Recombinant Wnt3a and Wnt5a elicit macrophage cytokine production and tolerization to microbial stimulation via Toll-like receptor 4.

An increasing number of studies address the roles of Wnt proteins in shaping leukocyte functions. Recombinant Wnt3a and Wnt5a, prototypical activators of β-Catenin-dependent and -independent Wnt signaling, respectively, are widely used to investigate the effects of Wnt proteins on myeloid cell functions. Recent reports describe both proinflammatory and immunemodulatory effects of Wnt3a and Wnt5a on macrophages, DCs, and microglia. The underlying molecular mechanisms for this divergence are unclear. We show here that recombinant Wnt3a- and Wnt5a-induced cytokine production from murine C57BL/6 macrophages was dependent on TLR4 and inhibited by Polymyxin B. Similarly, impairment of TLR-induced cytokine production upon preexposure to Wnt proteins was TLR4 dependent. The extent of Wnt3a- and Wnt5a-induced inflammatory gene expression greatly varied between Wnt protein lots. We conclude that cytokine responses and TLR tolerization induced by recombinant Wnt proteins are likely explained by contaminating TLR4 agonists, although we cannot fully exclude that Wnt proteins have an intrinsic capacity to signal via TLR4. This study emphasizes the need for careful, independent verification of Wnt-mediated cellular responses.