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Received 16 Feb 2015 | Accepted 27 Jul 2015 | Published 10 Sep 2015

Jae Kyu Ryu1, Mark A. Petersen1,2, Sara G. Murray1, Kim M. Baeten1, Anke Meyer-Franke1, Justin P. Chan1, Eirini Vagena1, Catherine Bedard1, Michael R. Machado1, Pamela E. Rios Coronado1, Thomas Prodhomme3,4, Israel F. Charo5, Hans Lassmann6, Jay L. Degen7, Scott S. Zamvil3,4 & Katerina Akassoglou1,3,4

Autoimmunity and macrophage recruitment into the central nervous system (CNS) are critical determinants of neuroinammatory diseases. However, the mechanisms that drive immunological responses targeted to the CNS remain largely unknown. Here we show that brinogen, a central blood coagulation protein deposited in the CNS after bloodbrain barrier disruption, induces encephalitogenic adaptive immune responses and peripheral macrophage recruitment into the CNS leading to demyelination. Fibrinogen stimulates a unique transcriptional signature in CD11b antigen-presenting cells inducing the recruitment and local

CNS activation of myelin antigen-specic Th1 cells. Fibrinogen depletion reduces Th1 cells in the multiple sclerosis model, experimental autoimmune encephalomyelitis. Major histocompatibility complex (MHC) II-dependent antigen presentation, CXCL10- and CCL2-mediated recruitment of T cells and macrophages, respectively, are required for brinogen-induced encephalomyelitis. Inhibition of the brinogen receptor CD11b/CD18 protects from all immune and neuropathologic effects. Our results show that the nal product of the coagulation cascade is a key determinant of CNS autoimmunity.

1 Gladstone Institute of Neurological Disease, University of California, San Francisco, California 94158, USA. 2 Division of Neonatology, Department of Pediatrics, University of California San Francisco, San Francisco, California 94143, USA. 3 Department of Neurology, University of California San Francisco, San Francisco, California 94143, USA. 4 Program in Immunology, University of California San Francisco, San Francisco, California 94143, USA. 5 Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, California 94158, USA. 6 Centre for Brain Research, Medical University of Vienna, Vienna A-1090, Austria. 7 Division of Experimental Hematology, Cincinnati Childrens Hospital Research Foundation and University of Cincinnati College of Medicine, Cincinnati, Ohio 45229, USA. Correspondence and requests for materials should be addressed to S.S.Z. (email: mailto:[email protected]

Web End [email protected] ) or K.A. (email: mailto:[email protected]

Web End [email protected] ).

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Blood coagulation protein brinogen promotes autoimmunity and demyelination via chemokine release and antigen presentation

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9164

Disruption of the homeostatic balance between the vasculature and the brain is a sustained and often early feature of neurologic diseases and traumatic insults to the

central nervous system (CNS). Understanding how bloodbrain barrier (BBB) disruption instigates and amplies immune and degenerative responses leading to brain pathology and loss of function would be instrumental in the design of novel treatments for neurologic diseases. Fibrinogen (coagulation factor I) is a major component in the blood that upon BBB disruption enters the CNS and is deposited as insoluble brin1. Although soluble brinogen in the bloodstream is not proinammatory, activation of the coagulation cascade results in the formation of brin associated with exposure of cryptic epitopes that transform brinogen from a blood factor to a potent activator of innate immunity1. In multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), BBB disruption and brin deposition are detected in the white matter along with microglial activation before T-cell inltration and the onset of demyelination26. Indeed, increased coagulation activity leading to brin formation occurs early in neuroinammation before demyelination4, and is highly upregulated in active MS plaques7. Moreover, injection of soluble brinogen in the healthy brain results in brin formation5. Fibrin deposition is abundant not only in early but also active and chronic MS lesions, and correlates with demyelination and T-cell inltration3,810. Genetic or pharmacologic depletion of brinogen decreases microglial activation and axonal damage and attenuates neurologic signs in EAE5,7,1113 and other MS models14,15. While studies in EAE mice support a role for brin in the activation of microglia in myelinated areas, its role in the initiation and propagation of myelin-targeted adaptive immune responses is unknown. Moreover, despite the vast literature on BBB disruption and activation of the coagulation cascade in brain diseases, there is currently no model of neuroinammation induced by a coagulation factor.

Here we developed a model of coagulation-driven demyelination to directly assess the role of BBB disruption and brin in the induction of CNS autoimmunity and demyelination. Surprisingly, introduction of brinogen into the healthy CNS was sufcient to induce activation of adaptive immunity targeted to CNS myelin antigens leading to demyelination. The effect of brinogen as an initiator of CNS autoimmunity was rst substantiated in vivo, where a single stereotactic injection of brinogen in the corpus callosum induced recruitment and local differentiation of myelin antigen-specic Th1 cells leading to demyelination. In accordance, endogenous brinogen was required for T-helper 1 (Th1) cell activation in EAE. Microarray analysis in cell autonomous systems of antigen-presenting cells (APCs) uncovered a unique transcriptional signature for brin that links the coagulation cascade with M1-type activation of APCs associated with induction of antigen presentation and release of leukocyte recruiting chemokines. Rescue experiments to genetically deplete the brin-specic immune responses in APCs demonstrated that secretion of the monocyte chemoattractant protein-1 (MCP-1/ CCL2) and the C-X-C motif ligand-10 (CXCL10), together with MHC II-dependent antigen presentation, are essential molecular mediators for brin-induced autoimmune responses in the CNS. Furthermore, we showed that brin induces CNS autoimmune responses not via its prohaemostatic functions, but due to CD11b/CD18 receptor activation in APCs. Our results show for the rst time that a component of the coagulation cascade induces autoimmunity, attributes the CNS effects of coagulation to the proinammatory effects of brin and introduces brinogen-induced encephalomyelitis (FIE) as a novel coagulation-driven, autoimmune-mediated model for the study of novel mechanisms and therapies for brain inammation.

ResultsFibrinogen in the CNS induces inammatory demyelination. To determine whether brinogen extravasation is sufcient to induce CNS pathology in vivo, we stereotactically injected brinogen at its physiological plasma concentration into the corpus callosum, the largest CNS white matter tract associated with disability in MS16. Surprisingly, this single injection of brinogen spontaneously induced local demyelination within 7 days (Fig. 1a). Microglial activation was rapidly induced 1 day after brinogen injection and preceded demyelination (Fig. 1a). Activation of innate immunity regulates autoimmune responses including T-cell recruitment and differentiation17,18. Fibrinogen injection induced inltration of both CD4 and CD8 T-cell populations in the CNS, but not in the spleen (Fig. 1a,b;

Supplementary Fig. 1). T-cell inltration occurred as early as 1 day after injection and markedly increased 3 and 7 days after injection (Fig. 1a), thus also preceding demyelination. Injection of brinogen in the spinal cord ventral column also induced inammatory demyelination (Supplementary Fig. 2). Neuropathologic alterations were not observed after injection of control articial cerebrospinal uid (ACSF) in either the brain or spinal cord (Fig. 1; Supplementary Fig. 2). Although microglia show a diffuse response in the cortex along the needle track 1 day after ACSF injection, this response resolves and no alterations are observed either in the cortex or the corpus callosum (Fig. 1). While wild-type (WT) plasma induced demyelination similar to brinogen, injection of plasma from brinogen-decient (Fib / ) mice19, which contains all plasma proteins except brinogen, led to an 82% reduction in demyelination compared with WT plasma (Supplementary Fig. 3). Injection of the plasma proteins albumin or high-molecular-weight kininogen had no effect (Supplementary Fig. 3).

Activation of the coagulation cascade converts brinogen to proinammatory brin primarily due to exposure of a cryptic epitope in the brinogen g chain (g377395), which binds to

CD11b/CD18 integrin (Mac-1, complement receptor 3, aMb2)20,21. The interaction of brin with CD11b/CD18 is genetically targeted in Fibg390396A mice, in which brinogen has been mutated to lack the CD11b/CD18-binding motif, but retains normal clotting function22. Injection of plasma derived from Fibg390396A mice resulted in a 70% reduction in demyelination compared with WT plasma (Supplementary Fig. 3), suggesting that the interaction of brin with CD11b/ CD18 is required for the induction of demyelination. Since a portion of brinogen in the plasma is known to bind extracellular matrix proteins and growth factors, we also produced and tested recombinant brinogen, which is clottable and hydrodynamically indistinguishable from plasma brinogen, with the exception that it did not carry other plasma-derived factors23. Similar to plasma brinogen, recombinant brinogen also induced demyelination and microglial activation (Supplementary Fig. 4). These results suggest that brinogen is a major component in the plasma that in the healthy CNS white matter triggers T-cell recruitment and demyelination even in the absence of pre-existing inammatory or myelin abnormalities.

