RESEARCH ARTICLE

Alterations in nuclear structure promote lupus autoimmunity in amouse modelNamrata Singh1, Duncan B. Johnstone2, Kayla A. Martin3, Italo Tempera3, Mariana J. Kaplan4 andMichael F. Denny5,*

ABSTRACTSystemic lupus erythematosus (SLE) is an autoimmune disordercharacterized by the development of autoantibodies that recognizecomponents of the cell nucleus. The vast majority of lupus researchhas focused on either the contributions of immune cell dysfunction orthe genetics of the disease. Because granulocytes isolated fromhuman SLE patients had alterations in neutrophil nuclear morphologythat resembled the Pelger–Huet anomaly, and had prominent mis-splicing of mRNA encoding the nuclear membrane protein lamin Breceptor (LBR), consistent with their Pelger–Huet-like nuclearmorphology, we used a novel mouse model system to test thehypothesis that a disruption in the structure of the nucleus itself alsocontributes to the development of lupus autoimmunity. The lupus-prone mouse strain New Zealand White (NZW) was crossed withc57Bl/6 mice harboring a heterozygous autosomal dominantmutation in Lbr (B6.Lbric/+), and the (NZW×B6.Lbric)F1 offspringwere evaluated for induction of lupus autoimmunity. Only female(NZW×B6.Lbric)F1 mice developed lupus autoimmunity, whichincluded splenomegaly, kidney damage and autoantibodies. Kidneydamage was accompanied by immune complex deposition, andperivascular and tubule infiltration of mononuclear cells. The titers ofanti-chromatin antibodies exceeded those of aged femaleMRL-Faslpr

mice, and were predominantly of the IgG2 subclasses. The anti-nuclear antibody staining profile of female (NZW×B6.Lbric)F1 serawas complex, and consisted of an anti-nuclear membrane reactivitythat colocalized with the A-type lamina, in combination with ahomogeneous pattern that was related to the recognition ofhistones with covalent modifications that are associated with geneactivation. An anti-neutrophil IgM recognizing calreticulin, but notmyeloperoxidase (MPO) or proteinase 3 (PR3), was also identified.Thus, alterations in nuclear structure contribute to lupusautoimmunity when expressed in the context of a lupus-pronegenetic background, suggesting a mechanism for the developmentof lupus autoimmunity in genetically predisposed individuals that isinduced by the disruption of nuclear architecture.

KEY WORDS: Nucleus, Chromatin, Histone modifications,Calreticulin, Lamina, Autoantibody

INTRODUCTIONSystemic lupus erythematosus (SLE) is regarded as a failure of theimmune system to maintain tolerance to self-antigens (Tsokos,2011; Choi et al., 2012). Despite steady advances identifying theimportance of the immune system and inflammatory mediators(Deng and Tsao, 2010; Lopez de Padilla and Niewold, 2016),including type I interferons (Elkon and Stone, 2011; Elkon andWiedeman, 2012), in mediating the progression and severity of thedisease, the underlying mechanisms driving the initiation anddevelopment of lupus autoimmunity remain unresolved. SLE ischaracterized by the presence of autoantibodies recognizingcomponents of the cell nucleus, and is thought to originate fromloss of tolerance to chromatin that spreads to other components,including histones and DNA. Although immune system dysfunctionand the associated loss of tolerance to nuclear autoantigens remainsan area of intense investigation, the potential role for alterations inthe structure of the nucleus itself remains unexamined.

Alterations in nuclear structure have been reported previously incells isolated from SLE patients; however, their presence iscommonly attributed to a consequence of disease activity ratherthan a contributor to disease incidence. Cell aspirates from the bonemarrow of individuals with lupus have a variety of abnormalities(Voulgarelis et al., 2006; Oka et al., 2008; Papageorgiou et al.,2013), and alterations in nuclear morphology secondary to SLE isan exclusion in the diagnosis of myelodysplastic disorders.Similarly, alterations in the nuclear morphology of neutrophils insystemic circulation have been identified (Hacbarth and Kajdacsy-Balla, 1986; Bennett et al., 2003; Denny et al., 2010; Singh et al.,2014). Given the nature of SLE as an autoimmune disorder thattargets the cell nucleus, an alternative model for induction of thedisease is that alterations in nuclear structure promote thedevelopment of an immune response that initially targets aberrantnuclei, which then spreads to include additional components of thenucleus.

The nucleus is stabilized by a network of proteins containedwithin the lamina (Gotzmann and Foisner, 1999; Holaska et al.,2002), and components of the nuclear lamina have been identifiedas targets of autoimmunity (Reeves et al., 1987; Courvalin et al.,1990; Chou and Reeves, 1992; Senecal et al., 1999). Lamins A andC are anchored to the nuclear membrane by scaffold proteins,including the lamina-associated polypeptides, whereas B-typelamins are anchored by lamin B receptor (LBR) (Gotzmann andFoisner, 1999; Holmer and Worman, 2001; Worman, 2005;Gruenbaum and Medalia, 2015). LBR spans the inner nuclearmembrane and has a DNA-binding region that associates withheterochromatin to maintain its localization at the membranemargins (Smith and Blobel, 1993; Ye and Worman, 1994;Received 20 February 2016; Accepted 1 June 2016

1Internal Medicine, University of Iowa, Iowa City, IA 52242, USA. 2Section ofNephrology, Internal Medicine, Temple University School of Medicine, Philadelphia,PA 19140, USA. 3Department of Microbiology/Immunology, Fels Institute for CancerResearch, Temple University, Philadelphia, PA 19140, USA. 4Systemic AutoimmunityBranch,National Institute of Arthritis andMusculoskeletal and SkinDiseases, NationalInstitutes of Health, Bethesda, MD 20892, USA. 5Section of Rheumatology, TempleUniversity School of Medicine, Philadelphia, PA 19140, USA.

*Author for correspondence (mikedenny@verizon.net)

M.F.D., 0000-0002-7874-1744

This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,distribution and reproduction in any medium provided that the original work is properly attributed.

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Pyrpasopoulou et al., 1996; Polioudaki et al., 2001; Makatsori et al.,2004; Solovei et al., 2013). Impaired expression of LBR results inautosomal dominant disruptions in nuclear structure that include analteration in neutrophil nuclear morphology called the Pelger-Huetanomaly (Hoffmann et al., 2002; Worman, 2005). In mice, Lbr iswithin the Sle1 lupus susceptibility interval on chromosome 1derived from the New Zealand White (NZW) strain (Morel et al.,1997, 2001; Mohan et al., 1998; Shultz et al., 2003). Given the roleof Lbr in stabilizing nuclear structure and its ability to bindchromatin, it is conceivable that disruptions in the Lbr gene mightcontribute to the development of autoimmunity, particularly ingenetically predisposed individuals. A mouse model was developedin which a heterozygous defect in Lbr mRNA splicing wasexpressed in the context of a hemizygous lupus-prone NZW geneticbackground. Disruption of Lbr promoted the development oflupus autoimmunity in female offspring, consistent with nuclearalterations contributing to the disease when expressed in asusceptible genetic background.

