Histone deacetylase inhibitors modulate renal disease in the MRL-lpr/lpr mouse (original) (raw)
TSA and SAHA decrease expression of IFN-γ mRNA and protein. Splenocytes from diseased (24-week-old) MRL-lpr/lpr mice spontaneously express high concentrations of specific cytokines, including IFN-γ, IL-12, IL-6, and IL-10, compared with younger (10-week-old) mice (27). For this reason, splenocytes derived from mice at 24 weeks were used for concentration-response analysis of unstimulated cells. To determine whether TSA can downregulate spontaneously produced IFN-γ mRNA, splenocytes from diseased (24-week-old) MRL-lpr/lpr mice were treated with 0–500 ng/ml of TSA over 18 hours. The 18-hour time interval was selected based on our recent data demonstrating optimal downregulation of CD154 and IL-10 transcripts in human SLE T cells by TSA at 18 hours (16). TSA at low concentrations (100 ng/ml) had little effect or even possibly increased IFN-γ mRNA levels as measured by semiquantitative RT-PCR. At 300 ng/ml TSA, there was downregulation of IFN-γ mRNA, while at 500 ng/ml TSA, suppression of IFN-γ transcription was almost complete (Figure 1a).
Downregulation of IFN-γ transcript and protein levels by TSA and SAHA. (a) TSA (300–500 ng/ml) decreases the level of IFN-γ mRNA relative to GAPDH mRNA in splenocytes from 24-week-old MRL-lpr/lpr mice. (b) Splenocytes from 10-week-old MRL-lpr/lpr mice were incubated in the absence or presence of 300 ng/ml TSA or 10 μM SAHA for 18 hours. Splenocytes were then stimulated with ConA (10 μg/ml) for 18 hours. In lanes 3 and 5, TSA or SAHA was added at the same time as ConA and cultured for 18 hours. IFN-γ mRNA levels relative to GAPDH are shown. (c) Based on densitometric scanning of the gel in b, this graph depicts the fold change of IFN-γ mRNA in cells cultured as described above. A representative of three independent experiments is shown. (d) Splenocytes from 10-week-old mice were cultured in the presence of vehicle, LPS, LPS plus TSA, LPS plus SAHA, ConA, ConA plus TSA, or ConA plus SAHA for 72 hours. The concentrations of TSA and SAHA were 300 ng/ml and 10 μM, respectively. This graph depicts the amount of IFN-γ protein secretion. The bar represents the mean ± SEM of three independent experiments.
To determine whether TSA or SAHA downregulates ConA-induced IFN-γ mRNA expression, splenocytes from younger, predisease 10-week-old MRL-lpr/lpr mice were stimulated for 18 hours with the mitogen. Splenocyte mitogen activation upregulated IFN-γ mRNA content approximately 2.5-fold but did not alter GAPDH mRNA expression (Figure 1b, lane 1 vs. lane 2, and Figure 1c). In contrast, when splenocytes were preincubated with TSA or SAHA for 18 hours, upregulation of IFN-γ transcript was markedly attenuated compared with cells not exposed to these inhibitors. Concentration-titration experiments revealed that the optimal concentration of SAHA for IFN-γ transcript suppression was 10 μM (data not shown). Thus, both TSA and SAHA inhibit IFN-γ mRNA upregulation by splenocytes from 10-week-old predisease MRL-lpr/lpr mice in response to mitogenic stimulation.
The marked decrement of IFN-γ transcript by these inhibitors prompted us to quantify IFN-γ protein secretion in splenocyte culture supernatants. Over 72 hours, ConA, but not LPS, stimulated splenocytes from 10-week-old MRL-lpr/lpr mice to secrete a mean (± SEM) 1,563.2 ± 88.3 pg/ml of IFN-γ (Figure 1d). ConA-stimulated splenocytes cultured in the presence of either 10 μM SAHA or 300 ng/ml TSA did not secrete any detectable IFN-γ protein (Figure 1d). Thus, inhibition of IFN-γ transcription by TSA and SAHA blocks synthesis and secretion of IFN-γ protein by mitogen-stimulated splenocytes from 10-week-old MRL-lpr/lpr mice.
