Viral 50-triphosphate RNA and non-CpG DNA aggravateautoimmunity and lupus nephritis via distinctTLR-independent immune responses

Ramanjaneyulu Allam1, Rahul D. Pawar1, Onkar P. Kulkarni1,

Veit Hornung2, Gunter Hartmann2, Stephan Segerer1, Shizuo Akira3,

Stefan Endres4 and Hans-Joachim Anders1

1 Medical Policlinic, University of Munich, Germany2 Division of Clinical Pharmacology, Department of Medicine, University of Bonn, Bonn,

Germany3 Department of Host Defense, Research Institute for Microbial Diseases, Osaka University,

Osaka, Japan4 Department of Clinical Pharmacology, University of Munich, Munich, Germany

Certain viral nucleic acids aggravate autoimmunity through nucleic acid-specific TLR.

Viral 50-triphosphate RNA (3P-RNA) and double-stranded non-CpG DNA induce antiviral

immunity via TLR-independent pathways but their role in autoimmunity is unknown.

Transient exposure of 16-wk-old MRLlpr/lpr mice to 3P-RNA aggravated lupus nephritis by

increasing IFN signaling and decreasing CD41CD251 T cells. By contrast, transient

exposure to non-CpG DNA exacerbate lupus nephritis in association with splenomegaly,

lymphoproliferation, hypergammaglobulinaemia and increased B2201CD1381 plasma

cells. Both, 3P-RNA and non-CpG DNA increased glomerular complement factor C3c

deposits but both nucleic acid formats were less potent in aggravating renal pathology as

compared with CpG DNA. 3P-RNA and non-CpG DNA also localized to the glomerular

mesangial cells and activated cultured mesangial cells to produce IL-6. We conclude,

3P-RNA or non-CpG DNA both trigger autoimmune disease in MRLlpr/lpr mice by specifi-

cally activating adaptive immunity but similarly enhance inflammation on the tissue level.

Key words: Autoimmunity . Innate immunity . Lupus . Lupus nephritis . TLR

Introduction

Viral infection can aggravate disease activity of autoimmune

diseases like systemic lupus erythematosus (SLE) but the

molecular mechanisms remain elusive. Antiviral immune

responses might drive autoimmune tissue injury via type I IFN,

Th1 cytokines or CD81 T cells as these mediators regulate both

conditions [1, 2]. Antiviral immunity is triggered by conserved

viral nucleic acid formats via two classes of innate recognition

receptors. These are the nucleic acid-specific TLR, i.e. TLR3,

TLR7, TLR8 and TLR9, which detect viral RNA (TLR3, TLR7,

TLR8) or viral CpG DNA (TLR9) in intracellular endosomes

of antigen-presenting cells [3–7]. Signaling via these nucleic

acid-specific TLR involves the intracellular adaptor molecules

myeloid differentiation primary-response protein (MyD)-88

(TLR7, TLR8, TLR9) or translation initiation region domain-

containing adaptor protein-inducing-IFN beta TRIF (TLR3)

and subsequent activation of IFN-regulatory factors (IRF)

and NF-kB [3]. The second class of viral recognition

receptors includes the retinoic-acid-inducible gene (RIG)-I, and

melanoma-differentiation-associated gene (MDA)-5 that detect

viral RNA in the intracellular cytosol and induce type 1 IFN viaCorrespondence: Dr. H.-J. Anderse-mail: hjanders@med.uni-muenchen.de

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Eur. J. Immunol. 2008. 38: 3487–3498 DOI 10.1002/eji.200838604 Innate immunity 3487

the mitochondrial IFN-b promoter stimulator (IPS)-1. These

helicases have been identified to recognize viral dsRNA [8] and

single-stranded 50-triphosphate (3P)-RNA in the cytosol, respec-

tively [9, 10]. In addition, recently identified cytosolic DNA

receptor DAI and yet unknown cytosolic DNA receptors must

exist to mediate the immunostimulatory effects of viral dsDNA

[11–14]. TLR7, TLR8 and TLR9 are only expressed in a narrow

subset of immune cells, while almost all nucleated cells can

activate antiviral responses upon viral infection [15]. In fact,

TLR3 and TLR-independent recognition of viral RNA or dsDNA is

a potent inducer of type I IFN and proinflammatory cytokines in

non-immune cells [12, 16–19].

While the role of cytosolic and TLR-independent nucleic acid

recognition for antiviral immunity becomes increasingly recog-

nized, its potential role in activating disease activity of auto-

immunity and autoimmune tissue injury, similar to what has

been documented for the nucleic acid-specific TLR, remains

unknown [20–22]. We therefore hypothesized that exposure to

viral 3P-RNA or non-CpG DNA would modulate autoimmune

tissue injury in vivo and we questioned whether this would be

mediated via similar or different types of immune responses.

Results

3P-RNA and non-CpG DNA induce IL-6 independent ofMyd88 in vivo

Innate RNA and DNA recognition can involve TLR-dependent or

independent pathways [3]. Triphosphate RNA(3P-RNA) and non-

CpG DNA have both been reported to trigger cytokine release via

TLR-independent pathways in cultured DC [9, 11]. We replicated

the same results from cultured Myd88�/� and TLR9�/� BMDC

(data not shown). To extend these results in vivo we compared

serum IL-6 levels before and 6 h after a single injection of 3P-RNA

or non-CpG DNA to Myd88-deficient and wild-type mice. We

found that Myd88 was not required to induce IL-6 with either of

these two ligands in vivo (Fig. 1A).

