INTRODUCTION
Fracture healing starts with an inflammatory phase during which leukocytes infiltrate the blood collection surrounding the fracture site [
1,
2]. Animal studies suggest that this blood collection, which is generally referred to as fracture hematoma (FH), forms a reservoir of essential factors and cells that regulate downstream processes of bone repair. This is illustrated by the finding that transplantation of the FH into muscle tissue induced ectopic bone formation and angiogenesis in animal models [
3,
4]. Moreover, removal or repetitive irrigation of the FH impaired fracture healing in rats [
5,
6].
Although controlled local inflammation is essential for adequate fracture healing [
7], several animal studies have also shown that both local and systemic “hyper-inflammatory” conditions impair bone regeneration. For instance, injection of beta-glucan into the fracture site induces local hyper-inflammation and impairs fracture healing in rats [
8]. Moreover, intraperitoneal injection of lipopolysaccharides in rats induces systemic inflammation and negatively affects the outcome of bone repair [
9]. In addition, blunt chest injury, which is a model of trauma-induced systemic inflammation, also impairs fracture healing in rats [
10].
It is well known that severely injured patients have an increased risk of developing impaired fracture healing [
11,
12]. This not only has a significant impact on quality of life, but also carries a substantial economical burden to society [
13]. Based on the abovementioned animal studies, we hypothesized that the systemic immune response after major trauma contributes to the high incidence of impaired fracture healing in multitrauma patients [
1,
11]. The underlying mechanism remains unclear. However, experimental studies suggest that major trauma pre-activates neutrophils and induces increased influx of neutrophils towards sites of inflammation, such as the fracture hematoma [
10,
14,
15], and impairs bone healing.
Such a pathological role of neutrophils was supported by the finding that depletion of neutrophils improved the outcome of bone repair in rats [
16,
17]. However, systemic depletion of neutrophils would significantly compromise the hosts’ defense against pathogens.
Therefore, we tried to identify neutrophil chemoattractants within the sterile FH that may be blocked in the future without affecting chemotaxis of neutrophils towards sites of infection. As a first step, we tested whether neutrophil chemotaxis towards the human FH could be studied in vitro. Furthermore, we explored whether neutrophil chemotaxis towards the FH is mediated by IL-8 receptors CXCR1 and CXCR2, formylated peptide receptors (FPR), and complement receptor C5aR.
DISCUSSION
The current literature suggests that increased influx of neutrophils into the fracture hematoma (FH) during hyper-inflammatory conditions impairs fracture healing after major trauma [
1,
25]. Future therapies that inhibit influx of neutrophils into the FH without compromising the hosts’ defense against pathogens may therefore prevent impairment of bone healing in multitrauma patients. Our study shows that chemotaxis of neutrophils towards the FH can be studied
in vitro with Ibidi™ Chemotaxis
3D μ-Slides. We found that serum from the human FH significantly induces neutrophil chemotaxis, which was not affected by blocking the CXCR1 and CXCR2 receptors (Fig.
2e). In contrast, CHIPS induced a significant decrease in neutrophil chemotaxis towards the human FH
in vitro (Fig.
2f). CHIPS is an exoprotein produced by several strains of
S. aureus and is a potent inhibitor of neutrophil and monocyte chemotaxis towards C5a and formylated peptides like fMLF [
23]. It is known that tissue injury induces complement activation and release of C5a [
15,
26], as well as release of formylated peptides from mitochondria into the circulation [
27]. CHIPS exclusively binds directly to the C5aR and FPR1 and FPR2 receptors, thereby preventing their natural ligands from activating these receptors [
23,
28]. We additionally used a CHIPS mutant lacking the first N-terminal amino acid (CHIPSΔ1F), which has impaired or absent FPR but still intact C5aR-blocking activity [
24]. Our data shows that blocking C5aR with CHIPSΔ1F also significantly inhibits neutrophil chemotaxis towards the FH (Fig.
