Assessment of faithful interleukin-3 production by novel bicistronic interleukin-3 reporter mice

Highlights

• Reporter mice expressing IL-3 and enhanced green fluorescent protein (ZsGreen) were created.

• T cells in vitro showed faithful ZsGreen expression and retained endogenous IL-3.

• IL-3-producing CD4 T cells were identified following nematode infection.

• Nematode infection of reporter mice causes expected increases in basophils and mast cells.

Abstract

Interleukin-3 (IL-3) is an important hematopoietic growth factor and immunregulatory cytokine. Although activated T helper cells represent a main source of IL-3, other cell types have been reported to express this cytokine. However, precise identification and quantification of the cells that produce IL-3 in vivo have not been performed. Therefore, we used a CRISPR/Cas approach to engineer mice containing a bicistronic mRNA linking a readily identifiable reporter, enhanced green fluorescent protein (ZsGreen1), to IL-3 expression. To characterize these novel reporter mice, we first examined ZsGreen1 expression by CD4 T cells subsets primed and activated in vitro. We found that activated Th1 cells expressed ∼4-fold higher levels of ZsGreen1 as compared to Th0 and Th2 cells. Endogenous IL-3 expression remained intact although reporter Th1 cells secreted ∼33 % less IL-3 than similarly activated wild-type cells. To characterize the ability of reporter mice to accurately mark IL-3-producing cells in vivo, we infected mice with Nippostrongylus brasiliensis. Low but significant numbers of ZsGreen1⁺ CD4 T cells were detected in the mesenteric lymph nodes and lung following both primary and secondary infection. No difference in basophil and intestinal mast cell numbers were observed between infected reporter and wild-type mice indicating that reporter mice secreted IL-3 levels in vivo that results in IL-3-driven biological activities which are indistinguishable from those observed in corresponding wild-type mice. These IL-3 reporter mice will be a valuable resource to investigate IL-3-dependent immune responses in vivo.

Introduction

The cytokine interleukin-3 (IL-3) is well documented at promoting the in vitro differentiation and proliferation of murine hematopoietic progenitor cells and the survival and activation of mature myeloid cells, including mast cells, basophils, dentritic cells, neutrophils, eosinophils, macrophages, and erythrocytes [[1], [2], [3]]. A role for IL-3 in allergic inflammation is suggested by the ability of IL-3 to promote basophil and mast cell growth, differentiation, and mediator release [[4], [5], [6], [7], [8]]. In addition, IL-3 has been reported to enhance monocyte/macrophage activation and/or phagocytosis [[9], [10], [11]], promote the secretory function of eosinophils [12], stimulate endothelial cell proliferation, migration, and activation [13], modulate development of regulatory T (Treg) cells [14], and enhance the development and function of classical and plasmacytoid dendritic cells [15,16].

Despite the activities attributed to IL-3, mice lacking the IL-3 gene display intact steady-state hematopoiesis, including having normal numbers of tissue mast cells and basophils [17,18]. These findings, and the fact that IL-3 levels in the blood or tissues are low or undetectable in normal mice, suggest that IL-3 either is not involved in hematopoietic cell development under physiological conditions or is a component of a larger network of redundant cytokines. On the other hand, our experiments with IL-3 gene-deficient (knockout [KO]) mice have revealed either a net suppressive or stimulatory role for IL-3 during certain disease conditions. For example, we have shown that IL-3 KO mice show impaired T cell-dependent contact hypersensitivity responses to haptens [17], have an increased accumulation of eosinophils during ragweed-induced allergic peritonitis [19], mount attenuated mast cell and basophil responses to gastrointestinal nematode infection that result in compromised worm expulsion [7,18,20,21], and are more resistant to blood-stage malaria infection [22]. More recently, it has been reported that IL-3 deficiency protects mice against sepsis by reducing myeloid cell proinflammatory cytokine production, and that elevated levels of IL-3 correlate with increased mortality in human septic patients [1].

The main sources of IL-3 are activated T helper (Th) cells [23]. While the particular subsets of Th cells that produce IL-3 are not well defined, both Th1 and Th2 are known to secrete IL-3 [24]; Our results with Plasmodium-infected mice [22] and with contact hypersensitivity responses in mice [17] suggest that non-CD4 T cells may also represent a biologically significant source of IL-3 in vivo. Indeed, other cell types have been reported to express IL-3 such as mast cells [25,26], basophils [27], NKT cells [28], eosinophils [29], endothelial cells [30], innate response activator (IRA) B cells [1], and neurons and astrocytes in the brain [31].

The fact that many different cell types represent potential sources of IL-3 in vivo raises many important questions: (1) Does the nature, strength, site and stage of infection determine the type of cellular sources of IL-3? (2) Does the type of IL-3-producing cells determine the nature, quality, duration and outcome of the immune responses? (3) What are the molecular mechanisms underlying the expression of IL-3, and are these mechanisms cell-type and/or tissue-specific? The investigation of such questions requires faithful identification and characterization of IL-3-producing cells in vivo. Unfortunately, the problems inherent in existing methods for detecting the temporal and cell-type-specific patterns of IL-3 expression make answering these questions difficult or unfeasible. To overcome these problems, we have used a CRISPR/Cas approach to engineer mice containing a bicistronic mRNA linking a readily identifiable reporter, enhanced green fluorescent protein (ZsGreen1), to IL-3 expression. We characterize these IL-3-ZsGreen1 reporter (3Gr) mice using both in vitro T cell assays and in vivo infection with Nippostrongylus brasiliensis (N.b.). These novel reporter mice represent a valuable resource to examine the potential and known involvement of IL-3 in various disease models.