Differential regulation of macrophage inflammatory activation by fibrin and fibrinogen

Abstract

Fibrin is a major component of the provisional extracellular matrix formed during tissue repair following injury, and enables cell infiltration and anchoring at the wound site. Macrophages are dynamic regulators of this process, advancing and resolving inflammation in response to cues in their microenvironment. Although much is known about how soluble factors such as cytokines and chemokines regulate macrophage polarization, less is understood about how insoluble and adhesive cues, specifically the blood coagulation matrix fibrin, influence macrophage behavior. In this study, we observed that fibrin and its precursor fibrinogen elicit distinct macrophage functions. Culturing macrophages on fibrin gels fabricated by combining fibrinogen with thrombin stimulated secretion of the anti-inflammatory cytokine, interleukin-10 (IL-10). In contrast, exposure of macrophages to soluble fibrinogen stimulated high levels of inflammatory cytokine tumor necrosis factor alpha (TNF-α). Macrophages maintained their anti-inflammatory behavior when cultured on fibrin gels in the presence of soluble fibrinogen. In addition, adhesion to fibrin matrices inhibited TNF-α production in response to stimulation with LPS and IFN-γ, cytokines known to promote inflammatory macrophage polarization. Our data demonstrate that fibrin exerts a protective effect on macrophages, preventing inflammatory activation by stimuli including fibrinogen, LPS, and IFN-γ. Together, our study suggests that the presentation of fibrin(ogen) may be a key switch in regulating macrophage phenotype behavior, and this feature may provide a valuable immunomodulatory strategy for tissue healing and regeneration.

Statement of significance: Fibrin is a fibrous protein resulting from blood clotting and provides a provisional matrix into which cells migrate and to which they adhere during wound healing. Macrophages play an important role in this process, and are needed for both advancing and resolving inflammation. We demonstrate that culture of macrophages on fibrin matrices exerts an anti-inflammatory effect, whereas the soluble precursor fibrinogen stimulates inflammatory activation. Moreover, culture on fibrin completely abrogates inflammatory signaling caused by fibrinogen or known inflammatory stimuli including LPS and IFN-γ. Together, these studies show that the presentation of fibrin(ogen) is important for regulating a switch between macrophage pro- and anti-inflammatory behavior.

Keywords: Fibrin; Fibrinogen; Macrophage; Polarization.

Fig. 1

Composition of collagen-fibrin gels affects fiber architecture and macrophage cytokine secretion. (A) Representative backscatter images of composite collagen-fibrin gels with indicated mg/ml collagen: mg/ml fibrin concentrations. Scale bar: 25 μm. (B) Graph of TNF-α (left) and IL-10 (right) secretion by macrophages cultured on 2 mg/ml collagen:fibrin composite gels with indicated fibrin concentrations. Values are mean ± SEM of n = 3 biological replicates and asterisks denote p < 0.05 by one way ANOVA followed by Tukey’s HSD when comparing with the 0 mg/ml fibrin condition.

Fig. 2

Presentation of fibrin(ogen) modulates macrophage cytokine secretion. (A) Schematic of experimental conditions examining the effect of fibrin and fibrinogen on macrophage activation. (B) Graph of TNF-α (left) and IL-10 (right) secretion by macrophages cultured on different concentrations of fibrin or stimulated with different doses of soluble fibrinogen for 36 h. Values are mean ± SEM of n = 3 biological replicates and asterisks denote p < 0.05 by Student’s t-test followed by false discovery rate correction for multiple comparisons for comparisons within each concentration group. (C) Schematic of experimental conditions to examine the combined effect of fibrin and fibrinogen on macrophage activation. (D) Graph of TNF-α (left) and IL-10 (right) secretion by macrophages cultured on 2 mg/ml fibrin or TCP surface, and stimulated with different doses of soluble fibrinogen for 36 h. Values are mean ± SEM of n = 3 biological replicates and asterisks denote p < 0.05 by Student’s t-test followed by false discovery rate correction for multiple comparisons for comparisons within each concentration group.

Fig. 3

Fibrin inhibits fibrinogen-induced inflammatory signaling. (A) Column-wise mean normalized Z-score heat map of cytokines secreted by macrophages after culture on 2 mg/ml fibrin gels with or without 4 mg/ml fibrinogen, or cultured on tissue culture plastic and stimulated with 4 mg/ml fibrinogen, 1 ng/ml LPS/IFN-γ, 10 ng/ml IL-4/IL-13, or 1 ng/ml of LPS and 10 ng/ml IL-4/IL-13 for 18 h. (B) Graphs of selected cytokines secreted by macrophages cultured on 2 mg/ml fibrin with and without 4 mg/ml soluble fibrinogen (F and F + Fg) or cultured on TCP with and without 4 mg/ml soluble fibrinogen (Control and Fg) for 4 and 18 h. See Supplemental Materials for information on statistical comparisons and experimental replicates.

Fig. 4

Fibrin and fibrinogen differentially regulate markers associated with macrophage polarization. (A) Column-wise Z-score normalized heat map of mean gene expression by macrophages after culture on 2 mg/ml fibrin gels with or without 2 mg/ml fibrinogen, or cultured on TCP and stimulated with 2 mg/ml fibrinogen, 1 ng/ml LPS/ IFN-γ (L + I), 10 ng/ml IL-4/IL-13 (4 + 13), or 1 ng/ml of LPS and 10 ng/ml IL-4/IL-13 (L + 4 + 13) for 4 h. (B) Graphs of expression levels relative to Gapdh of selected gene expressed by macrophages cultured on 2 mg/ml fibrin with and without 2 mg/ml soluble fibrinogen (F and F + Fg) or cultured on TCP with and without 2 mg/ml soluble fibrinogen (Control and Fg) for 4 h. See Supplemental Materials for information on all genes and conditions shown in A and statistical comparisons. (C) Representative fluorescence images of macrophages cultured in the indicated conditions for 18 h and stained for arginase-1 (red), iNOS (green) or nuclei (blue). Scale bar: 25 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Actin distribution and cell shape are modulated by fibrin(ogen). Representative fluorescent images of phalloidin (green) and Hoechst (blue)-stained macrophages cultured on glass or 2 mg/ml fibrin and stimulated with 4 mg/ml fibrinogen for 4 or 18 h. Scale bar: 25 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Effect of fibrin and fibrinogen on macrophage activation by LPS and cytokines. Graph of TNF-α (left) and IL-10 (right) secretion of macrophages seeded on TCP surface or 2 mg/ml fibrin gels, with and without 2 mg/ml fibrinogen, and further stimulated with 1 ng/ml LPS/IFN-γ, 10 ng/ml IL-4/IL-13 or 1 ng/ml of LPS and 10 ng/ml IL-4/IL-13 (LPS/IL-4/IL-13). Values are mean ± SEM of n = 3 biological replicates; asterisk groups denote p < 0.05 by one way ANOVA followed by Tukey’s HSD test for comparisons within each stimulation group.