Non-CpG DNA induces serum cytokines in nephriticMRLlpr/lpr mice

In order to test the effects of transient exposure to 3P-RNA and

non-CpG DNA in mice with pre-existing autoimmune disease we

repeatedly injected 16-wk-old nephritic MRLlpr/lpr mice with

3P-RNA and non-CpG DNA. Pilot studies with different doses of

3P-RNA and non-CpG DNA revealed that a single dose of either

20mg 3P-RNA or 50mg non-CpG DNA, but not higher doses,

potently increased IL-6 or CCL2 serum levels in 16-wk-old

MRLlpr/lpr mice (Fig. 1B). As this response was not dose-dependent

we assumed the doses tested to be at the plateau-level of the dose

response. Based on these data we injected 16-wk-old MRLlpr/lpr

mice with either 5% glucose, 20mg of 3P-RNA, 50mg of non-CpG

DNA or 40mg CpG DNA for 2 wk. Serum cytokine levels were

determined 3 h after the last injection. Only non-CpG DNA

significantly increased serum TNF and IL-12 levels at 18 wk,

although not as high as compared with CpG DNA (Fig. 2). By

contrast, 3P-RNA and non-CpG DNA had no effect on levels of

IFN-a and IFN-g, but IFN-g was significantly induced by CpG DNA

(Fig. 2). Together, 3P-RNA and non-CpG DNA are less potent than

CpG DNA to increase serum levels of TNF, IFN-a, IFN-g, IL-12, IL-6

and TNF in nephritic MRLlpr/lpr mice.

Non-CpG DNA but not 3P-RNA induces lympho-proliferation in MRLlpr/lpr mice

Repeated exposure to CpG DNA can trigger significant lympho-

proliferation [23], hence, we assessed the weight of spleens

and mesenteric lymphnodes at the time of sacrifice in all mice.

Non-CpG DNA but not 3P-RNA increased the weight of spleen

and mesenteric lymphnodes in MRLlpr/lpr mice to the same extent

Figure 1. Serum IL-6 response in Myd88 knockout mice and dosestudies in MRLlpr/lpr mice. (A) C57BL/6 mice with (black bars) or withoutMyD88 (white bars) were injected with a single dose of 3P-RNA, non-CpG DNA or CpG DNA. After 6 h, serum samples were obtained and IL-6levels were determined by ELISA. Values are means7SEM from threemice in each group, each performed in duplicate. Note that non-CpGDNA and 3P-RNA injected Myd88-deficient mice significantly increaseserum IL-6 levels 6 h after injection as compared with time point zerobut no response to CpG DNA. (B) Different concentrations of 3P-RNAand non-CpG DNA were injected intraperitoneally into 12-wk-oldMRLlpr/lpr mice as indicated. Serum IL-6 (B) and CCL2 (C) weremeasured at 6 h after injection by ELISA. Data represent means7SEMfrom four mice in each group. Comparison between different doseswere analyzed by ANOVA and post hoc Bonferroni correction, �po0.05.N.d. 5 not detectable.

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as CpG DNA (Fig. 3A and B). Non-CpG DNA increased the

percentage of splenic CD4/CD8 double negative T cells and

B220/CD138 double positive plasma cells in MRLlpr/lpr mice

(Fig. 4A and B). Furthermore, we assessed the activation of

CD11c1 DC by flow cytometry for MHC class II and CD40 surface

expression. 3P-RNA and non-CpG DNA did not increase surface

expression of MHC II and CD40 as it was observed with CpG DNA

(Fig. 4C and D). Thus, non-CpG DNA but not 3P-RNA induces

lymphoproliferation in nephritic MRLlpr/lpr mice.

Spleen mRNA expression of inflammatory mediatorsand transcription factors

How do 3P-RNA and non-CpG DNA affect the expression of

inflammatory mediators? 3P-RNA but not non-CpG DNA induced

Mx1, a marker of type I IFN signaling, in spleens of MRLlpr/lpr

mice, while IFN-g and TNF mRNA were not induced (Fig. 5).

Neither 3P-RNA nor non-CpG DNA induced IL-6 and CCL2 mRNA

as seen with CpG DNA (Fig. 5). The mRNA levels of TBX21 and

GATA2, respective markers of Th1 or Th2 responses, were rather

decreased upon 3P-RNA exposure (Fig. 5). 3P-RNA and non-CpG

DNA did not significantly affect the mRNA expression of RIG-I,

MDA5 (and DAI, data not shown) but both nucleic acids reduced

IPS-1 mRNA levels (Fig. 5). 3P-RNA suppressed Foxp3 mRNA,

crucial regulator in the development and function of regulatory

T cells [24]. This was consistent with reduced numbers of CD4/

CD251 T cells in 3P-RNA-treated MRLlpr/lpr mice (Table 1). IL-15

mRNA levels were not altered by any of the three ligands (Fig. 5).

Do 3P-RNA and non-CpG DNA modulate Th17-dependent

autoimmunity [25, 26]? Both, the Th17-related factors Rorc

and IL-23a, were found not to be induced in spleens of MRLlpr/lpr

mice (data not shown). Thus, 3P-RNA induces type I IFN-

dependent MX1 signaling and impairs Foxp3-driven regulatory T

cells in spleens of MRLlpr/lpr mice.

