Small nuclear RNA and associated lupus autoantigens activateB cells and dendritic cells via Toll-like receptor-7 (TLR-7);therefore, TLR-7 may represent a potential therapeutic targetin lupus. MRL lpr mice were administered an injection of eithersaline or synthetic oligodeoxynucleotides with immunoregulatorysequences (IRS) that specifically block signaling via TLR-7(IRS 661) or via TLR-7 and TLR-9 (IRS 954, which uses a activesequence from IRS 661 along with a TLR-9 inhibitory sequence)from weeks 11 to 24 of age. IRS 661 and IRS 954 both significantlyreduced the weight of spleen and lymph nodes as well as serumlevels of TNF as compared with saline-treated MRL lpr mice.Only IRS 661 but not IRS 954 significantly reduced serum levelsof IL-12p40, anti-dsDNA IgG2a, IgG2b, and anti-Smith IgG. BothIRS localized to the kidney after intraperitoneal injectionand significantly improved the activity index and chronicityindex for lupus nephritis in MRL lpr mice. This was associatedwith significant reduction of renal glomerular and interstitialmacrophage infiltrates and the number of interstitial T cells.Autoimmune lung injury was also attenuated with IRS 661 andIRS 954. These data demonstrate that TLR-7 antagonism, initiatedafter the onset of autoimmunity, can prevent autoimmune kidneyand lung injury in MRL lpr mice. Concomitant blockade of TLR-9with IRS 954 neutralized the effect of TLR-7 blockade on dsDNAIgG2a, dsDNA IgG2b, and Smith antigen autoantibodies but hadneither additive nor opposing effects on autoimmune lung andkidney injury. Hence, TLR-7 is proposed as a novel and potentialtherapeutic target in systemic lupus erythematosus.
Systemic lupus erythematosus (SLE) is characterized by spontaneouslymphoproliferation, expansion of autoreactive B and T cells,and production of polyclonal autoantibodies against numerousnuclear antigens (1,2). Although the role of nuclear autoantigensfor the pathogenesis of immune complex disease and T cell–mediatedautoimmune tissue injury is generally accepted, their role asendogenous immune adjuvants remains under debate. Studies fromMarshak-Rothstein's laboratory (3,4) first suggested that chromatinor ssRNA components of SLE immunocomplexes can activate Toll-likereceptor-9 (TLR-9) or TLR-7 in intracellular endosomes of Bcells, respectively. Thereby, such nucleic acid–containingimmune complexes were shown to activate autoreactive B cellsand autoantibody production independent from T cell control.In fact, immune complexes that are prepared from sera of patientswith lupus also activate TLR-9 on human dendritic cells, aneffect that is sensitive to DNAse digestion (5). However, datafrom Medzhitov's group (6) showed that self-DNA cannot be detectedby endosomal TLR-9 unless a chimeric TLR-9 is "relocated" tothe cell surface. This study rather suggested that the endosomallocalization of TLR-9 represents a mechanism to prevent immunestimulation by self-DNA.
In vivo studies remain the appropriate tool to test the significanceof such in vitro phenomena. For example, Tlr7 overexpressionis associated with antinuclear autoantibody production and lupus-likedisease in mice (7,8). Contrary, backcrossing Tlr7-deficientmice into lupus-prone MRLlpr/lpr mice is associated with lesslymphoproliferation, less activation of plasmacytoid dendriticcells, and less autoimmune lung and kidney injury (9). A detailedcomparison of antinuclear antibodies in Tlr7-deficient and wild-typemice revealed that TLR-7 contributes to the production of antibodiesagainst the Smith antigen of Sm-RNP RNA (9). This is interestingbecause the lupus autoantigen U1snRNP RNA was identified asan endogenous ligand for TLR-7 (10,11). Furthermore, 564 Igitransgenic mice produce large amounts of anti-RNA, -DNA, and-nucleosome antibodies of the IgG2a and IgG2b isotype that causenephritis, a phenomenon that was abrogated in Tlr7-deficientmice (12).
The contribution of TLR-9 to the pathogenesis of lupus mustinvolve different mechanisms. Several groups backcrossed Tlr9-deficientmice into lupus-prone mouse strains and found the autoimmunetissue injury to be aggravated as compared with their respectivewild-type strains of mice (9,13–15). Attempts to identifythe causal mechanism have not been successful, but obviouslylack of TLR-9 is associated with less chromatin-specific autoantibodiesand with a higher activation state of plasmacytoid dendriticcells (9). These findings challenge the concept of a generalproinflammatory role of endogenous TLR-9 ligands in the pathogenesisof lupus (16). However, targeting these TLR may still representa valuable option for the treatment of lupus (17,18). This isindicated by the therapeutic effects of chloroquine in patientswith lupus, an inhibitor of endosomal acidification and TLRactivation (19). However, because TLR-7 and TLR-9 deficiencyhave opposing effects on experimental lupus, it becomes necessaryto dissect therapeutic blockade of these TLR by specific antagonists.Such antagonists were identified by Barrat et al. (20), whocharacterized the inhibitory effects of synthetic oligodeoxynucleotideswith immunoregulatory sequences (IRS) on TLR-7 signaling invitro. On the basis of this evidence of a proinflammatory roleof TLR-7 for lupus, we hypothesized that antagonism of TLR-7would have beneficial effects on experimental lupus.
