Inactivation of NuRD Component Mta2 Causes Abnormal T Cell Activation and Lupus-like Autoimmune Disease in Mice
2008; Elsevier BV; Volume: 283; Issue: 20 Linguagem: Inglês
10.1074/jbc.m801275200
ISSN1083-351X
AutoresXiangdong Lu, Kovalev Gi, Hua Chang, Eric M. Kallin, Geoffrey Knudsen, Xia Li, Nilamadhab Mishra, Phillip Ruiz, En Li, Lishan Su, Yi Zhang,
Tópico(s)Immune Cell Function and Interaction
ResumoDynamic changes in chromatin structure through ATP-dependent remodeling and covalent modifications on histones play important roles in transcription regulation. Among the many chromatin modifiers identified, the NuRD (nucleosome remodeling histone deacetylase) complex is unique because it possesses both nucleosome remodeling and histone deacetylase activities. To understand the biological function of the NuRD complex, we generated a knock-out mouse model of the Mta2 (metastasis-associated protein 2) gene, which encodes a NuRD-specific component. Mta2 null mice exhibited partial embryonic lethality. The surviving mice developed lupus-like autoimmune symptoms including skin lesions, bodyweight loss, glomerulonephritis, liver inflammation, and production of autoantibodies. Transplantation of bone marrow cells from Mta2 null mice recapitulated some of the symptoms including skin lesion and bodyweight loss in the recipient mice. Mta2 null T lymphocytes showed normal development but hyperproliferation upon stimulation, which correlates with hyperinduction of interleukin (IL)-2, IL-4, and interferon (IFN)-γ. T cell hyperproliferation, but not other autoimmune symptoms, was observed in T cell-specific Mta2 knock-out mice. Mta2 null T cells produced more IL-4 and IFN-γ under Th2 activation conditions, but normal levels of IL-4 and IFN-γ under Th1 activation conditions. Furthermore, we found that IL-4 is a direct target gene of Mta2. Our study suggests that Mta2/NuRD is involved in modulating IL-4 and IFN-γ expression in T cell immune responses, and gene expression in non-T cells plays an important role in controlling autoimmunity. Dynamic changes in chromatin structure through ATP-dependent remodeling and covalent modifications on histones play important roles in transcription regulation. Among the many chromatin modifiers identified, the NuRD (nucleosome remodeling histone deacetylase) complex is unique because it possesses both nucleosome remodeling and histone deacetylase activities. To understand the biological function of the NuRD complex, we generated a knock-out mouse model of the Mta2 (metastasis-associated protein 2) gene, which encodes a NuRD-specific component. Mta2 null mice exhibited partial embryonic lethality. The surviving mice developed lupus-like autoimmune symptoms including skin lesions, bodyweight loss, glomerulonephritis, liver inflammation, and production of autoantibodies. Transplantation of bone marrow cells from Mta2 null mice recapitulated some of the symptoms including skin lesion and bodyweight loss in the recipient mice. Mta2 null T lymphocytes showed normal development but hyperproliferation upon stimulation, which correlates with hyperinduction of interleukin (IL)-2, IL-4, and interferon (IFN)-γ. T cell hyperproliferation, but not other autoimmune symptoms, was observed in T cell-specific Mta2 knock-out mice. Mta2 null T cells produced more IL-4 and IFN-γ under Th2 activation conditions, but normal levels of IL-4 and IFN-γ under Th1 activation conditions. Furthermore, we found that IL-4 is a direct target gene of Mta2. Our study suggests that Mta2/NuRD is involved in modulating IL-4 and IFN-γ expression in T cell immune responses, and gene expression in non-T cells plays an important role in controlling autoimmunity. Dynamic changes in chromatin structure through ATP-dependent