Increased RNase expression reduces inflammation and prolongs survival in TLR7 transgenic mice

Abstract

TLR7 activation is implicated in the pathogenesis of systemic lupus erythematosus (SLE). Mice that overexpress TLR7 develop a lupus-like disease with autoantibodies and glomerulonephritis and early death. To determine whether degradation of the TLR7 ligand, RNA, would alter the course of disease, we created RNase A transgenic (Tg) mice. We then crossed the RNase Tg to TLR7 Tg mice to create TLR7 x RNase double Tg (DTg) mice. DTg mice had a significantly increased survival associated with reduced activation of T and B lymphocytes and reduced kidney deposition of IgG and C3. We observed massive hepatic inflammation and cell death in TLR7 Tg mice. In contrast, hepatic inflammation and necrosis were strikingly reduced in DTg mice. These findings indicate that high concentrations of serum RNase protect against immune activation and inflammation associated with TLR7 stimulation and that RNase may be a useful therapeutic strategy in the prevention or treatment of inflammation in SLE and, possibly, liver diseases.

Introduction

Systemic lupus erythematosus is a potentially fatal disease caused by immune complex (IC) deposition in the kidneys and other organs. Recently, it was discovered that, not only do IC cause tissue injury through activation of FcgR on myeloid cells and activation of the complement cascade (1), but they also enter plasmacytoid dendritic cells (pDC) to stimulate the production of type 1 interferon (T1 IFN) through activation of TLR (2). In mouse models of lupus, there is strong evidence to suggest that activation of TLR7, a receptor for single stranded RNA, plays a pivotal role in promoting lupus. This evidence includes marked attenuation of disease in MRL/lpr mice deficient in TLR7 (but not TLR9) (3), identification an additional copy of TLR7 as being responsible for the accelerating effect of the Yaa mutation in BXSB mice (4, 5) and generation of a lupus-like disease in mice that have a knock in of TLR7 (6) (hereafter referred to as TLR7 Tg mice).

Since RNA is the ligand for TLR7 and since RNase treatment of apoptotic or necrotic extracts markedly reduces stimulation of T1 IFN by pDC in vitro (reviewed in (7), we asked whether RNase would attenuate the expression of lupus in vivo. To this end we created a mouse that constitutively secreted bovine RNase and crossed the RNase transgenic (Tg) to TLR7 knock in mice. Overexpression of RNase in TLR7 Tg mice resulted in a reduction in splenomegaly, reduced numbers of activated B and T cells, fewer immune deposits in the kidney, reduced liver inflammation, and increased survival.

Material and Methods

Creation of bovine RNase transgenic mice

Since the RNase gene contains no introns, bovine RNase was amplified from bovine genomic DNA by PCR using 5′-AATCCCGGGTCATCATGGCTCTGAAGTCC-3′ and 5′-GGACTAGTGGTAGAGACCTACACTGAAGCATCAA-3′ as primers. The amplified bovine RNase gene was cloned into PCRII-TOPO vector (Invitrogen, Life Technologies, Carlsbad, CA) and then subcloned into Alb1L3NB-3 vector (kindly provided by Richard Palmiter, University of Washington) that utilizes the human albumin promoter resulting in hepatic expression of transgenes (8). Following sequence confirmation, the DNA fragment containing the albumin promoter and bovine RNase gene was transfected into the ES cells from C57BL/6 (75%)C3H (25%) mice and selected ES injected into blastocytes to generate transgenic founders (called JLC mice). Founders were backcrossed to pure C57BL/6 (B6) mice for 5 generations to generate the RNase transgenic line used in these studies. The same founder line for all studies reported. When comparing different genotypes, that is in crosses with other transgenic mice, we used the same F1 and littermate controls for the experiments.

Quantitation of RNase concentrations and activity by ELISA and Single Radial Enzyme Diffusion (SRED)

RNase concentrations in serum were quantified by an in house sandwich ELISA. In brief, ELISA plates were coated with a polyclonal anti-bovine RNase antibody (Abcam, Cambridge, MA) and detected with a biotinylated polyclonal anti-bovine RNase antibody (Rockland Immunochemicals, Gilbertsville, PA) followed by HRP-strepavidin (Biolegend, San Diego, CA) and substrate. Sera were tested at a 1/50 dilution. Bovine RNase (DNase free, Life Technologies) was used to create a standard curve. Functional RNase activity was quantified by SRED (9) using poly-C (Sigma) as a substrate. The gel was incubated for 4 hours in a moist chamber at 37°C and then stained with ethidium bromide for 30 min on ice. The size of the rings was read under UV light and quantified using Carestream Molecular Imaging software (Kodak).

