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International Immunology Advance Access originally published online on June 23, 2006
International Immunology 2006 18(8):1211-1219; doi:10.1093/intimm/dxl067
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Toll-like receptor 9-independent aggravation of glomerulonephritis in a novel model of SLE

Philipp Yu1,6, Ute Wellmann2, Sandra Kunder3, Leticia Quintanilla-Martinez3, Luise Jennen3, Neil Dear4, Kerstin Amann5, Stefan Bauer1,6, Thomas H. Winkler2 and Hermann Wagner1

1 Institute of Medical Microbiology, Immunology and Hygiene, Technical University of Munich, Trogerstrasse 4a, 81675 Munich, Germany
2 Nikolaus-Fiebiger-Center for Molecular Medicine, University Erlangen-Nürnberg, Glückstrasse 6, 91054 Erlangen, Germany
3 Institute of Pathology, GSF-National Research Center for Environment and Health, Ingolstädter Landstrasse1, 85764 Neuherberg, Germany
4 Division of Clinical Sciences, University of Sheffield, Royal Hallamshire Hospital, Beech Hill Road, Sheffield S10 2RX, UK
5 Division of Nephropathology, Institute for Pathology, University Erlangen-Nürnberg Krankenhausstrasse 8-10, 91054 Erlangen, Germany
6 Institute of Immunology, Philipps-University Marburg, Hans-Meerwein Strasse 3, 35037 Marburg, Germany

Correspondence to: P. Yu; E-mail: philipp.yu{at}staff.uni-marburg.de


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
The generation of anti-DNA auto-antibodies is characteristic for the human autoimmune condition systemic lupus erythematosus (SLE) and its animal models. However, the contribution of the toll-like receptor (TLR) system of innate immunity receptors and, in particular, TLR9 to this B cell-mediated autoimmune process remains controversial. Here we report that in a novel murine model of SLE, based on hyper-reactive B cell activation mediated by mutant phospholipase Cg2, the genetic deficiency of TLR9 does not protect from spontaneous anti-DNA auto-antibody formation and glomerulonephritis. On the contrary, disease induction is aggravated and additional nucleolar antibody specificity develops in autoimmune TLR9-deficient mice. In vitro studies demonstrate that, in autoimmune-prone mice, dual signaling via the B cell receptor and non-CpG DNA results in synergistic B cell activation in a TLR9-independent manner. These results suggest that engagement of a TLR9-independent DNA activation pathway may promote autoimmunity, while TLR9 signaling can ameliorate SLE-like immune pathology in vivo.

Keywords: autoimmunity, B cells, innate immunity, systemic lupus erythematosus


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Anti-DNA auto-antibody production is a hallmark for the inflammatory autoimmune disease systemic lupus erythematosus (SLE) (1, 2). Although it is successfully used as a phenotypic marker for the human disease and for murine models, the signals that render inert double-stranded (ds) self-DNA immunogenic are virtually unknown. One possibility may be a loss of tolerance to self-antigen DNA complexes (3) or the de novo acquisition of high-affinity anti-DNA specificity during somatic hyper-mutation of memory B cells (4). Numerous studies suggest that unidentified genetic factors affect the development of SLE. To address the complex genetics of SLE, it is necessary to study SLE-like murine models (5, 6). This allows dissection of the interaction of signals that lead to the breakdown of self-tolerance and complex inflammatory autoimmune disease.

We recently reported a novel murine model for SLE, named Ali5, which is based on a gain-of-function mutation in the phospholipase Cg2 (Plc{gamma}2) gene. Plc{gamma}2 is a central signaling enzyme in B cells and other immune cells that generates inositol Tris-phosphate, thus controlling calcium responses and cell activation (7).

Mutant mice on a C57BL/6 genetic background demonstrate features common to human SLE, such as B cell hyperactivity, increased Ca2+ flux in B cells, anti-DNA antibody formation and immune complex glomerulonephritis. Other phenotypic similarities between SLE and the Ali5 model are dermatitis, arthritis and neurological abnormalities (P. Yu, unpublished data), which appear to be controlled by unknown genes (6).

It has been firmly established that the immune response relies on the quality and magnitude of Toll-like receptor (TLR) family signaling (8). The endosomal nucleic acid-recognizing receptors TLR7, -8 and -9 are potent activators of both the innate and the adaptive arms of the immune system (9, 10). In protective immunity, the finding that bacterial DNA containing non-methylated CpG motifs is able to activate the immune system provided a central link between these two arms. Activation through the TLR9 leads to cytokine release, up-regulation of activation markers and proliferation (9, 11).

The situation is complicated by the finding that eukaryotic genomic DNA, which is heavily methylated, as well as non-CpG oligodeoxynucleotides (ODN) and genomic DNA immune complexes are also able to activate TLR9 signaling (12), if delivered to intracellular compartments (13). It is important to note that the release of the usually sequestered auto-antigen DNA as a chromatin complex by apoptotic cells seems to be a fundamental step in disease induction. This has been studied in mice with impaired apoptotic cell clearance or DNAse II deficiency. In the latter, notably, the pathology is independent of TLR9 (14, 15). The use of TLR9-deficient mice indicates the existence of a TLR-independent sensing mechanism that can activate innate immunity in response to endogenous DNA (13, 16).