Fibrinogen induces M1-type activation of APCs. Genome-wide microarray analysis either in the corpus callosum after brinogen injection or in cell autonomous systems of brin-stimulated microglia or bone marrow-derived macrophages (BMDMs) revealed a unique brin transcriptional signature enriched in genes regulating immune responses, particularly those required to induce activation of T cells by APCs24, such as MHC II, CD86 and IL-12, and recruitment of T cells and peripheral macrophage into the CNS2527, such as CXCL10 and CCL2

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9164 ARTICLE

ACSF Fibrinogen

****

Demyelination

(% of area)

LFB PAS

Toluidine blue

MBP lba-1

CD3 DAPI

1 d 3 d 7 d

**** ****

****

CD3+ T cells/0.5 mm2 Iba-1+ cells/0.5 mm2

120

100 80 60 40 20

***

****

ACSF Fibrinogen ACSF Fibrinogen

1.9% 8.8% 0.7% 2.3%

CD4

Brain

CD8

Brain

CD3 CD3

Figure 1 | Induction of T-cell recruitment and inammatory demyelination by a single brinogen injection in the CNS. (a) Demyelination (LFB/PAS and toluidine blue), microglial activation and demyelination (MBP/Iba-1), and T-cell inltration (CD3) in the corpus callosum of mice injected with brinogen compared with ACSF control. Scale bar, 100 mm (top panel); 10 mm (second panel); 100 mm (third panel); 80 mm (bottom panel). Representative histological sections from day 7 after injection are shown. Data are presented as means.e.m. (n 56 mice per time point). *Po0.05, ***Po0.001, ****Po0.0001

(two-way ANOVA and Bonferronis multiple comparisons test). (b) FACS analysis of T cells isolated from the brain (corpus callosum) 7 days after brinogen or ACSF injection stained with CD3, CD4 and CD8 (n 3 independent experiments; each experiment generated from pooled brain cells from

n 34 mice). ANOVA, analysis of variance; d, days; FACS, uorescence-activated cell sorting; LFB, Luxol fast blue.

(Fig. 2ad; Supplementary Tables 13). Other immune response genes, such as complement components, lipocalin and proteins involved in iron binding and oxidative stress, were also increased. Fibrin induced M1-type activation and induction of antigen-presenting genes in both primary microglia and BMDMs (Fig. 2c; Supplementary Fig. 5). Consistent with these ndings, protein and gene expression of MHC class II and CD86 were also induced in brin-exposed BMDMs, and were inhibited by anti-CD11b treatment (Fig. 2d; Supplementary Fig. 6). Lipopolysaccharide (LPS) was used as a positive control (Fig. 2d; Supplementary Fig. 6). In agreement, brinogen injection in the corpus callosum induced MHC class II and CXCL10 in Iba-1 cells (Supplementary Fig. 7a,b). Similar to brinogen, injection of

WT plasma in the corpus callosum induced CXCL10 expression, while plasma derived from Fib / or Fibg390396A mice showed a signicant reduction (Supplementary Fig. 7c). Strikingly, MHC

II / (ref. 28), Cxcl10 / (ref. 29) and RAG2 / gc / (ref. 30) mice, which lack T, B and natural killer cells, had a marked reduction in demyelination after brinogen injection

compared with WT mice (Fig. 2e). Cxcl10 / mice also had 54% less T-cell inltration than WT mice after brinogen injection (Supplementary Fig. 8). Overall, these results suggest that brin is a potent activator of APCs that triggers demyelination via T-cell recruitment into the CNS.

Fibrinogen promotes recruitment of encephalitogenic T cells. To examine whether brinogen could induce T-cell responses against myelin antigens in vivo, we tested its effects in 2D2 transgenic mice constitutively expressing T-cell antigen receptors (TCRs) specic for myelin oligodendrocyte glycoprotein (MOG)31. After brinogen injection in the corpus callosum, T-cell inltration and demyelination in 2D2 mice were 46% and 51% higher, respectively, compared with WT mice (Fig. 3a). In contrast, in control OT-II transgenic mice constitutively expressing TCRs specic for ovalbumin (OVA), an antigen not present in the CNS32, T-cell inltration after brinogen injection was 77% and 66% less than in 2D2 or WT mice, respectively (Fig. 3a). Consistent with this result, demyelination was also

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a b

ACSF

Fibrinogen

ACSF

Fibrinogen

Brain white matter Primary microglia

Untreated

Fibrin

GO term ( biological process)

GO term (biological process)

Immune responseDefense responseResponse tp TNFAntigen processing and presentation Regulation of NF-KB import into nucleus Regulation of chemokine production

Leukocyte chemotaxis

Immune response

Response to wounding

Immune effector process

Response to cytokine stimulus

Regulation of neurological system process

Positive regulation of transport

Regulation of cell death

Regulation of vesicle-mediated transport

Regulation of cellular localization

P value

>0.05 <0.0005

Regulation of cell proliferation Integrin-mediated signalling pathway Positive regulation of endocytosis PhagocytosisCytokine metabolic process Purinergic receptor signalling pathway Positive regulation of JNK cascade Lymphocyte homeostasis Lipoprotein metabolic process Response to fatty acid

P value

>0.05 <0.0005

Expresssion (log2)

5 7.5

Expresssion (log2)

5 7.5

APCs: M1 genes

Untreated

Fibrin

LFB PAS

BMDM APCs

Immune system process

Fibrin Untreated

Fibrin

GO term (biological process)

Cd69 Cd86 Tlr2 lcam1 ll1b

ll12b ll18Tnf TnfSF10

Cxcl1 Cxcl9 Cxcl10 Cxcl11 Ccl4 Ccl5

Ccl20 Gbp2

Gbp4 lrg1 lrf1 lrf7 Socs3

Nos2 Pde4b Cfb

ll6

Regulation of cytokine production Regulation of immune system process

Leukocyte differentiation

Leukocyte cellcell adhesion

Myeloid cell differentiation

Cytokine secretion

Response to interferon-

Response to interleukin-12 Response to interleukin-1Cell adhesion mediated by integrin Response to type I interferon Microtubule-based movement Hormone transportHistamine secretion

IL-12 Cxcl10

Ccl2

CD40

CD86

IFN-

Receptors Cytokines, chemokines Others Signalling

Untreated

Expresssion (log2)

5 7.5 10

MHC II/

Expresssion (log2)

5 7.5

P value

>0.05 <0.0005

Rag2/ c/

Expresssion (log2)

5 7.5

Untreated Fibrin+lgG

Fibrin+anti-CD11b LPS

7.35% 38.3%

16.8% 64.5%

***

****

***

MHC II/

Rag2/ c/

CD86

CD86RNA relative

expression

Fibrin lgG

Anti-CD11b

8 6 4 2

Demyelination

(% of area)

+ + +

Fibrinogen

CD11b

Figure 2 | Adaptive immunity and antigen presentation are required for brinogen-induced demyelination. (a) Affymetrix microarray gene expression analysis and enrichment of gene ontology (GO) of brinogen-injected or ACSF-injected corpus callosum at 12 h post injection. Heatmaps of the 142 genes with Z1.5 change in expression between ACSF and brinogen. (b) Affymetrix microarray gene expression analysis of brin-stimulated rat primary

microglia at 6 h in vitro. Heatmap and GO analysis of gene expression proles of 1,342 genes with Z1.5 change in expression between unstimulated and

brin treatment. (c) Affymetrix microarray gene expression analysis of brin-stimulated mouse APCs at 6 h in vitro. Heatmap and GO analysis of gene expression proles of key genes with Z1.5 change in expression (left). Heatmap of the M1-related genes with Z2.0 change in expression after brin

stimulation (right). (d) FACS analysis of CD86 expression in APCs after brin stimulation. LPS was used as positive control. Anti-CD11b antibody treatment reduces CD86 expression in APCs. Real-time PCR analysis of CD86 gene expression in BMDMs after brin stimulation treated with anti-CD11b or IgG isotype control antibody. Data are presented as means.e.m. (n 4 independent experiments; right). **Po0.01, ***Po0.001, ****Po0.0001 (one-way

ANOVA). (e) Demyelination (LFB/PAS) in the corpus callosum of WT, MHC II / or RAG2 / gc / mice 7 days after brinogen injection. Representative images are shown. Scale bar, 100 mm. Data are presented as means.e.m. (n 6 mice per group). **Po0.01, ***Po0.001 (one-way

ANOVA and Bonferronis multiple comparisons test). ANOVA, analysis of variance; FACS, uorescence-activated cell sorting; LFB, Luxol fast blue.

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9164 ARTICLE

WT 2D2 OTII

LFB PASCD3DAPI

CD3+ T cells/0.5 mm2

****

* **

0 WT 2D2 OTII Fibrinogen

***

150 120

90 60 30

Demyelination

(% of area)

25 20 15 10

5 0

WT 2D2 OTII Fibrinogen

BMDMs

+ Fibrin

+ Anti-CD11b, lgG + MOG3555

Co-cultured with naive 2D2 CD4+ T cells

BrdU assay

* *

BrdU+ CD4+

T cells (%)

50 40 30 20 10

Fibrin

+ + +

Anti-CD11b

lgG

LPS

WT (C57BL/6)

CFSE-labelled CD4+ 2D2 T cells injection

Fibrinogen/ACSF injection

Brain collected/ FACS analysis

ACSF Fibrinogen

CFSEhi

1 d

0 d

6 d

84.2%

% Of max

CFSEIow

CFSEIow36.8%

CFSEhi63.2%

15.7%

CFSE

Rag1/

0 d

2 d

5 d

BMDMs In vitro + Fibrin+ MOG 3555

+ OVA 323339

Co-cultured with 2D2 or OTII CD4+

T cells

CFDA-SE Fibrin MOG3555+

Fibrin + MOG3555+

Rag1/

Infiltrated CD4+ T cells

in corpus callosum

Fibrinogen injection

(103) *10

CFDA-SE labelled 2D2 or OTII CD4+ T cells injection

Brain collected

Fibrin

MOG 3555

OVA 323339

2D2 T cells ODII T cells

+ +

Figure 3 | Extravascular brinogen drives myelin-specic T cells into the CNS. (a) T-cell inltration (CD3) and demyelination (LFB/PAS) in the corpus callosum of WT, 2D2 and OT-II mice 7 days after brinogen injection. Scale bars, 100 mm (top panel); 200 mm (bottom panel). Quantication of CD3 Tcells and demyelination at 7 days after brinogen injection. Data are presented as means.e.m. (n 6 mice per group). *Po0.05, **Po0.01, ***Po0.001,

