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The Journal of Immunology, 2007, 178: 6941-6948.
Copyright © 2007 by The American Association of Immunologists, Inc.
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p300 Protein Acetyltransferase Activity Suppresses Systemic Lupus Erythematosus-Like Autoimmune Disease in Mice1

Nicole Forster*, Sven Gallinat*, Jadwiga Jablonska{dagger}, Siegfried Weiss{dagger}, Hans-Peter Elsässer{ddagger} and Werner Lutz2,*

* Institute of Molecular Biology and Tumor Research, University of Marburg, Marburg, Germany; {dagger} Helmholtz Centre for Infection Research, Molecular Immunology, Braunschweig, Germany; and {ddagger} Institute of Cytobiology and Cytopathology, University of Marburg, Marburg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Conditional knock-in mice expressing a histone acetyltransferase-deficient version of the transcriptional coregulator p300 exclusively in B lymphocytes die prematurely with full penetrance. The mice develop an autoimmune disease similar to systemic lupus erythematosus in its pathological manifestations, such as splenomegaly, glomerulonephritis, vasculitis, deposition of immune complexes, and production of autoantibodies against dsDNA. Aged mice show a severe reduction of transitional and marginal zone B cells and generate aberrant mature B cells. These B cells show diminished proliferation in response to stimulation of the BCR, but respond normally to other stimuli. Yet, the mice mount a normal primary immune response against a T-dependent Ag. In contrast, the memory response is impaired. In addition, serum Ig levels, in particular IgG2b, are increased. We conclude that p300 acetyltransferase activity is essential for maintaining self-tolerance of B lymphocytes. These findings support the concept of treating lupus with inhibitors of protein deacetylases and point to B cells as a critical target of these drugs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Reversible acetylation of proteins by acetyltransferases (histone acetyltransferase, HATs)3 and deacetylases (histone deacetylase, HDACs) regulates a large number of physiological processes (1). Imbalanced activity of HATs and HDACs has been implicated in several diseases, including cancer (2). Consequently, HDAC inhibitors, a family of drugs that can reestablish this balance, show promising results in cancer therapy (3). The p300 protein is one of >15 known mammalian HATs (4, 5). Many transcription factors interact with and recruit p300 to target promoters, where p300 acts as a transcriptional coregulator (6, 7). Among the substrates of p300 acetyltransferase (AT) activity are the four core histones, as well as a rapidly growing number of transcription factors, including NF-{kappa}B and Bcl-6 (8, 9). Thus, via acetylation, p300 can modulate both the activity of transcription factors and the chromatin structure of their target genes. However, p300 displays additional biochemical activities that contribute to its coregulator function, including direct contact with the basal transcription machinery (10), E3/E4 ubiquitin ligase activity (11), and an autonomous transcription-repression domain (12). Indeed, not all functions of p300 require its AT activity (13, 14). A major challenge regarding p300 is to determine which of its multiple biochemical activities, interaction partners, and substrates are relevant for a particular function in vivo.

Mice heterozygous for a mutant allele of p300, encoding a protein that specifically lacks AT activity, show multiple developmental defects, including delayed muscle differentiation, and die during embryonic development with full penetrance (14, 15). Because the majority of mice that are heterozygous for a null allele of p300 are viable, AT-deficient p300 acts in a dominant-negative manner (15). Indeed, autoacetylation is required to dissociate p300 from the promoter and to allow the binding of TFIID, suggesting that AT-deficient p300 stalls the process of transcriptional activation midway, essentially freezing the promoter in an inactive configuration (16).

Many of the transcription factors that regulate hemopoiesis physically interact with p300 (10). Murine embryonic stem cells deficient for p300 are impaired in hemopoietic differentiation (17). Loss of p300 in hemopoietic stem cells results in a reduction of pre- and pro-B cells, whereas loss of p300 after the pro-B cell stage has only minor effects on B cell differentiation (18). Mice that are homozygous for a point mutation in the KIX domain of p300, an interaction surface for numerous transcription factors, such as c-Myb (19), show defects in multiple hemopoietic lineages, including a deficiency of B cells (20). In this study, we address the role of p300-dependent protein acetylation in B cells in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Mice carrying the p300AS-neo allele on a C57BL/6 background (14, 15) were mated with CD19-cre mice on a 129/Sv background (21) to obtain double-heterozygous animals. In most cases, mice homozygous for CD19-cre were used to increase the fraction of double-heterozygous animals. Mice heterozygous for p300AS-neo, but lacking CD19-cre, were derived from matings between p300+/AS-neo mice and heterozygous CD19-cre mice. Mice of all genotypes used in this study were on the same mixed 129/Sv x C57BL/6 background. In all experiments shown, the control mice were either of the same litters or age- and sex-matched to the p300+/AS-neoCD19-cre mice. Mice developed a similar pathology when housed in individually ventilated cages or conventionally. All animal experiments have been approved by the Regierungspräsidium Giessen.

