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Activation of toll-like receptor-9 induces progression of renal disease in MRL-Fas(lpr) mice ¹

HANS-JOACHIM ANDERS², VOLKER VIELHAUER, VACLAV EIS, YVONNE LINDE, MATTHIAS KRETZLER, GUILLERMO PEREZ DE LEMA, FRANK STRUTZ^*, STEFAN BAUER, MARK RUTZ, HERMANN WAGNER, HERMANN-JOSEF GRÖNE and DETLEF SCHLÖNDORFF¹

Nephrological Center, Medical Policlinic, Ludwig-Maximilians-University Munich, Germany;
^* Department of Nephrology and Rheumatology, University Medical Center, Göttingen, Germany;
Institute of Medical Microbiology, Immunology and Hygiene, Technische Universität München, Munich, Germany; and
Division of Molecular and Cellular Pathology, German Cancer Research Center, Heidelberg, Germany

²Correspondence: Medizinische Poliklinik der LMU, Pettenkoferstr. 8a, 80336 Munich, Germany. E-mail: hjanders{at}med.uni-muenchen.de

SPECIFIC AIMS

It has been demonstrated that interaction of bacterial CpG-DNA with toll-like receptor (TLR) -9 on B cells stimulates autoantibody secretion, which might contribute to the pathogenesis of systemic lupus erythematosus. We investigated the effects of exogenous CpG-oligodeoxynucleotides (ODN) or DNA derived from Escherichia coli on systemic autoimmunity in nephritic MRL^lpr/lpr mice 16–18 wk of age.

PRINCIPAL FINDINGS

1. E. coli DNA and CpG-ODN increase serum DNA autoantibodies in MRL^lpr/lpr mice
We investigated the effects of bacterial DNA or CpG-ODN on anti-DNA IgG_2a and IgG_2a antibody production in MRL^lpr/lpr mice. Bacterial DNA derived from E. coli and CpG-ODN induced a significant increase in serum IgG_2a DNA autoantibodies vs. saline- or GpC-ODN-treated MRL^lpr/lpr controls. There was a trend toward increased DNA autoantibodies of the IgG₁ isotype, arguing for a shift toward a Th1 response.

2. TLR9 is expressed on macrophages and dendritic cells in nephritic lesions of MRL^lpr/lpr mice
To examine whether exogenous DNA could have local effects on nephritic lesions of MRL^lpr/lpr mice, we determined the renal expression of TLR9. RT-PCR for TLR9 showed renal TLR9 expression in kidneys from all MRL^lpr/lpr mice but not in non-nephritic kidneys of wild-type MRL mice. Immunostaining localized TLR9 positive cells in the glomerular tuft, in periglomerular infiltrates around glomerular crescents, in interstitial infiltrates, and in perivascular fields of arterioles with vasculitic lesions in nephritic MRL^lpr/lpr mice. Injection of 3'-rhodamine-labeled CpG-ODN localized to glomeruli in a diffuse capillary and mesangial staining pattern (Fig. 1 B, left) or to single mononuclear cells in glomeruli (Fig. 1C , right) in a cytoplasmic staining pattern comparable to the immunostaining pattern for TLR9. Costaining with antibodies for macrophages (F4/80, Fig. 1C ) and dendritic cells (CD11c, Fig. 1D ) revealed 3'-rhodamine/marker double positive cells (glomeruli: Fig. 1C, D , interstitium: Fig. 1F, G ). Rhodamine-labeled DNA derived from E. coli did bind to infiltrating interstitial and glomerular cells but not to glomerular matrix (Fig. 1H ). In nephritic kidneys of MRL^lpr/lpr mice, exogenous CpG-DNA and E. coli DNA bind specifically to glomerular and interstitial macrophages and dendritic cells but not to intrinsic renal cells.

