Journal of Neuroimmunology 166 (2005) 132 – 143
`
`www.elsevier.com/locate/jneuroim
`
`Detoxication enzyme inducers modify cytokine production in rat
`mixed glial cells
`
`Anne Wierinckx a,1, John Breve´ a, Dominique Mercier a,2, Marianne Schultzberg b,
`Benjamin Drukarch a, Anne-Marie Van Dam a,*
`
`aInstitute for Clinical and Experimental Neurosciences, Department of Medical Pharmacology,
`VUmc, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands
`bNeurotec Department, Division of Experimental Geriatrics, Karolinska Institute, Novum, SE-14186 Stockholm, Sweden
`
`Received 9 March 2005; accepted 27 May 2005
`
`Abstract
`
`interleukin-1h (IL-1h),
`factor-a (TNFa) as well as
`tumor necrosis
`interleukin-6 (IL-6),
`Pro-inflammatory cytokines, e.g.
`neurotoxic molecules such as nitric oxide (NO), that are produced and released by activated glial cells, play an important role in
`inflammation and oxidative stress occurring during Multiple Sclerosis (MS). Reduction of these processes could therefore be of
`therapeutic interest. Dimethylfumarate (DMF) and sulforaphane (SP) are well known for their detoxicating properties. Furthermore,
`they have anti-inflammatory effects as shown clinically by the treatment of inflammatory skin diseases. However, their detoxication
`and anti-inflammatory action on brain-derived cells
`is unknown.
`In the present
`study we have studied, within the same
`concentration range, the anti-inflammatory and detoxicating effects of DMF and SP on the production and release of mediators of
`inflammation and detoxication from lipopolysaccharide (LPS) activated primary co-cultures of rat microglial and astroglial cells.
`DMF and SP attenuated the LPS-induced production and release of TNFa, IL-1h, IL-6 and NO. In addition, DMF and SP increase
`both mRNA level and activity of NAD(P)H:quinone reductase (NQO-1), a detoxication enzyme, as well as the cellular glutathione
`content. We conclude that DMF or SP simultaneously can (1) reduce mediators of inflammation and (2) enhance detoxication
`enzymes in LPS stimulated co-cultures of astroglial and microglial cells. This double-sided effect could potentially be of therapeutic
`interest.
`D 2005 Elsevier B.V. All rights reserved.
`
`Keywords: Interleukin-1h; Interleukin-6; Tumor necrosis factor-a; Nitric oxide; Dimethylfumarate; Sulforaphane
`
`* Corresponding author. Tel.: +31 20 444 8095; fax: +31 20 444 8100.
`E-mail addresses: a.wierinckx@laposte.net (A. Wierinckx),
`jjp.breve@vumc.nl (J. Breve´), dominique.mercier@igmm.cnrs.fr
`(D. Mercier), schultzberg@nuerotec.ki.se (M. Schultzberg),
`b.drukarch@vumc.nl (B. Drukarch), amw.vandam@vumc.nl
`(A.-M. Van Dam).
`1 Present address: Unite´ Inserm U433-Neurobiologie Expe´rimentale et
`Physiopathologie Faculte´ Laennec, 8, rue G. Paradin 69372 Lyon cedex 08,
`France. Tel.: +33 4 72 91 34 92; fax: +33 4 72 68 49 55.
`2 Present address: CNRS-UMR 5535-IGMM, 1919 route de Mende, 34293
`Montpellier cedex 5, France. Tel.: +33 4 67 36 05; fax: +33 67 04 02 31.
`
`0165-5728/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
`doi:10.1016/j.jneuroim.2005.05.013
`
`1. Introduction
`
`The etiology of chronic inflammatory, neurodegener-
`ative diseases such as Multiple Sclerosis (MS) is not well
`understood. Neuropathologically,
`the presence of
`infil-
`trated immune cells and activated glial cells is evident
`(Benveniste, 1997; Heneka and Feinstein, 2001). During
`inflammation these cells produce a variety of pro-
`inflammatory mediators including interleukin-1h (IL-1h),
`interleukin-6 (IL-6) and tumor necrosis factors-a (TNFa)
`as well as neurotoxic molecules such as nitric oxide (NO).
`All of these are known to contribute directly or indirectly
`to cellular dysfunction and/or cell death associated with
`neurodegenerative processes (Hantraye et al., 1996;
`
`Sawai (IPR2019-00789), Ex. 1035, p. 001
`
`

