Activated protein C inhibits neutrophil extracellular trap formation in vitro and activation in vivo

Abstract

Activated protein C (APC) is a multifunctional serine protease with anticoagulant, cytoprotective, and anti-inflammatory activities. In addition to the cytoprotective effects of APC on endothelial cells, podocytes, and neurons, APC cleaves and detoxifies extracellular histones, a major component of neutrophil extracellular traps (NETs). NETs promote pathogen clearance but also can lead to thrombosis; the pathways that negatively regulate NETosis are largely unknown. Thus, we studied whether APC is capable of directly inhibiting NETosis via receptor-mediated cell signaling mechanisms. Here, by quantifying extracellular DNA or myeloperoxidase, we demonstrate that APC binds human leukocytes and prevents activated platelet supernatant or phorbol 12-myristate 13-acetate (PMA) from inducing NETosis. Of note, APC proteolytic activity was required for inhibiting NETosis. Moreover, antibodies against the neutrophil receptors endothelial protein C receptor (EPCR), protease-activated receptor 3 (PAR3), and macrophage-1 antigen (Mac-1) blocked APC inhibition of NETosis. Select mutations in the Gla and protease domains of recombinant APC caused a loss of NETosis. Interestingly, pretreatment of neutrophils with APC prior to induction of NETosis inhibited platelet adhesion to NETs. Lastly, in a nonhuman primate model of Escherichia coli-induced sepsis, pretreatment of animals with APC abrogated release of myeloperoxidase from neutrophils, a marker of neutrophil activation. These findings suggest that the anti-inflammatory function of APC at therapeutic concentrations may include the inhibition of NETosis in an EPCR-, PAR3-, and Mac-1-dependent manner, providing additional mechanistic insight into the diverse functions of neutrophils and APC in disease states including sepsis.

Keywords: Mac-1; PAR3; activated protein C; cell biology; cell signaling; coagulation factor; endothelial protein C receptor; hemostasis; neutrophil; neutrophil extracellular traps; protein C; thrombosis.

Figure 1.

Neutrophils bind APC. Acid-washed glass coverslips were coated with denatured BSA (5 mg/ml), 20 μg/ml IgG, 20 μg/ml fibronectin (FN), or 100 μg/ml immobilized APC for 1 h, followed by PBS wash and then blocked with denatured BSA (5 mg/ml). A, purified human neutrophils (2 × 10⁶/ml) were plated on the coverslips for 1 h at 37 °C, followed by washing and fixation. After imaging, cell adhesion was counted in ImageJ and quantified as cell per field of view (FOV). B, neutrophils (2 × 10⁶/ml) were plated on fibronectin-coated coverslips and were treated with HBSS or PMA (10 nm) for 3 h at 37 °C. Cell samples were washed and vehicle (HBSS buffer) or APC (300 nm) was then incubated for 15 min with the cell samples at 37 °C. Samples were then fixed with 4% PFA and incubated overnight with primary antibodies. The samples were then incubated with Hoechst 33342 (1:1000) and secondary antibody Alexa Fluor 488 goat anti-mouse (Invitrogen) (1:500). Images were normalized to secondary antibody-alone images. Shown above (C) are representative images of APC-positive staining in the presence of NETs and neutrophils *, p < 0.05 versus BSA. Data are mean ± S.E. n=3.

Figure 2.