On the other hand, a series of experiments directly examining the B cell receptor (BCR) and TLR signaling have established that the activity of autoreactive B cells is influenced by cooperation between adaptive and innate activation mechanisms. It has been examined in transgenic models whether BCR and TLR9 signaling cooperate in the development of B cell-mediated autoimmunity. The results suggested that DNA-mediated TLR9 signaling might be an essential second signal that, together with the BCR signal, drives auto-antibody production (3, 17, 18). This ‘two-receptor paradigm’ of TLR-mediated cellular activation has become instrumental in our understanding of the connection between innate and adaptive immune responses (19).

To investigate if this mechanism is operative in vivo, we generated autoimmune-prone Plcg2Ali5 mice on a TLR9-deficient genetic background. Here we show that the development of autoimmune pathology and anti-DNA antibody formation were not dependent on TLR9. Surprisingly, and contrary to the current paradigm, anti-DNA auto-antibody formation increased and kidney pathology was more severe in TLR9-deficient autoimmune mice. Furthermore, the absence of TLR9 allows the development of anti-nucleolar antibodies in TLR9-deficient autoimmune mice. To address the mechanism of B cell activation in vivo, we performed in vitro B cell activation studies. They suggest that a necessary secondary signal can be provided by non-CpG oligonucleotides (ODNs) without TLR9 activation but in conjunction with hyper-reactive BCR signals.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
The phenotype of Plcg2+/Ali5 mutant mice has been previously described (6). C3H Ali5 female mice were mated to C57BL/6J wild-type male mice for six generations. TLR9-deficient mice were obtained from S. Akira, Osaka University (11), and backcrossed on the C57BL/6J background for at least 10 generations. All animal experiments were conducted in accordance to German animal experimentation regulations.

DNA and other TLR ligands
The ODNs 1668 (5'-tccatgacgttcctgatgct-3), 1720 (5'-tccatgagcttcctgatgct-3), NAO-1 (5'-gctcatgagcttcctgatgctg-3) and AP-1 (5'-gcttgatgactcagccggaa-3) were purchased from TIB Molbiol (Berlin, Germany) as endotoxin-free phosphorothioate (PTO) backbone oligos. The TLR ligand LPS was used at 10 µg ml–1 (Sigma–Aldrich, Taufkirchen, Germany).

Cell stimulation and proliferation and FACS assay
Spleen cells were isolated from pools of Plcg2Ali5/+-TLR9-deficient (n = 2), Plcg2Ali5/+-TLR9-deficient (n = 2), Plcg2Ali5/+-TLR9WT (n = 4), Plcg2WT-TLR9 deficient (n = 2) and Plcg2WT-TLR9WT (n = 4) mice. B220+ splenocytes were positively enriched by MACS according to the manufacturer's guidelines (Miltenyi Biotec). B cells were incubated for 18–24 h with 12.5 µg ml–1 of DNA in a volume of 200 µl as indicated in figures. Complexes of DNA with DOTAP (Roth, Germany) were prepared according to the manufacturer's instructions. Briefly, 5 µg of DNA in 50 µl of HBS (20 mM HEPES, 150 mM NaCl, pH 7.4) was combined with 10 µg of DOTAP in 50 µl of HBS. After 15 min of incubation, 100 µl of complete DMEM medium was added to the mixture of DNA and DOTAP. Cells in 100 µl of complete medium were incubated with 100 µl of DNA DOTAP complex in 96-well culture plates. For proliferation assays, 1 x 105 B cells per well in round bottom 96-well plates were incubated with the following stimuli: LPS (20 µg ml–1, Sigma), anti-IgM F(ab)2 (5 mg ml–1, Jackson ImmunoResearch), ODNs with and without DOTAP (Roth). Incubation of primary B cells was done for 72 h in triplicates with the last 14 h pulsed with 1 µCi per well [3H]dT (Pharmacia). Proliferation experiments were repeated twice with similar results.

Anti-DNA antibody ELISA and anti-nuclear antibody immunofluorescence analysis
Detection of anti-DNA auto-antibodies was done with UV-pre-activated ELISA plates (Maxisorp, Nunc). Plates were coated with 50% heat-denatured and 50% untreated calf thymus DNA (Sigma) diluted to 5 mg ml–1 in DNA-coating buffer (10 mM Tris–HCl, 150 mM NaCl) over night. After standard blocking and washing the plasma samples were diluted (starting at 1:50) in 1:2 serial dilution steps in PBS-1% BSA. Bound antibodies were detected with goat anti-mouse IgG heavy chain-specific antibodies, directly labeled with HRP (Dianova, Germany). The mice were C57BL/6 Plcg2Ali5/+ mice and wild-type Plcg2+/+ on a TLR9-deficient background or on a TLR9WT background. Mice were between 4 and 6 months old. The values given are titers of the specific dilutions at half maximal optical density compared with a standard serum of MRL/lpr mice.

