Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Oct 24;10:2148.
doi: 10.3389/fimmu.2019.02148. eCollection 2019.

Disruption of Neutrophil Extracellular Traps (NETs) Links Mechanical Strain to Post-traumatic Inflammation

Affiliations
Free PMC article

Disruption of Neutrophil Extracellular Traps (NETs) Links Mechanical Strain to Post-traumatic Inflammation

Shailesh Agarwal et al. Front Immunol. .
Free PMC article

Abstract

Inflammation after trauma is both critical to normal wound healing and may be highly detrimental when prolonged or unchecked with the potential to impair physiologic healing and promote de novo pathology. Mechanical strain after trauma is associated with impaired wound healing and increased inflammation. The exact mechanisms behind this are not fully elucidated. Neutrophil extracellular traps (NETs), a component of the neutrophil response to trauma, are implicated in a range of pro-inflammatory conditions. In the current study, we evaluated their role in linking movement and inflammation. We found that a link exists between the disruption and amplification of NETs which harbors the potential to regulate the wound's response to mechanical strain, while leaving the initial inflammatory signal necessary for physiologic wound healing intact.

Keywords: NET; inflammation; movement; neutrophils; trauma.

Figures

Figure 1
Figure 1
Mechanical or chemical disruption of NETs augments inflammation and induces pathologic wound healing. (A) Mice undergo tendon transection and either remain mobile or are immobilized via splint; (B) H&E staining of mobile hindlimb 1 week post-injury intense blue staining represent infiltrated cells casting (4x left; 10x right); (C) H&E staining of immobile hindlimb 1 week post-injury; (D) Immobile hindlimbs (n = 6) have significantly reduced normalized neutrophil (1.0 vs. 0.27, p < 0.05) and macrophage (1.0 vs. 0.26, p < 0.05) counts compared with mobile hindlimbs (n = 6) 48 h after injury; (E) Immobile hindlimbs (n = 3) have significantly reduced normalized neutrophil (1.0 vs. 0.08, p < 0.05) and macrophage counts (1.0 vs. 0.13, p < 0.05) compared with mobile hindlimbs (n = 3) 1 week after injury; (F) H3-Cit immunostaining and DAPI showing NETs in the mobile hindlimb 48 h after injury (40x); (G) H3-Cit immunostaining and DAPI showing NETs in the immobile hindlimb 48 h after injury (40x); (H) Experimental strategy with DNase I; (I) DNase I significantly increases normalized neutrophil (1.0 vs. 6.39, p < 0.05) and macrophage (1.0 vs. 3.0, p < 0.05) counts in the immobile hindlimb (n = 8) 48 h after injury; (J) DNase I does not increase normalized neutrophil (1.0 vs. 1.04, p = 0.87) and macrophage (1.0 vs. 0.81, p = 0.23) counts in the mobile hindlimb (n = 5) 48 h after injury; (K) H3-Cit immunostaining and DAPI showing NETs in the DNase I-treated immobile hindlimb (40x). All in vivo studies had n ≥ 3/group. Scale bars are 200 μm. *p < 0.05.
Figure 2
Figure 2
Mechanically disrupted NETs induce NETosis. (A) In vitro experimental set-up to evaluate whether mechanical disruption of NETs can induce NETosis; (B) Representative images from PMA-induced NETs to demonstrate types of NETs; (C) 2° NETs with mechanical disruption of 1° NETs; (D) 2° NETs without mechanical disruption of 1° NETs; (E) Number of 2° NETs normalized to the number of DAPI+ cells per hpf (46.6 vs. 20.2, p < 0.05); (F) Number of compound 2° NETs normalized to the number of all NETs per hpf (92.7 vs. 35.8, p < 0.05); (G) Number of DAPI+ nuclei normalized to the number of 2° NETs per hpf (2.9 vs. 1.8, p < 0.05). Scale bars are 200 μm. All in vitro studies had n = 15 hpf/group. *p < 0.05.
Figure 3
Figure 3
NET-induced NETosis augments inflammation. (A) In vivo experimental design with Cl-Amidine; (B) Cl-Amidine significantly reduces normalized neutrophil (1.0 vs. 0.31, p < 0.05) and macrophage (1.0 vs. 0.32, p < 0.05) counts in the immobile hindlimb of DNase I-treated mice (n = 4) 48 h after injury vs. untreated immobile plus DNase I-treated controls (n = 3); (C) Cl-Amidine significantly reduces normalized neutrophil (1.0 vs. 0.40, p < 0.05) and macrophage (1.0 vs. 0.31, p < 0.05) counts in the mobile hindlimb (n = 5) 48 h after injury vs. untreated mobile controls (n = 5); (D) Cl-Amidine does not reduce normalized neutrophil (1.0 vs. 0.96, p = 0.79) or macrophage (1.0 vs. 1.35, p = 0.08) counts in the immobile hindlimb without DNase I (n = 5) 48 h after injury vs. untreated immobilized controls (n = 5); (E) H3-Cit immunostaining and DAPI showing NETs in the mobile hindlimb 48 h after injury; (F) H3-Cit immunostaining and DAPI showing NETs in the mobile hindlimb with Cl-Amidine 48 h after injury; (G) In vitro experimental set-up with Cl-Amidine; (H) H3-Cit and DAPI showing 2° NETs with or without Cl-Amidine after mechanical disruption of 1° NETs; (I) Number of 2° NETs normalized to the number of DAPI+ cells per hpf with Cl-Amidine (46.6 vs. 9.1, p < 0.05); (J) Number of compound 2° NETs normalized to the number of all NETs per hpf with Cl-Amidine (92.7 vs. 5.4, p < 0.05); (K) Number of DAPI+ nuclei normalized to the number of 2° NETs per hpf with Cl-Amidine (2.9 vs. 0.9, p < 0.05); (L) In vivo experimental-set up with delayed Cl-Amidine; (M) Cl-Amidine significantly reduces normalized neutrophil (1.0 vs. 0.27, p < 0.05) and macrophage (1.0 vs. 0.20, p < 0.05) counts in the immobile hindlimb of DNase I-treated mice 48 h after injury vs. immobile DNase I-treated controls; (N) Cl-Amidine significantly reduces normalized neutrophil (1.0 vs. 0.06, p < 0.05) and macrophage (1.0 vs. 0.14, p < 0.05) counts in the mobile hindlimb 48 h after injury vs. untreated mobile controls. Scale bars are 200 μm. All in vivo studies had n ≥ 3/group. All in vitro studies had n = 15 hpf/group. *p < 0.05.
Figure 4
Figure 4
NETs induce NETosis in human neutrophils. (A) Human neutrophils in vitro demonstrating the increase in NET formation after Mechanical Disruption of primary (PMA-induced) NETs. (B) Demonstration of the effect of ODN2088 on NET-induced NETosis of human neutrophils in vitro. (C) Quantification of increased NETosis after disruption of primary NETs; this effect is abrogated by ODN2088. Scale bars are 300 μm. ****p < 0.0001, by one-way ANOVA.
Figure 5
Figure 5
NETs induce NETosis through TLR activity. (A) In vitro experimental design with ODN-2088; (B) H3-Cit and DAPI showing 2° NETs with or without ODN-2088 after mechanical disruption of 1° NETs; (C) Number of 2° NETs normalized to the number of DAPI+ cells per hpf with ODN-2088 (46.6 vs. 11.0, p < 0.05); (D) Number of compound 2° NETs normalized to the number of all NETs per hpf with ODN-2088 (92.7 vs. 0.90, p < 0.05); (E) Number of DAPI+ nuclei normalized to the number of 2° NETs per hpf with ODN-2088 (2.9 vs. 0.60, p < 0.05); (F) In vivo experimental set-up with ODN-2088; (G) ODN-2088 significantly reduces normalized neutrophil (1.0 vs. 0.27, p < 0.05) and macrophage (1.0 vs. 0.41, p < 0.05) counts in the mobile hindlimb 48 h after injury vs. untreated mobile controls; All in vivo studies had n ≥ 3/group. All in vitro studies had n = 15 hpf/group. Scale bars are 200 μm. *p < 0.05.