APC treatment inhibits NETosis. Acid-washed glass coverslips were coated with 20 μg/ml fibronectin and then blocked with denatured BSA (5 mg/ml). Purified human neutrophils (2 × 10⁶/ml) were plated on the coverslips for 30 min at 37 °C, then incubated with APC (300 nm) for 30 min at 37 °C. Samples were then washed once with PBS and subsequently treated with HBSS, platelet secretome, or PMA (10 nm) for 3 h at 37 °C. All samples were then fixed with 4% PFA. Samples were incubated overnight with polyclonal mouse anti-myeloperoxidase antibody (MPO) (1:100) and rabbit anti-citrullinated histone 3 antibody (H3Cit) (1:250). Samples were then incubated with Hoechst 33342 (1:1000) and secondary antibodies Alexa Fluor 488 goat anti-rabbit and 546 goat anti-mouse IgG (Invitrogen) (1:500). Images were normalized to secondary antibody-alone (vehicle control) images. A, representative images of neutrophils and NETs with DNA, MPO, and H3Cit staining are shown. B, images were then analyzed in a custom MATLAB program to quantify each pixel-positive signal as area DNA per image. *, p < 0.05 versus DMSO + platelet secretome. #, p < 0.001 versus DMSO + PMA. Data are mean ± S.E. n = 4.

Figure 3.

APC treatment inhibits PMA-induced NETosis. Acid-washed glass coverslips were coated with 20 μg/ml fibronectin and then blocked with denatured BSA (5 mg/ml). Purified human neutrophils (2 × 10⁶/ml) were plated on the coverslips for 30 min at 37 °C. A and B, they were then incubated with increasing concentrations of APC (30, 100, and 300 nm), protein C (300 nm), and thrombin (300 nm) in the presence or absence of PPACK (40 μm) for 30 min at 37 °C. Samples were washed and then subsequently treated with HBSS or PMA (10 nm) for 3 h at 37 °C. C, purified human neutrophils (2 × 10⁶/ml) were plated on the coverslips for 30 min at 37 °C, then incubated with increasing concentrations of APC (30, 75, 100, 150, and 300 nm) for 30 min at 37 °C. Samples were then washed once with PBS and subsequently treated with HBSS or PMA (10 nm) for 3 h at 37 °C. All samples were then fixed with 4% PFA. Samples were incubated overnight with polyclonal rabbit anti-neutrophil elastase antibody (1:100). The samples were then incubated with Hoechst 33342 (1:1000) and secondary antibody Alexa Fluor 546 goat anti-rabbit IgG (Invitrogen) (1:500). Images were normalized to secondary antibody-alone images. Shown in A are representative images of PMA-induced NETs in the presence of increasing concentrations of coagulation factors. Images were analyzed in a custom MATLAB program to quantify each pixel-positive signal as area DNA per image, shown in B and C. *, p < 0.001 versus vehicle + PMA. Data are mean ± S.E. n = 3.

Figure 4.

APC inhibition of NETosis is abrogated by protein platelet activating factor and α1-antitrypsin. Acid-washed glass coverslips were coated with 20 μg/ml fibronectin and then blocked with denatured BSA (5 mg/ml). Purified human neutrophils (2 × 10⁶/ml) were plated on the coverslips for 30 min at 37 °C and allowed to adhere before APC treatments. A, cells were washed once with PBS and incubated with APC (100 nm) for 30 min at 37 °C. B, to test for inhibition of APC, cells were washed once with PBS and incubated with buffer (vehicle) or platelet activating factor (PAF) (100 nm) for 10 min at 37 °C prior to incubation with APC (100 nm) for 30 min at 37 °C. C, surface-bound cells were incubated with APC (300 nm) and thrombin (300 nm) in the presence or absence of α1-antitrypsin (α1-AT) (20 μm) for 30 min at 37 °C. Samples were then washed once with PBS and subsequently treated with HBSS or PMA (10 nm) for 3 h at 37 °C. They were then fixed with 4% PFA. The samples were incubated overnight with polyclonal rabbit anti-citrullinated histone H3 antibody (1:250) and were then incubated with Hoechst 33342 (1:1000) and secondary antibody Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen) (1:500). Images were normalized to secondary antibody-alone images and analyzed in a custom MATLAB program to quantify each pixel-positive signal as the area of DNA per image. *, p < 0.05 versus vehicle + PMA. #, p < 0.05 versus APC + PMA. Data are mean ± S.E. n = 3.

Figure 5.