Hypergammaglobulinaemia and DNA autoantibodiesin MRLlpr/lpr mice

TLR signaling contributes to B-cell activation and the production

of selected autoantibodies in lupus [27]. Hence, we meticulously

analyzed the effects of 3P-RNA and non-CpG DNA on serum IgG

levels and a number of common lupus autoantibodies. Consistent

with its effect on spleen plasma cells only non-CpG DNA

significantly increased serum levels of total IgG including all

isotypes and that of rheumatoid factor (Table 1). Critidiae luciliae

kinetoplast dsDNA binding was used to detect and quantify anti-

dsDNA IgG. A digital quantitative analysis of C. luciliae

kinetoplast positivity revealed that both 3P-RNA- and non-CpG

DNA-injected MRLlpr/lpr mice had little but statistically significant

higher levels of anti-dsDNA IgG as compared with vehicle

controls (Fig. 6A). This was associated with increased homo-

genous ANA staining pattern on Hep2 cells (Fig. 6B). Increased

anti-dsDNA IgG antibodies were observed with both ligands also

Figure 2. Serum cytokine levels in MRLlpr/lpr mice. Serum cytokinelevels of 18-wk-old MRLlpr/lpr mice for TNF, IFN-g, IL-6, IL-12, IFN-a weremeasured by ELISA. Data represent means7SEM from 10 to 12 mice ineach group. Comparison between different agonists and vehicle wereanalyzed by ANOVA and post hoc Bonferroni correction, �po0.05 versusvehicle.

Figure 3. Spleen and lymphnodes in MRLlpr/lpr mice. Weights ofspleens (A) and of mesenterial lymphnodes (B) were determined inall mice at the end of the study. Comparison between differentagonists and vehicle were analyzed by ANOVA and post hoc Bonferronicorrection, �po0.05 versus vehicle, ��po0.01 versus vehicle.

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by ELISA (Table 1). 3P-RNA and non-CpG DNA had no effect on

serum levels of anti-Sm IgG (Table 1). Together, transient

exposure to 3P-RNA and non-CpG DNA both specifically

enhanced the production of dsDNA autoantibodies but not anti-

Sm IgG. Only non-CpG DNA increases IgG and rheumatoid factor

in sera of MRLlpr/lpr mice.

3P-RNA and non-CpG DNA both increase glomerularIgG and complement deposits

Higher levels of circulating dsDNA autoantibodies are usually

associated with more severe immune complex-mediated renal

injury. In fact, 3P-RNA and non-CpG DNA both increased

glomerular IgG and complement factor C3c staining as compared

with control mice (Fig. 7A and B, Table 2). Hence, increased

glomerular IgG and C3c deposits were rather associated with the

consistent effect of 3P-RNA and non-CpG DNA on dsDNA autoanti-

body production than with hyperglobulinaemia, which was selec-

tively induced by non-CpG DNA. From the above results one

could predict that 3P-RNA and non-CpG DNA also aggravate

glomerular pathology, i.e. lupus nephritis, in MRLlpr/lpr mice. Control

MRLlpr/lpr mice had diffuse proliferative glomerulonephritis with

moderate mesangial hypercellularity, increase of mesangial matrix,

and few periglomerular inflammatory cell infiltrates at week 18

(Fig. 8). 3P-RNA and non-CpG DNA injections both increased

disease activity of lupus nephritis as evaluated by the histomorpho-

logical activity and chronicity scores for lupus nephritis (Fig. 8

and Table 2). These effects were less severe and specifically were

not associated with glomerular crescents or aggravated albuminuria

as seen with CpG DNA injections (Table 2). Aggravation of

renal pathology was associated with some increase in the numbers

of glomerular and interstitial macrophages and T cells (Fig. 8), but

again these effects were less prominent as with CpG DNA. 3P-RNA

also increased the number of Ki-67 positive proliferating cells in the

renal interstitium but not in tubular epithelial cells. Taken together,

transient exposure to 3P-RNA and non-CpG DNA aggravates

immune complex disease and proliferative lupus nephritis in

MRLlpr/lpr mice.

3P-RNA and non-CpG DNA localize to the kidney afterintravenous injection

TLR-independent recognition of viral nucleic acids is not

restricted to immune cells [12, 15, 17–19]. Hence, 3P-RNA and

non-CpG DNA might elicit local immunostimulatory effects in

renal cells and infiltrating immune cells in addition to their

systemic effects. To test this hypothesis we injected fluorescently

labeled 3P-RNA and non-CpG DNA into 18-wk-old MRLlpr/lpr

mice and harvested the kidney for fluorescence microscopy 2 h

later. Labeled 3P-RNA and non-CpG DNA were both found in the

kidney and colocalized with glomerular cells and tubular

epithelial cells in a cytoplasmic staining pattern (Fig. 7C and

D). Obviously, 3P-RNA and non-CpG DNA localize to the

cytosolic compartment of renal cells suggesting that they might

elicit local effects in addition to their effects on autoimmunity.

