Background. Myelosuppression is a common condition during chemotherapy. Bone-associated mesenchymal stem cells (BA-MSCs) play an essential role in the composition of the hematopoietic microenvironment and support hematopoietic activity. However, chemotherapy-induced damage to BA-MSCs is rarely studied. Recent studies have shown that platelets promote the wound-healing capability of MSCs by mitochondrial transfer. Therefore, this study is aimed at investigating the chemotherapy-induced damage to BA-MSCs and the therapeutic effect of platelet-derived mitochondria. Material/Methods. We established in vivo and in vitro BA-MSC chemotherapy injury models using the chemotherapy agent 5-fluorouracil (5-FU). Changes in the mitochondrial dynamics were detected by transmission electron microscopy, and the expression of mitochondrial fusion and fission genes was analyzed by qRT-PCR. In addition, mitochondrial functions were also explored by flow cytometry and luminometer. Platelet-derived mitochondria were incubated with 5-FU-damaged BA-MSCs to repair the injury, and BA-MSC functional changes were examined to assess the therapy efficacy. The mechanism of treatment was explored by studying the expression of mitochondrial fission and fusion genes and hematopoietic regulatory factor genes in BA-MSCs. Results. Stimulation with 5-FU increased the apoptosis and suppressed cell cycle progression of BA-MSCs both in vivo and in vitro. In addition, 5-FU chemotherapy inhibited the hematopoietic regulatory ability and disrupted the mitochondrial dynamics and functions of BA-MSCs. The mitochondrial membrane potential and ATP content of 5-FU-injured BA-MSCs were decreased. Interestingly, when platelet-derived mitochondria were transferred to BA-MSCs, the 5-FU-induced apoptosis was alleviated, and the hematopoietic regulatory ability of 5-FU-injured BA-MSCs was effectively improved by upregulating the expression of mitochondrial fusion genes and hematopoietic regulatory factor genes. Conclusion. BA-MSCs were severely damaged by 5-FU chemotherapy both in vivo and in vitro. Meanwhile, platelet-derived mitochondria could attenuate the 5-FU-induced injury to BA-MSCs, which provides future research directions for exploring the treatment strategies for chemotherapy-injured BA-MSCs and establishes a research basis for related fields.

Inflammatory caspase-11/4/5 recognize cytosolic LPS from invading Gram-negative bacteria and induce pyroptosis and cytokine release, forming rapid innate antibacterial defenses. Since extracellular or vacuole-constrained bacteria are thought to rarely access the cytoplasm, how their LPS are exposed to the cytosolic sensors is a critical event for pathogen recognition. Hemolysin is a pore-forming bacterial toxin, which was generally accepted to rupture cell membrane, leading to cell lysis. Whether and how hemolysin participates in non-canonical inflammasome signaling remains uncovered. Here, we show that hemolysin-overexpressed enterobacteria triggered significantly increased caspase-4 activation in human intestinal epithelial cells (IECs). Hemolysin promoted LPS cytosolic delivery from extracellular bacteria through dynamin-dependent endocytosis. Further, we revealed that hemolysin was largely associated with bacterial outer membrane vesicles (OMVs) and induced rupture of OMV-containing vacuoles, subsequently increasing LPS exposure to the cytosolic sensor. Accordingly, overexpression of hemolysin promoted caspase-11 dependent IL-18 secretion, gut inflammation, and enterocyte pyroptosis in orally-infected mice, which was associated with restricting bacterial colonization in vivo. Together, our work reveals a concept that hemolysin promotes noncanonical inflammasome activation via liberating OMVs for cytosolic LPS sensing, which offers insights into innate immune surveillance of dysregulated hemolysin via caspase-11/4 in intestinal antibacterial defenses. Significance: Sensing of lipopolysaccharide (LPS) in the cytosol triggers non-canonical inflammasome-mediated innate responses. Recent work revealed that bacterial outer membrane vesicles (OMVs) enables LPS to access the cytosol for extracellular bacteria. However, since intracellular OMVs are generally constrained in endosomes, how OMV-derived LPS gain access to the cytosol remains unknown. Here, we reported that hemolysin largely bound with OMVs and entered cells through dynamin-dependent endocytosis. Intracellular hemolysin significantly impaired OMVs-constrained vacuole integrity and increased OMV-derived LPS exposure to the cytosolic sensor, which promoted non-canonical inflammasome activation and restricted bacterial gut infections. This work reveals the role of hemolysin in promoting non-canonical inflammasome activation and alerting host immune recognition, which provides insights into the more sophisticated biological functions of hemolysin upon infection.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common drug-resistant bacteria that cause community and hospital infections. As one of the most common “superbugs” and the pathogen with the highest global incidence of hospital-acquired infections, MRSA has developed resistance to multiple antibiotics, posing a serious threat to public health. The rapid emergence and spread of multidrug resistance have increased the urgent need for new antimicrobial strategies and agents to combat MRSA-associated infections. In recent years, platelets have been widely recognized to play an important role in human immune defense. We have previously reported that platelets inhibit MRSA by inducing hydroxyl radical (OH ^● )-mediated apoptosis-like cell death. To further explore the platelet antibacterial mechanism, supernatants from co-culture of platelets and MRSA in vitro were used for proteomic analysis. Based on our observations using confocal and immunoelectron microscopy, we found a previously unrecognized platelet antimicrobial peptide, dermcidin (DCD), in the alpha ( α -) granules. Furthermore, after co-culturing with MRSA in vitro , activated platelets secreted large amounts of DCD. Additionally, we confirmed that DCD displayed anti-MRSA activity in a concentration-dependent manner and contributed to the inhibition of MRSA growth by platelets in vitro . Our findings provide important insights into the immune defense functions of platelets. IMPORTANCE: The emerging multidrug resistance in many pathogenic bacterial species poses a serious problem worldwide. Methicillin-resistant Staphylococcus aureus (MRSA), one of the most common gram-positive pathogenic bacteria, has also evolved strains with multidrug resistance. This calls for the urgent development of novel and effective treatments or bactericidal agents to mitigate this issue in clinical settings. In this study, we identified for the first time a previously unrecognized platelet antimicrobial peptide, DCD, which impedes the proliferation of MRSA and promotes the antibacterial effect of platelets on MRSA. Our findings enrich our understanding of platelet physiological function and antibacterial mechanisms and provide new insights into the development of novel natural antimicrobial agents for controlling infections.

The destruction of red blood cells by classical complement activation can form erythrocyte ghosts in immune-mediated hemolysis. However, the mechanisms involved in clearing these membrane remnants remain to be elucidated. In this study, a combination of in vivo models, in vitro macrophage co-culture systems, proteomic screening, and functional validation assays was employed to investigate the cellular processing of erythrocyte ghosts. We demonstrated that erythrocyte ghosts are engulfed by macrophage phagocytosis. Besides, a significant downregulation of CD47, a critical “don’t-eat-me” signal, is observed on the membranes of erythrocyte ghosts due to ubiquitin-proteasome-mediated degradation, facilitating their phagocytic removal by macrophages. Moreover, the E3 ubiquitin ligase MARCH 1 was identified as the principal mechanistic regulator governing the loss of CD47. These findings uncover a critical pathway for the efficient clearance of hemolytic remnants, offer alternative perspectives on post-hemolytic immune homeostasis, and suggest potential therapeutic avenues for reducing complications associated with hemolysis.