No abstract

Mast cells have essential effector and immunoregulatory functions in IgE-associated allergic disorders and certain innate and adaptive immune responses, but the role of miRNAs in regulating mast cell functions is almost completely unexplored. To examine the role of the activation-induced miRNA miR-221 in mouse mast cells, we developed robust lentiviral systems for miRNA overexpression and depletion. While miR-221 favored mast cell adhesion and migration towards SCF or antigen in trans-well migration assays, as well as cytokine production and degranulation in response to IgE-antigen complexes, neither miR-221 overexpression, nor its ablation, interfered with mast cell differentiation. Transcriptional profiling of miR-221-overexpressing mast cells revealed modulation of many transcripts, including several associated with the cytoskeleton; indeed, miR-221 overexpression was associated with reproducible increases in cortical actin in mast cells, and with altered cellular shape and cell cycle in murine fibroblasts. Our bioinformatics analysis showed that this effect was likely mediated by the composite effect of miR-221 on many primary and secondary targets in resting cells. Indeed, miR-221-induced cellular alterations could not be recapitulated by knockdown of one of the major targets of miR-221. We propose a model in which miR-221 has two different roles in mast cells: in resting cells, basal levels of miR-221 contribute to the regulation of the cell cycle and cytoskeleton, a general mechanism probably common to other miR-221-expressing cell types, such as fibroblasts. Vice versa, upon induction in response to mast cell stimulation, miR-221 effects are mast cell-specific and activation-dependent, contributing to the regulation of degranulation, cytokine production and cell adherence. Our studies provide new insights into the roles of miR-221 in mast cell biology, and identify novel mechanisms that may contribute to mast cell-related pathological conditions, such as asthma, allergy and mastocytosis.

MicroRNAs (miRNAs) constitute a class of molecules regulating gene expression in many different cell types, including cells of the mammalian immune system. Indeed, changes in miRNA expression patterns have been implicated in various physiological and pathological processes. Mast cells (MCs) are hematopoietic cells that originate in the bone marrow and migrate into the tissues, where they mature and reside. They have an important immunoregulatory and effector role in IgE-associated allergic disorders, as well as in certain innate and adaptive immune responses. An effective way to explore the functions of miRNAs in murine MCs includes the modification of miRNA expression in primary bone marrow-derived mast cells (BMMCs), followed by the analysis of the phenotypic consequences of such perturbation. In this chapter, we describe how to differentiate BMMCs and transduce them with lentiviruses. As an example, we expressed miR-221 and miR-222, which showed stable expression in BMMCs and acted as post-transcriptional regulators of c-Kit expression.