Phosphothioate IRS Oligodeoxyribonucleotides and Other TLR Ligands
The following endotoxin-free oligodeoxyribonucleotides (ODN;TIB Molbiol, Berlin, Germany) were used for in vitro or in vivostudies: IRS 661 5'-TGCTTGCAAGCTTGCAAGCA-3'; IRS 954 5'-TGCTCCTGGA-GGGGTTGT-3';and CpG-DNA 1668 5'-TCGATGACGTTCCTGATGCT-3'. Control ODNs were5'-TCCTGCAGGTTAAGT-3' and 5'-TCCTGGCGGAAAAGT-3'.
Studies with Spleen Monocytes
Adherent spleen monocytes were prepared from 10-wk-old femaleMRLlpr/lpr mice and kept in culture as described previously(21). Cells were stimulated with either medium or various concentrationsof imiquimod (Sequoia Research Products Ltd., Oxford, UK) orCpG-DNA, Lps, and poly (I:C) (Invivogen, San Diego, CA) withor without inhibitory oligos as indicated. Supernatants wereharvested after 24 h for ELISA.
Animal Studies
Ten-week-old female MRLlpr/lpr mice were obtained from JacksonLaboratories (Bar Harbor, MA) and were kept in filter-top cagesunder a 12-h light and dark cycle. Autoclaved water and standardchow (Sniff, Soest, Germany) were available ad libitum. Forassessment of the distribution of injected IRS 661, 100 µgof 3'-rhodamine–labeled IRS 661 was injected intraperitoneallyinto 16-wk-old MRLlpr/lpr mice. Tissues were collected 2 h afterinjection and subjected to further analysis as recently described(21). Various groups of mice were treated with saline, IRS 661,or IRS 954 at a dosage of 40 µg on alternate days fromweeks 11 to 24 of age. Blood samples were collected after 3h of the last injection by retro-orbital puncture. Mice werekilled by cervical dislocation under anesthesia with inhaledether. Blood and urine samples were collected from each mouseat the end of the study period, and the urine protein/creatinineratio was determined as described previously (21). All experimentalprocedures were performed according to the German animal careand ethics legislation and were approved by the local governmentauthorities.
Autoantibody Analysis Antinuclear Antibodies.
Antinuclear antibodies (ANA) were determined by incubation ofserum samples (1:200) with Hep-2 slides (Biosystems S.A. CostaBrava, Barcelona, Spain). The fluorescence patterns of ANA wereclassified as described previously (9). Serum of 6- to 10-wk-oldC57BL/6 mice was used as negative control. Crithidia luciliaeimmunofluorescence on specific slides (BioRad Laboratories,Munich, Germany) was performed with 1:100 dilution of serumand scored for the intensity of kinetoplast DNA from 0 to 4as described previously (9).
Anti-dsDNA.
NUNC maxisorp ELISA plates were coated with poly-l-lysine (Trevigen,Gaithersburg, MD) and mouse embryonic stem cell dsDNA. Afterincubation with mouse serum, dsDNA-specific IgG, IgG1, IgG2a,IgG2b, and IgG3 were detected by ELISA (Bethyl Labs, Montgomery,TX).
Anti-Smith.
NUNC maxisorp ELISA plates were coated with Smith (Sm) antigen(Immunovision, Springdale, AR). The Sm IgG (Y12) antibody (GeneTex,San Antonio, TX) was used for standard. A horseradish peroxidase–conjugatedgoat anti-mouse IgG (Rockland, Gilbertsville, PA) was used fordetection as described previously (9). The same procedure wasfollowed for anti-SmRNP as for anti-Sm except that the ELISAplates were captured with Sm-RNP complex (Immunovision) insteadof Sm antigen.
Rheumatoid Factor.
ELISA plates were coated with 10 µg/ml rabbit IgG (JacksonImmunoresearch, West Grove, PA) overnight at 4°C. Serumsamples were diluted 1:100; C57BL/6 10-wk mouse serum was usedas negative control. Horseradish peroxidase–conjugatedanti-mouse IgG was used as secondary antibody.
Cytokine Levels
Serum cytokine levels and cell culture supernatants were determinedusing the following commercial ELISA kits for IL-6, IL-12p40(BD OptEiA, San Diego, CA), TNF- (Biolegend, San Diego, CA),and IFN- (PBL Biomedical Labs, Piscataway, NJ) following theprotocols provided by the manufacturers.