****Po0.0001 by one-way ANOVA and Bonferronis multiple comparisons test. (b) BMDMs treated with brin alone or in the presence of anti-CD11b or isotype IgG antibody control were co-cultured with naive 2D2 CD4 Tcells. BrdU proliferation assay in response to MOG3555 peptide after brin stimulation and anti-CD11b treatment. LPS was used as positive control. Data are presented as means.e.m. (n 34 independent experiments, *Po0.05, **Po0.01 by

one-way ANOVA and Bonferronis multiple comparisons test). (c) WTrecipient mice received CFSE-labelled CD4 CD62L 2D2 Tcells (0 d) and 1 day later brinogen or ACSF were injected in the corpus callosum. CFSE-labelled 2D2 T cells were isolated from the brains of brinogen- or ACSF-injected mice 6 days later and analysed by FACS. FACS plots and quantication showing CFSE dilution (CFSElow) indicate proliferation of 2D2 T cells in brinogen-injected brain.

Data are representative of two independent experiments, each from pooled brain cells from n 4 mice. (d) Naive CD4 2D2 or control OT-II T cells were

co-cultured with BMDMs treated with the indicated peptides, MOG3555 or OVA323339 alone or peptide in the presence of brin. CFDA-SE-labelled 2D2 or

OT-II T cells were transferred into Rag1 / mice injected with brinogen in the corpus callosum. CFDA-SE T cells (green) in the corpus callosum (dashed line) of Rag1 / mice. Scale bar, 100 mm. Quantication shows increased 2D2 T cells after co-culture with BMDMs treated with both brin and MOG3555, compared with brin and OVA323339 or MOG3555 alone. Data are presented as means.e.m. (n 4 mice per group, *Po0.05 by non-parametric

MannWhitney U-test). ANOVA, analysis of variance; d, days; FACS, uorescence-activated cell sorting; LFB, Luxol fast blue.

signicantly reduced in OT-II mice (Fig. 3a). Fibrin-treated BMDMs signicantly increased the proliferation of MOG3555-treated CD4 2D2 T cells in a CD11b-dependent manner, further suggesting that brin-induced activation of APCs promotes myelin-specic T-cell activation (Fig. 3b). LPS was used as a positive control (Fig. 3b).

Fibrinogen promotes CNS expansion of encephalitogenic T cells. In addition to macrophages, microglia contribute to antigen presentation and T-cell activation during CNS autoimmune diseases33. This prompted us to investigate whether brin-induced microglial activation also inuenced the activation of encephalitogenic T cells. Co-culture of microglia with naive 2D2

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T cells in the presence of MOG peptide increased antigen-specic cell proliferation (Supplementary Fig. 9). Importantly, brin-stimulated microglia were more efcient APC for encephalitogenic T-cell proliferation than unstimulated microglia (Supplementary Fig. 9). On the basis of these ndings, we asked whether brin induced activation of encephalitogenic T cells in the CNS. We labelled naive MOG-specic 2D2 T cells with carboxyuorescein diacetate succinimidyl ester (CFSE) to monitor lymphocyte proliferation in vivo34,35. CFSE-labelled 2D2 T cells were adoptively transferred into brinogen- or control ACSF-injected WT mice and donor T cells were reisolated from the brain at 6 days after the transfer (Fig. 3c). MOG-specic 2D2 T cells proliferated in the brains of brinogen-injected recipients, compared with ACSF-injected controls (Fig. 3c), suggesting local T-cell activation. We next examined whether brin-induced activation of APCs promotes encephalitogenic T-cell trafcking into the CNS. Naive 2D2 or control OT-II T cells were co-cultured with BMDMs in the presence of MOG3555 or

OVA323339, respectively. We adoptively transferred stimulated 2D2 or OT-II T cells into Rag1 / recipients that had been injected with brinogen and assessed migrated

T cells in the corpus callosum after 3 days. Accumulation of 2D2 T cells in the corpus callosum was observed when donor 2D2 T cells were stimulated with their cognate antigen in the presence of brin-treated BMDMs, compared with only MOG3555-treated cells (Fig. 3d). In contrast, brin-stimulated

BMDMs did not promote the accumulation of OT-II T cells in the corpus callosum of Rag1 / recipients that had been injected with brinogen (Fig. 3d). These results suggest that brin signalling via CD11b/CD18 primes APCs, which facilitates the local expansion of antigen-specic encephalitogenic T cells within the CNS.

Fibrinogen activates endogenous myelin antigen-specic T cells. Local CNS activation of antigen-specic T cells in the brain drives autoimmune reactions18,36. Using the I-Ab MOG3555

tetramer to detect in situ myelin-specic CD4 T cells37,38, we examined whether brinogen entry in the CNS induces the local activation of myelin antigen-specic T cells. After 7 days post injection, isolated lymphocytes from draining lymph nodes were cultured with MOG3555 peptide (Fig. 4a). Ex vivo staining with MOG3555 tetramer of cells from draining lymph nodes showed a small but detectable fraction of tetramer-positive CD4 T cells only in brinogen-injected, but not in ACSF-injected mice. No tetramer-positive cells were detected on incubation with control I-Ab OVA323339 tetramer (Fig. 4b), suggesting that brinogen induced myelin antigen-specic T-cell activation. Interestingly, only CD4 T cells isolated from the cervical lymph nodes of brinogen-injected, but not ACSF-injected mice showed increased bromodeoxyuridine (BrdU) incorporation on MOG3555 stimulation (Fig. 4c). To examine T-cell responses in the SJL/J mouse strain, lymphocytes were isolated from draining lymph nodes of brinogen-injected SJL/J mice and cultured with different proteolipid protein (PLP) peptides, including PLP139151 and PLP178191, which are immunodominant epitopes in the SJL/J mouse strain. CD4 T cells of brinogen-injected SJL/

J mice did not show signicant proliferation in response to PLP139

151 or to ACSF (Supplementary Fig. 10), suggesting that perhaps isolation and expansion of a higher number of T cells from the draining lymph node and the CNS would be required to study responses to other myelin antigens after induction of FIE in other strains. In our study, MOG3555 tetramer binding (Fig. 4b) and

MOG3555-induced BrdU incorporation (Fig. 4c) demonstrate the presence of functional autoreactive myelin antigen-specic CD4 T cells in mice with brinogen-induced inammatory demyelination.

Fibrin induces Th1-cell differentiation via CD11b/CD18. T-cell differentiation into either Th1 or Th17 cells is central to the induction of autoimmune demyelination, and is pertinent to MS pathogenesis39,40. In a co-culture system of CD4 T cells with

BMDMs stimulated with a brin preparation, we observed elevated expression of Th1 (T-bet), but not Th2 (Gata-3) or Th17 (Rorc) transcription factors (Fig. 5a). In accordance, the frequency of interferon-g (IFN-g) CD4 T cells, but not interleukin (IL)-4CD4 T cells, was higher when co-cultured with brin-stimulated BMDMs (Fig. 5b). Treatment of brin-stimulated BMDMs with an antibody against CD11b (M1/70) reduced the frequency of IFN-g CD4 T cells and the gene expression of IFN-g compared with treatment with isotype control antibody (Fig. 5b). Fibrin stimulation of BMDMs increased expression of the Th1-promoting cytokine IL-12p40 (ref. 41), which was markedly blocked by anti-CD11b treatment (Fig. 5b). Co-culture of BMDMs stimulated with kininogen did not induce Th1-cell differentiation (Supplementary Fig. 11). Introduction of brinogen in the CNS increased expression of the Th1 transcription factor, T-bet, but not the Th2, Th17 or regulatory T-cell transcription factors, Gata-3, Rorc and FoxP3, respectively (Fig. 5c). In accordance, brinogen injection increased gene expression of the Th1 cytokine IFN-g and

IL-12p40, while it had no effect on the expression of IL-4 (Th2) and IL-17F (Th17) (Fig. 5c). T cells isolated from the brinogen-injected corpus callosum also displayed increased IFN-g-expressing cells among inltrated CD4 and CD8 T cells by6.19- and 28.2-fold, respectively (Fig. 5d). In contrast, brinogen injection did not signicantly increase the number of IL-4 or

IL-17 in CD4 and CD8 T cells as compared with ACSF injection (Fig. 5d). Fibrinogen induced local Th1 polarization specically in the brain, as no changes were observed in the spleen (Fig. 5d). In accordance, depletion of brinogen in EAE reduced the number and proliferation of IFN-g T cells, while no effects were observed in IL-4 T cells (Fig. 5e). Overall, these ndings introduce the novel concept that a plasma-derived coagulation factor, brinogen, may be a key molecular pathway to selectively induce release of Th1-polarizing cytokines in APCs and increase the effector function of encephalitogenic T cells.

