Effects of IFNβ1a and IFNβ1b treatment on the ex

Authors: Natalia LubinaDąbrowska Adam Stepień Grzegorz Sulkowski Beata DąbrowskaBouta Józef Langfort Małgorzata Chalimoniuk

Publish Date: 2017/03/15

Volume: 65, Issue: 4, Pages: 325-338

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Abstract

The aim of this study was to investigate the effects of interferon IFNβ1a and IFNβ1b treatment on inflammatory factors and myelin protein levels in the brain cortex of the Lewis rat experimental autoimmune encephalomyelitis EAE animal model of multiple sclerosis To induce EAE rat were immunized with inoculums containing spinal cord guinea pig homogenized in phosphatebuffered saline and emulsified in Freund’s complete adjuvant containing 110 µg of the appropriate antigen in 100 µl of an emulsion and additionally 4mg/ml Mycobacterium tuberculosis H37Ra The rats were treated three times per week with subcutaneous applications of 300000 units IFNβ1a or IFNβ1b The treatments were started 8 days prior to immunization and continued until day 14 after immunization The rats were killed on the 14th day of the experiment EAE induced dramatic increase in interleukin IL1β IL6 and tumor necrosis factor TNFconcentrations and inducible nitric oxide synthase iNOS expression in the brain which closely corresponded to the course of neurological symptoms and the loss of weight Both IFNβ1b and IFNβ1a treatments inhibited the proinflammatory cytokines IL6 IL1β TNFα and IFNγ decreased the activation of astrocytes increased the myelin protein level in the brain cortex and improved the neurological status of EAE rats by different mechanisms IFNβ1a reduced iNOS expression at least in part by the enhancement of IL10 while IFNβ1b diminished IL10 concentration and did not decrease EAEinduced iNOS expressionMultiple sclerosis MS is a chronic inflammatory neurodegenerative disease which is characterized by demyelination and remyelination and neuronal damage Holz et al 2000 Stadelmann 2011 Stüve and Oksenberg 2010 with onset of disease typically occurring between the ages of 20 and 40 years Sospedra and Martin 2005 Demyelinating lesions are often found in the white matter of the brain stem the spinal cord and the cerebellum Compston and Coles 2008 After a course of the relapsingremitting phase of the disease most MS patients enter a phase characterized by progressive neurodegeneration associated with an irreversible variety of physical disabilities Ireland and Monson 2011 Lucchinetti et al 2000 Trapp et al 1999 Pathological and magnetic resonance imaging studies indicate that axonal damage predominantly develops in the early stage of MS as a consequence of inflammatory process Bendfeldt et al 2009 Comi 2000 which leads to the most numerous ~85 of cases relapsingremitting form of the disease Weiner 2008A considerable infiltration by macrophages monocytes and lymphocytes into central nervous system during MS induces secretion of many activated microglia and astrocytes proinflammatory cytokines including interleukin IL1β tumor necrosis factor TNFα and IL6 Aguzzi et al 2013 Hartung et al 1995 which are involved in the production of oxidative radicals Merrill and Benveniste 1996 and expression of the inducible nitric oxide synthase iNOS Willenborg et al 1999 Elevated levels of aforementioned proinflammatory cytokines in the plasma cerebrospinal cord and brain cortex were found in patients with MS Giovannoni and Thorpe 2001 Navikas et al 1996a b Rieckmann et al 1995 Sharief and Hentges 1991 and a positive correlation was found between the levels and the disease’s activity and severity Navikas et al 1996b Rieckmann 1995 Sharief and Hentges 1991 A similar effect ie elevation of proinflammatory cytokines including IFNγ IL1β TNFα and IL6 was observed in experimental autoimmune encephalomyelitis EAE an animal model of MS Schneider et al 2009 Sulkowski et al 2013 Tanuma et al 1997 The increase in the production of cytokines with proinflammatory potential is generally accompanied by concomitant increase in the production of cytokines with antiinflammatory/immunoregulatory properties among which TGFβ and IL10 play a dominant role Imitola et al 2005 Thus disordered balance