remodeling and covalent histone modifications play important roles in regulating gene expression. Studies in recent years have identified many ATP-dependent chromatin remodeling and histone modifying enzymes (1Margueron R. Trojer P. Reinberg D. Curr. Opin. Genet. Dev. 2005; 15: 163-176Crossref PubMed Scopus (606) Google Scholar, 2Martin C. Zhang Y. Nat. Rev. Mol. Cell. Biol. 2005; 6: 838-849Crossref PubMed Scopus (1603) Google Scholar, 3Cairns B.R. Curr. Opin. Genet. Dev. 2005; 15: 185-190Crossref PubMed Scopus (134) Google Scholar). Among them, the NuRD (nucleosome remodeling histone deacetylase) complex is of special interest because it possesses both nucleosome remodeling and histone deacetylase activities (4Feng Q. Zhang Y. Curr. Top. Microbiol. Immunol. 2003; 274: 269-290PubMed Google Scholar). The major components of the mammalian NuRD complex include Mi-2β, Mta2 (metastasis-associated protein 2), HDAC1/2, 3The abbreviations used are: HDAC, histone deacetylase; KO, knockout; WT, wild type; TKO, T cell-specific knockout; BMT, bone marrow transplantation; SCID, severe combined immunodeficiency; ChIP, chromatin immunoprecipitation; SLE, systemic lupus erythematosus; IL, interleukin; IFN, interferon; TCR, T cell receptor; ELISA, enzyme-linked immunosorbent assay; LN, lymph node; mAb, monoclonal antibody; FACS, fluorescence-activated cell sorter; dsDNA, double stranded DNA; ES cell, embryonic stem cell. RbAp46/48, and Mbd3 (5Xue Y. Wong J. Moreno G.T. Young M.K. Cote J. Wang W. Mol. Cell. 1998; 2: 851-861Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar, 6Zhang Y. LeRoy G. Seelig H.P. Lane W.S. Reinberg D. Cell. 1998; 95: 279-289Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar). HDAC1/2 and RbAp46/48 form a deacetylase core complex that exists in both NuRD and the Sin3A histone deacetylase complex (6Zhang Y. LeRoy G. Seelig H.P. Lane W.S. Reinberg D. Cell. 1998; 95: 279-289Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar, 7Zhang Y. Iratni R. Erdjument-Bromage H. Tempst P. Reinberg D. Cell. 1997; 89: 357-364Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar, 8Avitahl N. Winandy S. Friedrich C. Jones B. Ge Y. Georgopoulos K. Immunity. 1999; 10: 333-343Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). However, Mi-2β, Mbd3, and Mta2 appear to be unique for the NuRD complex (4Feng Q. Zhang Y. Curr. Top. Microbiol. Immunol. 2003; 274: 269-290PubMed Google Scholar, 9Bowen N.J. Fujita N. Kajita M. Wade P.A. Biochim. Biophys. Acta. 2004; 1677: 52-57Crossref PubMed Scopus (252) Google Scholar). Mi-2β is an ATP-dependent nucleosome remodeling enzyme (10Wang H. Cao R. Xia L. Erdjument-Bromage H. Borchers C. Tempst P. Zhang Y. Mol. Cell. 2001; 8: 1207-1217Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar). Studies in Drosophila and Caenorhabditis elegans indicate that Mi-2β is involved in Hox gene silencing and somatic cell differentiation (11Kehle J. Beuchle D. Treuheit S. Christen B. Kennison J.A. Bienz M. Muller J. Science. 1998; 282: 1897-1900Crossref PubMed Scopus (306) Google Scholar, 12Unhavaithaya Y. Shin T.H. Miliaras N. Lee J. Oyama T. Mello C.C. Cell. 2002; 111: 991-1002Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). In mammalian cells, Mi-2β has been shown to interact with a master lymphocyte transcription factor Ikaros (13Hollander M.C. Sheikh M.S. Bulavin D.V. Lundgren K. Augeri-Henmueller L. Shehee R. Molinaro T.A. Kim K.E. Tolosa E. Ashwell J.D. Rosenberg M.P. Zhan Q. Fernandez-Salguero P.M. Morgan W.F. Deng C.X. Fornace Jr., A.J. Nat. Genet. 1999; 23: 176-184Crossref PubMed Scopus (442) Google Scholar). Mbd3 is a member of the Mbd (methyl-CpG-binding domain) containing protein family. It interacts with Mbd2, which in turn can recruit the NuRD complex to repress transcription of methylated DNA (8Avitahl N. Winandy S. Friedrich C. Jones B. Ge Y. Georgopoulos K. Immunity. 