Immunostimulatory DNA activates production of type I interferons and interleukin-6 in equine peripheral blood mononuclear cells in vitro

Veterinary Immunology and Immunopathology 107 (2005) 265–279 www.elsevier.com/locate/vetimm

Immunostimulatory DNA activates production of type I interferons and interleukin-6 in equine peripheral blood mononuclear cells in vitro Eva Wattrang *, Mikael Berg 1, Mattias Magnusson 2 Section of Veterinary Immunology and Virology, Department of Molecular Biosciences, Swedish University of Agricultural Sciences, SE-75123 Uppsala, Sweden Received 7 February 2005; received in revised form 15 April 2005; accepted 10 May 2005

Abstract This study aimed to evaluate different nucleic acid preparations as cytokine inducers in equine cells. To induce cytokine production, bacterial plasmid DNA or short synthetic oligodeoxyribonucleotides (ODN), with or without the transfection reagent lipofectin, were added to cultures of purified equine peripheral blood mononuclear cells (PBMC). Cytokine activity was detected with bioassays in cell culture supernatants after 24 h of induction and cytokine mRNA expression was detected using RT-PCR at 6 h post induction. For IFN-a/b it was found that both plasmid DNA and phosphodiester ODN, containing an unmethylated CpG-motif, were able to induce IFN production in the presence of lipofectin but not without. The levels of IFN varied with individuals and were often quite low. Moreover, methylation or removal of the CpG sequence completely abolished IFN induction. CpG-containing ODN with poly-guanine (G) sequences in the 50 and 30 ends induced considerably higher levels of IFN, especially when the polyG sequences had a phosphorothioate backbone. ODN with poly-G sequences also had the ability to induce IFN in the absence of lipofectin but the levels of IFN induced were radically reduced compared to those induced with lipofectin. In contrast to IFN, which was only detected upon induction, low spontaneous IL-6 production was observed in unstimulated control cultures. Nevertheless, plasmid DNA and CpG-containing ODN were able to increase the IL-6 production threefold. All the IFN inducing ODN also induced IL-6 production and the levels of IL-6 induced seemed influenced by addition of lipofectin and presence of poly-G sequences in the same way as was observed for the IFN-production. However, a complete phosphorothioate ODN with a central CpG-motif and poly-C sequences, that did not induce IFN, readily induced IL-6 both in the presence and absence of lipofectin. In addition, there was also evidence that some ODN induced increased expression of IL-12p40 mRNA. To conclude, equine PBMC were able to recognize CpG-DNA and respond with both IFN-a/b and/or IL-6 production. The levels of cytokine

* Corresponding author. Present address: Department of Parasitology, National Veterinary Institute, SE-75189 Uppsala, Sweden Tel.: +46 18 674034; fax: +46 18 674304. E-mail address: [email protected] (E. Wattrang). 1 Present address: Section of Parasitology and Virology, Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, SE-75007 Uppsala, Sweden. 2 Present address: Department of Rheumatology and Inflammation Research, The Sahlgrenska Academy, Go¨teborg University, SE-413 46 Gothenburg, Sweden. 0165-2427/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2005.05.001

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induced, and sometimes which cytokine induced, varied with, e.g., CpG-motifs used, the presence of poly-G sequences, ODN backbone chemistry and presence of lipofectin. # 2005 Elsevier B.V. All rights reserved. Keywords: CpG-DNA; Equine; IFN; IL-6

1. Introduction It is, by now well known that different forms of nucleic acid may be potent inducers of immune responses. Indeed, the identification of foreign nucleic acid is considered a part of the innate immune defence against invading microorganisms and this recognition is thought to be based on certain types of nucleotide sequences especially prevalent in microbial genomes (Krieg, 2002). Among such sequences, DNA containing unmethylated CpG dinucleotides is by far the most studied type of immunostimulatory DNA and has been shown to exert a variety of effects on the immune system. For instance, CpG-DNA induces B-cell proliferation, NK-cell activation and production of cytokines that e.g., potentiate antigen presentation and favours the development of Th1-cells (Klinman, 2004; Krieg, 2002). However, the optimal CpG-motif, i.e., a CpG dinucleotide plus one or two flanking bases at it’s 50 and 30 -side, seem to differ both for the induced immune function (Krieg, 2002) and between species studied (Bauer et al., 2001; Rankin et al., 2001; Takeshita et al., 2001). Although the stimulatory motifs may vary, the ability to recognize and react to CpG-DNA has, apart from in man and mouse, also been described in a number of other mammals, e.g., pigs, cattle, sheep, horses, dogs, cats and rabbits (Domeika et al., 2004; Magnusson et al., 2001a; Rankin et al., 2001), birds (He et al., 2003) and fish (Jorgensen et al., 2003; Lee et al., 2003). Beside studies of the natural involvement of immunostimulatory DNA in immunity to infections, the possible use of CpG-DNA in prophylaxis and therapeutics has also been exploited. For instance in DNA vaccination, it is now believed that CpG-motifs in the plasmids used to introduce the genes of protective antigens in the vaccinate are indeed responsible for the Th1-adjuvant effects observed with this procedure (Tighe et al., 1998). Moreover, short synthetic oligodeoxyribonucleotides (ODN) have been introduced in vaccine formulations to make