Serological analysis

ANA were detected by indirect immunofluorescence using Hep-2 slides as substrate at a dilution of 1/50. IgG Anti-RNA antibodies were detected by ELISA as described (10) using yeast RNA (10ug/ml) (Sigma) as antigen. The purified mAb, BWR4 (11, 12) was used to create a reference standard curve. Serum IgG anti-RNA subclasses were analyzed using the same ELISA but developed with subclass specific antibodies (Sigma) as previously described (13) except that for all subclass analysis, anti-IgG2c rather than IgG2a was used as appropriate for the B6 background. For IgG2b, the mAb H564 (kindly provided by Teresa Imanishi-Kari) (14) was used to create a standard curve but for other subclasses, selected TLR7 Tg serum (for IgG1 and IgG2c) or H564 serum (IgG3) with the highest OD value was used to create a standard curve. Total serum IgG in 4 month old mice was quantified by a sandwich ELISA as described (13). To detect anti-bovine RNase antibodies, plates were coated with bovine RNase (5 μg/ml) and sera from 4 month old mice tested at a dilution of 1/50.

Flow cytometry analysis and flow sorting

Spleen samples were pressed through a 40-micron cell strainer to generate a single cell suspension that was depleted of red blood cells by treatment with Ack Lysing Buffer. For flow based sorting following sacrifice, splenocytes were incubated with 200 μg DNase (Sigma-Aldrich, St Louis. MO) and 8 mg type IV collagenase (Worthington Biochemical Corp, Lakewood, NJ). After 25 minutes, cell dissociation buffer (Invitrogen) was added to 15% final volume for an additional 5 minutes. For surface staining, ~2 × 10⁶ cells were stained with one of the following Ab as indicated in the figures: APC anti-mouse TCRβ chain antibody, anti-mouse CD69 PE (ebioscience, San Diego, CA), PE anti-mouse CD45RB antibody, PE anti-mouse CD44 antibody, percp/cy5.5 anti-mouse CD19 antibody, PE anti-mouse CD86 antibody, Alexa Fluor 488 anti-mouse CD80 antibody. Myeloid cells were identified by Alexa Fluor 647 anti-mouse CD11c, FITC anti-mouse CD11b antibody. All antibodies were from Biolegend except where indicated. Samples were incubated at 4°C for 30 minutes. Data were acquired using a FACSCanto (BD Biosciences, San Diego, CA) and analyzed using FlowJo software (TreeStar Software, Ashland, OR). In all cases, doublets were excluded by gating before gating on live cells using FSC and SSC. To evaluate cytokine responses to TLR agonists, splenocytes from WT and TLR7 Tg mice were stimulated with the TLR7 or TLR4 ligands, gardiquimod (200–800 ng/ml) or LPS (6–10 ng/ml) respectively, and simultaneously treated with GolgiStop. After 2–3 hrs, cells were stained with mAb against CD11b, Ly6C, Ly6G and TNF. Cells to 95% or greater purity were sorted using either a FACSAria or FACSVantage (BD Biosciences).

RNA preparation and quantitative real-time PCR

Total RNA was isolated from sorted cells using the RNeasy mini kit with on-column DNAse treatment (Qiagen, Valencia, CA, USA). First-strand cDNA was generated using 25 ng RNA with the high-capacity cDNA RT-kit) using random primers (Applied Biosystems, Foster City, CA, USA). Reactions in duplicate (20 μl) were run on an ABI StepOne Plus using the primers shown in Supplementary Table 2 and a two-stage cycle of 95°C for 15 s and 60°C for 1 min repeated for 40 cycles followed by a dissociation stage. Threshold cycle (CT) values were determined by setting a constant threshold at 0.2, and fold changes in gene expression were then calculated using the 2-ΔΔC T method or relative expression method (Pfaffl et al., 2002). The standard curve showed similar amplification efficiencies for each gene and that template concentrations were within the linear dynamic range for each of primer set.

Pathology

Kidneys and livers were preserved in 10% formalin and embedded in paraffin and were also snap-frozen in liquid nitrogen and TissueTek OCT compound (Sakura Finetek, Torrance, CA) and stored at −70°C. Paraffin embedded sections were stained with H&E or with Periodic Acid Schiff stain. Quantitation of glomerular tuft area, macrophage infiltration using the antibody, Mac-2 and immunfluorescence staining with goat anti-mouse IgG, IgG subclasses (Santa Cruz Biotechnology, Santa Cruz, CA), and C3 (Cappel, Westchester, PA) were performed as described (13).

