RESUMEN
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.
Asunto(s)
Basófilos/inmunología , Hipersensibilidad/inmunología , Inflamación/inmunología , Animales , Ratones Endogámicos C57BL , Piel/inmunologíaRESUMEN
Although IL-4 is long associated with CD4 Th2 immune responses, its role in Th2 subset development in non-lymphoid tissues is less clear. We sought to better define IL-4's role in CD4 Th2 responses by using transgenic mice that express a dual IL-4 AmCyan/IL-13 DsRed (IL-4AC/IL-13DR) fluorescent reporter on an IL-4-sufficient or IL-4-deficient background. Using primary Th2 immune response models against house dust mite or Nippostrongylus brasiliensis (Nb) allergens, we examined the requirement for IL-4 by each of the defined Th2 subsets in the antigen draining lymph node, skin, and lung tissues. In the lymph node, a CXCR5+PD-1+ T follicular helper (Tfh) and a CXCR5loPD-1lo Th2 subset could be detected that expressed only IL-4AC but no IL-13DR. The number of IL-4AC+ Tfh cells was not affected by IL-4 deficiency whereas the number of IL-4AC+ Th2 cells was significantly reduced. In the non-lymphoid dermal or lung tissues of allergen primed or Nb-infected mice, three strikingly distinct T cell subsets could be detected that were IL-4AC, or IL-4AC/IL-13DR, or IL-13DR CD4. The IL-4- and IL-4/IL-13-expressing subsets were significantly reduced in IL-4-deficient mice, while the numbers of IL-13-expressing CD4 T cells were not affected by IL-4 deficiency indicating that other factors can play a role in directing the development of this Th2 subtype. Taken together, these data indicate that the appearance of IL-4-expressing Tfh cells in the lymph node is not dependent on IL-4 while the appearance of IL-4-expressing Th2 subsets in the lymph node and IL-4, IL-4/IL-13-expressing Th2 subsets in skin and lung tissues of antigen primed mice is significantly IL-4 dependent.