Histology, immunohistology, and immunofluorescence

Tissues were fixed in Carnoy’s fixative or 10% formalin and embedded in paraffin. Sections 4-µm thick were stained with H&E. For detection of Ig deposits, tissues were frozen in isopentane cooled by liquid nitrogen. Cryosections were air-dried for 30 min, fixed in acetone for 15 min at 4°C, rehydrated in PBS, blocked with 3% goat serum for 1 h, and stained with a FITC-conjugated goat anti-mouse Ig that detects both H and L chains (Dianova) at a dilution of 1/200 in PBS/3% goat serum.

For the staining of marginal zone B cells, spleens were snap frozen in Optimal Cutting Temperature Compound (Tissue-Tek; Sakura). Cryosections 12-µm thick were air-dried for 2 h at room temperature and fixed in acetone (2 min at –20°C). Slides were rehydrated in PBS, blocked with 0.05% BSA and Fc block, and then stained with the following reagents diluted in PBS: mouse anti-mouse IgMb PE (BD Pharmingen), rat anti-mouse metallophilic macrophage (MOMA) 1 biotin (BMA Biomedicals), rabbit anti-mouse Laminin 1 {gamma}-chain (ImmunDiagnostic), goat anti-rabbit AlexaFluor 488 (Molecular Probes), and streptavidin-Cy5 (Molecular Probes). After staining and washing, slides were dried, mounted with Neo-Mount (Merck), and analyzed using a LSM 510 META laser scanning confocal microscope (Zeiss). Images were processed with LSM5 Image Browser (Zeiss) and Adobe Photoshop 7.

For indirect immunofluorescence of Crithidia luciliae, we used a clinical assay system (Euroimmun), except that the secondary Ab reactive against human Abs was replaced by a FITC-conjugated goat anti-mouse Ig, detecting both H and L chains (Dianova) at a dilution of 1/200 in PBS/3% goat serum.

Flow cytometry and cell sorting

Single-cell suspensions of bone marrow, spleen, and lymph nodes were blocked with Fc block, stained with PE- and FITC-conjugated mAbs, and analyzed or sorted on a FACSCalibur (BD Biosciences). The following Abs were used: clone RA3-6B2 (CD45R), clone B3B4 (CD23), clone 11-26c.2a (IgD), clone 7G6 (CD21/CD35), clone 17A2 (CD3), clone H1.2F3 (CD69), clone AA4.1 (C1qRp; eBioscience), and clone 1B4B1 (IgM; Southern Biotechnology Associates). Magnetic cell sorting was performed with CD45R microbeads using two rounds of positive selection (Miltenyi Biotec). Apoptotic cells were detected by staining of permeabilized cells with a FITC-conjugated Ab recognizing activated caspase 3 (BD Biosciences).

ELISA

For detection of autoantibodies, we used the anti-dsDNA ELISA kit (Euroimmun) according to the instructions of the supplier, except that the secondary Ab reactive against human Abs was replaced by an anti-mouse peroxidase conjugate (A0412; Sigma-Aldrich) at a dilution of 1/10,000. For mouse Ig isotype-specific ELISA, the SBA Clonotyping System/HRP was used (Southern Biotechnology Associates). Ig isotype concentrations of individual serum samples were calculated using a mouse Ig reference serum (Immunology Consultants Laboratory). For the cardiolipin ELISA, we used the anti-cardiolipin ELISA kit (Euroimmun) with the same modification as described above for the anti-dsDNA ELISA kit.

Quantitative RT-PCR

Total cytoplasmic RNA was isolated with the TRIzol reagent and was reverse-transcribed using MoMuLV Reverse Transcriptase and random primers. PCR amplifications were performed in duplicate on an Applied Biosystems 7000 in the presence of SYBR green. Differences in expression between different samples were calculated according to the {Delta}{Delta}Ct relative quantitation method (Applied Biosystems, User Bulletin 2). Primers for the S16 gene encoding a ribosomal protein were used to control for differences in RNA input. Primer sequences are available upon request.

T-dependent B cell response

Mice were injected i.p. with 10 µg of chicken OVA in PBS/Imject Alum (Pierce) at days 0 and 22. Serum titers of OVA-specific Igs were measured by ELISA, using a nonisotype-specific secondary Ab. An OVA-specific polyclonal Ab (ab17293; Abcam) served as a positive control.

