Systemic Immune Modulation Alters Local Bone Regeneration in a Delayed Treatment Composite Model of Non-Union Extremity Trauma

Abstract

Bone non-unions resulting from severe traumatic injuries pose significant clinical challenges, and the biological factors that drive progression towards and healing from these injuries are still not well understood. Recently, a dysregulated systemic immune response following musculoskeletal trauma has been identified as a contributing factor for poor outcomes and complications such as infections. In particular, myeloid-derived suppressor cells (MDSCs), immunosuppressive myeloid-lineage cells that expand in response to traumatic injury, have been highlighted as a potential therapeutic target to restore systemic immune homeostasis and ultimately improve functional bone regeneration. Previously, we have developed a novel immunomodulatory therapeutic strategy to deplete MDSCs using Janus gold nanoparticles that mimic the structure and function of antibodies. Here, in a preclinical delayed treatment composite injury model of bone and muscle trauma, we investigate the effects of these nanoparticles on circulating MDSCs, systemic immune profiles, and functional bone regeneration. Unexpectedly, treatment with the nanoparticles resulted in depletion of the high side scatter subset of MDSCs and an increase in the low side scatter subset of MDSCs, resulting in an overall increase in total MDSCs. This overall increase correlated with a decrease in bone volume (P = 0.057) at 6 weeks post-treatment and a significant decrease in mechanical strength at 12 weeks post-treatment compared to untreated rats. Furthermore, MDSCs correlated negatively with endpoint bone healing at multiple timepoints. Single cell RNA sequencing of circulating immune cells revealed differing gene expression of the SNAb target molecule S100A8/A9 in MDSC sub-populations, highlighting a potential need for more targeted approaches to MDSC immunomodulatory treatment following trauma. These results provide further insights on the role of systemic immune dysregulation for severe trauma outcomes in the case of non-unions and composite injuries and suggest the need for additional studies on targeted immunomodulatory interventions to enhance healing.

Keywords: MDSCs; S100A8/A9; immune dysregulation; musculoskeletal trauma; non-union.

Figure 1

Single cell RNA sequencing of MDSC depletion in vitro. (A) Clustering of integrated tSNE plots from the SNAb-treated and untreated cells enriched for MDSCs. (B) Overlay of tSNE plots from SNAb-treated and untreated groups showing differences in cell populations. (C) Gene expression for immunosuppressive MDSCs gene markers (S100a9, IL1b, Arg1, and Junb) pre- and post-SNAb treatment. (D) Gene expression for macrophage gene markers (CD68 and Adgre1) pre- and post-SNAb treatment.

Figure 2

Characterization of circulating immune cells after injury (Day 0) and treatment (Week 8). (A–G) Significant changes in immune cell populations were observed in rats after injury. T cells, T helper cells, and cytotoxic T cells were significantly decreased in injured rats (A–C), while MDSCs and monocytes were significantly elevated (E,G). Data are mean ± SEM, n = 7 to 13 per group. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 as indicated.

Figure 3

Characterization of circulating immune cells after treatment with synthetic nanoparticle antibodies (SNAbs). (A–G) Significant changes in immune cell populations were observed in rats at multiple timepoints after SNAb injections. T cells, T helper cells, and cytotoxic T cells were significantly decreased in SNAb-injected rats (A–C), while MDSCs and monocytes were significantly elevated (E,G). Data are mean ± SEM, n = 7 to 13 per group. 1,2 correspond to significant differences between the naive group and the 1x SNAbs group (1) or the 2x SNAbs group (2), with P < 0.05.

Figure 4

MDSCs at 24 h post-SNAb treatment in the composite trauma model. (A) Flow cytometry dot plots showing two MDSC sub-populations. Systemic levels of total MDSCs (B) and the two MDSC subsets (C) at 24 h post-SNAb treatment in the composite trauma model. Significance was determined using a one-way ANOVA. ***P < 0.001; ****P < 0.0001 as indicated; n = 5 to 11 per group.

Figure 5

Functional regeneration may be impaired following treatment with synthetic nanoparticle antibodies (SNAbs). (A,B) Total bone volume (A) and bone mineral density (B) of newly formed bone, as quantified by in vivo µCT at week 14 and ex vivo µCT at week 20. (C,D) Mechanical strength (C) and stiffness (D) of regenerated femurs as quantified by ex vivo torsional testing to failure at week 20. The dotted lines indicate mechanical properties of intact bone. *P < 0.05 as indicated.

Figure 6

Circulating immune cells correlate with bone regeneration in rats treated with synthetic nanoparticle antibodies (SNAbs). (A–E) Immune cell populations from peripheral blood at multiple timepoints significantly correlate with week 20 bone volumes as measured by ex vivo µCT. T cells, T helper cells, and cytotoxic T cells were positively correlated with bone regeneration (A–C), while MDSCs and monocytes were negatively correlated (D,E). Pearson correlation analyses performed, n = 7–8 per time point, slope of linear regression significantly nonzero for all data shown (P < 0.05).

Figure 7

Single cell RNA sequencing in the composite trauma model. (A) Integrated clustering of PBMCs from naïve and composite trauma rats at 5 days post-injury. (B) Gene expression of MDSC gene markers. (C) MDSC clusters identified in (A) were re-integrated and re-clustered for more in-depth analysis. Three subclusters were identified. (D) Comparison of MDSC percentages for clusters 0, 1, and 2 in composite trauma model rats versus naïve rats. (E) Density plots (top) and violin plots (bottom) of S100a8 expression (left) and S100a9 expression (right).