TSA and SAHA downregulate expression of IL-12p40 and IL-12p35 mRNA, and IL-12p40 protein. We next investigated whether TSA downregulates transcription of IL-12 assaying for both IL-12p35 and IL-12p40 subunits. Splenocytes from diseased, 24-week-old MRL-lpr/lpr mice had constitutive expression of both IL-12 subunit transcripts (Figure 2a). Concentrations of TSA greater than 100 ng/ml completely suppressed both transcripts (Figure 2a).
Downregulation of IL-12 transcript and protein levels by TSA and SAHA. (a) Increasing concentrations of TSA (0–500 ng/ml) progressively decrease levels of IL-12p40 and IL-12p35 mRNA relative to GAPDH mRNA in splenocytes from 24-week-old MRL-lpr/lpr mice. (b) Splenocytes from 10-week-old MRL-lpr/lpr mice were incubated in the absence or presence of 300 ng/ml TSA or 10 μM SAHA for 18 hours. Splenocytes were then stimulated with LPS (100 ng/ml) plus IFN-γ (100 IU/ml) for 6 or 18 hours. IL-12p40 and IL-12p35 mRNA levels relative to GAPDH are shown. (c and d) Based on densitometric scanning of the gel in b, these graphs depict the fold change of IL-12p40 (c) and IL-12p35 (d) mRNA in cells cultured as described above. A representative of three independent experiments is shown. (e) Splenocytes from 10-week-old mice were cultured in the presence of vehicle, TSA, LPS plus IFN-γ, LPS plus IFN-γ plus TSA, LPS plus IFN-γ plus SAHA, or SAHA for 24 hours. The concentrations of TSA and SAHA were 300 ng/ml and 10 μM, respectively. This graph depicts the amount of IL-12p40 protein secretion. The bar represents the mean ± SEM of three independent experiments.
To address whether TSA and or SAHA downregulates LPS- and IFN-γ–induced IL-12p35 and p40 transcripts, splenocytes from 10-week-old MRL-lpr/lpr mice were stimulated in the presence or absence of TSA and SAHA for 18 hours. Splenocytes from younger mice were used because these cells express little if any constitutive IL-12p35 and p40 transcripts compared with splenocytes from older mice (Figure 2b, lane 1). Splenocytes were then stimulated with LPS and IFN-γ for 6 and 18 hours, respectively. Figure 2b demonstrates that, at 6 hours, there was a 9- and 3.5-fold increase of IL-12p40 and p35 transcripts, respectively, in the absence of the inhibitors relative to GAPDH transcript, as measured by densitometry. Similarly, at 18 hours, there was a 6- and 1.5-fold increase of IL-12p40 and p35 mRNA, respectively, relative to GAPDH mRNA in the absence of inhibitors. When splenocytes from 10-week-old mice were activated in the presence of 300 ng/ml TSA or 10 μM SAHA, IL-12p35 and p40 transcripts were undetectable (Figure 2, b–d).
In the absence of stimulation, splenocytes from 10-week-old MRL-lpr/lpr mice did not secrete detectable IL-12p40 protein during 24 hours. When splenocytes were activated with LPS and IFN-γ for 24 hours, the mean (± SEM) IL-12p40 secretion was 45.3 ± 9.1 pg/ml. However, preincubation of the splenocytes with TSA and SAHA resulted in significantly lower IL-12p40 protein production over 24 hours (mean ± SEM TSA, 1.5 ± 1.4 pg/ml; SAHA, 9.1 ± 1.9 pg/ml; P = 0.003 by ANOVA) (Figure 2e). Taken together, these results demonstrate that inhibition of p40 transcripts by TSA and SAHA leads to marked downregulation of IL-12p40 protein secretion by stimulated MRL-lpr/lpr splenocytes.