Activation of primary B cells and incorporation of 3H-labeled thymidine

Naive B cells were enriched from splenic single-cell suspensions by magnetic depletion of CD43+ cells using MS columns (Miltenyi Biotec). A total of 105 cells was plated in triplicate in 96-well plates and cultured in RPMI 1640/5% sodium pyruvate/50 µM 2-ME/10% FCS. The indicated stimuli were added for 64 h (or 36 h in the case of the TLR9 agonist). During the last 16 h (or 12 h in the case of the TLR9 agonist), cells grew in the presence of 1 µCi of 3H-labeled thymidine. Cells were then harvested to measure incorporated radioactivity.

Statistical analyses

Survival curves were calculated using GraphPad Prism software. For all other statistical analyses, the two-tailed Mann-Whitney U test was used.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Mice expressing AT-deficient p300, specifically in B cells, die prematurely

Mice carrying the knock-in allele p300AS-neo encode a mutant p300 protein in which two amino acids in the AT domain have been mutated (14, 15). The mutant protein p300AS lacks detectable AT activity. In addition, p300AS-neo is a conditional allele, because it is expressed only after removal of the neo gene from an intron (14, 15). Mating of p300+/AS-neo mice with CD19-cre mice, expressing Cre-recombinase specifically in B lymphocytes (21), resulted in Cre-mediated deletion of neo exclusively in B lymphocytes (CD45R+) of double-heterozygous animals (Fig. 1, A and B). Deletion of neo was observed both in CD45R+ splenocytes and in the bone marrow where the CD19 promoter is active as early as the pro-B cell stage, but not in splenocytes depleted of B cells. B lymphocytes from p300+/AS-neoCD19-cre mice expressed similar levels of both the wild-type (WT) and the mutant allele, whereas expression of p300AS was not detectable in B lymphocytes from control mice (Fig. 1C). We have previously shown that the deletion of the neo gene from p300AS-neo results in full derepression of the mutant allele (14). Therefore, the relative expression of the mutant and WT allele can be used as a surrogate measure of recombination efficiency. Allele-specific quantitative RT-PCR of p300+/AS-neoCD19-cre mice showed that the expression of p300AS in mature, naive B cells (CD43-depleted splenocytes) of double-heterozygous mice is, on average, >95% of the WT allele (95.4%, SD = 4.6; n = 3), confirming efficient recombination (data not shown).


Figure 1
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  		FIGURE 1. B cell-specific expression of AT-deficient p300 causes premature death. A, Scheme of the experimental system used. See text for details. B, B cell-specific removal of neo by Cre-recombinase. Genomic DNA was prepared from magnetically purified CD45R+ B cells, splenocytes magnetically depleted for CD45R+ cells (CD45R–), and bone marrow cells (BM) of 8-wk-old mice of different genotypes; deletion of the loxP-flanked neo gene was detected by PCR with primers that distinguish the WT allele p300wt and the recombined allele p300AS; allele p300AS-neo is not detected under the conditions used. C, B cell-specific expression of p300AS. Allele-specific RT-PCR to measure expression of p300 (wt) and p300AS (AS) in magnetically purified B cells from the spleen of control and double-heterozygous mice. D, Staining of bone marrow cells of 12-wk-old female littermates of different genotypes with Abs against CD45R and sIgM, distinguishing pre- and pro-B cells (IgM–/CD45R+), immature B cells (IgM+CD45R+), and recirculating B cells (IgM+CD45Rhigh). Percentages of pre-/pro-, immature, and recirculating B cells in the control mouse are 15.7, 3, and 4.8%. The corresponding numbers for the p300+/AS-neoCD19-cre animal are 13.7, 2.1, and 1.4%. E, Survival curves of p300+/AS-neoCD19-cre (n = 31), p300+/AS-neo (n = 12), and CD19-cre control mice (n = 41). Mice of all three genotypes were on the same mixed genetic background (129/Sv x C57BL/6). The p values are <0.0001 for the comparison of p300+/AS-neoCD19-cre with CD19-cre and 0.0012 for the comparison of p300+/AS-neoCD19-cre with p300+/AS-neo, respectively.

 
In young p300+/AS-neoCD19-cre mice with impaired p300-dependent protein acetylation, B cell differentiation was normal, except for a reduction of recirculating B cells in the bone marrow (Fig. 1D). The observation that B cells expressing AT-deficient p300 did not show a defect of early differentiation in the bone marrow is consistent with the absence of a differentiation phenotype in mice lacking p300 in B cells (22). Yet, on a mixed 129/Sv x C57BL/6 genetic background, p300+/AS-neoCD19-cre died prematurely with full penetrance (Fig. 1E). Mice heterozygous for p300+/AS-neo, but negative for Cre-recombinase, on the same mixed genetic background had a normal life span.