A semi-quantitative score of H&E stained liver sections was given by a pathologist blinded to sample identity. This included a count of inflammatory foci (greater than 20 cells/focus) per 5 random 10x fields, and a relative lesion severity score (range 0–5) based on size, distribution and presence of bridging between foci, presence of bile duct hyperplasia and hepatocellular necrosis. Cleaved caspase-3 and the macrophage antigen F4/80 were detected by polyclonal rabbit anti-mouse (Biocare Medical, Concord, CA) and monoclonal rat anti-mouse (Life Technologies) antibodies respectively. Immunohistochemical procedures were performed on a Leica Bond automated immunostainer using HRP conjugated secondary antibodies. Numbers of caspase-3 positive cells per mm of liver section area were expressed as positive cells per mm. Digital images were captured using a Nikon Digital Sight camera system with NIS Elements software. Color and contrast of entire images were standardized using NIS Elements software. No selective corrections were performed

Statistical analysis

Statistical significance between groups was determined by ANOVA with Tukey’s multiple comparison test, Mann-Whitney U test or a Wilcoxon signed-rank test. A p value of <0.05 was considered significant. Differences in proportions were calculated by the Chi square test. Graphs and statistical analyses were performed using Prism software (Graphpad Software) or SigmaStat (Systat Software).

Results

Creation of single and double transgenic mice that overexpress RNase

TLR7.1 Tg mice express TLR7 mRNA at 8–16 fold higher than WT control and develop a lupus-like disease characterized by immune activation, splenomegaly and glomerulonephritis associated with the production of anti-RNA autoantibodies (6). Since it is presumed that ss-RNA is the ligand that drives TLR7 activation in these mice, we developed a strategy to degrade extracellular RNA in TLR7 Tg mice. First, we generated wild type mice on the C57/BL/6 (B6) background that overexpressed bovine RNase A under control of the albumin promoter such that RNase A was secreted from the liver. These mice were born in normal Mendelian ratios, were healthy and fertile. Bovine RNase was detected in the serum at concentrations of ~25 ng/ml in Tg mice, and single radial enzyme diffusion (SRED) analysis revealed potent functional activity (Fig. 1a, b). Based on these results, the specific activity of RNase in the serum of DTg mice was calculated as 0.05 U/μg. Since this is lower than the commercial bovine RNase standard (1 U/μg) this may be explained by optimization of the recombinant enzyme or serum factors that attenuate RNase activity.

Figure 1. Expression of RNase in RNase transgenic (RNase Tg) and double (RNase x TLR7, DTg) transgenic mice.

a, c. The concentration of transgene encoded bovine RNase in mouse serum was quantified by a sandwich ELISA (not cross reactive with mouse RNase) using commercial bovine RNase to create a standard curve. b, RNase functional activity was quantified by single radial enzyme diffusion (SRED) as described in Methods. Upper panel, serum from a normal wild type C57BL/6 (B6) and two RNase Tg mice were tested. Lower panel, one B6 and two randomly selected DTg mice were tested neat and at the dilutions shown. In the top row, commercial RNase was used as a positive control and expressed as functional activity (Units where 1 Unit is equivalent to 0.118 Kunitz Unit). Mice were 14–16 weeks of age.

Examination of spleen size and lymphocyte proportions and activation revealed no differences compared with non transgenic wild type mice (Suppl Fig. 1). The RNase Tg mice were crossed with TLR7.1 Tg mice to yield RNase x TLR7 double transgenic (DTg) mice. Introduction of the RNase Tg to the TLR7 Tg mice also appeared to have no detrimental effects on litter size, and DTg mice were born in the expected ratios. DTg mice had serum RNase concentrations and activity similar to the RNase Tg mice (Fig. 1b, c). Based on SRED functional analysis, RNase activity in the DTg mice was estimated at ~5 fold WT B6 mice (Fig. 1b, lower panel). The normal growth, maturation, fertility and numbers of immune cells in RNase Tg mice indicate that the overexpression of RNase in the RNase Tg mice had no apparent adverse effects. No IgG antibodies to bovine RNase were detected in TLR7 Tg or DTg mice (n=10 in each group, not shown).

Overexpression of RNase partially restores immune expansion and attenuates B and T cell activation

When we evaluated immune activation in RNase x TLR7 DTg mice at 3.5–4.0 mo of age, we observed that spleen weight was significantly reduced in DTg as compared to TLR7 Tg mice (Fig. 2a). Consistent with the reduction in spleen weight, the striking increase in the numbers of myeloid cells in TLR7 Tg mice (23-fold higher compared to B6 mice) was reduced to 12-fold in DTg mice whereas the numbers of T and B cells were very similar between the two strains (Fig. 2b). The reduction in the numbers of myeloid cells in DTg mice resulted in partial restoration of the normal proportions of T and B cells in the spleen (Fig. 2c). Despite the increase in the proportions of B and T cells in DTg mice, the percentages of B and T cells that were activated were significantly reduced as determined by the expression of CD69 and CD80 (B cells) and CD69 and CD44 (T cells) (Fig. 2d).

