Diverse innate stimuli activate basophils through pathways involving Syk and IκB kinases.

Mature basophils play critical inflammatory roles during helminthic, autoimmune, and allergic diseases through their secretion of histamine and the type 2 cytokines interleukin 4 (IL-4) and IL-13. Basophils are activated typically by allergen-mediated IgE cross-linking but also by endogenous "innate" factors. The aim of this study was to identify the innate stimuli (cytokines, chemokines, growth factors, hormones, neuropeptides, metabolites, and bacterial products) and signaling pathways inducing primary basophil activation. Basophils from naïve mice or helminth-infected mice were cultured with up to 96 distinct stimuli and their influence on basophil survival, activation, degranulation, and IL-4 or IL-13 expression were investigated. Activated basophils show a heterogeneous phenotype and segregate into distinct subsets expressing IL-4, IL-13, activation, or degranulation markers. We find that several innate stimuli including epithelial derived inflammatory cytokines (IL-33, IL-18, TSLP, and GM-CSF), growth factors (IL-3, IL-7, TGFß, and VEGF), eicosanoids, metabolites, TLR ligands, and type I IFN exert significant direct effects on basophils. Basophil activation mediated by distinct upstream signaling pathways is always sensitive to Syk and IκB kinases-specific inhibitors but not necessarily to NFAT, STAT5, adenylate cyclase, or c-fos/AP-1 inhibitors. Thus, basophils are activated by very diverse mediators, but their activation seem controlled by a core checkpoint involving Syk and IκB kinases.

Basophils promote barrier dysfunction and resolution in the atopic skin.

BACKGROUND: The type 2 cytokines IL-4 and IL-13 promote not only atopic dermatitis (AD) but also the resolution of inflammation. How type 2 cytokines participate in the resolution of AD is poorly known. OBJECTIVE: Our aim was to determine the mechanisms and cell types governing skin inflammation, barrier dysfunction, and resolution of inflammation in a model of AD. METHODS: Mice that exhibit expression of IL-4, IL-13, and MCPT8 or that could be depleted of basophils or eosinophils, be deficient in IL-4 or MHC class II molecules, or have basophils lacking macrophage colony-stimulating factor (M-CSF) were treated with calcipotriol (MC903) as an acute model of AD. Kinetics of the disease; keratinocyte differentiation; and leukocyte accumulation, phenotype, function, and cytokine production were measured by transepidermal water loss, histopathology, molecular biology, or unbiased analysis of spectral flow cytometry. RESULTS: In this model of AD, basophils were activated systemically and were the initial and main source of IL-4 in the skin. Basophils and IL-4 promoted epidermal hyperplasia and skin barrier dysfunction by acting on keratinocyte differentiation during inflammation. Basophils, IL-4, and basophil-derived M-CSF inhibited the accumulation of proinflammatory cells in the skin while promoting the expansion and function of proresolution M2-like macrophages and the expression of probarrier genes. Basophils kept their proresolution properties during AD resolution. CONCLUSION: Basophils can display both beneficial and detrimental type 2 functions simultaneously during atopic inflammation.

MR1-dependent immune surveillance of the skin contributes to pathogenesis and is a photobiological target of UV light therapy in a mouse model of atopic dermatitis.

BACKGROUND: Mucosal-associated invariant T (MAIT) cells are unconventional T cells which recognize microbial metabolites presented by the major histocompatibility complex class I-related molecule MR1. Although MAIT cells have been shown to reside in human and murine skin, their contribution to atopic dermatitis (AD), an inflammatory skin disease associated with barrier dysfunction and microbial translocation, has not yet been determined. METHODS: Genetic deletion of MR1 and topical treatment with inhibitory MR1 ligands, which result in the absence and functional inhibition of MAIT cells, respectively, were used to investigate the role of MR1-dependent immune surveillance in a MC903-driven murine model of AD. RESULTS: The absence or inhibition of MR1 arrested AD disease progression through the blockade of both eosinophil activation and recruitment of IL-4- and IL-13-producing cells. In addition, the therapeutic efficacy of phototherapy against MC903-driven AD could be increased with prior application of folate, which photodegrades into the inhibitory MR1 ligand 6-formylpterin. CONCLUSION: We identified MAIT cells as sentinels and mediators of cutaneous type 2 immunity. Their pathogenic activity can be inhibited by topical application or endogenous generation, via phototherapy, of inhibitory MR1 ligands.