In addition, anti-dsDNA auto-antibodies were measured with an ELISA exactly as described (20). The results of this anti-dsDNA ELISA were consistent with the results from a commercial kit using plasmid DNA as substrate (ReCombi ANA Profile, Pharmacia, Freiburg, Germany). Additional antigens tested with this kit were U1RNP, Sm, SS-A/Ro, SS-B/La, Scl-70, CENP-B and Jo-1. For the detection of anti-histone antibodies, a mixture of H1, H2A, H2B, H3 and H4 (Roche, Mannheim, Germany) was used. Anti-nucleosome antibodies were detected with a commercial kit from Orgentec (Mainz, Germany) adapted for murine antibody detection.

Anti-nuclear antibody (ANA) detection was done by indirect immunofluorescence on Hep-2 cells. Briefly, slides with fixed Hep-2 cells (EUROIMMUN, Luebeck, Germany) were incubated with 1:50 diluted plasma and bound IgG was detected with FITC-labeled goat anti-mouse IgG (Dianova, Hamburg, Germany). Slides were evaluated by a reader blinded for the genotype of the mice.

Histology, immunohistochemistry, FACS and electron microscopy
Kidney and spleen samples were fixed in 4% formalin. Paraffin-embedded tissue sections (3 µm) were stained with H&E, Masson's Trichrome and PAS and investigated using light microscopy at various magnifications a semi-quantitative scoring system. Glomerular, tubulointerstitial and vascular changes were scored from – to +++++ according to the following scheme: –, no changes; +, mild changes, i.e. mild mesangial hypercellularity; ++, moderate focal–segmental changes, i.e. hypercellularity in less than 50% of glomeruli, segmental pattern; +++, moderate diffuse changes, i.e. hypercellularity in more than 50% of glomeruli, whole glomerulus involved; ++++, marked diffuse changes, i.e. hypercellularity and intra-capillary proliferation in more than 50% of glomeruli and +++++, most pronounced changes, i.e. diffuse hypercellularity of nearly all glomeruli with even some crescents as signs of activity. The investigator was blinded to the animal group. Spleen sections were analyzed for the expression of B220/CD45R (BD Bioscience, Germany) by using an automated immunostainer (Ventana Medical Systems, USA) following the manufacturer's protocol. Kidney tissue was fixed in 3% glutaraldehyde and examined by transmission electron microscopy. FACS of spleen cells was done using anti-CD21–FITC and anti-CD23–PE antibody (BD Biosciences, Germany) staining of lymphocyte-gated cells.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Plcg2Ali5/+-TLR9-deficient mice develop anti-DNA auto-antibodies and anti-nucleolar antibodies
The presence of anti-DNA antibodies is one sign of failed immunological tolerance (21). We reasoned that intercrossing of autoimmune-prone Plcg2Ali5/+ with TLR9-deficient mice would provide us with a test system to evaluate the contribution of TLR9 to the activation of autoreactive B cells in vivo. Although other nuclear antigens, e.g. RNA, might be involved in glomerulonephritis, the DNA anti-DNA antibody complexes seem to be most important in the development of disease (22, 23).

Total IgG levels were not significantly different between Plcg2Ali5/+ mice compared with wild-type mice, although a trend toward elevated levels was apparent. The total IgG levels were Plcg2+/+-TLR9+/+: 1.1 ± 0.8 mg ml–1 (n = 5), Plcg2+/+-TLR9–/–: 1.5 ± 1.0 mg ml–1 (n = 4), Plcg2Ali5/+-TLR9+/+: 2.5 ± 1.9 mg ml–1 (n = 11) and Plcg2Ali5/+-TLR9–/–: 2.8 ± 1.4 mg ml–1 (n = 14).

In order to analyze plasma anti-DNA titers of Plcg2Ali5/+-TLR9-deficient mice, we choose a standard antigen-specific ELISA setup. For the initial analysis, we used a mixture of heat-denatured single-stranded (ss) and native ds DNA (Fig. 1A). In our hands, and in accordance with previous published studies, the direct coating of polystyrene ELISA plates with pure calf thymus DNA results in a very sensitive and specific assay (24).


Figure 1
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  		Fig. 1 Plcg2Ali5/+-TLR9-deficient mice develop anti-DNA auto-antibodies. (A) Plasma levels of anti-DNA antibody of the IgG isotype were measured in 4- to 6-month old Plcg2Ali5/+-TLR9-deficient mice and their controls by antigen-specific ELISA. Each dot represents a mouse. Values are titers at half maximal OD compared with a standard MRL/lpr serum. The bar shows the mean titers of the positive mice. (B) Anti-nucleosomal, anti-dsDNA and anti-histone auto-antibodies in plasma of Plcg2Ali5/+-TLR9+/+-competent (closed triangle) Plcg2Ali5/+-TLR9-deficient (open triangle) mice as determined by ELISA. A serum pool from 4- to 5-month old MRL/lpr mice was analyzed for comparison (x). Median values of groups of mice are indicated. Statistics are P-values of Fisher's exact test and n.s. is non-significant.