use of their adjuvant effects and several beneficial effects have been observed (Klinman, 2004). In veterinary species, enhanced antibody responses and evidence of stronger Th1 induction have for example been monitored in sheep and cattle (Ioannou et al., 2002; Mena et al., 2002; Zhang et al., 2003). Apart from vaccination, ODN have also been used as nonspecific immune stimulants both prophylactic, inducing increased resistance to viral and bacterial infections as well as to non-infectious inflammatory bowel disease (Jorgensen et al., 2003; Obermeier et al., 2003; Ray and Krieg, 2003) and post-exposure, inducing improved recovery from retroviral infection (Olbrich et al., 2002). The effects of ODN preparations are dependent on a number of factors. First, as mentioned above the nucleotide sequence may determine species specificity and type of activation and in this context both the number and localization of CpG motifs (Klinman and Currie, 2003), as well as 30 sequence modifications such as addition of polyguanosine (poly-G) sequences (Dalpke et al., 2002; Zimmermann et al., 2003) may alter the biological activity of ODN (Krieg, 2002). Moreover, the type of nucleotide backbone chemistry may affect the activating capacity of an ODN where ODN with a phosphorothioate backbone generally are considered more active than the natural phosphodiester form. This effect is probably due to phosphorothioate ODN being more resistant to nucleases (Agrawal et al., 1995) and some observed negative side effects with this type of ODN, e.g., lymphadenopathy (Lipford et al., 2000), are most likely due to their persistence in tissues. High doses of intravenous phosphorothioate ODN may also induce acute toxicities through complement activation (Henry et al., 2002). Regarding the horse, lymphocyte proliferation in response to CpG-containing ODN has been demonstrated (Rankin et al., 2001). Furthermore, horses have been shown to respond relatively well to DNA vaccination (Fischer et al., 2003; Giese et al., 2002; Lopez et al., 2003; Soboll et al., 2003) which may be

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due to recognition of CpG-motifs. Many of the effects exerted by CpG-DNA have in other species been shown to depend on its ability to induce production of different cytokines. For instance, type I interferons (IFN), IFN-g, interleukin (IL)-1, IL-6, IL-10, IL-12 and IL-18 can all be induced by CpG-DNA (Krieg, 2002). Indeed, the induction of type I IFN is considered of special importance for the adjuvant effect of immunostimulatory DNA (Cho et al., 2002; Van Uden et al., 2001) which may include cross-priming of CTL (Beignon et al., 2003). In order to efficiently exploit the use of ODN to improve adjuvants in equine vaccines, the effects of ODN on the equine immune system need to be further characterized, for instance with respect to cytokine induction. The present study aimed to evaluate induction of cytokine production, in particular type I IFN, by immunostimulating DNA in equine cells. Therefore, the bacterial plasmid pcDNA3 and fourteen different synthetic ODNs were assessed for capacity to induce IFN and IL-6 production in purified equine PBMC in vitro. The detection and quantification of IFN and IL-6 biological activity was combined with detection of IFN-a, IFN-b, IL-6, IL-12p35 and IL-12p40 mRNA expression.

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2. Material and methods 2.1. Animals, blood sampling and PBMC purification Thirteen, clinically healthy horses, 2–24 years of age, were used in the present study (approved by the Ethical Committee for Animal Experiments, Uppsala, Sweden). The horses comprised 8 mares and 5 geldings of the following breeds: 6 standardbred trotters, 3 Swedish warmbloods, 1 Swedish draft, 1 Swedish warmblood—draft cross, 1 Connemara pony and 1 Thoroughbred. Blood samples were collected from the jugular vein into evacuated glass tubes with 143 USP heparin (BD Vacutainer, Meylan, France). PBMC were purified from heparinized leukocyte rich plasma by centrifugation on Ficoll-PaqueTM PLUS (Amersham Biosciences AB, Uppsala, Sweden), washed in phosphate buffered saline and re-suspended in RPMI 1640 (Bio Whittaker Europe, Verviers, Belgium) with 20 mM HEPES buffer, 2 mM L-glutamine, 200 IU penicillin/ml, 100 mg streptomycin/ml and 5  105 M 2-mercaptoethanol, i.e., growth medium, supplemented with 10% foetal calf serum (FCS; Myoclone, Gibco Paisley, UK).

Table 1 Nucleotide sequences and concentrations of ODNs used to induce cytokine production in equine PBMC and the number of horses responding with IFN production (IFN+) out of the number of horses tested with each ODN in the absence (lipo) or presence (+lipo) of lipofectamine ODN H I HI HA IT HA I T Hmet Imet HmetImet 2216 2243 2216pd 2243pd MM1 D19 D25 E

Sequence 50 to 30 1,2

TTT TCA ATT CGA AGA TGA AT ATT CAT CTT CGA ATT GAA AA2 Double strand of H and I TTT TCA ATT CAA AGA TGA AT2 ATT CAT CTT CTA ATT GAA AA2 Double strand of HA and IT TTT TCA ATT Me5CGA AGA TGA AT2 ATT CAT CTT Me5CGA ATT GAA AA2 Double strand of Hmet and Imet ggG GGA CGA TCG TCg ggg gG3,4 ggG GGA GCA TGC TCg ggg gG3,4 GGG GGA CGA TCG TCG GGG GG5 GGG GGA GCA TGC TCG GGG GG ggG GTC ATC GAT GAg ggg gG5 ggT GCA TCG ATG CAG ggg gg6 GGT GCA TCG ATG CAG GGG GG6,7 ccc ccc ccc ccc aac gtt ccc ccc ccc ccc8

Concentration (mg/ml)

IFN+/tested  lipo

IFN+/tested + lipo

25 25 25 25 25 25 25 25 25 3 3 3 3 3 3 3 5

0/5 0/5 0/7 nt nt nt nt nt nt 9/13 0/11 3/9 nt 7/10 3/8 5/8 0/7

11/13 12/13 11/13 0/7 0/7 0/7 0/7 0/7 0/7 10/10 6/8 5/5 0/7 7/7 5/5 7/7 0/7

Phosphodiester nucleotides are indicated in upper case letters and phosphorothioate nucleotides in lower case. CpG motifs, their modifications and methylated cytosines (Me5) are underlined. Selected references for the ODNs are indicated in superscript numbers and are 1Sato et al. (1999), 2 Magnusson et al. (2001b), 3Krug et al. (2003), 4Jarrossay et al. (2001), 5Domeika et al. (2004), 6Kamstrup et al. (2001), 7Verthelyi et al. (2001), 8 own ODN complimentary to oligo-B in Sonehara et al. (1996). nt, not tested.