1999; 10: 333-343Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 14Feng Q. Zhang Y. Genes Dev. 2001; 15: 827-832PubMed Google Scholar). Recent studies have also shown that Mbd3 is required for pluripotency of embryonic stem cells (15Kaji K. Caballero I.M. Macleod R. Nichols J. Wilson V.A. Hendrich B. Nat. Cell. Biol. 2006; 8: 285-292Crossref PubMed Scopus (298) Google Scholar, 16Kaji K. Nichols J. Hendrich B. Development. 2007; 134: 1123-1132Crossref PubMed Scopus (133) Google Scholar). In mammalian cells, Mta2 belongs to the Mta protein family, which also includes Mta1 and Mta3. In vitro studies demonstrated that MTA2 positively regulates HDAC activity (8Avitahl N. Winandy S. Friedrich C. Jones B. Ge Y. Georgopoulos K. Immunity. 1999; 10: 333-343Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The C. elegans Mta2 homolog, egl-27 together with other NuRD component homologs have been shown to antagonize the Ras signaling pathway during vulval development (17Solari F. Ahringer J. Curr. Biol. 2000; 10: 223-226Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). In addition to Mta2, Mta3 has also been shown to form a complex with other NuRD components and play important roles in invasive growth of breast cancer cells through repressing Snail gene transcription, and in B cell differentiation through the Bcl-6 transcription repressor (9Bowen N.J. Fujita N. Kajita M. Wade P.A. Biochim. Biophys. Acta. 2004; 1677: 52-57Crossref PubMed Scopus (252) Google Scholar, 18Fujita N. Jaye D.L. Kajita M. Geigerman C. Moreno C.S. Wade P.A. Cell. 2003; 113: 207-219Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar). Mta1 gene overexpression has been associated with cancer metastasis (19Toh Y. Oki E. Oda S. Tokunaga E. Ohno S. Maehara Y. Nicolson G.L. Sugimachi K. Int. J. Cancer. 1997; 74: 459-463Crossref PubMed Scopus (161) Google Scholar, 20Toh Y. Kuwano H. Mori M. Nicolson G.L. Sugimachi K. Br. J. Cancer. 1999; 79: 1723-1726Crossref PubMed Scopus (113) Google Scholar), although whether it functions together with other NuRD components is not clear. Compared with Mta1 and Mta3, Mta2 is more ubiquitously expressed. It is likely that different Mta family members form different NuRD-like complexes with distinct functions. Chromatin remodeling and histone modifications have been shown to play crucial roles in transcription regulation in the immune system (21Smale S.T. Fisher A.G. Annu. Rev. Immunol. 2002; 20: 427-462Crossref PubMed Scopus (139) Google Scholar). Previous studies have established that during T helper (Th) cell differentiation, expression of specific transcription factors, such as T-bet and GATA3, and cytokines, such as IFN-γ and IL-4, are regulated at the chromatin level (21Smale S.T. Fisher A.G. Annu. Rev. Immunol. 2002; 20: 427-462Crossref PubMed Scopus (139) Google Scholar, 22Ansel K.M. Lee D.U. Rao A. Nat. Immunol. 2003; 4: 616-623Crossref PubMed Scopus (366) Google Scholar, 23Lee G.R. Kim S.T. Spilianakis C.G. Fields P.E. Flavell R.A. Immunity. 2006; 24: 369-379Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). For example, histone hyperacetylation has been observed at the IFN-γ regulatory region in Th1 cells and at the IL-4 regulatory region in Th2 cells (24Avni O. Lee D. Macian F. Szabo S.J. Glimcher L.H. Rao A. Nat. Immunol. 2002; 3: 643-651Crossref PubMed Google Scholar, 25Messi M. Giacchetto I. Nagata K. Lanzavecchia A. Natoli G. Sallusto F. Nat. Immunol. 2003; 4: 78-86Crossref PubMed Scopus (303) Google Scholar). DNA demethylation at IL-4 promoters and the regulatory region have also been observed in Th2 differentiation (23Lee G.R. Kim S.T. Spilianakis C.G. Fields P.E. Flavell R.A. Immunity. 