B cell-specific expression of AT-deficient p300 causes splenomegaly, nephritis, and vasculitis

Pathological analysis of morbid p300+/AS-neoCD19-cre mice revealed that splenomegaly sometimes was accompanied by hepatomegaly and lymphadenopathy (Fig. 2, A and B, and data not shown). Enlarged spleens showed increased extramedullary hemopoiesis, with abundant islands of granulopoiesis and a 4- to 20-fold increase in the number of megakaryocytes compared with control spleens, and lacked normal tissue organization into red and white pulp (Fig. 2, D and E, and data not shown). The kidneys of p300+/AS-neoCD19-cre mice were presented with interstitial nephritis and glomerulonephritis (Fig. 2C). The glomeruli were enlarged and often filled with homogeneously staining protein deposits. Signs of vasculitis, such as paravascular cell infiltration and thickening of the artery walls, were detected in the kidney, as well as in other organs including the spleen, liver, and lungs (Fig. 2, C and E–G).


Figure 2
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  		FIGURE 2. B cell-specific expression of AT-deficient p300 causes splenomegaly, nephritis, and vasculitis. A, Spleen weights in p300+/AS-neoCD19-cre (n = 22) and CD19-cre control mice (n = 27) at an age of 4–6 mo. On average, the spleens of double-heterozygous animals of this age were 4.5 times as big as the spleens of control animals (mean spleen weight, 734 mg vs 164 mg; p < 0.0001). B, Spleen of a control mouse (left) and enlarged spleen of a p300+/AS-neoCD19-cre mouse (900 mg; right). C, H&E-stained kidney section of a p300+/AS-neoCD19-cre mouse with renal pathology including sclerotic glomeruli, nephritis, and fibrosis. D and E, H&E-stained sections of the normal-sized spleen of a CD19-cre control mouse (D) and an enlarged spleen of a p300+/AS-neoCD19-cre mouse (E). Note the loss of normal tissue architecture and severe vasculitis (arrowheads point to the central arteries of the white pulp). F, H&E-stained section of a liver from a p300+/AS-neoCD19-cre mouse showing infiltrating mononuclear cells surrounding a blood vessel. G, H&E-stained section of the lung of a p300+/AS-neoCD19-cre mouse showing infiltrating cells around a vessel (indicated by arrowheads). Scale bars, 100 µm.

 
Mice with reduced p300 AT activity in B cells develop a systemic lupus erythematosus (SLE)-like autoimmune disease

The pathological findings suggested that the mice might have developed a systemic autoimmune disease. Indeed, indirect immunofluorescence revealed deposits of immune complexes in the glomeruli and the liver of p300+/AS-neoCD19-cre mice (Fig. 3, A and B). Autoantibodies against dsDNA, a hallmark of SLE, were detected in the sera of aged p300+/AS-neoCD19-cre mice but in none of the control mice (OD at 450 nm > 0.3; p < 0.0001; Fig. 3, C and D). Renal pathology correlated with the production of autoantibodies, because all mice with nephritis produced dsDNA-specific Abs. Of 10 mice with anti-dsDNA reactivity tested, all produced IgM, and 9 of 10 also produced IgG anti-dsDNA Abs (Fig. 3E).


Figure 3
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  		FIGURE 3. Reduced p300 AT activity in B cells causes SLE-like autoimmune disease. A and B, Immune complex deposits in the glomeruli (A) and liver (B) of p300+/AS-neoCD19-cre mice. Cryosections of kidneys and livers of a morbid p300+/AS-neoCD19-cre mouse and an age-matched control were incubated with a FITC-labeled Ab against mouse Ig H and L chains. Scale bars, 50 µm (kidney sections) and 100 µm (liver sections). C, p300+/AS-neoCD19-cre mice produce autoantibodies. dsDNA-specific Abs in p300+/AS-neoCD19-cre mice (AS) and age-matched CD19-cre control mice (wt) were measured by ELISA. The horizontal line represents median OD levels. At the time of serum collection, the p300+/AS-neoCD19-cre mice were without signs of disease (average age at serum collection: 8.8 mo). D, Detection of dsDNA-specific Abs in the serum of mice that had been positive by ELISA using Crithidia immunofluorescence. Scale bar, 10 µm. Shown is the result for a serum producing an OD of roughly 0.8 in the ELISA (Fig. 3C). E, Isotypes of dsDNA-specific Abs in the serum of p300+/AS-neoCD19-cre mice (n = 10) measured by ELISA. Strongly increased OD values, indicating dsDNA-specific Abs of that particular isotype are highlighted in bold. F and G, Female p300+/AS-neoCD19-cre mice die earlier than males of the same genotype. Survival curves of male (n = 18) and female (n = 13) p300+/AS-neoCD19-cre mice on a mixed 129/Sv x C57BL/6 background (F) and of male (n = 11) and female (n = 11) p300+/AS-neoCD19-cre mice on a mixed 129/Sv x FVB/N background (G). H, Increased number of activated lymphocytes in p300+/AS-neoCD19-cre mice. Splenocytes from CD19-cre mice (wt) and p300+/AS-neoCD19-cre mice (AS) at an age of 3–8 mo were double stained for the activation marker CD69 and either a B cell (CD19) or a T cell marker (CD3). Horizontal lines, Median percentage of activated lymphocytes.