TSA and SAHA downregulate expression of IL-6 mRNA and protein. To determine whether TSA or SAHA effects Th2 cytokine production, splenocytes from diseased, 24-week-old MRL-lpr/lpr mice were treated with increasing concentrations of TSA over 18 hours. As shown in Figure 3a, lane 1, significantly increased, constitutive IL-6 transcript was present in splenocytes from 24-week-old mice (28) compared with 10-week-old mice (Figure 3b, lane 1). As shown in Figure 3a, TSA downregulated IL-6 mRNA in unstimulated splenocytes from 24-week-old MRL-lpr/lpr mice beginning at a concentration of 100 ng/ml.
Downregulation of IL-6 transcript and protein levels by TSA and SAHA. (a) Increasing concentrations of TSA (0–500 ng/ml) progressively decrease levels of IL-6 relative to GAPDH mRNA in splenocytes from 24-week-old MRL-lpr/lpr mice. (b) Splenocytes from 10-week-old MRL-lpr/lpr mice were incubated in the absence or presence of 300 ng/ml TSA or 10 μM SAHA for 18 hours. Splenocytes were then stimulated with LPS (100 ng/ml) plus IFN-γ (100 IU/ml) for 6 or 18 hours. IL-6 mRNA levels relative to GAPDH are shown. (c) Based on densitometric scanning of the gel in b, this graph depicts the fold change of IL-6 mRNA in cells cultured as described above. A representative of three independent experiments is shown. (d) Splenocytes from 10-week-old mice were cultured in the presence of vehicle, LPS plus IFN-γ, LPS plus IFN-γ plus TSA, or LPS plus IFN-γ plus SAHA for 72 hours. The concentrations of TSA and SAHA were 300 ng/ml and 10 μM, respectively. This graph depicts the amount of IL-6 protein secretion. The bar represents the mean ± SEM of three independent experiments.
To address whether TSA and SAHA downregulate LPS- and IFN-γ−induced IL-6 transcript content, splenocytes from 10-week-old MRL-lpr/lpr mice were treated in the absence or presence of TSA or SAHA for 18 hours before LPS and IFN-γ stimulation for 6 and 18 hours, respectively. As depicted in Figure 3, b and c, IL-6 mRNA was upregulated 18-fold at 6 hours and tenfold at 18 hours relative to GAPDH. In contrast, there was no detectable IL-6 mRNA present in TSA- or SAHA-pretreated splenocytes from 10-week-old mice (Figure 3b, lanes 4–7). The downregulation of IL-6 mRNA was paralleled by a lack of detectable secretion of IL-6 protein in culture supernatants of splenocytes from 10-week-old mice (Figure 3d). When splenocytes from 10-week-old mice were cultured with LPS and IFN-γ for 72 hours, there was a mean (± SEM) 34.4 ± 7.6 pg/ml of IL-6 protein present in culture supernatants. Addition of TSA or SAHA with LPS and IFN-γ for 72 hours completely blocked IL-6 protein secretion. Taken together, these results demonstrate that both TSA and SAHA downregulate IL-6 mRNA transcription and IL-6 protein secretion by stimulated MRL-lpr/lpr splenocytes from 10-week-old mice.
TSA and SAHA decrease expression of IL-10 mRNA and protein. To investigate whether TSA downregulates other Th2 cytokines, such as IL-10, splenocytes from 24-week-old MRL-lpr/lpr mice were treated with increasing concentrations of TSA. TSA downregulated IL-10 mRNA at a concentration of 300 ng/ml (Figure 4a). To address whether TSA and SAHA downregulate LPS- and IFN-γ–induced IL-10 transcript content, splenocytes from predisease, 10-week-old MRL-lpr/lpr mice were treated in the absence or presence of TSA and SAHA for 18 hours before LPS and IFN-γ stimulation for 6 and 18 hours, respectively. Figure 4, b and c, demonstrates that IL-10 mRNA was upregulated approximately fivefold at 6 hours and approximately threefold at 18 hours relative to GAPDH transcript. There was no detectable IL-10 mRNA when the splenocytes were pretreated with either TSA or SAHA (Figure 4b, lanes 4–7).
