C107-12 The E3 Ubiquitin Ligase ARIH2 Regulates the Phospho-ERK-NFkB-NLRP3 Axis to Prevent LPS-induced Acute Lung Injury

Rationale Acute Respiratory Distress Syndrome (ARDS) and Acute Lung Injury (ALI), often triggered by severe bacterial infections, remain major causes of morbidity and mortality in critically ill patients. The core pathology involves uncontrolled inflammation driven by bacterial components like Lipopolysaccharide (LPS), activating the Toll-like receptor 4 (TLR4) pathway, leading to a surge of pro-inflammatory cytokines and massive immune cell infiltration. Ariadne RBR E3 Ubiquitin Ligase 2 (ARIH2) is a known immunoregulator that limits NFκB signaling, but its specific function and mechanism of action in LPS-induced ALI remain entirely unexplored. We investigated ARIH2’s immunomodulatory role in ALI to identify potential therapeutic targets. Methods For in vitro investigation, we manipulated ARIH2 expression in murine RAW264.7 macrophages using AAV6.2-ARIH2 (overexpression) and siARIH2 (knockdown), followed by LPS stimulation. Western blotting quantified key inflammatory intermediates. For the in vivo study, C57BL/6 mice were pre-treated intranasally with AAV6.2-ARIH2 or a control vector (AAV6.2-EGFP) before LPS challenge. ALI severity was assessed through lung histology (H&E), analysis of bronchoalveolar lavage fluid (BALF) for total protein content and immune cell populations (via flow cytometry), and Multiplex Luminex assays for pro-inflammatory cytokines in lung homogenates. Results In vitro, ARIH2 knockdown significantly amplified LPS-mediated increases in activated signaling proteins, including pERK1/2, pNFκB, and the inflammasome component NLRP3. Conversely, ARIH2 overexpression potently inhibited the activation of these pathways. In vivo, ARIH2 overexpression conferred robust protection against ALI, evidenced by: 1) markedly preserved alveolar-capillary barrier integrity, indicated by significantly lower total protein content in BALF; 2) histological improvement and a sharp reduction in total immune cell infiltration in both lung tissue and BALF, particularly affecting the double-negative T (DNT) cell population; and 3) a significant reduction in key pro-inflammatory cytokines in lung homogenates. Mechanistically, this protection was confirmed by the inhibition of the pERK1/2, pNFκB, and NLRP3 inflammatory markers in the lung tissue. Conclusions Our findings establish ARIH2 as a critical, intrinsic negative regulator of the severe inflammatory response central to ALI. Its protective function is mediated by its ability to control the core pERK-NFκB-NLRP3 signaling axis, effectively dampening the cytokine storm and limiting destructive immune cell recruitment. This study unveils ARIH2 as a promising novel therapeutic target for developing urgently needed, targeted anti-inflammatory strategies to treat ALI and other acute pulmonary inflammatory conditions. This abstract is funded by: The Ohio State University Startup (PG100125)