The fibrin-derived gamma377-395 peptide inhibits microglia activation and suppresses relapsing paralysis in central nervous system autoimmune disease

Abstract

Perivascular microglia activation is a hallmark of inflammatory demyelination in multiple sclerosis (MS), but the mechanisms underlying microglia activation and specific strategies to attenuate their activation remain elusive. Here, we identify fibrinogen as a novel regulator of microglia activation and show that targeting of the interaction of fibrinogen with the microglia integrin receptor Mac-1 (alpha(M)beta(2), CD11b/CD18) is sufficient to suppress experimental autoimmune encephalomyelitis in mice that retain full coagulation function. We show that fibrinogen, which is deposited perivascularly in MS plaques, signals through Mac-1 and induces the differentiation of microglia to phagocytes via activation of Akt and Rho. Genetic disruption of fibrinogen-Mac-1 interaction in fibrinogen-gamma(390-396A) knock-in mice or pharmacologically impeding fibrinogen-Mac-1 interaction through intranasal delivery of a fibrinogen-derived inhibitory peptide (gamma(377-395)) attenuates microglia activation and suppresses relapsing paralysis. Because blocking fibrinogen-Mac-1 interactions affects the proinflammatory but not the procoagulant properties of fibrinogen, targeting the gamma(377-395) fibrinogen epitope could represent a potential therapeutic strategy for MS and other neuroinflammatory diseases associated with blood-brain barrier disruption and microglia activation.

Figure 1.

Fibrinogen directly activates microglia resulting in cytoskeletal rearrangements and increased phagocytosis. (A) IsoB4/DAPI immunostained microglia cultured on immobilized fibrinogen have increased cell body size adopting an amoeboid morphology (right). Untreated primary microglia show small cell bodies and thin, bipolar processes (left). LPS-treated cells show activated morphology characterized by cell body swelling (middle). Bar, 26 μm; inset, 21 μm. (B) Quantitation of microglia activation reveals a dramatic increase upon fibrinogen stimulation. (C) Fibrinogen-stimulated microglia showed increased phagocytosis of fluorescent Escherichia coli as compared with untreated microglia. LPS served as a positive control. (D) Deconvolution microscopy of primary microglia revealed significant rearrangements of the cytoskeleton upon treatment with fibrinogen. Microglia were stained with antibodies to actin and β-tubulin in red, and the nucleus was stained blue with DAPI. Bar, 4.4 μm.

Figure 2.

Fibrinogen directly activates microglia via the Mac-1 integrin receptor. (A) Fibrinogen-induced morphologic activation of microglia is blocked by the addition of a rat anti-CD11b neutralizing antibody (M1/70). Rat IgG (control) did not change the effects of fibrinogen in microglia activation. Bar, 39 μm; inset, 17 μm. (B) Quantitation of microglia activation reveals that the Mac-1 neutralizing antibody blocks fibrinogen-induced activation but not LPS activation of microglia. (C) Increased microglia phagocytosis upon fibrinogen stimulation is blocked by the addition of a PI3K inhibitor (LY294002) and diminished in the presence of a CD11b neutralizing antibody. A control anti-TLR4 or IgG showed no reduction in fibrinogen-stimulated phagocytosis. (D) Western blots show increased active RhoA and Akt activation upon fibrinogen stimulation of microglia. In both assays, LPS served as a positive control. (E) Confocal microscopy demonstrates the spatial interaction between CD11b⁺ cells (green) and fibrin (red) in spinal cords from PLP_139-151–immunized mice. (F) Confocal double immunofluorescence shows fibrin (green) surrounding an activated microglia (red) in a human early demyelinating plaque of acute MS. Bars: E, 6.7 μm; F, 15 μm.

Figure 3.

Fibrin depletion using ancrod reverses relapsing paralysis and reduces microglia activation in EAE. (A) Immunofluorescence of control (left) and fibrin-depleted (right) spinal cord with antibodies against fibrinogen (red) and CD11b (green). Activated CD11b⁺ cells colocalize with fibrin deposition in the control spinal cord (yellow). Bar, 8.4 μm. (B) Clinical scores of PLP_139-151–immunized mice. Fibrin depletion begins after the first paralytic episode. Fibrin-depleted mice (red circles; n = 8) did not develop first or second relapses as compared with control mice (black circles; n = 10). (C) Rotarod motor strength and coordination test. Fibrin-depleted mice outperformed immunized control untreated mice on day 25 after immunization. Data are represented as the mean clinical score and are mean ± SEM.