2f). Previous studies have shown that systemic antagonism of the C5aR improves fracture healing after major trauma in rats [
15]. It is tempting to speculate that systemic C5aR antagonism prevents increased influx of neutrophils into the FH and thereby reduces the deleterious effect of major trauma on fracture healing.
In our
in vitro experiments, we were unable to completely block neutrophil chemotaxis towards the FH using CHIPS or CHIPSΔ1F. One possible explanation for this effect is that the concentrations of blocking antibodies were insufficient to completely block all receptors. Also, several additional neutrophil chemoattractants may be present within the FH that do not exert their effect through CXCR1/2, FPR, or C5aR. Neutrophils possess several receptors that detect chemoattractants, such as chemokines, complement components, and several other chemotactic lipids and peptides [
29]. Nineteen chemokine receptors have been identified so far, which include seven CXC receptors (CXCR1–7), ten CCR (CCR1–10), one CX
3CR (CX
3CR1), and one CR (XCR1) receptor [
30]. Neutrophils are traditionally known to express only a very limited number of chemokine receptors and mainly express CXCR1 and CXCR2 in healthy individuals [
31]. CXCR1 and CXCR2 are used by neutrophils to recognize N-terminal ELR (glutamic acid-leucine-arginine) motif-containing CXC chemokines. Human CXCR1 binds to CXCL8 (interleukin-8/IL-8) and CXCL6 (granulocyte chemotactic protein-2) [
20,
29], as well as the ECM breakdown product N-acetyl PGP [
32]. These three factors can also bind to CXCR2. However, CXCR2 is more promiscuous and binds different additional CXC chemokines, including CXCL1 (growth regulated oncogene-alpha/GRO-α), CXCL2 (GRO-β), CXCL3 (GRO-γ), CXCL5 (epithelial cell-derived neutrophil activating peptide-78/ ENA-78), and CXCL7 (neutrophil activating protein-2/GCP-2) [
29]. Our study implies that these CXCR1 and CXCR2 ligands are not relevant in migration of neutrophils towards the FH
in vitro. However, although neutrophils in healthy individuals mainly express CXCR1 and CXCR2 [
31], it has been shown that infiltrated neutrophils from patients with chronic inflammatory lung diseases and rheumatoid arthritis express additional chemokine receptors on their surface,
i.e., CCR1, CCR2, CCR3, CCR5, CXCR3, and CXCR4 [
31]. Moreover, major trauma induces the release of several neutrophil subsets into the peripheral circulation, including young banded neutrophils and hyper-segmented neutrophils, which exhibit different properties and receptor expressions compared to mature neutrophils from healthy individuals [
33]. Future studies may focus on the role of these neutrophil subsets in fracture healing and determine whether neutrophils within the FH express other chemokine receptors compared to neutrophils isolated from peripheral blood of healthy donors.
Another chemotactic factor for neutrophils is leukotriene B4 (LTB4), which is recognized by a high-affinity receptor (BLT1) and a low-affinity receptor (BLT2) [
34]. Animal studies have shown that LTB4 mediates neutrophil influx after experimental spinal cord injury [
35]. It is tempting to speculate that LTB4 also mediates neutrophil influx into other types of sterile tissue injury, such as bone injury. An additional chemoattractant for neutrophils is platelet-activating factor (PAF), which is a phospholipid that is bound by the PAF receptor (PAFR) [
36]. Little is known about the role of PAF in tissue injury although animal studies did show that inactivation of PAF by PAF acetylhydrolase significantly decreased neutrophil influx in a rabbit model of myocardial ischemia/reperfusion injury [
37]. Future studies should investigate to which extent the abovementioned factors are also relevant in chemotaxis of neutrophils towards the FH.
In summary, our study shows that chemotaxis of neutrophils towards the FH can be studied in vitro with Ibidi™ Chemotaxis3D μ-Slides. We found that serum from the human FH significantly induces chemotaxis, which was not affected by blocking CXCR1 and CXCR2. In contrast, CHIPS and CHIPSΔ1F, which blocks C5aR, induced a significant decrease in chemotaxis of neutrophils towards the FH. These findings may aid the development of therapies that prevent impairment of fracture healing after major trauma.