Reactive oxygen species (ROS) generated by neutrophils provide a frontline defence against invading pathogens. We investigated the supportive effect of tonsil-derived mesenchymal stem cells (TMSCs) on ROS generation from neutrophils using promyelocytic HL-60 cells. Methods: Differentiated HL-60 (dHL-60) cells were cocultured with TMSCs isolated from 25 independent donors, and ROS generation in dHL-60 cells was measured using luminescence. RNA sequencing and real-time PCR were performed to identify the candidate genes of TMSCs involved in augmenting the oxidative burst of dHL-60 cells. Transcriptome analysis of TMSCs derived from 25 independent donors revealed high levels of procollagen C-endopeptidase enhancer 2 (PCOLCE2) in TMSCs, which were highly effective in potentiating ROS generation in dHL-60 cells. In addition, PCOLCE2 knockdown in TMSCs abrogated TMSC-induced enhancement of ROS production in dHL-60 cells, indicating that TMSCs increased the oxidative burst in dHL-60 cells via PCOLCE2. Furthermore, the direct addition of recombinant PCOLCE2 protein increased ROS production in dHL-60 cells. These results suggest that PCOLCE2 secreted by TMSCs may be used as a therapeutic candidate to enhance host defences by increasing neutrophil oxidative bursts. PCOLCE2 levels in TMSCs could be used as a marker to select TMSCs exhibiting high efficacy for enhancing neutrophil oxidative bursts.

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Background and Purpose Paracetamol (acetaminophen)‐induced hepatotoxicity is the leading cause of drug‐induced liver injury worldwide. Autophagy is a degradative process by which various cargoes are collected by the autophagic receptors such as p62/SQSTM1/Sequestosome‐1 for lysosomal degradation. Here, we investigated the protective role of p62‐dependent autophagy in paracetamol‐induced liver injury. Experimental Approach Paracetamol‐induced hepatotoxicity was induced by a single i.p. injection of paracetamol (500 mg·kg−1) in C57/BL6 male mice. YTK‐2205 (20 mg·kg−1), a p62 agonist targeting ZZ domain, was co‐ or post‐administered with paracetamol. Western blotting and immunocytochemistry were performed to explore the mechanism. Key Results N‐terminal arginylation of the molecular chaperone calreticulin retro‐translocated from the endoplasmic reticulum (ER) was induced in the livers undergoing paracetamol‐induced hepatotoxicity, and YTK‐2205 exhibited notable therapeutic efficacy in acute hepatotoxicity as assessed by the levels of serum alanine aminotransferase and hepatic necrosis. This efficacy was significantly attributed to accelerated degradation of ubiquitin (Ub) conjugates as well as damaged mitochondria (mitophagy) and endoplasmic reticulum (ER‐phagy). In primary murine hepatocytes treated with paracetamol, YTK‐2205 induced the co‐localization of p62+LC3+ phagophores to the sites of mitophagy and ER‐phagy. A similar activity of YTK‐2205 was observed with N‐acetyl‐p‐benzoquinone imine, a putative toxic metabolite of paracetamol in Hep3B cells. Conclusion and Implications Our results elucidated that p62‐dependent autophagy plays a key role in the removal of cytotoxic materials such as damaged mitochondria in paracetamol‐induced hepatotoxicity. Small molecule ligands to p62 may be developed into drugs to treat this pathological condition.