`

`A. Wierinckx et al. / Journal of Neuroimmunology 166 (2005) 132 – 143
`
`133
`
`Hooper et al., 1997; Oshima et al., 1996; Simonian and
`Coyle, 1996; Takahashi et al., 1997). In MS for example,
`cytokines and reactive oxygen species (ROS,
`including
`NO) are present in perivascular T-cells, macrophages and
`in surrounding activated glial cells. It is shown that their
`presence leads to demyelination,
`functional motor dis-
`turbances and finally axonal loss (Penkowa and Hildago,
`2003; Bar-Or et al., 1999; Brosnan et al., 1995; Cannella
`and Raine, 1995).
`Neuro-inflammation and oxidative stress, as detected in
`MS,
`leads to alterations in the activity/functionality of
`resident brain cells.
`In a chronic situation,
`it has a
`progressive effect on the course of the disease, emphasizing
`the importance of reducing the level of inflammation and
`reactive oxygen species within the brain. Several naturally
`occurring and synthetic compounds, including dimethylfu-
`marate (DMF) and sulforaphane (SP), are known to present
`detoxicating properties in mammalian cells. These com-
`pounds do not scavenge free radicals, but elevate the
`production of
`relevant detoxication enzymes including
`NAD(P)H:quinone reductase (NQO-1) and/or stimulate
`glutathione synthesis (Basten et al., 2002; Dringen et al.,
`1998; Gao et al., 2001; Khanna et al., 1998; McMahon et
`al., 2001; Nelson et al., 1999; Prochaska and Fernandes,
`1993; Spencer et al., 1990).
`Interestingly, DMF and SP have also been shown to
`affect inflammatory processes. DMF has been used for the
`systemic treatment of psoriasis and reduces itching and
`scaling and prevents extension of the disease (Nieboer et al.,
`1989). Mechanistically, DMF reduces cytokine expression
`in lymphocytes (de Jong et al., 1996; Ockenfels et al.,
`1998), has an anti-proliferative effect on keratinocytes (Thio
`et al., 1994) and,
`inhibits monocyte differentiation into
`dendritic cells (Zhu and Mrowietz, 2001). SP down-
`regulates bacterial lipopolysaccharide (LPS)-mediated pro-
`duction of several inflammatory proteins (e.g. iNOS, COX-
`2 and TNFa) in cultured macrophages (Heiss et al., 2001).
`Recently, Wu and collaborators have shown decreases in
`NFnB nuclear translocation and infiltration of activated
`macrophages in kidneys of spontaneous hypertensive rats
`fed with dried broccoli sprouts containing glucoraphanin,
`metabolized into SP (Wu et al., 2004).
`The compounds DMF and SP may represent promising
`therapeutic targets to enhance detoxication and reduce
`inflammation,
`if both actions occur within the same
`concentration range of the compound used. In the present
`study, we question whether DMF and SP show detoxication
`and anti-inflammatory activities in LPS-activated brain-
`derived glial cells. Until now, little is known of effects of
`DMF and SP on brain-derived cells. To address this question,
`rat mixed microglia and astrocytes were treated with LPS to
`stimulate production of pro-inflammatory mediators (e.g. IL-
`1h, TNFa, IL-6) and reactive oxygen species (e.g. NO).
`Subsequently, the anti-inflammatory effects of DMF and SP
`on IL-1h, TNFa, IL-6 and NO production by the glial cells
`were studied. In addition, the detoxicating effects of DMF
`
`and SP were studied by measuring glutathione (GSx) levels
`and NQO-1 activity in the cells.
`
`2. Materials and methods
`
`2.1. Mixed glial cell culture
`
`Primary co-cultures of astroglial and microglial cells were
`prepared from newborn (2 day-old) Wistar rats (Harlan CPB,
`Zeist, The Netherlands) (Vincent et al., 1996). Cerebral
`cortices were cleared from adhering meninges and blood
`vessels, and dissociated using 0.25% trypsin (Sigma
`Chemical Co., St Louis, MO) in phosphate-buffered saline
`(PBS). Cells (0.5 106/well) were plated in poly-l-lysine
`(15 Ag/ml; Sigma)-coated 12 wells plates (Nunclon, Ham-
`-
`strop, Denmark) and incubated at 37
`C in humidified air
`containing 5% CO2. The culture medium consisted of
`Dulbecco’s modified Eagles medium (DMEM)-F10 (Gibco,
`Life Technologies, Breda, The Netherlands), supplemented
`with 10% v/v heat-inactivated fetal calf serum (FCS)
`(Gibco), 2 mM l-glutamine (Sigma), 37.5 U/ml streptomy-
`cin (Sigma) and 100 U/ml penicillin (Sigma). The medium
`was changed 1 day after seeding. Cultures consisted mainly
`of microglial and astroglial cells with less than 0.01%
`oligodendrocytes. Experiments were carried out after 7 days
`of culture, when microglia showed short ramifications and
`cultures were confluent.
`
`2.2. Treatment of glial cells
`
`In a first series of experiments, co-cultures were incubated
`for 1, 2, 4, 6, 8, 16, 24, 48 or 72 h with LPS (100 ng/ml;
`Escherichia coli serotype 055-B5, Westphal, Difco, Detroit,
`MI) or media alone to determine the time-dependent
`cytokine production by activated glial cells. Culture super-
`natants were collected, divided into aliquots and stored at
` 20
`-
`C until assayed. The cells were lysed with a RNA
`extraction buffer (Promega Corporation, USA) and stored at
` 80
`-
`C. In subsequent series of experiments, astroglial –
`microglial cell co-cultures were incubated for 4 and 24 h
`with LPS (100 ng/ml). Incubation with LPS was carried out
`in the absence (vehicle: 0.0006% DMSO or 50 AM HCL) or
`presence of DMF (5, 15 or 30 AM in 0.0006% DMSO) or SP
`(1, 5 or 15 AM in 50 AM HCL). All conditions were tested in
`triplo and the experiments were repeated at least twice.
`Treatment of the cells did neither result in reduced cell
`viability nor in altered cellular protein levels within the
`timeframe of the experiments (data not shown). After
`incubation, culture supernatants were collected, divided into
`aliquots and stored at 20
`-
`C. Cells were lysed with a RNA
`extraction buffer (Promega Corporation) and stored at 80
`-
`C or lysed with Tris/EDTA and sonificated for NQO-1
`activity and stored at 20
`-
`C. For GSx measurements,
`cells were lysed with 2.5% salicilyc acid (SSA) and stored
`at 20
`-
`C.
`
`Sawai (IPR2019-00789), Ex. 1035, p. 002
`
`