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2.2. Cytokine induction For cytokine induction, different types of ODN (Table 1), the bacterial plasmid pcDNA3 (Invitrogen, San Diego, CA, USA) and live Sendai virus were used. ODN E was purchased from KEBO Lab (Spa˚ nga, Sweden) all other ODN were purchased from Cybergene AB (Stockholm, Sweden) and all ODN were used in desalted form, dissolved in H2O. In ODN with methylated cytosines, 5-methylcytosine was used. For induction, most ODN were only used in single stranded form but when used in double stranded form, hybridization was performed according to Magnusson et al. (2001b). The final concentrations of each ODN in the PBMC cultures are indicated in Table 1. Choice of ODN concentrations was based on optimal IFN-inducing capacity based on serial dilutions with equine PBMC (data not shown). The plasmid pcDNA3 was used in endotoxin-free (<0.05 EU/ml medium in the induction cultures) preparations as described by Magnusson et al. (2001a), at a final concentration of 0.5 mg/ml culture medium. Sendai virus was propagated in embryonated hens’ eggs and used as chorioallantoic fluid diluted in growth medium at a final dilution of 1:1000. ODN and pcDNA3 were used with and without pretreatment with lipofectin (Life Technologies, Paisley, UK) as described by Magnusson et al. (2001a). In brief, lipofectin was incubated in FCS-free growth medium for 45 min at room temperature, thereafter the inducer was added and the mixture incubated for a further 15 min at room temperature before addition to PBMC cultures at a final concentration of 5 mg lipofectin/ml. Induction cultures for cytokine analysis, were set up in triplicate wells of flat bottomed 96-well plates (Nunc, Roskilde, Denmark) with 100 ml of PBMC suspension at 10  106 cells/ml and 100 ml of inducer preparation giving a final concentration of 5  106 PBMC/ml. Plates were incubated at 378 C, 7% CO2 in a humid atmosphere for 24 h where after cell culture supernatants were collected and stored at 20 8C until cytokine analysis. Induction cultures for RT-PCR analysis were set up according to the same principle using 500 ml aliquotes of PBMC and inducer preparations, respectively, in 24-well plates (Nunc). After 6 h incubation at 37 8C PBMC were harvested in TRIzol (Life Technologies; see below) and cell culture

supernatants collected. Identical induction cultures were incubated for a full 24 h and supernatants collected as described above. 2.3. Cytokine bioassays IFN-activity was detected by inhibition of viral cytopathic effect in a bioassay using Madin-Darby bovine kidney cells and vesicular stomatitis virus as previously described for equine samples (JensenWaern et al., 1998; Wattrang et al., 2003). Induction culture supernatants were titrated by 2-fold dilutions from 1:4 in this assay and the IFN content in each sample was calculated by defining 1 unit IFN as the amount protecting 50 % of the cells in one well from lysis. Laboratory standards of equine leukocyte IFN (Jensen-Waern et al., 1998) and human IFN-a were included on every test plate to calibrate the assay. IL-6 activity was detected with the murine IL-6 dependent cell-line B9 essentially as earlier described for equine samples (Wattrang et al., 2003) but with some modifications. In brief, washed B9 cells (kindly donated by Dr. K. van Reeth, Ghent University, Belgium) were plated in 100 ml volumes at a concentration of 5  104 cells/ml growth medium in flat bottomed 96-well plates (Nunc). Induction culture supernatants were titrated in 2-fold dilutions between 1:8 and 1:640 and serial dilutions of human IL-6 (4– 0.06 U/ml; Roche Diagnostics GmbH, Mannheim, Germany) were included as standard in every assay. Test plates were incubated for 96 h at 37 8C where after B9 viability was determined by reduction of tetrazolium salts into formazan derivates with the EZ4U kit (Biomedica GmbH, Vienna, Austria) according to the manufacturers’ instructions. The IL-6 content in the samples was calculated by comparison with the standard curve. To verify IL-6 as the inducer of B9 proliferation, a monoclonal rat anti-mouse IL-6 receptor a chain (CD 126) antibody (anti-IL-6R; D7715A7, BD PharMingen, Erembodegem, Belgium) was used to inhibit proliferation as described earlier (Wattrang et al., 2003). 2.4. RNA extraction, cDNA synthesis and RT-PCR RNA was isolated from PBMC in 24-well induction cultures after 6 h of incubation as described above. Both non-adherent and adherent cells in each well

