Nucleic acid sensing promotes inflammatory monocyte migration through biased coagulation factor VIIa signaling

Abstract

Protease activated receptors (PARs) are cleaved by coagulation proteases and thereby connect hemostasis with innate immune responses. Signaling of the tissue factor (TF) complex with factor VIIa (FVIIa) via PAR2 stimulates extracellular signal-regulated kinase (ERK) activation and cancer cell migration, but functions of cell autonomous TF-FVIIa signaling in immune cells are unknown. Here, we show that myeloid cell expression of FVII but not of FX is crucial for inflammatory cell recruitment to the alveolar space after challenge with the double-stranded viral RNA mimic polyinosinic:polycytidylic acid [Poly(I:C)]. In line with these data, genetically modified mice completely resistant to PAR2 cleavage but not FXa-resistant PAR2-mutant mice are protected from lung inflammation. Poly(I:C)-stimulated migration of monocytes/macrophages is dependent on ERK activation and mitochondrial antiviral signaling (MAVS) but independent of toll-like receptor 3 (TLR3). Monocyte/macrophage-synthesized FVIIa cleaving PAR2 is required for integrin αMβ2-dependent migration on fibrinogen but not for integrin β1-dependent migration on fibronectin. To further dissect the downstream signaling pathway, we generated PAR2S365/T368A-mutant mice deficient in β-arrestin recruitment and ERK scaffolding. This mutation reduces cytosolic, but not nuclear ERK phosphorylation by Poly(I:C) stimulation, and prevents macrophage migration on fibrinogen but not fibronectin after stimulation with Poly(I:C) or CpG-B, a single-stranded DNA TLR9 agonist. In addition, PAR2S365/T368A-mutant mice display markedly reduced immune cell recruitment to the alveolar space after Poly(I:C) challenge. These results identify TF-FVIIa-PAR2-β-arrestin-biased signaling as a driver for lung infiltration in response to viral nucleic acids and suggest potential therapeutic interventions specifically targeting TF-VIIa signaling in thrombo-inflammation.

Graphical abstract

Figure 1.

FVII is required for Poly(I:C)-induced monocyte/macrophage recruitment to lung exudates. (A) FVII plasma levels in F7^flflLysM^cre vs in littermate F7^flfl mice determined by clotting assay. (B) F7 mRNA and FVII-secreted protein in isolated peritoneal macrophages from F7^flflLysM^cre and F7^flflCx3Cr1^cre mice in comparison with littermate F7^flfl controls. (C) Schematic overview of the standard experimental model of lung inflammation induced by intranasal Poly(I:C) application. (D) Schematic model of PAR2 activation by the TF-FVIIa-FXa complex involving the endothelial protein C receptor (EPCR). (E) Cell counts in BAL in unchallenged and Poly(I:C)-treated F10^flflLysM^cre and littermate F10^flfl mice. (F) Schematic overview of PAR2 activation by the TF-FVIIa complex involving integrins. (G) Cell counts in BAL in unchallenged and Poly(I:C)-treated F7^flflLysM^cre and F7^flfl littermate control mice. (H) Cell counts in BAL in F7^flflCx3Cr1^cre and F7^flfl mice. (I) Cell counts in BAL of mice 24 hours after receiving 1 or 2 doses of Poly(I:C). Mean ± standard deviation (SD); the 2-way ANOVA with the Sidak multiple comparison test (E,G,I) or the unpaired Student t test (H). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. Mo, monocytes; MՓ, macrophages.

Figure 2.

PAR2 cleavage is required for Poly(I:C)-dependent monocyte/macrophage recruitment to the lungs. (A) Schematic overview of PAR2 mutation PAR2^G37I and PAR2^R38 and their cleavage resistance to different proteases. (B) Cell count in BAL in unchallenged and Poly(I:C) treated of F2rl1^G37I, F2rl1^R38E and WT mice. (C) Flow cytometry gating strategy to identify different cell populations in whole lung cell suspensions, pregated on viable, single CD45⁺ cells. (D) Flow cytometry analysis of whole lung cell suspensions from F2rl1^R38E and WT mice after Poly(I:C) treatment. Mean ± SD; the 2-way ANOVA with the Sidak multiple comparison test (panel A) or the unpaired Student t test (panel C). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. SSC, side scatter; MHCII, major histocompatibility complex class II; NK cells, natural killer cells.

Figure 3.

Myeloid FVII deficiency and PAR2 cleavage insensitivity regulate circulating inflammatory monocytes. (A) Flow cytometry analysis of whole lung cell suspension after Poly(I:C) treatment of F7^flflLysM^cre compared with that of F7^flfl littermate control mice. (B) Flow cytometry gating strategy of blood samples pregated on viable, single CD45⁺ cells. (C) Monocytes in blood after intranasal Poly(I:C) treatment of F7^flflLysM^cre and F7^flfl littermate control mice or (D) F2rl1^R38E and WT mice analyzed by flow cytometry. Mean ± SD; the unpaired Student t test ∗P < .05; ∗∗P < .01.

Figure 4.