 
Because SLE predominantly affects women (23), we compared male and female p300+/AS-neoCD19-cre mice. On the mixed 129/Sv x C57BL/6 background, female mice had a shorter life expectancy than males (median survival of 29.4 wk for females compared with 42.5 wk for males; p = 0.0393; Fig. 3F). p300+/AS-neoCD19-cre mice on a different genetic background (mixed 129/Sv x FVB/N) also died prematurely with similar pathological findings (data not shown). However, on this background, clinical disease developed later, with a median survival of 73 wk (p = 0.0131; n = 22). Notably, the sex bias was much more pronounced on the 129/Sv x FVB/N background, indicating that under genetic conditions that result in slower disease, the influence of sex differences is stronger (p < 0.0001; Fig. 3G).

During an autoimmune disease, the mutual stimulation of B and T cells is believed to create a positive feedback loop, resulting in chronic activation of autoreactive lymphocytes (24, 25). Indeed, the fraction of B cells and T cells with an activated phenotype (CD69+) was strongly increased in the spleen of p300+/AS-neoCD19-cre mice (p = 0.0101 for B cells and p = 0.0003 for T cells), suggesting that T cells contribute to disease pathology in p300+/AS-neoCD19-cre mice (Fig. 3H).

Aberrant maturation of B cells with reduced p300 AT activity

Because expression of AT-deficient p300 in p300+/AS-neoCD19-cre mice is restricted to B cells, we analyzed B cell differentiation in double-heterozygous mice at the time clinical disease was first noticed. Flow cytometric analyses of bone marrow B cells did not reveal significant differences to age-matched CD19-cre control mice except for the reduction of recirculating B cells already noted in young animals (data not shown). In the spleen, the relative numbers of CD3+ T cells and CD45R+ B cells were similar in mice with the different genotypes (data not shown). However, cells with high surface (s) IgM were selectively reduced in p300+/AS-neoCD19-cre mice, whereas CD45R+/sIgM– B cells were increased (Fig. 4A). Consistent with the reduced number of sIgMhigh cells, a smaller fraction of B cells showed a transitional phenotype (AA4.1+) in p300+/AS-neoCD19-cre mice (Fig. 4B; p = 0.0012). However, total numbers of transitional B cells were similar in CD19-cre and p300+/AS-neoCD19-cre mice (2.6 ± 0.96 x 106 vs 3 ± 1.86 x 106 AA4.1+ cells; n = 6; p = 0.94). Consistent with a reduced number of sIgMhigh cells, we did not detect increased numbers of B1 B cells (sIgMhigh; CD43+) in the spleen (data not shown). Marginal zone (MZ) B cells (sIgMhigh/CD21high) were strongly reduced in p300+/AS-neoCD19-cre mice (Fig. 4C). This result was confirmed by immunohistology, showing loss of both MZ B cells and (MOMA+, and, to a lesser extent, also MZ macrophages (ERTR9+; data not shown), in p300+/AS-neoCD19-cre mice (Fig. 4D). An analysis of p300+/AS-neoCD19-cre mice at different ages showed that these changes in the B cell compartment predated the production of autoantibodies (data not shown). The number of sIgD+ mature B cells was normal (Fig. 4E), suggesting that a reduction of p300 AT activity does not prevent the maturation of follicular B cells. Staining of splenocytes from the same mice for CD23, another marker of mature B cells, gave a very different picture. Whereas the majority of splenic B cells from control mice stained positive for CD23, CD23+ cells were absent or reduced in the spleens of aged p300+/AS-neo CD19-cre mice (Fig. 4F). Thus, aged p300+/AS-neoCD19-cre mice produce aberrant mature B cells that are sIgD+, but lack CD23, indicating that mice with reduced p300-dependent AT activity show an age-dependent loss of part of the normal B cell differentiation program.