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(approximately 5  106) were lysed in a total volume of 1 ml TRizol and stored at 80 8C until RNA isolation. RNA isolation was performed by phenol/ chloroform extraction and isopropyl alcohol precipitation according to the TRIzol manufacturers’ protocol. The isolated RNA was dissolved in DEPC-treated H2O and complementary DNA (cDNA) was synthesized using SuperScriptTM II RNase H-reverse transcriptase (Invitrogen) according to the manufacturers’ protocol. For this reaction a novel oligo (dT) primer was designed (50 -CCT GAC CCA ACC AGT AGA CCA TTT TTT TTT TTT TTT TTT TN-30 ), introducing 21 new bases at the end of the synthesized cDNA. To avoid amplification of contaminating genomic DNA, a primer with bases identical to the 21 inserted bases was used for amplification of cDNA in the first RT-PCR reaction. In this reaction, 0.4 mM of each specific forward primer (Table 2) and 1.2 mM of a backward primer for the new bases (50 -TGA CCC AAC CAG TAG ACC A-30 ) were added together with 2 ml of cDNA template in a 15 ml total volume containing 2.0 mM MgCl2, 0.25 mM dNTP, PCRbuffer and 0.3 ml Taq polymerase (MBI Fermetas, Lithuania). The PCR comprised 35 cycles of 30 s

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denaturation at 95 8C, 30 s of annealing at 60 8C and 90 s of elongation at 72 8C and a final 8 min of elongation at 72 8C. The products from this reaction were then used as templates in separate nested PCR reactions for each product tested for. In the nested PCR, 0.5 ml template was added to 0.4 mM of specific nested forward and backward primer, respectively (Table 2) in a 25 ml total volume containing 25 mM MgCl2, 0.25 mM dNTP, PCR-buffer and 0.5 ml Taq polymerase (MBI Fermentas). Denaturation was 30 s at 95 8C, and elongation was 30 s at 72 8C in each cycle with a final 8 min of elongation at 72 8C, the number of cycles and annealing temperatures for each specific nested PCR reaction are indicated in Table 2. Messenger RNA expression of all cytokines tested for was analysed with the nested PCR procedure while bactin expression was analysed using a single round of PCR only. The PCR primers were designed from nucleic acid sequences available in GenBank/EMBL data bases and, with the exception for IFN-a/b, chosen to give products spanning at least one intron when aligned to the corresponding human gene sequences. For the four equine IFN-a subtypes sequenced (Himmler et al., 1986), primers were designed to

Table 2 RT-PCR primers for equine cytokines IFN-a, IFN-b, IL-6, IL-12p40, IL-12p35 and equine b-actin Primer

Sequence 50 to 30

Annealing temperature (8C)

IFN-a F IFN-a nestF IFN-a nestB

CAA CAC AAG GGT CTT GAT GCT C CCA TGA GAC GAT CCA ACA GAT C CAT GAT TTC TGC TCT GAC CAT C

60 60 60

IFN-b F IFN-b nestF IFN-b nestB

GTG AAC TAT GAC TTG CTT CGG CGA GGA CAC AAT GAA CTT CC CTC CTC CAT TAT TTC CTC CAG

60 65 65

IL-6 F IL-6 nestF IL-6 nestB

TGG TGA TGG CTA CTG CTT TC AGC AAG TGT GAA AAC AGC AAG GGT ACT GAT CTG CTT AGT CT

60 60 60

IL-12p40 F IL-12p40 nestF IL-12p40 nestB

GTT GTA GAA TTG GAT TGG TAC GAC CTT TCT AAA ATG TGA GGC CTT ATA CTC CCT GTC GTC CAC

60 60 60

IL-12p35 F IL-12p35 nestF IL-12p35 nestB

AAT GTT CCA GTG CCT CAA CC TCA CAA AAG ACA AGA GCA GC TCA TGG CCT TGA ACT CCA C

60 55 55

15

240

b-actin F b-actin B

GAC ATG GAG AAG ATC TGG CA ATG TCA CGC ACG ATT TCC CT

60 60

30

389

F, forward primer; B, backward primer; nest, nested primer.

No. of cycles in final PCR

15

Product length in final PCR (bp)

277

GenBank no.

A33683 A33697 A33699, A33687 A33685

10

10

229

215

ECU64794 AF041975 AF005227 Y11129

15

187 Y11130

AF035774

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react with all four sequences. The PCR products were separated by electrophoresis on 2 % agarose gels containing ethidium bromide and visualized with UV-light.

3. Results 3.1. IFN induction by immunostimulatory DNA The different nucleic acid preparations were tested for ability to induce IFN production in cultures of purified equine PBMC (Table 1, Fig. 1). Because a large individual variation in the levels of IFN produced was observed for all inducers (e.g., between 16 and 512 U/ml for Sendai virus, n =13), and the horses showed the same pattern as ‘‘high’’ or ‘‘low’’ responders irrespective of inducer, IFN data was expressed as percentage of the IFN activity induced by

the Sendai virus control. All inducers were tested with PBMC from at least five different donors and care was taken to test all ODN with PBMC obtained from several ‘‘high responders’’. No IFN activity was detected in medium or lipofectin control cultures of PBMC from any of the horses. To test if bacterial plasmid DNA had the ability to induce IFN production in equine PBMC, the plasmid pcDNA3 was used with and without pre-treatment with lipofectin (Fig. 1). Indeed, in combination with lipofectin pcDNA3 induced IFN production in PBMC from all but one of 13 tested horses, while the plasmid alone did not induce any IFN activity (n = 6). To facilitate studies of the role of nucleotide sequence in the induction of IFN by DNA, short synthetic ODN were evaluated as IFN inducers (Table 1, Fig. 1). Phosphodiester ODN H and ODN I, each containing one central unmethylated CpGmotif, are complementary to each other and were used

Fig. 1. IFN activity expressed as percentage of Sendai virus control in cell culture supernatants from equine PBMC cultured for 24 h in the presence of the indicated inducers, with or without lipofectin (lipo). Data are presented as mean values with 95% confidence intervals, so that non-overlapping confidence intervals indicate statistically significant differences. The number of horses tested with each inducer is 5 (exact numbers are given in Section 3.1 and Table 1).