Figure 2. DTg mice have a reduction in spleen size, myeloid expansion and T and B cell activation.

Mice were sacrificed around 3.5 months of age and (a) the spleen weight determined. b Flow cytometry of splenocytes was performed using the phenotypic markers TCR-beta (T cells), CD19 (B cells) and CD11b (myeloid cells) and the fold increase of T, B and myeloid cells calculated as a ratio of the number of cells in TLR7 or DTg mice/the number of cells in WT B6 control mice. In c, the relative percentage of T, B and myeloid cells was determined by flow cytometry analysis. In d, the % of B and T cells that expressed the activation markers CD69 and CD80 (B cells) or CD69 and CD44 (T cells) are shown.

Since we observed that B cell activation was reduced in DTg mice, we next looked for serologic differences between TLR7 Tg and DTg mice. There were no differences between the two strains in total serum IgG concentrations (median +/− SD values of 3161+/− 2084 and 3143+/− 2045 in TLR7 and DTg mice respectively, n=10 per group, p=non significant). TLR7 Tg mice develop anti-RNA antibodies that produce cytoplasmic and nucleolar staining by immunofluorescence (6). We asked whether expression of RNase affected the levels of anti-RNA autoantibodies in a second large cohort of DTg mice for the survival study. As shown in Fig. 3a, there was no significant difference in anti-RNA autoantibody concentration in DTg mice compared to littermate controls. When younger (2 month old) mice were evaluated or the differences between percentage positive in the two strains were evaluated, no statistically significant differences were observed (not shown). Consistent with these observations, no obvious reduction in intensity or alteration in the pattern of immunofluorescence ANA staining were detected in the DTg mice (not shown). We next evaluated the relative levels of IgG anti-RNA subclass antibodies in older TLR7 and DTg mice. First, in TLR7 Tg mice, we observed that the concentrations of total IgG anti-RNA (Fig. 3a) and IgG2b anti-RNA were similar whereas the other subclasses showed either no (IgG3) or only modest (IgG1 and IgG2c) elevations compared to B6 mice (Fig. 3b). When we compared subclass levels between TLR7 and DTg mice, there was a modest, but statistically significant increase in IgG1 anti-RNA in DTg compared to TLR7 Tg mice but no other statistical differences in subclass distribution (Fig. 3b). We have previously used the ratio between IgG1 and 2a (c) as an indirect measure of CD4 Th cell skewing and nephrogenicity (13, 15). As shown in Fig. 3c, there was a significant reduction in the IgG2c/IgG1 ratio in DTg compared to TLR7 Tg mice. Together, these findings suggest an alteration in Th skewing affecting subclass distribution in DTg mice but that the Th1 associated subclass, IgG2b (16), remained the dominant anti-RNA subclass in both TLR7 Tg and DTg mice.

Figure 3. IgG anti-RNA autoantibodies persist in DTg mice but show alterations in subclass distributions and DTg mice have a significant improvement in survival.

TLR7 and DTg mice (n=25 and 19 mice respectively) were bled monthly. Total IgG (a) and IgG subclass (b, at 10–12 months of age) anti-RNA antibodies were quantified by ELISA as described in Methods. Anti-RNA mAb were used to construct standard curves for total IgG and IgG2b anti-RNA and the results expressed as μg equivalents. For IgG1, IgG2c and IgG3, no anti-RNA mAb were available so results are expressed as O.D. Since IgG3 anti-RNA was uniformly low in TLR7 mice, a serum from H564 mouse was used as a positive control (Δ). Differences in anti-RNA antibody levels between TLR7 and DTg were not statistically significant except where indicated. c. The same mice as in a. were followed for survival and the Kaplan-Meier survival curve plotted over a 12 month period. Significance was determined by the logrank test using GraphPad Prizm software.

RNase overexpression leads to improvement in survival and reduced immune deposits in the kidneys in TLR7 Tg mice

Since we had observed improvement in some, but not all, measures of immune function in DTg mice, we compared survival between DTg and TLR7.1 littermate controls in a 2^nd large cohort of mice. As shown in Fig. 3c, there was a highly significant difference in survival of DTg mice compared to littermate controls. At 7 months, 50% of TLR7.1 littermate controls had died whereas only 13% of DTg were dead. This finding indicates that despite the lack of effect on anti-RNA antibody titers in this strain, overexpression of RNase exerted a strong therapeutic effect.

The reasons why TLR7.1 mice die prematurely is not entirely clear although severe anemia, thrombocytopenia and glomerulonephritis could play a part (6). To determine whether red cell and platelet counts were positively impacted by RNase therapy, we performed blood counts but found no significant differences between in the two strains (results not shown). With regard to renal function, <10% of mice had >1+ proteinuria over the time of observation and there were no significant differences in proteinuria between DTg and TLR7 Tg mice (not shown).

