Granzyme B-inhibitor serpina3n induces neuroprotection in vitro and in vivo

Journal of Neuroinflammation

Fabrizio Giuliani³  R. Chris Bleackley²  Bradley Kerr¹  Camille Olechowski¹  Katia Carmine-Simmen²  Yohannes Haile³ 

[1] Department of Anesthesiology and Pain Medicine, University of Alberta, Edmonton, Canada;Department of Biochemistry, University of Alberta, Edmonton, Canada;Department of Medicine, Division of Neurology, University of Alberta, 4C Kaye Edmonton Clinic, Edmonton T6G 1Z1, Alberta, Canada

关键词: Neurodegeneration;    Granzyme B;    serpina3n;    EAE;    Multiple sclerosis;   

Others  :  1227070
DOI  :  10.1186/s12974-015-0376-7

 received in 2015-05-01, accepted in 2015-08-16,  发布年份 2015

【 摘 要 】

Background

Multiple sclerosis (MS) is an autoimmune inflammatory and neurodegenerative disease of the central nervous system (CNS). It is widely accepted that inflammatory cells play major roles in the pathogenesis of MS, possibly through the use of serine protease granzyme B (GrB) secreted from the granules of cytotoxic T cells. We have previously identified GrB as a mediator of axonal injury and neuronal death. In this study, our goal was to evaluate the effect of GrB inhibition in the human system in vitro, and in vivo in EAE using the newly isolated GrB-inhibitor serpina3n.

Methods

We used a well-established in vitro model of neuroinflammation characterized by a co-culture system between human fetal neurons and lymphocytes. In vivo, we induced EAE in 10- to 12-week-old female C57/BL6 mice and treated them intravenously with serpina3n.

Results

In the in vitro co-culture system, pre-treatment of lymphocytes with serpina3n prevented neuronal killing and cleavage of the cytoskeletal protein alpha-tubulin, a known substrate for GrB. Moreover, in EAE, 50 μg serpina3n substantially reduced the severity of the disease. This dose was administered intravenously twice at days 7 and 20 post EAE induction. serpina3n treatment reduced axonal and neuronal injury compared to the vehicle-treated control group and maintained the integrity of myelin. Interestingly, serpina3n treatment did not seem to reduce the infiltration of immune cells (CD4 ⁺and CD8 ⁺T cells) into the CNS.

Conclusion

Our data suggest further studies on serpina3n as a potentially novel therapeutic strategy for the treatment of inflammatory-mediated neurodegenerative diseases such as MS.

【 授权许可】

   
2015 Haile et al.

【 图 表 】

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5.