Morphologic Analysis
From all mice, kidneys were fixed in 10% buffered formalin,processed, and embedded in paraffin. Two-micrometer sectionsfor periodic acid-Schiff stains were prepared following routineprotocols. The severity of the renal lesions was graded usingthe indices for activity and chronicity as described for humanlupus nephritis (22). Immunostaining was performed on eitherparaffin-embedded or frozen sections as described previously(21) using the following primary antibodies: Anti-mouse Mac-2(1:50; Cedarlane, Burlington, ON, Canada), anti-mouse CD3 (1:100;Serotec, Oxford, UK), anti-mouse IgG1 (1:100, M32015; CaltagLaboratories, Burlingame, CA), anti-mouse IgG2a (1:100, M32215;Caltag), and anti-mouse C3c (1:200, GAM/C3c/FITC; Nordic ImmunologicalLaboratories, Tilburg, Netherlands). Negative controls includedincubation with a respective isotype antibody. For quantitativeanalysis, glomerular cells were counted in 10 cortical glomeruliper section. Semiquantitative scoring of complement C3c depositsfrom 0 to 3 was performed on 10 cortical glomerular sectionsas described previously (23).
Flow Cytometry
Flow cytometry of splenocytes was performed as described previously(23). The following primary antibodies were used to identifyT cell subsets: FITC-labeled hamster anti-mouse CD3 (clone 145–2C11;BD Biosciences, Heidelberg, Germany), allophycocyanin-labeledrat anti-mouse CD4 (clone RM4–5; BD Biosciences), peridininchlorophyll–labeled rat anti-mouse CD8 (clone 53 to 6.7;BD Biosciences), peridinin chlorophyll–labeled rat IgG2a(clone R35–95; BD Biosciences), and allophycocyanin-labeledrat IgG2a (clone R35–95; BD Biosciences), and PE-labeledrat IgG2a (clone R35–95; BD Biosciences) were used asisotype controls, respectively.
Real-Time Quantitative (TaqMan) Reverse Transcription–PCR
Real-time reverse transcription–PCR on RNA that was isolatedfrom cultured cells or renal tissue was performed as describedpreviously (21). Controls that consisted of ddH2O were negativefor target and housekeeper genes. Oligonucleotide primer (300nM) and probes (100 nM) were from PE Biosystems (Weiterstadt,Germany) and used as follows: Ccr2 forward primer 5'-CCTTGGGAATGAGTAACTGTGTGA-3',reverse primer 5'-ACA-AAGGCATAAATGACAGGATTAATG-3', 6 FAM 5'-TGACAAGCACTTAGACCAGGC-CATGCA-3';Ccr5 forward primer 5'-CAAGACAATCCTGATCGTGCAA-3', reverse primer5'-TCCTACTCCCAAGCTGCATAGAA-3', 6 FAM 5'-TCTATACCCGATCCAC-AGGAGAACATGAAGTTT-3'.Primers for Ccl2, Ccl5, and 18s rRNA were predeveloped TaqManassay reagent from PE Biosystems.
Statistical Analyses
Statistics were done using GraphPad Prism 4.03 version (GraphPad,San Diego, CA). Data were expressed as means ± SEM. Datawere analyzed using unpaired two-tailed t test for comparisonbetween two groups. One-way ANOVA followed by post hoc Bonferronitest was used for multiple comparisons. For nonparametric analysisof two groups, two-tailed Fisher exact test and Mann-WhitneyU test were performed.
IRS 661 Inhibits the Imiquimod-Induced Release of IL-6 and TNF- and IRS 954 Inhibits the Imiquimod- and CpG-ODN–Induced Release of IL-6 and TNF- in Spleen Monocytes from MRLlpr/lpr Mice
Oligodeoxynucleotides with IRS specifically block TLR-7–(IRS661) or TLR-7/TLR-9–mediated IFN- production (IRS 954)in vitro (20). To ascertain the rationale to use these IRS inMRLlpr/lpr mice, we stimulated spleen monocytes from MRLlpr/lprmice with respective TLR-7 or TLR-9 ligands (imiquimod or CpG-ODN)(24,25). IRS 661 dose-dependently blocked imiquimod-inducedproduction of IL-6 and TNF- (Figures 1A and 2A). IRS 954 butnot IRS 661 dose-dependently inhibited CpG-DNA–inducedproduction of IL-6 and TNF- (Figures 1B and 2B). IRS 954 butnot IRS 661 showed significant dose-dependent inhibition ofIL-6 and TNF- production when the cells were incubated withimiquimod plus CpG-DNA (Figures 1C and 2C). By contrast, neitherIRS affected IL-6 or TNF- production induced by LPS or pI:CRNA (Figures 1D and 2D). The same set of experiments were repeatedfor the two control oligos. Neither of the control oligos showedany inhibition of the IL-6 and TNF- release (Figures 1, E throughH, and 2, E through H). These data confirm that IRS 661 andIRS 954 act as specific antagonists for either TLR-7–orTLR-7/TLR-9–induced activation of monocytes in vitro.