 
No anti-DNA antibodies were detected in wild-type or TLR9-deficient mice (Fig. 1A). However, C57BL/6 Plcg2Ali5/+ mice between the ages of 4 and 6 months developed high titers of anti-DNA antibodies of the IgG isotype both in the TLR9 wild-type and TLR9-deficient backgrounds (Fig. 1A). The penetrance of anti-DNA antibody production was ~50%. These results were confirmed by an ELISA that specifically detects anti-dsDNA (Fig. 1B). Anti-DNA antibody measurements suggested that the anti-DNA response is similar or even higher in the double-mutant Plcg2Ali5/+-TLR9-deficient mice (Fig. 1A and B)

The Crithidia luciliae anti-dsDNA antibody test was negative for all samples tested, except for an MRL/lpr-positive control and one Plcg2Ali5/+-TLR9-deficient sample (out of 14), probably due to the low sensitivity of this assay (data not shown). In order to further analyze the influence of TLR9 on the auto-antigen specificity of the autoreactive B cell response, we analyzed various antigens that play a role in human SLE diagnostics. The antibody response against U1RNP, Sm, SS-A/Ro, SS-B/La, Scl-70, CENP-B and Jo-1 was all negative (data not shown). Two other important auto-antigen specificities tested were anti-nucleosome and anti-histone antibodies. Whereas, autoimmune Plcg2Ali5/+ mice with or without TLR9 did not show anti-histone reactivity, the anti-nucleosome response was significantly weaker in Plcg2Ali5/+-TLR9-deficient animals, compared with the Plcg2Ali5/+-TLR9-competent mice (Fig. 1B). Therefore, Plcg2Ali5/+ mice did not develop a broad specificity for nuclear auto-antigens, but had a rather restricted repertoire of anti-DNA and anti-nucleosome antibodies.

As a complementary assay to define the antigen specificity of the auto-antibodies, we performed a fluorescent ANA assay using Hep-2 cells. Homogenous nuclear staining correlates with anti-dsDNA antibodies, whereas a coarsely speckled nuclear staining pattern is observed with antibodies directed against RNA-containing antigens such as snRNPs or Sm. Plasma from seven out of 11 Plcg2Ali5/+-TLR9+/+ mice showed the homogenous pattern of nuclear staining, without any other staining pattern. Most importantly, the plasma from 12 out of 14 Plcg2Ali5/+-TLR9-deficient mice still displayed a double-staining pattern—a homogenous pattern of staining characteristic for anti-dsDNA antibodies, thus confirming the results from the anti-DNA ELISA but also an accompanying clear anti-nucleolar staining (Fig. 2A and B). In contrast, only two out of 11 Plcg2Ali5/+-mice displayed this staining pattern. A speckled/granular staining was rarely observed as only one Plcg2Ali5/+-TLR9+/+-competent mouse displayed this pattern and none of the Plcg2Ali5/+-TLR9-deficient mice (Fig. 2B).


Figure 2
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  		Fig. 2 Plcg2Ali5/+-TLR9-deficient plasma shows anti-DNA and nucleolar staining patterns. (A) ANAs were determined in sera (1:50) from 6- to 8-month old mice. Lower left shows homogenous pattern of a representative plasma from a Plcg2Ali5/+-TLR9+/+ mouse. Bottom right is the staining pattern of a representative Plcg2Ali5-TLR9-deficient plasma displaying a nucleolar pattern superimposed on a homogenous nuclear staining. (B) Plasma ANAs were classified as nuclear homogenous, nuclear speckled/granular or nucleolar staining patterns. Black bars indicate Plcg2Ali5/+-TLR9+/+ (n = 11) and white bars indicate Plcg2Ali5/+-TLR9-deficient plasma samples (n = 14). *P = 0.015 (Fisher's exact test).

 
In summary, two independent assays indicate that DNA-recognizing auto-antibodies of the IgG isotype have been formed in autoimmune-prone Ali5 mice. The anti-DNA antibody formation in Ali5 mice proves to be completely independent of TLR9 in vivo. Despite the lack of a significant influence on anti-DNA antibody formation, we could demonstrate that TLR9 does have an influence on the auto-antibody specificity, by supporting anti-nucleosome reactivity but inhibiting the development of anti-nucleolar antibodies.

Pathology of Plcg2Ali5/+-TLR9-deficient mice: glomerulonephritis and splenomegaly
While anti-DNA antibodies of low affinity are present in healthy individuals, an unknown mechanism can alter the affinity or quantity of the anti-DNA antibodies and result in the development of pathogenic immune complexes. Most often the kidney succumbs to autoimmune-driven glomerulonephritis. Recently, a role of TLR9 in glomerulonephritis has been proposed (25).