Histologic sections from kidney tissue at 14–16 weeks stained with the Periodic Acid Schiff (PAS) reagent showed mild expansion of mesangial regions that was qualitatively similar between TLR7 and DTg mice. Glomeruli were without prominent inflammatory cell infiltration, sclerosis, hypercellularity, or histologic evidence of prominent immune complex deposition such as intracapillary accumulations of “hyaline thrombi” or subendothelial capillary wall deposits of the type seen in severe proliferative lupus nephritis. Although no statistically significant differences in IgG and C3 deposition was observed by indirect immunofluorescence in DTg mice at 3½ months, we observed significantly less IgG and C3 deposition in mice sacrificed at the termination of the survival study (Fig. 4). Semiquantitative analysis of immunofluorescence staining (scale 0–4) was total IgG: 2.8 +/− 0.15 and 1.2 +/− 0.15; C3 2.8 +/−0.14 and 1.6 +/−0.24 for TLR7 Tg and DTg respectively (p<0.005 for both IgG and C3, n=7–9 mice per group). In view of alterations in the serum concentrations of anti-RNA subclass antibodies in DTg mice, we examined subclass distribution of IgG in their kidneys by antibodies validated in a previous study (13). IgG1 and IgG3 staining was similar to the B6 control staining (not shown). However IgG2b and IgG2c were consistently detected in TLR7 Tg mice but significantly reduced in intensity in the DTg mice (Fig. 4). Semiquantitative analysis was IgGb: 2.2 +/− 0.13 and 0.8 +/−0.06; IgG2c 2.3 +/− 0.26 and 0.8 +/−0.14 for TLR7 Tg and DTg respectively (n=4–7 mice per group, p<0.005 for both). In summary, TLR7.1 DTg mice survived longer than their single Tg counterparts and had a reduction in total IgG and C3 deposition in their kidneys associated with reduced deposits of the complement fixing isotypes, IgG2b and IgGc at a late time point in their disease.

Figure 4.

Representative frozen sections from kidneys obtained from mice aged 12 months (n= 4–9 mice per group) were stained by indirect immunofluorescence for IgG, IgG subclasses and C3. Glomerular staining for C3 is delineated by white arrowheads. Semiquantitative assessment of staining intensity is reported in Results. IgG1 and IgG3 staining was minimal and not different between B6, TLR7 and DTg mice (not shown).

RNase overexpression rescues TLR7 Tg mice from severe hepatic inflammation

Recently, Fukui et al (17) engineered a mutation, D34A, that leads to a selective loss of TLR9 binding but enhanced TLR7 binding to Unc93B1 resulting in markedly enhanced TLR7 signaling. The most striking pathology in the D34A mutant was observed in the liver where severe inflammation and patchy necrosis was observed (17). Some inflammation in the liver, but not the lung, was previously noted in TLR7 Tg mice (6). Since the hepatic inflammation and necrosis was considered the most likely cause of death in Unc93B D34A mutant mice and we also detected elevated liver transaminases in TLR7 Tg moribund mice as did Fukui et al (17), we compared the liver pathology in TLR7 single and double Tg mice.

Hepatic lesions in TLR7 Tg mice aged 12–14 weeks were distinct and typically characterized by large, dense and frequently confluent accumulations of primarily mononuclear inflammatory cells within portal and periportal locations but also, on occasion, surrounding central veins (Fig. 5 and Suppl Table 1). In portal regions, inflammatory cell accumulation was accompanied by disruption of the limiting plate and replacement of adjacent hepatic parenchymal cells by dense, F4/80 antigen positive accumulations of tissue macrophages intermixed with lymphocytes, some fibroblasts and neutrophils (Fig. 5B, E, K, L). Hemosiderin laden macrophages and pooled erythrocytes were also common in many inflammatory foci suggesting chronic microvascular disruption. In some areas, adjacent portal triads were bridged or partially bridged by this chronic/active inflammatory process. Bile duct hyperplasia was common within severely affected portal regions (Fig. 5E) of these mice. Hepatocellular apoptosis and necrosis typically involved individual cells within the region of the limiting plate and at the margin of larger inflammatory foci (Fig. 5E, G). In contrast, DTg mice had substantially less severe lesions as evidenced by the presence of a lower lesion severity score and fewer inflammatory cell foci compared to TLR7 Tg mice (Fig. 5C, F, M and Suppl Table 1). The lessened severity of the lesions was due to smaller, more widely scattered inflammatory cell accumulations and mild to nonexistent bile duct hyperplasia or bridging of portal regions. Scattered foci of extramedullary hematopoiesis were present within livers of both TLR 7.1 and DTg mice - whereas cell death was much more prominent in TLR7 Tg mice as determined by staining with antibody to activated caspase3 (Fig. 5H–J). TLR7 Tg and DTg mice had 3.45 +/− 1 versus 0.85+/−0.51 caspase 3 positive cells per mm² respectively, p=0.04).