【 参考文献 】

• [1]Compston A, Coles A: Multiple sclerosis. Lancet. 2008, 372:1502-1517.
• [2]Keegan BM, Noseworthy JH: Multiple sclerosis. Annu Rev Med. 2002, 53:285-302.
• [3]Kobelt G, Berg J, Atherly D, Hadjimichael O: Costs and quality of life in multiple sclerosis—a cross-sectional study in the United States. Neurology. 2006, 66:1696-1702.
• [4]Hauser SL, Oksenberg JR: The neurobiology of multiple sclerosis: genes, inflammation, and neurodegeneration. Neuron. 2006, 52:61-76.
• [5]Trapp BD, Nave KA: Multiple sclerosis: an immune or neurodegenerative disorder? Annu Rev Neurosci. 2008, 31:247-269.
• [6]Kniesel U, Wolburg H: Tight junctions of the blood-brain barrier. Cell Mol Neurobiol. 2000, 20:57-76.
• [7]Engelhardt B, Ransohoff RM: The ins and outs of T-lymphocyte trafficking to the CNS: anatomical sites and molecular mechanisms. Trends Immunol. 2005, 26:485-495.
• [8]Pittock SJ, Lucchinetti CF: The pathology of MS: new insights and potential clinical applications. Neurologist. 2007, 13:45-56.
• [9]Lu JQ, Fan Y, Mitha AP, Bell R, Metz L, Moore GR, et al.: Association of alpha-synuclein immunoreactivity with inflammatory activity in multiple sclerosis lesions. J Neuropathol Exp Neurol. 2009, 68:179-189.
• [10]Frischer JM, Bramow S, Dal-Bianco A, Lucchinetti CF, Rauschka H, Schmidbauer M, et al.: The relation between inflammation and neurodegeneration in multiple sclerosis brains. Brain. 2009, 132:1175-1189.
• [11]Giuliani F, Goodyer CG, Antel JP, Yong VW: Vulnerability of human neurons to T cell-mediated cytotoxicity. J Immunol. 2003, 171:368-379.
• [12]Boivin WA, Cooper DM, Hiebert PR, Granville DJ: Intracellular versus extracellular granzyme B in immunity and disease: challenging the dogma. Lab Invest. 2009, 89:1195-1220.
• [13]Haile Y, Pasychniyk D, Turner D, Bleackley RC, Giuliani F. CD4+CD25+CD127dimFoxp3+ T cells are cytotoxic for human neurons. J Leukoc Biol. 2011;89:927-34.
• [14]Haile Y, Simmen KC, Pasichnyk D, Touret N, Simmen T, Lu JQ, et al.: Granule-derived granzyme B mediates the vulnerability of human neurons to T cell-induced neurotoxicity. J Immunol. 2011, 187:4861-4872.
• [15]Bots M, Medema JP: Granzymes at a glance. J Cell Sci. 2006, 119:5011-5014.
• [16]Kaiserman D, Bird CH, Sun J, Matthews A, Ung K, Whisstock JC, et al.: The major human and mouse granzymes are structurally and functionally divergent. J Cell Biol. 2006, 175:619-630.
• [17]Ewen CL, Kane KP, Bleackley RC: A quarter century of granzymes. Cell Death Differ. 2012, 19:28-35.
• [18]Neumann H, Medana IM, Bauer J, Lassmann H: Cytotoxic T lymphocytes in autoimmune and degenerative CNS diseases. Trends Neurosci. 2002, 25:313-319.
• [19]Bien CG, Vincent A, Barnett MH, Becker AJ, Blumcke I, Graus F, et al.: Immunopathology of autoantibody-associated encephalitides: clues for pathogenesis. Brain. 2012, 135:1622-1638.
• [20]Henkart PA: Mechanism of lymphocyte-mediated cytotoxicity. Annu Rev Immunol. 1985, 3:31-58.
• [21]Podack ER, Konigsberg PJ: Cytolytic T cell granules. Isolation, structural, biochemical, and functional characterization. J Exp Med 1984, 160:695-710.
• [22]Redmond MJ, Letellier M, Parker JM, Lobe C, Havele C, Paetkau V, et al.: A serine protease (CCP1) is sequestered in the cytoplasmic granules of cytotoxic T lymphocytes. J Immunol. 1987, 139:3184-3188.
• [23]Bredemeyer AJ, Lewis RM, Malone JP, Davis AE, Gross J, Townsend RR, et al.: A proteomic approach for the discovery of protease substrates. Proc Natl Acad Sci U S A. 2004, 101:11785-11790.
