Neutrophil and NETosis Modulation in Traumatic Heterotopic Ossification

Objective: To characterize the role of neutrophil extracellular traps (NETs) in heterotopic ossification formation and progression and to use mechanical and pharmacological methods to decrease NETosis and mitigate heterotopic ossification (HO) formation. Summary Background Data: Traumatic heterotopic ossification (HO) is the aberrant osteochondral differentiation of mesenchymal progenitor cells following traumatic injury, burns, or surgery. While the innate immune response has been shown to be necessary for HO formation, the specific immune cell phenotype and function remains unknown. Neutrophils, one of the earliest immune cells to respond following HO inducing injuries, can extrude DNA, forming highly inflammatory neutrophil extracellular traps. We hypothesized that neutrophils and NETs would be diagnostic biomarkers and therapeutic targets for the detection and mitigation of HO. Methods: C57BL6J mice underwent burn/tenotomy (BT) (a well-established mouse model of HO) or a non-HO-forming sham injury. These mice were either 1) ambulated ad libitum, 2) ambulated ad libitum with daily intraperitoneal hydroxychloroquine (HCQ), ODN-2088 (both known to affect NETosis pathways), or control injections, or 3) had the injured hind limb immobilized. Single-cell analysis was performed to analyze neutrophils, NETosis, and downstream signaling following the HO-forming injury. Immunofluorescence (IF) microscopy was used to visualize NETosis at the HO site and neutrophils were identified using flow cytometry. Serum and cell lysates from HO sites were analyzed using ELISA for MPO-DNA and ELA2-DNA complexes to identify NETosis. Micro-CT (uCT) was performed on all groups to analyze the HO volume. Results: Molecular and transcriptional analyses revealed the presence of NETs within the HO injury site, which peaked in the early phases after injury. These NETs were highly restricted to the HO site, with gene signatures derived from both in vitro NET induction and clinical neutrophil characterizations showing a high degree of NET “priming” at the site of injury, but not in neutrophils in the blood or bone marrow. Cell-cell communication analyses revealed that this localized NET formation coincided with high levels of Toll-like receptor (TLR) signaling specific to neutrophils at the injury site. Reducing the overall neutrophil abundance within the injury site, either pharmacologically through treatment with hydroxychloroquine (HCQ), the TLR9 inhibitor OPN-2088, or mechanical treatment with limb offloading, results in mitigation of HO formation. Conclusions: These data provide a further understanding of the ability of neutrophils to form NETs at the injury site, clarify the role of neutrophils in HO, and identify potential diagnostic and therapeutic targets for HO mitigation.