View larger version (25K): [in this window] [in a new window]   Figure 1. Renal distribution of injected CpG-ODN. 3 h after intravenous injection of 3'-rhodamine-labeled CpG-ODN to MRL wild type mice positive signals localized to proximal tubular cells but not to glomeruli (A). In MRLlpr/lpr mice, labeled CpG-ODN localized to glomeruli with a diffuse capillary and mesangial staining pattern (left, B) or a more restricted pattern (right, C). Costaining with macrophage (F4/80) and dendritic cell (CD11c) specific antibodies identified both cell types to bind labeled CpG-DNA in the glomerulus [red rhodamine-CpG-signal on the left, green F4/80 (C) or CD11c (D) signal in the middle, yellow colocalization signal on the right]. In MRLlpr/lpr mice, rhodamine-positive mononuclear cells were found in the interstitium and showed a cytoplasmic staining pattern in single cells (left and right, E) that, after costaining with F4/80 and CD11c antibodies, showed both cell types to bind labeled CpG-DNA in the interstitium [red rhodamine-CpG-signal on the left, green F4/80 (F) or CD11c (G) signal in the middle, yellow colocalization signal on the right]. Injection of 3'-rhodamine-labeled DNA derived from E. coli showed single cell signals in glomeruli and intersitial field. Injection of 3'-rhodamine alone did not result in renal staining, indicating that the glomerular and tubular uptake was independent of the fluorochrome itself (not shown).

3. Bacterial DNA and CpG-ODN aggravate renal disease in MRL^lpr/lpr mice
Saline-treated MRL^lpr/lpr mice had diffuse proliferative GN with little periglomerular cell infiltration. Injection of bacterial DNA derived from E. coli or CpG-ODN enhanced mesangial cell number and matrix. Crescent formation corresponding to extracellular cell proliferation and global glomerulosclerosis was seen (Fig. 2 B). CpG-DNA-induced aggravation to crescentic glomerulonephritis was associated with severe proteinuria (Fig. 2A ) and grade 2 renal vasculitis in 50% of CpG-ODN-treated animals and in 72% E. coli DNA-treated animals compared with 0% GpC-ODN controls. CpG-ODN but not E. coli DNA increased the amount of glomerular macrophages compared with GpC-ODN- and saline-injected controls, but the low amount of glomerular lymphocytes was not affected (Fig. 2D ). The amount of interstitial fibroblasts increased in kidneys of E. coli DNA and CpG-ODN-treated mice compared with saline and GpC controls (Fig. 2E ). Real-time RT-PCR from renal isolates showed that CpG-ODN induced a significant increase in renal collagen I mRNA expression compared with GpC-ODN- and saline-injected controls (Fig. 2F ). In MRL control mice CpG-ODN had no effect on the extent of urinary protein and normal glomerular, vascular, or interstitial histology.

View larger version (35K): [in this window] [in a new window]   Figure 2. CpG-ODN and E. coli DNA aggravate proteinuria and histopathological changes in MRLlpr/lpr mice. A) Proteinuria was assessed using the Bradford assay on urine samples.(*P<0.05, n=7–9 in each group). B) Activity and chronicity score used to classify proliferative lupus nephritis in humans assessed the extent of renal damage in MRLlplr/lpr mice. Treatment with control GpG-ODN did not affect the parameters of disease activity (black bars) and chronicity (gray bars) vs. saline-injected controls. E. coli DNA and CpG-ODN aggravated disease activity and chronicity compared with GpC-ODN-treated mice (*P=0.0013 and 0.0007, respectively; n=7–9 in each group). In wild-type mice, neither CpG- nor GpC-ODN caused renal disease. C) The glomerular and interstitial cell infiltrate was characterized by immunohistochemistry. The glomerular infiltration was counted per glomerulus (gray bars) and the interstitial infiltration per high-power field (black bars) (n=7–9 in each group). D) E. coli DNA and CpG-ODN markedly increased interstitial infiltration of CD3 positive lymphocytes vs. GpC-ODN- or saline-treated controls (*P<0.0005). Glomerular counts of CD3 positive cells were low and did not differ between per group. E) The amount of FSP1 positive interstitial fibroblast was assessed by high-power field in 7–9 mice/group (*P=0.001). F) Renal collagen I mRNA expression was determined by real-time RT-PCR of total renal isolates (*P<0.05, n=5 in each group).