`

`134
`
`A. Wierinckx et al. / Journal of Neuroimmunology 166 (2005) 132 – 143
`
`Table 1
`Rat primers and accession numbers of the rat DNA sequences used to design the primers
`
`Name
`
`GAPDH
`IL-1h
`IL-6
`iNOS
`NQO-1
`TNFa
`
`Primer FW
`5V-GAACATCATCCCTGCATCCA-3V
`5V-AAAGAAGAAGATGGAAAAGCGGTT-3V
`5V-CCCCAACTTCCAATGCTCTC-3V
`5V-AACTTGAGTGAGGAGCAGGTTGA-3V
`5V-AACGTCATTCTCTGGCCAATTC-3V
`5V-CCACACCGTCAGCCGATT-3V
`
`Primer Rev
`5V-CCAGTGAGCTTCCCGTTCA-3V
`5V-GGGAACTGTGCAGACTCAAACTC-3V
`5V-AGATGAGTTGGATGGTCTTGGTC-3V
`5V-CGCACCGAAGATATCCTCATGA-3V
`5V-GCCAATGCTGTACACCAGTTGA-3V
`5V-TCCTTAGGGCAAGGGCTCTT-3V
`
`Accession number
`
`NM_017008
`NM_031512
`NM_012589
`NM_012611
`NM_017000
`AJ002278
`
`2.3. Nitrite assay
`
`2.5. Rat IL-1b ELISA
`
`The nitrite concentration in the culture supernatant is
`used as a measure of NO production. Nitrite was measured
`by a colorimetric assay based on the Griess reaction, as
`described by Ding et al. (Ding et al., 1988). Briefly, 100 Al
`supernatant was mixed with 100 Al Griess reagent, consist-
`ing of 1% sulfanilamide (Jansen chimica, Geel, Belgium),
`0.1% naphtylethylene diamine dihydrochloride (Merck,
`Darmstadt, Germany), and 2.5% H3PO4 in water, and
`incubated at room temperature (RT)
`for 10 min. The
`absorbance was measured in a microtiter plate reader at
`550 nm (ICN Flow, MS2 Reader,
`ICN Biomedicals,
`Zoetermeer, The Netherlands). Sodium nitrite (Merck)
`dissolved in culture medium was used as the standard.
`Detection limit of the assay was 1 AM nitrite.
`
`2.4. Rat IL-6 ELISA
`
`IL-6 concentrations were measured in culture super-
`natant using an enzyme-linked immunosorbent assay
`(ELISA) specific for
`rat
`IL-6 (Rees et al., 1999a).
`Immunoaffinity-purified sheep anti-rat
`IL-6 antibodies
`were used as coating and detecting antibodies (S206/
`B1 : 2 mg/ml and 1 : 1000 dilution, respectively; NIBSC
`Potter Bar, UK). Recombinant rat IL-6 (NIBSC) diluted in
`culture medium/HPE (CLB, Amsterdam, The Netherlands)
`(50% v/v) was used as the standard. Detection limit of the
`assay was 8 pg IL-6/ml.
`
`IL-1h was measured using a
`The concentration of
`sandwich ELISA specific for rat IL-1h (Safieh-Garabedian
`et al., 1995). Purified polyclonal sheep anti-rat IL-1h
`antibodies were used as coating and detecting antibodies
`(S1002(110700): 1 Ag/ml and 1 : 1000 dilution, respectively;
`NIBSC). Standard curves, included in duplicate on each
`plate, were made with rat recombinant IL-1h (NIBSC)
`diluted in culture medium/HPE (CLB) (50% v/v) to measure
`IL-1h in the supernatant. Detection limit of the assay was 10
`pg IL-1h/ml.
`
`2.6. Rat TNFa ELISA
`
`The concentration of TNFa was measured using a
`rat TNFa (Rees et al.,
`sandwich ELISA specific for
`1999b). Purified polyclonal sheep anti-rat TNFa antibodies
`were used as coating and detecting antibodies
`(S54(050900): 1 Ag/ml and 1 : 500 dilution, respectively;
`NIBSC). Standard curves, included in duplicate on each
`plate, were made with rat recombinant TNFa (NIBSC)
`diluted in culture medium/HPE (CLB) (50% v/v). Detection
`limit of the assay was 10 pg TNFa/ml.
`
`2.7. NQO-1 enzyme activity measurement
`
`Cells were washed with PBS and lysed in ice cold 25
`mM Tris – HCL and 1 mM EDTA (pH = 7.4). After 5 s of
`
`Table 2
`IL-1h, IL-6, TNFa and nitrite levels produced by mixed astroglial – microglial cells
`
`Protein
`
`1 h
`
`IL-1b (pg/ml)
`Control
`< dl
`LPS
`< dl
`
`2 h
`
`4 h
`
`6 h
`
`8 h
`
`24 h
`
`48 h
`
`72 h
`
`< dl
`3.53
`
`T
`
`0.55
`
`< dl
`12.77
`
`T
`
`1.44
`
`< dl
`30.80
`
`T
`
`3.97
`
`< dl
`36.57
`
`T
`
`9.13
`
`< dl
`75.83
`
`T
`
`5.91
`
`< dl
`83.33
`
`T
`
`3.79
`
`< dl
`123.13
`
`T
`
`10.61
`
`T
`
`T
`
`T
`
`T
`
`IL-6 (pg/ml)
`Control
`< dl
`LPS
`< dl
`
`TNFa (pg/ml)
`Control
`< dl
`LPS
`139
`
`T
`
`Nitrite (lM)
`Control
`< dl
`LPS
`< dl
`
`< dl
`< dl
`
`< dl
`392.70
`
`T
`
`18.23
`
`< dl
`870.93
`
`T
`
`92.55
`
`< dl
`1019.83
`
`88.78
`
`< dl
`3711.67
`
`88.50
`
`< dl
`5164.60
`
`245.22
`
`< dl
`5775.50
`
`451.42
`
`62.13
`
`< dl
`1167
`
`T
`
`96.42
`
`< dl
`3110
`
`T
`
`97.54
`
`< dl
`3612
`
`T
`
`152.26
`
`< dl
`3716.1
`
`T
`
`292.10
`
`< dl
`4274.267
`
`T
`
`165.39
`
`< dl
`1918.6
`
`T
`
`97.92
`
`< dl
`1275.6
`
`T
`
`101.33
`
`< dl
`< dl
`
`< dl
`< dl
`
`< dl
`< dl
`
`< dl
`< dl
`
`< dl
`6.97
`
`T
`
`0.51
`
`< dl
`24.10
`
`T
`
`1.23
`
`< dl
`35.17
`
`T
`
`1.00
`
`< dl = below detection limit.
`
`Sawai (IPR2019-00789), Ex. 1035, p. 003
`
`