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both in single stranded (ss) and double stranded (ds) form. Single stranded ODN H and ODN I as well as ds ODN HI induced IFN production in PBMC from most donors when used with lipofectin, while these ODN without pre-incubation with lipofectin did not induce any IFN activity. To study the role of the unmethylated CpG-motif in this induction the CpG-motifs of ODN H and ODN I were altered, either by substitution of the C to an A in ODN H (ODN HA) or of the G to a T in ODN I (ODN IT) or by methylation of the cytosine (ODN Hmet and ODN Imet). These modified ODN were used in combination with lipofectin. All these modifications abolished the IFN induction irrespective whether the ODN were used in ss or ds form (Table 1, Fig. 1). Compared to the Sendai virus control, the IFN responses induced with pcDNA3 or ODNs H and I in combination with lipofectin were relatively low (for pcDNA3 approximately 30%, and for ss ODN H, ss ODN I and ds ODN HI approximately 10% of the Sendai virus induced responses). The one horse not responding to lipofected pcDNA3 nor to lipofected H or I was an overall ‘‘low responder’’, only producing 16 U IFN/ml in response to Sendai virus compared to the mean Sendai virus induced response of 157  38 U IFN/ml (1 S.E., n = 13). Thus, both plasmid DNA and CpG containing phosphodiester ODN were able to induce IFN production in equine PBMC but only in the presence of lipofectin and an unmethylated CpG-motif seemed necessary for the induction. To test if a more efficient IFN induction in equine PBMC could be achieved, possibly also in the absence of lipofectin, ODNs with other and/or increasing numbers of CpG-motifs, sequence alterations such as poly-G sequences and altered nucleotide backbone chemistry were used, e.g., chimeric ODN with phosphorothioate poly-G sequences with 3 (ODN 2216) or 1 (ODN MM1 and ODN D19) CpG-motifs and a complete phosphorothioate ODN with poly-C sequences and 1 CpG-motif (ODN E; Table 1, Fig. 1). In combination with lipofectin, ODN 2216, ODN MM1 and ODN D19 induced IFN production in PBMC from all tested horses and the IFN responses induced were as high as, or for some horses higher than, those induced with the Sendai virus control. Interestingly, these particular ODN were also in some cases able to induce IFN production without

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lipofectin but, the levels of IFN induced by ODN 2216, ODN MM1 and ODN D19 alone were considerably lower than those induced by the corresponding ODN in combination with lipofectin. To address the importance of CpG-motifs in the IFN induction by ODN 2216, 2 of its 3 CpG-motifs were inverted to GpC, i.e., ODN 2243. ODN 2243 did not have the ability to induce IFN without lipofectin but in PBMC from some individuals, ODN 2243 in combination with lipofectin induced IFN albeit the levels of IFN produced were considerably lower than those induced by ODN 2216. To evaluate the role of the phosphorothioate backbone of the poly-G sequences for the IFN induction, ODN 2216, ODN 2243 and ODN D19 were used as complete phosphodiester ODNs, i.e., ODN 2216pd, ODN 2243pd and ODN D25 (Table 1, Fig. 1). Indeed, ODN 2216pd and ODN D25 induced IFN production without lipofectin in PBMC from some horses while ODN 2243pd did not induce IFN production. Moreover, the complete phosphorothioate ODN E failed to induce IFN production either in combination with lipofection or without. Thus, differences in CpG-motifs and/or other sequence alterations seemed important for the levels of IFN induced but all CpG-containing ODN did not induce IFN production. Furthermore, induction of IFN in the absence of lipofectin was possible and poly-G sequences in phosphorothioate form seemed beneficial but not essential for this capacity. 3.2. IL-6 induction by immunostimulatory DNA Some of the nucleic acid preparations were also tested for their ability to induce IL-6 production in cultures of equine PBMC (n = 6; Fig. 2). A background of IL-6 activity (mean 39  20 U IL-6/ml) was observed in the medium controls of PBMC from all the tested horses. Therefore, to reduce the individual and possible inter-assay variation, IL-6 data were expressed as percent of the medium control. To test if nucleic acid in the form of plasmid or phosphodiester ODN could alter the IL-6 production of cultured equine PBMC, pcDNA3 and ssODN H and I in combination with lipofectin were used (Fig. 2). All these nucleic acid preparations induced an average 2fold increase in IL-6 activity compared to the medium control while lipofectin alone did not induce any

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Fig. 2. IL-6 activity expressed as percentage of medium controls in cell culture supernatants from equine PBMC (n = 6) cultured for 24 h in the presence of the indicated inducers, with or without lipofectin (lipo). Data are presented as mean values with 95% confidence intervals, so that non-overlapping confidence intervals indicate statistically significant differences.

