Fibrinogen regulates the cytotoxicity of mycobacterial trehalose dimycolate but is not required for cell recruitment, cytokine response, or control of mycobacterial infection

Abstract

During inflammatory responses and wound healing, the conversion of soluble fibrinogen to fibrin, an insoluble extracellular matrix, long has been assumed to create a scaffold for the migration of leukocytes and fibroblasts. Previous studies concluded that fibrinogen is a necessary cofactor for mycobacterial trehalose 6,6'-dimycolate-induced responses, because trehalose dimycolate-coated beads, to which fibrinogen was adsorbed, were more inflammatory than those to which other plasma proteins were adsorbed. Herein, we investigate roles for fibrin(ogen) in an in vivo model of mycobacterial granuloma formation and in infection with Mycobacterium tuberculosis, the causative agent of tuberculosis. In wild-type mice, the subcutaneous injection of trehalose dimycolate-coated polystyrene microspheres, suspended within Matrigel, elicited a pyogranulomatous response during the course of 12 days. In fibrinogen-deficient mice, neutrophils were recruited but a more suppurative lesion developed, with the marked degradation and disintegration of the matrix. Compared to that in wild-type mice, the early formation of granulation tissue in fibrinogen-deficient mice was edematous, hypocellular, and disorganized. These deficiencies were complemented by the addition of exogenous fibrinogen. The absence of fibrinogen had no effect on cell recruitment or cytokine production in response to trehalose dimycolate, nor was there a difference in lung histopathology or overall bacterial burden in mice infected with Mycobacterium tuberculosis. In this model, fibrin(ogen) was not required for cell recruitment, cytokine response, or response to infection, but it promoted granulation tissue formation and suppressed leukocyte necrosis.

FIG. 1.

Subcutaneous TDM matrices in WT mice are primarily neutrophilic at early time points but later become pyogranulomatous to granulomatous, while Fib KO matrices become progressively suppurative. WT and Fib KO mice (n = 4 to 5 per time point) were injected subcutaneously in the scruff with 300 μl of TDM-coated 90-μm-diameter microspheres suspended within Matrigel. Matrices were removed for analysis at days 4 (A and E), 7 (B and F), and 12 (C, D, and G). Representative samples were fixed, sectioned, and stained with H&E. Photomicrographs represent leukocytic infiltrates in WT matrices (A to D) and in Fib KO matrices (E to G) typical for the depicted time point and are representative of three separate experiments. Multinucleated giant cells (black arrows) are present in panel C. An area showing neutrophil degeneration and necrosis, as well as peripheral eosinophilic ghost cells, as observed sporadically in 12-day-old TDM matrices from WT mice, is shown in panel D. The nuclear streaming of recruited neutrophils (red arrows) is prominent in panel F. Original magnification, ×1,000; bar = 50 μm.

FIG. 2.

Subcutaneous TDM matrices in Fib KO are grossly similar to those of the WT, with slight delays in neovascularization. TDM matrices from WT (A to C) and Fib KO (D to F) mice were removed at days 4 (A and D), 7 (B and E), and 12 (C and F).

FIG. 3.

Granulation tissue and capsule formation in KO matrices is delayed and impaired but can be complemented by the addition of exogenous fibrinogen. Sections of TDM matrices were stained using Masson's trichrome stain to show the deposition of collagen (blue) during capsule formation. The capsule surrounding WT (A) and Fib KO (D) matrices at 4 days is loosely organized and histologically similar. Well-developed granulation tissue surrounds 7-day-old WT matrices (B), and by 12 days (C), the capsule is composed primarily of collagen. (E) In Fib KO matrices, however, the granulation tissue response at 7 days is disorganized, edematous, and hypocellular, similar to the appearance at 4 days. (F) The addition of 500 μg/ml fibrinogen to the KO matrices allows well-organized granulation tissue to surround the matrix at 7 days. Asterisks indicate the interface between Matrigel and capsule. Original magnification, ×200.

FIG. 4.

Fibrinogen suppresses TDM-induced neutrophil necrosis in vitro. Bone marrow-derived neutrophils were purified and stimulated by TDM, vehicle control (n-hexane), or 60 μM staurosporine for 2 h in the presence or absence of 500 μg/ml fibrinogen and then stained using propidium iodide (PI) and annexin V to determine the percentage of the neutrophil population that is necrotic or apoptotic, respectively, by flow cytometry. (A) TDM induces a significantly higher percentage of PI-positive cells above that of the vehicle (n-hexane) control, and this percentage is significantly reduced in the presence of fibrinogen. (B) Staurosporine-induced neutrophil apoptosis, but not necrosis, also is significantly reduced in the presence of fibrinogen. Data from triplicate samples from a representative experiment are shown as means ± standard deviations, and asterisks indicate statistically significant differences (P < 0.05).

FIG. 5.

TDM induces NETs in the absence of fibrinogen. Sections of 7-day-old TDM matrices from Fib KO mice were labeled using rabbit anti-human neutrophil elastase and mouse anti-human histone H1, followed by Alexa Fluor 555 goat anti-rabbit and Alexa Fluor 488 goat anti-mouse antibodies and the DNA stain Draq5, and then examined by confocal immunofluorescence microscopy. (A) Histone H1 is observed along the surface of the streaming DNA. (C) Neutrophil elastase is observed within intact neutrophils (arrows) as well as along the streaming neutrophil DNA. (E) Draq5 staining indicates that this streaming material is composed of DNA. (B and D) Negative control sections were stained only with secondary antibody for H1 and elastase, respectively.