Characterizing The Role Of Neutrophils In A Mouse Model Of Fibrodysplasia Ossificans Progressiva
Wesley Huang, BS, Amanda Huber, PhD, Charles Hwang, BS, Nicole Patel, BS, Chase Pagani, BA, Noelle Visser, MS, Kaetlin Vasquez, MS, Nicole Edwards, PhD, Karen Kessell, BS, Shuli Li, MD/PhD, Haichun Pan, PhD, Yuji Mishina, PhD, Benjamin Levi, MD.
University of Michigan, Ann Arbor, MI, USA.
Purpose: Fibrodysplasia Ossificans Progressiva (FOP) is a debilitating congenital disease that causes heterotopic ossification (HO) of soft tissue during flares or after injury. Inflammation is crucial for the pathogenesis of disease and previous studies have demonstrated a characteristic acute influx of inflammatory cells including neutrophils. Additionally, there are no current biomarkers to identify flares. Neutrophils possess the unique ability to extrude their chromatin with degradative enzymes, termed neutrophil extracellular traps (NETs). Given the demonstrated ability for NETs and their byproducts to propagate further inflammation, specifically in diseases with characteristic spontaneous “flares”, like Rheumatoid Arthritis (RA), we hypothesize that neutrophils and NETs play a role in, and may serve as an early biomarker for FOP flares.
Methods: Neutrophils at post-injury day 1 and 3 from a tamoxifen-inducible cardiotoxin-mediated FOP mouse model (Fig A), mice with the R206H genetic mutation in the Activin A receptor (ACVR1), were analyzed via single-cell RNA sequencing (scRNA). FOP neutrophil phagocytic and reactive oxygen species (ROS)-generating ability of peripheral and bone marrow granulocytes, respectively, (n=3/group) were measured. Immunofluorescent (IF) microscopy (n=2-4/group/time point) of NETosis markers citrullinated histone H3 (H3cit) and myeloperoxidase (MPO) at the site of HO formation was performed. Neutrophils were stimulated to NET in order to quantify (n=2-3/group) the sensitivity of neutrophils to NET in FOP and WT neutrophils. Finally, human plasma samples from FOP (n=10) and non-FOP controls (n=6) were analyzed for NETs by ELISA.
Results: Our scRNA data in FOP neutrophils demonstrated an association with Gene Ontology (GO) terms “immune system process” and “response to stimulus” while WT neutrophils associated with “biological regulation,” suggesting FOP neutrophils are more primed for an inflammatory response to stimuli. Further, KEGG pathway analysis demonstrated “TLR signaling” as one of the top 10 upregulated pathways. There was no significant difference in phagocytic ability (p=0.30) or ROS-generating ability (p=0.14) in FOP vs WT neutrophils (Fig B). IF demonstrated that NETs are found along the sheaths of fascial planes (Fig C). FOP neutrophils generate significantly more NETs (p=<0.0001) when stimulated with calcium ionophore (A23187) (Fig D). There was a trend of increased cfDNA in plasma from FOP patients compared to non-FOP controls that did not reach significance (p=0.12).
Conclusion: These studies demonstrate that FOP neutrophils may by hyper-responsive to external stimuli, and are more “primed” to undergo NETosis, even prior to NETosis induction. NETs were present in fascial planes, locally at sites where HO eventually forms in FOP. scRNA studies indicate that TLR signaling may play an important role in FOP neutrophils. Together, our data in FOP neutrophils suggest important differences from non-FOP neutrophils and that NETosis may play a pathogenic role in the formation of HO.
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