In our model we observed diffuse proliferative immune complex glomerulonephritis in nine out of 11 Plcg2Ali5/+-TLR9-deficient mice positive for anti-DNA antibodies as well as five out of five Plcg2Ali5/+-TLR9+/+ mice, but none of the wild-type controls (n = 5) or TLR9-deficient mice (n = 7). This indicates that TLR9 is not essential for the development of glomerular injury. On the contrary, the scoring of histopathological kidney disease activity revealed that the mean score of 1.8 for Plcg2Ali5/+-TLR9+/+ mice is significantly lower than 3.8 for Plcg2Ali5/+-TLR9-deficient mice (Fig. 3). Mice with glomerulonephritis showed the classic morphological pattern of proliferative glomerulonephritis with enlarged and hypercellular glomeruli and thickening of glomerular basement membrane due to immune complex deposition (Fig. 4A). The intra-capillary proliferation was generalized and diffuse. Perivascular leukocytic infiltration and fibrinoid necrosis were also detected (data not shown). Of note, in animals with heavy glomerulonephritis also, marked tubulointerstitial damage with tubular dilatation, interstitial inflammation and scarring were observed. Marked alterations of intra-renal arteries were not seen. Electron microscopy revealed visceral epithelial cell foot process swelling and effacement (so-called fusion) together with thickening of the glomerular basement membrane (Fig. 4B).


Figure 3
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  		Fig. 3 Scoring of glomerulonephritis disease activity of 4- to 6-month old Plcg2Ali5/+-TLR9+/+ and Plcg2Ali5/+-TLR9-deficient mice. For definition of 1+ to 5+, see Methods. Statistical analysis shows P-value of Student's t-test.

 

Figure 4
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  		Fig. 4 Histology and electron microscopy of Plcg2Ali5/+-TLR9-deficient mice. (A) Representative sections of glomeruli stained with PAS. The glomeruli from the wild-type and TLR9-deficient mice show no alterations. In contrast, the Plcg2Ali5/+-TLR9+/+ and Plcg2Ali5/+-TLR9-deficient mice show an intra-capillary proliferative glomerulonephritis. Note that the Plcg2Ali5/+-TLR9-deficient mouse has a severe proliferative glomerulonephritis with necrosis and hyalinosis (arrow), which probably represents insudation of plasma proteins into the damaged regions of the glomerulus. (B) Electron microscopy of the corresponding glomeruli. The two figures on the left show a normal glomerular capillary wall. The base membrane (BM), endothelium (END) and foot processes (FP) of the epithelial cells are normal. In contrast, both the Plcg2Ali5/+-TLR9+/+ and Plcg2Ali5/+-TLR9-deficient mice show swelling and effacement (so-called fusion) of visceral epithelial cell foot processes together with thickened base membrane secondary to deposits of immune complexes. (C) Immunohistochemistry of the spleen. The normal morphology of the white pulp is depicted for a wild-type mouse. Staining with B220 highlights the B cell follicles (BF). The negative cells represent the T cell area surrounding the central arteriole (arrow). TLR9-deficient mice show ill-developed B cell follicles and Plcg2Ali5/+-TLR9-deficient mice show only dispersed B cell staining. (D) FACS analysis of marginal zone and follicular B cell populations. The CD21high CD23low is characteristic for marginal zone B cells and CD21intermediate CD23high for FBCs. Numbers are percentages of gated cells.

 
The second pathological change we observed was splenomegaly and changes in the follicular structure of the spleen in Plcg2Ali5/+-TLR9-deficient mice. The spleen (S in mg) and body weight (bw in g) of seven mice per group was determined: Plcg2+/+-TLR9+/+ (S = 85.7 ± 15.1 mg and bw = 32.2 ± 4.8 g), Plcg2+/+-TLR9 deficient (S = 112.9 ± 41.5 mg and bw = 30.7 ± 3.5 g), Plcg2Ali5/+-TLR9+/+ (S = 118.6 ± 35.8 mg and bw = 32.1 ± 4.4 g), Plcg2Ali5/+-TLR9 deficient (S = 222.9 ± 43.9 mg and bw = 28.7 ± 3.5 g).

Histological examination revealed a mild increase in extra-medullary granulopoiesis in the red pulp (data not shown). While primary and secondary B cell follicles and T cell periarteriolar lymphocytic sheaths are normal in wild-type and Plcg2Ali5/+ mice, B cell follicle formation was already partially impaired in TLR9-deficient mice. Surprisingly, in three out of three Plcg2Ali5/+-TLR9-deficient mice analyzed, an absence of primary and secondary B cell follicle structure was observed (Fig. 4C). In addition, the normal distinct borders between T cell and B cell zones in the white pulp were lost (data not shown). This change in intra-splenic B cell distribution is only partially reflected in a FACS analysis using CD23 and CD21 as established markers for marginal zone/follicular B cell (FBC) subset discrimination. Although Ali5 mice either with or without the TLR9 gene have clearly reduced FBC percentages compared with wild-type and TLR9-deficient mice, we could not see a significant further disruption of B cell subsets in Plcg2Ali5/+-TLR9-deficient mice by FACS analysis (Fig. 4D).