• [24]Russell JH, Ley TJ: Lymphocyte-mediated cytotoxicity. Annu Rev Immunol. 2002, 20:323-370.
• [25]Barry M, Bleackley RC: Cytotoxic T lymphocytes: all roads lead to death. Nat Rev Immunol. 2002, 2:401-409.
• [26]Agrawal SM, Silva C, Wang J, Tong JP, Yong VW: A novel anti-EMMPRIN function-blocking antibody reduces T cell proliferation and neurotoxicity: relevance to multiple sclerosis. J Neuroinflammation. 2012, 9:1742-2094. BioMed Central Full Text
• [27]Havla J, Kleiter I, Kumpfel T: Bridging, switching or drug holidays—how to treat a patient who stops natalizumab? Ther Clin Risk Manag 2013, 9:361-369.
• [28]Wuthrich C, Popescu BF, Gheuens S, Marvi M, Ziman R, Denq SP, et al.: Natalizumab-associated progressive multifocal leukoencephalopathy in a patient with multiple sclerosis: a postmortem study. J Neuropathol Exp Neurol. 2013, 14:14.
• [29]Diotti RA, Nakanishi A, Clementi N, Mancini N, Criscuolo E, Solforosi L, et al.: JC polyomavirus (JCV) and monoclonal antibodies: friends or potential foes? Clin Dev Immunol. 2013, 967581:25.
• [30]Marta M, Giovannoni G: Disease modifying drugs in multiple sclerosis: mechanisms of action and new drugs in the horizon. CNS Neurol Disord Drug Targets. 2012, 11:610-623.
• [31]Sipione S, Simmen KC, Lord SJ, Motyka B, Ewen C, Shostak I, et al.: Identification of a novel human granzyme B inhibitor secreted by cultured sertoli cells. J Immunol. 2006, 177:5051-5058.
• [32]Ang LS, Boivin WA, Williams SJ, Zhao H, Abraham T, Carmine-Simmen K, et al.: Serpina3n attenuates granzyme B-mediated decorin cleavage and rupture in a murine model of aortic aneurysm. Cell Death Dis. 2011, 8:88.
• [33]Hsu I, Parkinson LG, Shen Y, Toro A, Brown T, Zhao H, et al.: Serpina3n accelerates tissue repair in a diabetic mouse model of delayed wound healing. Cell Death Dis. 2014, 5:e1458.
• [34]Musgrave T, Benson C, Wong G, Browne I, Tenorio G, Rauw G, et al.: The MAO inhibitor phenelzine improves functional outcomes in mice with experimental autoimmune encephalomyelitis (EAE). Brain Behav Immun. 2011, 25:1677-1688.
• [35]Olechowski CJ, Truong JJ, Kerr BJ: Neuropathic pain behaviours in a chronic-relapsing model of experimental autoimmune encephalomyelitis (EAE). Pain. 2009, 141:156-164.
• [36]Benson CA, Wong G, Tenorio G, Baker GB, Kerr BJ: The MAO inhibitor phenelzine can improve functional outcomes in mice with established clinical signs in experimental autoimmune encephalomyelitis (EAE). Behav Brain Res. 2013, 252:302-311.
• [37]Zastepa E, Fitz-Gerald L, Hallett M, Antel J, Bar-Or A, Baranzini S, et al.: Naive CD4 T-cell activation identifies MS patients having rapid transition to progressive MS. Neurology. 2014, 82:681-690.
• [38]Luessi F, Siffrin V, Zipp F: Neurodegeneration in multiple sclerosis: novel treatment strategies. Expert Rev Neurother. 2012, 12:1061-1076.
• [39]Coronado MJ, Brandt JE, Kim E, Bucek A, Bedja D, Abston ED, et al.: Testosterone and interleukin-1beta increase cardiac remodeling during coxsackievirus B3 myocarditis via serpin A 3n. Am J Physiol Heart Circ Physiol. 2012, 302:H1726-1736.
• [40]Zipp F, Aktas O: The brain as a target of inflammation: common pathways link inflammatory and neurodegenerative diseases. Trends Neurosci. 2006, 29:518-527.
• [41]Berke G: The binding and lysis of target cells by cytotoxic lymphocytes: molecular and cellular aspects. Annu Rev Immunol. 1994, 12:735-773.