Figure 4.

Fib^γ390-396A mice have reduced clinical scores and microglia activation in EAE. (A) Clinical scores of MOG_35-55–immunized mice. Fib^γ390-396A mice (blue triangles; n = 29) show statistically significant lower clinical scores (*, P < 0.01) than Fib^γWT control mice (black squares; n = 28). (B) Individual clinical scores from Fib^γ390-396A and Fib^γWT mice on days 20 (average clinical score, 3.3 ± 0.22 for Fib^γWT [n = 19] vs. 2.1 ± 0.32 for Fib^γ390-396A [n = 20]; P < 0.01) and 31 after immunization (average clinical score, 2.8 ± 0.25 for Fib^γWT [n = 15] vs. 1.5 ± 0.23 for Fib^γ390-396A [n = 20]; P < 0.001). (C) Histological analysis of spinal cord sections stained with hematoxylin/eosin revealed increased inflammation in the Fib^γWT mice versus the Fib^γ390-396A mice. (D) Survival curve of Fib^γ390-396A and Fib^γWT mice after MOG_35-55 immunization. Fib^γ390-396A had a greater survival rate than the Fib^γWT mice. (E) Rotarod analysis of Fib^γ390-396A and Fib^γWT mice on day 13. Fib^γ390-396A significantly outperformed their wild-type counterparts in a behavioral test designed to assess motor skill function. n = 7 mice per group. **, P < 0.05. (F) Increased activation of IsoB4⁺ cells in Fib^γWT mice as compared with Fib^γ390-396A mice. Bar, 58 μm. Data are represented as the mean clinical score and are mean ± SEM.

Figure 5.

The fibrin-derived γ^377-395 peptide inhibits microglia activation. (A) Microglia activation upon fibrinogen stimulation is attenuated in the presence of γ^377-395. Bar, 33 μm; inset, 29 μm. (B) Quantitation of microglia activation reveals that γ^377-395 diminishes fibrinogen-stimulated microglia activation but has no effect on LPS activation. (C) Treatment of microglia with γ^377-395 blocks fibrinogen-induced Akt activation in vitro. LPS activation of Akt is unaffected by γ^377-395 treatment.

Figure 6.

The fibrin-derived γ^377-395 peptide suppresses EAE. (A) Clinical scores from γ^377-395 peptide–vaccinated mice. Mice were immunized with γ^377-395 peptide before EAE induction with PLP_139-151 peptide. Vaccinated mice (blue triangles; n = 15) had significantly reduced clinical scores as compared with control mice (black squares; n = 15). (B) Clinical scores from PLP_139-151–immunized mice where the γ^377-395 peptide was administered every day intranasally after the peak of the initial paralytic episode. γ^377-395–treated mice (blue triangles; n = 14) did not show a relapse at approximately day 30 as compared with the controls (black squares; n = 13). *, P < 0.05; **, P < 0.01. Data are represented as the mean clinical score and are mean ± SEM. Immunostaining for Mac-3 and iNOS (inset) on day 30 after immunization shows dramatic reduction of microglia activation in the γ^377-395 peptide–treated mice after EAE induction (D) when compared with control (C). Bar, 20 μm; inset, 10 μm. (E) Quantitation shows a twofold reduction in Mac-3 (P < 0.05) and a 4.2-fold reduction in iNOS (P < 0.01) after γ^377-395 peptide treatment, whereas there are no major differences in T cell infiltration.

Figure 7.

The fibrin-derived γ^377-395 peptide does not affect in vivo coagulation or fibrin polymerization. In vivo clotting time (A) and prothrombin times (B) were assayed in the presence of 30 μg γ^377-395 peptide, the dose administered in vivo daily, and 90 μg γ^377-395 peptide. Neither dose affected blood clotting times. (C) In vitro fibrin polymerization was examined in the presence of the γ^377-395 peptide. GPRP, a known inhibitor of fibrin formation, significantly attenuated fibrin formation, whereas the γ^377-395 peptide had no effect on fibrin polymerization.

Figure 8.

Proposed model for the role of fibrin-induced activation of microglia in inflammatory demyelination. BBB disruption permits the leakage of fibrinogen, the high affinity ligand for Mac-1, in the CNS parenchyma. Fibrinogen is converted to fibrin and functions as the spatial signal to induce local activation of microglia via activation of the Mac-1 integrin receptor and induction of signaling pathways downstream of Mac-1, such as Akt and Rho, resulting in phagocytosis that could determine the extent of tissue damage in inflammatory demyelination.