Downregulation of IL-10 transcript and protein levels by TSA and SAHA. (a) TSA (300–500 ng/ml) decreases the levels of IL-10 mRNA relative to GAPDH mRNA in splenocytes from 24-week-old MRL-lpr/lpr mice. (b) Splenocytes from 10-week-old MRL-lpr/lpr mice were incubated in the absence or presence of 300 ng/ml TSA or 10 μM SAHA for 18 hours. Splenocytes were then stimulated with LPS (100 ng/ml) plus IFN-γ (100 IU/ml) for 6 or 18 hours. IL-10 mRNA levels relative to GAPDH are shown. (c) Based on densitometric scanning of the gel in b, this graph depicts the fold change of IL-10 mRNA in cells cultured as described above. A representative of three independent experiments is shown. (d) Splenocytes from 10-week-old mice were cultured for 72 hours in the presence of vehicle, LPS, LPS plus TSA, LPS plus SAHA, ConA, ConA plus TSA, ConA plus SAHA, LPS plus IFN-γ, LPS plus IFN-γ plus TSA, or LPS plus IFN-γ plus SAHA. The concentrations of TSA and SAHA were 300 ng/ml and 10 μM, respectively. This graph depicts the amount of IL-10 protein secretion. The bar represents the mean ± SEM of three independent experiments.
To determine the effect of TSA and SAHA on IL-10 protein secretion, splenocytes from 10-week-old MRL-lpr/lpr mice were stimulated with ConA, LPS, or LPS plus IFN-γ for 72 hours in the absence or presence of TSA or SAHA. As shown in Figure 4d, splenocytes stimulated with ConA, LPS, or LPS plus IFN-γ secreted a mean (± SEM) of 31.8 ± 4.5, 32.3 ± 1.9, or 34.8 ± 4.9 pg/ml, respectively, of IL-10 protein. When cells were pretreated with TSA or SAHA, there was no detectable IL-10 secretion. Taken together, these results reveal that both TSA and SAHA downregulate induced IL-10 mRNA transcription and protein secretion by splenocytes from 10-week-old MRL-lpr/lpr mice.
TSA and SAHA induce accumulation of acetylated histones. We propose that one mechanism by which HDIs suppress transcription of cytokine genes in MRL-lpr/lpr splenocytes is by accumulation of acetylated histones, resulting in chromatin remodeling. To test this possibility, we quantified acetylated histones H3 and H4 in HDI-treated splenocytes. Splenocytes from 10-week-old MRL-lpr/lpr mice were cultured with increasing concentrations of TSA or SAHA for 18 hours; nuclear histones were isolated and separated by SDS-PAGE, and transferred to Immobilon for immunoblotting with anti–acetylated H3 and H4 Ab’s. In Figure 5, immunoblot analysis of H3 and H4 histones prior to incubation with TSA or SAHA revealed background acetylation. Exposure of cells to TSA or SAHA for 18 hours resulted in approximately three- or twofold increased accumulation of acetylated H3 and H4 histones, respectively. Coomassie blue staining of the gel revealed no change in total H3 and H4 histones when cells were exposed to TSA or SAHA. These findings indicate that inhibition of HDACs by TSA and SAHA increases acetylation of H3 and H4 histones in splenocytes from MRL-lpr/lpr mice.
Western immunoblot analysis of acetylated histones H3 and H4 in 24-week-old MRL-lpr/lpr splenocytes. Histones were isolated by acid extraction from cells cultured with different doses of TSA or SAHA for 18 hours. Acetylation was detected using anti–acetylated H3 and H4 Ab’s. A parallel gel stained with Coomassie blue is shown as a control for protein loading in each lane (66–68).
TSA and SAHA do not increase cell death. To determine whether increasing doses of TSA and SAHA decrease cell viability, thus explaining the inhibition of cytokine production, splenocytes from 24-week-old mice were cultured for 24 hours with increasing concentrations of TSA (50–500 ng/ml) or SAHA (5–10 μM). The cell viability was determined by trypan blue exclusion, MTT assay, and cell cycle analysis. The effect of different concentrations of TSA and SAHA on splenocyte cell viability is summarized in Figure 6. There was no evidence for decreased cell viability in TSA- or SAHA-treated cells compared with vehicle-treated cells, even at the highest concentrations tested.