`

`A. Wierinckx et al. / Journal of Neuroimmunology 166 (2005) 132 – 143
`
`135
`
`sonification, cytosolic supernatants were collected, ali-
`quoted and stored at 20
`-
`C prior to use.
`The enzymatic activity of NQO-1 was determined
`spectrophotometrically by measuring the NADPH-depend-
`ent, menadiol-mediated reduction of 3-(4,5-dimethylthiazol-
`2yl)-2,5-diohenyltetrazolium bromide (MTT) as described
`before (Prochaska and Santamaria, 1988) with minor
`modifications (van Muiswinkel et al., 2000).
`
`rabbit IgGs (1 : 100; Jackson Immunoresearch Laboratories)
`and Alexa fluor 488 labelled streptavidin (1 : 100; Molecular
`Probes, or biotinylated donkey anti-rabbit IgGs (1 : 100;
`Jackson Immunoresearch Laboratories) and FITC labelled
`donkey anti-goat IgGs (1 : 25; Nordic, Denmark). Finally,
`the cells double-labelled for NQO-1 and GFAP were
`incubated with Texas Red labelled avidin (1 : 100; Vector
`Laboratories).
`
`2.8. Cellular localization of NQO-1
`
`2.9. Glutathione level measurement
`
`Mixed glial cells were cultured in a 12 wells plate and
`treated for 24 h with LPS (100 ng/ml), LPS and DMF (30
`AM) or LPS and sulforaphane (15 AM). After treatment, the
`cells were washed with PBS and fixed for 20 min with 4%
`paraformaldehyde. The cells were rinsed with TBS and
`subsequently incubated for 1 h at room temperature with a
`rabbit anti-rat NQO-1 antibody (MS-14; Dr. M. Schultzberg,
`Karolinska Institute, Stockholm, Sweden), diluted 1 : 10,000
`in TBS containing 0.1% Tween-20,
`followed by an
`incubation with biotinylated goat anti-rabbit IgGs (1 : 100;
`Jackson Immunoresearch Laboratories, USA) and finally an
`incubation with ABC (1 : 400; Vector Laboratories, USA).
`After a final wash in Tris/HCl, a nickel-enhanced DAB
`reaction was performed using H2O2 as a substrate to identify
`NQO-1 positive cells.
`A double-labeling immunocytochemical procedure was
`performed to determine which glial cell
`type expresses
`NQO-1. Therefore, the cells were incubated with a mixture
`of either rabbit anti-rat NQO-1 (1 : 10,000) and biotinylated
`mouse anti-rat ED-1 (1 : 250; gift from Prof. Dr. C.D.
`Dijkstra, Department of Molecular Cell Biology, VUmc,
`Amsterdam) or rabbit anti-rat NQO-1 (1 : 10,000) and goat
`anti-rat GFAP (1 : 100; Santa Cruz). After washing, the cells
`were incubated with either Texas Red labelled goat anti-
`
`To measure the total intracellular glutathione (GSx = 1
`GSH + 2 GSSG) content
`in cell
`lysates, we used a
`spectrophotometrical assay as described previously (Dru-
`karch et al., 1996). The amount of GSx in a cell lysate was
`determined according to a standard curve of 0, 1, 2, 3, 4 and
`5 AM GSx in 2.5% SSA, and calculated as Amol/mg protein.