Figure 4
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  		FIGURE 4. Aberrant differentiation of B cells in aged p300+/AS-neoCD19-cre mice. A, p300+/AS-neoCD19-cre mice lack B cells with high sIgM expression. Splenocytes were stained with the indicated Abs and analyzed by flow cytometry. Shown are splenocytes from 6-mo-old, female littermates of different genotypes. The p300+/AS-neoCD19-cre mouse had been clinically inconspicuous until the day of analysis, when it appeared terminally ill. The dot blots in A, C, E, and F show splenocytes from the same pair of mice. They are representative of a large number of p300+/AS-neo CD19-cre mice ages 5–12 mo, both with and without nephritis and signs of morbidity. B, Reduced number of transitional B cells in the spleen of p300+/AS-neoCD19-cre mice (AS) compared with CD19-cre mice (wt). Splenic lymphocytes from mice at an age of 3–6 mo were stained for CD45R and AA4.1 (C1qRp), a marker of transitional B cells. Numbers, Percentage of splenic B cells that stained positive for AA4.1. Horizontal lines, Median percentage of transitional B cells. C and D, p300+/AS-neoCD19-cre mice have reduced numbers of MZ B cells. Splenocytes were stained with the indicated Abs and analyzed by flow cytometry (C). Immunohistology of the splenic MZ of CD19-cre (top) and p300AS-neoCD19-cre (bottom) littermates at an age of 6.5 mo (D). At the time of analysis, the spleen of the p300AS-neoCD19-cre mouse weighed 430 mg. Roughly one-half of the spleen appeared histologically to be normal, whereas the other half displayed a loss of tissue architecture. The immunofluorescence shown is from an area with normal histology. Laminin, which identifies the splenic MZ sinus, is stained in green, metallophilic MZ macrophages (MOMA-1+) in blue, and B cells (IgM+) in red. MZ B cells are the IgM+ cells located on the outer rim of the MZ sinus. Scale bars, 50 µm. E and F, p300+/AS-neoCD19-cre mice produce an aberrant population of mature B cells that are IgD+, but CD23–. Splenocytes were stained with the indicated Abs and analyzed by flow cytometry. The dot blots are representative of a large number of p300+/AS-neoCD19-cre mice, ages 5–12 mo both with and without signs of morbidity. Based on the analysis of many mice of different ages, CD23+ cells are gradually lost with increasing age in p300+/AS-neoCD19-cre mice.

 
Mice with B cell-selective reduction of p300 AT activity mount a normal primary immune response, but show an impaired memory response

Because p300+/AS-neoCD19-cre mice produced aberrant mature B cells, we analyzed the function of these cells in vivo. Total Ig levels were, on average, 1.6-fold higher in mice with reduced p300 AT activity at an age of 10 mo (Fig. 5A). By far, the biggest difference was seen for IgG2b, whose serum concentration was increased on average 3.7-fold in p300+/AS-neoCD19-cre mice. Because the sIgD+CD23– B cells of p300+/AS-neoCD19-cre mice are competent for the production of serum Igs, we measured the humoral immune response of these mice to a T-dependent Ag. Before immunization with chicken OVA, the p300+/AS-neoCD19-cre mice produced, on average, 2-fold more OVA-specific Igs than control mice (Fig. 5B). However, the median primary response 14 days after immunization was similar in p300+/AS-neoCD19-cre mice and control mice. In contrast, the memory response upon boosting of the mice with OVA was reduced on average 3-fold in p300+/AS-neoCD19-cre mice. Thus, mice with reduced p300 AT activity in B cells can mount a normal primary immune response but show an impaired memory response.


Figure 5
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  		FIGURE 5. Influence of reduced p300 AT activity on humoral immune response. A, Serum levels of Ig isotypes in unimmunized, healthy mice. CD19-cre (wt; n = 11) and p300+/AS-neoCD19-cre (AS; n = 10) mice at an age of 10 mo were bled, and serum concentrations of Ig isotypes were determined by ELISA. Total Ig levels were calculated from the values obtained for individual isotypes. Horizontal lines, Median serum levels. B, p300+/AS-neoCD19-cre mice mount a normal primary immune response in vivo, but show an impaired memory response. CD19-cre (wt; n = 11) and p300+/AS-neoCD19-cre (AS; n = 10) mice at an age of 10 mo were immunized with the T-dependent Ag chicken OVA. Twenty-two days later, the animals were boosted with the same Ag. OVA-specific Igs were measured in serum harvested at days 0, 7, 14, and 38 (16 days after the second immunization). The lowest level of OVA-specific Ig measured (serum of one of the control mice at day 0) was set to 1. Horizontal lines, Median relative Ig levels.

 
Reduced p300 AT activity impairs proliferation in response to B cell receptor signaling

BCR signaling controls all major cell fate decisions of B lymphocytes, including positive and negative selection (26). Therefore, we measured the response of primary naive B cells to BCR engagement and other stimuli. Proliferation of p300+/AS B cells was similar to that of control B cells in response to LPS, IL-4, anti-CD40, and ODN1668, an agonist of TLR9 (Fig. 6A). In contrast, p300+/AS B cells showed diminished proliferation in response to BCR engagement by anti-IgM. Simultaneous treatment with anti-IgM and anti-CD40 or IL-4 largely restored proliferation in response to BCR stimulation. These data suggest that reduced p300-dependent protein acetylation selectively impairs proliferation of mature B cells in response to BCR signaling.


