Blood Coagulation Protein Fibrinogen Promotes Autoimmunity and Demyelination Via Chemokine Release and Antigen Presentation
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ARTICLE Received 16 Feb 2015 | Accepted 27 Jul 2015 | Published 10 Sep 2015 DOI: 10.1038/ncomms9164 OPEN Blood coagulation protein fibrinogen promotes autoimmunity and demyelination via chemokine release and antigen presentation 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 Prod’homme3,4, 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 cri- tical 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 tran- scriptional signature in CD11b þ 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. 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 Children’s 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: [email protected]) or K.A. (email: [email protected]). NATURE COMMUNICATIONS | 6:8164 | DOI: 10.1038/ncomms9164 | www.nature.com/naturecommunications 1 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9164 isruption of the homeostatic balance between the Results vasculature and the brain is a sustained and often early Fibrinogen in the CNS induces inflammatory demyelination. Dfeature of neurologic diseases and traumatic insults to the To determine whether fibrinogen extravasation is sufficient to central nervous system (CNS). Understanding how blood–brain induce CNS pathology in vivo, we stereotactically injected barrier (BBB) disruption instigates and amplifies immune and fibrinogen at its physiological plasma concentration into the degenerative responses leading to brain pathology and loss of corpus callosum, the largest CNS white matter tract associated function would be instrumental in the design of novel treatments with disability in MS16. Surprisingly, this single injection of for neurologic diseases. Fibrinogen (coagulation factor I) is a fibrinogen spontaneously induced local demyelination within major component in the blood that upon BBB disruption enters 7 days (Fig. 1a). Microglial activation was rapidly induced 1 day the CNS and is deposited as insoluble fibrin1. Although soluble after fibrinogen injection and preceded demyelination (Fig. 1a). fibrinogen in the bloodstream is not proinflammatory, activation Activation of innate immunity regulates autoimmune responses of the coagulation cascade results in the formation of fibrin including T-cell recruitment and differentiation17,18. Fibrinogen associated with exposure of cryptic epitopes that transform injection induced infiltration of both CD4 þ and CD8 þ T-cell fibrinogen from a blood factor to a potent activator of innate populations in the CNS, but not in the spleen (Fig. 1a,b; immunity1. In multiple sclerosis (MS) and experimental Supplementary Fig. 1). T-cell infiltration occurred as early as autoimmune encephalomyelitis (EAE), BBB disruption and 1 day after injection and markedly increased 3 and 7 days after fibrin deposition are detected in the white matter along with injection (Fig. 1a), thus also preceding demyelination. Injection of microglial activation before T-cell infiltration and the onset of fibrinogen in the spinal cord ventral column also induced demyelination2–6. Indeed, increased coagulation activity leading inflammatory demyelination (Supplementary Fig. 2). Neuro- to fibrin formation occurs early in neuroinflammation before pathologic alterations were not observed after injection of control demyelination4, and is highly upregulated in active MS plaques7. artificial cerebrospinal fluid (ACSF) in either the brain or spinal Moreover, injection of soluble fibrinogen in the healthy brain cord (Fig. 1; Supplementary Fig. 2). Although microglia show a results in fibrin formation5. Fibrin deposition is abundant not diffuse response in the cortex along the needle track 1 day after only in early but also active and chronic MS lesions, and ACSF injection, this response resolves and no alterations are correlates with demyelination and T-cell infiltration3,8–10. Genetic observed either in the cortex or the corpus callosum (Fig. 1). or pharmacologic depletion of fibrinogen decreases microglial While wild-type (WT) plasma induced demyelination similar activation and axonal damage and attenuates neurologic signs in to fibrinogen, injection of plasma from fibrinogen-deficient EAE5,7,11–13 and other MS models14,15. While studies in EAE (Fib À / À ) mice19, which contains all plasma proteins except mice support a role for fibrin in the activation of microglia in fibrinogen, led to an 82% reduction in demyelination compared myelinated areas, its role in the initiation and propagation of with WT plasma (Supplementary Fig. 3). Injection of the plasma myelin-targeted adaptive immune responses is unknown. proteins albumin or high-molecular-weight kininogen had no Moreover, despite the vast literature on BBB disruption and effect (Supplementary Fig. 3). activation of the coagulation cascade in brain diseases, there is Activation of the coagulation cascade converts fibrinogen to currently no model of neuroinflammation induced by a proinflammatory fibrin primarily due to exposure of a cryptic coagulation factor. epitope in the fibrinogen g chain (g377–395), which binds to Here we developed a model of coagulation-driven demyelina- CD11b/CD18 integrin (Mac-1, complement receptor 3, 20,21 tion to directly assess the role of BBB disruption and fibrin in the aMb2) . The interaction of fibrin with CD11b/CD18 is induction of CNS autoimmunity and demyelination. Surprisingly, genetically targeted in Fibg390–396A mice, in which fibrinogen introduction of fibrinogen into the healthy CNS was sufficient to has been mutated to lack the CD11b/CD18-binding motif, but induce activation of adaptive immunity targeted to CNS myelin retains normal clotting function22. Injection of plasma derived antigens leading to demyelination. The effect of fibrinogen as an from Fibg390–396A mice resulted in a 70% reduction in initiator of CNS autoimmunity was first substantiated in vivo, demyelination compared with WT plasma (Supplementary where a single stereotactic injection of fibrinogen in the corpus Fig. 3), suggesting that the interaction of fibrin with CD11b/ callosum induced recruitment and local differentiation of myelin CD18 is required for the induction of demyelination. Since antigen-specific Th1 cells leading to demyelination. In accor- a portion of fibrinogen in the plasma is known to bind dance, endogenous fibrinogen was required for T-helper 1 (Th1) extracellular matrix proteins and growth factors, we also cell activation in EAE. Microarray analysis in cell autonomous produced and tested recombinant fibrinogen, which is clottable systems of antigen-presenting cells (APCs) uncovered a unique and hydrodynamically indistinguishable from plasma fibrinogen, transcriptional signature for fibrin that links the coagulation with the exception that it did not carry other plasma-derived cascade with M1-type activation of APCs associated with factors23. Similar to plasma fibrinogen, recombinant fibrinogen induction of antigen presentation and release of leukocyte also induced demyelination and microglial activation recruiting chemokines. Rescue experiments to genetically deplete (Supplementary Fig. 4). These results suggest that fibrinogen is the fibrin-specific immune responses in APCs demonstrated that a major component in the plasma that in the healthy CNS white secretion of the monocyte chemoattractant protein-1 (MCP-1/ matter triggers T-cell recruitment and demyelination even in the CCL2) and the C-X-C motif ligand-10 (CXCL10), together with absence of pre-existing inflammatory or myelin abnormalities. MHC II-dependent antigen presentation, are essential molecular mediators for