RESULTSGranulocytes from human SLE patients have defects in LBRmRNA splicingPatients with SLE have neutrophils with lobulated or ovoid nuclei(Hacbarth and Kajdacsy-Balla, 1986; Bennett et al., 2003;Voulgarelis et al., 2006; Denny et al., 2010; Singh et al., 2014).This alteration in neutrophil nuclear morphology in SLEresembles the Pelger–Huet anomaly (Fig. 1A), an autosomaldominant genetic disorder that results in nuclear alterations due toimpaired expression of the nuclear membrane protein LBR(Hoffmann et al., 2002; Shultz et al., 2003; Olins et al., 2008).Low-density granulocytes (LDGs) and polymorphonuclear cells(PMNs) isolated from SLE patient samples were examined fordefects in LBR mRNA expression. Defective LBR mRNA splicingin SLE neutrophils was apparent in the cDNA amplicons betweenexons 7 to 14 (Fig. 1B), displaying a laddering pattern consistentwith exon skipping. The PCR products were subcloned andsequenced to determine which LBR exons were prone to mis-splicing in PMNs isolated from SLE patients. Of the 29 LBRplasmids sequenced from five healthy controls, 20 were properlyspliced. In contrast, only 7 of 37 LBR subclones obtained fromfour pairs of LDGs and PMNs isolated from SLE patients encodedintact LBR mRNA. Because the incidence of mis-spliced LBRamplicons was similar between LDGs and PMNs, these sampleswere analyzed together for splicing errors. LBR exon 10 wasskipped in 29 mis-spliced cDNA amplicons from SLE patients(Fig. 1C), and frequently occurred in combination with skippingof exon 9 and/or exon 12. Thus, the splicing of the nuclearenvelope protein LBR is disrupted in PMNs from SLE patients,consistent with their Pelger–Huet-like nuclear morphology; thesefindings support a model in which alterations in nuclear structureare present in SLE patients.

Nuclear alterations promote the development of lupusautoimmunity in a lupus-prone backgroundTo evaluatewhether a disruption in nuclear structure can contribute tothe development of lupus autoimmunity, lupus-prone NZW micewere crossed with non-autoimmune-prone c57Bl/6 mice that areheterozygous for a mutation in Lbr that causes an impairment inmRNA splicing (B6.Lbric/+ mice). Because the Lbric mutationinduces an autosomal dominant disruption in nuclear structure(Hoffmann et al., 2002; Shultz et al., 2003), and a hemizygous NZWbackground is sufficient to promote autoimmunity, the (NZW×B6.

Lbric)F1 offspring tested whether the combination of an alteration innuclear structure and a lupus-prone genetic background results inautoimmunity. Female (NZW×B6.Lbric)F1 mice displayed markedsplenomegaly at 9 months of age relative to their (NZW×B6)F1littermates (Fig. 2A), but the spleen size of themale (NZW×B6.Lbric)F1 and (NZW×B6)F1 mice did not differ (Fig. 2B). This is in contrastto the gradual reduction in spleen size in B6 mice with impaired Lbrexpression (Verhagen et al., 2012). Whereas complete loss of Lbrexpression causes runting (Shultz et al., 2003), the body weight of(NZW×B6.Lbric)F1 mice did not differ from (NZW×B6)F1 (Fig. 2B).The development of kidney damage also had a strict female sex bias,with formation of inflammatory foci readily detectable by Masson’sTrichrome staining of female (NZW×B6.Lbric)F1 kidney sections(Fig. 2C). Female (NZW×B6.Lbric)F1 mice had moderate-to-severeglomerulosclerosis and tubulointerstitial fibrosis (Fig. 2D),accompanied by robust perivascular infiltration of mononuclearcells (Fig. 2E). Glomerular deposition of immune complexes wasalso present in female (NZW×B6.Lbric)F1 mice (Fig. 2F), but not infemale (NZW×B6)F1 littermate controls (Fig. S1), or in F1 male miceof either Lbr genotype (not shown). Thus, only female (NZW×B6.Lbric)F1 mice had splenomegaly and kidney damage – pathologicalfeatures that are associated with the induction of lupus autoimmunity.

To confirm that the loss of a single copy of Lbr was sufficient toinduce a disruption in nuclear structure in the (NZW×B6)F1 mousemodel system, the nuclei of isolated white blood cells were stainedwith DAPI, and neutrophils were identified by counterstaining witha fluorescently labeled antibody specific for the neutrophil surfacemarker Ly6G (Fig. 3A,B). The Ly6G-positive cells of (NZW×B6)F1 mice of either sex possessed the ring-shaped nuclear morphologythat is characteristic of mouse neutrophils (Fig. 3A,B). In markedcontrast, the neutrophils of (NZW×B6.Lbric)F1 mice displayedprominent alterations in nuclear morphology (Fig. 3A,B), which isconsistent with the impairment in Lbr expression (Hoffmann et al.,2002; Shultz et al., 2003; Verhagen et al., 2012). The disruptionin the nuclear morphology of neutrophils establishes the autosomaldominant alteration in nuclear structure, which is attributable toimpaired Lbr expression in mice that inherited the Lbric mutation.

Female (NZW×B6.Lbric)F1 mice had serum titers of anti-chromatin that exceeded those of aged female MRL-Faslpr mice(Fig. 3C). Despite the comparatively high titers of anti-chromatinattained in the female (NZW×B6.Lbric)F1 mice, the distribution ofanti-double-stranded DNA did not differ from the littermate controlfemale (NZW×B6)F1 mice (not shown). Only two female(NZW×B6.Lbric)F1 mice, with relative anti-chromatin titers overthreefold greater than those attained in the female MRL-Faslpr

serum standard, developed a detectable titer of anti-double-strandedDNA autoreactivity (not shown). Although titers of anti-chromatinwere observed in a few of the female (NZW×B6)F1 mice, only oneattained a level comparable to MRL-Faslpr (Fig. 3C). Male mice didnot develop anti-chromatin antibodies (not shown). IgG2a, IgG2b,IgG2c and IgM were prevalent anti-chromatin immunoglobulinsubtypes in the (NZW×B6.Lbric)F1 sera, and anti-chromatin IgG1and IgG3 were not detected (Fig. 3D). Similarly, female (NZW×B6.Lbric)F1 serum had strong anti-nuclear antibody (ANA) reactivity,displaying a combination of anti-nuclear membrane reactivity andhomogenous staining (Fig. 3E; Fig. S2). The nuclear membranestaining profile of female (NZW×B6.Lbric)F1 mice was distinctfrom the purely homogenous pattern of female MRL-Faslpr mice(Fig. 3E). Female (NZW×B6)F1 littermates displayed little or noanti-nuclear reactivity (Fig. S2), and male mice did not develop anti-nuclear antibodies, irrespective of Lbr genotype (Fig. S3).Therefore, the induction of anti-nuclear autoimmunity required an

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alteration in nuclear structure in a lupus-prone background, and wasrestricted to female mice.

Autoantigens recognized by female (NZW×B6.Lbric)F1 serainclude modified histonesThe strong anti-chromatin titer and the homogenous ANA stainingcomponent of the female (NZW×B6.Lbric)F1 sera suggested anti-histone immunoreactivity. Serum samples from female (NZW×B6.Lbric)F1 mice that were previously identified as ANA-positive weresubjected to counterstaining with putative nuclear antigens.Because counterstaining human epithelial type 2 (HEp-2) cells fortotal histones was uninformative, the colocalization of the mousesera ANA reactivity with specific histone modifications wasexamined in greater detail (Fig. 4A). Female (NZW×B6.Lbric)F1serum staining colocalized with several histone H3 modificationsthat are associated with gene expression, including H3K4me3,H3K27ac and H3K9ac (Fig. 4B; Fig. S4). In contrast, histonemodifications associated with gene repression, such as H3K27me3and H3K9me3, did not colocalize (not shown). This selectiverecognition of activation-associated histone modifications wasconfirmed by sequential re-precipitation of modified H3 fromfemale (NZW×B6.Lbric)F1 serum immunoprecipitates ofbiotinylated histones (Fig. 4C). Female (NZW×B6.Lbric)F1 serumpreferentially recovered histone modifications associated with geneactivation (Fig. 4C), consistent with the colocalization patternobserved by fluorescence microscopy (Fig. 4A,B; Fig. S4). Female

(NZW×B6.Lbric)F1 serum immunoblotting of low-molecular-massproteins of mouse embryonic fibroblast (MEF) cytoplasmic andnuclear extracts confirmed the recognition of histones (Fig. 4D).A prominent band at 16 kDa in the nuclear extracts was alsorecognized by female MRL-Faslpr sera. The nuclear fraction alsohad bands at 33 kDa, consistent with hnRNP-A2/RA33 (Fig. 4D)(Monneaux et al., 2001). The serum immunoblots using female(NZW×B6)F1 serum did not detect any cytosolic or nuclear proteins(Fig. S5). Taken together, these data demonstrate that (NZW×B6.Lbric)F1 mice develop an anti-nuclear autoimmune response thatselectively recognizes specific modified histones; however, theinduction of autoimmunity is restricted to females.

Because nuclear alterations resulting from impaired expression ofLbr affect the regulation of gene expression (Solovei et al., 2013)and cell survival (Verhagen et al., 2012), it is possible that the defectin Lbr expression generates additional sources of autoantigens dueto alterations in chromatin homeostasis. Because the pronouncedchanges in immune cell number (Fig. 2A,B) and tissue recruitment(Fig. 2D,E) that are present in female (NZW×B6.Lbric)F1 micewould obfuscate the analysis of chromatin immunogenicity, a directexamination of the auto-antigenic profile of chromatin isolated fromwild-type B6 and B6.Lbric/+ was conducted. Chromatin isolatedfrom the liver of female wild-type B6 and B6.Lbric/+ mice wasimmunoblotted with sera from two female (NZW×B6.Lbric)F1 mice(Fig. 4E). The serum immunoblots of chromatin isolated from thenon-autoimmune-prone wild-type B6 mice displayed only weak

Fig. 1. LBR splicing defects in PMNs isolated from SLE patients. (A) Differential staining of normal density neutrophils isolated from a healthy individual, andthe LDGs from two SLE patients, demonstrates the Pelger–Huet-like nuclear morphology of SLE neutrophils. Imagemagnification, 400×. (B) PCR amplification ofexons 7 to 14 of human LBR cDNA prepared from control PMNs, SLE normal density PMNs and LDGs. The horizontal lines above the sample lanes reflectautologous pairs of LDGs and normal density PMNs isolated in parallel. The arrow indicates the position of properly spliced human LBR. (C) Subcloning andsequencing of human LBR cDNA amplicons verified the mis-splicing of mRNA, and identified extensive skipping of LBR exon 10, both alone and in combinationwith exons 9 and/or 12. Statistical significance assessed by Fisher’s exact test, one-tailed.

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and limited immunoreactivity (Fig. 4E). In marked contrast, the B6.Lbric/+ liver chromatin had multiple intensely reactive bands,consistent with a robust enhancement of chromatin immunogenicityresulting from impaired Lbr expression, even when present in thenon-autoimmune-prone B6 genetic background. The profile ofinduction of serum immunoreactivity to B6.Lbric/+ liver chromatinresembled that of nuclear extracts isolated from primary B6 MEFcultures, in which the major epitopes were modified histones at∼16 kDa and a set of 33-kDa bands that were also detected byMRL-Faslpr serum (Fig. 4D). However, unlike the nuclear extractsof MEFs grown in vitro, a prominent additional band at 60 kDa wasdetected in B6.Lbric/+ mouse liver chromatin isolated ex vivo. Theseresults demonstrate that the disruption of Lbr expression inducesalterations in chromatin regulation that are sufficient to enhance itspotential auto-antigenicity.

Disruption of Lbr inducesautoreactivity to theA-type lamina,but not lamin A/C itselfThe sera of female (NZW×B6.Lbric)F1 mice also displayed anti-nuclear membrane reactivity, which was absent in the MRL-Faslpr

sera (Fig. 3C). Because components of both the A-type and B-type

nuclear lamina are identified targets of autoimmunity (Reeves et al.,1987; Courvalin et al., 1990; Chou and Reeves, 1992; Senecal et al.,1999), the colocalization of the anti-nuclear membrane componentof female (NZW×B6.Lbric)F1 mouse serum with proteins containedwithin the lamina was examined (Gotzmann and Foisner, 1999;Holaska et al., 2002). HEp-2 cells were co-stained with ANA-positive female (NZW×B6.Lbric)F1 serum and rabbit antibodiesrecognizing lamin A/C, lamin B1 and LBR (Fig. 5A; Fig. S6).Although all of the nuclear structural proteins were detected inHEp-2 cells, only lamin A/C colocalized with the nuclear membranereactivity of female (NZW×B6.Lbric)F1 serum (Fig. 5A,B; Fig. S6).The recognition of lamin A/C was examined directly byimmunoblotting (NZW×B6.Lbric)F1 serum immunoprecipitatesfrom MEFs for lamin A/C (Fig. 5C). Lamin A/C was recovered inthe control immunoprecipitates, but was not detected in the female(NZW×B6.Lbric)F1 mouse serum immunopreciptates, nor inimmunoprecipitates from the negative control of MRL-Faslpr

serum, or non-specific mouse IgG. Likewise, immunoblottingwith female (NZW×B6.Lbric)F1 sera did not detect lamin A/Cimmunoprecipitates (not shown). (NZW×B6.Lbric)F1 serumimmunoblotting of high-molecular-mass proteins of cytoplasmic

Fig. 2. Development of splenomegaly and kidney damage in female (NZW×B6.Lbric)F1 mice. (A) Intact spleens isolated from four female (NZW×B6.Lbric)F1

mice and one female (NZW×B6)F1 control. The spleens of three female (NZW×B6.Lbric)F1 mice are enlarged, whereas the size of the fourth is similar to that of the

control. (B) Mean body weight (y-axis) and splenic mass (x-axis) in male (triangles) and female (circles) mice of the NZW×B6 (filled symbols) and NZW×B6.Lbric

(open symbols) genotype. Error bars represent s.e.m. Asterisk (*) indicates significant difference spleen weight in female (NZW×B6.Lbric)F1 mice relative tofemale (NZW×B6)F1 littermates (Student’s t-test, two-tailed, P

and nuclear extracts prepared fromMEFs revealed a∼220 kDa bandrecognized predominantly in the cytoplasmic fraction, and a120 kDa band in the nuclear fraction (Fig. 5C), confirming thelack of lamin A/C immunoreactivity. No high-molecular-massproteins were detected in immunoblots using MRL-Faslpr serum(not shown), or female (NZW×B6)F1 serum (Fig. S5). Thus, infemale (NZW×B6.Lbric)F1 mice, disrupting the expression of Lbraffects the organization of the B-type lamina, but induces thedevelopment of autoimmunity directed against components of theA-type lamina, but lamin A/C itself is not the target.

Anti-neutrophil autoreactivity mediated by IgM is directedagainst calreticulin, but not MPO or PR3Because mature hematopoietic cells do not express lamin A/C underresting conditions, and do not have an A-type lamina (Rober et al.,1990; Broers et al., 1997), they would be predicted to lack theanti-nuclear membrane component of (NZW×B6.Lbric)F1 serumstaining identified in HEp-2 cells. To confirm that the anti-nuclearmembrane component in (NZW×B6.Lbric)F1 sera recognized theA-type lamina, ethanol-fixed human polymorphonuclear cells wereexamined by fluorescence microscopy. Quite unexpectedly, female(NZW×B6.Lbric)F1 sera displayed robust, and selective, reactivitytoward the nuclei of ethanol-fixed neutrophils (Fig. 6A). Segmentedmature neutrophils displayed prominent nuclear staining, but thebilobed eosinophils lacked any appreciable staining. (Fig. 6A;Fig. S7). Weak and patchy staining of eosinophils was detected athigher magnification (Fig. 6B), but it was clearly distinct from the

intense staining of neutrophils. Variable staining of monocyteswithin the peripheral blood mononuclear fraction was alsoobserved, but it was also distinct from the prominent staining ofisolated neutrophils (Fig. S7). Anti-neutrophil antibodies inautoimmune diseases are defined by their perinuclear (perinuclearanti-neutrophil cytoplasmic antibodies; pANCA) or cytoplasmic(cytoplasmic anti-neutrophil cytoplasmic antibodies; cANCA)staining patterns using ethanol-fixed neutrophils. The strongstaining of the neutrophil nuclei in the sera of female (NZW×B6.Lbric)F1 mice did not colocalize with myeloperoxidase (MPO;pANCA) or proteinase 3 (PR3; cANCA) (Fig. 6C, and not shown).The lack of anti-MPO and anti-PR3 reactivity was confirmed byELISA (not shown). Thus, the anti-neutrophil response in female(NZW×B6.Lbric)F1 mice is not due to pANCA or cANCA.

Female (NZW×B6.Lbric)F1 serum immunoblotting of neutrophillysates identified a protein of ∼48 kDa that was detected byimmunoblotting with serum from several mice (Fig. 7A). BecauseTrim21 and calreticulin are autoantigens within this size range(Kishore et al., 1997; Oke andWahren-Herlenius, 2012), neutrophilimmunoprecipitates of Trim21 and calreticulin were serum-immunoblotted. Immunoblotting with the sera of 6 of 11 female(NZW×B6.Lbric)F1 mice detected calreticulin, but Trim21 was notdetected (Fig. 7B, and not shown). Although the calreticulinimmunoreactivity was apparent, the overall intensity of theimmunoblots was weak and fuzzy, suggesting that the secondaryantibody provided poor recognition of the mouse primary antibody.To test this possibility, the calreticulin immunoprecipitates were

Fig. 3. Development of lupus autoantibodies in female (NZW×B6.Lbric)F1 mice. (A,B) Disruption of neutrophil nuclear morphology in (NZW×B6.Lbric)F1

mice. White blood cells isolated from female (A) and male (B) (NZW×B6.Lbric)F1 and (NZW×B6)F1 micewere stained with FITC-conjugated anti-Ly-6G to identifyneutrophils, and the nuclei counter-stained with DAPI. The ring-shaped nuclear morphology of (NZW×B6)F1 mice is characteristic of mouse neutrophils, whereasthe disorganized nuclear morphology of (NZW×B6.Lbric)F1 neutrophils is consistent with the autosomal dominant disruption in Lbr. (C) Anti-chromatin ELISAassays of serum from female (NZW×B6)F1 (filled circles) and (NZW×B6.Lbr

ic)F1 mice (open circles). Serum was screened initially for anti-chromatin IgG titersrelative to female MRL-Faslpr. (D) Sera from six anti-chromatin IgG-positive (NZW×B6.Lbric)F1 mice and one anti-chromatin-negative (NZW×B6)F1 mouse weresubsequently screened for anti-chromatin titers of IgG1, IgG2a, IgG2b, IgG2c, and IgM relative to female MRL-Fas

lpr. (E) Distinct anti-nuclear antibody (ANA)staining patterns of the HEp-2 cell line with sera from female (NZW×B6.Lbric)F1 (left panel) and MRL-Fas

lpr mice (right panel). Additional images for female(NZW×B6.Lbric)F1 mice, and female (NZW×B6)F1 are presented in Fig. S2. Anti-nuclear antibody staining for male mice is presented in Fig. S3. Imagemagnification, 1000×.

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blotted with mouse sera as before, then probed with secondaryantibodies specific for IgA, IgG or IgM. The IgM-specificsecondary antibody greatly facilitated the detection of calreticulinin the serum immunoblots (Fig. 7B). As before, Trim21was notdetected. The IgM specificity of the autoreactive calreticulinantibodies was confirmed by immunoblotting purified calreticulinwith female (NZW×B6.Lbric)F1 sera and secondary antibodiesspecific for IgA, IgG, or IgM (Fig. 7C). Sera from female(NZW×B6.Lbric)F1 mice, and aged female MRL-Fas

lpr mice,were also examined for autoantibodies recognizing calreticulin byELISA (Fig. 7D). Using this more sensitive assay system, 9 of the 11female (NZW×B6.Lbric)F1 mice had detectable anti-calreticulinIgM titers, whereas MRL-Faslpr mice had predominantly an IgGresponse (Fig. 7D). The recognition of calreticulin in intactneutrophils was addressed using paraformaldehyde-fixed cells andfluorescence microscopy. The anti-neutrophil reactivity in female(NZW×B6.Lbric)F1 sera was detected by an anti-mouse IgMsecondary antibody, which clearly co-localized with rabbit anti-calreticulin staining (Fig. 7E). Neither anti-mouse IgA nor IgGyielded a detectable signal (Fig. 7F), confirming results of the serum

immunoblots of calreticulin immunoprecipitates (Fig. 7B) andpurified calreticulin (Fig. 7C). Thus, female (NZW×B6.Lbric)F1mice developed IgM antibodies recognizing calreticulin.

DISCUSSIONNuclear alterations contribute to the development of lupusautoimmunityLupus is characterized by the presence of autoantibodiesrecognizing the cell nucleus. Considerable research effort hasfocused on aberrant immune responses in development of thedisease (Tsokos, 2011; Choi et al., 2012), and several componentsof the nuclear lamina are targets of autoimmunity (Lassoued et al.,1988; Chou and Reeves, 1992; Senecal et al., 1999), yet a modelproposing that alterations in the structure of the nucleus itselfcontribute to the development of anti-nuclear autoimmunityremained unexamined. The present study demonstrates thatnuclear alterations promote autoimmunity when expressed inconjunction with a lupus-prone genetic background. Anautosomal dominant alteration in Lbr splicing, when expressed inthe context of a hemizygous lupus-prone NZW genetic background,

Fig. 4. Female (NZW×B6.Lbric)F1 mice develop anti-nuclear autoantibodies recognizing histone H3 modifications associated with gene activation.(A) Co-localization of anti-nuclear reactivity and the activation-associated histone H3 modifications H3K4me3 (left panel), H3K27ac (middle panel), and H3K9ac(right panel). Anti-nuclear antibody (ANA) staining in the female (NZW×B6.Lbric)F1 serum was detected with an Alexa Fluor 488-conjugated goat anti-mouseantibody, anti-modified H3 histones were detected by an Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody. Nuclei were counterstained with DAPI.Image magnification, 1000×. The individual red, green and blue color channels demonstrating the co-localization of the homogenous component of anti-nuclearautoantibody reactivity withmodified histones are presented in Fig. S4. (B) Digital enlargement of the individual cells indicated in A. Anti-nuclear stainingmediatedby the (NZW×B6.Lbric)F1 serum is shown in the green channel, and the indicated modified histone is counterstained in the red channel. The yellow overlap signalrepresents nuclear co-localization of mouse serum immunoreactivity with anti-modified H3 histones. (C) Histones purified from HeLa cells were biotinylatedin vitro, immunoprecipitated with (NZW×B6.Lbric)F1 mouse sera, collected on Protein A/G sepharose beads, washed, and eluted. The contents of the recoveredhistone extracts, consisting of histones that were specifically recognized by (NZW×B6.Lbric)F1 mouse sera, were sequentially re-precipitated with rabbit serarecognizing H3K4me3, H3K27ac, H3K9ac, H3K4me, H3K27me3, H3K9me3. Samples prepared from histone extracts collected from each step before, during,and after the re-precipitation process were blotted using peroxidase-conjugated streptavidin and enhanced chemiluminescence. (D) Cytoplasmic and nuclearextracts prepared from 1×106 B6 mouse embryonic fibroblasts (MEFs) were resolved using a 12% SDS-PAGE gel, and immunoblotted with sera from two female(NZW×B6.Lbric)F1 mice, and pooled sera from aged female MRL-Fas

lpr mice. Serum staining from a representative female (NZW×B6)F1 mouse, and verificationof cytoplasmic and nuclear fractionation, is presented in Fig. S5. (E) Chromatin (5 µg) was isolated from the liver of female wild-type B6 and B6.Lbric/+ mice, andimmunoblotted with serum from two female (NZW×B6.Lbric)F1 mice.

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elicited the induction of several autoantibodies, including anti-modified histones, IgG2 antibodies recognizing chromatin, and IgMdirected against calreticulin, as well as moderate-to-severe kidneydisease and splenomegaly, that was sex-restricted to only the femaleF1 progeny. This profile of autoantibody development, organdamage, and female sex-bias resembles lupus autoimmunity inhumans. Although previous studies reported that impaired expressionof Lbr in the B6 background did not promote autoimmunity(Verhagen et al., 2012), combining the impairments in Lbr with thelupus-prone NZW genetic background induced robust lupusautoimmunity. Thus, alterations in nuclear structure might not besufficient to induce autoimmunity in the absence of a lupus-pronegenetic background. Since neutrophils from SLE patients displaydisruptions in Lbr splicing and a corresponding Pelger–Huet-likenuclear morphology, similar nuclear alterations might contribute tothe development of lupus autoimmunity in individuals with aninherited genetic risk.

Potential mechanisms for the female sex bias in thedevelopment of lupus autoimmunityThe restriction of lupus autoimmunity to female (NZW×B6.Lbric)F1 mice mimics human SLE. Current models propose that thisfemale sex bias is related to the influence of steroid hormones onimmune system function or cell death pathways (Jog and Caricchio,

2013; Sakiani et al., 2013; Tan et al., 2015; Trigunaite et al., 2015).Female steroid hormones potentiate pro-inflammatory immuneresponses mediated by, at least in part, estrogen receptor-dependentsignaling pathways (Sakiani et al., 2013; Tan et al., 2015), whereasandrogens provide anti-inflammatory influences (Trigunaite et al.,2015). In addition, sex-steroid hormones contribute to fundamentaldifferences in the execution of programmed cell death, which resultsin a predisposition for activation of pro-inflammatory pathwaysassociated with necrosis in females, and immunologically silentapoptotic pathways in males (Jog and Caricchio, 2013). However,the roles for nuclear structure and Lbr in regulating cell responses tosex-steroid hormone receptor activation are not known; likewise, thedysregulation of circulating levels of sex-steroid hormone that aresecondary to alterations in nuclear structure mediated by impairedLbr expression has not been examined.

The genetic model utilized herein cannot exclude the possibilitythat the Y chromosome encodes genes that reconstitute aspects ofLbr function to prevent the development of lupus autoimmunity inmales. Although the Lbric mutation was sufficient to induce analteration in nuclear morphology in the neutrophils of both male andfemale (NZW×B6.Lbric)F1 mice, only the females developedautoimmunity. This suggests that males can compensate for some,but not all, of the effects of impaired Lbr expression, possibly due tothe unique profile of structural proteins that comprise the neutrophil

Fig. 5. Female (NZW×B6.Lbric)F1 mice develop anti-nuclear autoantibodies recognizing the A-type lamina. (A) Co-staining of HEp-2 cells with female(NZW×B6.Lbric)F1 serum and lamin A/C (left panel), LBR (middle panel), and lamin B1 (right panel). Anti-nuclear antibody (ANA) staining in the serum wasdetected with an Alexa Fluor 488-conjugated goat anti-mouse antibody, lamina proteins were labeled with the indicated rabbit anti-serum and an Alexa Fluor594-conjugated goat anti-rabbit secondary antibody. Nuclei were counterstained with DAPI. Image magnification, 1000×. The individual red, green and bluecolor channels demonstrating the co-localization of the nuclear membrane component of anti-nuclear autoantibody reactivity with the A-type lamina are presentedin Fig. S6. (B) Digital enlargement of the individual cells indicated in A. Anti-nuclear antibody staining mediated by the (NZW×B6.Lbric)F1 serum is shown in thegreen channel, and the indicated nuclear envelope protein is counterstained in the red channel. The nuclear membrane colocalization of mouse serum withthe anti-lamin A/C is represented by the yellow overlap signal. (C) Lamin A/C immunoblots of 5×106 MEFs immunoprecipitated with a mouse lamin A/Cmonoclonal antibody, the sera from two female (NZW×B6.Lbric)F1 mice (Serum 1 and 2), or pooled aged femaleMRL-Fas

lpr sera. Non-specific mouse IgG (mIgG)served as a serum control. (D) Cytoplasmic and nuclear extracts prepared from 1×106 MEFs were resolved on a 7.5% SDS-PAGE gel, and immunoblotted withsera from two female (NZW×B6.Lbric)F1 mice.

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nucleus (Olins and Olins, 2005); or that the mechanisms thatmediate the functional redundancy in males are tissue- or cell-selective and not expressed in neutrophils, as reflected by theextensive levels of heterochromatin condensation and genesilencing found in mature neutrophils (Olins and Olins, 2005;Olins et al., 2008).The Y chromosome encodes over 100 genes, including proteins

that bind nucleic acids and regulate nuclear structure (Jangravi et al.,2013), as well as the male-restricted histone demethylases KDM5Dand UTY (Wang et al., 1995; Warren et al., 2000; Iwase et al., 2007;Walport et al., 2014). UTY demethylates the repression-associatedmodified histone H3K27me3 (Walport et al., 2014), whereasKDM5D demethylates the activation-associated modified histoneH3K4me3 (Iwase et al., 2007), a prominent target for anti-nuclearautoimmunity in female (NZW×B6.Lbric)F1 mice. Thus, analternative model consistent with the female sex bias in lupus isthat the additional genomic complexity provided the Y chromosomeallows males to reconstitute defects in nuclear regulation.

Induction ofmultiple targets of anti-nuclear autoimmunity infemale (NZW×B6.Lbric)F1 miceThe ANA staining pattern of female (NZW×B6.Lbric)F1 micerepresented a combination of anti-nuclear membrane andhomogenous nuclear labeling. The homogeneous nuclear stainingwas attributed to recognition of modified histones, and the reactivitywas selective for histones possessing modifications related to geneactivation and apoptosis (Liu et al., 2012), including acetylation

(van Bavel et al., 2010; Pieterse et al., 2015). It is not yet knownwhether these histone modifications were detected directly, or if aco-associated mark was recognized. The recognition of acetylatedhistones might have important ramifications for the therapeutic useof histone deacetylase inhibitors (Pieterse et al., 2015; Tan et al.,2010). Although short-term treatment with histone deacetylaseinhibitors might be beneficial as a cancer therapy, prolonged orchronic administration might elicit drug-induced lupus ingenetically predisposed individuals (Nambiar et al., 2002;Gottlicher et al., 2001; Bleck and Smith, 1990). Thus, a potentialassociation between epigenetic modulation and the induction oflupus may merit close scrutiny as agents targeting the epigenomeare developed and tested in clinical trials.

The anti-nuclear membrane reactivity detected by female(NZW×B6.Lbric)F1 serum co-localized with the A-type lamina,but it was not attributable to recognition of lamin A/C itself,indicating that additional nuclear proteins mediate the nuclearmembrane staining. Although hematopoietic cells lack an A-typelamina under resting conditions (Rober et al., 1990; Broers et al.,1997), T-lymphocytes transiently re-express components of theA-type lamina upon stimulation (Gonzalez-Granado et al., 2014),suggesting that aberrant reorganization of the nucleus followingT-lymphocyte stimulation might facilitate the development of anti-nuclear membrane antibodies.

It is difficult to reconcile the seemingly paradoxical finding ofautoreactivity toward the A-type lamina as the impairment in Lbrdisrupts the B-type lamina; however, histone modifications also

Fig. 6. Anti-neutrophil serum reactivity develops in (NZW×B6.Lbric)F1 mice. (A) Fluorescence microscopy of ethanol-fixed human PMNs stained with DAPI(top panel), and female (NZW×B6.Lbric)F1 mouse serum and an Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (bottom panel). Broken circlesindicate DAPI-positive bilobed eosinophils that were not readily detected by the female (NZW×B6.Lbric)F1 mouse serum. Image magnification, 100×.(B) High-power magnification of ethanol-fixed PMN staining. Neutrophils displayed strong nuclear staining, but eosinophils had comparatively weak and patchyperinuclear staining. Image magnification, 400×. The preferential staining of isolated neutrophils relative to peripheral blood mononuclear cells and isolatedmonocytes is demonstrated in greater detail in Fig. S7. (C) Ethanol-fixed PMNs were co-stained with female (NZW×B6.Lbric)F1 mouse serum (green) and anti-MPO (red), nuclei were counterstained with DAPI (blue). The mouse serum staining did not colocalize with MPO. Image magnification, 1000×.

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support physical associations with nuclear envelope proteins tomaintain nuclear stability (Gotzmann and Foisner, 1999; Polioudakiet al., 2001; Makatsori et al., 2004; Harr et al., 2015). EHMT2/G9amethyltransferase produces H3K9me2, which promotes nuclearstability through an association with lamin A/C (Harr et al., 2015),and is a component of a regulatory complex with Lbr and theheterochromatin-binding protein HP1 (Ye et al., 1997; Polioudakiet al., 2001; Makatsori et al., 2004; Tachibana et al., 2005).H3K9me2 also mediates gene silencing during stem celldifferentiation (Wen et al., 2009; Hawkins et al., 2010; Harr et al.,2015) and embryonic development (Tachibana et al., 2002), andreduced levels of H3K9 methylation facilitate production of theactivation-associated modified histones H3K9ac (Renneville et al.,2015) and H3K4me3 (Binda et al., 2010), two prominentautoantigens in female (NZW×B6.Lbric)F1 mice. In humans,EMHT2/G9a is in the well-established lupus-susceptibilityinterval embedded in the human leukocyte antigen (HLA) locusat 6p21.31 (Brown et al., 2001), and high-resolution fine-mappingof the HLA interval identified a polymorphic variant within a Myb-family binding site in the EMHT2/G9a enhancer as a prominentlupus risk allele (Kim et al., 2012; Armstrong et al., 2014).Although rs558702 is in the second intron of the complementprotein 2 (C2) gene, it is positioned less than 5 kb upstream of the

transcriptional start site for EMHT2/G9a, providing an alternativemechanism for HLA-associated lupus susceptibility throughdisruption of EMHT2/G9a regulation and epigenetic alterationsthat affect the A-type lamina.

Female (NZW×B6.Lbric)F1 mice develop an IgM thatrecognizes calreticulinIn addition to autoantibodies targeting the cell nucleus, there was anunanticipated induction of antibodies recognizing calreticulin.Calreticulin is widely expressed, and is involved in calciumbuffering, protein folding, and shuttling between the endoplasmicreticulum and the plasma membrane (Coe and Michalak, 2009).Although it is unclear how impaired expression of Lbr wouldfacilitate the development of IgM autoantibodies recognizingcalreticulin, the presence of anti-calreticulin autoantibodies hasbeen described in SLE patients (Kishore et al., 1997; Sanchez et al.,2000), and is associated with increased risk of fetal heart conductionblock (Orth et al., 1996; Suzuki et al., 2005). The humanCALR geneis encoded on chromosome 19, which is prone to genomicalterations in abnormal neutrophils isolated from SLE patients(Singh et al., 2014), and somatic insertion/deletion mutations inexon 9 of calreticulin have been identified in myeloproliferativeneoplasms with non-mutated JAK2 kinase (Klampfl et al., 2013;

Fig. 7. Anti-calreticulin IgM mediates the anti-neutrophil reactivity in (NZW×B6.Lbric)F1 mice. (A) Triton X-100-soluble extracts of human PMNs wereresolved on a 10% SDS-PAGE gel and immunoblotted with female (NZW×B6.Lbric)F1 mouse serum. (B) Immunoprecipitates of Trim21 and calreticulin wereprepared from 20×106 human PMNs, resolved on a 10% SDS-PAGE gel, serum immunoblotted, and detected with peroxidase-conjugated goat anti-mousesecondary antibodies recognizing IgG (H+L) (left panel) or IgM (right panel). Exposure time for development of the IgM signal was∼1/10 that of IgG (H+L). Controlblots (far right panels) verified recovery of Trim21 and calreticulin in immunoprecipitates. (C) Calreticulin purified from bovine liver was serum immunoblotted anddetected with peroxidase-conjugated goat anti-mouse secondary antibodies recognizing IgA (left panel), IgG (middle panel), or IgM (right panel). The location ofthe calreticulin standard immunoblot using a calreticulin monoclonal antibody is indicated by the arrowhead. (D) IgG (H+L) (left panel) and IgM (right panel) anti-calreticulin ELISA assay of individual female (NZW×B6.Lbric)F1 or MRL-Fas

lprmouse sera. Dashed line represents the mean plus 2 standard deviations from n=7wild-type B6 mice. (E) Paraformaldehyde-fixed PMNs were co-stained with female (NZW×B6.Lbric)F1 mouse serum (green) and rabbit anti-calreticulin (red),nuclei were counterstained with DAPI (blue). The co-localization of mouse serum staining and calreticulin was detected using an anti-IgM secondary antibody(merge: yellow signal). Image magnification, 400×. (F) Paraformaldehyde-fixed PMNs were co-stained with female (NZW×B6.Lbric)F1 mouse serum (green) andrabbit anti-calreticulin (red), nuclei were counterstained with DAPI (blue). Neither the anti-IgG (top panel), nor anti-IgA (bottom panel) secondary antibodiescolocalized the mouse serum staining with calreticulin. Image magnification, 400×.

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Nangalia et al., 2013; Rumi et al., 2014). A relationship betweenlupus autoimmunity, myeloproliferative disorders and calreticulinmutations is reminiscent of the incidence of scleroderma incancer patients with RNA polymerase IIIA mutations (Josephet al., 2014).

Environmental factors that mediate nuclear alterationsmight contribute to lupus autoimmunityAlthough the present study used a genetic model to induce nuclearalterations in a lupus-prone genetic background, the relationshipbetween nuclear structure and the development of anti-nuclearautoimmunity suggests a fundamental role for cell biology in theelusive non-genetic components of SLE. Environmental factorsproposed to trigger lupus autoimmunity (viral infections, drugs, UVirradiation and cancer) also induce alterations in nuclear structure.The nuclear envelope is a physical barrier for viral entry into thenucleus, and must be breached for successful infection of a host cell(Okada et al., 2005; Kobiler et al., 2012; Porwal et al., 2013), andthese virally mediated alterations in nuclear structure coincide withan interferon alpha-driven anti-viral immune response (Elkon andStone, 2011; Levy et al., 2011; McNab et al., 2015). Similarly, thealterations in nuclear structure that occur in cancer cells mightprovide a link to autoimmunity (Foster et al., 2010; Hutchison,2014). Taken together, environmental factors that alter the structureof the nucleus itself might provide crucial contributions to thedevelopment of anti-nuclear autoimmunity in individuals with aninherited genetic risk.

MATERIALS AND METHODSMiceNZWand B6.Lbric/+ mice (Jackson Labs) were housed in specific pathogen-free conditions, and handled in accordance with the guidelines of theTemple University IACUC. NZWand B6.Lbric/+micewere bred to generate(NZW×B6.Lbric) and (NZW×B6)F1 littermates. To prevent biasing of theexperimental results evaluating the incidence of lupus autoimmunity in theF1 progeny, all mice were assessed for the development of serumautoantibodies recognizing chromatin and the cell nucleus prior togenotyping. The mouse Lbric allele was genotyped by Bgl1 restrictionfragment polymorphism analysis (Shultz et al., 2003). The F1 progeny wereborn in predicted ratios, and (NZW×B6.Lbric)F1 mice did not display theichthyosis phenotype of B6.Lbric/ic mice (Shultz et al., 2003).

Evaluation of glomerulonephritis and kidney damageGlomerular immune complex deposition was analyzed by fluorescencemicroscopy (Denny et al., 2006). Pathological evaluation of kidney damagewas assessed in paraffin-embedded sections (4 µm) stained with Masson’sTrichrome, and conducted in a blinded manner by a nephrologist withexperience evaluating mouse kidney sections (D.B.J.). The non-parametricMann–Whitney test was used for statistical comparisons of tissue damagescores, and the Student’s t-test compared white blood cell infiltration, withstatistical significance of P

Southern Biotech). Immunoprecipitates of lamin A/C were prepared fromHeLa lysates in RIPA buffer (Millipore) containing protease andphosphatase inhibitors (Pierce) and rabbit anti-lamin A/C (1:500;GTX101127, Genetex).

Sequential immunoprecipitations were performed on histones isolated fromHeLa cells using the Histone Isolation Kit (Active Motif). HeLa cells werecultured inDulbecco’smodifiedEaglemediumwith 10% fetal bovine serumat37°C in a humidified 5% CO2 atmosphere in media containing penicillin andstreptomycin.Confluentmonolayers ofHeLacells were dislodged bydigestionin trypsin-EDTA solution, washed in fresh culture media, and collected bycentrifugation. Purified histones (∼5 µg) were diluted in ddH2O and labeledwith biotin-NHS (Pierce) at room temperature for 1 h before quenching with1%TritonX-100 lysis buffer.Mouse serum immunoprecipitateswere preparedby pre-coating Protein A/G beads with a rabbit anti-mouse bridging antibody.The pre-coated beads were incubated with mouse serum for 2 h at 4°C in 1%Triton X-100 lysis buffer, washed three times, and incubated with thebiotinylated histone extracts overnight. After three washes with Triton X-100buffer, the serum-adsorbed biotinylated histones were eluted with 0.1 ml of0.5×SDS-PAGE sample buffer and diluted tenfold with 1% Triton X-100 lysisbuffer. Sequential re-precipitations were conducted by incubating the biotin-labeled histone extract with a series of affinity-purified rabbit antibodiesrecognizing specific histone H3 modifications at a final dilution of 1:250(H3K4me3, H3K27ac, H3K9ac, H3K4me, H3K27me3, H3K9me3; all fromActive Motif). Following each round of histone re-precipitation, the clearedbiotinylated histone extract was subsequently re-precipitated with a differentanti-modified histone antibody. The re-precipitated biotinylated histones wereeluted in 2×SDS-PAGE sample buffer, and western blotted using HRP-conjugated streptavidin (Southern Biotech).

Preparation of nuclear and cytoplasmic extractsMouse embryonic fibroblasts (MEFs) were cultured in Dulbecco’s modifiedEagle mediumwith 10% fetal bovine serum at 37°C in a humidified 5%CO2atmosphere in media containing penicillin and streptomycin. Confluentmonolayers of MEFs were dislodged by digestion in trypsin-EDTAsolution, washed in fresh culture media, and collected by centrifugation.MEF cell pellets were washed once in cold hypo-osomotic lysis buffer(10 mM Tris, 10 mM NaCl, 3 mMMgCl2, pH 7.5) containing protease andphosphatase inhibitors (Pierce), resuspended in 0.45 ml of the same buffer,incubated at 4°C on a rotator for 15 min, lysed by addition of NP-40 at a finalconcentration of 0.05%, and incubated again at 4°C on the rotator for15 min. Lysis was confirmed by Trypan Blue staining, and the nucleipelleted at 500×g for 10 min. The supernatant was collected as thecytoplasmic fraction and the nuclear pellet was re-extracted, the cytoplasmicfractions were pooled, the pelleted nuclei were extracted into 2×SDS-PAGEsample buffer, and insoluble debris was cleared from each fraction bycentrifugation at 21,000×g. Fractionation of nuclear proteins was verified byanti-histone H3 immunoblotting using affinity purified rabbit antibodies(1:5000; ab1791, Abcam).

Isolation of chromatin from mouse liverMouse liver chromatin was prepared by a modification of the proceduredescribed by Burlingame and Rubin (1990). The liver of 9-month-oldfemale B6 and B6.Lbric/+ mice was homogenized in a blender with fivevolumes of ice-cold Homogenization buffer (1.2 M sucrose, 3 mM MgCl2,0.6 mM CaCl2, 40 mMNaCl, 10 mM sodium acetate pH 6.0, with 200 mMPMSF), and strained through cheesecloth. The homogenate was centrifugedat 750×g for 10 min at 4°C, the supernatant discarded, the pelletresuspended in the original volume of Homogenization buffersupplemented with 0.1% Triton X-100, re-homogenized with a blender,and centrifuged 750×g for 10 min at 4°C. The supernatant was discarded,the recovered isolated nuclei were extracted with two rounds of blenderhomogenization in 50 ml of ice-cold Buffer A (0.25 M sucrose, 0.25%Triton X-100, 10 mM sodium acetate pH 6.0, with 200 mM PMSF), andcollected by centrifugation at 1500×g for 20 min at 4°C. The extracted pelletwas washed twice by resuspension and blender homogenization in 50 ml ofice-cold Buffer B (0.25 mM sucrose, 1 mM CaCl2, 1 mM Trizma pH 8.0,with 200 mM PMSF), followed by centrifugation at 1500×g for 20 min at4°C. The pellet of isolated chromatin was resuspended in 1.0 ml of Buffer B,

the nucleic acid and protein content was determined by absorbance at260 nm and 280 nm, respectively, and stored in aliquots at −80°C.

Human samplesThe Institutional Review Boards at Temple University and the University ofMichigan approved this study, and subjects gave informed consent inaccordance with the Declaration of Helsinki. SLE patients fulfilled therevised American College of Rheumatology criteria (Tan et al., 1982).PMNs and low-density granulocytes (LDGs) were isolated as previouslydescribed (Denny et al., 2010; Singh et al., 2014), and nuclear morphologywas assessed by Hema3 staining (Thermo Scientific).

Neutrophils from healthy donors were enriched by depletion ofeosinophils from the PMN fraction using magnetic bead-assisted cellsorting. Eosinophils were labeled with a biotinylated anti-CD23 antibody(10 ng/ml; clone BU38, Ancell) on ice for 30 min, washed with cold cellisolation buffer (0.5% BSA, 1 mM EDTA, in PBS), and incubated for30 min with a suspension of anti-biotin paramaganetic microbeads(Miltenyi) on ice, and removed using a magnetic column according tomanufacturer’s guidelines. Monocytes were isolated from Ficoll-Hypaquepreparations of peripheral blood mononuclear cells by incubation with acocktail of mouse biotinylated monoclonal antibodies recognizing CD3(clone UCHT1), CD7 (clone 3A1e), CD19 (clone BU12), CD79b (cloneSN8/3A2-2E7), CD15 (clone AHN1.1), CD56 (clone ANC7C7), andglycophorin A (clone A63-B/C2) (all supplied by Ancell) on ice for 30 min,washed with cold cell isolation buffer, and labeled with anti-biotin-coatedparamagnetic beads (Miltenyi). A magnetic column (Miltenyi) was used tocollect the enriched monocyte preparations.

RNA was isolated using TriPure reagent (Roche) and cDNAs wereproduced using oligo-dT primed reverse-transcription with SuperscriptII(Life Technologies). Human LBR cDNA was amplified using Perfect TaqPolymerase (5-Prime) and the primer pairs: Exon 7 forward GTTGTAG-AAGGAACGCCTC; Exon 14 reverse CGGTAGGGCACACGGTGACA-GTAC. PCR products were subcloned into pCR2.1 (Life Technologies) forsequencing by dideoxynucleotide termination (GeneWiz). Exon skippingwas evaluated by Fisher’s exact test, one-tailed.

AcknowledgementsThis project was initiated using B6.Lbric/+ mice that were kindly provided by Dr KatrinHoffmann (Institute of Human Genetics, Martin Luther University of Halle-Wittenberg, Germany), and the authors are grateful for her contribution. The authorsare also grateful for the support of Dr Philip Cohen. The technical assistanceprovided by Ms Yuxuan Zhen and Ms Roshanak Razmpour, and the comments ofDrs Brendan Hilliard andMarc Monestier are greatly appreciated. We are thankful forthe unrestricted access to the Department of Anatomy and Cell Biology MicroscopyFacility at Temple University provided courtesy of Dr Mary Barbe. Aspects of thisresearch were presented by N.S. at the 2015 Annual Meeting of the AmericanCollege of Rheumatology, San Francisco, CA, in partial fulfillment of the researchrequirements for the Rheumatology Fellows Training Program at Temple University.M.J.K. is the Chief of the Systemic Autoimmunity Branch at NIAMS, the opinionsexpressed in this article are the author’s own and do not reflect the view of theNational Institutes of Health, the Department of Health and Human Services, or theUnited States government.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsN.S.: conception, design, execution and interpretation of the findings beingpublished, and drafting and revising the article. D.B.J.: execution and interpretationof the findings being published, and drafting and revising the article. K.A.M.: design,execution and interpretation of the findings being published, and drafting andrevising the article. I.T.: design and interpretation of the findings being published,and drafting and revising the article. M.J.K.: conception, design, and interpretationof the findings being published, and drafting and revising the article. M.F.D.:conception, design, execution and interpretation of the findings being published, anddrafting and revising the article.

FundingThis work was supported by the National Institutes of Health, provided by a NationalInstitute of Arthritis and Musculoskeletal and Skin Diseases New Investigator Grant[grant number R03-AR061026 to M.F.D.], the Alliance for Lupus Research Target

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Identification in Lupus Grant to M.F.D., and the Department of Medicine at TempleUniversity Faculty Research Development Award to M.F.D.

Supplementary informationSupplementary information available online athttp://dmm.biologists.org/lookup/doi/10.1242/dmm.024851.supplemental

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RESEARCH ARTICLE Disease Models & Mechanisms (2016) 9, 885-897 doi:10.1242/dmm.024851

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