Abstract Rationale Acute lung injury (ALI) is characterized by increased vascular permeability and excessive inflammatory responses, often triggered by bacterial pathogens such as methicillin-resistant Staphylococcus aureus (MRSA). Endothelial cells (EC) are critical regulators of vascular integrity and immune signaling during lung injury. Lysosomal proteases, including cathepsin S (CTSS), have been implicated in the pathogenesis of inflammatory diseases such as acute respiratory distress syndrome (ARDS). However, whether CTSS mediates lung endothelial injury remains largely unclear. This study aimed to investigate the role of CTSS in MRSA-induced endothelial barrier disruption and inflammation, and to evaluate the therapeutic potential of targeting CTSS expression or activity in the context of ALI. Methods Bulk RNA sequencing was performed on human lung microvascular endothelial cells (HLMVEC) and human pulmonary artery endothelial cells (HPAEC) following treatment with heat-killed MRSA (2.5 × 108 CFU/mL for 18 hours). CTSS protein expression was evaluated by western blotting (WB) in MRSA-treated lung EC and in bronchoalveolar lavage (BAL) fluid from C57BL/6 mice exposed to MRSA (2.5 × 108 CFU/mouse, intratracheally, 18 hours). To investigate the functional role of CTSS, its expression was silenced using siRNA, or its activity was inhibited using BI-1915 (opnMe, Boehringer Ingelheim) prior to MRSA exposure. In separate experiments, recombinant CTSS (rCTSS) was applied to lung EC. Inflammatory markers were quantified by WB and ELISA. Endothelial barrier integrity was assessed using electric cell-substrate impedance sensing (ECIS) and immunofluorescence staining for VE-cadherin and claudin-5. Results RNA-seq analysis identified CTSS as one of the most upregulated genes in both HLMVEC and HPAEC. Protein expression of CTSS was significantly elevated in lysates and supernatants from MRSA-treated lung EC, as well as in BAL fluid from mice subjected to a MRSA-induced ALI model. Silencing CTSS via siRNA markedly reduced MRSA-induced expression of VCAM-1 and IL-6 in lung EC. Similarly, pharmacological inhibition of CTSS activity in EC treated with MRSA attenuated pro-inflammatory signaling (VCAM-1 expression and IL-6 release) and decreased endothelial permeability, as measured by ECIS. Furthermore, rCTSS disrupted endothelial barrier integrity (as determined by ECIS) and caused disassembly of VE-cadherin and claudin-5 junctions. Conclusion MRSA-induced upregulation of CTSS in lung EC contributes to increased endothelial permeability and pro-inflammatory signaling. These findings highlight CTSS as a potential therapeutic target for mitigating vascular dysfunction and inflammation in ALI. This abstract is funded by: American Lung Association, Innovation Award #1445152 (EL) and NIH R01 HL167518 (SD)
Letsiou et al. (Fri,) studied this question.