CD62L on blood basophils: a first pre-treatment predictor of remission in severe lupus nephritis.

BACKGROUND: Basophils were recently shown to contribute to lupus nephritis (LN). This study assessed blood basophil activation markers (BAMs) for the diagnosis of LN severity and as pre-treatment prognostic markers of the response to treatment in patients with severe LN. METHOD: The diagnostic study included all the patients of a monocentric prospective observational cohort built with consecutive patients diagnosed with LN on the basis of a renal biopsy. The prognostic study selected patients of this cohort according to the following inclusion criteria: ≥18 years old, Class III or IV A ± C ± Class V or pure Class V LN at the time of inclusion and treated with an induction treatment for LN. Clinical data and BAMs were obtained at the time of the kidney biopsy. LN remission status was recorded 12 months after induction therapy initiation. Associations between baseline data and histological severity of LN or LN remission were assessed using logistic regression. RESULTS: No significant association was found between BAMs and the histological severity of LN in 101 patients. Among the 83 patients included in the prognostic study, 64 reached renal remission. CD62L expression on blood basophils at baseline was independently negatively associated with remission at 12 months [odds ratio = 0.26, 95% confidence interval 0.08-0.82, P = 0.02 for quantitative CD62L expression >105 (geometric fluorescent intensity) gMFI]. CD62L <105 gMFI was associated with a probability of 0.87 of LN remission in the next 12 months after the start of induction therapy. CONCLUSION: Pre-treatment CD62L expression on blood basophils could be a first predictive biomarker of renal response to induction therapy at 12 months in patients with severe LN.

Cyclosporine A impairs nucleotide binding oligomerization domain (Nod1)-mediated innate antibacterial renal defenses in mice and human transplant recipients.

Acute pyelonephritis (APN), which is mainly caused by uropathogenic Escherichia coli (UPEC), is the most common bacterial complication in renal transplant recipients receiving immunosuppressive treatment. However, it remains unclear how immunosuppressive drugs, such as the calcineurin inhibitor cyclosporine A (CsA), decrease renal resistance to UPEC. Here, we investigated the effects of CsA in host defense against UPEC in an experimental model of APN. We show that CsA-treated mice exhibit impaired production of the chemoattractant chemokines CXCL2 and CXCL1, decreased intrarenal recruitment of neutrophils, and greater susceptibility to UPEC than vehicle-treated mice. Strikingly, renal expression of Toll-like receptor 4 (Tlr4) and nucleotide-binding oligomerization domain 1 (Nod1), neutrophil migration capacity, and phagocytic killing of E. coli were significantly reduced in CsA-treated mice. CsA inhibited lipopolysaccharide (LPS)-induced, Tlr4-mediated production of CXCL2 by epithelial collecting duct cells. In addition, CsA markedly inhibited Nod1 expression in neutrophils, macrophages, and renal dendritic cells. CsA, acting through inhibition of the nuclear factor of activated T-cells (NFATs), also markedly downregulated Nod1 in neutrophils and macrophages. Silencing the NFATc1 isoform mRNA, similar to CsA, downregulated Nod1 expression in macrophages, and administration of the 11R-VIVIT peptide inhibitor of NFATs to mice also reduced neutrophil bacterial phagocytosis and renal resistance to UPEC. Conversely, synthetic Nod1 stimulating agonists given to CsA-treated mice significantly increased renal resistance to UPEC. Renal transplant recipients receiving CsA exhibited similar decrease in NOD1 expression and neutrophil phagocytosis of E. coli. The findings suggest that such mechanism of NFATc1-dependent inhibition of Nod1-mediated innate immune response together with the decrease in Tlr4-mediated production of chemoattractant chemokines caused by CsA may contribute to sensitizing kidney grafts to APN.