3P-RNA and non-CpG DNA induce IL-6 release inglomerular mesangial cells

To test whether 3P-RNA and non-CpG DNA can activate murine

mesangial cells be exposed cultured primary mesangial cells to

both ligands. 3P-RNA and non-CpG DNA activated mesangial

cells to produce IL-6 in a dose-dependent manner as assessed by

ELISA after 24 h of stimulation (Fig. 9). By contrast, mesangial

cells did not respond to CpG DNA stimulation as they lack the

Figure 4. Spleen cell subsets in MRLlpr/lpr mice. Spleen cell subsetswere determined by flow cytometry as follows: (A) CD31/CD4�/CD8�

T cells by staining CD41 and CD81 for CD31 gated T cells; the meanpercentage of CD31/CD4�/CD8� T cells from the round marked lowerleft quadrant were shown in right side graph; (B) B2201CD1381 plasmacells, mean percentage of B2201CD1381 cells from the round markedupper right quadrant were shown in right side graph. (C) Percentage oftotal splenic CD11c1 DC expressing MHC-II; (D) percentage of totalsplenic CD11c1 DC that express CD40. �po0.05 versus vehicle.Comparison between different agonists and vehicle were analyzed byANOVA and post hoc Bonferroni correction, �po0.05 versus vehicle.

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Figure 5. Spleen mRNA expression of proinflammatory mediators and transcription factors. Total spleen mRNA from ten mice in each group wasused for real-time RT-PCR. The primers for target mRNA used are listed in methods. Data are expressed as means7SEM of the ratio from the targetmRNA per respective 18s rRNA expression. Comparison between different agonists and vehicle were analyzed by ANOVA and post hoc Bonferronicorrection, �po0.05 versus vehicle, ��po0.01 versus vehicle n 5 10 per group.

Table 1. Serum autoantibodies and spleen lymphocytes in 18-wk-old MRLlpr/lpr micea)

Vehicle Non-CpG DNA 3P-RNA CpG DNA

Serum IgG (mg/mL) 871 1672�� 971 871

Serum IgG1 (mg/mL) 15.072.6 22.071.6� 13.072.2 14.071.5

Serum IgG2a (mg/mL) 4.070.4 8.971.7� 6.071.1 6.071.3

Serum IgG2b (mg/mL) 2.070.2 5.070.8� 2.070.3 1.070.3

Anti-dsDNA IgG (mg/mL) 144722 203737 200730 206735

Anti-dsDNA IgG1 (mg/mL) 1676 108756 104737� 85736

Anti-dsDNA IgG2a (mg/mL) 4627108 6717142 5557105 5827159

Anti-dsDNA IgG2b (mg/mL) 1073 43713� 46721 60721�

Anti-Sm IgG (mg/mL) 674 873 572 472

Rheumatoid factor (od 450 nm) 0.470.1 0.770.1� 0.470.1 0.470.1

CD41 % of cells in spleen 53.770.7 48.271.7 53.378.4 48.971.6

CD81 % of cells 16.371.1 14.871.1 12.970.9 17.071.3

CD41CD251 % of cells 2.170.2 2.170.2 1.470.2 1.670.1

a) Comparison between different agonists and vehicle were analyzed by ANOVA and post hoc Bonferroni correction �po0.05 versus vehicle.

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expression of TLR9 [21]. Thus, 3P-RNA and non-CpG DNA

activate glomerular mesangial cells to produce IL-6.

Discussion

Transient exposure to viral nucleic acids can trigger disease

activity in SLE via TLR-dependent pathways but the role of TLR-

independent viral nucleic acid recognition in autoimmunity is

hypothetical. We used the model of spontaneous SLE-like

autoimmunity in MRLlpr/lpr mice to study the effects of

intermittent exposure to 3P-RNA and non-CpG DNA, synthetic

ligands to RIG-I and recently identified cytosolic dsDNA receptor

DAI or yet unknown cytosolic dsDNA receptors, in vivo [9–11,

13–15]. Here we provide first evidence that 3P-RNA and non-CpG

DNA can aggravate both autoimmunity and autoimmune tissue

injury in nephritic MRLlpr/lpr mice and also these ligands

exacerbrate disease through different types of immune responses.

Antiviral host defense requires activation of innate immunity

including the local production of type I IFN. We have previously

shown that transient exposure to viral dsRNA or imiquimod both

aggravates lupus nephritis in MRLlpr/lpr mice in association with

enhanced IFN-a signaling [21, 22]. While imiquimod triggers

endosomal TLR7 signaling via MyD88/IRF-7 [28] viral dsRNA

can signal either via endosomal TLR3/TRIF/IRF-3 or by

activating both cytosolic RIG-like helicases [29, 30]. 3P-RNA

specifically interacts with RIG-I [9, 10]. RIG-I expression in lupus

nephritis was reported [31]. We used the immunostimulatory

ssRNA triphosphate oligonucleotide to mimic viral 3P-RNA,

because its 19 bases were identified to be the minimal length to

efficiently induce IFN-a in monocytes 3P-RNA [9]. Injections with

3P-RNA enhanced MX1 mRNA expression in spleens and kidneys

of MRLlpr/lpr mice indicating a type I IFN signature although an

increase in IFN-a serum levels was not evident. This is in line with

the observation that autoimmunity in MRLlpr/lpr mice depends on

IFN-g rather than on type I IFN as compared with autoimmune

NZB/NZW mice [32, 33]. In the spleen IFN-a is mainly produced

by plasmacytoid DC in a TLR-dependent manner [18] but

3P-RNA can activate RIG-I-dependent type I IFN production and

also in non-immune cells [15, 18, 19]. Injected 3P-RNA localized

Figure 6. Lupus autoantibodies in MRLlpr/lpr mice. (A) Anti-dsDNA antibodies were assayed by binding to C. luciliae kinetoplast dsDNA and weredetected by immunofluorescence. IgG staining to C. luciliae dsDNA is shown in green (left), and DAPI staining of DNA is shown in blue (middle).White arrows indicate the kinetoplast. Specific anti-dsDNA antibodies are identified by colocalization of IgG and DAPI staining in the kinetoplastand appear in mixed color (right). Specific anti-dsDNA staining of C. luciliae kinetoplasts mean intensity was shown in lower panel. Two-tailedFisher’s exact test was used for non-parametric analysis. �po0.05 versus vehicle. ��po0.01 versus vehicle. (B) Serum anti-nuclear antibodies weredetected by Hep2 slide staining as described in the Materials and methods. Staining patterns were classified as homogenous, speckled orcytoplasmic. Data shown on the right lower panel represent the staining pattern present in percent of mice of each group as indicated.