alteration in IL-6 activity. To assess the effects of nucleotide sequence and backbone chemistry on the IL-6 production a different ODNs were used (Fig. 2). ODN 2216, ODN 2216pd and ODN MM1 induced on average 6- to 8-fold increases in IL-6 activity when used in combination with lipofectin and about half of this when used without. ODN D19 and ODN D25 induced 10- to 15-fold increases in IL-6 activity when used in combination with lipofectin, while they seemed to have more limited effects when used alone. ODN 2243 seemed to have little effect on IL-6 production either with or without lipofectin. Interestingly, phosphorothioate ODN E, that did not induce IFN production, induced an average 4-fold increase of IL-6 activity either with or without lipofectin. In order to verify that B9 proliferation was induced by IL-6 and not for instance by residual nucleic acid in the cell culture supernatants, an anti-IL-6R antibody was used to neutralize the effects of IL-6. A total of 12 samples, from two horses induced with ODN MM1, ODN D19 and ODN E with and without lipofectin,

with a range of 1838-33 U IL-6/ml in the bioassay were used. Using the anti-IL-6R antibody to neutralise the effects of the natural human IL-6 standard the most effective inhibition was achieved with 16-4 U IL-6 (72  2%, 73  6%, 72  1%, 63  1% and 37  3% inhibition by anti-IL-6R at 16, 8, 4, 2 and 1 U human IL-6/ml, respectively; mean  1S.D., n = 2). The cell culture supernatants were diluted to fit into this range, effectively containing approximately 3 U IL-6/ml (range 5.4-1.1 U IL-6/ml), and the inhibition achieved was 67  11% (mean  1S.D., n = 12). Thus, the neutralisation achieved with anti-IL-6R in the equine cell culture supernatants showed a similar pattern as that of natural human IL-6 and confirmed that B9 proliferation was induced by IL-6 in the samples. Taken together, CpG-DNA induced increased IL-6 production in cultured equine PBMC and it seemed that different CpG-motifs and other sequence alterations influenced the levels of IL-6 induced. Moreover, there was not a complete correlation between high IFN induction and high IL-6 induction.

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3.3. Induction of cytokine mRNA expression by immunostimlatory DNA In order to verify the origin of biological activity detected in the IFN and IL-6 bioassays, and obtain indications on the induction of IL-12, RT-PCR was used to detect IFN-a, IFN-b, IL-6, IL-12p35 and IL-2p40 mRNA expression in PBMC stimulated with different nucleic acid preparations. The mRNA expression was analyzed at 6 h of induction using PBMC from two horses, A and B (Fig. 3), and corresponding cell culture supernatants were tested for IFN and IL-6 activity at 6 and 24 h of induction.

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At 6 h of induction, low levels of IFN activity were only detected in supernatants from cultures induced with Sendai virus (16 U IFN/ml both horse A and B), ssODN H and I in combination with lipofectin (2 U IFN/ml horse B only), pcDNA3 in combination with lipofectin (4 U IFN/ml, horse B only) and ODN 2216 in combination with lipofectin (6 and 16 U IFN/ml horse A and B, respectively). At 24 h of induction the IFN responses in cell culture supernatants from both horses (Fig. 3) showed a similar pattern with the different inducers as the overall average of all tested horses described earlier (Fig. 1). The RT-PCR verified IFN mRNA expression in PBMC from cultures where 8 or more units of IFN activity were detected at 24 h of

Fig. 3. IFN-a, IFN-b, IL-6, IL-12p40, IL-12p35 and b-actin mRNA expression in equine PBMC cultured for 6 h in the presence of the indicated inducers, with or without lipofectin (lipo). Results from PBMC isolated from two horses are shown, in (A) a 9-year-old Thoroughbred gelding and in (B) a 14-year-old Swedish Warmblood mare. IFN and IL-6 activity in corresponding cultures at 24 h of culture are also given.

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culture (Fig. 3). The two tested horses produced similar levels of IFN in the Sendai virus control cultures and as expected this virus induced expression of both IFN-a and IFN-b. All the IFN inducing nucleic acid preparations appeared also to induce both IFN-a and IFN-b mRNA expression, although when only low levels of bioactive IFN was induced the expression seemed to be predominantly IFN-a. When testing for IL-6 activity in cell culture supernatants at 6 h of induction, approximately 25-30 U IL-6/ml was detected for both horses in all cultures regardless of inducer. At 24 h of culture however, the IL-6 activity in cell culture supernatants from both horses (Fig. 3) showed a similar pattern with the different inducers as the overall average of all tested horses described earlier (Fig. 1). The RT-PCR analysis confirmed the expression of IL-6 mRNA in all cultures although the levels of expression seemed not completely consistent with the levels of bioactivity. For IL-12, mRNA expression of both p35 and p40 was detected in all cultured PBMC (Fig. 3) but an increased expression of IL-12p40 was indicated in cultures where high levels of IFN were induced, e.g., those stimulated with ODN 2216 in combination with lipofectin. Interestingly, IL-12p35 mRNA seemed to be expressed in two isoforms.

4. Discussion The present study shows that immunostimulatory DNA, both in the form of bacterial plasmid and synthetic ODN, can induce both type I IFN and IL-6 production in equine cells. The induction appeared dependent on the presence of unmetylated CpG-motifs but the levels of cytokines induced seemed to vary with different CpG-motifs and/or other sequence alterations as well as the nucleic acid backbone chemistry. PBMC from most horses responded with IFN production when the plasmid pcDNA3 was used as inducer but only in combination with the transfection agent lipofectin. Likewise, ODN H and ODN I in ss or ds form induced IFN production in the majority of individuals in the presence of lipofectin. This is in analogy with human and porcine PBMC cultures were both pcDNA3 and ODN H and ODN I only induces IFN when combined with lipofectin (Domeika et al., 2004; Magnusson et al., 2001a,b; Vallin et al., 1999).