Figure 1. Immunoregulatory sequence (IRS) 661 and IRS 954 specifically block imiquimod- and CpG-DNA–induced IL-6 release in spleen monocytes from MRLlpr/lpr mice in vitro. Spleen monocytes from MRLlpr/lpr mice were incubated with imiquimod and IRS 661 at various concentrations as indicated or standard medium without supplements for 24 h as indicated. (A through C) Spleen monocytes were incubated with either imiquimod or CpG-DNA alone or imiquimod plus CpG-DNA. (D) Cells were stimulated with either ultrapure LPS or pIC-RNA. In A through D, IL-6 production was measured in supernatants by ELISA. Data are means ± SEM from two identical experiments. *P < 0.05 versus imiquimod or CpG stimulation, by one-way ANOVA followed by post hoc Bonferroni test.
Figure 2. IRS 661 and IRS 954 specifically inhibit imiquimod- and CpG-DNA–induced TNF- release in spleen monocytes from MRLlpr/lpr mice in vitro. Spleen monocytes from MRLlpr/lpr mice were incubated with imiquimod and IRS 661 at various concentrations as indicated or standard medium without supplements for 24 h as indicated. (A through C) Spleen monocytes were incubated with either imiquimod or CpG-DNA alone or imiquimod plus CpG-DNA. (D) Cells were stimulated with either ultrapure LPS or pIC-RNA. In A through D, TNF- production was measured in supernatants by ELISA. Data are means ± SEM from two identical experiments. *P < 0.05 versus imiquimod or CpG stimulation, by one-way ANOVA followed by post hoc Bonferroni test.
IRS Block Imiquimod- or CpG-ODN–Induced IL-12p40 Serum Levels in MRLlpr/lpr Mice
In view of the in vitro efficacy of IRS 661 and IRS 954 to blockimiquimod- or CpG-DNA–induced cytokine production in spleenmonocytes, we questioned whether these effects also apply toMRLlpr/lpr mice in vivo. Five groups of 14-wk-old MRLlpr/lprmice (n = 5 to 8) received intraperitoneal injections with asingle dose of saline, 25 µg of imiquimod, or 40 µgof CpG-DNA in the presence or absence of a single dose of IRS661 or IRS 954 (40 µg) 30 min before the administrationof the agonist. Serum IL-12p40 levels were determined after3 h of agonist administration by ELISA. Imiquimod and CpG-DNAsignificantly induced the serum levels of IL-12p40 as comparedwith saline-injected control mice (Figure 3). IRS 661 and IRS954 both significantly reduced the imiquimod-induced inductionof IL-12p40. IRS 954 significantly reduced the CpG-DNA–inducedinduction of IL-12p40. Thus, 40 µg of IRS 661 and IRS954 was observed to block TLR-7 or TLR-9 signaling in vivo.
Figure 3. IRS 661 and IRS 954 block imiquimod- and CpG-DNA–induced IL-12p40 production in vivo. Groups of 14-wk-old female MRLlpr/lpr mice (n = 5) were administered an injection of saline or a single dose of 25 µg of imiquimod (imi), 40 µg of CpG-DNA (CpG), or 40 µg of IRS as indicated. Serum IL-12p40 levels were determined after 3 h by ELISA. Data are means ± SEM. *P < 0.05 versus saline; #P < 0.05 versus imiquimod; P < 0.05 versus CpG-DNA.
Serum Cytokine Levels of MRLlpr/lpr Mice Administered Injections of IRS from Weeks 11 to 24
TLR-7 and TLR-9 signaling activates the expression of proinflammatorycytokines and type I interferons (26). Therefore, we examinedwhether injections with IRS 661 or IRS 954 from weeks 11 to24 of age affected the serum levels of these mediators in MRLlpr/lprmice by ELISA. In fact, both IRS 661 and IRS 954 significantlyreduced the serum levels of TNF as compared with saline-injectedMRLlpr/lpr mice (Table 1). IL-6 levels were also decreased withIRS 954; the trend did not reach statistical significance forIRS 661. Contrary, serum IL-12p40 levels were found to be reducedsignificantly only with IRS 661 injections but not with IRS954. IFN- levels were not affected by either IRS.