Figure 6
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  		FIGURE 6. Proliferation and apoptosis of p300+/AS B cells in response to different stimuli. A, p300+/AS B cells are specifically impaired in their proliferative response to BCR engagement. Primary naive B cells from CD19-cre control mice and p300+/AS-neoCD19-cre mice at an age of 4–5 mo, without signs of morbidity, enriched from the spleen by magnetic depletion of CD43+ cells, were stimulated with LPS (10 µg/ml), anti-IgM (20 µg/ml), IL-4 (2 ng/ml), anti-CD40 (10 µg/ml), or combinations thereof, and with 1 µM of the phosphorothioate oligodeoxynucleotide ODN1668, an agonist of TLR9, ODN1720 lacking the CpG motif of ODN1668, and a control ODN with an AP-1 binding site (56 57 ). Proliferation was measured at day 3 (day 2 for cells stimulated with ODNs) by incorporation of 3H-labeled thymidine. Measurements were performed in triplicate. Error bars, SD of triplicate samples. B, Transitional, but not mature p300+/AS B cells, show increased apoptosis after stimulation of the BCR. Primary naive B cells from CD19-cre control mice and p300+/AS-neoCD19-cre mice were left untreated or were treated with 10 µg/ml anti-IgM for 16 h. Cells were then stained with Ab AA4.1 to distinguish immature and mature B cells and with an Ab directed against activated caspase 3. The numbers represent the fraction of apoptotic cells in the immature and mature B cell populations, respectively. Cells were also stained immediately after purification to determine the level of apoptosis in freshly isolated naive B cells. Error bars, SD of triplicate samples.

 
To distinguish whether the reduced incorporation of [3H]thymidine in p300+/AS B cells resulted from reduced proliferation or enhanced apoptosis, we measured the extent of apoptosis of transitional and mature B cells before and after stimulation with anti-IgM. Immediately after isolation from the spleen, the fraction of transitional and mature B cells undergoing apoptosis was similar in CD19-cre and p300+/AS-neoCD19-cre mice. After 16 h, transitional B cells from p300+/AS-neo CD19-cre mice showed increased apoptosis compared with transitional B cells from control mice, both in the presence and absence of anti-IgM (Fig. 6B). In contrast, apoptosis was similar in mature B cells of both genotypes, indicating that mature p300+/AS B cells are not more susceptible to apoptosis than control cells (Fig. 6B).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
By using mice carrying a p300 allele with a point mutation that specifically disrupts AT activity, but does not affect other biochemical activities of the p300 protein, and by restricting the phenotypic manifestation of this mutation to B cells, we have identified AT-dependent functions of p300 in B cells in vivo. We draw two major conclusions from our work: 1) a primary genetic defect restricted to B cells can trigger systemic autoimmune disease and 2) p300, via its AT activity, suppresses autoimmune disease.

Several lines of evidence indicate that B cells are necessary for the development of systemic autoimmunity. In particular, lupus-prone MRL/lpr mice, a model of spontaneous SLE, will not develop autoimmune manifestations when they are deficient in B cells (27), and depletion of B cells is effective in the treatment of SLE (28). Our findings support a critical role of B lymphocytes in the initiation of a SLE-like autoimmune disease and suggest that other disease-associated events, such as T cell activation, are secondary to a defect in B cells. As yet, it is not known which of the various effector functions of B cells actually contribute to the disease (29, 30, 31). The production of serum autoantibodies is not essential, since autoimmunity in the MRL/lpr mouse model can be induced with genetically manipulated B cells that are unable to produce serum Abs (32). In fact, production of autoantibodies and renal disease can be genetically separated in mice (33).

More than 10 distinct checkpoints in the bone marrow and spleen enforce self-tolerance of B cells (34). These checkpoints include L chain editing, induction of anergy, negative selection at various stages, and exclusion of mature B cells from follicles, as well as death of germinal center B cells, as a consequence of BCR signaling or competition for follicular T cell help. As yet, we do not know which of these checkpoints is compromised in p300+/AS-neo CD19-cre mice and how reduced p300 AT activity leads to the various changes in the B cell compartment of p300+/AS-neoCD19-cre mice and autoimmune disease. One defect of B cells with reduced p300 AT activity is their reduced response to BCR signaling. The strength of the BCR signal controls negative selection of B cells, and mutations that change the threshold for activation of BCR signaling can affect self-tolerance (35, 36, 37). For example, lack of the tyrosine kinase Lyn, which results in reduced BCR signaling strength, causes autoimmune disease in mice (38).