`
`2.10. RNA extraction
`
`Cells were homogenized in 175 Al of SV RNA Lysis
`Buffer (SV Total RNA Isolation System, Promega Corpo-
`ration, USA). Total RNA was extracted according to the
`manufacturer’s protocol. The RNA samples were eluted in
`sterile water and concentrations were measured at A260 with
`a spectrophotometer (SPECTRAmax 250, Molecular Devi-
`ces, USA). The purity of RNA was valued on by a ratio
`A260 / A280 of 1.9 – 2.1.
`
`2.11. Reverse transcription
`
`One microgram of total RNA was reverse transcribed
`using the Avian Myeloblastosis Virus (AMV) Reverse
`Transcriptase (Reverse Transcription System, Promega
`Corporation). The absence of contaminating genomic
`
`Table 3
`Relative mRNA expression of IL-1h, IL-6, TNFa, iNOS and NQO-1 measured in mixed astroglial – microglial cells
`
`mRNA
`
`1 h
`
`2 h
`
`4 h
`
`6 h
`
`8 h
`
`24 h
`
`48 h
`
`72 h
`
`IL-1b (Relative expression to GAPDH, in percent)
`T
`T
`Control
`0.46
`0.03
`0.64
`0.002
`0.67
`T
`T
`LPS
`77.23
`1.56
`130.15
`6.32
`233.55
`
`T
`T
`
`0.03
`3.76
`
`0.26
`163.62
`
`T
`T
`
`0.003
`3.43
`
`0.11
`21.21
`
`T
`T
`
`T
`T
`
`0.07
`2.62
`
`0.01
`0.93
`
`T
`T
`
`T
`T
`
`0.30
`151.4
`
`0.09
`19.85
`
`0.13
`5.26
`
`0.07
`151.07
`
`0.01
`1.4
`
`0.04
`19.19
`
`T
`T
`
`T
`T
`
`0.004
`5.39
`
`0.08
`118.33
`
`0.01
`0.67
`
`0.01
`13.72
`
`T
`T
`
`T
`T
`
`0.01
`5.22
`
`0.16
`119.12
`
`0.001
`1.00
`
`0.03
`11.14
`
`T
`T
`
`T
`T
`
`0.02
`4.88
`
`0.001
`0.6
`
`IL-6 (Relative expression to GAPDH, in percent)
`T
`T
`Control
`0.21
`0.0001
`0.22
`0.02
`T
`T
`LPS
`1.35
`0.002
`16.07
`1.03
`
`0.11
`67.7
`
`TNFa (Relative expression to GAPDH, in percent)
`T
`T
`Control
`0.62
`0.05
`0.65
`0.03
`0.43
`T
`T
`LPS
`51.29
`2.49
`64.96
`3.50
`39.89
`
`T
`T
`
`0.03
`2.14
`
`0.30
`9.09
`
`iNOS (Relative expression to GAPDH, in percent)
`T
`T
`Control
`0.0075
`0.0008
`0.0123
`0.004
`0.0166
`T
`T
`LPS
`0.04
`0.01
`0.76
`0.07
`10.95
`
`T
`T
`
`0.0009
`0.85
`
`0.0093
`8.41
`
`NQO-1 (Relative expression to GAPDH, in percent)
`T
`T
`Control
`1.55
`0.05
`1.69
`0.04
`1.61
`T
`T
`LPS
`1.61
`0.01
`1.70
`0.01
`1.45
`
`T
`T
`
`0.07
`0.06
`
`1.81
`1.29
`
`T
`T
`
`T
`T
`
`T
`T
`
`0.03
`0.46
`
`0.33
`9.64
`
`0.0006
`0.32
`
`0.0040
`3.77
`
`0.04
`0.04
`
`1.68
`1.43
`
`T
`T
`
`T
`T
`
`T
`T
`
`0.04
`0.29
`
`0.14
`3.05
`
`0.001
`0.04
`
`0.0002
`12.05
`
`0.04
`0.06
`
`1.80
`3.66
`
`T
`T
`
`T
`T
`
`T
`T
`
`0.01
`0.14
`
`0.14
`1.49
`
`0.0001
`0.12
`
`0.0002
`4.75
`
`0.16
`0.09
`
`1.10
`4.57
`
`T
`T
`
`T
`T
`
`T
`T
`
`0.01
`0.05
`
`0.12
`0.92
`
`0.0002
`0.10
`
`0.0005
`3.89
`
`0.09
`0.15
`
`1.95
`4.56
`
`T
`T
`
`T
`T
`
`T
`T
`
`0.01
`0.07
`
`0.0001
`0.51
`
`0.27
`0.44
`
`Sawai (IPR2019-00789), Ex. 1035, p. 004
`
`