Table 1. Serum factors and lymphoproliferation in 24-wk-old MLR lpr/lpr micea
IRS 661 But Not IRS 954 Reduces Serum Autoantibody Levels in MRLlpr/lpr Mice
TLR-7 and TLR-9 were shown to have distinct effects on the productionof antinuclear antibodies in MRLlpr/lpr mice (9). Therefore,we assessed whether injections with IRS 661 and IRS 954 fromweeks 11 to 24 of age affected serum IgG and autoantibody levels.Serum levels of IgG1, IgG2a, IgG2b, and IgG3 were not significantlyaffected by either IRS. Antinuclear antibody staining on Hep2cells revealed positive nuclear signals in each MRLlpr/lpr mouseof all groups (Figure 4A). In saline-treated MRLlpr/lpr mice,a homogeneous nuclear staining was most common. Positivity ofcondensed chromosomal areas of mitotic cells was indicativeof autoantibodies directed against chromatin (data not shown).A speckled nuclear staining pattern was less frequent, and acytoplasmic staining pattern was not observed. In IRS 661–treatedMRLlpr/lpr mice, the homogeneous pattern was less frequent andspeckled pattern was somewhat more frequent as compared withsaline-treated mice. IRS 954 injections were associated withan equal distribution of homogeneous and speckled nuclear stainingpatterns (Figure 4A). Antinuclear antibodies specific for dsDNAwere studied by the specific binding to kinetoplast DNA of Crithidialuciliae. IRS 661 but not IRS 954 significantly reduced theindirect immunofluorescence intensity of mouse serum (at a dilutionof 1:100) incubated with Crithidia luciliae substrates (Figure 4B).Assessing the binding of serum to polylysine-linked dsDNA byELISA is another method to determine the specificity of antinuclearantibodies. IRS 661 but not IRS 954 significantly reduced serumlevels of dsDNA-specific IgG2a, IgG2b, and anti-Sm IgG antibodiesas compared with saline-treated MRLlpr/lpr mice (Table 1). Anti-SmIgG and anti-Sm RNP IgG were reduced with IRS 661 treatmentto the same extent (latter not shown). Anti-dsDNA IgG1 and IgG3levels as well as rheumatoid factors were not affected by eitherIRS. These findings are suggestive of an inhibitory effect ofIRS 661 on the production of anti dsDNA IgG2a and IgG2b antibodies,which are known to induce glomerular pathology in MRLlpr/lprmice by activating complement (27,28). It is interesting thatglomerular deposits of IgG2a and complement factor C3c weresignificantly reduced in both IRS 661–and IRS 954–injectedMRLlpr/lpr mice (Figure 5, Table 2). Together these data indicatethat injections with IRS 661 but not with IRS 954 reduce theproduction of selected autoantibodies. Glomerular complementactivation was reduced with either IRS, independent of circulatingIgG2a isotype levels, suggesting that IRS can modulate immunecomplex deposition and complement activation independent ofcirculating autoantibody levels in MRLlpr/lpr mice.
Figure 4. IRS 661 and IRS 954: Antinuclear antibodies and glomerular immune complex deposits in MRLlpr/lpr mice. (A) Serum antinuclear antibodies were detected by Hep2 slide staining as described in the Materials and Methods section. Staining patterns were classified as homogeneous, speckled, or cytoplasmic. Positive staining of mitotic chromatin was assessed independent of the other staining patterns. Data shown on the right represent the staining pattern present in percentage of mice of each group as indicated. (B) Serum antibodies against Crithidia luciliae kinetoplast DNA were analyzed as described in the Materials and Methods section. Data represent staining intensity of sera from individual MRLlpr/lpr mice from each group. *P < 0.01 versus saline by two-tailed Mann Whitney U test.
Figure 5. Glomerular immune complex and C3c deposits in MRLlpr/lpr mice. Paraffin-embedded renal sections were stained for IgG1 and IgG2a as indicated. In saline-treated mice, IgG1 and IgG2a localize to the mesangium and the glomerular capillary wall. Note that IRS 661–and IRS 954–treated MRLlpr/lpr mice show less IgG2a deposits. Frozen renal sections were stained for complement factor C3c. Note the robust glomerular complement activation in saline-treated MRLlpr/lpr mice, which is hardly detectable in IRS 661–and IRS 954–treated MRLlpr/lpr mice. Images are representative of 10 mice in each group. Magnification, x400.