In equine cells, the use of modified ODN H and ODN I where the central CpG-motif was either substituted by other bases or altered by methylation of the cytosine, completely abolished IFN induction showing the importance of the unmethylated CpG-motif for this induction. Also in porcine PBMC, these alterations of ODN H and ODN I severely reduced IFN induction (Domeika et al., 2004). In contrast, with human PBMC only the substitution of bases in the CpG-motif used in ODN HA and ODN IT reduced IFN production while ssODN H and ODN I with GpC-motifs or ODN I with methylation of the cytosine retained some of their IFN inducing capacity (Magnusson et al., 2001b). Thus, there seem to be species differences in the requirement of an unmethylated CpG-motif for IFN induction by ODN H and ODN I. The importance of different CpG-motifs and other sequence and/or nucleic acid backbone modifications were studied using a number of ODN previously evaluated for type I IFN induction in other species. ODN 2216 has been proven a potent IFN-a/b inducer in human, murine and porcine plasmacytoid dendritic cells (PDC; Domeika et al., 2004; Hartmann et al., 2003; Jarrossay et al., 2001; Kerkmann et al., 2003; Krug et al., 2001; Okada et al., 2003; Rothenfusser et al., 2001) and in ovine PBMC (Mena et al., 2003; Nichani et al., 2004). ODN MM1, ODN D19 and ODN D25 induced IFN-a in porcine PBMC (Domeika et al., 2004) and ODN D19 also induced IFN-a in human PDC (Marshall et al., 2003). ODN E induced IFN in leukocytes isolated from murine bone marrow (M.-L. Eloranta, personal communication). On the whole, equine PBMC stimulated with ODN 2216, ODN MM1, ODN D19 and ODN D25 combined with lipofectin produced higher levels of IFN compared to those induced by ODN H and ODN I, which is in analogy with results observed with porcine cells (Domeika et al., 2004). Common for the former four inducers was the presence of poly-G sequences that, in all but ODN D25, consisted of phosphorothioate instead of phosphodiester nucleotides. Poly-G sequences may form quarternary structures that facilitate cellular uptake of ODN, possibly by binding to scavenger receptors (Bartz et al., 2004; Kimura et al., 1994; Lee et al., 2000; Pearson et al., 1993; Pisetsky, 1996). Moreover, the use of phosphorothioate nucleotides may also have contributed to the efficiency of these ODN since this backbone

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chemistry both renders the ODN more resistant to nuclease activity (Agrawal et al., 1995; Krieg, 2002) and may increase cellular uptake (Zhao et al., 1993). In addition, ODN 2216 contained three CpG-motifs while ODN MM1, D19 and D25 only contained one central CpG-motif. It, thus seemed that the additional CpG-motifs in ODN2216 had little effect on the levels of IFN induced compared to the other ODNs with a similar delineation. It has been shown that increased numbers and diversity of CpG-motifs were beneficial for the immune stimulatory capacity of ODN in human PBMC (Klinman and Currie, 2003), but in this case the CpG-motif of ODN MM1, ODN D19 and ODN D25 might simply have been better recognized than those of ODN 2216. However, when the number of CpG motifs in ODN 2216 were reduced to one, i.e., ODN 2243, it lost most of its IFN inducing capacity, showing that for this particular ODN additional CpGmotifs indeed conferred better IFN induction. Also in human cells ODN 2216 induced high levels of IFN-a while ODN 2243 failed to induce this cytokine (Krug et al., 2001). However, solely the presence of an unmethylated CpG was not sufficient for type I IFN induction in equine PBMC as no IFN-activity was observed after induction with ODN E which indicates the importance of the different CpG-motifs and further flanking sequences. By the use of RT-PCR as a complement to the IFNbioassay, the expression of both IFN-a and IFN-b mRNA after induction with plasmid DNA and ODN (ODN H, ODN I and ODN 2216) was demonstrated. The IFN-a mRNA expression seemed, however, to dominate as in some cases when low levels of IFN activity were induced, no IFN-b mRNA expression could be detected. This would be in analogy with results obtained with human PDC where ODN 2216 induced approximately 100-fold higher levels of IFNa than IFN-b (Krug et al., 2001). An enhanced cytokine inducing capacity was observed for the majority of nucelic acid preparations used in the present study when combined with lipofectin. Indeed, for IFN induction many of the inducers were highly dependent on lipofectin. The mechanisms behind the requirement of lipofectin for the in vitro induction of IFN have not been clarified but one may speculate that this cationic lipid facilitates cellular uptake (Bennett et al., 1992; Hartmann et al., 1998) of the inducer and/or protects it from

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degradation by nucleases (Thierry and Dritschilo, 1992). However, lipofectin might not be necessary if the inducers were administered to horses in vivo, since studies in pigs show that nucleic acid inducers requiring lipofectin for in vitro induction of IFN, i.e., poly I:C and pcDNA3 (Magnusson et al., 2001a), may induce IFN in vivo without this treatment (Johansson et al., 2002a; Wattrang et al., 1997). Moreover, IFN induction without lipofectin was also possible in the equine PBMC cultures when ODN 2216, ODN 2216pd, ODN MM1, ODN D19 and ODN D25 were used. The increased cellular uptake and/or protection from nucleases mediated by the poly-G sequences might thus have allowed sufficient amounts of these ODN to be recognized by the IFN producing cells even in the absence of lipofectin. The levels of IFN induced by ODNs without lipofectin were, however, much lower than those induced by the same ODN in the presence of lipofectin. This is contrary to results obtained with porcine PBMC, where the levels of IFN induced with ODN 2216, ODN 2216pd, ODN D19 and ODN D25 showed little or no difference when the ODNs were used with or without lipofectin (Domeika et al., 2004). On average the levels of IFN produced by equine PBMC in response to many of the nucleic acid inducers seemed rather low, for instance in comparison with the viral inducer. Also in comparison with results reported for human and porcine PBMC (Ba˚ ve et al., 2003; Domeika et al., 2004; Magnusson et al., 2001a; Magnusson et al., 2001b), the levels of IFN induced by the equine PBMC seemed low, albeit comparison of IFN bioactivity units between species may be misleading. Taken together it seems that the equine cells on the whole displayed rather low and/or different patterns of IFN responses to the nucleic acid inducers. If these differences are due to species differences in the recognition of CpG-motifs, as shown for other immune functions (Bauer et al., 2001; Rankin et al., 2001; Takeshita et al., 2001) or a general low IFN production in the horse as a species or even to the selection of individuals used, remains to be determined. The induction of IL-6 by nucleic acids was more difficult to interpret than the clear on–off induction of type I IFN since the mere culture of the PBMC triggered IL-6 production. It is well know in several species that cell culture procedures induce IL-6 production in PBMC, for instance shown at mRNA