2006; 24: 369-379Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 26Fields P.E. Lee G.R. Kim S.T. Bartsevich V.V. Flavell R.A. Immunity. 2004; 21: 865-876Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Even though it is well established that histone modification and chromatin remodeling play important roles in lymphocyte differentiation and activation, little is known about the identity of the corresponding enzymes. Several recent reports have indicated a role of the NuRD complex in these processes. For example, deficiency in Mbd2, a NuRD-interacting methyl-CpG-binding protein, results in abnormal Th cell differentiation and abnormal IL-4 expression (27Hutchins A.S. Mullen A.C. Lee H.W. Sykes K.J. High F.A. Hendrich B.D. Bird A.P. Reiner S.L. Mol. Cell. 2002; 10: 81-91Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Another NuRD interacting protein, Ikaros, has been shown to set thresholds for T cell activation and TCR-mediated T cell differentiation (8Avitahl N. Winandy S. Friedrich C. Jones B. Ge Y. Georgopoulos K. Immunity. 1999; 10: 333-343Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). However, a direct link between NuRD and T cell function has yet to be established. To understand the in vivo function of Mta2/NuRD, we have generated Mta2 knock-out mice. Mta2 null mice exhibit multiple phenotypes including partial embryonic lethality, development defects, and more interestingly, a lupus-like autoimmune disease. This report focuses on characterizing the autoimmune phenotypes and T lymphocyte function in adult Mta2 null mice. Our data revealed the important role of Mta2/NuRD in regulating IL-4 and IFN-γ expression during Th2-prone immune response and in regulating gene expression in non-T cells to control autoimmunity. Generation of Mice Carrying Mta22lox and Mta21lox Alleles and Manipulation of Mice—The Mta2 targeting vector included PGK-Neo and MC1-tk expression cassettes for positive and negative selection, respectively. Two FRT sites were inserted to each side of the PGK-Neo minigene for future removal of the expression cassette by FLP recombinase. Two loxP sites were inserted in introns 3 and 11, respectively, to flank the region including the PGK-Neo cassette and the genomic sequence from exons 4 to 11. Linearized Mta2 targeting vector was electroporated into J1 ES cells (129SvJ) and selected in the presence of G418 and FIAU (1-(2′-deoxy-2′-fluoro-β-d-arabinofuranosyl)-5′-iodouracil). Correctly targeted ES cell clones identified by PCR screens were expanded and injected into C57Bl/6 blastocysts to obtain chimeric mice, which were then bred to C57Bl/6 to produce C57Bl/6/129SvJ hybrid F1 progeny carrying the Mta22lox allele. Mice heterozygous for the Mta22lox allele were crossed with EIIa Cre transgenic mice to generate mice carrying the Mta21lox allele in which Cre recombinase-mediated loxP recombination had occurred and removed the PGK-Neo cassette as well as the genomic sequence from exons 4 to 11. The primers (set a in Fig. 1B) for genotyping Mta2 wild-type (WT) and Mta22lox alleles are the 5′ sequence 5′-GCTGAAGCAGACAGCAAAC-3′ and 3′ sequence, 5′-CATGCCAGGTTTTGAACCC-3′. PCR was performed as follows: 94 °C at 3 min followed by 35 cycles of: 94 °C for 30 s, 55 °C for 30 s, 72 °C for 45 s, and then one cycle at 72 °C for 7 min. A 390-bp fragment is derived from the mutant allele and a 335-bp fragment is derived from the wild-type allele. The primers (set b in Fig. 1B) for genotyping the Mta21lox allele are the 5′ sequence, 5′-GCTGACAGTAATGCTCGTGAGT-3′ and the 3′ sequence, 5′-ATGCTTCTCACTGAGCTACAGC-3′, and the fragment is 1.2-kb. PCR was performed as follows: 94 °C for 3 min followed by 35 cycles of: 94 °C for 30 s, 60 °C for 45 s, 72 °C