Effects of different concentrations of TSA or SAHA on cell viability. Cell viability was assessed by trypan blue exclusion (a), MTT method (b), and cell cycle analysis by propidium iodide staining (c). The bars represent the mean ± SD of triplicates.
TSA treatment of MRL-lpr/lpr mice decreases spleen weight. Based on in vitro evidence that TSA downregulates Th1 and Th2 cytokine production by MRL-lpr/lpr splenocytes, we tested the hypothesis that TSA would delay or prevent the progression of lupus activity. We assessed several indicators of lupus activity that reflect the underlying immunopathology of the disease. The first was spleen size, because MRL-lpr/lpr mice develop massive splenomegaly and lymphadenopathy with disease progression. At age 19 weeks, five animals from each group were sacrificed, and the mean spleen weights were determined. Compared with vehicle-treated controls (0.41 ± 0.06 g), TSA-treated mice had significantly smaller spleens (0.28 ± 0.08 g, P < 0.05) (Figure 7).
Spleen weights of MRL-lpr/lpr mice receiving daily injections of either TSA (0.5 mg/kg BW in DMSO) or vehicle (DMSO) for 5 weeks beginning at 14 weeks of age (n = 5, P < 0.05).
TSA decreases the number of CD3+B220+ cells in spleen. To investigate whether TSA treatment differentially affects splenocyte subsets that might account for the decrease in spleen size in TSA-treated mice, we initially assessed changes in splenic architecture by standard evaluation of H&E-stained frozen splenic sections. No differences in overall splenic architecture were noted between the two groups (data not shown). We next determined by immunofluorescence whether TSA treatment affected the percentage of specific T cell subsets in the spleen, using mean fluorescence as an indicator of the presence of a particular cell type. We observed a trend toward increased fluorescence for CD4, CD8, and CD19 in the spleens of the TSA treated group, but no significant differences between the two groups as to mean fluorescence. Using confocal microscopic colocalization of cells that were both CD3+ and B220+, there was an overall reduction in the percentage of double-positive cells (control 26.1% ± 3% vs. TSA 12.4% ± 7%, P < 0.05). We interpret these findings to indicate, based on spleen size, that there was an overall reduction in the number of all splenic cellular subsets, and that there was a specific reduction in the number of CD3+/B220+ T cells.
TSA has no effect on autoantibody production. MRL-lpr/lpr mice develop hypergammaglobulinemia and produce high concentrations of serum autoantibodies directed against several autoantigens, including dsDNA and GBM. To determine whether treatment with TSA modifies autoantibody production, serum levels of anti-dsDNA and anti-GBM autoantibodies were quantified. The levels of both anti-dsDNA and anti-GBM autoantibodies in TSA-treated mice and controls were similar at ages 14 and 19 weeks (Figure 8, a and b). IgG isotype levels were also not affected by TSA treatment (Figure 8c). Thus, TSA treatment does not affect serum autoantibody production or IgG isotype levels.
Serum anti-dsDNA and anti-GBM Ab levels in MRL-lpr/lpr mice receiving daily injections of either TSA (0.5 mg/kg BW in DMSO) or vehicle (DMSO) measured by ELISA at 14 and 19 weeks of age. (a) Anti-dsDNA levels in sera from MRL-lpr/lpr mice. Data are the OD380 at 1:100 serum dilution in each group with 5 μg/ml double-stranded calf thymus DNA as antigen. (b) Anti-GBM Ab levels in sera from MRL-lpr/lpr mice. Data are the OD380 at 1:100 sera dilution in each group with 50 μg/ml rat GBM as antigen. (c) Total IgG and IgG isotype levels in sera from MRL-lpr/lpr mice.
