Neutrophil and NETosis Modulation in Traumatic Heterotopic Ossification

Abstract

Objective: To characterize the role of neutrophil extracellular traps (NETs) in heterotopic ossification (HO) formation and progression and to use mechanical and pharmacological methods to decrease NETosis and mitigate HO formation. Background: Traumatic HO is the aberrant osteochondral differentiation of mesenchymal progenitor cells after 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 remain unknown. Neutrophils, one of the earliest immune cells to respond after HO-inducing injuries, can extrude DNA, forming highly inflammatory NETs. 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 (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, 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 after the HO-forming injury. Immunofluorescence 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 enzyme-linked immunosorbent assay for myeloperoxidase-DNA and ELA2-DNA complexes to identify NETosis. Micro-computerized tomography 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 signaling specific to neutrophils at the injury site. Reducing the overall neutrophil abundance within the injury site, either pharmacologically through treatment with hydroxychloroquine, the toll-like receptor 9 inhibitor OPN-2088, or mechanical treatment with limb offloading, results in the 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.

Itaconate-producing neutrophils regulate local and systemic inflammation following trauma

“…When nearing the end of their lifespan, neutrophils typically cycle back to the bone marrow for turnover through the concomitant downregulation of CXCR2 and upregulation of CXCR4 ( 13 ). While some neutrophils are likely to terminate within the injury tissue through the formation of neutrophil extracellular traps (NETs) ( 31 , 32 ), a portion of neutrophils may cycle back to the bone marrow following homing to sites of injury. Expression analysis of Cxcr2 and Cxcr4 in our neutrophil population from the marrow, blood, and injury site showed the expected changes in receptor expression ( Figure 4D ).…”

Intersections of Fibrodysplasia Ossificans Progressiva and Traumatic Heterotopic Ossification

“…In a similar manner to FOP, tHO has shown to be at least in part driven by inflammatory processes, many of which overlap with FOP inflammatory mechanisms. Like FOP patients, individuals with tHO have shown evidence of increased immune cell presence as well as increased inflammatory cytokines such as IL-3, IL-6, IL-10, and MCP-1 following blast and penetrating combat injuries [ 184 , 185 , 186 , 187 ]. Burn/tenotomy HO mouse models have also shown early increased levels of IL-6 and IL-1α in mice that form HO [ 187 ].…”

“…Like FOP patients, individuals with tHO have shown evidence of increased immune cell presence as well as increased inflammatory cytokines such as IL-3, IL-6, IL-10, and MCP-1 following blast and penetrating combat injuries [ 184 , 185 , 186 , 187 ]. Burn/tenotomy HO mouse models have also shown early increased levels of IL-6 and IL-1α in mice that form HO [ 187 ]. Recent studies have shown that the NF-κB signaling pathway plays an important role in tHO; when the NF-κB cascade was blocked, HO formation significantly decreased [ 188 ].…”

Interaction between immuno-stem dual lineages in jaw bone formation and injury repair

“…TGF-β produced by immune cells in HO exacerbates the disease, by inducing MSC migration and osteogenic differentiation ( MAO et al, 2020 ). Recent studies have shown that neutrophils are involved in HO injury and that toll-like receptor signaling is highly expressed in cells at the site of injury ( NUNEZ et al, 2023 ). Toll-like receptor signaling regulates BMSC function ( NEMETH et al, 2010 ).…”