for 90 s, and then one cycle at 72 °C for 7 min. A 1.5-kb fragment is derived from the Mta21lox allele. The Mta2 null and control mice described in this report were on a mixed genetic background (129SvJ and C57Bl/6). EIIa-Cre (28Lakso M. Pichel J.G. Gorman J.R. Sauer B. Okamoto Y. Lee E. Alt F.W. Westphal H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5860-5865Crossref PubMed Scopus (909) Google Scholar) transgenic mice were purchased from the Jackson Laboratory and the LckCre transgenic mice were obtained from the laboratory of Zhuang Yuan at Duke University (24Avni O. Lee D. Macian F. Szabo S.J. Glimcher L.H. Rao A. Nat. Immunol. 2002; 3: 643-651Crossref PubMed Google Scholar). The mouse maintenance and experiments were done following the University of North Carolina Institutional Animal Care and Use Committee approved protocols. The skin and liver tissues from control and Mta2 knock-out mice were fixed in formalin for 20–24 h and embedded in paraffin. Tissue sections (5 μm) were stained with hematoxylin and eosin (H&E) for morphological examination. ELISA—Serum was collected from Mta2 null and control mice of different ages. Serum anti-dsDNA, anti-Sm, anti-SSA, and anti-SSB antibodies levels were measured using ELISA kits from Alpha Diagnostics International Inc. (San Antonio, TX). Flow Cytometry Analysis of Lymphoid Population—Age-matched conventional Mta2 null, T cell-specific null, and age- and sex-matched control mice were used for analysis. Single cell suspensions of thymocytes, spleen cells, and lymph node (LN) cells were stained with fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, peridinin chlorophyll protein (PerCP)-, and allophycocyanin (APC)-conjugated monoclonal antibodies and analyzed with FACSCalibur (BD Biosciences). All the antibodies used for immunostaining were purchased from BD Biosciences. Data were analyzed using SUMMIT software. T Cell Proliferation, Th Polarization, and Cytokine Assay—T cell proliferation assay was performed using a previously described protocol (29Kovalev G.I. Franklin D.S. Coffield V.M. Xiong Y. Su L. J. Immunol. 2001; 167: 3285-3292Crossref PubMed Scopus (47) Google Scholar). Briefly, cervical, umbilical, and axial lymph nodes from each mouse were harvested and pooled. About 5 × 105 LN cells or 1 × 105 purified CD4+CD25- T cells were plated in each well of a 96-well plate. The plate was previously coated with goat anti-hamster antibody (10 μg/ml). Thirty-six to 48 h later, the cells were pulsed with [3H]thymidine and harvested after 12 h of incubation. For cytokine production assay, about 2 × 106 anti-CD3/anti-CD28 mAb-stimulated LN cells were plated in a 24-well plate and cultured for 4 days before harvesting of the supernatant. The cytometric bead array was performed according to manufacturer's instruction (BD Biosciences). For Th differentiation, CD4+ T cells were purified from pooled spleen and LN cells using the CD4+ purification kit follow up AutoMACS™ cell separation (Miltenyi Biotec Inc., Auburn, CA). 2 × 106 CD4+ cells were stimulated with anti-CD3 and anti-CD28 mAb, as described, in the presence of recombinant human IL-2 (100 units/ml) (nonpolarized condition). In addition, for Th1 differentiation, cells were stimulated in the presence of recombinant mouse IL-12 (5 ng/ml) and anti-IL-4 (4 μg/ml) (Pharmingen). For Th2 differentiation, cells were stimulated in the presence of mouse IL-4 (50 ng/ml), anti-IL-12 (5 μg/ml), and anti-IFN-γ (4 μg/ml) mAbs (Pharmingen). After 7 days of culture, cells were washed and stimulated with phorbol 12-myristate 13-acetate (10 ng/ml) and ionomycin (1 μm) for 6 h in the presence of BD GolgiPlug containing brefeldin A (Pharmingen) for the last 4 h. IL-4 and IFN-γ expression was assessed by intracellular staining. Harvested cells were stained with anti-CD4 mAb, permeabilized with BD FACS Permeabilizing Solution 2 (BD Bioscience), and stained with anti-IL4 and anti-IFN-γ mAbs. Samples were fixed with 1% paraformaldehyde and analyzed by FACSscan (BD Bioscience). 