C3-dependent mechanism of microglial priming relevant to multiple sclerosis - PubMed (original) (raw)

C3-dependent mechanism of microglial priming relevant to multiple sclerosis

Valeria Ramaglia et al. Proc Natl Acad Sci U S A. 2012.

Abstract

Microglial priming predisposes the brain to neurodegeneration and affects disease progression. The signal to switch from the quiescent to the primed state is unknown. We show that deleting the C3 convertase regulator complement receptor 1-related protein y (Crry) induces microglial priming. Mice that were double-knockout for Crry and either C3 or factor B did not show priming, demonstrating dependence on alternative pathway activation. Colocalization of C3b/iC3b and CR3 implicated the CR3/iC3b interaction in priming. Systemic lipopolysaccharide challenge overactivated primed microglia with florid expression of proinflammatory molecules, which were blocked by complement inhibition. Relevance for neurodegenerative disease is exemplified by human multiple sclerosis (MS) and by experimental autoimmune encephalomyelitis (EAE), a model of MS. In human MS, microglial priming was evident in perilesional white matter, in close proximity to C3b/iC3b deposits. EAE was accelerated and exacerbated in Crry-deficient mice, and was dependent on C activation. In summary, C3-dependent microglial priming confers susceptibility to other challenges. Our observations are relevant to progression in MS and other neurological diseases exacerbated by acute insults.

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Conflict of interest statement

Conflict of interest statement: V.R. and F.B. are shareholders of Regenesance BV, a biopharmaceutical spin-off company that develops therapeutics that affect complement activation.

Figures

Fig. 1.

Fig. 1.

Alternative pathway-dependent microglial phenotype in Crry−/− spinal cord. (A_–_E) CD11b immunostaining in spinal cord of WT (A and Inset), Crry−/− (B and Inset), Crry−/−C3−/− (C), Crry−/−fB−/− (D), and fH−/− mice (E). (F) Quantification of CD11b immunostaining in brain and spinal cord of WT (n = 7), Crry−/− (n = 7), Crry−/−C3−/− (n = 3), Crry−/−fB−/− (n = 3), and fH−/− mice (n = 4). Values are given as means ± SD. Error bars are for n, number of animals. *P < 0.001 determined by one-way ANOVA. wm, white matter; gm, gray matter. (Scale bar: A_–_E, 100 μm; Insets, 50 μm.)

Fig. 2.

Fig. 2.

Alternative pathway activation in Crry−/− spinal cord. (A_–_C) Immunostaining for C3b/iC3b neoepitope, showing deposition in Crry−/− (B) but not in WT (A) or fH−/− (C) spinal cords. (D_–_F) Colocalization of C3b/iC3b with the microglial marker CD11b in Crry−/− mice. (Scale bar: A_–_C, 100 μm; D_–_F, 10 μm.) (G_–_L) Relative C3 (G), fB (H), fD (I), DAF (J), fH (K), and Crry (L) mRNA expression in spinal cords of WT (n = 7), Crry−/− (n = 7), and fH−/− (n = 4) mice. Values are normalized to the expression of β-actin and given as percentage (means ± SD) of WT control levels. Error bars are for n, number of animals. *P < 0.05 determined by one-way ANOVA.

Fig. 3.

Fig. 3.