between pro and antiinflammatory mediators may lead to induction and progression of MS/EAE Brosnan and Raine 1996There are two structural forms of IFNβ ie IFNβ1b and IFNβ1a that are used therapeutically They demonstrate a good efficacy in longterm relapsingremitting of MS RRMS therapy Bendfeldt et al 2010 Stępień et al 2013 and also to some extent in the treatment of secondary progressive MS European Study Group on Interferon β1b in Secondary Progressive MS 1998 However the biological activities of IFNβ1b and IFNβ1a are not the same It is known that the IFNβ1a has a higher biological potency in its antiviral properties Antonetti et al 2002 Following this notion these two IFNβ variants altered diversely the plasma cytokine profile of RRMS patients following 3year therapy despite similar improvement of neurological status and marked reduction of the annual relapse rate in a majority of RRMS patients with mild to moderate disability Stępień et al 2013Growing evidence demonstrates that the inflammatory process is most active at the beginning of MS Comi et al 2001 and takes part in degeneration of the myelin sheath of nerve cells As one of the plausible modes of IFNβ action in responsive patients is antiinflammatory effect Graber et al 2007 Liu et al 2010 Ransohoff et al 1991 Rio and Montalban 2005 Salama et al 2003 the improved results of IFNβ therapy could be especially expected from early treatment of MS when the inflammatory process initiates This issue is poorly investigated in the clinical practice and relatively little is known about inflammatory process leading to demyelination as well as efficacy of IFNβ1b and IFNβ1a treatment on this process at the beginning of MS when disease in most patients may be ongoing subclinically To better recognize this issue we have investigated the effects of IFNβ1a and IFNβ1b monotherapies on selected serum cytokines and nitrite levels in the early phased of EAE rats This study was also aimed to investigate the influence of IFNβ1a and IFNβ1b treatment on the iNOS and myelin protein levels indicated by MOG and CNPase levels and their possible adjustment by regulation of selected pro and antiinflammatory cytokines in the cerebral cortex in rats subjected to EAEEightweekold female Lewis rats 183 ± 10 g were supplied by the animal house of the Mossakowski Medical Research Centre Polish Academy of Sciences All procedures involving rodent care and experimentation were carried out in accordance with the European Communities Council Directive 86/609/EEC for the Care and Use of Laboratory Animals All protocols were approved by the 4th Local Ethics Committee for Animal Experiments National Medicines Institute Warsaw Poland Rats were housed in a temperaturecontrolled room with a 12h light/dark cycle and free access to water and food including the Ssniff® R2 complete diet for rat breeding Ssniff Spezialdoten GmbH Soest GermanyTo induce EAE female Lewis rats were immunized with inoculums which contained spinal cord guinea pig homogenized in phosphatebuffered saline and emulsified in Freund’s complete adjuvant which contained 110 µg of the appropriate antigen in 100 µl of emulsion and 4mg/ml Mycobacterium tuberculosis H37Ra Kerschensteiner et al 2004 Meyer et al 2001 The animals were observed daily and monitored for neurological deficits with clinical severity scores and weight The clinical scores of EAE were assigned according to the following criteria—0 asymptomatic 1 complete loss of tail tone 2 hind limb paraplegia 3 complete hind limb paralysis 4 hind limb paralysis with forelimb involvement and 5 moribund/dead Kerschensteiner et al 2004 Meyer et al 2001 Four different experimental groups of animals were used control EAE EAE treated with IFNβ1a and EAE treated with IFNβ1bThe treatments of IFNβ1a or IFNβ1b were started 8 days after immunization and continued until day 14 after immunization Wender et al 2001 The rats with EAE were treated three times per week with subcutaneous applications of 300000 units of IFNβ1a Biogen IDEC LTD Berkshire UK or IFNβ1b Bayer Schering Pharma Berlin Germany The rats were euthanized on the 14th day of the experiment

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