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to glomerular cells and tubular epithelial cells in kidneys of

MRLlpr/lpr mice cytosolic staining pattern and cultured mesangial

cells produced IL-6 upon 3P-RNA and non-CpG DNA stimulation.

Consistently, RIG-I expression was reported to localize to

mesangial cells in human lupus nephritis [32]. Kidneys

of 3P-RNA-injected mice expressed increased levels of CCL2 (data

not shown), which was consistent with the moderate increase in

glomerular and interstitial macrophage and T-cell infiltrates,

Figure 7. Glomerular immune complex deposits and distribution of 3P-RNA and non-CpG DNA in kidneys of MRLlpr/lpr mice. (A) Immunostainingfor total IgG was performed on paraffin-embedded renal sections from of 18-wk-old MRLlpr/lpr mice of all groups. Original magnification � 400.(B) Frozen sections of the same kidneys were used for immunostaining for complement factor C3 with a FITC-labeled antibody (green). Cell nucleiare stained with DAPI (blue). Original magnification � 400. (C) and (D) Rhodamine-labeled 3P-RNA and non-CpG DNA were intravenously injectedinto 16-wk-old MRLlpr/lpr mice. Renal tissue was harvested 2 h later and frozen sections underwent fluorescence microscopy. Rhodamin-labelednucleic acids appear in red. FITC phalloidin staining appears in green and DAPI-stained cell nuclei are blue. Glomeruli are encircled. Originalmagnification �200.

Table 2. Parameters of lupus nephritis in 18-wk-old MRLlpr/lpr micea)

Vehicle Non-CpG DNA 3P-RNA CpG DNA

Proteinuria (mg/mg)

Albumin/creatinine ratio 33711 37714 67739 108755

Glom. deposit scores (0–3)

IgG 1.270.1 1.570.1 1.970.2 2.170.2�

C3c 0.870.1 1.570.1� 1.870.1� 1.870.1�

Histological scores

Activity index 6.371.5 12.472.0� 11.071.7� 15.471.7�

Chronicity index 0.670.5 2.570.9� 1.870.6� 3.670.9�

Cellular response (cells/glom. or hpf)

Glom. Mac21 (cells/glom.) 6.970.9 8.570.8� 10.170.9� 15.371.6�

Glom. CD31 (cells/glom.) 0.770.1 1.470.2� 1.270.2 2.270.2�

Glom. Ki671 (cells/glom.) 2.370.4 2.570.2 2.170.2 4.670.2�

Interst. Mac21 (cells/hpf) 8.071.3 10.371.0 17.473.4 29.374.6�

Interst. CD31 (cells/hpf) 11.671.8 16.071.9 17.173.0 34.674.1�

Interst. Ki671 (cells/hpf) 5.871.4 6.971.2 10.471.6 35.371.5�

Tubular Ki671 (cells/hpf) 13.973.1 12.571.7 10.571.4 40.273.8�

a) �po0.05 versus vehicle.

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because the CC-chemokine CCL2 mediates recruitment of these

cells to the glomerular and interstitial compartment in MRLlpr/lpr

mice [34, 35]. 3P-RNA also induced the production of dsDNA

autoantibodies and subsequent glomerular IgG and complement

deposits although 3P-RNA did not trigger plasma cell expansion

and hypergammaglobulinaemia. Whether the discrepancy

between 3P-RNA-induced autoantibody production and lack of

plasma cell proliferation relates to the suppressive effect of 3P-

RNA on Foxp3 mRNA expression and the numbers of spleen

CD4CD251 T cells or to another yet undefined mechanism

remains to be determined. Thus, 3P-RNA aggravates lupus

nephritis by enhancing systemic autoimmunity and by aggravat-

ing local inflammation. 3P-RNA induces IFN-a signaling, dsDNA

autoantibody production and aggravates immune complex

disease, renal CCL2 expression and immune cell infiltrates.

Viral DNA can induce immunity via TLR9-dependent as well

as TLR9-independent pathways [36, 37]. In SLE both pathways

can be activated by self-DNA that is released from apoptotic cells

or bound to immune complexes [16, 17, 38–41]. TLR9-depen-

dent recognition of CpG DNA causes B-cell proliferation and

repeated injections with CpG DNA cause significant lymphopro-

liferation in mice [23, 42]. In MRLlpr/lpr mice transient exposure

to CpG DNA increases the production of DNA autoantibodies and

fosters crescentic glomerulonephritis [20]. The role of TLR9 in

Figure 8. Histopathology of kidney MRLlpr/lpr mice. Paraffin-embedded sections were stained with PAS, CD3, Mac-2 and Ki-67 as indicated (originalmagnification � 200 to � 400). Note that 3P-RNA, non-CpG DNA and CpG-treated MRLlpr/lpr mice show higher periglomerular, and interstitialinflammatory cell infiltrates as compared with vehicle-treated MRLlpr/lpr mice.