PD-L1- and IL-4-expressing basophils promote pathogenic accumulation of T follicular helper cells in lupus.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by anti-nuclear autoantibodies whose production is promoted by autoreactive T follicular helper (TFH) cells. During SLE pathogenesis, basophils accumulate in secondary lymphoid organs (SLO), amplify autoantibody production and disease progression through mechanisms that remain to be defined. Here, we provide evidence for a direct functional relationship between TFH cells and basophils during lupus pathogenesis, both in humans and mice. PD-L1 upregulation on basophils and IL-4 production are associated with TFH and TFH2 cell expansions and with disease activity. Pathogenic TFH cell accumulation, maintenance, and function in SLO were dependent on PD-L1 and IL-4 in basophils, which induced a transcriptional program allowing TFH2 cell differentiation and function. Our study establishes a direct mechanistic link between basophils and TFH cells in SLE that promotes autoantibody production and lupus nephritis.

AMG853, A Bispecific Prostaglandin D2 Receptor 1 and 2 Antagonist, Dampens Basophil Activation and Related Lupus-Like Nephritis Activity in Lyn-Deficient Mice.

Systemic lupus erythematosus is a complex autoimmune disease during which patients develop autoantibodies raised against nuclear antigens. During the course of the disease, by accumulating in secondary lymphoid organs (SLOs), basophils support autoreactive plasma cells to amplify autoantibody production. We have recently shown that murine lupus-like disease could be controlled by 10 days of oral treatment with a combination of prostaglandin D2 (PGD2) receptor (PTGDR) antagonists through the inhibition of basophil activation and recruitment to SLOs. Importantly, inhibiting solely PTGDR-1 or PTGDR-2 was ineffective, and the development of lupus-like disease could only be dampened by using antagonists for both PTGDR-1 and PTGDR-2. Here, we aimed at establishing a proof of concept that a clinically relevant bispecific antagonist of PTGDR-1 and PTGDR-2 could be efficient to treat murine lupus-like nephritis. Diseased Lyn-deficient female mice received treatment with AMG853 (vidupiprant, a bispecific PTGDR-1/PTGDR-2 antagonist) for 10 days. This led to the dampening of basophil activation and recruitment in SLOs and was associated with a decrease in plasmablast expansion and immunoglobulin E (IgE) production. Ten days of treatment with AMG853 was consequently sufficient in reducing the dsDNA-specific IgG titers, circulating immune complex glomerular deposition, and renal inflammation, which are hallmarks of lupus-like disease. Thus, bispecific PTGDR-1 and PTGDR-2 antagonists, such as AMG853, are a promising class of drugs for the treatment or prevention of organ damage in systemic lupus erythematosus.

CT-M8 Mice: A New Mouse Model Demonstrates That Basophils Have a Nonredundant Role in Lupus-Like Disease Development.

Tissue-specific mouse models are essential tools to decipher the role of each cell compartment and/or their expressed genes in the pathophysiology of diseases. Here, we describe a new knock-in mouse model allowing expression of both the fluorescent protein tdTomato and the CRE recombinase selectively in the basophil compartment under the control of the Mcpt8 gene. These "CT-M8" mice did not show any abnormalities in their peripheral distribution of major immune cell populations nor their basophil function. CT-M8 mice allowed the identification of basophils by immunofluorescence and flow cytometry and basophil-specific Cre-mediated floxed gene deletion. Breeding of our CT-M8 mice with the ROSA26flox-stop-DTA mice led to the generation of basophil-deficient mice with no detectable abnormalities in other cell compartments. These mice were then used to document basophil involvement in systemic lupus erythematosus (SLE) pathophysiology since we previously reported by transient depletion of these cells during the course of an ongoing disease that they support and amplify autoantibody production in two distinct lupus-like mouse models (Lyn-/- and pristane-induced). Here, constitutive basophil deficiency prevented pristane-induced lupus-like disease development by limiting autoantibody titers and renal damages. Therefore, basophils have a nonredundant role in pristane-induced lupus-like disease and are involved in both its induction and amplification. This CT-M8 new mouse model will allow us to finely decipher the role of basophils and their expressed genes in health and disease.

