IBR1: A Novel Endogenous dsRNA Binding to the Helicase Domain of IFIH1 and Its Role in LPS-induced IFIH1 Activation and Macrophage M1 Polarization in ARDS [RNA-seq]
Uncontrolled inflammatory responses driven by macrophages and monocytes play a crucial role in perpetuating lung injury and the severity of acute respiratory distress syndrome (ARDS). Previous studies have identified IFIH1 as a key regulator of macrophage M1 polarization in pneumonia-induced ARDS. IFIH1 is a cytosolic receptor responsible for recognizing viral double-stranded RNA (dsRNA) and activating inflammatory and antiviral signaling pathways. Interestingly, our observations indicate that IFIH1 can also be activated by lipopolysaccharide (LPS), a component of bacterial cell walls. However, the precise molecular mechanism underlying LPS-induced IFIH1 activation remains unclear. In our preliminary investigations, we have discovered a novel transcript, named IFIH1-binding RNA1 (IBR1), which is upregulated by LPS and gram-negative bacilli in monocytes and macrophages. We have confirmed that IBR1 interacts with the IFIH1 protein, forming a complex. Further experiments have revealed that IBR1, acting as an endogenous dsRNA, binds to the helicase domain of IFIH1 due to its local double-stranded structure. This specific interaction serves as the underlying molecular mechanism responsible for LPS-induced IFIH1 activation and subsequent macrophage M1 polarization in ARDS. Importantly, our observations indicate a significant upregulation of IBR1 expression in ARDS patients with gram-negative bacilli-induced pneumonia. Understanding the molecular mechanisms driving IFIH1 activation and macrophage M1 polarization in ARDS is of great importance for developing new therapeutic strategies to alleviate lung inflammation and improve patient outcomes. Further research is warranted to elucidate the precise roles of IBR1 and its potential as a therapeutic target in the management of ARDS. Overall design: Bone Marrow-Derived Macrophage (BMDM) isolation, differentiation, and identification: Male C57BL/6 mice, aged 8 weeks, were euthanized by cervical dislocation. BMDMs were isolated from the bone marrow using established methods, including density gradient centrifugation and cell differentiation in the presence of M-CSF. The differentiated BMDMs were identified by flow cytometry analysis using macrophage-specific markers.  RIP-seq for IFIH1-bound RNA complexes: RNA extracts from the isolated BMDMs were subjected to RNA-binding protein immunoprecipitation (RIP) using the Magna RIPTM kit. The IFIH1-bound RNA complexes were enriched by incubating the RNA extracts with IFIH1 antibody-bound beads and RIP immunoprecipitation buffer. The purified RNAs from the IFIH1-bound complexes were then sequenced using Illumina Sequencing Technology, with 5% input RNA as a negative control to reduce noise.