These findings demonstrate that in the absence of TLR9, a B cell-driven autoimmune process comprising anti-DNA and anti-nucleolar antibodies ultimately results in glomerulonephritis with aggravated kidney pathology. Furthermore, the lack of TLR9, in conjunction with the autoimmunity-inducing Plcg2 mutation, seems to disrupt the splenic B cell architecture and promotes splenomegaly. These data imply a novel potential role for TLR9 signaling in B cell development.

In vitro B cell activation of splenic B cells from autoimmune Plcg2Ali5/+-TLR9-deficient mice
As the previous data suggest that alternative B cell activation signaling pathways might exist in vivo that are independent of TLR9, we tested how B cells from Plcg2Ali5/+-TLR9-deficient mice respond to a dual signal provided by BCR and DNA in vitro.

For in vitro DNA challenge, we chose PTO-stabilized ODNs containing a CpG motif as bona fide TLR9 ligands. For comparison, we used PTO non-CpG ODN AP-1 which has been described to have a low level of TLR9-dependent activity (26). Activation was assessed by measuring B cell proliferation. As our model employs non-transgenic mice, the frequency of auto-antibody-producing B cells is very low so that antigen-specific auto-antibody production cannot be measured in vitro.

The mitogenic activity of microbial stimuli mediated via TLRs is well known. To examine if TLR9-independent signals encoded by DNA might influence the BCR-driven autoimmunity observed in Ali5 mice, we stimulated splenic B cells with various ODNs. For maximum stability and signal strength, we used ss PTO-stabilized ODN as model reagents for DNA activation. Delivery of phosphodiester ODN did not result in proliferation, presumably due to rapid degradation of these ODNs (data not shown). Similarly, DOTAP-forced, joint activation of B cells with genomic ds calf thymus DNA and anti-IgM did not result in measurable cytokine (IL-6) induction or proliferation in wild-type or mutant mice (data not shown).

Control stimulation with anti-IgM or in combination with the liposome carrier DOTAP resulted only in low proliferation with a higher background in the Plcg2Ali5/+-TLR9-deficient B cells (Fig. 5). The TLR9 deficiency did not influence the TLR4-mediated LPS response in otherwise normal B cells. Surprisingly, B cells from Plcg2Ali5/+ mice responded differently to LPS depending on their TLR9 status. While the TLR9-competent Plcg2Ali5/+ displayed a reduced response to LPS, the B cells from Plcg2Ali5/+-TLR9-deficient mice exhibited an enhanced response to LPS (Fig. 5). To fully understand this phenomena, the cross-talk between the TLR4 and TLR9 signaling pathways needs to be studied.


Figure 5
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  		Fig. 5 In vitro B cell activation of splenic B cells from autoimmune Plcg2Ali5/+-TLR9-deficient mice. MACS-sorted B220+-positive splenic B cells were stimulated for 20 h with the indicated reagents. Bars are standard deviation. Legends: Plcg2WT-TLR9WT (gray), Plcg2WT-TLR9-deficient (hatched), Plcg2Ali5/+-TLR9WT (open) and Plcg2Ali5/+-TLR9-deficient (black) mice. Proliferation assay of [3H]thymidine ([3H]TdR)-labeled splenic B cells. The cells were stimulated in triplicates with the indicated stimuli for 72 h including 1 µM [3H]TdR for the last 16 h. Values are counts per minute (c.p.m.). Statistical analysis was done by Student's t-test. *P ≤ 0.001.

 
As expected 1668 CpG ODN only induced vigorous proliferation in TLR9-containing cells (Fig. 5). In agreement with published data, we observed an increase in proliferation when the dual signal of CpG ODN and anti-IgM was administered. The Plcg2Ali5/+ B cells responded with reduced proliferation to the single CpG ODN, compared with the wild-type cells, but they also respond with increased proliferation to the dual anti-IgM and CpG signal.

Consistent with previous data, the single signal of the non-CpG ODN AP-1 did not lead to proliferation. Application of the non-CpG ODN with DOTAP or without DOTAP but with anti-IgM only confirmed a hyperreactivity of the Plcg2Ali5-TLR9-deficient B cells, as seen with LPS.

The combination of the non-CpG ODN delivered by DOTAP with an anti-IgM leads to strong proliferation of Plcg2Ali5/+-containing B cells (Fig. 5). Moreover this combination of signals also drives wild-type B cells into proliferation. However, the effect on normal B cells seems to be TLR9 dependent, as TLR9 deficiency ablates responsiveness.

Plcg2Ali5/+ B cells proliferate strongly even in the absence of the TLR9. The strength of the proliferation induced is similar to the Plcg2Ali5 B cells from TLR9+/+ mice and reaches ~40% of the maximum proliferation induced by anti-IgM and the CpG ODN 1668 in TLR9+/+ B cells (Fig. 5).

