Sepsis is a life-threatening condition with limited therapeutic options, characterized as excessive systemic inflammation and multiple organ failure. Macrophages play critical roles in sepsis pathogenesis. Metabolism orchestrates homeostasis of macrophages. However, the precise mechanism of macrophage metabolism during sepsis remains poorly elucidated. In this study, we identified the key role of zinc fingers and homeoboxes (Zhx2), a ubiquitous transcription factor, in macrophage glycolysis and sepsis by enhancing 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (Pfkfb3) expression. Mice with myeloid Zhx2-specific deletion (abbreviated as MKO) showed more resistance to cecal ligation and puncture and LPS-induced sepsis, exhibiting as prolonged survival, attenuated pulmonary injury, and reduced level of proinflammatory cytokines, such as TNF-α, IL-6, and IL-1β. Interestingly, Zhx2 deletion conferred macrophage tolerance to LPS-induced glycolysis, accompanied by reduced proinflammatory cytokines and lactate. Consistently, treatment of glycolytic inhibitor 2-deoxyglucose almost completely abrogated the protection of mice from LPS-induced sepsis initiated by Zhx2 deletion in macrophages. RNA sequencing and chromatin immunoprecipitation assays confirmed that Zhx2 enhanced transcription of Pfkfb3, the glycolysis rate-limiting enzyme, via binding with Pfkfb3 promoter. Furthermore, Pfkfb3 overexpression not only rescued the reduction of macrophage glycolysis caused by Zhx2 deficiency, displaying as extracellular acidification rates and lactate production but also destroyed the resistance of mice to LPS-induced sepsis initiated by transfer of bone marrow–derived macrophages from MKO mice. These findings highlight the novel role of transcription factor Zhx2 in sepsis via regulating Pfkfb3 expression and reprogramming macrophage metabolism, which would shed new insights into the potential strategy to intervene sepsis.

Background: Liver cancer stem cells (CSCs) are critical determinants of HCC relapse and therapeutic resistance, but the mechanisms underlying the maintenance of CSCs are poorly understood. We aimed to explore the role of tumor repressor Zinc-fingers and homeoboxes 2 (ZHX2) in liver CSCs. Methods: CD133 + or EPCAM + stem-like liver cancer cells were sorted from tumor tissues of HCC patients and HCC cell lines by flow cytometry. In addition, sorafenib-resistant cells, tumor-sphere forming cells and side population (SP) cells were respectively cultured and isolated as hepatic CSCs. The tumor-initiating and chemoresistance properties of ZHX2-overexpressing and ZHX2-knockdown cells were analyzed in vivo and in vitro. Microarray, luciferase reporter assay, chromatin immunoprecipitation (ChIP) and ChIP-on-chip analyses were performed to explore ZHX2 target genes. The expression of ZHX2 and its target gene were determined by quantitative RT-PCR, western blot, immunofluorescence and immunohistochemical staining in hepatoma cells and tumor and adjacent tissues from HCC patients. Results: ZHX2 expression was significantly reduced in liver CSCs from different origins. ZHX2 deficiency led to enhanced liver tumor progression and expansion of CSC populations in vitro and in vivo. Re-expression of ZHX2 restricted capabilities of hepatic CSCs in supporting tumor initiation, self-renewal and sorafenib-resistance. Mechanically, ZHX2 suppressed liver CSCs via inhibiting KDM2A-mediated demethylation of histone H3 lysine 36 (H3K36) at the promoter regions of stemness-associated transcription factors, such as NANOG, SOX4 and OCT4. Moreover, patients with lower expression of ZHX2 and higher expression of KDM2A in tumor tissues showed significantly poorer survival. Conclusion: ZHX2 counteracts stem cell traits through transcriptionally repressing KDM2A in HCC. Our data will aid in a better understanding of molecular mechanisms underlying HCC relapse and drug resistance.

T cell Ig and mucin domain-containing molecule 3 (Tim-3) has been found to play important roles in autoimmune diseases, but whether Tim-3-mediated engulfment of apoptotic cells is involved in systemic lupus erythematosus (SLE) remains to be elucidated. In this study, we verified the role of human Tim-3 (hTim-3) as the receptor of phosphatidylserine (PS) in human embryonic kidney (HEK)293 cells, which initiated the engulfment of apoptotic cells. Both IgV and the mucin domain of Tim-3 were crucial in the phagocytosis of apoptotic cells, and there existed the key cytoplasmic domain for signal transduction. Alanine at 111, locating around the FG-CC' loop of hTim-3, was necessary for its engulfment of apoptotic cells. In accordance, Tim-3 on CD14 cells negatively correlated with the percentage of peripheral apoptotic cells in control subjects. However, although Tim-3 was significantly increased on CD14 cells in SLE patients, peripheral apoptotic cells remained much higher than those in control subjects. Tim-3 on CD14 cells showed positive correlation with percentage of apoptotic cells and level of dsDNA, indicating the involvement of Tim-3 in SLE. Accordingly, soluble Tim-3 (sTim-3) was significantly increased in plasma of SLE patients, which might contribute to higher expression of a disintegrin and metalloproteinase (ADAM)-10. Pretreatment with both plasma from SLE patients and recombinant sTim-3 greatly inhibited hTim-3-initiated phagocytosis of apoptotic cells. Furthermore, anti-Tim-3 antibody depletion of plasma from SLE patients reversed the decreased phagocytosis of apoptotic cells. Collectively, our data suggest that sTim-3 might play inhibitory roles in impaired Tim-3-mediated clearance of apoptotic cells in SLE.

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