Figure 9. IL-6 production in glomerular mesangial cells from MRLlpr/lpr

mice. Primary mesangial cells were prepared from MRLlpr/lpr mice andstimulated with 3P-RNA, non-CpG DNA and CpG DNA as described inthe Materials and methods. IL-6 production was determined in super-natants after 24 h by ELISA. Data represent means7SEM from threeindependent experiments each performed in duplicate. Comparisonbetween different agonists and vehicle were analyzed by ANOVA andpost hoc Bonferroni correction, �po0.05 versus medium. N.d. 5 notdetectable.

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SLE is still unclear because lack of TLR9 aggravates SLE in

MRLlpr/lpr mice despite a reduction of dsDNA autoantibodies

[43–46]. Hence, other pathways might be responsible for DNA

autoantibody production. In the present study this 45 bases long,

double-stranded, phosphodiester non-CpG DNA induced plasma

cell expansion, lymphoproliferation and dsDNA autoantibodies in

MRLlpr/lpr mice similar to CpG DNA. Stetson and Medzhitov [11]

have described overlapping but unique gene expression programs

by CpG- and non-CpG DNA in DC, e.g. in terms of IL-12 induc-

tion. Consistent with these data, CpG DNA strongly induced

serum IL-12 levels and reduced spleen GATA3 mRNA expression

in MRLlpr/lpr mice, which was not observed in non-CpG DNA-

injected MRLlpr/lpr mice. IL-12 production has been implicated

with crescent glomerular lesions in murine glomerulonephritis. In

fact, non-CpG DNA did not induce crescentic glomerulonephritis

and did not induce IL-6 and CCL2 mRNA in kidneys as did CpG

DNA. This may explain why non-CpG DNA was less potent in

aggravating lupus nephritis as compared with CpG DNA.

However, the induction of hypergammaglobulinaemia, rheuma-

toid factors and dsDNA autoantibodies was associated with

increased glomerular IgG and C3c deposits and glomerular

pathology. Obviously, Myd88-dependent recognition of CpG DNA

and Myd88-independent recognition of non-CpG DNA share the

potential to trigger B-cell activation in vivo. CpG DNA and dsDNA

differ in terms of shifting T-cell function to the Th1-type, which is

associated with crescent formation in mice.

In summary, the antiviral immune responses triggered by

3P-RNA and non-CpG DNA differently modulate autoimmunity

but both aggravate autoimmune tissue injury such as lupus-like

nephritis in MRLlpr/lpr mice. These data extend the previous

finding that TLR-independent recognition of RNA and DNA

initiate overlapping but still different gene expression patterns

[11]. RNA and DNA both enhance the production of dsDNA

autoantibodies, the glomerular deposition of IgG and subsequent

complement activation in the glomerulus. However, the immu-

nostimulatory effects of 3P-RNA and non-CpG DNA differ in

terms of type I IFN signaling, Foxp3-mediated regulatory T cells

and plasma cell proliferation in MRLlpr/lpr mice. These data

suggest that viral 3P-RNA and dsDNA differently modulate

autoimmunity but still both aggravate autoimmune tissue injury,

e.g. by activating non-immune cells at the tissue level.

Materials and methods

Animals and experimental protocol

Ten-week-old female MRLlpr/lpr mice were obtained from

Harlan Winkelmann (Borchen, Germany) and kept in

filter top cages under a 12 h light and dark cycle. Water and

standard chow (Sniff, Soest, Germany) were available

ad libitum. All experimental procedures have been approved by

the local government authorities. Mice were distributed into

four groups, each group consisting of 10–14 mice. From weeks

16 to 18 of age, groups of 12 MRLlpr/lpr mice received

intraperitoneal injections every other day with either 5%

glucose or 20 mg phosphodiester 3P-RNA (50-PPP-GAAAAGGGGA-

CACACACACACACACACACAC-30) dissolved in transfection

agent in vivo-JetPEITM (Polyplus-transfection, IIIkirch, France)

according to the manufacturer’s recommendations. 3P-RNA,

devoid of TLR7 activation, was prepared as previously described

[9]. A third group of MRLlpr/lpr mice was injected with

50 mg phosphodiester non-CpG dsDNA (sense 50-TACAGATCTAC-

TAGTGATCTATGACTGATC TGTACATGATCTACA-30). Non-CpG

DNA was generated by annealing complementary single

strands of DNA as previously described [11] dissolved in JetPEI.

JetPEI was used to facilitate the uptake of both ligands

into the cellular cytosol in vivo. As a positive control for the

immunostimulatory effects of DNA, a group of mice were

injected with 40mg of the immunostimulatory A-class phosphothio-

ate CpG-ODN 1668, 50-TCG ATG ACG TTC CTG ATG CT-30

(TIB Molbiol, Berlin, Germany) dissolved in saline as described

[20]. Phosphorothioate or phosphodiester backbones of

synthetic CpG oligonucleotides can modulate their binding

affinity to DNA autoantibodies [47]. Neither of the aforementioned

ODN bound to serum IgG from C57BL/6 mice but all of them

bound to serum IgG of MRLlpr/lpr mice. Binding affinity was

strongest for phosphorothioate CpG DNA particularly at lower

concentrations. Binding affinity of phosphodiester non-CpG DNA

was less potent and was not much affected by complexing non-CpG

DNA to JetPEI. At a concentration of 10mg/mL the binding

affinity of all nucleic acid formats tested was almost identical (data

not shown). All mice were sacrificed by cervical dislocation

at the end of week 18 of age. Endotoxin levels of all synthetic

nucleic acids were negligible as tested by the Limulus Assay.