In summary, these in vitro experiments reveal a potential mechanism by which non-CpG DNA might activate autoreactive B cells. The mechanism is not dependent on TLR9, the DNA component must be efficiently transported into the inner cell compartments and an accompanying hyperactive BCR signal is mandatory for activation.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The morbidity associated with SLE is substantial and our lack of understanding of the pathomechanism might be the reason for a lack of new therapies for SLE in the last 30 years (27). In particular, the striking specificity of anti-nuclear antibodies remains enigmatic. To our knowledge, no theoretical model for the initiation of autoimmunity, e.g. molecular mimicry or bystander activation, can give a satisfactory explanation as to why and how SLE occurs. However, it is without doubt that genetic components are required to synergize with environmental events to break tolerance against nuclear antigens (28).

In this context, previous studies support the hypothesis that TLR9 is an important signal component in activation of autoreactive B cells. The capacity of DNA and DNA-associated auto-antigens to activate autoreactive B cells via sequential engagement of the BCR and TLR9 has been called the two-receptor paradigm (3). The original model employs rheumatoid factor transgenic B cells, which proliferate in vitro to IgG–(CpG)–DNA immunocomplexes in a TLR9-dependent way (3, 17, 18). A practical implication from these studies is that the innate arm of immunity might be a potential therapeutic target to cure autoimmunity (19). The idea is to use TLR9-inhibitory ODNs to suppress activation of autoreactive B cells or IFN-{alpha}-producing plasmacytoid DCs (29, 30).

In vivo, the two-receptor paradigm is supported by data from a model employing SLE-prone MRL/lpr mice deficient for the TLR9 gene. Here, TLR9 deficiency results in a complete inhibition of anti-DNA antibody formation but does not result in amelioration of kidney immunpathology (31). In apparent contradiction to the current paradigm are recent experiments that use the identical mouse model, with the exception of a complete backcross of the TLR9 deficiency to the MRL/lpr background. Wu and Peng demonstrated that kidney pathology, lymphoadenopathy and anti-DNA auto-antibodies are not absent, but actually more severe in TLR9-deficient mice. Thus, it was concluded that TLR9 might not promote but rather protect against murine lupus (32). Adding complexity to the picture of TLR signaling in humoral autoimmunity are MyD88-deficient MRL/lpr mice which do not develop anti-nuclear antibodies and glomerulonephritis (33).

These contradictory data place TLRs in the center of the interest to understand and manipulate the aberrant B cell biology of SLE-like autoimmunity.

The present study was undertaken to analyze the in vivo function of TLR9 in a novel SLE-like autoimmune setting. The use of the Plcg2Ali5 model is of particular importance because genetic data suggest that SLE might not only be a multigenetic disease but also consist of different diseases that share a common phenotype. As long as the exact signaling pathways in SLE are not known, it is better not to reduce the analysis to a single mouse model, but rather compare the analysis of different mouse models for SLE in order to overcome the obstacles in our understanding of the autoimmune mechanisms involved.

We have recently shown that ENU-driven mouse mutagenesis is able to identify new key regulators in inflammatory autoimmune disease (6). The SLE model Plcg2Ali5/+ is compatible with human SLE being a disease that has its roots in hyperactive BCR signal transduction (1). Our data imply that a pathological BCR signal strength is essential for the development of SLE-like autoimmunity, but not TLR9. Using an alternative SLE-like mouse model, our data support the hypothesis that TLR9 signaling might be protective against lupus-like pathology in vivo (32). Most importantly the pathology of autoimmune glomerulonephritis is significantly more severe in Plcg2Ali5/+-TLR9-deficient mice than in Plcg2Ali5/+-TLR9+/+-autoimmune mice. In accordance with this finding is the occurrence of splenomegaly and impaired B cell follicle architecture only in Plcg2Ali5/+-TLR9-deficient mice (21). An alternative explanation is that splenomegaly is caused by cells reminiscent of the SHP-1-deficient non-B non-T cell population in motheaten mice (34). The expansion of similar cells could be responsible for the disruption of the splenic structure, resulting in masking of the B cell follicular structure. An in-depth analysis of a novel function of TLR9 in maintenance of the structure of the spleen or in the suppression of non-lymphoid cell expansion is currently underway.

Auto-antigen specificity of SLE-prone B cells is extremely complex and its effect on the course of disease is poorly understood. The genetic manipulation of TLR9 allows to correlate its in vivo function on pathology and antigen specificity. Based on the results obtained, we can conclude that the lack of TLR9 does not inhibit anti-DNA antibody formation. This is of considerable interest because it proves that usually inert genomic DNA can become a target of the adaptive B cell response in vivo, independent of CpG motif-driven TLR9 signals. These findings are in conflict with data from MRL/lpr-TLR9-deficient mice where the anti-DNA antibody production is completely abrogated (31), but confirm the results of Wu and Peng (32).