Table 2. Autoimmune tissue injury in 24-wk-old MLR lpr/lpr micea
Distribution of Labeled IRS 661 and IRS 954 in Kidney of Autoimmune MRLlpr/lpr Mice
Do IRS 661 and IRS 954 mediate local effects in nephritic kidneysof MRLlpr/lpr mice? This would require that they localize tothe kidney after intraperitoneal injection, which was studiedby assessment of the distribution of rhodamine-labeled IRS 661and IRS 954 in nephritic kidneys of 16-wk-old MRLlpr/lpr mice.After injection, IRS 661 was detected in a glomerular as wellas a tubular staining pattern (Figure 6A). Co-staining withan anti-mouse IgG antibody showed that labeled IRS 661 partiallyco-localized with glomerular IgG deposits (Figure 6A). However,tubular (Figure 6B, left) and glomerular (Figure 6B, right)IRS 661–positive granules that were negative for IgG werealso observed, suggesting the uptake of IRS 661 into endosomalcompartments of glomerular and tubular epithelial cells. Co-stainingwith a Mac-2 antibody showed that IRS 661 was also taken upby 52% of glomerular and 63% of interstitial macrophages (Figure 6C).The distribution of IRS 954 after intraperitoneal injectionwas identical to that of IRS 661 (data not shown). NephriticMRLlpr/lpr mice that were administered an injection of rhodamineonly did not show any renal deposits (data not shown). Thesefindings show that injected IRS 661 and IRS 954 localize tothe kidney and are taken up by glomerular cells, tubular epithelialcells, and intrarenal macrophages.
Figure 6. Localization of IRS 661 and IRS 954 in kidneys of MRLlpr/lpr mice after intraperitoneal injection. Rhodamine-labeled IRS 661 and IRS 954 were intraperitoneally injected into 16-wk-old MRLlpr/lpr mice, and renal tissue was harvested 2 h later. (A) Fluorescence imaging of frozen sections showed uptake of labeled IRS 661 in glomeruli (encircled) in a mesangial and capillary staining pattern as well as in tubular epithelial cells. Co-staining with IgG showed partial co-localization of IRS 661 with glomerular IgG deposits. (B) At higher magnification, granular deposits of IRS 661 in tubular epithelial cells (left) and glomerular cells (right) can be seen. (C) Co-staining with a FITC-labeled Mac-2 antibody identified IRS 661 to co-localize with renal macrophages (arrow), whereas other renal macrophages remain IRS 661 negative. Magnification, x400 in A and C.
IRS 661 and IRS 954 Reduce Lymphoproliferation and Protect MRLlpr/lpr Mice from Autoimmune Tissue Injury
On the basis of the reported phenotype of Tlr7-deficient MRLlpr/lprmice (9) and our own data with IRS 661, a course of injectionswith IRS 661 from weeks 11 to 24 of age might have beneficialeffects on experimental lupus of MRLlpr/lpr mice. Given thephenotype of Tlr9-deficient MRLlpr/lpr mice (9), the effectsof IRS 954 on experimental lupus were less predictable. IRS661 and IRS 954 significantly reduced the weight of spleensand mesenteric lymph nodes in MRLlpr/lpr mice as compared withsaline-treated controls (Table 1). IRS 661 and IRS 954 alsoreduced the numbers of CD4/CD8–double-negative CD3+ Tcells in spleen, whereas the numbers of spleen CD4+ cells andCD8+ cells remained unaffected (Table 1). These data indicatethat both IRS reduce lymphoproliferation and the expansion ofCD4/CD8–double-negative "autoreactive" T cells in MRLlpr/lprmice. Morphometric analysis of the kidney revealed a significantreduction of the activity and chronicity scores for lupus nephritisin IRS 661–or IRS 954–treated MRLlpr/lpr mice (Table 2,Figure 7). Both IRS 661 and IRS 954 also significantly reducedthe numbers of glomerular and interstitial Mac-2 macrophagesand of interstitial CD3 lymphocytes (Table 2, Figure 7). Leukocyterecruitment depends on local chemokine production, and chemokinesare triggered by local TLR signaling via the NF-B signalingpathway. We determined mRNA levels of the inflammatory chemokinesCCL2 and CCL5 and their respective chemokine receptors CCR2and CCR5 in kidneys of all treatment groups. Both IRS 661 andIRS 954 reduced mRNA levels of all of these markers in kidneysof MRLlpr/lpr mice (Figure 8), indicating that IRS-induced preventionof severe lupus nephritis was associated with decreased intrarenalinflammation. Proteinuria is a clinically important marker oflupus nephritis, but usually it shows a high interindividualvariability in female MRLlpr/lpr mice. Therefore, the trendto decreased proteinuria in IRS 661–and IRS 954–treatedMRLlpr/lpr mice did not reach statistical significance. Furthermore,we evaluated extrarenal autoimmune tissue injury in lungs ofMRLlpr/lpr mice. Lungs of IRS-treated MRLlpr/lpr mice showedless peribronchiolar and perivascular inflammatory cell infiltratescompared with saline-treated controls (Table 2, Figure 7). Togetherthese data show that IRS 661 and IRS 954 both reduced autoimmunetissue injury in lungs and kidneys of MRLlpr/lpr mice.