Fibrinogen induces macrophage recruitment into the CNS. Recruitment of CCR2 peripheral monocytes regulate the severity of demyelination26,42, and are essential for EAE progression to paralytic disease43,44. We injected brinogen into Ccr2RFP/ Cx3cr1GFP/ mice, which differentially label resident microglia (green uorescent protein (GFP) positive (GFP ), green) and inammatory monocytes (red uorescent protein (RFP) positive (RFP ), red)42. RFP cells were detected at 3 days after injection and their numbers remained elevated at 7 days after injection (Fig. 6a). GFP cells rapidly accumulated 1 day after brinogen injection and gradually increased up to 7 days post injection (Fig. 6a), suggesting that microglial activation preceded peripheral macrophage cell inltration. No RFP cells were found in the corpus callosum after ACSF injection. These results suggest that in addition to T-cell inltration, extravascular brinogen also triggers recruitment of inammatory monocytes into the CNS.

Fibrinogen increased Ccl2 in the corpus callosum in vivo, and after stimulation of primary microglia and APCs (Fig. 2ac). Interestingly, the local upregulation of CCL2 appeared to be instrumental in FIE. Indeed, peripheral macrophage recruitment, T-cell inltration and demyelination were reduced following brinogen injection into Ccr2RFP/RFP mice lacking the CCL2 receptor (Fig. 6b). Like puried brinogen, WT plasma was a potent inducer of Ccl2 and Cxcl10 expression, whereas injection of

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WT (C57BL/6)

Fibrinogen injection

Lymphocytes isolated from LN + MOG3555

FACS analysis MOG3555 tetramer + BrdU

Cervical lymph nodes

CD3+CD4+ ACSF Fibrinogen

0.014%

0.004% 0.007%

7 d

0 d

7 d

0.056%

l-Ab MOG3 3555

l-Ab OVA 323339

100

***

I-Ab MOG 3535+

CD4 T cells

ACSF

Fibrinogen

Cervical lymph nodes

ACSF

103

Fibrinogen

105

1.72% 4.74%

105

104

103

102 0

0 105

104

BrdU+ CD4+ T cells (%)

104

BrdU1.72%

BrdU4.74%

BrdU

102

103

104

105

102

103

CD4

ACSF

Fibrinogen

Figure 4 | Fibrinogen drives accumulation of myelin antigen-specic T cells. (a) Experimental design diagram: C57BL/6 mice were stereotaxically injected with brinogen or ACSF in the corpus callosum. Seven days after injection, lymphocytes were prepared from draining lymph nodes and stimulated with MOG3555 for 7 d. I-Ab MOG3555 tetramer was used to detect myelin-specic CD4 Tcells in brinogen-injected WTmice. I-Ab OVA323339 tetramer was used as a negative control for MOG3555 tetramer staining. (b) Flow cytometry analysis of I-Ab MOG3555 tetramer stained CD4 T cells 7 days after

MOG3555 stimulation. No tetramer-positive cells were detected with I-Ab OVA323339 tetramer. Graph shown number of I-Ab MOG3555 tetramer stained CD4 T cells. Data are presented as means.e.m., n 89, with each sample being pooled from 23 mice from three independent experiments.

***Po0.001 (non-parametric MannWhitney U-test). (c) Proliferation analysis of BrdU incorporation in CD4 T cells of ACSF- and brinogen-injected mice, stimulated with MOG3555 for 7 days. Data are presented as means.e.m. (four independent experiments with pooled cells from 23 mice per experiment for ACSF and brinogen), *Po0.05 (non-parametric MannWhitney U-test). d, days.

either Fib / or Fibg390-396A plasma resulted in their signicant reduction (Fig. 6c; Supplementary Fig. 7c). Overall, these results suggest that brinogen-induced upregulation of Ccl2 via CD11b/ CD18 facilitates the recruitment of peripheral macrophages and contributes to the induction of inammatory demyelination.

CD11b inhibition rescues the brinogen CNS effects. To further examine whether CD11b/CD18 signalling is required for

brinogen-induced demyelination, we injected brinogen into the corpus callosum of CD11b-decient mice (Itgam / )45.

Microglial activation, T-cell inltration, demyelination and expression of Cxcl10, Ccl2, T-bet, IFN-g and IL-12p40 were signicantly decreased in Itgam / mice after brinogen injection, compared with WT (Fig. 7), suggesting the requirement for CD11b/CD18 signalling in brinogen-induced adaptive immune activation. Expression of OX-40, a co-

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a b

***

IFN-+ CD4+

T cells (%)

BMDMs

+ Fibrin

+ Anti-CD11b, lgG

Co-cultured with naive WT CD4+ T cells

Analysis

Untreated Fibrin0.21%

1.39% 5.58%

0.26%

CD4+

IL-4+ CD4+

T cells (%)

0.4

0.3

0.2

0.1

IL-4

Untreated Fibrin

Fibrin+lgG Fibrin+anti-CD11b

0.22%

6.04% 3.36%

0.20%

RNA relative

expression

IL-12p40RNA

relative expression

5 4 3 2 1 0

0 T-bet

Gata-3

Rorc

IFN-

Fibrin

IgG Anti-CD11b

+ + +

2.5

2.0

1.5

1.0

0.5

***

ACSF

Fibrinogen

Brain ACSF

Fibrinogen

Spleen

Spleen T cells (103 )

ACSF Fibrinogen

RNA relative expression

T-bet

0 IFN-

IL-12p40

Gata-3

IL-4

Rorc

lL-17F

lL-23p19

Foxp3

0 IFN- IL-4 IL-17 IFN- IL-4 IL-17

CD4+ T cells CD8+ T cells

2.5

2.0

1.5

1.0

0.5

0 IFN- IL-4 IL-17 IFN- IL-4 IL-17

CD4+ T cells CD8+ T cells

Th1 Th2 Th17 Treg

CNS infiltrated T cells (102 )

PLPSaline Ancrod7.75%

0.73% 0.66%

IFN-+ CD4+ T cells (%)

IL-4+ CD4+ T cells (%)

8 6 4 2 0

Saline Ancrod

4.75%

IFN-

IL-4

PLP

1.00.80.60.40.2 0

% Changing in BrdU

incorporation

25 20 15 10

5 0

Figure 5 | Fibrin induces activation of innate immunity via CD11b/CD18 to induce Th1-cell differentiation. (a) BMDMs treated with brin or in the presence of anti-CD11b or isotype IgG antibody control were co-cultured with naive WT CD4 T cells and analysed for gene expression or by FACS. Gene expression analysis of transcriptional factors indicative of Th1, Th2, Th17 and Treg cells in CD4 Tcells co-cultured with brin-stimulated BMDMs. Data are presented as means.e.m. (n 4 independent experiments, *Po0.05 by non-parametric MannWhitney U-test). (b) Gated percentage of IFN-g- or

IL-4-expressing CD4 T cells after co-culture with brin-stimulated BMDMs in the presence of rat IgG isotype control antibody or anti-CD11b antibody. Data are presented as means.e.m. (n 3 independent experiments, *Po0.05, **Po0.01, ***Po0.001 by one-way ANOVA and Bonferronis multiple

comparisons test). Real-time PCR analysis of Th1-inducing cytokine IL-12p40 in brin-stimulated BMDMs in the presence or absence of anti-CD11b antibody. Data are presented as means.e.m. (n 34 independent experiments, *Po0.05, **Po0.01 by one-way ANOVA and Bonferronis multiple

comparisons test). (c) Real-time PCR analysis of transcription factors and cytokines indicative of Th1, Th2, Th17 and Treg cells in the corpus callosum at 3 days after ACSF or brinogen injection. Data are presented as means.e.m. (n 35 mice per group). *Po0.05 (non-parametric MannWhitney

U-test). (d) Comparison of IFN-g-, IL-4- and IL-17-producing cells in inltrated CD4 and CD8 Tcells isolated from the brains and spleens after ACSF or brinogen injection at day 7 post injection. Data are presented as means.e.m. (n 3 independent experiments, *Po0.05, ***Po0.001 by one-way

ANOVA and Bonferronis multiple comparisons test). (e) IFN-g and IL-4 expression in CD4 lymph node T cells isolated from saline- or brin-depleted (ancrod) mice after PLP139151-induced EAE and restimulated in vitro with PLP139151. Data are presented as means.e.m. (n 6 mice per group, *Po0.05,

**Po0.01 by non-parametric MannWhitney U-test). ANOVA, analysis of variance; FACS, uorescence-activated cell sorting; Treg, regulatory T cell.

