Pikfyve Regulates Lysosomal Biogenesis and Functions in Macrophages

Macrophages are professional phagocytes essential for host defense and tissue homeostasis. These functions are critically dependent on the proper biogenesis and degradative activity of lysosomes. Through the biosynthetic pathway, lysosomes receive newly synthesized lysosomal enzymes, which are necessary for the degradation of endogenous and exogenous materials obtained via endocytosis or autophagy pathways. Recent studies identified the endosomal phosphoinositide PI(3,5)P2 as a critical regulator of the trafficking pathways along the endolysosomal system. PI(3,5)P2 is synthesized by the lipid kinase PIKfyve in mammals. In our previous work, we demonstrated that PIKfyve is vital in the lysosomal biogenesis and homeostasis in platelets and PIKfyve deficiency could lead to arterial thrombosis and inflammation in vivo. However, despite the critical importance of lysosomal biogenesis and functions in macrophages, the role of PIKfyve in these cells is unknown.

To address the role of PIKfyve in macrophages, we generated mice lacking PIKfyve in their macrophages by pairing PIKfyve^fl/fl mice with mice that are transgenic for the myeloid-specific LysM-Cre. As expected, macrophage expression of PIKfyve was normal in wild-type mice, partially reduced in the PIKfyve^fl/+ LysM-Cre mice, and undetectable in the PIKfyve^fl/fl LysM-Cre mice. To validate the tissue specificity of LysM-Cre, PIKfyve^fl/fl LysM-Cre mouse was crossed with the Cre-dependent YFP reporter mouse. LysM-Cre induced YFP expression was present predominantly in the monocytes and neutrophils, and minimally in lymphocytes. As they matured, PIKfyve^fl/fl LysM-Cre mice developed abdominal distention due to severe hepatosplenomegaly. Histological analysis of their liver and spleen demonstrated tissue infiltration of cells with numerous cytoplasmic vacuoles. These vacuolated cells stained with markers of neutrophils and macrophages by immunostaining. Immunophenotyping analysis of white blood cells from the peripheral blood demonstrated elevated counts of monocytes and neutrophils but decreased number of lymphocytes in the PIKfyve^fl/fl LysM-Cre mice. In addition, PIKfyve^fl/fl LysM-Cre mice had increased levels of inflammatory cytokines compared to control mice. Together, these data indicate that PIKfyve deficiency in myeloid cells induce inflammatory responses and suggest that PIKfyve in myeloid cells is essential to prevent pathological inflammatory responses.

To investigate the effects of PIKfyve ablation in lysosome biogenesis and functions in macrophages, we examined the macrophages isolated from the bone marrow and spleen of control and PIKfyve^fl/fl LysM-Cre mice. Immunofluorescence analysis showed enlarged vacuoles staining with LAMP-1 (marker of late endosomes and lysosomes) in PIKfyve-null macrophages. Immunoblotting analyses of total lysates from PIKfyve-null macrophages revealed excessive levels of lysosomal proteins including LAMP-1 and Cathepsin D. In spite of the increased levels of lysosomal proteins, intralysosomal proteolysis was defective. This demonstrates that PIKfyve is essential not only for macrophage lysosomal biogenesis but also for the degradative function in lysosomes.

Intriguingly, PIKfyve deficiency was associated with reduced phosphorylation of the transcription factor TFEB, which is key regulator of the lysosomal gene expression. We found that compared to wild-type macrophages, PIKfyve-null macrophages had significantly reduced levels of the phosphorylated forms of TFEB and primarily expressed the dephosphorylated form of TFEB. These findings provide a clear link between PIKfyve, lysosome biogenesis, and TFEB.

Taken together, our study demonstrates that PIKfyve is essential in both the biogenesis and function of macrophage lysosomes. Furthermore, our work demonstrates a previously unrecognized connection between phosphoinositide signaling, the transcription factor TFEB, and lysosomes in professional phagocytic cells. Whether this link is perturbed in human diseases would be an interesting direction of investigation.