Figure 5. DTg mice have a marked reduction in hepatic inflammation and necrosis relative to TLR7 Tg mice.

Representative liver sections from control (B6), TLR7.1 and DTg mice. Sections of WT liver (A-10x; D-40x) showing normal hepatic structure. A normal portal triad is centrally placed in plate D. Liver from TLR7 Tg mice demonstrates bridging between triads by highly cellular inflammatory and stromal elements (B-10x). At 40x (E) portal triads are fully surrounded by tissue macrophages (some of which contain brown hemosiderin pigment) admixed with lymphocytes, neutrophils and fibrous tissue elements. Bile duct hyperplasia (b), limiting plate necrosis and apoptosis of individual hepatocytes occur in severely affected triads (E). DTg mice have a similar type of inflammatory hepatitis, although much less severe and rarely bridges adjacent triads (C-10x; F-40x). Similarly, bile duct hyperplasia (b), limiting plate necrosis and apoptosis are less common and milder in degree. The lower severity of hepatic inflammation in DTg mice is reflected by a lower lesion severity score of 2.1+/−1.1 (compared to 4.3 +/− 0.8 for TLR7 Tg, n=6 per group, p=0.006) and fewer inflammatory cell foci (8.2+/−5.8 per random 10X field compared to 32.6 +/− 5.8 for TLR7 Tg mice, p=0.001). See Methods for definitions and Supplementary Table 1 for analysis of individual mice. Classical shrunken apoptotic hepatocytes (arrows) are common typically near margins of inflammatory foci (G-40x) and revealed by staining with antibody to activated caspase-3 (light brown in H–J, 40x). F4/80 staining of the inflammatory foci in TLR7.1 mice (B-10x; E-40x and K-10x) revealed intense staining surrounding triads and central veins (dark brown, L-10x).

Inflammatory monocytes produce TNF in response to TLR7 stimulation

Since myeloid cells were prominent in the liver infiltrates as determined by positive staining with F4/80 (Fig. 5L), we first addressed whether TLR7 expression was increased in peripheral myeloid cells. We found that flow sorted splenic inflammatory monocytes (CD11bhigh Ly6Chigh Ly6Gnegative) and neutrophils (CD11bhigh Ly6Ghigh Ly6Chigh) obtained from TLR7 Tg mice, expressed 5–10-fold more TLR7 mRNA compared to WT B6 mice, equivalent to TLR7 expression levels in pDC (18) or B cells (6) in this strain. Of considerable interest, the splenic neutrophils, but even more so the inflammatory monocytes defined by surface markers described in Methods, expressed much higher levels of genes encoding granule proteins associated with neutrophils: myeloperoxidase, cathepsin G, proteinase 3 and elastase compared to wild type (Fig. 6a).

Figure 6. Myeloid cells from TLR7 Tg mice express high levels of proteases and produce TNF on activation.

a. Splenic neutrophils and inflammatory monocytes were purified by flow cytometry, RNA isolated and QPCR for the genes shown performed on mice aged 3 months as described in Methods. Results are expressed relative to the ribosomal 18S RNA control. b. Total spleen cells from WT and TLR7Tg mice were stimulated with the TLR7 or TLR4 ligands, gardiquimod (800 ng/ml) or LPS (10 ng/ml) respectively. After 3 hrs, cells were stained with mAb against CD11b, Ly6C, Ly6G and TNF. Inflammatory monocytes (CD11bhigh Ly6Chigh Ly6Gnegative) and neutrophils (CD11bhigh Ly6Ghigh Ly6Chigh) were identified (left panel) and the intracellular expression of TNF in the populations examined by flow cytometry. A representative result is shown in the middle panel (where the percentage positive refers to TNF positive in that cell type) and the average of 3 experiments in the right panel. c. QPCR for TNF was compared in 10 week old WT, TLR7 Tg and DTg mice. * p<0.05.