Figure 7. Lupus nephritis and lung injury in MRLlpr/lpr mice. (A) Renal sections were stained with periodic acid-Schiff (PAS), Mac-2, and CD3 as indicated. Note that IRS 661–and IRS 954–treated MRLlpr/lpr mice show less periglomerular and interstitial inflammatory cell infiltrates as compared with saline-treated MRLlpr/lpr mice. (B) Lung sections taken from 24-wk-old MRLlpr/lpr mice of all groups were stained with PAS. Note that IRS 661–and IRS 954–treated MRLlpr/lpr mice show less peribronchiolar and perivascular inflammatory cell infiltrates as compared with saline-injected MRLlpr/lpr mice. Images are representative of 10 mice in each group. Magnification, x200.
Figure 8. Renal mRNA expression of CC-chemokines and their chemokine receptors. The mRNA expression of CCL2 and CCL5 and their respective chemokine receptors CCR2 and CCR5 was determined by real-time reverse transcription–PCR from total RNA from kidneys of 24-wk-old MRLlpr/lpr mice. For analysis, RNA was pooled from 10 mice of each group. mRNA levels for saline-, IRS 661–, and IRS 954–treated MRLlpr/lpr mice are expressed in relation to the respective 18S rRNA expression of each kidney.
Nuclear lupus autoantigens trigger autoantibody production andthe release of proinflammatory mediators by activating TLR-7and TLR-9 in B cells and dendritic cells in vitro (3–5,10,11,20,29).Blocking TLR-9 signaling with injections of specific ODN canameliorate lupus nephritis in MRLlpr/lpr mice (21,30); therefore,we hypothesized that antagonism of TLR-7 or TLR-7 plus TLR-9may be as effective in vivo.
TLR7 Blockade with IRS 661
Injections with IRS 661 were started from week 11 of age andsubstantially reduced autoimmune tissue lung and kidney injuryat week 24 of age, which is consistent with the phenotype ofTlr7-deficient MRLlpr/lpr mice (9). These data support a roleof TLR-7 for the mechanisms that foster the progression of autoimmunetissue injury in MRLlpr/lpr mice (e.g., the expansion of theCD4/CD8–double-negative autoreactive T cell population,immune complex–mediated local complement activation, andthe local inflammatory response involving macrophage and T cellrecruitment [1,2,31]). In fact, IRS 661 significantly reducedthe number of CD4/CD8–double-negative T cells in spleen,a population that continuously expands in MRLlpr/lpr becauseof the inability to delete autoreactive and activated T cellsin mice via the interaction of Fas with the Fas ligand in thesemice (32). Again, our data from IRS 661–injected MRLlpr/lprmice are consistent with the data reported from Tlr7-deficientMRLlpr/lpr mice (9).
Autoantibody production is another characteristic of MRLlpr/lprmice and contributes to autoimmune tissue injury via immunecomplex formation and local complement activation. It is interestingthat TLR-7 is particularly required to generate anti-Sm RNPIgG (9), and serum levels of anti-Sm RNP IgG were also reducedwith injection of IRS 661. Furthermore, we found that IRS 661reduced the serum levels of anti-dsDNA IgG2a and IgG2b. In fact,the glomerular deposits of IgG2a and complement factor C3c bothwere reduced in IRS 661–treated mice, indicating a rolefor TLR-7 in the production of selected nephritogenic autoantibodiesand immune complex glomerulonephritis in MRLlpr/lpr mice. Bycontrast, by using identical assay systems, our data and thosereported by Christensen et al. (9) consistently show that blockadeor lack of TLR-7 does not substantially reduce homogeneous nuclearand mitotic ANA staining of Hep2 cells. As a third mechanism,autoimmune tissue injury in MRLlpr/lpr mice is mediated by CC-chemokine–drivenmacrophage and T cell infiltrates. For example, in nephriticMRLlpr/lpr mice, infiltrating macrophages and nonimmune renalcells produce CCL2 and CCL5, which triggers additional macrophageand T cell recruitment into the kidney (33). Because CC-chemokinesare produced upon TLR-7 activation in macrophages, blockingintrarenal TLR-7 signaling may reduce the local production ofCC-chemokines and subsequent recruitment of immune cells tothe nephritic kidney. We demonstrate that when IRS 661 is injectedinto nephritic MRLlpr/lpr mice, it localized to tubular epithelialcells and intrarenal macrophages, but only the latter expressTLR-7 (34). In fact, IRS 661 reduced the intrarenal productionof CCL2 and CCL5 and renal macrophage and lymphocyte infiltratesin MRLlpr/lpr mice. Together, the beneficial effects of TLR-7blockade with IRS 661 on autoimmune tissue injury of MRLlpr/lprmice were associated with a reduction of CD4/CD8–double-negativeT cells, the production of selected autoantibodies, and renalproduction of proinflammatory chemokines that are known to mediateimmune cell recruitment into the nephritic kidney.