`

`136
`
`A. Wierinckx et al. / Journal of Neuroimmunology 166 (2005) 132 – 143
`
`DNA and the efficiency of the RT reactions were controlled
`by PCR on RNA and cDNA using GAPDH Forward and
`Reverse primers (Table 1).
`
`2.12. Quantitative PCR
`
`The cDNA synthesized was assayed using quantitative
`real time PCR (SYBR Green PCR, ABI 7700, PE Applied
`Biosystem, California, USA). With this method, direct
`detection of PCR products is monitored by measuring the
`increase in fluorescence caused by the binding of SYBR
`Green to double stranded DNA. The resulting relative
`increase in reporter fluorescent dye emission was monitored
`in real time during PCR amplification using the Sequence
`Detection System (ABI PRISM 7700 Sequence Detection
`system and software, PE Applied Biosystems).
`Primers (Table 1) were designed from rat corresponding
`sequences using Primer Express Software (PE Applied
`Biosystems).
`Quantitative PCR was carried out in a final volume of 20
`Al with 6.5 ng of cDNA, 3 mM MgCl2, 0.2 mM dATP,
`dCTP and dGTP, 0.4 mM dUTP, 0.75 mM of each primer,
`
`0.3 U of AmpliTaq Gold DNA and 0.12 U of Amperase
`uracil-N-glycosylase (all reagents from PE Applied Bio-
`-
`-
`system). PCR conditions were: 50
`C for 2 min, 95
`C for
`-
`-
`10 min followed by 45 cycles of 95
`C for 15 s and 60
`C
`for 1 min.
`Threshold cycle (Ct) values provide an index of the
`mRNA level. The level of GAPDH mRNA was used as an
`internal standard to control the amplification variations due
`to differences in the starting mRNA concentrations. The
`relative expression level of cytokine mRNA for each tissue
`was computed from the Ct values obtained for the gene of
`interest and GAPDH using the following formula:
`Relative mRNA expression of gene of interest (in percent
`compared to the GAPDH)
`¼ 2 Ct
`ð
`
`interest geneCt GAPDH
`
`Þ
`
`2.13. Statistical analysis
`
`Using the NCSS 2000 statistical program (NCSS,
`Kaysville, Utah, US),
`two-way ANOVA was performed
`
`Fig. 1. Effects of DMF and SP on lipopolysaccharide (LPS)-induced production of IL-1h in mixed glial cells. Cells were treated with LPS (100 ng/ml) for 4 and
`24 h. IL-1h protein was measured in the media after 4 and 24 h and IL-1h mRNA was measured after 4 h. a, effect of DMF on IL-1h protein level, b, effect of
`SP on IL-1h protein level, c, effect of DMF on IL-1h mRNA level, d, effect of SP on IL-1h mRNA level. Data are expressed as percentage of levels measured
`T
`in cultures co-incubated with LPS and solvent (control), and represent mean
`S.E.M. of 2 – 3 independent experiments (n = 6 – 12). *P < 0.01 vs. control.
`
`MYLAN PHARMS. INC. EXHIBIT 1035 PAGE 5
`
`Sawai (IPR2019-00789), Ex. 1035, p. 005
`
`

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`A. Wierinckx et al. / Journal of Neuroimmunology 166 (2005) 132 – 143
`
`137
`
`followed by a t-test for independent measurements (Fish-
`er’s LSD test). When the data did not follow a normal
`distribution,
`the analysis was performed on log-trans-
`formed data. P values < 0.01 were considered to indicate
`a significant difference.
`
`3. Results
`
`3.1. Time-dependent induction of IL-1b, IL-6, TNFa, iNOS,
`nitrite and NQO-1
`
`to measure
`time point(s)
`To establish the optimal
`inflammatory mediators produced by glial cells after LPS
`stimulation, the astroglial – microglial cell co-cultures were
`treated for 1 up to 72 h with LPS.
`At the protein level, TNFa was the first cytokine to be
`detected in the media after 1 h of LPS stimulation, IL-1h
`was detectable after 2 h, IL-6 was elevated after 4 h and
`nitrite after 24 h (Table 2). The concentration of TNFa
`reached its maximum at 24 h after LPS treatment. However,
`
`IL-1h, IL-6 and NO increased progressively until the end
`(72 h) of the experiment.
`The maximal mRNA levels for IL-1h, IL-6 and iNOS
`were observed after 4 h of LPS stimulation after which they
`attenuated, except
`for
`iNOS mRNA which remained
`elevated (Table 3). The mRNA level of TNFa reached its
`maximum after 2 h and decreased after 6 h (Table 3). NQO-
`1 mRNA was induced by LPS after 24 h to reach a plateau at
`48 h (Table 3).
`Based on these data, protein levels were measured at 4
`and 24 h after LPS incubation and mRNA levels at 4 h after
`LPS stimulation in all further experiments. Nitrite was
`measured at 24 h only.
`
`3.2. Effect of the compounds on LPS-induced IL-1b, IL-6
`and TNFa and nitrite levels
`
`The LPS-induced IL-1h protein levels measured in the
`culture supernatant were 15 pg/ml at 4 h after LPS
`treatment and 80 pg/ml at 24 h after LPS treatment. At 4 h
`after co-incubation of the cells, DMF suppressed slightly,
`
`Fig. 2. Effects of DMF and SP on lipopolysaccharide (LPS)-induced production of IL-6 by mixed glial cells. Cells were treated with LPS (100 ng/ml) for 4 and
`24 h. IL-6 protein was measured in the media after 4 and 24 h, and IL-6 mRNA was measured after 4 h. a, effect of DMF on IL-6 protein level, b, effect of SP
`on IL-6 protein level, c, effect of DMF on IL-6 mRNA level, d, effect of SP on IL-6 mRNA level. Data are expressed as percentage of levels measured in
`T
`cultures co-incubated with LPS and solvent (control), and represent mean
`S.E.M. of 2 – 3 independent experiments (n = 6 – 12). *P < 0.01 vs. control.
`
`Sawai (IPR2019-00789), Ex. 1035, p. 006
`
`