TF-FVIIa-PAR2 signaling promotes migration on fibrinogen. (A) Migration of peritoneal macrophages and BM monocytes from C57BL/6N mice stimulated with Poly(I:C) (25 μg/mL) on fibrinogen-coated filters in the presence of inhibitory αMβ2 (M1/70, 25 μg/mL) antibody or control IgG. (B) Migration of macrophages and monocytes from Itgb1^flflLysM^cre and littermate Itgb1^flfl WT control mice stimulated with Poly(I:C) on fibronectin-coated filters. (C-E) Migration of macrophages and monocytes from F2rl1^R38E, F7^flflLysMcre, F7^flflCX3CR1cre, F10^flflLysMcre, and WT control mice stimulated with Poly(I:C). (F) Inhibition of migration on fibrinogen with PAR2 antagonist AZ3451 (1 μM). (G) Migration of macrophages and monocytes from C57BL/6N mice with inhibitors anti-TF21E10 (25 μg/ml) or 5L15 (50 nM). (H) Fold induction of Poly(I:C) induced migration with and without 5 nM mouse FVII (mFVII) added to macrophages from F7^flflLysMcre mice. Mean ± SD; the 1-way ANOVA with the Tukey multiple comparison test (A,F,G); the 2-way ANOVA with the Sidak multiple comparison test (B,C,D,E,H). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

Figure 5.

Poly(I:C) stimulates migration and type 1 IFN induction via MAVS. (A) Schematic overview of Poly(I:C) signaling pathways via TLR3 or MAVS. (B) Migration of peritoneal macrophages from C57BL/6J mice stimulated with Poly(I:C) (25 μg/mL) on fibrinogen-coated filters in the presence of TLR3/dsRNA complex inhibitor (27 μM) (C) Migration of peritoneal macrophages from C57BL/6J WT mice and Mavs^–/– mice stimulated with Poly(I:C) (25 μg/ml) or CpG-B (5 μM) on fibrinogen-coated filters. (D) Gene induction of TNFα, TF, IFNα and IFNβ by Poly(I:C) (10 μg/mL) of macrophages plated on fibrinogen from F7^flflLysM^cre mice in comparison to littermate F7^flfl controls. (E) Migration of peritoneal macrophages on fibrinogen-coated filters from F7^flflLysM^cre and F7^flflCx3Cr1^cre mice in comparison to littermate F7^flfl controls stimulated with CpG-B (5 μM). (F) FXa generation with anti-TF 43D8, anti-TF 21E10, or control mouse IgG2a on MC38 cells (G) IFNα and IFNβ secreted into the cell supernatant by peritoneal macrophages from C57BL6J mice treated with anti-TF 43D8 IgG2a (50 μg/mL) or control mouse IgG2a (50 μg/mL). (H) Migration of peritoneal macrophages from C57BL/6J mice stimulated with Poly(I:C) (25 μg/mL) on fibrinogen-coated filters in the presence of anti-TF 43D8 IgG2a (50 μg/mL) or control mouse IgG2a (50 μg/mL). (I) Secreted TNFα, IFNα, and IFNβ in the cell supernatant of WT peritoneal macrophages plated on fibrinogen-coated plates and stimulated with Poly(I:C) (10 μg/mL) and treated with p38 inhibitor (SB 203580, 250 nM) or with ERK inhibitor (CAS 1049738-54-6 25 μM). (J) Migration of peritoneal macrophages stimulated with Poly(I:C) (25 μg/mL) on fibrinogen-coated filters with p38 inhibitor (SB 203580, 250 nM) or with ERK inhibitor (CAS 1049738-54-6 25 μM). Mean ± SD; the 1-way ANOVA with the Tukey multiple comparison test (B,I,J); the 2-way ANOVA with the Sidak multiple comparison test (C,D,E,G,H). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

Figure 6.

PAR2-β-arrestin signaling drives Poly(I:C)-induced monocyte/macrophage recruitment to the lungs. (A) Schematic overview of downstream PAR2 signaling pathways via recruitment of β-arrestin or G-proteins. (B) IFNα and IFNβ secreted into the cell supernatant from macrophages from F2rl1^S365A/T368A compared with WT mice on fibrinogen-coated plates. (C) Representative microscopic staining comparing the phosphorylation of ERK from peritoneal macrophage from F2rl1^S365A/T368A and WT mice after Poly(I:C) stimulation (25 μg/mL). (D) Nuclear and cytosolic quantification of pERK with Citation5. (E) Migration of macrophages and monocytes from F2rl1^S365A/T368A and WT mice on fibrinogen- or fibronectin-coated filters after Poly(I:C) stimulation (25 μg/mL). (F) Migration of macrophages from F2rl1^S365A/T368A and WT mice on fibrinogen- or fibronectin-coated filters after CpG-B stimulation (5 μM). (G) Quantification of different monocyte and macrophage subsets by flow cytometry of whole lung cell suspensions after intranasal Poly(I:C) treatment of F2rl1^S365A/T368A and WT mice. (H) Cell counts in BAL in unchallenged and Poly(I:C)-treated F2rl1^S365A/T368A and C57BL/6N mice. Mean ± SD; the 2-way ANOVA with the Sidak multiple comparison test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. PKC, protein kinase C.