Neutrophils contribute to αPD1-induced latent myocardial injury via NET/NETosis axis: exploration of potential patho-mechanism, diagnostic biomarkers and therapeutic targets in ICIs-associated myocard
Y Chen, L Cheng, J GeAbstract
Background
Immune checkpoint inhibitors (ICIs) associated myocarditis (ICIAM) is a lethal complication caused mainly by cytotoxic CD8(+) T cells. Neutrophil extracellular traps (NETs) formation (NETosis), albeit an innate immune-mechanism, has also been implicated in various autoimmune diseases, yet its contribution to ICIAM remains unexplored.
Objective
This study aims to examine the role of NET/NETosis in the latent or overt ICIAM (OICIAM/LICIAM) induced by anti-programmed cell death protein-1 antibody (αPD1), and to explore the potential values of NET/NETosis in early detection and treatment targets of ICIAM.
Methods
The study first performed scRNA-seq of PBMCs from LICIAM patients and RNA-seq of myocardial tissues from melanoma-bearing mice with LICIAM and OICIAM to identify the differential expression genes (DEGs) and enrich potential pathway related to ICIAM. Then further assess the transcriptional and expression alterations of NET/NETosis axis in LICIAM mice, LICIAM mice treated with PF-1355 alone or in combination with PMA, with RT-qPCR, western blotting, and histopathological studies. Patients with LICIAM/OICIAM were recruited to essay serum biomarkers related to NET/NETosis and pyroptosis.
Results
Transcriptomic enrichment results from cancer patients and tumor-bearing mice with LICIAM identified the DEGs including CXCR1, GZMB, and CAPZB in humans, as well as Ly6g and Padi4 in mice, with pathway analyses consistently implicating neutrophils activation and NET/NETosis. Myocardial tissues from LICIAM mice demonstrated significantly increased fluorescence intensities of MPO and CitH3, accompanied by elevated mRNA levels of NETosis-related genes (Mpo, Ly6g, Padi4) and neutrophil-related chemokines (Cxcl2, Il17, Ifnγ) (all P<0.0001). Concurrently, the transcriptional and expressional level of NLRP3 inflammasome-pyroptosis pathway was activated. Treatment with the MPO inhibitor PF-1355 effectively reduced these changes, which could be totally reversed by PMA co-treatment, thereby confirming mechanistic involvement. In the clinical cohort with control (n=20), LICIAM (n=20), and OICIAM (n=20) groups, NETosis biomarkers (CitH3 and cfDNA) were strongly correlated with ICIAM patients, especially in LICIAM group (all R>0.80), while the conventional myocardial injury marker hs-cTnT showed only a moderate-to-strong association (all R≤0.75), suggesting that NETosis-related biomarkers may offer additional value to hs-cTnT for the early detection of LICIAM.
Conclusion
This study demonstrates that NET/NETosis contributes to ICIAM, particularly LICIAM, blocking NET/NETosis may be promising for ICIAM treatment, and assessing NETosis-related biomarkers may provide additional values to cardiac troponins for LICIAM detection. However, these findings based on animal experiments warrant further clinical verifications.central graphical abstract