• [42]Chaitanya GV, Kolli M, Babu PP. Granzyme-b mediated cell death in the spinal cord-injured rat model. Neuropathology. 2009;29:270-79.
• [43]Chaitanya GV, Schwaninger M, Alexander JS, Babu PP: Granzyme-b is involved in mediating post-ischemic neuronal death during focal cerebral ischemia in rat model. Neuroscience. 2010, 165:1203-1216.
• [44]Cullen SP, Brunet M, Martin SJ: Granzymes in cancer and immunity. Cell Death Differ. 2010, 17:616-623.
• [45]Wang T, Allie R, Conant K, Haughey N, Turchan-Chelowo J, Hahn K, et al.: Granzyme B mediates neurotoxicity through a G-protein-coupled receptor. Faseb J. 2006, 20:1209-1211.
• [46]Phares TW, Stohlman SA, Hinton DR, Atkinson R, Bergmann CC: Enhanced antiviral T cell function in the absence of B7-H1 is insufficient to prevent persistence but exacerbates axonal bystander damage during viral encephalomyelitis. J Immunol. 2010, 185:5607-5618.
• [47]Richter K, Hausmann J, Staeheli P: Interferon-gamma prevents death of bystander neurons during CD8 T cell responses in the brain. Am J Pathol. 2009, 174:1799-1807.
• [48]Sobottka B, Harrer MD, Ziegler U, Fischer K, Wiendl H, Hunig T, et al.: Collateral bystander damage by myelin-directed CD8+ T cells causes axonal loss. Am J Pathol. 2009, 175:1160-1166.
• [49]Bredemeyer AJ, Townsend RR, Ley TJ: Use of protease proteomics to discover granzyme B substrates. Immunol Res. 2005, 32:143-153.
• [50]Trapp BD, Peterson J, Ransohoff RM, Rudick R, Mork S, Bo L: Axonal transection in the lesions of multiple sclerosis. N Engl J Med. 1998, 338:278-285.
• [51]Shriver LP, Dittel BN: T-cell-mediated disruption of the neuronal microtubule network: correlation with early reversible axonal dysfunction in acute experimental autoimmune encephalomyelitis. Am J Pathol. 2006, 169:999-1011.
• [52]Lassmann H: Multiple sclerosis pathology: evolution of pathogenetic concepts. Brain Pathol. 2005, 15:217-222.
• [53]Peterson JW, Trapp BD: Neuropathobiology of multiple sclerosis. Neurol Clin. 2005, 23:107-129.
• [54]Geurts JJ, Calabrese M, Fisher E, Rudick RA: Measurement and clinical effect of grey matter pathology in multiple sclerosis. Lancet Neurol. 2012, 11:1082-1092.
• [55]Gold R, Linington C, Lassmann H: Understanding pathogenesis and therapy of multiple sclerosis via animal models: 70 years of merits and culprits in experimental autoimmune encephalomyelitis research. Brain. 2006, 129:1953-1971.
• [56]Linkermann A, Qian J, Lettau M, Kabelitz D, Janssen O: Considering Fas ligand as a target for therapy. Expert Opin Ther Targets. 2005, 9:119-134.
• [57]Prakash MD, Munoz MA, Jain R, Tong PL, Koskinen A, Regner M, et al.: Granzyme B promotes cytotoxic lymphocyte transmigration via basement membrane remodeling. Immunity. 2014, 41:960-972.
• [58]Zamanian JL, Xu L, Foo LC, Nouri N, Zhou L, Giffard RG, et al.: Genomic analysis of reactive astrogliosis. J Neurosci. 2012, 32:6391-6410.
• [59]Mun-Bryce S, Rosenberg GA: Gelatinase B modulates selective opening of the blood-brain barrier during inflammation. Am J Physiol. 1998, 274:R1203-1211.
• [60]Toft-Hansen H, Buist R, Sun XJ, Schellenberg A, Peeling J, Owens T: Metalloproteinases control brain inflammation induced by pertussis toxin in mice overexpressing the chemokine CCL2 in the central nervous system. J Immunol. 2006, 177:7242-7249.
• [61]Agrawal SM, Silva C, Tourtellotte WW, Yong VW: EMMPRIN: a novel regulator of leukocyte transmigration into the CNS in multiple sclerosis and experimental autoimmune encephalomyelitis. J Neurosci. 2011, 31:669-677.