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Ccr2RFP/+ Cx3cr1GFP/+

ACSF

ACSF, 1 d

Fibrinogen

Fibrinogen, 1 d Fibrinogen, 7 d 2.0

1.5

1.0

0.5

8 6 4 2 0

100 Ccr2RFP/+

Ccr2RFP/RFP

GFP intensityRFP intensity

** *

***

0 1 d 3 d 7 d

1 d 3 d 7 d

****

Ccr2RFP/+ Ccr2RFP/RFP

15 12

9 6 3 0

Ccr2 DAPI

LFB PAS

CD3+ T cells

/ 0.5 mm2

Fibrinogen

Demyelination

(% of area)

Fibrinogen

** *

Ccl2RNA relative

expression

0 ACSF

Fib 390396A

Fib/

Plasma

Figure 6 | Fibrinogen induces recruitment of peripheral macrophages into the CNS via CD11b/CD18-mediated upregulation of CCL2. (a) Inltration of peripheral Ccr2 macrophages (RFP, red) in the corpus callosum 7 days after brinogen injection in Ccr2RFP/Cx3cr1GFP/ . Scale bar, 200 mm. Quantication of RFP (top graph) and GFP (bottom graph) intensity in the same sections of corpus callosum of ACSF- or brinogen-injected Ccr2RFP/ Cx3cr1GFP/mice on days 1, 3 and 7. Data are presented as means.e.m. (n 46 mice per time point). *Po0.05, **Po0.01, ***Po0.001, ****Po0.0001 (two-way ANOVA and

Bonferronis multiple comparisons test). (b) Inltration of peripheral Ccr2 macrophages (RFP, red) in the corpus callosum 7 days after brinogen injection in

Ccr2RFP/ - and Ccr2-decient (Ccr2RFP/RFP) mice at 7 days post-brinogen injection. Scale bar, 50 mm. Quantication of inltrated CD3 T cells (top graph)

and demyelinated area (bottom graph) in the corpus callosum of Ccr2RFP/ - and Ccr2-decient (Ccr2RFP/RFP) mice 7 days after brinogen injection. Data are presented as means.e.m. (n 67 mice per group). **Po0.01 (non-parametric MannWhitney U-test). (c) Real-time PCR analysis of Ccl2 gene expression

in corpus callosum 12 h after injection of ACSF and plasma obtained from WT, Fibg390396A Fib / or mice. Data are presented as means.e.m. (n 4 mice

per group). *Po0.05, **Po0.01 (one-way ANOVA and Bonferronis multiple comparisons test). ANOVA, analysis of variance; d, days.

stimulatory molecule expressed in activated T cells46, increased by B30-fold is on CNS-inltrating T cells after brinogen injection (Fig. 8a). OX-40-labelled T-cell activation was signicantly reduced in Itgam / mice after brinogen injection by B60% (Fig. 8a), suggesting that brinogen induces local CNS T-cell activation in a CD11b-dependent manner. Consistent with these results, in mice challenged by brinogen injection into the corpus callosum pharmacologic inhibition of CD11b by intracerebroventricular delivery of M1/70 reduced expression of Cxcl10 and Ccl2 (Fig. 8b), and decreased T-cell and peripheral macrophage inltration (Fig. 8c), when compared with

isotype control IgG-treated cohorts. Taken together, these data suggest that the introduction of brinogen into CNS tissues induces peripheral immune cell recruitment, T-cell activation and demyelination via a CD11b/CD18-dependent mechanism.

DiscussionThis study establishes the fundamental role of the coagulation cascade as a driver of adaptive immune responses and the mechanisms by which extravasation of a blood clotting factor into the brain white matter can induce autoimmunity, demyelination and peripheral macrophage recruitment into the CNS. Our results

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lba-1+ cells/0.5 mm2

120

100

CD3+ T cells/0.5 mm2

Demyelination

(% of area)

0 WT ltgam/

ltgam/

**** **

ACSF Fibrinogen

Cxcl10RNA relative expression

Ccl2RNA

relative expression

ltgam/

*** **

2.5

2.0

1.5

1.0

0.5

** **

T-betRNA

relative expression

IFN- RNA relative expression

IL-12p40RNA

relative expression

20 ** **

0WT

ltgam/

Figure 7 | Genetic inhibition of CD11b blocks brinogen-induced microglial activation and inammatory demyelination. (a) Quantication of microglial activation (IBA-1), T-cell inltration (CD3) and demyelination (LFB/PAS) 7 days after brinogen injection in the corpus callosum of Itgam / or control

WT mice (n 6). Data are presented as means.e.m. *Po0.05, **Po0.01 (non-parametric MannWhitney U-test). (b) Fibrinogen-induced gene

expression of Cxcl10 and Ccl2 is reduced in the corpus callosum of Itgam / mice compared with WT control. Results are means.e.m. of 67 mice per group, **Po0.01, ****Po0.0001 (two-way ANOVA and Bonferronis multiple comparisons test). (c) Fibrinogen-induced gene expression of T-bet, IFN-g and IL-12p40 is reduced in the corpus callosum of Itgam / mice compared with WT control (n 58 mice). Data are presented as means.e.m.

**Po0.01, ***Po0.001 (two-way ANOVA and Bonferronis multiple comparisons test). ANOVA, analysis of variance; d, days; LFB, Luxol fast blue.

suggest that brin, the nal product of the coagulation cascade, constitutes a sustained, non-diffusible and geographically constrained immunological molecular switch that triggers the inopportune local activation of resident APCs to induce the proliferation, recruitment and local activation of myelin antigen-specic Th1 cells in the CNS. The introduction of brinogen in myelinated areas in the healthy CNS led to the four major ndings (Fig. 8d): (i) brinogen leakage in the CNS is a major plasma protein that induces CNS autoimmune responses and demyelination; (ii) brinogen is a key inducer of recruitment of both encephalitogenic T cells and peripheral macrophages into the CNS via CXCL10 and CCL2 chemokine secretion, respectively; (iii) activation of the coagulation cascade in the CNS favours Th1-cell differentiation via brin-induced upregulation of IL-12; and (iv) the effects of coagulation to CNS autoimmunity are primarily due to the proinammatory, CD11b-mediated effects of brin, thus dissecting the benecial prohaemostatic roles of coagulation from its damaging effects in CNS disease. Fibrinogen is not only sufcient to induce T-cell activation and demyelination in the healthy CNS, but is also required for T-cell activation after peripheral autoimmune activation as shown on depletion of endogenous brinogen in EAE. These new ndings, together with our prior studies in EAE showing (i) early activation of coagulation4 and (ii) reduction of neurological

signs, microglial activation, demyelination and axonal damage on brinogen depletion5,11,14, bolster the signicance of coagulation activity in the development of neuroinammatory lesions and establish brinogen as a key component of the coagulation cascade with pleiotropic functions in CNS innate and adaptive immunity.

In EAE, T cells enter the CNS after local activation by perivascular macrophages at the leptomeninges and their initial local CNS activation determines the clinical outcome of the autoimmune response18. Fibrin is localized in the leptomeninges at EAE pre-onset and induces early perivascular clustering of microglia and meningeal macrophages5. Moreover, in marmoset EAE early BBB leakage is associated with perivascular inammatory cufng and parenchymal microglial activation, but precedes demyelination6. Therefore, it is possible that within the CNS brin functions as an instructive signal enabling antigen-presenting properties in resident perivascular macrophages and thus facilitating T-cell entry, proliferation and activation. MOG35

55 tetramer binding in the cervical lymph nodes (Fig. 4b) and MOG3555-induced BrdU incorporation (Fig. 4c) demonstrate the presence of functional autoreactive myelin antigen-specic CD4

T cells in C57BL/6 mice injected with brinogen in the corpus callosum. Future studies will characterize the responses of T cells to other myelin antigens of not only C57BL/6 mice, but also in the

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WT Itgam/

** ***

ACSF

OX40 DAPI

OX40+ T cells/0.5 mm2

0 WT Itgam/

b c

ACSF

Fibrinogen

25 20 15 10

5 0

*** *

** * *

CD3 intensity

Cxcl10RNA relative expression

Ccl2RNA

relative expression

8 6 4 2 0

2.01.51.00.5 0

Fibrinogen

IgG Anti-CD11b

Ccr2-RFP intensity

ACSF ACSF

Fibrinogen

Fibrinogen IgGIgG

IgG Anti-CD11bAnti-CD11b

Anti-CD11b

+ +

Fibrinogen

+ + +

+ +

+ + +

Coagulation cascade

BBB disruption

Fibrinogen

Fibrin

Chemokine release CXCL10, MCP-1

Cytokine secretion IL-12

Peripheral immune cell recruitment

CD11b+ microglia

APCs

CD11b+ peripheral macrophage

IL-12 T cells Th1

Autoimmunity

Demyelination

Figure 8 | Inhibition of CD11b blocks brinogen-induced local T-cell activation, chemokine gene expression and peripheral inammatory cell recruitment. (a) Fibrinogen-induced local T-cell activation (OX-40) in the corpus callosum of WT mice is signicantly reduced in Itgam / mice (n 4).

Representative images are shown. Data are presented as means.e.m. **Po0.01, ***Po0.001 (one-way ANOVA and Bonferronis multiple comparisons test). Scale bar, 100 mm. (b) In vivo pharmacologic blockade of CD11b by intracerebroventricular delivery of anti-CD11b antibody reduces brinogen-induced

Cxcl10 and Ccl2 gene expression, compared with isotype IgG control antibody. Data are presented as means.e.m. (n 5 per group). *Po0.05, **Po0.01,

***Po0.001 (one-way ANOVA and Bonferronis multiple comparisons test). (c) Quantication of inltrated CD3 T cells and RFP macrophages in the corpus callosum 7 days after injection of brinogen in WT mice treated with anti-CD11b or IgG isotype control antibody. Data are presented as means.e.m. (CD3, n 67 mice per group; RFP, n 7 mice per group). *Po0.05, **Po0.01 (non-parametric MannWhitney U-test). (d) Proposed

model for the role of brin, the nal product of the coagulation cascade, in the development of CNS autoimmunity. On BBB disruption, brinogen extravagates into the CNS and is converted to brin upon activation of coagulation. Fibrin, the high-afnity plasma-derived ligand for CD11b/CD18, activates CNS resident innate immune cells (microglia and perivascular macrophages) to stimulate chemokine release leading to recruitment of peripheral inammatory macrophages/monocytes and T cells. Fibrin also induces antigen-presenting properties and provides instructive signals (such as IL-12) for inducing Th1-cell differentiation. Fibrin-induced microglial activation, recruitment of peripheral macrophages and T-cell activation lead to inammatory demyelination. ANOVA, analysis of variance.

