Inhibition of neutrophil infiltration and NETosis ameliorates sepsis-induced cardiac injury and reduces myocardial inflammation and apoptosis

Aims: Sepsis-induced cardiomyopathy (SIC) lacks effective targeted therapies. This study investigated whether inhibiting neutrophil infiltration and neutrophil extracellular traps (NETosis) preserves cardiac function and mitigates inflammation and Apoptosis in sepsis.

Methods: We performed analyses of gene-expression microarray datasets and single-cell RNA Sequencing to define the SIC landscape, and generated cardiac RNA Sequencing in LPS-treated versus control mice to validate microarray signatures. In vitro, we used two complementary models, purified NETs applied to HL-1 cardiomyocytes and HL-1-neutrophil coculture to induce NETs, ROS, inflammation, and Apoptosis,to evaluate NET-targeting interventions, including CI-amidine, PAD4 inhibition; Sivelestat, neutrophil Elastase inhibition; DNase I, NET degradation. To define NET subtype, we verified that PMA-induced NETs were NADPH oxidase-dependent by demonstrating apocynin sensitivity, and we further showed that the mitochondrial ROS scavenger Mito-TEMPO reduced NET-induced ROS in HL-1 cells. In vivo, mice were assigned to control, CLP, or LPS groups; CLP/LPS mice received vehicle or a NET inhibitor. Echocardiography, plasma LDH/CK-MB, IL-1β/6/TNF-α, cardiac NETs (Ly6G/PAD4, MPO/NE IF) and Apoptosis (TUNEL, Bax/Bcl-2/cleaved Caspase-3) were blindly assessed.

Results: Datasets-based analyses highlighted immune activation with programmed cell death enrichment, prominently featuring NETosis pathways. Cardiac RNA Sequencing in LPS hearts confirmed upregulation of neutrophil/NET-related signatures. Mechanistically, the induced NETs were NADPH oxidase-dependent. In vitro, both purified NETs intervention and HL-1-neutrophil coculture were used to elicit ROS, inflammatory signaling, and Apoptosis, and NET-inducing conditions were consistently attenuated by netosis-targeted interventions. CLP and LPS animal model produced early declines in EF/FS, rises in LDH/CK-MB and proinflammatory cytokines, increased cardiac neutrophil infiltration and NET burden, and enhanced cardiomyocyte Apoptosis. The neutrophil-targeted interventions reduced cardiac neutrophils and NETs, lowered IL-1β/IL-6/TNF-α, decreased TUNEL positivity and apoptotic signaling, and preserved systolic function.

Conclusions: Our findings indicate that NETosis and neutrophil infiltration are therapeutically actionable contributors in SIC. Strategies that inhibit NET formation, degrade extracellular DNA, or limit neutrophil recruitment were associated with reduced myocardial inflammation and Apoptosis and partial preservation of systolic function. These results provide a theoretical basis for early diagnosis, timely intervention, and improved prognosis in SIC.

Apoptosis; Inflammation; Neutrophil extracellular traps; Neutrophils; Sepsis-induced cardiomyopathy.