1 × 105 cells from each polarization condition were harvested and analyzed for IL-4 expression by real-time PCR. Reconstituted SCID Mice—To reconstitute SCID mice, bone marrow (BM) cells from 11-week-old Mta2 WT and Mta2 null mice were injected into irradiated (250R) SCID-NOD mice (5 × 106 bone marrow cells per mouse). LN T cells in the reconstituted mice were harvested at 8 weeks post-reconstitution, and standard FACS and T cell proliferation assays were performed. Quantitative Real-time PCR— Total RNA was extracted using the Qiagen RNeasy mini kit as per the manufacturer's instructions. RNAs were denatured for 3 min at 70 °C and cDNAs were synthesized in Ambion RT buffer with random decamers and 100 units of Moloney murine leukemia virus reverse transcriptase (Invitrogen) for 1 h at 42 °C followed by 10 min at 95 °C to inactivate the enzyme. Real-time PCR analysis was performed using TaqMan primer/probe mixtures (Applied Biosystems) as recommended by the manufacturer and analyzed on an Applied Biosystems 7900HT system. Chromatin Immunoprecipitation (ChIP) Assay—The ChIP assay was modified from a previously described protocol (24Avni O. Lee D. Macian F. Szabo S.J. Glimcher L.H. Rao A. Nat. Immunol. 2002; 3: 643-651Crossref PubMed Google Scholar). The peripheral T cells were purified by the CD4+ T cell purification kit (Miltenyi Biotec Inc., Auburn, CA). IL-4 PCR primers were: 1 forward GCCAATCAGCACCTCTCTTC, 1 reverse, TAAAGCCTCATTCCATGGTC; 2 forward, CATCGCTACACCTCCCAC, 2 reverse, CCTTGGTTTCAGCAACTTTAAC. Statistics—Chi-square tests were used for genotype distribution analysis. T tests were used for statistical analysis of mouse bodyweight, different type of T cell analysis, and T cell proliferation assay. Wilcoxon rank test was used for survival analysis. Generation of the Mta2 Mutant Mice—Although the composition and biochemical properties of the mammalian NuRD complex have been extensively characterized (4Feng Q. Zhang Y. Curr. Top. Microbiol. Immunol. 2003; 274: 269-290PubMed Google Scholar), its biological function is not well understood. To gain insight into its function, we generated a mouse model in which a component of the NuRD complex is deleted by gene targeting. We chose to inactivate Mta2 because the protein encoded by this gene appears to be present only in the NuRD complex. We made a conditional gene targeting vector in which Mta2 exons 4–11 were flanked by two loxP sites (Fig. 1A). Cre-mediated recombination at these two loxP sites resulted in deletion of exons 4–11 and a frameshift in exon 12. As a result, the Mta2 mutant allele would only encode the N-terminal 63 amino acids of the Mta2 protein (Fig. 1A). To investigate the function of Mta2 in whole animal, the Mta22lox/+ mice were crossed with EIIa-Cre transgenic mice. The Mta21lox mice carrying one completely recombined allele (equal to conventional heterozygous knock-out mice) were selected for breeding. The Mta21lox mice will be referred to as Mta2 heterozygous mice and the Mta21lox/1lox mice will be referred to as Mta2 null or Mta2 conventional null mice in this report. As shown in Fig. 1B, the Mta22lox, Mta21lox, and Mta2 wild-type allele can be distinguished by PCR. Western blot analysis of protein extracts derived from Mta2 knockout mice confirmed the lack of Mta2 protein (Fig. 1C). Mta2 Knock-out Mice Exhibit Multiple Phenotypes Including Lupus-like Autoimmune Diseases—Inactivation of the Mta2 gene caused embryonic and perinatal lethality in about half of Mta2 null