TSA decreases protein secretion. To determine whether TSA altered the progression of renal disease, we quantified albumin excretion by ELISA in urine specimens collected over 24-hour intervals between ages 14 and 19 weeks. The MRL-lpr/lpr mice in the TSA-treated group at the initiation of treatment at age 14 weeks had a mean urine-albumin excretion of 374 ± 252 μg per mouse per day. At 19 weeks of age, mice treated with TSA had a mean albumin excretion of 94 ± 87 μg per mouse per day (P = 0.009). Thus, TSA treatment decreased proteinuria when given early in disease. In contrast, vehicle-treated 19-week-old mice had a significant increase in albuminuria (765 ± 350 μg/mouse/day) compared with pretreated 14-week-old mice (298 ± 191 μg/mouse/day). These results demonstrate that TSA treatment significantly reverses early disease manifestations and prevents progression of proteinuria with age in MRL-lpr/lpr mice (Figure 9).
Urinary-albumin excretion by MRL-lpr/lpr mice receiving daily injections of either TSA (0.5 mg/kg BW in DMSO) or vehicle (DMSO) for 5 weeks beginning at 14 weeks of age. Data are the 24-hour urinary albumin excretion (μg/mouse/day) in each group. At 19 weeks of age, four of the nine untreated mice had albumin excretion above 1 mg/d, while none of the nine TSA-treated mice had albuminuria above 1 mg/d (P < 0.05 at 19 weeks).
TSA modifies renal pathology and renal score. To further assess effects of TSA treatment on disease progression, kidney sections obtained at necropsy at 19 weeks were examined by standard immunohistologic techniques for evidence of glomerular inflammation, immune complex deposition, and complement fixation. Figure 10a shows a representative kidney section from TSA- and vehicle-treated mice, stained with H&E. The glomeruli from the vehicle-treated mouse show characteristic histopathologic evidence of diffuse proliferative GN, including hypercellularity, mesangial proliferation, and glomerular sclerosis. In contrast, the kidney section from the TSA-treated mouse shows minimal signs of inflammation or cellular proliferation. Based on light-microscopy pathologic assessment, the renal pathology index of TSA-treated mice was significantly lower than that of vehicle-treated mice at 19 weeks of age (Figure 10b). Immunofluorescence microscopy, however, revealed no significant difference in the intensity or distribution of staining for IgG or C3, indicating that treatment with TSA did not prevent immune complex deposition or complement fixation in the glomerulus (Figure 11, a and b). Analysis of IgG isotype deposition in the glomeruli (IgG1, IgG2a, and IgG3) revealed no difference in staining between the two groups (data not shown). Thus the significant decrease in proliferative renal disease was evident in the TSA-treated mice even though TSA did not affect IgG or C3 deposition.
(a) Representative kidney sections stained with H&E from an MRL-lpr/lpr mouse receiving daily injections of either TSA (0.5 mg/kg BW in DMSO) or vehicle (DMSO) for 5 weeks beginning at 14 weeks of age. At the time of sacrifice (19 weeks), the kidneys were removed and then sectioned before staining with H&E. (b) The kidney slides were graded for glomerular inflammation, proliferation, crescent formation, and necrosis. Scores from 0 to 3+ were assigned for each of these features and then added together to yield a final renal score.
Immunohistochemical analysis of IgG and C3 deposition in the kidneys of MRL-lpr/lpr mice receiving daily injections of either TSA (0.5 mg/kg BW in DMSO) or vehicle (DMSO) for 5 weeks beginning at 14 weeks of age. (a) Representative section of a kidney stained for IgG fluorescence. (b) Representative section of a kidney stained for C3 fluorescence.
Toxicity. The toxicity of TSA in vivo was assessed by monitoring of body weight, food intake, and behavior in treated mice compared with control mice. There were no notable differences between the groups. No deaths occurred in either the TSA-treated or the vehicle-group during the treatment period. At the age of 19 weeks, there was no significant difference in the weights of treated (35.6 ± 1.3 g) and control mice (34.4 ± 1.3 g). However, small subcutaneous ulcers developed at the site of injection in both the TSA- and the vehicle-treated groups. These ulcers most likely resulted from DMSO.