Macrophage-intrinsic STAT3 drives LPS-induced acute lung injury: allosteric blockade by K116 and genetic validation...

Acute lung injury (ALI) is a life-threatening condition driven by uncontrolled pulmonary inflammation and macrophage-mediated cytokine release. Signal transducer and activator of transcription 3 (STAT3) is a central mediator of macrophage inflammatory activation. Although prior work demonstrated that targeting the STAT3 coiled-coil domain (CCD) with small-molecule inhibitors (e.g., K134) attenuates ALI, whether this protection depends strictly on macrophage-intrinsic STAT3, rather than off-target effects on pulmonary epithelial or endothelial cells, has never been e established by definitive in vivo genetic evidence. To address this, we used macrophage-specific STAT3-conditional knockout mice (STAT3^cKO; Lyz2-Cre × STAT3loxP/loxP) and wild-type C57BL/6 mice in an LPS-induced ALI model. Mice were treated with K116, a STAT3 CCD allosteric inhibitor, administered intraperitoneally at 2 h and 12 h after LPS exposure. Lung injury was assessed using histopathology, bronchoalveolar lavage fluid (BALF) analysis, ELISA, flow cytometry, and Western blotting. We found that STAT3^cKO mice were significantly protected from LPS-induced lung injury, exhibiting reduced inflammatory cell infiltration, decreased BALF protein and nitrite levels, and suppressed pro-inflammatory cytokine production. In wild-type mice, K116 effectively reversed established lung injury and inhibited macrophage activation by suppressing STAT3 phosphorylation at Y705 and S727 through CCD-mediated allosteric modulation. Importantly, K116 failed to confer additional protection in STAT3^cKO mice, phenocopying the effects of genetic deletion and demonstrating that its therapeutic efficacy is dependent on macrophage-intrinsic STAT3. Together, these findings identify macrophage-intrinsic STAT3 as a key driver of LPS-induced ALI and establish CCD-directed allosteric inhibition as a mechanistically validated and potentially translatable therapeutic strategy for inflammatory lung injury.