Spinal cord expression of inflammatory mediators is up-regulated in Crry−/− mice after systemic LPS challenge. (A_–_F) Relative mRNA expression of IL-1β (A), TNF-α (B), IL-6 (C), Cox-2 (D), TGF-β1 (E), and iNOS (F) in spinal cords of WT and Crry−/− mice before (−LPS; WT n = 3; Crry−/− n = 4) and after (+LPS; WT n = 5; Crry−/− n = 8) systemic challenge with LPS. Some groups were pretreated at 12 h earlier with sCR1 (+sCR1; WT n = 4; Crry−/− n = 4) or PBS (+PBS; WT n = 3; Crry−/− n = 3). Values are normalized to expression of β-actin and given as percentage (means ± SD) of WT controls. Asterisks indicate statistically significant differences determined by one-way ANOVA. Error bars are for n, number of animals. (G_–_J) Immunohistochemical staining for IL-1β. IL-1β microglial immunoreactivity is indicated by arrows. (Scale bar: G_–_J, 50 μm.) (K) Western blot analysis of plasma from Crry−/− mice before and after systemic treatment with one dose (20 mg/kg) of sCR1. Plasma from WT and untreated Crry−/− mice is included as additional controls. C3 intact α-chain is shown (arrow). (L) Densitometry of Western blot analysis. Values are expressed as mean ± SD of the integrated optical density (IOD) for the immunoreactive band. Error bars are for n, number of animals. Asterisks indicate statistically significant differences determined by one-way ANOVA.

Fig. 4.

Fig. 4.

Microglial morphology and C3b/iC3b immunoreactivity in normal-appearing MS tissue. (A and B) Immunostaining for the HLA-DP-DQ-DR microglial marker showing increased immunoreactivity and thick, ramified microglial morphology in normal-appearing MS tissue (B) compared with control white matter (A). (C) A cluster of thickly ramified microglial cells (in black) on a proteolipid protein (PLP)-positive nerve fiber (in light brown) in normal-appearing tissue surrounding an MS lesion. (D) Transverse view of a microglial cluster (in black) showing thick microglial ramifications around a central core. (E) A C3b/iC3b-positive segment of nerve fiber located in intact MS tissue. (F) Transverse view of E. (G and H) Double-immunostaining for HLA-DP-DQ-DR (green) and C3b/iC3b (red) in intact MS tissue showing partial colocalization (in yellow), suggesting discrete areas of contact between the C3b/iC3b localized on the nerve fiber and the HLA-DP-DQ-DR microglial marker.

Fig. 5.

Fig. 5.

Induction and characterization of EAE in Crry−/− mice. (A) Clinical disease score for EAE, showing earlier onset and increased disease severity in Crry−/− mice (n = 6) compared with WT (n = 4). All animals were killed by day 21. Values are expressed as mean ± SD. Error bars are for n, number of animals. *P < 0.05 determined by Mann–Whitney U nonparametric test. (B_–_M) Inflammation and myelin loss were assessed by H&E staining (B_–_D), Iba-1 immunostaining of macrophages (E_–_G), and IL-1β immunostaining (H_–_J) and LFB staining (K_–_M) of myelin in WT (B, E, H, and K) and Crry−/− (C, F, I, and L) EAE spinal cords. Perivascular infiltration, Iba-1–positive microglia/macrophages, IL-1β deposition, and demyelination were evident in Crry−/− spinal cords (arrows in C). Arrowheads in K and L indicate a blood vessel. (Scale bars: 100 μm.) Quantification of perivascular infiltration (D), Iba-1 staining (G), IL-1β deposition (J), and perivascular LFB staining (M) in Crry−/− mice compared with WT. Values are expressed as mean ± SD. Error bars are for n, number of animals. *P < 0.05 in D and M; *P < 0.01 in G and J; determined by two-tailed Student's t test.

Fig. 6.

Fig. 6.

Model of microglial priming. In the normal situation, a peripheral event, such as an infection, is the first hit to the brain (via mechanisms yet to be defined and discussed in the text), activating resting microglia to produce inflammatory mediators affecting neuronal function and producing sickness behavior or delirium. In pathological situations such as MS, a first hit (disease) primes microglia for overactivation in response to a second hit. In normal-appearing brain tissue, accumulation of C3b/iC3b on microglia, either secondary to preexisting neurodegeneration or caused by a primary (possibly genetic) dysregulation of C3 activation, is the first hit that primes microglial cells. Under these circumstances, a peripheral injurious event, such as an infection, is the second hit to the brain that overactivates primed microglia to produce an elevated amount of inflammatory mediators, determining severe sickness behavior and prolonged delirium leading to disease progression.

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