EPCR, PAR-3, and Mac-1 are required for APC-mediated inhibition of NETosis. Acid-washed glass coverslips were coated with 20 μg/ml fibronectin and then blocked with denatured BSA (5 mg/ml). Purified human neutrophils (2 × 10⁶/ml) were plated on the coverslips and allowed to adhere for 30 min at 37 °C. Cells were washed once with PBS. A, cells were incubated with CD11b blocking antibody (ab8878) (10 μg/ml), CD18 blocking antibody (CBL158) (1 μg/ml), EPCR blocking antibody (RCR-252) (10 μg/ml), or PAR3 blocking antibody (ab-5598) (25 μg/ml) for 10 min at 37 °C prior to incubation with APC (300 nm) for 30 min at 37 °C. B, cells were incubated with PAR3 peptides P3K or P3R (50 μm) representative of cleavage at Lys-38 (P3K) or Arg-41 (P3R) in either the presence or absence of APC (300 nm) for 30 min at 37 °C. Samples were then washed once with PBS and subsequently treated with HBSS or PMA (10 nm) for 3 h at 37 °C. Samples were then fixed with 4% PFA and were incubated overnight with polyclonal rabbit anti-neutrophil elastase antibody (1:100) or polyclonal rabbit anti-citrullinated histone H3 antibody (1:250). The samples were then incubated with Hoechst 33342 (1:1000) and secondary antibody Alexa Fluor 546 goat anti-rabbit IgG (Invitrogen) (1:500). Images were normalized to secondary antibody-alone images and analyzed in a custom MATLAB program to quantify each pixel-positive signal as the area of DNA per image. *, p < 0.001 versus vehicle + PMA. #, p < 0.05 versus APC + PMA. Data are mean ± S.E. n = 3.

Figure 6.

APC-mediated inhibition of NETosis requires Gβγ signaling to inhibit PKC and PI3K. Acid-washed glass coverslips were coated with 20 μg/ml fibronectin and then blocked with denatured BSA (5 mg/ml). Purified human neutrophils (2 × 10⁶/ml) were plated on the coverslips, and allowed to adhere for 30 min at 37 °C. Cells were washed once with PBS. A, cells were incubated with PI3K inhibitor wortmannin (100 nm), PLC inhibitor U73122 (5 nm), the inactive analog U73343 (5 nm), or JAK/STAT inhibitor cucurbitacin I (500 nm) for 30 min at 37 °C. B, cells were incubated with PKC inhibitors GF109203X (5 μm) and Ro31–8220 (380 μm), PLC inhibitor U73122 (5 nm), or Gβγ inhibitor gallein (10 μm) for 30 min at 37 °C prior to incubation with APC (300 nm) for 30 min at 37 °C. Samples were then washed once with PBS and subsequently treated with HBSS, autologous platelet secretome, or PMA (10 nm) for 3 h at 37 °C. Samples were then fixed with 4% PFA and incubated overnight with polyclonal rabbit anti-citrullinated histone H3 antibody (1:250). The samples were then incubated with Hoechst 33342 (1:1000) and secondary antibody Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen) (1:500). Images were normalized to secondary antibody-alone images and analyzed in a custom MATLAB program to quantify each pixel-positive signal as the area of DNA per image. *, p < 0.001 versus vehicle + PMA. #, p < 0.001 versus APC + PMA. Data are mean ± S.E. n = 3.

Figure 7.

Identification of APC residues required for APC-mediated inhibition of NETosis. A, structural scheme for APC with residue mutations numbered beginning with the active site Ser-360 in the protease domain. B–E, acid-washed glass coverslips were coated with 20 μg/ml fibronectin and then blocked with denatured BSA (5 mg/ml). Purified human neutrophils (2 × 10⁶/ml) were plated on the coverslips for 30 min at 37 °C, then incubated with 300 nm APC or APC mutants for 30 min at 37 °C. Samples were then washed once with PBS subsequently treated with HBSS or PMA (10 nm) for 3 h at 37 °C. They were then fixed with 4% PFA and were incubated overnight with polyclonal rabbit anti-neutrophil elastase antibody (1:100). Samples were then incubated with Hoechst 33342 (1:1000) and secondary antibody Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen) (1:500). Images were normalized to secondary antibody-alone images and analyzed in a custom MATLAB program to quantify each pixel-positive signal as the area of DNA per image. B, compares wtAPC versus rhAPC used to make all mutants, where all mutations within the active site are presented in C, the charged exosite mutations are presented in D, and the Gla domain mutations in APC are presented in E. *, p < 0.001 versus vehicle + PMA. #, p < 0.05 versus rhAPC + PMA. δ, p < 0.05 versus L38D + PMA. φ, p < 0.05 versus S252A + PMA. Data are mean ± S.E. n = 3.

