 in the Il4 locus. Our results indicate that HS2 is the target of GATA-3 in regulating chromosomal modification of the Il4 locus and is independent of the Il5 and Il13 loci.

The transcription factor E4BP4 regulates the production of IL-10 and IL-13 in CD4+ T cells.

The immunoregulatory cytokine interleukin 10 (IL-10) is expressed mainly by T helper type 2 (T(H)2) cells but also by T(H)1 cells during chronic infection. Here we observed plasticity in the expression of IL-10 and IL-13 after chronic T(H)1 stimulation; furthermore, the expression of Il10 and Il13 was regulated by the transcription factor E4BP4. Chronically stimulated E4BP4-deficient (Nfil3(-/-); called 'E4bp4(-/-)' here) T(H)1 cells, regulatory T cells (T(reg) cells) and natural killer T cells (NKT cells) had attenuated expression of IL-10 and IL-13. Enforced expression of E4bp4 initiated the production of IL-10 and IL-13 by conventional T(H)1 cells. E4bp4(-/-) T(H)2 cells showed impairment of IL-10 production with no effect on IL-13. Our results indicate that E4BP4 has multiple functions in controlling the plasticity of IL-13 in T(H)1 cells and IL-10 in T(H)1 cells, T(H)2 cells, T(reg) cells and NKT cells.

Breadth of Antibody Responses during Influenza Virus Infection and Vaccination.

Influenza viruses are a major public health problem, causing severe respiratory diseases. Vaccines offer the effective protective strategy against influenza virus infection. However, the systemic and adaptive immune responses to infection and vaccination are quite different. Inactivated vaccines are the best available countermeasure to induce effective antibodies against the emerged virus, but the response is narrow compared with potential breadth of virus infection. There is solid evidence to indicate that antibody responses to natural infection are relatively broad and exhibit quite different immunodominance patterns. Furthermore, T follicular helper cells (TFH) and germinal center (GC) responses play a central role in generating broad protective antibodies. In this review, we discuss recent advances on the contribution of TFH and GC responses to the breadth of antibody responses.

Basophil-derived interleukin-4 controls the function of natural helper cells, a member of ILC2s, in lung inflammation.

Allergic asthma is an inflammatory disease characterized by lung eosinophilia controlled by type 2 cytokines. Cysteine proteases are potent triggers of allergic inflammation by causing barrier disruption in lung epithelial cells inducing the elevation of interleukin-5 (IL-5) and IL-13 from natural helper (NH) cells, a member of ILC2s, which leads to lung eosinophilia. In this study, we found that basophils play a crucial role in NH cell-mediated eosinophilic inflammation induced by protease allergens. Conditional deletion of basophils caused a resolution of the papain-induced eosinophilia and mucus production. Resolution of eosinophilia was also observed in mice lacking IL-4 specifically in basophils, indicating that basophil-derived IL-4 enhanced expression of the chemokine CCL11, as well as IL-5, IL-9, and IL-13 in NH cells, thus attracting eosinophils. These results demonstrate that IL-4 from basophils has an important role in the NH-derived cytokine and chemokine expression, subsequently leading to protease allergen-induced airway inflammation.

The enzyme Cyp26b1 mediates inhibition of mast cell activation by fibroblasts to maintain skin-barrier homeostasis.

Mast cells (MCs) mature locally, thus possessing tissue-dependent phenotypes for their critical roles in both protective immunity against pathogens and the development of allergy or inflammation. We previously reported that MCs highly express P2X7, a receptor for extracellular ATP, in the colon but not in the skin. The ATP-P2X7 pathway induces MC activation and consequently exacerbates the inflammation. Here, we identified the mechanisms by which P2X7 expression on MCs is reduced by fibroblasts in the skin, but not in the other tissues. The retinoic-acid-degrading enzyme Cyp26b1 is highly expressed in skin fibroblasts, and its inhibition resulted in the upregulation of P2X7 on MCs. We also noted the increased expression of P2X7 on skin MCs and consequent P2X7- and MC-dependent dermatitis (so-called retinoid dermatitis) in the presence of excessive amounts of retinoic acid. These results demonstrate a unique skin-barrier homeostatic network operating through Cyp26b1-mediated inhibition of ATP-dependent MC activation by fibroblasts.

Involvement of Activation of Mast Cells via IgE Signaling and Epithelial Cell-Derived Cytokines in the Pathogenesis of Pollen Food Allergy Syndrome in a Murine Model.

Murine models to elucidate the pathogenesis of pollen food allergy syndrome (PFAS), characterized by oral hypersensitivity symptoms induced by specific foods in patients previously sensitized with a pollen, are lacking. The study aimed to examine PFAS pathogenesis in a novel murine model. Birch pollen-immunized mice were orally administered apple extract, and oral symptoms were evaluated based on oral rubbing frequency following the challenge. The birch pollen-immunized mice orally challenged with apple extract exhibited PFAS-like symptoms, including oral rubbing and positive reaction of swelling by the prick test. The apple extract administered with a protease inhibitor reduced the oral rubbing frequency, which was also significantly reduced in the immunized Fcer1a -/- and mast cell-deficient mice compared with the immunized control mice. The oral rubbing frequency, serum IgE levels, and Th2-cytokine production by the cervical lymph node cells were significantly reduced in the immunized Il-33 -/- and thymic stromal lymphopoietin receptor-deficient (Crlf2 -/-) mice as compared with the immunized wild-type mice. IL-33 and thymic stromal lymphopoietin involve the pathogenesis of PFAS. The apple-extract stimulation did not lead to increased Th2-cytokine production in the oral mucosa or number of group 2 innate lymphoid cells or eosinophils. PFAS involves an early-phase response by mast cell degranulation via IgE signaling after the cross-reactivity of Bet v 1-specific IgE and the food allergen, and exacerbation of allergic symptom via proteases in food; PFAS does not involve a late phase with local Th2/eosinophilic inflammation in the oral mucosa. This novel murine model might be used for elucidating the pathogenesis and assessing new therapeutic strategies for PFAS.