Sex-dependent effects of peptidylarginine deiminases on neutrophil function and long-term outcomes after spinal cord injury

Abstract Traumatic spinal cord injury (SCI) initiates an influx of peripheral immune cells to the spinal cord parenchyma that compound tissue damage and restrict functional recovery. Neutrophils infiltrate the spinal cord within the first day after injury, releasing extracellular traps (NETs) comprised of decondensed DNA, modified histones, and granule enzymes, that can worsen tissue damage. Peptidylarginine demininases (PADs), particularly PAD4, have been indicated as mediators of NET formation by facilitating the decondensation of nuclear chromatin via histone citrullination. Though PADs have been shown to be regulated by sex hormones, sex-differences in PAD regulation of neutrophil function in the context of CNS injury have yet to be explored. In this work, we investigated the role of PADs in recovery after SCI using Cl-amidine, a pan-PAD inhibitor. Strikingly, Cl-amidine treated mice exhibited sex-dependent changes to motor function, body weight, and white matter sparing after SCI. Acutely, Cl-amidine treated mice had reduced NET accumulation in the blood and decreased spinal cord neutrophil granularity. Analysis of publicly available scRNA-seq data revealed that female bone marrow neutrophils exhibited elevated Padi4 expression relative to their male counterparts. We then utilized Padi4 knockout ( Padi4 -/- ) mice to assess the role of PAD4 in long-term recovery of male and female mice after SCI. While we observed no changes in motor recovery, a sex-dependent effect on tissue sparing was observed with Padi4 deficiency. These data are the first description of sex differences in PAD-mediated neutrophil function after SCI and highlight the importance of inclusion of both sexes in pre-clinical research.