In view of the prominent myeloid expansion, liver infiltration and high TLR7 expression in myeloid cells in TLR7 Tg mice, we asked whether myeloid cells from TLR7 Tg mice responded abnormally to TLR7 agonists. We focused on TNF rather than IFN-a because TLR7 Tg mice show only modest increases in interferon response genes (see Fig. 5 in (6) and TNF rather than type 1 IFN is strongly associated with liver inflammation and hepatocyte death as demonstrated in many other situations (19, 20). Inflammatory monocytes, but not neutrophils, from bone marrow and spleen obtained from TLR7 Tg mice responded with significantly higher levels of TNF compared to WT cells following stimulation with gardiquimod (TLR7 agonist) but not LPS (TLR4 agonist) (Fig. 6b). Similar findings were observed with inflammatory monocytes obtain from DTg mice (not shown). No difference in IL-6 expression was observed (not shown). Significantly, TNF mRNA expression was elevated in the livers of TLR7 Tg mice but significantly reduced in DTg mice (Fig. 6c). Together, these findings implicate TNF in liver injury and show reduced expression of this cytokine in DTg mice.

Discussion

We observed that when the lupus-prone mouse strain, TLR7 Tg, overexpressed RNase, it was partially protected from inflammation in the kidney and, more strikingly, in the liver and had a significant improvement in survival. Overexpression of RNase itself had no obvious adverse effects as determined by clinical manifestations or early mortality in RNase Tg mice. Furthermore, detailed evaluation of immune cellular composition in this strain appeared to be normal. Although variant forms of RNase such as frog (Rana pipiens) RNase (Onconase) have been used as a chemotherapeutic drug in certain types of cancer (21), RNase A, that is normally present in the circulation, is not cytotoxic (22). This is explained by differences in the binding of Onconase and RNase A to the cell surface membrane as well as the fact that RNase A, but not Onconase, is bound by the cytosolic RNase inhibitor (RNAI) with femtomolar affinity and efficiently neutralized (23).

Unc93B1 is an endoplasmic reticulum resident protein that controls TLR3, 7 and 9 transport as evidenced by the loss of function of these TLRs in ‘3d’ mice with the H412R missense mutation (24). In contrast, the D34A mutation results in a loss of ligand binding to TLR9, but increased activation of TLR7 (17). The striking similarity between the phenotype of mice with the Unc93B1 D34A mutation and the TLR7 Tg mice used in the current study, including myeloid expansion, anemia, thrombocytopenia and mild glomerulonephritis, indicates that it is the enhanced response to TLR7 ligand rather than overexpression of TLR7 that causes disease in these genetically altered strains of mice.

Similar to findings in the Unc93B1 D34A mice (17), we observed severe inflammation and patchy necrosis in the livers of TLR7 Tg mice which was likely the major contributor to death. In the DTg mice however, hepatic inflammation was markedly attenuated and fewer dying cells were detected. Whether local overexpression of RNase by hepatocytes is necessary for the beneficial effects of the enzyme in the liver will need to be tested in the future by alternative modes of RNase delivery.

What accounts for the severe hepatic injury in TLR7 Tg mice? Behrens et al (25) recently showed that repetitive TLR9 stimulation causes macrophage activation and hepatitis and that this syndrome was predominately caused by innate immune system activation. Since myeloid cells were prominent in the liver infiltrates, these cells are likely major contributors to liver injury. Significantly, we observed that both inflammatory monocytes and neutrophils isolated in the periphery expressed 5–10-fold more TLR7 mRNA compared to WT B6 mice, indicating that they may be more sensitive to TLR7 ligands. Indeed, inflammatory monocytes from TLR7 Tg mice produced significantly higher levels of TNF following stimulation with a TLR7 ligand. While TNF alone may not be sufficient to kill hepatocytes, it primes Kupffer cells and neutrophils to release cytotoxic mediators (26, 27) and is strongly implicated in hepatic injury in ischaemia reperfusion injury, (28). Consistent with the reduction in inflammation in the livers of DTg mice, TNF mRNA expression was significantly reduced in the livers of DTg mice implying that a reduction in the physiologic ligand for TLR7, RNA, led to both reduced expansion and activation of myeloid cells in the livers of DTg mice.

Precisely how innate immune cells are activated in the liver of TLR7 Tg mice remains to be determined. Dying hepatocytes as a potential source of RNA were readily demonstrated in the livers of TLR7 Tg mice so they likely perpetuate TLR7 activation. Neutrophils, that were readily identifiable by their characteristic morphology release both DNA and RNA which, when bound to cathelicidin (LL37), are potent stimulators of TLR9 and TLR7 respectively (29). Finally, apoptotic CD8 T cells that also die in the liver (30) may contribute to the load of dying cells in the liver and stimulate cytokine production in cells that are hyperresponsive to TLR7.

