NETosis is present and influences tumors in Lymphangioleiomyomatosis models 2309907

Abstract

Abstract Introduction Lymphangioleiomyomatosis (LAM) is a cystic lung disease primarily found in women whereby, over time, small thin-walled cysts take over normal alveolar space, leading to progressive lung function loss. Cysts are caused by estrogen-sensitive smooth muscle LAM tumor cells with mutations in the tuberous sclerosis gene complex (TSC), which leads to constitutive activation of mTORC1 signaling and increased LAM cell proliferation. Our lab proposed that LAM cells originate from the estrogen sensitive myometrium of the uterus and created a mouse model whereby TSC2 knockout in the uterus leads to uterine LAM tumors that metastasize to the lungs. With this model, we demonstrated that neutrophils promote this tumor growth in part through release of neutrophil elastase. Here we examined the potential role of NETosis, a process of releasing modified chromatin and neutrophils serine proteases like neutrophil elastase, in promoting LAM tumor cell growth. Methods We analyzed RNAseq on neutrophils treated with and without estradiol to understand the role of estrogen in neutrophil function. We also generated NETs from bone marrow derive murine neutrophils and stimulated mouse uterine cells. Finally, we used immunofluorescence looking for citrullinated histones and myeloperoxidase in TSC2 null mouse uteri versus littermate control uteri, in xenografts generated from TSC2 null ELT3 rat myometrial cells, and in human LAM lung samples versus controls. Results Neutrophil degranulation and immunity signaling pathways were upregulated in neutrophils treated with estrogen. Mouse uterine cells responded to NETs by upregulating expression of genes associated with cell survival and EMT. NETosis was detected in both the uterine tumor of our mouse model and in human lung samples from patients with LAM in comparison to controls. Conclusion Evidence suggests that NETosis could be contributing to the release of neutrophil elastase in TSC2 null tumor microenvironments, exacerbating tumor growth. Funding Source LAM Foundation DOD NIH Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)