Figure 8.

APC inhibits platelet adhesion to NETs and LDH release. A and B, acid-washed glass coverslips were coated with 20 μg/ml fibronectin and then blocked with denatured BSA (5 mg/ml). Purified human neutrophils (2 × 10⁶/ml) were plated on the coverslips and allowed to adhere for 30 min at 37 °C. Cells were washed once with PBS and incubated with HBSS or 300 nm APC 30 min at 37 °C. Samples were then washed once with PBS and subsequently treated with HBSS or PMA (10 nm) for 3 h at 37 °C. The samples again were washed once with PBS and incubated with autologous platelets (2 × 10⁷/ml) and allowed to adhere for 45 min at 37 °C. Samples were then washed once with PBS and fixed with 4% PFA and were incubated overnight with polyclonal rabbit anti-CD41 antibody (1:100). They were then incubated with Hoechst 33342 (1:1000) and secondary antibody Alexa Fluor 488 goat anti-rabbit (Invitrogen) (1:500). Images were normalized to secondary antibody-alone images and analyzed in a custom MATLAB program to quantify each pixel-positive signal as (A) the area of FITC (CD41) per image. In (B) each pixel positive for both CD41 and DNA signals per image was quantified. *, p < 0.001 versus vehicle + PMA. C, purified human neutrophils (4 × 10⁵/ml) were incubated in solution with 300 nm APC for 30 min at 37 °C. Samples were then incubated with HBSS or PMA (20 nm, final) and incubated for 3 h at 37 °C. 10× lysis buffer was added to duplicate columns and incubated for 45 min at 37 °C. Supernatants from samples and the LDH-positive controls were transferred to new 96-well plates prior to incubation with reaction mixture for 30 min at 37 °C. The reaction was stopped and absorbance was measured at 490 nm and 680 nm. Percent cytotoxicity was quantified per the manufacturer's instructions (Thermo Fisher). *, p < 0.05 versus vehicle + PMA. Data are mean ± S.E. n = 3.

Figure 9.

Infusion of APC reduces plasma levels of MPO in an LD₁₀₀E. coli baboon model of sepsis. In this established baboon model of sepsis, Papio c. anubis-treated animals were initially dosed with 3 mg/kg bolus of APC at time (T) − 10 min. From T − 0 min to T + 320 min, APC was infused intravenously at 16 μg/kg/min. A lethal dose (1–2 × 10¹⁰ cfu/kg) of E. coli, ATCC 12701 serotype O86:K61(B7) was infused intravenously over 2 h (from T − 0 to T − 120 min). Blood samples were obtained at 0, 2, 4, 8, and 24 h and plasma samples were isolated. MPO in plasma samples was determined using Fluoro MPO (fluorescent myeloperoxidase detection kit) (Cell Technology) per the manufacturer's instructions. Data are mean ± S.E. LD₁₀₀ animals n = 8; LD₁₀₀ + APC animals n = 4.

Figure 10.

Schematic overview for APC-mediated inhibition of NETosis. In vitro, select stimuli including cytokines, bacteria, the PKC-agonist PMA, and the platelet secretome induce PKC-dependent NETosis. Our observations show a mechanism whereby proteolytically active APC requires Mac-1, EPCR, and PAR3 to induce intracellular, cytoprotective signaling that results in the downstream inhibition of NET formation.