mice. Fewer than 50% of Mta2 null mice (in 129/C57/B6 mixed genetic background) survived to adulthood (Table 1). Backcross of the Mta2 null mice onto a C57/B6 background caused more severe embryonic lethality. The Mta2 knock-out embryos exhibited defects in axial skeleton, skin, and craniofacial structure with different penetrance (data not shown). The Mta2 null mice that survived postnatally exhibited smaller body size and female infertility.TABLE 1Inactivation of the Mta2 gene causes partial embryonic lethality Numbers and percentages of newborn mice with three possible Mta2 genotypes (+/+, +/–, and –/–) from Mta2+/– breeding pairs or male Mta2–/– and female Mta2+/– breeding pairs. The percentages of newborn Mta2–/– mice are significantly lower than Mendelian distribution (p < 0.01), indicating some Mta2–/– mice died at embryonic stage.Mta2 genotype+/++/––/–Mta2+/– × Mta2+/– Number5317126 Percentage21.2%68.4%10.4%Mta2–/– × Mta2+/– Number9026 Percentage77.6%22.4% Open table in a new tab Adult Mta2 mice also developed erosive skin lesions at multiple locations with high penetrance. More than 90% of the Mta2 null mice showed periorbital erosion by 1 month of age (Fig. 2, A and B). From 4 months of age, ∼75% of Mta2 null mice developed additional skin lesions in areas surrounding the mouth and nose, and about 30% of Mta2 null mice showed skin lesions in the neck region (Fig. 2A, upper panels). Histological sections of skin from the neck lesions of the Mta2 mutant mice revealed hyperkeratosis and acanthosis of epidermal cells, as well as a chronic inflammatory response (Fig. 2A, compare two lower panels). The periorbital skin from null mice also showed hyperkeratosis and inflammation. In some regions there were immature hair follicles and a reduced number of hair follicles although frank alopecia was not evident at these early stages. Often there was evidence of dermal and subcutaneous inflammation (panniculitis) and the overall changes could be characterized as a chronic spongiotic dermatitis. The similarity between the skin lesions of Mta2 null mice and those of mice with lupus-like autoimmune disease (30Kanauchi H. Furukawa F. Imamura S. J. Investig. Dermatol. 1991; 96: 478-483Abstract Full Text PDF PubMed Scopus (41) Google Scholar, 31Lomvardas S. Thanos D. Cell. 2002; 110: 261-271Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) implies possible defects in the immune system of the Mta2 null mice. A histological examination of internal organs revealed that most Mta2 null mice developed global mesangial cell proliferation in kidney (Fig. 2B, compare upper panels) and some of them also showed a significant lymphocyte-rich chronic inflammatory cell infiltration (Fig. 2B, compare lower panels). These renal abnormalities are consistent with histopathological changes occurring with glomerulonephritis seen in murine lupus models. Consistent with above symptoms observed in Mta2 null kidneys, about 40% of adult Mta2 knock-out mice showed moderate (>30 mg/dL) or high (>100 mg/dL) urine protein levels (n = 20). Control mice showed only trace or negative urine protein levels. This phenotype is more severe in female null mice than in the male null mice. In addition, liver inflammation was also found in more than half of the adult Mta2 null mice. The lymphocyte infiltration was generally limited to the portal regions (Fig. 2C). Consistent with injury to hepatocytes in the Mta2 null mice, ELISA results showed that the serum alanine aminotransferase activity level is increased when compared with that of wild-type and heterozygous littermates (Fig. 2D). However, the activity levels of serum aspartate aminotransferase and alkaline phosphatase did not exhibit a significant difference between wild-type and Mta2 null