4. CpG-ODN stimulate renal chemokine and CCR5 expression in MRL^lpr/lpr mice
We recently showed that CpG-ODN increases chemokine and chemokine receptor expression in J774 macrophages in vitro and in a model of apoferritin-induced murine glomerulonephritis. We analyzed whether the uptake of CpG-ODN by renal macrophages and dendritic cells in nephritic kidneys of MRL^lpr/lpr mice was associated with increased local chemokine expression, as this could contribute to the enhanced leukocyte accumulation and renal damage. To localize the source of renal chemokine mRNA expression, we performed in situ hybridization and immunostaining for CCL2/MCP-1 and CCL5/RANTES. At 18 wk, single spots of CCL5/RANTES and CCL2/MCP-1 mRNA expression were noted within the glomerular tuft and along Bowmann’s capsule of some glomeruli as well as in focal interstitial areas of saline- and GpC-ODN-treated MRL^lpr/lpr mice. In contrast, CpG-ODN-treated MRL^lpr/lpr mice showed marked CCL2/MCP-1 and CCL5/RANTES mRNA expression colocalized with interstitial leukocytic cell infiltrates and glomerular crescents. CCL2/MCP-1 mRNA expression by cortical tubules was only occasionally detected with CpG- and GpC-ODN, arguing against tubular epithelial cells being a major source of CCL2/MCP-1 expression in kidneys of nephritic MRL^lpr/lpr mice of this age. The distribution and induction of renal CCL2/MCP-1 and CCL5/RANTES mRNA expression by CpG-ODN injection were confirmed by immunostaining for the respective chemokines.

Because the expression of CCL5/RANTES was found to be markedly induced by CpG-DNA and because CCR5 positive T cell infiltration may be associated with progression of human renal disease, we determined the expression of renal CCR5 mRNA expression by in situ hybridization. CCR5 colocalized with leukocytic cell infiltrates in the interstitium of GpC-ODN-treated MR-L^lpr/lpr mice at wk 18. CpG-ODN induced a marked increase of interstitial CCR5 mRNA expression in a spatial association with interstitial and periglomerular cell infiltrates.

5. Chloroquine blocks CpG-ODN-induced CCL5/RANTES mRNA expression in spleen monocytes from MRL^lpr/lpr mice
To confirm CpG-ODN-induced CCL5/RANTES expression in vitro, we used adherent monocytes from spleens of MRL^lpr/lpr mice. TLR9 expression of these cells was confirmed by RT-PCR. After stimulation with CpG- or GpC-ODN for 10 h, CCL5/RANTES mRNA expression was determined by real-time RT-PCR. CpG-ODN induced a fourfold increase of CCL5/RANTES mRNA expression in primary monocytes of MRL^lpr/lpr mice compared with stimulation with GpC-ODN. Interaction of CpG-DNA with TLR9 in macrophages has been localized to endosomes. In fact, chloroquine completely blocked CpG-ODN-induced CCL5/RANTES mRNA expression in primary monocytes from MRL^lpr/lpr mice indicating that CpG-ODN-induced chemokine expression involves endosomal TLR9 activation in MRL^lpr/lpr mice.

CONCLUSIONS AND SIGNIFICANCE

Administration of CpG-DNA can aggravate many disease models by inducing an immune response of the Th1 type. In SLE, CpG-DNA may have additional effects, as it was demonstrated that B cells derived from MRL^lpr/lpr mice are activated to secrete autoantibodies, presumably by cross-linking of TLR9 and Ig receptors with immune complexes that contain IgG and CpG-DNA. The relevance of these data for SLE is supported by our observation that in autoimmune MRL^lpr/lpr mice CpG-ODN or CpG-DNA stimulated the adaptive immune response to DNA with a predominance of IgG_2a DNA autoantibodies.

The question arises of whether CpG-DNA-TLR9 interaction could occur locally in the nephritic kidney and exacerbate disease. In nephritic kidneys of MRL^lpr/lpr mice, macrophages and dendritic cells (but not intrinsic renal cells) express TLR9 and bind circulating CpG-DNA. Activation of TLR9 induces gene expression of inflammatory cytokines and chemokines, and macrophages isolated from MRL^lpr/lpr mice expressed CCL5/RANTES mRNA upon stimulation with CpG-ODN in vitro. That chloroquine, a drug used to treat SLE in humans, can block CpG-DNA-induced CCL5/RANTES mRNA expression in vitro supports the hypothesis that TLR9-mediated immune cell activation is involved in the pathogenesis of human SLE. In vivo CpG-DNA induced progression to severe nephritis and renal fibrosis.

We conclude that TLR9 activation triggers disease activity of systemic autoimmunity, e.g., lupus nephritis. Adaptive and innate immune mechanisms contribute to the CpG-DNA-induced progression of lupus nephritis, which may help to explain infection-exacerbated disease in patients with SLE.

View larger version (33K): [in this window] [in a new window]   Figure 3. Schematic diagram.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0646fje;

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