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level with equine PBMC (Giguere and Prescott, 1999) or at protein level with human PBMC (Marshall et al., 2003). It was, however, clear that CpG-DNA could induce increased production of IL-6 in the cultured equine cells and in particular some of the ODN with poly-G sequences induced quite high levels of IL-6 activity. This type of ODN with poly-G sequences, referred to as ‘‘A-class’’ ODN, is often considered a poor inducer of IL-6 especially when compared to ‘‘B-class’’ ODN that readily activates IL-6 production (Verthelyi et al., 2001). Nevertheless, both ODN 2216 and ODN D19 have been shown to induce IL-6 production in human PBMC and/or pDC (Hartmann et al., 2003; Jarrossay et al., 2001; Marshall et al., 2003; Verthelyi et al., 2001; Vollmer et al., 2004) and in human cells both these ODN induce the IL-6 production in pDC or at least require the presence of pDC for IL-6 induction (Hartmann et al., 2003; Marshall et al., 2003). In murine splenocytes on the other hand, ODN 2216 induced very little IL-6 production (Vollmer et al., 2004). In the present study, cytokine induction was also monitored at mRNA level with a novel RT-PCR methodology particularly designed for detection mRNA expression of intronless genes such as the type I IFNs. In this respect, the method indeed proved very useful as a sensitive test for IFN-a/b mRNA detection eliminating the problems of contaminating genomic DNA. IFN-a/b mRNA expression was detected in cultures where as little as 8 U of antiviral activity was measured with the bioassay. That lower levels of IFN activity remained undetected at mRNA level is probably due to the induction of either more type I IFNs, e.g., IFN-v, and/or more IFN-a subtypes in the cell culture supernatants, that were detected in the bioassay but not in the PCR. The IFN-a PCR primers used were designed to recognise the four equine IFN-a subtypes described (Himmler et al., 1986) but it is likely that horses, like other mammals, have a large number of IFN-a subtypes and all of them might not be equally well detected in the PCR. Among the other genes tested for, IL-12p35 mRNA was, as expected (Giguere and Prescott, 1999; Ainsworth et al., 2003; Watford et al., 2003), constitutively expressed in the PBMC, and there was no indication of alterations in this expression induced by the immunostimulatory DNA. Interestingly, the PCR primers amplified two different length

fragments of IL-12p35 that probably reflected the expression of different isoforms of the gene. Expression different isoforms due to differential splicing of IL-12p35 transcripts have been described in human (Johansson et al., 2002b), murine (Babik et al., 1999) and porcine (Johansson et al., 2003) cells. For IL-12p40 mRNA a low grade expression was also detected in all of the samples from the cultured PBMC which is in analogy with earlier observations with cultured equine PBMC (Giguere and Prescott, 1999). An increased expression of IL-12p40 mRNA was, however, indicated in some of the cultures where also high levels of IFN activity were detected, e.g., ODN 2216 in combination with lipofectin. ODN 2216 has been shown to induce production of IL-12p40 protein by murine thymic DC but production of the IL12 heterodimer p70 was not detected (Okada et al., 2003). Futhermore, ODN 2216 did not induce IL12p70 in human PBMC or human pDC, respectively (Jarrossay et al., 2001; Rothenfusser et al., 2001). It has also been suggested that Toll like receptor ligands, such as CpG-DNA, alone are insufficient to induce production of IL-12 or IL-23 heterodimers and often induce expression of only IL-12p40 (Ho¨ lscher, 2004). With the current methodology it was, however, not possible to determine if the increased expression of IL-12p40 mRNA indicated in the present study resulted in production of IL-12 or IL-23 heterodimers or IL-12p40 homodimers. Taken together, the present study clearly shows that equine leukocytes are capable to respond with both type I IFN and IL-6 production upon stimulation with CpG-DNA. The presence of an unmethylated CpG-motif was necessary but not sufficient for this induction. Type of CpG-motif and other sequence modifications were important for the quality of cytokine responses and the equine cells also showed differences in cytokine responses compared to human, murine and porcine systems. Thus, species differences must be taken into consideration when the immune activating properties of a particular ODN is evaluated.

Acknowledgements This study was supported by grants from the Intervet research foundation (Sweden), The Swedish Horserace Totalizator Board and the Gunnar Philipsson scholar-

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ship. The authors wish to thank all the horse owners for their invaluable contributions, Lisbeth Fuxler for expert help with the IFN bioassay, Dr. David Morrison for statistical advice and professor Caroline Fossum for scientific support and discussions.

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