TLR-7/TLR-9 Blockade with IRS 954
Given the opposing effects of Tlr7 and Tlr9 deficiency in MRLlpr/lprmice (9,14), the effects of IRS 954 were less predictable. Injectionswith IRS 954 from weeks 11 to 24 of age improved kidney andlung disease in MRLlpr/lpr mice, and no additive effects wereobserved as compared with IRS 661. The effects of IRS 954 onlupus nephritis and lung injury are comparable to what has beenobserved with oligonucleotide antagonists that are specificfor TLR-9 only in the same lupus model (21) or in NZB/NZW mice(30). These findings support the concept that recognition ofendogenous DNA and RNA molecules via TLR-7 and TLR-9 contributeto the progression of autoimmune tissue injury. This conceptdeveloped from studies that showed that lupus autoantigens inimmune complexes with IgG prepared from autoimmune mice or patientswith lupus can activate B cells and dendritic cells in vitro(3–5,10,11). These in vitro experiments and in vivo studieswith TLR-9 antagonists are inconsistent with the aggravatedphenotype of Tlr9-deficient MRLlpr/lpr mice and have causedus to question the specificity of the antagonists and the assaysystems used for the analysis of serum autoantibodies (9,13–16).The data reported by Barrat et al. (20) and our own study demonstratedthe specificity of IRS 954 for TLR-7 and TLR-9. Furthermore,by applying identical assay systems as in the study reportedby Christensen et al. (9), we showed that IRS 954 has distincteffects on serum autoantibody levels as compared with IRS 661.For example, in contrast to IRS 661, IRS 954 did not affectserum dsDNA autoantibodies; TLR-9 antagonism provided by IRS954 "neutralized" the suppressive effect of IRS 661 on TLR-7–mediatedanti-dsDNA IgG2a, IgG2b, and anti-Sm IgG production. Furthermore,IRS 954 injections were associated with fewer mice with homogeneousnuclear staining on Hep2 cells, which is consistent with whathas been observed in Tlr9-deficient MRLlpr/lpr mice (9). Thefinding that IRS 954 did not reduce dsDNA autoantibodies ispotentially interesting because we previously found that ODN2114, blocking TLR-9 only, reduced these antibodies in MRLlpr/lprmice (21). Thus, the roles of TLR-7 and TLR-9 for the evolutionof specific autoantibodies may be even more complex and requiresa detailed analysis of immune cell subsets from TLR-7 and TLR-9double-knockout cells, which are not yet available.
Our data suggest that delayed onset of oligonucleotide-basedinhibition of TLR-7 reduces autoantibody production and preventsautoimmune tissue injury in experimental lupus. Combined blockadeof TLR-7 and TLR-9 has no additive effects. These data supportthe concept that endogenous ligands of TLR-7 contribute to thepathogenesis of autoantibody production and autoimmune tissueinjury in SLE and propose TLR-7 blockade as a novel therapeuticconcept for lupus.
This work was supported by grants from the Deutsche Forschungsgemeinschaft(AN372/8-1, GRK 1202), the Fritz Thyssen Foundation, the EUNetwork of Excellence "MAIN" (FP6-502935), and the EU IntegratedProject "INNOCHEM" (FP6-518167). S.S. was supported by a grantfrom the Else Kröner-Fresenius Stiftung and the DeutscheForschungsgemeinschaft (SE888/4-1).
Parts of this project were prepared as a doctoral thesis atthe Faculty of Medicine, University of Munich, by R.D.P.
The expert technical assistance of Dan Draganovic, Jana Mandelbaum,Ewa Radomska, and Stephanie Pfeiffer is gratefully acknowledged.We are grateful to Bruno Luckow for the generous gift of dsDNA.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Top
Abstract
Introduction
(Biolegend, San Diego, CA), and IFN-
B signaling pathway. We determined mRNA levels of the inflammatory chemokines CCL2 and CCL5 and their respective chemokine receptors CCR2 and CCR5 in kidneys of all treatment groups. Both IRS 661 and IRS 954 reduced mRNA levels of all of these markers in kidneys of MRLlpr/lpr mice (Figure 8), indicating that IRS-induced prevention of severe lupus nephritis was associated with decreased intrarenal inflammation. Proteinuria is a clinically important marker of lupus nephritis, but usually it shows a high interindividual variability in female MRLlpr/lpr mice. Therefore, the trend to decreased proteinuria in IRS 661–and IRS 954–treated MRLlpr/lpr mice did not reach statistical significance. Furthermore, we evaluated extrarenal autoimmune tissue injury in lungs of MRLlpr/lpr mice. Lungs of IRS-treated MRLlpr/lpr mice showed less peribronchiolar and perivascular inflammatory cell infiltrates compared with saline-treated controls (Table 2, Figure 7). Together these data show that IRS 661 and IRS 954 both reduced autoimmune tissue injury in lungs and kidneys of MRLlpr/lpr mice. 
P < 0.05 versus CpG-DNA.