The second important observation regarding auto-antigen specificity is that although TLR9 might be dispensable for anti-DNA antibody formation, it does seem to have a modulatory effect on the auto-antibody specificity. In the majority of Plcg2Ali5/+ mice the absence of TLR9 leads to the additional generation of anti-nucleolar antibodies. This is in line with TLR9 deficiency also having an effect on the auto-antibody specificity of MRL/lpr mice (31). However, the broadening of the auto-antigen specificity to anti-nucleolar antibody is observed in Plcg2Ali5, but not MRL/lpr mice. It is remarkable that in yet another SLE model Fc{gamma}RIIB-deficient mice the genetic accelerator locus Yaa has a similar effect on splenomegaly and also modifies the auto-antibody specificity to anti-nucleolar antibodies. Most noticeable is that the anti-nucleolar antibodies were also associated with an aggravation of the kidney pathology in this murine model (35). Anti-nucleolar antibodies react to several known nucleolar antigens including RNA polymerase I and nucleolin and are mostly correlated with scleroderma. Nevertheless, a subset of SLE patients (of African-American descent) develop anti-nucleolar antibody responses, which could be linked to a region on chromosome 11q14 (SLEH1) (36).

Finally, the modifier character of the TLR9 gene on the auto-antibody specificity is demonstrated by the strong reduction of anti-nucleosome antibodies in Plcg2Ali5-TLR9-deficient mice. This is interesting because, as discussed before, anti-dsDNA antibodies are not reduced. Because histones (as parts of the nucleosome) are not targets of auto-antibodies in the Ali5 model, we conclude that structural epitopes on nucleosomes may be TLR9-dependent auto-antigens in vivo.

Data from a number of laboratories and recently experiments by Wellmann et al. (4) have provided compelling evidence that B cells with an unknown non-DNA specificity acquire high-affinity DNA-binding capacity by somatic hyper-mutation. Indeed, this might circumvent the early negative selection processes and generate autoimmune anti-DNA B cells, which are sensitive to a second DNA-mediated signal that drives further B cell expansion. Our in vitro B cell experiments corroborate the hypothesis derived from the in vivo data that this signal does not necessarily have to be TLR9 mediated as proposed in the current two-receptor paradigm. We demonstrate that a non-CpG DNA component could have an activating quality and might substitute for TLR9 signals in TLR9-deficient mice. Indeed, if delivered efficiently into the inner cell compartment, a usually inert non-CpG ODN did synergize with a pathologically strong BCR signal and drive B cell activation. This in vitro mechanism might explain why the strong BCR signaling capacity of Plcg2Ali5/+ results in restricted auto-antigen specificity. One can postulate that the engagement of the BCR alone is not sufficient, otherwise a wider range of auto-antigen specificities would be present. Therefore, the possibility remains that apoptotic cells provide an unknown signal to render genomic DNA immunogenic or that DNA itself acts as the antigen as well as the postulated secondary activating signal. This second signal can be mediated by TLR9, but growing evidence suggests that an alternative DNA-mediated signaling system exists.

First, certain mutant mice with impaired apoptosis or uptake or processing of DNA reveal an ‘autoimmune’ anti-DNA antibody-related phenotype (14). This leads to the hypothesis that SLE is a disease of defective clearance of apoptotic cells and DNA. It is of particular interest that this activation is caused by a TLR9-independent sensing mechanism that activates innate immunity against endogenous DNA (15). In accordance with this is a recent finding that a novel pathway for IFN-ß responses of fibroblasts induced by ds B-form DNA exists, which is also not mediated by the TLR-system (37). However, in our in vitro system the B cells could not be activated by ds genomic DNA in conjunction with a hyperactive BCR signal. Possible explanation could be that either B cells do not use this mechanism or that the signaling threshold was not reached in vitro.

Finally, experiments with DCs from TLR9-deficient mice have suggested that non-CpG DNA activates these cells to produce a cytokine response (13, 19). These data and our experiments presented here for the lupus-prone model Plcg2Ali5 suggest that hyperactive BCR signaling, lack of sequestration of nuclear antigens in apoptotic cells and activation signals from endogenous DNA can promote SLE-like pathogenesis.

Because of the conflicting data from murine models, experiments with cells from SLE patients might address the role of TLR9-dependent and -independent signals in lupus. In this context, it is interesting to note that a human SLE genetics study did not identify an association of TLR9 with SLE (38). Nevertheless, understanding the complexity of human SLE will require the analysis of the interaction of innate and adaptive immune responses.


   Acknowledgements
 
We thank H. Drexler and E. Samson, J. Müller and C. Kloss for excellent technical assistance and A. Marshak-Rothstein and G. Häcker for critical reading of the manuscript. This work was supported by the NGFN2 grant (01GR0430). U.W., K.A. and T.H.W. are supported by the Deutsche Forschungsgemeinschaft through SFB 423, DFG SPP 1110 (S.B.) and SFB456 (H.W. and S.B.). We thank Ingenium Pharmaceuticals AG, Martinsried for providing us with Ali5 mice.


   Abbreviations
 
ANA, anti-nuclear antibody
BCR, B cell receptor
ds, double stranded
FBC, follicular B cell
[3H]TdR, [3H]thymidine
ODN, oligodeoxynucleotides
Plc, phospholipase c
PTO, phosphorothioate
SLE, systemic lupus erythematosus
ss, single stranded
TLR, Toll-like receptor
Received 24 March 2006, accepted 31 May 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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