IgE Autoreactivity in Atopic Dermatitis: Paving the Road for Autoimmune Diseases?

Atopic dermatitis (AD) is a common skin disease affecting 20% of the population beginning usually before one year of age. It is associated with the emergence of allergen-specific IgE, but also with autoreactive IgE, whose function remain elusive. This review discusses current knowledge relevant to the mechanisms, which leads to the secretion of autoreactive IgE and to the potential function of these antibodies in AD. Multiple autoantigens have been described to elicit an IgE-dependent response in this context. This IgE autoimmunity starts in infancy and is associated with disease severity. Furthermore, the overall prevalence of autoreactive IgE to multiple auto-antigens is high in AD patients. IgE-antigen complexes can promote a facilitated antigen presentation, a skewing of the adaptive response toward type 2 immunity, and a chronic skin barrier dysfunction and inflammation in patients or AD models. In AD, skin barrier defects and the atopic immune environment facilitate allergen sensitization and the development of other IgE-mediated allergic diseases in a process called the atopic march. AD is also associated epidemiologically with several autoimmune diseases showing autoreactive IgE secretion. Thus, a potential outcome of IgE autoreactivity in AD could be the development of further autoimmune diseases.

The Gastrointestinal Helminth Heligmosomoides bakeri Suppresses Inflammation in a Model of Contact Hypersensitivity.

Helminths regulate host immune responses to ensure their own long-term survival. Numerous studies have demonstrated that these helminth-induced regulatory mechanisms can also limit host inflammatory responses in several disease models. We used the Heligmosomoides bakeri (Hb) infection model (also known as H. polygyrus or H. polygyrus bakeri in the literature) to test whether such immune regulation affects skin inflammatory responses induced by the model contact sensitiser dibutyl phthalate fluorescein isothiocynate (DBP-FITC). Skin lysates from DBP-FITC-sensitized, Hb-infected mice produced less neutrophil specific chemokines and had significantly reduced levels of skin thickening and cellular inflammatory responses in tissue and draining lymph nodes (LNs) compared to uninfected mice. Hb-induced suppression did not appear to be mediated by regulatory T cells, nor was it due to impaired dendritic cell (DC) activity. Mice cleared of infection remained unresponsive to DBP-FITC sensitization indicating that suppression was not via the secretion of Hb-derived short-lived regulatory molecules, although long-term effects on cells cannot be ruled out. Importantly, similar helminth-induced suppression of inflammation was also seen in the draining LN after intradermal injection of the ubiquitous allergen house dust mite (HDM). These findings demonstrate that Hb infection attenuates skin inflammatory responses by suppressing chemokine production and recruitment of innate cells. These findings further contribute to the growing body of evidence that helminth infection can modulate inflammatory and allergic responses via a number of mechanisms with potential to be exploited in therapeutic and preventative strategies in the future.

Panel Design and Optimization for High-Dimensional Immunophenotyping Assays Using Spectral Flow Cytometry.

Technological advances in fluorescence flow cytometry and an ever-expanding understanding of the complexity of the immune system have led to the development of large (20+ parameters) flow cytometry panels. However, as panel complexity and size increase, so does the difficulty involved in designing a high-quality panel, accessing the instrumentation capable of accommodating large numbers of parameters, and analyzing such high-dimensional data. A recent advancement is spectral flow cytometry, which in contrast to conventional flow cytometry distinguishes the full emission spectrum of each fluorophore across all lasers, rather than identifying only the peak of emission. Fluorophores with a similar emission maximum but distinct off-peak signatures can therefore be accommodated within the same flow cytometry panel, allowing greater flexibility in terms of panel design and fluorophore detection. Here, we highlight the specific characteristics of spectral flow cytometry and aim to guide users through the process of building, designing, and optimizing high-dimensional spectral flow cytometry panels using a comprehensive step-by-step protocol. Special considerations are also given for using highly overlapping dyes, and a logical selection process for optimal marker-fluorophore assignment is provided. © 2020 by John Wiley & Sons, Inc.