CNS and draining lymph nodes of other mouse strains. Since interactions of T cells with APCs also continue during the peak of EAE18, brin-induced activation of autoimmune responses might play a role not only at the onset but also for the amplication and perpetuation of the autoimmune response.

Strikingly, our study shows that extravascular brinogen induces a transcriptional program of immune effectorcell

activation and recruitment that links innate with adaptive immunity. Upregulation of CXCL10 and IL-12p40, two immune modulators involved in T-cell recruitment and Th1-cell differentiation, occurs before the inltration of peripheral leukocytes, suggesting that brinogen-induced activation of CNS innate immunity appears to be a primary pathogenic event that precedes T-cell entry into the CNS. Since CCL2-dependent recruitment of

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peripheral monocytes is required for antigen-specic Th1 immune responses26, brin-induced recruitment of peripheral monocytes might contribute to Th1-cell differentiation. Indeed, the brin-induced IL-20p40 expression in both microglia and BMDMs observed here suggests that both resident and peripheral CD11b innate immune cells can be activated by brin to enable autoimmune responses. Therefore, an attractive scenario based on the present ndings is that weakening of the BBB either by peripheral brain autoimmune response or by exposure to high-risk environmental factor(s) that weaken the BBB, such as systemic inammation and infection, extravascular brin deposition and activation of coagulation in the CNS might be key upstream signals for the activation of innate and adaptive immune responses.

The time-course study in Ccr2RFP/ Cx3cr1GFP/ mice showed that the primary effect of brinogen is on the resident GFP microglia and secondarily on the peripheral RFP macrophages (Fig. 6). Indeed, morphologic changes in GFP-expressing microglia occur as early as 1 day after brinogen injection, while RFP-expressing macrophages are not detected in the CNS until 3 days after brinogen injection (Fig. 6a). Accordingly, increased chemokine gene expression in the corpus callosum is detected as early as 12 h after brinogen injection (Fig. 2a), before the inltration of peripheral macrophages (Fig. 6a) and T cells (Fig. 1a). Notably, pharmacologic inhibition of CD11b in the CNS by intracerebroventricular delivery of M1/70 reduced chemokine expression and decreased peripheral macrophage inltration (Fig. 8b,c), further suggesting that microglia are the primary cell target of brinogen in vivo. CD11b/CD18 is expressed in microglia and macrophages and regulates myelin phagocytosis11. It is possible that CD11b/CD18 engagement of extravascular brinogen induces activation of APC properties in innate immune cells in a hierarchical manner, rst by activating microglia and then peripheral macrophages after they enter the CNS. Since peripheral macrophages enter into the CNS in neurodegenerative diseases associated with BBB and brin deposition, such as stroke and Alzheimers disease47, brinogen might stimulate the recruitment of peripheral macrophages into the CNS in a wide spectrum of neurological diseases.

We established FIE as a novel experimental setting for exploring the cascade of pathogenic events that directly follow from the leakage of plasma proteins in the white matter in the absence of pre-existing peripheral immune activation or myelin pathology. We selected stereotactic delivery of brinogen as it is a common experimental method in viral- and toxin-induced demyelination48, as well as in established neurodegeneration models, such as kainic acid injection in the hippocampus. Moreover, stereotactic delivery evades any complexities associated with the imposed genetic expression, assembly and processing of all three chains of brinogen. FIE could be used to dissect the contribution of BBB disruption and the coagulation cascade in the CNS without the confounding factors of preexisting viral, cytokine, toxin or autoimmune events. Given its rapid 7-day disease course, FIE could also be used as a pharmacodynamic model to rapidly test the efcacy of novel treatments for inhibiting CNS-targeted innate and adaptive immune responses. Using albumin, kininogen and plasma derived from Fib / and Fibg377-395 mice, we show that brinogen is a major protein in the blood that drives sustained neuroinammatory responses in the CNS. It is possible that in addition to brinogen, other plasma proteins might play a role in brain pathology. In particular, other components of the coagulation cascade involved in brin formation and clot stabilization, such as thrombin, factors X, XII and XIII could be involved in inammatory responses49. Future studies will elucidate the relative contribution of blood proteins to brain

inammation and neurodegeneration. Our study demonstrates that brinogen induces demyelination using myelin-specic antibodies and histological stains that clearly show myelin loss. Demyelination can be associated with axonal damage in MS and Alzheimers disease5052. Future studies will show whether brinogen-induced demyelination is primary associated with preservation of axons, or whether it is accompanied by axonal damage.

Our results may have broad implications for the potential development of new therapeutic strategies for neuroinammatory diseases. Activation of CD11b resident CNS cells appear to be one of the earliest events leading to local CNS antigen presentation, amplication of myelin-reactive T-cell responses, inltration of peripheral macrophages and axonal damage5,17,53,54. Since CD11b/CD18 is a pleiotropic receptor55, specic inhibition of its pathogenic ligand brinogen would be a preferred upstream therapeutic strategy for suppressing the pathogenic cascade in neuroinammation over any global inhibition of CD11b/CD18. Selective disruption of CD11b/ CD18brinogen interface would also be expected to protect the brain from axonal damage and neurodegeneration5. Importantly, targeting the interaction of brinogen with CD11b would not affect the benecial functions of brinogen in haemostasis1,11. Since activation of innate immunity is a hallmark of neuroimmune and neurodegenerative diseases, inhibiting the interaction of brinogen with CD11b/CD18 could be benecial not only for suppressing autoimmunity, but also halting neurodegeneration. Identication of extravascular brinogen as a key regulator of CNS innate and adaptive immunity might allow us to develop novel therapies targeting early and late events in neuroinammatory diseases and potentially provide new treatment options.

Methods

Mice. C57BL/6 MHC II / (ref. 28), Cxcl10 / (ref. 29), Itgam / (ref. 45), Cx3cr1GFP/ (ref. 56), MOG-specic TCR transgenic mice (2D2)31 and SJL/J were purchased from the Jackson Laboratory. C57BL/6 Fib / (ref. 19), Fibg390396A mice22, OVA-specic TCR-transgenic mice (OT-II)32, RAG1 / (ref. 57),

RAG2 / gc / (ref. 30) and Ccr2RFP/RFP mice42 were also used. C57BL/6 Ccr2RFP/RFP mice were crossed with Cx3cr1GFP/GFP mice to generate Cx3cr1GFP/

Ccr2RFP/ mice. All animal experiments were performed on adult male mice at 1015 weeks of age. Mice were housed in the groups of ve per cage under standard vivarium conditions and a 12-h light/dark cycle. All animal protocols were approved by the Committee of Animal Research at the University of California, San Francisco, and in accord with the National Institutes of Health guidelines.

Stereotactic injections in the corpus callosum. Mice were anaesthetized with avertin and placed in a stereotactic apparatus. Plasma plasminogen-free brinogen (Calbiochem) was dissolved in endotoxin-free distilled water (HyClone), diluted to 5 mg ml 1 with ACSF. Fibrinogen (1 ml of 5 mg ml 1), ACSF, albumin (1 ml of 5 mg ml 1) or kininogen (1 ml of 0.1 mg ml 1) were slowly injected (0.3 ml min 1)

with a 10-ml Hamilton syringe attached to a 33-G needle into the brain at coordinates (anteroposterior, 1.0 mm; mediolateral, 1.0 mm; dorsoventral, 1.75 mm from the bregma, according to Paxinos and Watson) as described5. Plasma was isolated and injected as described5.

Intracerebroventricular delivery of antibody. Functional-grade puried anti-CD11b (M1/70; eBioscience), or isotype control IgG (eBioscience), was injected(0.2 ml min 1) with a 10-ml syringe attached to a 33-G needle into cerebral ventricle (anteroposterior, 2.0 mm; mediolateral, 0 mm, dorsoventral, 2.0 mm) 30 min before brinogen injection.

Stereotactic injections in the spinal cord. Mice were anaesthetized by intraperitoneal injection of ketamine (100 mg kg 1) and xylazine (15 mg kg 1).

A midline skin incision was made over the upper lumbar regions of spinal cord. The spinal column was secured via the mouse vertebral clamps xed in a stereo-taxic frame. The epidural space was exposed by disruption of the L1L2 interspinous ligament without laminectomy as described58. A pulled glass micropipette prelled with brinogen was inserted into the spinal cord at coordinates (0.3-mm lateral to the spinal midline, a depth of 0.9 mm from the

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spinal cord surface). Fibrinogen (1 ml of 5 mg ml 1) was injected (0.2 ml min 1) and the glass micropipette remained in place for 5 min before slowly withdrawn.

After surgery, the muscles and skin were sutured, and mice were allowed to recover.

Expression of recombinant brinogen. For the production of recombinant brinogen, freestyle HEK293T cells were transiently co-transfected with three eukaryotic expression vectors containing full-length complementary DNA (cDNA) for the three chains of brinogen, alpha (1.9 kb), beta (1.3 kb) and gamma (1.3 kb), according to the standard procedures. At day 4 post transfection, medium was collected and the supernatant was passed through a Vivaspin-20 concentrator with cutoff 100,000 molecular weight cutoff and was added to a Slide-A-Lyzer Dialysis cassette with cutoff 10,000 molecular weight cutoff for overnight dialysis in PBS buffer at 40 C with stirring. Expression of the recombinant brinogen protein was evaluated by SDSpolyacrylamide gel electrophoresis gel electrophoresis and Coomassie staining.

