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, TGFβ, 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 are rare circulating granulocytes activated by immunoglobulin E (IgE)-mediated cross-linking of the high affinity receptor for IgE (FcεRI), which induces their degranulation and synthesis of the type 2 cytokines, interleukin 4 (IL-4) and IL-13 in autoimmune, allergic diseases and helminthiases (1). Basophils are also sensitive to innate signals, including the cytokine IL-3, the alarmins IL-18, IL-33, and Thymic stromal Lymphopoietin (TSLP), the prostaglandin D2 (PGD2), ATP, and various chemokines or growth factors (1–4). Some of these stimuli might regulate basophil immunoregulatory functions during homeostasis (5). Secretion of IL-4 and histamine release by human basophils is sensitive to FK506 (a calcineurin inhibitor), while secretion of IL-13 is not affected, indicating that distinct signaling pathways regulate the expression of these type 2 cytokines (6–8). By contrast, activation of basophils by IgE or IL-18/33 involves distinct receptors and upstream signaling pathways but results in expression of both IL-4 and IL-13 (5, 7, 9–11). Despite common evolutionary ancestry, it is currently thought that IL-4 and IL-13 show divergent functions in immunity and physiology (12, 13). Basophil IL-4 secretion has been implicated in T helper type 2 (Th2) differentiation and M2 skewing. Basophils are an important source of IL-13 in the lungs, where they control the phenotype of alveolar macrophages during development (1, 5).The Il4/13 locus is subject to a differential regulation between distinct cell types but has been mostly studied in T cells. The locus contains elements regulated by cAMP, c-fos/AP-1, NFAT, NF-κB, GATA3, STAT5, and STAT6 that have been associated with the regulation of Il4/13 expression (6, 14–19). Basophils produce IL-4 and IL-13 after the cross-linking of their surface-bound IgE by antigen or by exposure to IL-3. IgE- or IL-3-mediated basophil activation are both controlled by the tyrosine kinase Syk (6, 20, 21). IgE-induced cytokine expression is also promoted by IκB kinase (IKK) through NF-κB-dependent or -independent signals (22, 23).To date, studies of murine basophils have focused on cultured bone marrow-derived basophils (BMBa) or primary bone marrow (BM) basophils, with circulating mature basophils too few in number for useful studies (6). We used B8 × 4C13R IL-4/IL-13 triple reporter mice (24) to follow and compare the ex vivo responses of primary mature basophils to 96 common stimuli and found that basophils were highly sensitive to type 2 and epithelial-derived cytokines and growth factors. Single-cell analysis revealed that basophils displayed distinct phenotypes upon stimulation with IL-3, expressing IL-4, and/or IL-13, Ly6C, or the degranulation marker CD63 (25). During helminth infection, basophils were found to be hyper- and hyporeactive to distinct stimuli (“Hp-basophils”). Purified Hp-basophils were also sensitive to homeostatic growth factors and antiviral response elements. Basophil reactivity was always sensitive to Syk and IKKs specific inhibitors. In conclusion, we found basophils to be sensitive to a complex array of distinct innate stimuli that worked through core common signaling pathways.