In principle, AT-deficient p300 could cause reduced proliferation in response to BCR signaling by either changing the expression of components of the BCR signaling pathway, or by impairing the activation of genes that respond to BCR signaling. Several transcription factors whose activity is controlled by BCR signaling, such as NF-{kappa}B and NF-AT, use p300 as a coactivator (39, 40). In addition, the activity of NF-{kappa}B is regulated by p300-dependent acetylation (9). We do not yet know whether AT-deficient p300 impairs the function of these proteins in B cells. Indeed, although many transcription factors produced in B cells use p300 as a coactivator, the expression of AT-deficient p300 in B cells did not affect gene expression globally. For example, p300 interacts with and acetylate members of the Smad family of transcription factors, which are downstream effectors of TGF-beta signaling (41). Mice lacking TGF-beta1 develop autoimmune manifestations similar to those seen in p300+/AS-neoCD19-cre mice (42). Yet, induction of TGF-beta target genes, such as Cd72 (43) by TGF-beta, was indistinguishable in p300+/AS and control B cells (N. Forster, S. Gallinat, J. Jablonska, S. Weiss, H.-P. Elsässer, and W. Lutz, unpublished results). Likewise, AT-deficient p300 did not impair the response to IL-4, which depends on the transcription factor Stat-6, although Stat-6 employs p300 for the regulation of target genes (44).

Some of the phenotypes resulting from impaired p300 AT activity resemble phenotypes observed in other mouse strains. For example, mice lacking the transcription factor Aiolos lack MZ B cells and show reduced numbers of recirculating B cells in the bone marrow (45, 46). Mice with a point-mutated, constitutively active Lyn show down-regulation of sIgM, impaired response to BCR, glomerulonephritis, and diminished numbers of MZ B cells, and produce autoantibodies (47). However, other phenotypes of p300+/AS-neoCD19-cre mice are different. For example, mice with constitutively active Lyn show a reduced primary response to a T cell-dependent Ag (47). In addition, their B cells show a reduced response, not only to BCR stimulation, but also to LPS, and on the same strain background used in this study, males die earlier than females.

Evidence from both humans and mice predicts that, in addition to numerous genes that predispose to autoimmune disease, there are other genes that can suppress SLE (48, 49). The p300 gene does not map to a known SLE susceptibility or suppressor locus in humans or mice. However, SLE is a highly heterogeneous disease, and mice with reduced p300 AT activity may model a subgroup of human SLE whose genetic basis cannot be revealed by linkage analysis (50, 51). Alternatively, analogous to the situation in cancer, reduced protein acetylation may be a common downstream event in human SLE, regardless of the primary genetic defect. In any case, the full penetrance of the disease indicates that a reduction of p300 AT activity in B cells generates a strong predisposition for autoimmune disease. This is reminiscent of the Rubinstein-Taybi syndrome, which results from inactivation of one allele of p300 or the close homolog CBP (52). Some patients carry missense mutations that affect only AT activity, showing that even moderate changes in the combined AT activity of p300 and CBP can have severe pathological consequences (53).

It was recently shown that epigenetic therapy with HDAC inhibitors can attenuate autoimmune manifestations in MRL/lpr mice (54). Indeed, splenocytes from these mice display global changes in posttranslational histone modifications, in particular hypoacetylation of histones H3 and H4 (55). Our work with a new mouse model of SLE, genetically unrelated to MRL/lpr, provides further evidence that SLE is indeed an epigenetic disease. These findings have important clinical implications: they strongly support the concept of using HDAC inhibitors in the treatment of SLE, and they point to B cells as a critical target of these drugs. In addition, our observation that p300 AT activity is essential for the maintenance of self-tolerance identifies p300 as one of the enzymes responsible for the epigenetic control of autoimmunity.


   Acknowledgments
 
We thank Klaus Rajewsky for the gift of CD19-cre mice; Antje Grzeschiczek for expertly organizing our mouse colony; Waltraud Ackermann for histology; Heiko Alfke and Boris Keil for magnetic resonance and x-ray images; Thomas Winkler for serum of MRL/lpr mice; Stefan Bauer for the TLR9 agonist; Elke Eckstein for blood analyses; Peter Barth and Annette Ramaswamy for help with pathological analyses; and Michael Lohoff, Bärbel Casper, Thomas Wirth, Serdar Sel, and Berit Schuhmann for technical advice. We thank Andreas Neubauer, Tarik Möröy, Hans-Martin Jäck, and Reinhard Voll for discussions and Martin Eilers for critical reading of this manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Deutsche Forschungsgemeinschaft (Grant Lu 194/2 and International Graduate School 767). Back

2 Address correspondence and reprint requests to Dr. Werner Lutz, Philipps-University, Marburg, Institute of Molecular Biology and Tumor Research, Marburg, Germany. E-mail address: lutz{at}imt.uni-marburg.de Back

3 Abbreviations used in this paper: HAT, histone acetyltransferase; HDAC, histone deacetylase; AT, acetyltransferase; s, surface; SLE, systemic lupus erythematosus; MZ, marginal zone; WT, wild type; MOMA, metallophilic macrophage. Back

Received for publication July 21, 2006. Accepted for publication March 16, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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