USP31 PROMOTES THE INFLAMMATORY RESPONSE OF HUMAN LUNG ORGANOIDS TO LIPOPOLYSACCHARIDE TREATMENT

ABSTRACT: Background: Acute lung injury (ALI) is a severe condition characterized by a high mortality rate, driven by an uncontrolled inflammatory response. Emerging evidence has underscored the crucial role of the ubiquitin system in ALI. However, because of their vast number, the specific functions of individual ubiquitination regulators remain unclear. Materials and methods: In this study, we established human lung organoids (HLOs) derived from human embryonic stem cells and subjected them to lipopolysaccharide (LPS) treatment to induce an inflammatory response, mimicking ALI. Subsequently, we detected the expression of inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukin 6, interleukin 18 (IL-18), and interleukin-1β (IL-1β), by quantitative polymerase chain reaction experiments. We also detected changes in the mRNA expression of several USPs before and after HLOs treatment and thus screened for USPs that had significant changes in HLOs after LPS stimulation. After screening for USP, we silenced the USP in HLOs and then subjected them to LPS treatment, and TNF-α, IL-6, IL-18, and IL-1β expressions were detected using quantitative polymerase chain reaction assays. Meanwhile, western blot was used to detect changes in NOD-, LRR-, and pyrin domain-containing 3 (NLRP3) and apoptosis-associated Speck-like protein containing a CARD (ASC) protein level in HLOs. Results: Through screening the expression of 40 ubiquitin-specific proteases (USPs), which are responsible for removing ubiquitination, we identified several USPs that exhibited differential expression in LPS-treated HLOs compared to untreated HLOs. Notably, USP31 emerged as the most significantly upregulated USP, and the knockdown of USP31 markedly attenuated the inflammatory response of HLOs to LPS treatment. Conclusions: USP31 may play a facilitating role in the inflammatory response during ALI.