Similar articles

See all similar articles

References

    1. Qian LW, Fourcaudot AB, Yamane K, You T, Chan RK, Leung KP. Exacerbated and prolonged inflammation impairs wound healing and increases scarring. Wound Repair Regen. (2016) 24:26–34. 10.1111/wrr.12381 - DOI - PubMed
    1. White ES, Mantovani AR. Inflammation, wound repair, and fibrosis: reassessing the spectrum of tissue injury and resolution. J Pathol. (2013) 229:141–4. 10.1002/path.4126 - DOI - PMC - PubMed
    1. Gurtner GC, Dauskardt RH, Wong VW, Bhatt KA, Wu K, Vial IN, et al. . Improving cutaneous scar formation by controlling the mechanical environment: large animal and phase I studies. Ann Surg. (2011) 254:217–25. 10.1097/SLA.0b013e318220b159 - DOI - PubMed
    1. Rustad KC, Wong VW, Gurtner GC. The role of focal adhesion complexes in fibroblast mechanotransduction during scar formation. Differ Res Biol Divers. (2013) 86:87–91. 10.1016/j.diff.2013.02.003 - DOI - PubMed
    1. Wong VW, Rustad KC, Akaishi S, Sorkin M, Glotzbach JP, Januszyk M, et al. . Focal adhesion kinase links mechanical force to skin fibrosis via inflammatory signaling. Nat Med. (2012) 18:148–52. 10.1038/nm.2574 - DOI - PMC - PubMed

Publication types

LinkOut - more resources

Feedback