mice (Fig. 2D). To further characterize the autoimmune disease observed in Mta2 null mice, we collected serums from mutant and control mice at different ages and examined them for the different autoantibodies. The Crithidia Luciliae assay demonstrated the presence of anti-dsDNA antibody in the Mta2 null serum (Fig. 3A). ELISA analysis showed significantly higher levels of anti-dsDNA, anti-Sm, anti-SSA, and anti-SSB antibodies (Fig. 3B). No difference in autoantibody level had been observed between Mta2 heterozygous mice and wild-type mice. Anti-dsDNA and anti-Sm antibodies are markers of human and murine systemic lupus erythematosus (SLE), and anti-SSA and anti-SSB antibodies are often evident with Sjogren disease or SLE. Furthermore, Mta2 null mice are smaller than their littermates. The extent of difference depends on mouse development stages. From perinatal to weaning stage, Mta2 null mice are about half to 2/3 size of the heterozygous or wild-type littermates. When they reach mature age (after 2 months old), both male and female null mice are about 75% of the size of their wild-type littermates (Fig. 3C). Mta2 null mice also have shortened lifespans when compared with their wild-type or heterozygous littermates (Fig. 3D). Interestingly, female knock-out mice exhibit a shorter lifespan than male knock-out mice. Given that 90% of SLE patients are female (32Whitacre C.C. Nat. Immunol. 2001; 2: 777-780Crossref PubMed Scopus (940) Google Scholar), and some lupus mouse models have shown more severe phenotype in females than in males (33Roubinian J.R. Talal N. Greenspan J.S. Goodman J.R. Siiteri P.K. J. Exp. Med. 1978; 147: 1568-1583Crossref PubMed Scopus (516) Google Scholar, 34Salvador J.M. Hollander M.C. Nguyen A.T. Kopp J.B. Barisoni L. Moore J.K. Ashwell J.D. Fornace Jr., A.J. Immunity. 2002; 16: 499-508Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), it is very likely that autoimmune-related symptoms are important factors causing early death in Mta2 null mice. However, our results do not exclude other factors in causing premature death. In summary, our observations strongly suggest that Mta2 null mice develop an autoimmune disease that resembles systemic lupus erythematosus in human. Bone Marrow Transplantation Recipient Mice Partially Recapitulate the Autoimmune Phenotypes— To test if the autoimmune phenotypes observed in the Mta2 null mice is caused by bone marrow derived from Mta2 null cells; we transferred Mta2 null bone marrow progenitor cells into SCID mice and monitored hematopoietic reconstitution over time. No significant defects were observed in lymphoid and myeloid reconstitution with Mta2 mutant progenitor cells (data not shown). However, recipient mice transplanted with Mta2 null hematopoietic stem and progenitor cell developed skin lesions and reduced body weight (Fig. 4, A and B), similar to Mta2 null mice. When mice were analyzed 8–12 weeks post bone marrow transplantation, normal T cells were present in the thymus and peripheral lymphoid organs (Fig. 4C). However, these Mta2 null T cells were hyperproliferative when stimulated with T cell mitogens (Fig. 4D). The phenotypes are, however, less severe compared with Mta2 null mice, and no obvious liver inflammation or glomerulonephritis was detected in the transplanted mice. No increased level of autoantibodies was observed either (data not shown). These results indicate that hematopoietic cells derived from Mta2 null bone marrow stem cells contribute to but are not sufficient to cause the autoimmune diseases. Therefore, nonhematopoietic cells must have also contributed to the autoimmune phenotypes of Mta2 null mice. T Cell Hyperproliferation in Mta2 Null Mice—The autoimmun
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