Histology and immunohistochemistry. Mice were transcardially perfused with 4% paraformaldehyde under avertin anaesthesia. Brains and spinal cord were removed, postxed, processes for parafn embedding. Luxol fast blue staining and immunohistochemistry were performed as described11,59,60. For toluidine blue staining, animals were intracardially perfused under deep ether anaesthesia with ice-cold 2% paraformaldehyde, 0.5% glutaraldehyde in 0.1 mol l 1 phosphate buffer, pH 7.2, for 1 min, followed by ice-cold 3% glutaraldehyde in 0.1 mol l 1 phosphate buffer for 5 min. Brains were removed, immersion-xed for 24 h in phosphate-buffered 3% glutaraldehyde, postxed in 2% osmium tetroxide solution and subsequently embedded in epoxy resin. Semi-thin sections were cut and stained with toluidine blue. For immunohistochemistry, sections were permeabilized in 0.1% Triton X-100, blocked with 5% bovine serum albumin and 5% normal donkey serum, and incubated for 24 h at 4 C with primary antibodies. Primary antibodies were rabbit anti-Iba-1 (1:1,000, Wako), rabbit anti-CD3 (1:1,000, Dako), rabbit anti-GFAP (1:500, Sigma), mouse anti-MBP (1:100, Covance), rat anti-mouse MHC class II (1:200, BMA Biomedicals) or goat anti-mouse CXCL10 (1:200, R&D system). Sections were rinsed in PBS with 0.1% Triton X-100 and incubated with secondary antibodies conjugated with Alexa Fluor 488 or 594 (1:200, Jackson Immunochemicals) for 1 h in the dark. Immunohistochemistry using the primary anti-MBP monoclonal antibody was performed with the MOM kit (Vector) according to the manufacturers instruction. After washing in PBS, sections were mounted on glass slides and coverslipped with Prolong Gold antifading agent (Invitrogen). Images were acquired using an Axioplan II epiuorescence microscope (Zeiss) equipped with dry Plan-Neouar objectives (10 0.3 numerical aperture (NA), 20 0.5 NA or 40 0.75 NA).

Quantication was performed on thresholded images using ImageJ by blinded observers.

Flow cytometry. Mice were perfused with saline and brain slices were prepared using the adult mouse brain slicer matrix (Zivic Instruments). Corpus callosal area was dissected from the slices and minced tissue was digested using collagenase IV (Roche) at 37 C for 30 min. Cell suspensions were ltered through a 70-mm-cell strainer (BD Falcon). Cell suspensions were prepared in RPMI-1640 (Invitrogen), supplemented with 5% (vol/vol) heat-inactivated fetal bovine serum (FBS, Invitrogen), 50 U penicillinstreptomycin (Invitrogen) and 50 mM b-mercaptoethanol (Invitrogen). Single-cell suspensions were incubated with Myelin Removal Beads (Miltenyi Biotec), and cells were collected by autoMACs Pro Separator (Miltenyi Biotec). Intracellular staining of splenocytes derived from mice undergoing MOG3555 EAE5,11 was used as positive control for uorescence-activated cell

sorting antibody staining. For cytokine analysis, cells were incubated for 4 h with phorbol 12-myristate 13-acetate (50 ng ml 1, Sigma), ionomycin (500 ng ml 1,

Sigma) and Golgi-Plug (BD Biosciences) and surface stained with anti-CD3-PE (eBioscience), anti-CD4-APC-Cy7 (eBioscience) and anti-CD8-PacBlue (eBioscience). Cells were then xed with Cytox/Cytoperm solution (BD Biosciences), and intracellular cytokine staining was performed with anti-IFN-g-

PE-Cy7 (eBioscience), anti-IL-4-Alexa 647 (eBioscience) and anti-IL-17a-FITC (eBioscience). All labelled antibodies were used at 1:300 dilutions. Flow cytometric analysis was performed on an LSR II (BD Biosciences). Data were analysed using the FlowJo software (Tree Star).

Detection of endogenous MOG antigen-specic T cells. Fibrinogen-injected brains and its draining lymph nodes were removed at 7 days post injection, and single-cell suspensions were prepared as described above. Prepared lymphocytes were stimulated with 20 mg ml 1 MOG3555 for 7 days. Endogeneous MOG antigen-specic T cells were identied with MOG3555-specic T-select I-Ab MOG3555

tetramer (MBL). I-Ab OVA323339 Tetramer (MBL) was used as a negative control. Cultured cells were resuspended in FCM buffer (2% FCS/0.05% NaN3/PBS) and incubated with FcR-blocking antibody for 5 min at room temperature. Cells were incubated with I-Ab MOG3555 tetramer for 60 min at 4 C followed by anti-CD4-

FITC incubation for 30 min at 4 C. After two washes with FCM buffer, cells were analysed with an LSR II.

T-cell CFSE staining and adoptive transfer. Naive 2D2 CD4 T cells were isolated from spleens and lymph nodes of 2D2 mice using CD4 CD62L T-cell isolation kits (Miltenyi Biotec). Isolated T cells were labelled with CFSE35 (Invitrogen) at room temperature. A total of 1 107 cells were transferred

intraperitoneally into recipient 24 h before ACSF and brinogen injection into the corpus callosum. On day 6, labelled T cells were isolated from the brains, and the frequency of CFSE (CFSElow)TCR-Vb11 CD4 T cells was analysed by ow cytometry. For analysis of CNS migratory activity of 2D2 or OT-II CD4 T cells, isolated naive 2D2 or OT-II CD4 T cells were co-cultured 3 days with BMDMs incubated with either 20 mg ml 1 MOG3555 or OVA323339 peptide together with or without brin. Cells were then labelled with CFDA-SE according to the manufacturers instructions (Invitrogen) and injected (1 107 cells per mouse)

into the Rag1 / recipient mice that had been injected with brinogen 2 days before. Three days after post-T-cell injection, mice were killed, and their brains were subjected to histological analysis.

Isolation of primary microglia. Microglia were prepared from neonatal rat or mouse pups at postnatal day (P) 23. The cortices were separated from meninges and minced with a sterile razor blade. Tissue pieces were transferred into 2.5% Trypsin solution (Life Technologies/Gibco) containing DNAse (SIGMA). After incubation at 37 C for 25 min the trypsin solution was removed, cortices were washed with 30% FBS in DPBS and serially triturated with 30% FBS in Dulbeccos phosphate-buffered saline (DPBS) (Life Technologies/Gibco) containing DNAse. The cell suspension was gently spun at 200g for 15 min and the pellet resuspended in DMEM (Life Technologies/Gibco), containing 10% FBS (Life Technologies/ Gibco), 100 units per millilitre penicillin and 100 mg ml 1 streptomycin (Life

Technologies/Gibco). Cells were plated into poly-D-lysine pre-coated T-75 asks at a density of 23 cortices per ask. On day 3 in vitro, fresh medium was added and cells were grown for 1 more day. On day 4 in vitro, asks were placed onto a shaker platform, preheated to 37 C and microglia cells were shaken off the cortical cell layer at 200 r.p.m. for 2 h. The medium containing mostly microglia cells was removed from the asks and cells were spun at 200g for 15 min. The cell pellets were gently resuspended in culture medium and the microglia density was adjusted to 5000 cells per microlitre.

Isolation of BMDMs. BMDMs were prepared as described61. In brief, bone marrow cells were isolated from tibia and femur of 10-week-old mice and cultured in RPMI-1640 (Invitrogen) supplemented with 10% (vol/vol) heat-inactivated FBS (Invitrogen), 50 U penicillinstreptomycin (Invitrogen), 50 mM b-mercaptoethanol and murine M-CSF (10 ng ml 1). On day 6, adherent BMDMs were harvested from plates by the addition of PBS containing 5 mM EDTA for experiments.

Fibrin stimulation of microglia or BMDMs. To prepare soluble brin62, human plasma brinogen (Calbiochem) was converted to brin by mixing with thrombin. Fibrin clots were cut into small pieces and fragmented by sonication. Clot fragments are ltered through sieves, their concentration was determined, and they were stored at 80 C. Soluble brin preparations were tested for LPS and

thrombin activity and were shown to have undetectable amounts of contaminants. Cultures were treated with soluble brin by adding soluble brin to the supernatant. To prepare brin-coated plates, a mixture of thrombin (1 U ml 1,

Sigma) and CaCl2 (7 mM, Sigma) in HEPES buffer was added into each well of tissue culture plates (TPP Techno Plastic Products AG, Switzerland) and subsequently 50 mg ml 1 of human plasma brinogen (Calbiochem) was added.

The plates were incubated for 1 h at 37 C. After incubation, solution was evaporated, and moisture retention was minimized by air ow through the dryer system. After washing the plates with PBS, microglia or BMDMs were seeded on brin-coated plates. All reagents were made in endotoxin-free water.