To assess the renal distribution of injected 3P-RNA and dsDNA in

vivo 50-rhodamine-labeled forms of 3P-RNA/JetPEI and non-CpG

DNA/JetPEI complexes were intravenously injected into MRLlpr/lpr

mice at 18 wk of age. Frozen renal sections were collected 2 h later,

stained with FITC-labeled phalloidin (Invivogen, San Diego, CA,

USA, 1:50) and 40,6-diamidino-2-phenylindol (DAPI, Vector

Laboratories, Burlingame, CA, USA) and subjected to immunofluor-

escence imaging. In some experiments single dose studies

were performed in C57BL/6 mice that were obtained from

Charles River, Sulzfeld, Germany, or in MyD88-deficient mice

that had been backcrossed for six generations to the same

background [48].

Evaluation of systemic lupus

The weight ratio of spleen and the bulk of mesenterial lymphnodes

to total body weight were calculated as markers of the

lupus-associated lymphoproliferative syndrome. Kidneys

from all mice were fixed in 10% buffered formalin, processed and

embedded in paraffin. Three micrometer sections for periodic

acid-Schiff stains and immunostaining were prepared following

routine protocols. The severity of the renal lesions was graded

using the indices for activity and chronicity as described

Eur. J. Immunol. 2008. 38: 3487–3498 Innate immunity 3495

& 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

for human lupus nephritis [49]. The following primary antibodies

were used for immunostaining: rat anti-Mac2 (macrophages,

Cederlane, Ontario, Canada, 1:50), anti-mouse CD3 (T cells,

1:100, clone 500A2, Serotec, Oxford, UK), anti-Ki-67 (cell

proliferation, Dianova, Hamburg, Germany, 1:25), anti-mouse IgG

(1:100, M32015, Caltag Laboratories, Burlingame, CA, USA), anti-

mouse C3c (complement, GAM/C3c/FITC, Nordic Immunological

Laboratories, Tilburg, The Netherlands, 1:200). Negative controls

included incubation with a respective isotype antibody. For

quantitative analysis glomerular cells were counted in 15 cortical

glomeruli per section. Glomerular Ig and C3c deposits were scored

from 0 to 3 on 15 cortical glomerular sections. Albuminuria was

determined by ELISA and expressed as urinary albumin/creatinine

ratio as described [22].

In vitro studies with BMDC and mesangial cells

BMDC cells were isolated from C57BL/6 wild-type mice or Myd88-,

TLR9-mutant mice backcrossed to the C57BL/6 background for at

least six generations as previously described [7, 48, 50]. BM isolates

were cultured with Flt3 ligand (Immuno Tools GmbH, Friesoythe,

Germany) for 1 wk as described [51]. For the preparation of

primary mesangial cells capsule and medulla of the kidney were

removed and the renal cortices were diced in cold phosphate

buffered saline, and sequentially passed through a series of stainless

steel sieves (150, 103, 63, 50 and 45mm) and treated with a 1mg/mL

solution of type IV collagenase (Worthington, Lakewood, NY, USA)

for 15min at 371C. Finally, the digested glomeruli were seeded into

(6-well plates with) RPMI 1640 containing 20% FCS, 1% ITS

(insulin, transferrine, selenium; Roche, Mannheim, Germany). After

five passages mesangial cells were positive for smooth muscle actin

and negative for cytokeratin 18. Cells were treated with either

medium or various concentrations of non-CpG DNA or 3P-RNA

complexed with lipofectamine as well as CpG DNA. Supernatants

were harvested after 24h for ELISA.

Cytokine ELISA

Serum and cell culture supernatant cytokine levels were

determined using commercial ELISA kits: IL-6, IL-12p70, IFN-g(all OptEiA, BD, Biosciences, Heidelberg, Germany), IFN-a (PBL

Biomedical Labs, NJ, USA), TNF-a (BioLegend, San Diego, CA,

USA) following the manufacturers protocols.

Flow cytometry

Splenocyte flow cytometry was performed as previously

described [51]. Anti-mouse CD3, CD4, CD8, CD25, B220 and

CD138 (BD) antibodies were used to detect CD31CD4�CD8�

double negative T cells, CD41CD251 regulatory T cells and

B2201CD1381 plasma cell populations in spleens. CD11c has

been stained to identify plasmacytoid and myeloid DC and their

activation was assessed by costaining for CD40 (BD) and MHCII

(eBioscience, San Diego, CA, USA). Respective isotype antibodies

were used to demonstrate specific staining of cell subpopulations.

Autoantibody analysis

Serum IgG isotype concentrations were determined as previously

described [52]. Anti-nuclear antibodies were determined by

incubating serum samples (1:200 dilutions) with Hep-2 slides

(Biosystems S.A, Barcelona, Spain). The fluorescent patterns

were classified as described [50]. Serum of 6–10-wk-old C57BL/6

mice was used as negative control. For anti-dsDNA, 1:100 diluted

serum was applied to fixed C. luciliae slides (Bio-Rad Labora-

tories, Munich, Germany). FITC-labeled goat anti-mouse IgG

(Molecular Probes, Leiden, The Netherlands) was used as a

detection reagent. C. luciliae DNA colocalized with DAPI and the

kinetoplast staining intensity was quantified from digital images

with Adobe Photoshop software (Adobe, San Jose, CA, USA).