`

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`
`but significantly LPS-induced IL-1h levels (Fig. 1a),
`whereas SP did not have any effect (Fig. 1b). At 24 h,
`IL-1h levels in the culture media were reduced by both
`DMF and SP (Fig. 1a, b).
`The LPS-induced IL-6 protein levels measured in the
`culture supernatant were 450 pg/ml at 4 h and 4200 pg/ml
`at 24 h after LPS treatment. Both DMF and SP reduce IL-
`6 levels in the media of LPS stimulated glia mix at 4 and
`24 h (Fig. 2a, b).
`LPS-induced TNFa protein levels measured in the
`culture supernatant were 2900 pg/ml at 4 h and 4100 pg/
`ml at 24 h after LPS treatment. DMF and SP affect TNFa
`production of LPS stimulated glia co-cultures at both time
`points measured (Fig. 3a, b).
`The LPS-induced nitrite levels measured in culture
`supernatant was 10 AM at 24 h after LPS treatment. Only
`the two highest doses of DMF and SP decreased nitrite
`LPS-induced nitrite levels measured at 24 h after incuba-
`tion (Fig. 4).
`
`3.3. Effect of the compounds on LPS-induced IL-1b, IL-6,
`TNFa and iNOS mRNA levels
`
`The mRNA levels of the cytokines and iNOS measured
`were detectable in non-LPS treated cells (Table 2) and were
`not modified by DMF or SP treatment (data not shown).
`LPS treatment dramatically enhanced cytokine and iNOS
`mRNA levels as shown in Table 2.
`Co-incubation of LPS with DMF inhibited the mRNA
`levels of all cytokines and of
`iNOS (Figs. 1c-4c).
`However, only the highest dose (15 AM) of SP reduced
`the transcription of LPS-induced inflammatory mediators
`(Figs. 1d-4d).
`
`3.4. Effect of the compounds on NQO-1 mRNA level and
`NQO-1 activity
`
`The average LPS-induced cellular NQO-1 activity was
`48 and 65 dE/min/mg of total protein at 4 and 24 h after LPS
`
`Fig. 3. Effects of DMF and SP on lipopolysaccharide (LPS)-induced production of TNFa by mixed glial cells. Cells were treated with LPS (100 ng/ml) for 4
`and 24 h. TNFa protein was measured in the media after 4 and 24 h, and TNFa mRNA was measured after 4 h. a, effect of DMF on TNFa protein level, b,
`effect of SP on TNFa protein level, c, effect of DMF on TNFa mRNA level, d, effect of SP on TNFa mRNA level. Data are expressed as percentage of levels
`T
`measured in cultures co-incubated with LPS and solvent (control), and represent mean
`S.E.M. of 2 – 3 independent experiments (n = 6 – 12). *P < 0.01 vs.
`control.
`
`Sawai (IPR2019-00789), Ex. 1035, p. 007
`
`

`

`A. Wierinckx et al. / Journal of Neuroimmunology 166 (2005) 132 – 143
`
`139
`
`Fig. 4. Effects of DMF and SP on lipopolysaccharide (LPS)-induced production of nitric oxide in mixed glial cells. Cells were treated with LPS (100 ng/ml)
`for 4 and 24 h. Nitrite was measured in the media after 24 h, and iNOS mRNA was measured after 4 h. a, effect of DMF on the nitrite level, b, effect of SP
`on the nitrite level, c, effect of DMF on the iNOS mRNA level, d, effect of SP on the iNOS mRNA level. Data are expressed as percentage of levels
`T
`measured in cultures co-incubated with LPS and solvent (control), and represent mean
`S.E.M. of 2 – 3 independent experiments (n = 6 – 12). *P < 0.01 vs.
`control.
`
`treatment, respectively. DMF and SP induced similar effects
`on NQO-1 mRNA level and activity (Fig. 5). At 4 h, the
`activity of NQO-1 was not modified by the presence of
`DMF or SP in cultures treated with LPS (Fig. 5a, b). After
`24 h of co-incubation a significant
`increase in NQO-1
`activity was observed with all doses of DMF and SP used
`(Fig. 5a,). In contrast to NQO-1 activity, NQO-1 mRNA was
`already increased at 4 h after co-incubation of the cells with
`LPS and either of the two compounds (Fig. 5c, d).
`
`activity of the detoxication enzyme NQO-1 was enhanced
`200% – 300% by DMF and SP, whereas the glutathione
`synthesis was maximally 60% enhanced, we studied the
`cellular localization of NQO-1 in the mixed glial cell
`culture. NQO-1 positive cells were clearly detected in cells
`with various morphological shapes (Fig. 6a). The subse-
`quent double labeling procedure showed that NQO-1 can be
`produced by both microglial and astroglial cell types (Fig.
`6b, c, d, e).
`
`3.5. Cellular localization of NQO-1
`
`3.6. Effects of the compounds on glutathione levels
`
`We and others observed in LPS-activated mixed glial
`cells, that pro-inflammatory mediators are mainly produced
`by microglial cells (Vincent et al., 1996, 1997; Nakamura et
`al., 1999; Ledeboer et al., 2000). As the production and
`
`In LPS activated glial cells, the GSx levels were 12.1
`Amoles/mg at 4 h and 16.1 Amoles/mg at 24 h. Co-
`incubation with DMF or SP for 4 h dramatically depleted
`GSx from the cells whereas after 24 h of co-incubation, both
`
`Sawai (IPR2019-00789), Ex. 1035, p. 008
`
`