The Basoph8 Mice Enable an Unbiased Detection and a Conditional Depletion of Basophils.

Basophils are granulocytes involved in parasite immunity and allergic diseases, known for their potent secretion of type 2 cytokines. Identifying their functions has proven to be controversial due to their relative rarity and their complex lineage phenotype. Here, we show that the expression of basophils lineage markers CD200R3 and FcεRIα is highly variable in inflammatory settings and hinders basophils identification by flow cytometry across multiple disease states or tissues. Fluorophore-conjugated antibody staining of these lineage markers strongly activates basophil type 2 cytokine expression, and represents a potential bias for coculture or in vivo transfer experiments. The Basoph8 is a mouse model where basophils specifically express a strong fluorescent reporter and the Cre recombinase. Basophils can be identified and FACS sorted unambiguously by their expression of the enhanced yellow fluorescent protein (eYFP) in these mice. We show that the expression of the eYFP is robust in vivo during inflammation, and in vitro on living basophils for at least 72 h, including during the induction of anaphylactoid degranulation. We bred and characterized the Basoph8xiDTR mice, in which basophils specifically express eYFP and the simian diphtheria toxin receptor (DTR). This model enables basophils conditional depletion relatively specifically ex vivo and in vivo during allergic inflammation and their detection as eYFP+ cells. In conclusion, we report underappreciated benefits of the commercially available Basoph8 mice to study basophils function.

Eosinophils Determine Dermal Thickening and Water Loss in an MC903 Model of Atopic Dermatitis.

Atopic dermatitis (AD) is a highly debilitating disease with significant health impacts worldwide. It has been a difficult disease to treat because of the wide spectrum of clinical manifestations. Therefore, the current clinical management strategies are nonspecific. Previous studies have documented that AD disease progression is precipitated by a combination of skin barrier dysfunction, itch, and immune dysregulation. However, the precise roles played by effector cells and cytokines have not been fully elucidated. To address this, we established a prolonged model of AD, using MC903. The phenotype of this MC903 model closely resembles the one observed in AD patients, including inflammatory parameters, barrier dysfunction, itch, and histopathological characteristics, thereby providing a platform to evaluate targets for the treatment of AD. This model exposed cells and cytokines that are critically associated with disease severity, including eosinophils, TSLP, and IL-4/IL-13. Indeed, eosinophil depletion significantly ameliorated AD pathology, most notably barrier dysfunction, to a similar extent as blocking of the IL-4/IL-13 axis by genetic deletion of STAT6. Thus, this study has identified eosinophils to be critical for the development and maintenance of AD, thereby proposing these effector cells as therapeutic targets for the treatment of AD.

Prostaglandin D2 amplifies lupus disease through basophil accumulation in lymphoid organs.

In systemic lupus erythematosus (SLE), autoantibody production can lead to kidney damage and failure, known as lupus nephritis. Basophils amplify the synthesis of autoantibodies by accumulating in secondary lymphoid organs. Here, we show a role for prostaglandin D2 (PGD2) in the pathophysiology of SLE. Patients with SLE have increased expression of PGD2 receptors (PTGDR) on blood basophils and increased concentration of PGD2 metabolites in plasma. Through an autocrine mechanism dependent on both PTGDRs, PGD2 induces the externalization of CXCR4 on basophils, both in humans and mice, driving accumulation in secondary lymphoid organs. Although PGD2 can accelerate basophil-dependent disease, antagonizing PTGDRs in mice reduces lupus-like disease in spontaneous and induced mouse models. Our study identifies the PGD2/PTGDR axis as a ready-to-use therapeutic modality in SLE.

Investigation of chimeric reads using the MinION.

