Prolonged Wnt3a exposure tolerizes macrophages to inflammatory stimuli.

Macrophages are highly plastic innate immune cells that have a broad range of phenotypic and functional roles in the body. The Wnt/β-catenin signaling pathway is known to play important roles in regulating the immune system, but the literature contains contradictory evidence for how Wnt impacts macrophages. Given the plasticity of macrophages, as well as the growing interest in utilizing Wnt inhibitors therapeutically, there is a need to better understand how Wnt signaling affects macrophage phenotype and function. We treated murine bone marrow derived macrophages with Wnt3a, LPS/IFN-γ, or IL-4 and measured gene/protein expression with bulk RNA sequencing, RT-qPCR, flow cytometry, and immunofluorescence to assess macrophage phenotype. RNA sequencing of macrophages treated continually for 5 days with Wnt3a demonstrated upregulation in genes associated with chemotaxis, cytokine activity, and both pro- and anti-inflammatory phenotypes. A time-course of Wnt3a treatment revealed acute upregulation of the inflammatory cytokines Il6, Tnf, and Il12b. Later timepoints showed upregulation of regulatory markers, such as Il10. Finally, re-treating with classic inflammatory cytokines revealed a Wnt-induced tolerant phenotype. In this study, we expanded upon past work to show that acute stimulation by Wnt3a induces inflammatory activation of macrophages in a time-dependent manner. Chronic stimulation with Wnt3a, as may be expected in a Wnt-ligand rich tissue microenvironment, caused macrophages to become tolerant to additional inflammatory stimuli and to upregulate markers of an anti-inflammatory phenotype. This study highlights the importance of considering time-dependent plasticity and regulatory feedback mechanisms in understanding macrophage phenotypes.