`

`140
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`A. Wierinckx et al. / Journal of Neuroimmunology 166 (2005) 132 – 143
`
`Fig. 5. Effects of DMF and SP on NQO-1 activity and mRNA level in lipopolysaccharide (LPS)-treated mixed glial cells. Cells were treated with LPS (100 ng/
`ml) for 4 and 24 h. Intracellular NQO-1 activity was measured after 4 and 24 h, NQO-1 mRNA was measured after 4 h. a, effect of DMF on NQO-1 activity, b,
`effect of SP on NQO-1 activity, c, effect of DMF on NQO-1 mRNA level, d, effect of SP on NQO-1 mRNA level. Data are expressed as percentage of levels
`T
`measured in cultures co-incubated with LPS and solvent (control), and represent mean
`S.E.M. of 2 – 3 independent experiments (n = 6 – 12). *P < 0.01 vs.
`control.
`
`compounds significantly increased the level of GSx in the
`cells (Fig. 7).
`
`4. Discussion
`
`Microglia and astrocytes are important sources of
`inflammatory mediators under neuropathological conditions
`(Hirsch et al., 2003, 1998; Liu and Hong, 2003; McGeer and
`McGeer, 1998) as well as under activated conditions in vitro
`(Gadient and Otten, 1997; Lee et al., 1993; Vincent et al.,
`1996). We have previously shown that interactions between
`activated glial cells determine the level of inflammatory
`mediators produced (Vincent et al., 1997, 1996). Therefore,
`a co-culture of astrocytes and microglia represents a relevant
`model to study neuro-inflammatory responses in vitro.
`In the present study, we observed for the first time that
`DMF and SP exert a dual action on brain-derived cells
`within the concentration range and timeframe measured, i.e.
`they reduce cytokine levels and, enhance NQO-1 mRNA
`
`and activity levels, as well as increase the cellular
`glutathione content of activated mixed glial cells.
`At first it was shown that LPS induced time-dependent
`mRNA production and subsequent release of IL-1h, IL-6,
`TNFa and NO. TNFa was produced and released within 1 h
`after LPS treatment, followed by IL-1h, IL-6 and finally
`NO. This sequence of cytokine and NO production fits
`nicely with previous in vitro observations (Nakamura et al.,
`1999), and allowed for the establishment of the optimal
`time-points to measure the above mentioned mediators after
`LPS treatment. Subsequently,
`the effects of
`the anti-
`inflammatory compounds DMF and SP were studied on
`mRNA production (at 4 h) and release of cytokines (4 and
`24 h) and NO (24 h). Co-incubation of mixed glial cells with
`LPS and either DMF or SP reduced cytokine and iNOS
`mRNA production as well as release of their concomitant
`proteins. DMF exerted the strongest effect on IL-6 followed
`by NO, TNFa and finally IL-1h production and release. SP
`reduced production and release of mentioned mediators in
`the same order, but mRNA levels were slightly less affected
`
`Sawai (IPR2019-00789), Ex. 1035, p. 009
`
`

`

`A. Wierinckx et al. / Journal of Neuroimmunology 166 (2005) 132 – 143
`
`141
`
`produced by both astrocytes and microglial cells in vitro is
`supported by the observation that glial cells in the brain are
`the main source of NQO-1 (van Muiswinkel et al., 2004). In
`addition, NQO-1 has been shown to be induced by DMF
`and SP demonstrating the potential value of these com-
`pounds as protective agents against chemical carcinogenesis
`and other forms of electrophile toxicity (Begleiter et al.,
`2001; Brooks et al., 2001; Cao et al., 2003; Kim et al., 2003;
`Kwak et al., 2001, 2003; Nelson et al., 1999; Otieno et al.,
`2000; Prochaska and Fernandes, 1993; Spencer et al., 1990).
`Our study now shows that the cellular glutathione content,
`depleted by DMF and SP at 4 h (Kim et al., 2003; Nelson et
`al., 1999), is significantly increased by both compounds
`after 24 h of treatment. Glutathione, produced by astrocytes
`(Dringen and Hirrlinger, 2003; Cooper and Kristal, 1997;
`Langeveld et al., 1996), plays a prominent role in protecting
`neurons from free radical-induced damage (Pompella et al.,
`2003). Indeed, because NQO-1 and glutathione are well
`known to protect numerous cells against redox cycling,
`oxidative stress and neoplasia (Jaiswal, 2000; Joseph et al.,
`1994), the strong induction of NQO-1 mRNA and activity,
`as well as the increase in glutathione content,
`in LPS-
`stimulated co-cultures of astrocytes and microglia by DMF
`and SP suggest they could constitute powerful anti-oxidant
`effe