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

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.