Novel mechanistic insights of inflammation and organ injury

Abstract A dysregulated host defense to bacterial pneumonia or sepsis often leads to overwhelming lung inflammation and potentially life-threatening conditions caused by organ damage. Circular RNA (circRNA) is a type of single- stranded RNA that forms a covalently closed continuous loop, unlike linear RNA. The expression of circRNAs in mammals is often conserved across species and shows tissue and cell specificity. CircRNA does not have 5' or 3' ends. The unique structure of circRNAs provides them with a longer half-life and more resistance to RNase R than linear RNAs, which makes them potential candidates for diagnostic biomarkers and therapeutic strategies. Via RNA sequencing and arrays, the highly induced circRNAs were determined after infectious stimuli in the lungs. Circular RNA-30884 (Circ30884) exhibits the greatest induction after various irritants, including LPS i.t., Streptococcus pneumoniae (Spn) i.t. (G+ bacteria), and Pseudomonas aeruginosa (PAE) i.t. (G- bacteria). Circ30884 is highly induced in lung MΦs, and robustly elevated in the extracellular vesicles (EVs) obtained from bronchial alveolar lavage fluid (BALF) and serum. Circ30884 is conserved between mice and humans and is derived from the xanthine dehydrogenase (XDH) gene via back-splicing. Modulating circ30884 does not impact its host gene XDH, suggesting that circ30884 carries a distinct function. Bacterial infection-induced N6- methyladenosine (m6A) of the host gene potentially regulated circ30884 biogenesis. Functionally, circ30884 promoted the secretion of proinflammatory cytokines and mΦ pyroptosis in vitro. For in vivo studies, we treated mice with the Exosome-cargo-antisense oligos (Exo-ASOs) for circ30884 via i.t. Deletion of circ30884 in lung MΦs in vivo using Exo-ASOs i.t. downregulated multiple inflammatory cytokines/chemokines in BALF and showed less lung inflammation in response to bacterial infections. On the other hand, circ30884 over-expression (OE) clones i.t. robustly induced lung inflammation. Mechanistically, overexpression of circ30884 promoted caspase-1 and gasdermin D cleavage and pore formation in AMs, suggesting induced pyroptosis by circ30884. Circ30884 physically bound with CSNK1α (CK1α), stabilized CK1α and subsequently modulated Wnt3a signaling and activated NLRP3 inflammasome in MΦs. CSNK1α is the kinase targeting NLRP3 at S803. Using computational modeling, we predicted the interacting motif sequences between Circ30884 and CK1α, providing a potentially precise target for the development of novel therapeutics. Our central hypothesis is that circ30884 facilitates bacterial infection-induced inflammation by enhancing mΦ pro-inflammatory activations and pyroptosis, via regulating NRLP3 inflammasome/ Gasdermin D activations. We propose three specific aims: Specific Aim I: To determine the expression and cellular function of circ30884 in mΦs in vitro. Specific Aim II: To dissect the molecular mechanisms of circ30884 on mΦ pyroptosis in vitro. Specific Aim III: To determine the role of circ30884 in inflammatory responses in vivo.