Substrate stiffness induces neutrophil extracellular trap (NET) formation through focal adhesion kinase activation

Abstract

Neutrophils predominate the early inflammatory response to tissue injury and implantation of biomaterials. Recent studies have shown that neutrophil activation can be regulated by mechanical cues such as stiffness or surface wettability; however, it is not known how neutrophils sense and respond to physical cues, particularly how they form neutrophil extracellular traps (NET formation). To examine this, we used polydimethylsiloxane (PDMS) substrates of varying physiologically relevant stiffness (0.2-32 kPa) and examined the response of murine neutrophils to untreated surfaces or to surfaces coated with various extracellular matrix proteins recognized by integrin heterodimers (collagen, fibronectin, laminin, vitronectin, synthetic RGD). Neutrophils on higher stiffness PDMS substrates had increased NET formation and higher secretion of pro-inflammatory cytokines and chemokines. Extracellular matrix protein coatings showed that fibronectin induced the most NET formation and this effect was stiffness dependent. Synthetic RGD peptides induced similar levels of NET formation and pro-inflammatory cytokine release than the full-length fibronectin protein. To determine if the observed NET formation in response to substrate stiffness required focal adhesion kinase (FAK) activity, which is down stream of integrin activation, FAK inhibitor PF-573228 was used. Inhibition of FAK using PF-573228 ablated the stiffness-dependent increase in NET formation and pro-inflammatory molecule secretion. These findings demonstrate that neutrophils regulate NET formation in response to physical and mechanical biomaterial cues and this process is regulated through integrin/FAK signaling.

Keywords: Focal adhesion kinase; Integrins; NET formation; NETosis; Neutrophil; Stiffness.

Fig. 1.

Increased substrate stiffness enhances neutrophil activation. Neutrophils were seeded on hydrogels of varying stiffness (E=0.2, 2, 8, 16, 32 kPa) at a density of 10,000/cm² for 4 hours prior to staining with DAPI for confocal microscopy. A) Representative images of stained nuclear material (NETs) on PDMS gels +/− standard deviation. B) Quantification of NET area and circularity by ImageJ software (n=60/variable). p < 0.05: # vs. 0.2 kPa, $ vs. 2 kPa, % vs. 8 kPa, & vs. 16 kPa.

Fig. 2.

Neutrophil inflammatory protein secretion increases in response to substrate stiffness. Neutrophils were cultured for 4 hours on PDMS hydrogels (n=6/group). Secreted interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), C-C motif ligand 2 (CCL2), CCL3, and myeloperoxidase (MPO) was measured in the conditioned media +/− standard deviation. p < 0.05: # vs. 0.2 kPa, $ vs. 2 kPa, % vs. 8 kPa, & vs. 16 kPa.

Fig. 3.

Loss of PAD4 activity attenuates stiffness-dependent NET formation. To inhibit PAD4 activity, neutrophils from control mice were pre-incubated with GSK484, or neutrophils were harvested from Padi4^−/− mice. Cells were then seeded on hydrogels of varying stiffness (E=0.2, 2, 8, 16, 32 kPa) at a density of 10,000/cm² for 4 hours prior to staining with DAPI for confocal microscopy +/− standard deviation. p < 0.05: # vs. 0.2 kPa, $ vs. 2 kPa, % vs. 8 kPa, & vs. 16 kPa from respective Ctrl, GSK484, or Padi4^−/−; p < 0.05: A vs. respective Ctrl, B vs. respective GSK484 from same stiffness.

Fig. 4.

Loss of PAD4 activity decreases stiffness-dependent changes in protein production. To inhibit PAD4 activity, neutrophils from control mice were pre-incubated with GSK484, or neutrophils were harvested from Padi4^−/− mice. Secreted IL-1β, IL-6, TNF-α, CCL2, CCL3, and MPO was measured in the conditioned media +/− standard deviation. p < 0.05: # vs. 0.2 kPa, $ vs. 2 kPa, % vs. 8 kPa, & vs. 16 kPa from respective Ctrl, GSK484, or Padi4^−/−-; p < 0.05: A vs. respective Ctrl, B vs. respective GSK484 from same stiffness.

Fig. 5.

Stiffness-dependent NET formation is dependent on adsorbed protein composition. Hydrogels were pre-incubated with type I collagen, fibronectin, laminin, or vitronectin at 5 or 10 μg/mL concentrations as indicated for 4 hours prior to staining with DAPI +/− standard deviation. p < 0.05: # vs. 0.2 kPa, $ vs. 2 kPa, % vs. 8 kPa, & vs. 16 kPa with same protein pre-incubation concentration; p < 0.05: A vs. respective 5 μg/mL, B vs. respective 10 μg/mL from same stiffness.

Fig. 6.

Stiffness-dependent neutrophil cytokine secretion is dependent on adsorbed protein composition. Secreted IL-1β, IL-6, TNF-α, CCL2, CCL3, C-X-C motif ligand 1 (CXCL1), and MPO was measured in the conditioned media.

Fig. 7.

Stiffness-dependent neutrophil activation occurs through integrin activation. Hydrogels of varying stiffness were pre-incubated with either fibronectin or soluble RGD domain at 5 or 10 μg/mL concentrations as indicated for 4 hours prior to A) NET morphological analysis or B) secreted protein quantification +/− standard deviation. p < 0.05: # vs. 0.2 kPa, $ vs. 2 kPa, % vs. 8 kPa, & vs. 16 kPa with same protein pre-incubation concentration; p < 0.05: A vs. respective 5 μg/mL fibronectin, B vs. respective 10 μg/mL fibronectin, C vs. respective 5 μg/mL RGD, D vs. respective 10 μg/mL RGD from same stiffness.

Fig. 8.

Loss of downstream focal adhesion kinase activity abrogates stiffness-dependent neutrophil activation. Hydrogels of varying stiffness were pre-incubated with either fibronectin or soluble RGD domain at 5 or 10 μg/mL concentrations, ± PF-573228 (FAK inhibitor) as indicated for 4 hours prior to A) NET morphological analysis or B) secreted protein quantification +/− standard deviation. p < 0.05: # vs. 0.2 kPa, $ vs. 2 kPa, % vs. 8 kPa, & vs. 16 kPa with same protein coating concentration; p < 0.05: A vs. Ctrl, B vs. fibronectin with the same substrate stiffness; p < 0.05: @ vs. PF-573228-untreated with the same substrate stiffness.

Figure 9.