Background: Mechanical ventilation (MV) can induce pulmonary fibrosis. This study aims to investigate whether MV-induced pulmonary fibrosis is associated with aerobic glycolysis and seeks to uncover the underlying mechanisms mediated by integrin β3-pyruvate kinase M2 (PKM2) pathway. Methods: PKM2 knockdown or inhibition, integrin β3 knockout or inhibition and wild-type mice were exposed to MV (20 mL/kg) for 2 h. Results: Mice exposed to MV exhibited increased expression of collagen deposition, and upregulation of α-smooth muscle actin and collagen I in lung tissues. Single cells analysis showed that MV-induced pulmonary fibrosis was associated with increased gene expression of integrin and glycolysis in pulmonary fibroblasts, as well as upregulation of glycolytic products tested by metabolomics. Meanwhile, increased protein level of integrin β3 and PKM2 was confirmed by western blot and immunohistochemistry. Double immunofluorescence staining and flow cytometric analysis showed increased number of fibronectin+/integrin β3+ and fibronectin+/PKM2+ fibroblasts in lung tissues. Furthermore, MV-induced aerobic glycolysis and pulmonary fibrosis were ameliorated after treatment with PKM2 knockdown-AAV and inhibition, or in integrin β3 knockout and inhibition mice. Conclusions: Integrin β3-PKM2 pathway-mediated aerobic glycolysis contributes to MV-induced pulmonary fibrosis. The inhibition of aerobic glycolysis targeting integrin β3-PKM2 pathway may be a promising treatment for MV-induced pulmonary fibrosis.
© The author(s).

Breast tumors display tremendous heterogeneity in part due to varying molecular alterations, divergent cells of origin, and differentiation. Understanding where and how this heterogeneity develops is likely important for effective breast cancer eradication. Insulin-like growth factor (IGF) signaling is critical for normal mammary gland development and function, and has an established role in tumor development and resistance to therapy. Here we demonstrate that constitutive activation of the IGF1 receptor (IGF1R) influences lineage differentiation during mammary tumorigenesis. Transgenic IGF1R constitutive activation promotes tumors with mixed histologies, multiple cell lineages and an expanded bi-progenitor population. In these tumors, IGF1R expands the luminal-progenitor population while influencing myoepithelial differentiation. Mammary gland transplantation with IGF1R-infected mammary epithelial cells (MECs) resulted in hyperplastic, highly differentiated outgrowths and attenuated reconstitution. Restricting IGF1R constitutive activation to luminal versus myoepithelial lineage-sorted MECs resulted in ductal reconstitutions co-expressing high IGF1R levels in the opposite lineage of origin. Using in vitro models, IGF1R constitutively activated MCF10A cells showed increased mammosphere formation and CD44+/CD24-population, which was dependent upon Snail and NFκB signaling. These results suggest that IGF1R expands luminal progenitor populations while also stimulating myoepithelial cell differentiation. This ability to influence lineage differentiation may promote heterogeneous mammary tumors, and have implications for clinical treatment.
Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.

Dendritic cells (DCs) are adept at cross-presentation and initiation of antigen-specific immunity. Clinically, however, DCs produced by in vitro differentiation of monocytes in the presence of exogenous cytokines have been met with limited success. We hypothesized that DCs produced in a physiological manner may be more effective and found that platelets activate a cross-presentation program in peripheral blood monocytes with rapid (18 hours) maturation into physiological DCs (phDCs). Differentiation of monocytes into phDCs was concomitant with the formation of an "adhesion synapse," a biophysical junction enriched with platelet P-selectin and monocyte P-selectin glycoprotein ligand 1, followed by intracellular calcium fluxing and nuclear localization of nuclear factor κB. phDCs were more efficient than cytokine-derived DCs in generating tumor-specific T cell immunity. Our findings demonstrate that platelets mediate a cytokine-independent, physiologic maturation of DC and suggest a novel strategy for DC-based immunotherapies.
Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) involves the reactivation of endogenous pluripotency genes and global DNA demethylation, but temporal resolution of these events using existing markers is limited. Here, we generate murine transgenic lines harboring reporters for the 5-methylcytosine dioxygenase Tet1 and for Oct4. By monitoring dual reporter fluorescence during pluripotency entry, we identify a sequential order of Tet1 and Oct4 activation by proximal and distal regulatory elements. Full Tet1 activation marks an intermediate stage that accompanies predominantly repression of somatic genes, preceding full Oct4 activation, and distinguishes two waves of global DNA demethylation that target distinct genomic features but are uncoupled from transcriptional changes. Tet1 knockout shows that TET1 contributes to both waves of demethylation and activates germline regulatory genes in reprogramming intermediates but is dispensable for Oct4 reactivation. Our dual reporter system for time-resolving pluripotency entry thus refines the molecular roadmap of iPSC maturation.
Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.

The generation of induced pluripotent stem cells (iPSCs) involves activation of the endogenous pluripotency circuitry and global DNA demethylation late in reprogramming, but temporal resolution of these events using existing markers is insufficient. Here, we generated murine transgenic lines harboring dual fluorescent reporters reflecting cell-state specific expression of the master pluripotency factor Oct4 and the 5-methylcytosine dioxygenase Tet1 . By assessing reprogramming intermediates based on dual reporter patterns, we identified a sequential order of Tet1 and Oct4 gene activation at proximal and distal regulatory elements following pluripotency entry. Full induction of Tet1 marks a pivotal late intermediate stage occurring after a phase of global gene repression, and preceding full activation of Oct4 along with late naive pluripotency and germline-specific genes. Sequential activation of Tet1 further distinguishes two waves of global DNA demethylation, targeting distinct genomic features and largely uncoupled from transcriptional changes, with dynamics unique to iPSC reprogramming. Moreover, we demonstrate that loss of Tet1 is compatible with reprogramming towards full Oct4 gene activation, but generates iPSCs with aberrant DNA methylation, chromosomal instability during lineage priming and defective differentiation potential. Therefore, the transcriptional logic of Tet1 expression signals a deterministic epigenetic roadmap towards generation of high quality iPSCs.