Following a nanopore sequencing run of PCR products of three amplicons less than 1kb, an abundance of reads failed quality control due to template/complement mismatch. A BLAST search demonstrated that some of the failed reads mapped to two different genes -- an unexpected observation, given that PCR was carried out separately for each amplicon. A further investigation was carried out specifically to search for chimeric reads, using separate barcodes for each amplicon and trying two different ligation methods prior to sample loading. Despite the separation of ligation products, chimeric reads formed from different amplicons were still observed in the base-called sequence. The long-read nature of nanopore sequencing presents an effective tool for the discovery and filtering of chimeric reads. We have found that at least 1.7% of reads prepared using the Nanopore LSK002 2D Ligation Kit include post-amplification chimeric elements. This finding has potential implications for other amplicon sequencing technologies, as the process is unlikely to be specific to the sample preparation used for nanopore sequencing.

Basophils contribute to pristane-induced Lupus-like nephritis model.

Lupus nephritis (LN), one of the most severe outcomes of systemic lupus erythematosus (SLE), is initiated by glomerular deposition of immune-complexes leading to an inflammatory response and kidney failure. Autoantibodies to nuclear antigens and autoreactive B and T cells are central in SLE pathogenesis. Immune mechanisms amplifying this autoantibody production drive flares of the disease. We previously showed that basophils were contributing to LN development in a spontaneous lupus-like mouse model (constitutive Lyn -/- mice) and in SLE subjects through their activation and migration to secondary lymphoid organs (SLOs) where they amplify autoantibody production. In order to study the basophil-specific mechanisms by which these cells contribute to LN development, we needed to validate their involvement in a genetically independent SLE-like mouse model. Pristane, when injected to non-lupus-prone mouse strains, induces a LN-like disease. In this inducible model, basophils were activated and accumulated in SLOs to promote autoantibody production. Basophil depletion by two distinct approaches dampened LN-like disease, demonstrating their contribution to the pristane-induced LN model. These results enable further studies to decipher molecular mechanisms by which basophils contribute to lupus progression.

Toll-Like Receptor 4, but Not Neutrophil Extracellular Traps, Promote IFN Type I Expression to Enhance Th2 Responses to Nippostrongylus brasiliensis.

The induction of Th2 responses is thought to be multifactorial, and emerge from specific pathways distinct from those associated with antagonistic antibacterial or antiviral Th1 responses. Here, we show that the recognition of non-viable Nippostrongylus brasiliensis (Nb) in the skin induces a strong recruitment of monocytes and neutrophils and the release of neutrophil extracellular traps (NETs). Nb also activates toll-like receptor 4 (TLR4) signaling with expression of Ifnb transcripts in the skin and the development of an IFN type I signature on helminth antigen-bearing dendritic cells in draining lymph nodes. Co-injection of Nb together with about 10,000 Gram-negative bacteria amplified this TLR4-dependent but NET-independent IFN type I response and enhanced the development of Th2 responses. Thus, a limited activation of antibacterial signaling pathways is able to boost antihelminthic responses, suggesting a role for bacterial sensing in the optimal induction of Th2 immunity.

Th2 responses are primed by skin dendritic cells with distinct transcriptional profiles.

The dendritic cell signals required for the in vivo priming of IL-4-producing T cells are unknown. We used RNA sequencing to characterize DCs from skin LN of mice exposed to two different Th2 stimuli: the helminth parasite Nippostrongylus brasiliensis (Nb) and the contact sensitizer dibutyl phthalate (DBP)-FITC. Both Nb and DBP-FITC induced extensive transcriptional changes that involved multiple DC subsets. Surprisingly, these transcriptional changes were highly distinct in the two models, with only a small number of genes being similarly regulated in both conditions. Pathway analysis of expressed genes identified no shared pathways between Nb and DBP-FITC, but revealed a type-I IFN (IFN-I) signature unique to DCs from Nb-primed mice. Blocking the IFN-I receptor at the time of Nb treatment had little effect on DC migration and antigen transport to the LN, but inhibited the up-regulation of IFN-I-induced markers on DCs and effectively blunted Th2 development. In contrast, the response to DBP-FITC was not affected by IFN-I receptor blockade, a finding consistent with the known dependence of this response on the innate cytokine TSLP. Thus, the priming of Th2 responses is associated with distinct transcriptional signatures in DCs in vivo, reflecting the diverse environments in which Th2 immune responses are initiated.