PARP3 promotes macrophage inflammation via mono ADP ribosylation of Ppia Glu140.

Background: Acute lung injury (ALI) carries significant mortality with limited targeted therapies. Macrophages drive early inflammatory propagation in ALI, exacerbating pulmonary inflammation. While ADP-ribosylation is a dynamic and reversible post-translational modification (PTM) associated with inflammatory diseases, its role in macrophage-mediated inflammation remains unclear.

Methods: Murine ALI model was established via intratracheal instillation with lipopolysaccharide (LPS). The ALI lung tissues and cultured mouse macrophage line (RAW264.7) treated with LPS were used to assess the expression of poly ADP-ribose polymerases (Parps). RNA sequencing (RNA-seq) identified differentially expressed genes (DEGs) following Parp3 knockdown (siParp3) in LPS-stimulated RAW264.7 cells, with subsequent pathway analysis was via transcription factors (TFs) profiling and gene ontology (GO) enrichment. In RAW264.7 cells, Parp3 and peptidyl-prolyl cis-trans isomerase A (Ppia) was modulated by siRNA or plasmid transfection. PARP3-Ppia interaction and ADP-ribosylation were assessed by immunoprecipitation. Modification alterations due to mutations at Ppia modification sites were assessed by immunoprecipitation. Enzyme-linked immune sorbent assay (ELISA) was used to quantify Ppia secretion. A mouse ALI model was used to evaluate the lung-protective and therapeutic effects of PARP3 inhibitor ME0328 by detecting inflammatory cytokines, phosphorylation of p65 and lung histopathology.

Results: LPS induced the expression of Parp3 in RAW264.7 cells and ALI lung tissues, correlating with elevated inflammatory cytokines. The 52 overlapping DEGs were mainly enriched in Toll-like receptor (TLR) signaling pathway. PARP3 promoted inflammation via NF-κB activation. ME0328 blocked NF-κB pathway activation in RAW264.7 cells and lung tissues. Immunoprecipitation confirmed that PARP3 interacted with Ppia. Ppia was modified with mono ADP-ribosylation. Ppia-E140 was the most inflammation related modification site. The mutation of E140 inhibited inflammatory response, mono ADP-ribosylation and secretion of Ppia. In vivo, ME0328 reduced inflammatory response, alleviated pulmonary edema and mitigated histopathological damage.

Conclusions: We identified the NF-κB as the downstream signaling pathway mediated by Ppia for PARP3 to promote macrophage inflammation. ME0328 alleviated pulmonary inflammation through the NF-κB signaling pathway. Our findings provide evidence that macrophage inflammation is associated with the mono ADP-ribosylation on Ppia. Understanding mono ADP-ribosylation regulation in macrophage from ALI may provide insight into the pro-inflammatory mechanisms and opportunities for effective therapeutic to treat acute lung injury.