Of considerable interest, both the inflammatory monocytes as well as the neutrophils from TLR7 Tg mice expressed much higher levels of mRNA encoding granulocyte proteases compared to wild type control mice. This signature is reminiscent of immature monocytes emerging from the bone marrow (www.immgen.org) and likely reflects abnormal myelopoiesis (18). Of clinical relevance, PBMC from SLE patients express a granulocyte signature which is explained by the presence of a low density granulocyte (LDG) fraction that comprises a mixed population of early granulocyte/monocyte precursors (myelocytes) (31, 32). These LDG produce inflammatory cytokines, including TNF, and are implicated in vascular cytotoxicity in SLE (32). Thus the inflammatory monocyte population described here, bears many similarities to the LDG subpopulation in SLE.

A surprising finding in this study was that there was no statistically significant change in anti-RNA antibody activity over time in DTg mice. This finding may be due to the fact that anti-RNA autoantibodies in TLR7.1 mice are only very modestly elevated (6) and varied considerably between mice. Low and variable levels of anti-RNA autoantibodies are also consistent with our findings that kidney damage was very mild in the TLR7 Tg mice. Although glomerulonephritis was previously thought to be an important contributor to death in TLR7 Tg mice, we observed that very few of these mice had evidence of severe nephritis on histology or impaired function as determined by proteinuria. Nevertheless, the reduction in immune deposits at late time points in DTg mice without a change in the total circulating anti-RNA autoantibodies titers suggested that either a different antibody specificity deposits in the glomeruli, that the circulating RNase may partially degrade the immune complex rendering it less efficient at tissue deposition and complement fixation and/or that there was a change in anti-RNA isotype.

A recent study by one of us (SB) determined that B cell activation in TLR7 Tg mice has a B cell intrinsic component but can also be driven by T cells (33). Transcript expression studies of TLR7 Tg follicular B cells revealed increased expression of IgG2b but not IgG2a(c) (33). Consistent with this observation, we found that most serum anti-RNA antibodies belong to the IgG2b subclass. When we compared subclass distribution of anti-RNA antibodies between TLR7 and DTg mice, we observed an increase in the IgG1 subclass and reduction in the IgG2c/IgG1 ratio of anti-RNA in the DTg mice suggesting a change in the Th1 to Th2 cell autoantibody drive. Coupled with the reduced B and T cell activation observed in DTg mice, these findings are consistent with partial degradation of antigen by RNase leading to alteration in TLR7 stimulation and possibly the affinity/avidity of antigen receptors impacting B cell maturation (34) and/or Th cell skewing (35, 36). We have, in fact, observed changes in B cell maturation in the spleens of DTg mice (manuscript in preparation). No significant alteration in the levels of IgG2b, 2c or 3 anti-RNA antibodies between TLR7 and DTg mice were seen. Since both IgG2c and IgG2b are considered to be driven by Th1 cells (16), yet the IgG2b subclass remained elevated in DTg mice, it suggests that other cytokines such as TGF-b and/or IL-6 may play a role in stimulating this autoantibody (37). Whereas IL-6 and TGF-b promote differentiation of Th17 cells, IL-17 deficiency did not reduce autoantibody production in TLR7 Tg mice (33). Future studies will be needed to address which APC (macrophage, DC, B cell), what cytokines and T cell subsets drive anti-RNA and how antigen degradation by RNase impact each of these components of the autoimmune response.

When we examined the subclasses deposited in the kidneys in older mice, we observed that there was very little deposition of IgG1 and 3 in either the TLR7 or DTg strains, but that there was a significant reduction in IgG2b and IgG2c deposition in DTg mice. Whereas a decrease in IgG2c staining can be explained by reduced serum levels in some mice (Fig. 3), the reduced renal deposition of IgG2b without a change in serum levels, is most consistent with the idea that that RNase partially degrades the immune complex rendering it less efficient at tissue deposition. Since both IgG2b and c are complement activating subclasses (16), their reduction in glomerular deposits explains the total reduction of IgG and C3 fixation in the kidney.

In conclusion, overexpression of RNase exerts a strong protective effect in a TLR7 driven mouse model with lupus-like features and liver inflammation and death. DTg mice had a reduction in spleen size, reduced myeloid cell expansion and reduced activation of B and T cells compared to TLR7 Tg mice. These observations provide evidence that prior to activation of endosomal TLR7, the RNA ligand is accessible to extracellular RNase. Precisely where RNA is released to impact lymphocyte function is uncertain, although ongoing studies suggest that one site is the spleen and influences B cell maturation (manuscript in preparation). These findings raise the possibility that treatment of established lupus-like or inflammatory liver disease with therapeutics to degrade RNA will be an effective strategy for treatment of SLE and other disorders where inflammation is driven by RNA that not only activates TLR7, but also TLR3, TLR8 and the RIG-I family (RLRs) (38).

Abbreviations used

ANA

antinuclear antibody

DTg

double transgenic

IC

immune complex

SRED

single radial enzyme diffusion

SLE

systemic lupus erythematosus

Tg

transgenic