T-cell proliferation assay. CD4 T cells were puried from the spleen with CD4 CD62L T-cell isolation kits (Miltenyi Biotec). CD4 T cells were mixed with BMDM at the ratio (1:5). T-cell proliferation was assessed by BrdU incorporation kit (BD Bioscience) during last 24 h of culture. For antigen-specic T-cell proliferation, CD4 T cells isolated from 2D2 mice were cultured with MOG3555

peptide (20 mg ml 1) for 4 days in the presence of BMDM treated with brin or primary mouse microglia plated on brin-coated plates or stimulated with LPS (200 ng ml 1) for 24 h. Cells were then xed, permeabilized and stained with

FITC-conjugated anti-BrdU (BrdU Flow Kits). For measurement of PLP antigen-specic CD4 T-cell proliferation, lymphocytes were isolated from the draining lymph nodes of SJL/J mice at 7 days after brinogen and ACSF injection into the corpus callosum. Lymphocytes were stimulated with PLP139151 peptide

(20 mg ml 1) and PLP178 191 peptide (20 mg ml 1), and IL-2 (10 ng ml 1) was

added every 2 days. BrdU incorporation was assessed as described above. For intracellular cytokine staining, CD4 T cells were stimulated with 20 mg ml 1

MOG3555 peptide for 72 h and restimulated with PMA and ionomycin for 4 h in the presence of Golgi-Plug (BD Bioscience), after which IFN-g-, IL-4- and IL-17-producing cells were analysed by intracellular staining.

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RNA isolation and quantitative PCR. RNA was isolated from brain samples with the RNAeasy kit (Qiagen), according to the manufacturers instructions. RNA was reverse-transcribed to cDNA using the GeneAmp RNA PCR Core kit (Applied Biosystems) and random hexamer primers. Real-time PCR analysis was performed using the Step One Plus (Applied Biosystems) and the Quantitect SYBR Green PCR kit (Qiagen) with 2 ml of cDNA template in a 25-ml reaction. Results were analysed using the Opticon 2 Software and the comparative CT method. Data are expressed as 2CT for the experimental gene of interest normalized to the housekeeping gene and presented as fold change relative to control. The gene-specic primers are listed in Supplementary Table 3.

Gene expression proling by microarray analysis. Microarray analysis was performed on cultured rat microglia treated with brin or BMDMs plated on brin-coated plates and brain tissues from ACSF- and brinogen-injected mice. Rat pups were used to isolate highly pure microglial cultures in sufcient numbers for microarray analysis. For brain tissue microarray analysis, corpus callosal area was dissected from brain slices prepared with brain slicer matrix (Zivic instruments). Total RNA was isolated using RNeasy Mini kit/RNeasy Lipid tissue mini kit (QIAGEN) according to the manufacturers instruction. Probes were prepared using NuGEN Ovation Pico WTA V2 kit and NuGEN Encore Biotin Module, and hybridized to Rat and Mouse Gene 1.0 ST GeneChip arrays (Affymetrix). Arrays were scanned using an Affymetrix GCS3000 scanner and Affymetrix Command Console software, and data were normalized using the RMA algorithm in Affymetrix Expression Console. Microarrays were normalized for array-specic effects using Affymetrixs Robust Multi-Array normalization. Normalized array values were reported on a log2 scale. For statistical analyses, we removed all array probe sets where no experimental groups had an average log2 intensity 43.0. This is a standard cutoff, below which expression is indistinguishable from background noise. Linear models were tted for each gene using the Bioconductor limma package in R63. Moderated t-statistics, fold change and the associated P values were calculated for each gene. To account for the fact that thousands of genes were tested, we reported false discovery rate (FDR)-adjusted values, calculated using the BenjaminiHochberg method64. FDR values indicate the expected fraction of falsely declared, differentially expressed (DE) genes among the total set of declared DE genes (that is, FDR 0.15 would indicate that B15% of the declared DE genes

were expected to be due to experimental noise instead of actual differential expression). The microarray data have been deposited in the Gene Expression Omnibus (GEO) database accession number GSE71084.

Induction of EAE and systemic brinogen depletion. EAE was induced in 8-week-old female SJL/J by subcutaneous immunization with 100 mg PLP139151

(HSLGKWLGHPDKF; Auspep Pty Ltd) as described11. Mice were depleted of brinogen with ancrod as described11. The mice received 2.4 U ancrod per day by mini-osmotic pump. In control animals, buffer-lled minipumps were implanted. On day 9 of immunization, draining lymph node T cells were obtained from EAE mice and cultured with PLP139151 peptide for the detection of intracellular IFN-g

and IL-4 in CD4 T cells and BrdU proliferation assay as described above.

Statistical analyses. The data are presented as means.e.m. Statistical calculations were performed using the GraphsPad Prism. Data distribution was assumed to be normal, but this was not formally tested. No statistical methods were used to predetermine sample size, but our sample sizes are similar to those reported previously5,11,59. Statistical signicance was determined with non-parametric two-sided MannWhitney U-test, one-way, or two-way, analysis of variance followed by Bonferroni post test (multiple comparisons). Mice and cells were divided into experimental groups in an unbiased manner. No randomization was used to assign groups or collect data. All animals survived until the end of the study and all data points were included in analysis. All histopathological analysis was performed by a blinded observer.

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Acknowledgements

We thank Noah Saederup and Eric M. Verdin for providing us with RAG2 / gc / and OT-II mice, respectively. Expression vectors for recombinant brinogen were kindly provided by Lundbeck. We thank Athena Soulika for discussions and sharing myelin removal protocols, Dimitrios Davalos for critical reading of the manuscript, Matthew Helmrick and Collin Spencer for expert technical assistance. We thank Robert Nelson and Jeff Stavenhagen for discussions. This publication was made possible with the help from the University of California San Francisco-Gladstone Institute of Virology and Immunology Center for AIDS Research (P30 AI027763) and the Mouse Pathology Core of the UCSF Helen Diller Family Comprehensive Cancer Center (CA082103). S.S.Z. received support from the NIH (R01 AI073737 and R01 NS063008), the NMSS (RG4768, RG5179 and RG5180), the Guthy Jackson Charitable Foundation and Maisin Foundation, M.A.P from the Pediatric Scientist Development Program fellowship, J.L.D from the NIH/NHLBI grant HL096126 and I.F.C. from the NIH/NHLBI grant HL102475. This work was supported by the National Multiple Sclerosis Society (NMSS) Postdoctoral Fellowship and the American Heart Association Fellowship to J.K.R., the Howard Hughes Medical Institute Medical Research Fellowship to S.G.M and by the NMSS RG4985 and NIH/NINDS R01 NS052189 grants to K.A.

Author contributions

J.K.R. designed experiments, performed in vitro and in vivo studies and analyzed data; M.A.P. performed histological analysis; S.G.M. performed quantitative RTPCR; K.B. analysed data and assisted with statistics and manuscript preparation; A.M.F. isolated primary microglia for microarray analysis; J.P.C., P.E.R.C. and M.R.M. performed histology and image analysis; E.V. produced recombinant brinogen; C.B. generated mouse lines and performed EAE; T.P. performed uorescence-activated cell sorting analysis;I.F.C. generated the Ccr2RFP/ mice; H.L. designed experiments, performed data analysis and neuropathology, and edited the manuscript. J.L.D. provided brinogen mutant mice, isolated plasma, designed experiments and edited the manuscript; S.S.Z. designed T cell experiments analyzed, and interpreted data and edited the manuscript; S.S.Z. and K.A. jointly supervised the T cell experiments; K.A. conceived the project, designed the study, analyzed and interpreted data. K.A. and J.K.R. wrote the manuscript with input from all authors.

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Autoimmunity and macrophage recruitment into the central nervous system (CNS) are critical determinants of neuroinflammatory diseases. However, the mechanisms that drive immunological responses targeted to the CNS remain largely unknown. Here we show that fibrinogen, a central blood coagulation protein deposited in the CNS after blood-brain barrier disruption, induces encephalitogenic adaptive immune responses and peripheral macrophage recruitment into the CNS leading to demyelination. Fibrinogen stimulates a unique transcriptional signature in CD11b⁺ antigen-presenting cells inducing the recruitment and local CNS activation of myelin antigen-specific Th1 cells. Fibrinogen depletion reduces Th1 cells in the multiple sclerosis model, experimental autoimmune encephalomyelitis. Major histocompatibility complex (MHC) II-dependent antigen presentation, CXCL10- and CCL2-mediated recruitment of T cells and macrophages, respectively, are required for fibrinogen-induced encephalomyelitis. Inhibition of the fibrinogen receptor CD11b/CD18 protects from all immune and neuropathologic effects. Our results show that the final product of the coagulation cascade is a key determinant of CNS autoimmunity.

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Blood coagulation protein fibrinogen promotes autoimmunity and demyelination via chemokine release and antigen presentation

Author

Ryu, Jae Kyu; Petersen, Mark A; Murray, Sara G; Baeten, Kim M; Meyer-franke, Anke; Chan, Justin P; Vagena, Eirini; Bedard, Catherine; Machado, Michael R; Coronado, Pamela E Rios; Prod'homme, Thomas; Charo, Israel F; Lassmann, Hans; Degen, Jay L; Zamvil, Scott S; Akassoglou, Katerina

Pages

8164

Publication year

2015

Publication date

Sep 2015

Publisher

Nature Publishing Group

e-ISSN

20411723

Source type

Scholarly Journal

Language of publication

English

DOI

https://doi.org/10.1038/ncomms9164

ProQuest document ID

1710669103

Blood coagulation protein fibrinogen promotes autoimmunity and demyelination via chemokine release and antigen presentation

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