Anti-Sm ELISA: NUNC maxisorb ELISA plates were coated with

Smith (Sm) antigen (Immunovision, Springdale, AR, USA)

overnight. An anti-Sm IgG (Y12) antibody (GeneTex, San

Antonia, TX, USA) was used as standard. HRP-conjugated goat

anti-mouse IgG (Rockland, Gilbertsville, PA, USA) was used for

detection as previously described [22]. Rheumatoid factor: ELISA

plates were coated with 10 mg/mL rabbit IgG (Jackson Immunor-

esearch, West Grove, PA, USA) overnight at 41C. Serum samples

were diluted 1:100 times, 10 wk C57BL/6 mouse serum was used

as negative control. HRP-conjugated anti-mouse IgG was used as

secondary antibody. For antibody binding assay ELISA plates

were coated overnight at 41C with CpG DNA, non-CpG DNA or

non-CpG DNA complexed with JetPEI and mouse genomic DNA

diluted to various concentrations in SSC solution. 1:100 diluted

sera from 20-wk-old MRLlpr/lpr or C57BL/6 mice were added for

1 h. After washing HRP-conjugated anti-mouse IgG was used as

secondary antibody. Reading was taken at OD 450 nm.

Real-time quantitative (TaqMan) RT-PCR

Real-time RT was performed on total spleen and renal mRNA as

previously described [22]. 18S rRNA was used as a housekeeper.

Controls consisting of ddH2O were negative for target and

housekeeper genes. Oligonucleotide primer (300 nM) and probes

(100 nM) were from Applied Biosystems (Darmstadt, Germany):

IL-6: ID Mm00446190_m1 FAM 50-AAATGAGAAAA

GAGTTGTGCAATGG-30,

Mx1: ID Mm00487796_m1 FAM 50-TGTACTGCTAAGTC

CAAAATTAAAG-30,

Ccl2: ID Mm00441242_m1 FAM 50-GCTCAGCCA

GATGCAGTTAACGCCC-30,

Ccl5: ID Mm01302428_m1 FAM 50-CCAATCTTGCAG

TCGTGTTTGTCAC-30,

Tbx21: ID Mm00450960_m1 FAM 50-GCAAGGACGGC

GAATGTTCCCATTC-30,

Eur. J. Immunol. 2008. 38: 3487–3498Ramanjaneyulu Allam et al.3496

& 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

Gata3: ID Mm00484683_m1 FAM 50-CCCACCACGGGAGC

CAGGTATGCCG-30,

FoxP3: ID Mm00475156_m1 FAM 50-ACCCAGCCACTC

CAGCTCCCGGCAA-30,

Rorc: ID Mm00441139_m1 FAM 50-CCCACACCTCA

CAAATTGAAGTGAT-30,

Ddx58/RIG-I ID Mm00554529_m1 FAM 50-CCAAACCA

GAGGCCGAGGAAGAGCA-30,

Ifih1/Mda-5 ID Mm00459183_m1 FAM 50-GACACCAGA

GAAAATCCATTTAAAG-30,

Ifn-g: ID Mm00801778_m1 FAM 50-CTATTTTAACT

CAAGTGGCATAGAT-30,

IPS-1 ID Mm00523168_m1FAM 50-AGTGACCAGGATC

GACTGCGGGCTT-30,

TNF ID Mm00443258_m1 FAM 50-GTCCCCAAAGGGATGA

GAAGTTCCC-30,

IL-23 ID Mm00518984_m1 FAM 50-CAAGGACAACAGC-

CAGTTCTGCTTG-30,

DAI Forward primer 50-CAG GGA AGC ACC CCT CTT AT-30,

Reverse primer 50-GAA TGA AGC TCC TGG GTC AG-30,

Core sequence 50-CCC CCA GAA GTG TCA ACC ACC ACT-30.

Acknowledgements: The work was supported by grants from the

Deutsche Forschungsgemeinschaft (AN372/9-1, GRK 1202,

SE888/4-1) and by the Else Kroner-Fresenius Foundation. We

thank Jana Mandelbaum, Ewa Radomska, Stephanie Pfeiffer and

Dan Draganovic for their expert technical assistance. Parts of this

project were prepared as a doctoral thesis at the Faculty of

Medicine, University of Munich, by R. A.

Conflict of interest: The authors declare no financial or

commercial conflict of interest.

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Abbreviations: IPS: IFN-b promoter stimulator � 3P-RNA: triphosphate

RNA � RIG: retinoic-acid-inducible gene � SLE: systemic lupus

erythematosus

Full correspondence: Dr. H.-J. Anders, Medizinische Poliklinik, Klinikum

der Universitat–Innenstadt, Pettenkoferstr. 8a, 80336 Munich, Germany

Fax: 149-89-218075860

e-mail: hjanders@med.uni-muenchen.de

Received: 11/6/2008

Revised: 1/8/2008

Accepted: 9/9/2008

Eur. J. Immunol. 2008. 38: 3487–3498Ramanjaneyulu Allam et al.3498

& 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu