SDS-PAGE was carried out according to Laemmli et al., except for using pre-cast 4–20% gradient gels (Bio-Rad). Gel proteins were transferred to nitrocellulose membranes using Trans-Blot Turbo transfer system and Trans-Blot Turbo transfer pack (Bio-Rad). Proteins were probed with anti-CXorf21 and antiactin antibodies (Novus Biotechnologies) and detected with alkaline phosphatase/nitro blue tetrazolium/5-bromo-5-chloro-3-indolyl phosphate system. Protein bands were quantified using densitometry (ImageJ).

### Lysosomal pH Determination

To detect differences in intracellular pH in live human male and female primary monocytes, dendritic cells, B cells, NK cells, and T cells, the LysoSensorTM Yellow/Blue DND-160 (Thermofisher) and pHrodo R Red AM Intracellular pH Indicator (Thermofisher) was used according to the manufacturer's suggested protocol. Briefly, primary cells were treated with LysoSensor reagents as ratiometric means for measuring lysosomal pH via fluorescence. The ratio of fluorescence allows for the adjustment for possible variability between particle uptake. To quantitate pH, primary cells plated on a 96-well plate were loaded with 5µM pHrodo R Red AM intracellular pH indicators for 30 min. Cells were then washed with a series of Live Cell Imaging MediaTM and standard buffers containing 10µM nigericin and 10µM valinomycin were added for 5 min in order to clamp intracellular pH values 4.5, 5.5, 6.5, and 7.5. We determined the mean cellular fluorescence in triplicate samples using a spectrophotometer (Synergy H1, Biotek). A standard curve for male or female samples showed a linear relationship between the intracellular pH and the relative fluorescence units.

# Statistics

Statistical analyses were carried out using T-test, one-way ANOVA with multiple comparisons, or Fisher's Exact test using GraphPad Prism 7.

#### RESULTS

#### Primary Female Human Monocytes Have a More Acidic Lysosomal pH Compared to Male Cells

CXorf21 mRNA and protein are expressed at higher levels in primary monocytes, CD19+ B cells, and Lymphoblastoid cell lines (LCLs) from female healthy subjects compared to male controls (**Figure 1**: Harris et al., unpublished). Based on the function of the CXorf21 protein and its interaction with SLC15A4, which is known to regulate lysosomal pH, we hypothesized that, with greater expression of the CXorf21 protein, lysosomal pH would be lower in female male monocytes. In order to assess a difference in lysosomal pH between male and female monocytes, we performed ex vivo lysosomal pH measurements. Following a 30-min incubation period of the cells with pH calibration buffers, a standard curve for both male and female (**Figure 2A**) primary monocytes to determine pH based on male and female (**Figure 2B**) relative fluorescence was generated. We found that unstimulated female monocytes have a significantly more acidic lysosomal pH (4.9) compared to male monocytes (5.6) (**Figure 2C**) (p = 0.0001 by Fisher's exact test).

anti-actin (42 kD) is shown as a loading control. Densitometry via Image J was

# Other Cell Types in Which CXorf21 Is Expressed Higher in Female Cells

used to quantitative optical density of protein bands.

This trend for lower pH in the lysosomes of female cells held true for both dendritic cells, where female DCs lysosomal pH was 4.9 and male DCs were 5.7 (p-value 0.044; **Figure 3**), as well as B lymphocytes, where female lysosomal pH was 5.0 and male lysosomal pH was 5.6 (p = 0.0447 by Fisher's exact test; **Figure 4**). These data suggest that female monocytes, DCs, and B cells, immune cells with increased CXorf21 and TLR7 expression, have a more favorable lysosomal processing environment compared to male cells, and may drive the robust TLRs/lysosomal-dependent immune response observed women compared to men (28).

## Lysosomal pH in Cells That do Not Express CXorf21

In order to assess the role of female overexpression of CXorf21 in lysosomal pH, we studied immune cells in which the expression of CXorf21 is absent. To that end, we isolated primary T cell and NK cells, two immune cells with minimal CXorf21 and TLR7 levels. We found, while their lysosomal pH was optimal for lysosomal signaling, there was no significant difference in pH between the sexes (**Figures 4D,E**).

# DISCUSSION

There are ∼80 autoimmune disease, the great majority of which affect women more than men. Both SLE and SS have a ratio of about 10 affected women for every one affected man (2). The sex bias in SLE is present among patients with childhood onset (29). In SLE and SS, more women are affected than men in older adulthood at ages where women are post-menopausal (30).   SS is much less well studied than SLE; however, again no explanation of the marked predilection for women has been made.

Based on data concerning X chromosome aneuploidies, we have proposed that the increased female risk of SLE and SS is the result of the X chromosome complement. Men with Klinefelter's syndrome (47,XXY) are enriched 15-fold in these diseases (23, 24). In addition, 47,XXX women are also found in excess among those with either SLE or SS, but not rheumatoid arthritis or primary biliary cirrhosis (25). Very rare abnormalities of the X chromosome among patients with SLE or SS, including partial triplications, as well as rare patients with Turner's syndrome and SLE may localize the effect to the X chromosome distal p arm (35, 36).

Of course, in cells with two or more X chromosomes all but one X is inactivated in order to equalize gene dose compared to male cells. However, X inactivation is not an all or none phenomenon with a significant fraction (up to 20%) of Xlinked human genes escaping X inactivation (22, 37). Thus, a key factor in the idea that an X chromosome gene dose effect mediates the sex bias of SLE and SS is the escape of X inactivation such that female cells have bi-allelic mRNA expression and potentially more functional protein of a given X-linked gene.

Two genes that escape X inactivation in immune cells, contain SLE-risk alleles, and have critical roles in production of interferon are CXorf21 and TLR7 (38). Thus, on this basis, these genes are candidates to mediate the X chromosome gene dose effect for the sex bias of SLE and SS. CXorf21 is a binding partner of another SLE-risk gene—Slc15a4. As noted above SLC15A4 is involved in transport of oligopeptides and hydrogen ions out of the lysosome, and knockout of Slc15a4 results in abrogation of TLR7 signaling as well as amelioration of murine lupus (17, 18, 20, 21). We have shown that CXorf21 protein is expressed exclusively in monocytes, B cells, and dendritic cells, and the protein levels are two–three-fold higher in female cells compared to male cells. In addition knockdown of CXorf21 with CRISPR-Cas resulted in abrogation of interferon, TNF-α and IL6 secretion after TLR7 activation in female cells. Furthermore, lysosomal pH increased, suggesting an environment less conducive to lysosomal signaling (Harris et al., unpublished).

Because lysosomal pH was affected by decreased expression of CXorf21 and because female cells express more CXorf21 than do male cells, we hypothesized that female cells expressing CXorf21 would have a more acidic pH than male cells. Therefore, we studied lysosomal pH in B cells, monocytes and dendritic cells from healthy human subjects. In fact, the present results demonstrate that female cells had a more acidic lysosomal pH than did these cells from male subjects. T lymphocytes and NK cells, which do not express CXorf21 at appreciable levels, did not have a pH difference between male and female derived cells. Thus, we conclude that a possible functional role of CXorf21 is regulation of lysosomal pH,

and that differing levels of expression between the sexes lead to distinct lysosomal pH. To our knowledge this is the first report of a lysosomal pH difference between male and female immune cells.

We have ventured to predict a mechanism of action for the uncharacterized protein CXorf21 as a short-chain dehydrogenase reductase. We put forth two plausible functions: (1) as a reductase CXorf21 utilizes NADPH to generate hydrogen ions for lysosome proton pump consumption (i.e., v-ATPase pump); or, (2) as a dehydrogenase generates NAPDH for lysosome superoxide production by the lysosomal-resident NOX2 complex. Both scenarios could result in changes in lysosomal pH.

What might be the functional consequences of this pH difference? Simply, altered (auto)antigen processing and presentation or modulation of endolysosomal resident TLR7 activity. As noted above, a recent report demonstrates that the X-linked TLR7 gene is bi-allelically expressed in immune cells (that is, escapes X inactivation), and has increased protein levels in female cells compared to male cells (38). In addition to an increase in lysosomal pH and abrogation of TLR7 signaling, our data using CXorf21 CRISPR-Cas knockdown show that there is a loss of TLR7 agonist-induced increased TLR7 expression (both mRNA and protein). Thus, CXorf21 is critically involved in TLR7 signaling, including a feedforward expression loop for TLR7. Thus, we propose increased expression of CXorf21, either because of the presence of two X chromosomes and the escape of X inactivation, or because the SLE-risk allele increases expression (18), leads to increased TLR7 signaling and increased interferon production.

The present study is limited, especially in regard to studying lysosomal pH and its regulation in regards to the pathogenesis and treatment of SLE. Endosomal TLR signaling, which leads to type 1 interferon production, is clearly important in SLE pathogenesis, both in humans and animal models (39). Furthermore, this signaling is exquisitely sensitive to changes in endolysosomal pH. CXorf21, Slc15a4, TLR7, and NCF1 (encodes the p47phox NOX2 subunit) all contain SLE risk alleles (4– 6). Published data discussed above demonstrate that SLC15A4 regulates endolysosomal pH, and data herein show that the protein product of CXorf21 also regulates this pH. NOX2 is activated by phosphorylation of its p47phox subunit by TLR7 signaling (40, 41). Meanwhile, TLR7 and NOX2 signaling are both regulated by endolysosomal pH, and activated NOX2 regulates endolysosomal pH (42, 43). Both CXorf21 and TLR7 escape X inactivation (22). Thus, determining how these genes, all of which are involved in endososomal TLR signaling and type 1 interferon production, impact the pathogenesis and sex bias of SLE will need a great deal more investigation. The treatment of SLE may also be impacted by the interaction of these genes, their SLE-associated alleles and their protein products. Hydroxychloroquine, an important mainstay of SLE therapy (44–49), has a variety of effects (50–54), including altering endolysosomal pH, antigen presentation, TLR signaling and cytokine production. Obviously, these mechanisms of action intersect with the TLR7 signaling pathway and the genes

units (RFUs) were measured with multi-well plate reader (Details in Material and Methods). (C) pH determined using pHrodo indication kit according to manufactures protocol. Male and Female (D) pan-T cells and (E) NK cell pH was determined as described above (standard curves and RFUs data not shown) Student's t-test was used to determine statistical difference in RFUs and pH that was converted into pH values via each standard curve; \*p < 0.05; ns=not significant; Error bar represent SEM.

discussed above. We have not addressed how the SLE-associated alleles in these four genes might influence the efficacy of hydroxychloroquine, which will likely require study of healthy controls as well as SLE patients with various combinations of these alleles.

These data demonstrate the function of SLC15A4 and CXorf21, which form a complex that regulates lysosomal pH, and in turn regulates TLR7 signaling. Furthermore, based on the differential expression of CXorf21 between the sexes, we have shown that lysosomal pH, a key factor in signaling in this cellular compartment, is different between men and women. This difference and the resulting functional immune differences may contribute to the X chromosome gene dose that underlies the sex bias of SLE and SS.

#### DATA AVAILABILITY

All datasets generated for this study are included in the manuscript and/or the supplementary files.

#### AUTHOR CONTRIBUTIONS

VH designed the experiments, carried out the experiments, wrote the first draft, edited the paper, and approved the final version. RS designed the experiments, wrote the manuscript, and approved the final version. IH designed the experiments, edited the paper, and approved the final version. BK carried out experiments, edited the paper, and approved the final version. KK designed the experiments, edited the paper, and approved the final version.

#### REFERENCES

#### FUNDING

This work was supported in part by grants from the Lupus Research Institute (370617), the US Department of Veterans Affairs (5I01BX001451), and the US National Institutes of Health (AR053734, AR053483, AI082714, GM104938), and NIAMS Training grant 5T32AR007534-32.

immunity for the initiation of autoimmune inflammation. Sci Rep. (2016) 6:24477. doi: 10.1038/srep24477

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Harris, Harley, Kurien, Koelsch and Scofield. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# A Variant of the Histone-Binding Protein sNASP Contributes to Mouse Lupus

#### Jiyu Ju1 , Jia Xu2 , Yaoqiang Zhu1 , Xiaoyan Fu1 , Laurence Morel 3 \* and Zhiwei Xu1,4 \*

*1 Department of Immunology, Weifang Medical University, Weifang, China, 2 Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States, 3 Immunology and Laboratory Medicine, Department of Pathology, College of Medicine, University of Florida, Gainesville, FL, United States, 4 Department of Anatomy and Cell Biology, College of Medicine, University of Florida, Gainesville, FL, United States*

The *Sle2c1rec1c* (*rec1c*) sublocus is derived from the mouse lupus susceptibility 2 (*Sle2*) locus identified in the NZM2410 model. Our current study dissected the functional characters and the genetic basis of the *rec1c* locus relative to lupus when co-expressed with the Fas*lpr* mutation, an established inducer of autoimmunity. The rec1c.lpr mice exhibited mild expansion of lymph nodes and had a normal T cell cellularity, but developed significantly kidney and lung inflammation, indicating that the *rec1c* amplifies *lpr*-induced autoimmune pathogenesis. A variant of somatic nuclear autoantigenic sperm protein (sNASP) was identified from the *rec1c* interval as a substitution of two consecutive amino acid residues in the histone-binding domain, resulting in an increased binding affinity to histone H4 and H3.1/H4 tetramer. To determine the role of the *sNASP rec1c* allele in mouse lupus, a novel strain was generated by introducing the *rec1c* mutations into the B6 genome. In this transgenic model, the *sNASP* allele synergized with the *lpr* mutation leading to moderate autoimmune phenotypes and aggravating inflammatory pathology alterations in kidney and lung that were similar to those observed in the rec1c.lpr mice. These results establish that the *sNASP* allele is a pathogenic genetic element in the *rec1c* sublocus, which not only promotes autoimmunity, but also exacerbates the inflammation reaction of end organs in mouse lupus pathogenesis. It also shows the complexity of the *Sle2c* locus, initially mapped as the major locus associated with B1a cell expansion. In addition to *Cdkn2c*, which regulates this expansion, we have now identified in the same locus a protective allele of *Csf3r*, a variant of Skint6 associated with T cell activation, and now a variant of *sNASP* that amplifies autoimmunity and tissue damage.

Keywords: mouse, lupus, lupus nephritis, genetics, NASP, histone-binding protein

# INTRODUCTION

Mouse models of systemic lupus erythematosus (SLE) have greatly contributed to the understanding of SLE pathogenesis, including by the identification of genetic pathways whose alterations lead to increased disease susceptibility or resistance (1). Although great efforts have been invested in the genetic analysis of spontaneous lupus mouse models, only a few lupus susceptibility genes have been identified with a putative causative etiology (2, 3). Although polymorphisms in these genes so far do not seem to be directly involved in human lupus, they fit into pathways that

#### Edited by:

*Michele Marie Kosiewicz, University of Louisville, United States*

#### Reviewed by:

*Stefania Gallucci, Temple University, United States Shaun William Jackson, Seattle Children's Research Institute, United States*

#### \*Correspondence:

*Laurence Morel<EMAIL_ADDRESS>Zhiwei Xu<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Autoimmune and Autoinflammatory Disorders, a section of the journal Frontiers in Immunology*

> Received: *08 November 2018* Accepted: *08 March 2019* Published: *02 April 2019*

#### Citation:

*Ju J, Xu J, Zhu Y, Fu X, Morel L and Xu Z (2019) A Variant of the Histone-Binding Protein sNASP Contributes to Mouse Lupus. Front. Immunol. 10:637. doi: 10.3389/fimmu.2019.00637*

**83**

have been associated with lupus or other rheumatic diseases (2). The murine lupus susceptibility locus Sle2 was identified on chromosome 4 as one of the three major loci associated with nephritis in the NZM2410 model (4). Sle2 expression on a nonautoimmune background in the B6.Sle2 congenic strain revealed that it regulates B cell hyperactivity (5) and B1a cell expansion (6), but is not sufficient for clinical disease. However, the coexpression of Sle2 with the lpr mutation in the Fas gene in the B6.Sle2.lpr mice resulted in more severe lupus nephritis and marked lymphadenopathy compared with B6.lpr mice (7).

The dissection of Sle2 revealed a complex genetic architecture, with three independent loci, Sle2a, Sle2b, and Sle2c, contributing to B1a cell expansion, with the NZB-derived Sle2c being the strongest contributor (8). We identified a hypomorph allele of Cdkn2c as responsible for the Sle2c B1a cell expansion (9, 10). Sle2c contains a suppressive Sle2c2 sublocus (11) that we have mapped to a missense mutation in the Csf3r gene encoding for the GCSF receptor and regulates the development of CD8a+ dendritic cells (11–13). In addition, we mapped the pathological phenotype synergizing with lpr to the centromeric portion of Sle2c, the Sle2c1 sublocus (7). Subsequently, we generated a series of shorter Sle2c1 intervals and investigated their epistatic interaction with lpr (14). Two non-overlapping subloci with non-redundant phenotypes were identified: The centromeric portion of Sle2c1, Sle2c1rec1a, which contains Cdkn2c, exerts a strong contribution to lupus autoimmunity without clinical phenotypes. A more telomeric sublocus named Sle2c1rec1d (rec1d) was associated with more severity of renal inflammation and lymphadenopathy, and higher frequency of dermatitis in the B6.Sle2c1rec1d.lpr (rec1d.lpr) mice than that in the B6.Sle2c1rec1a.lpr (rec1a.lpr) mice (14). It is reasonable to assume that the Sle2c1rec1c (rec1c) sublocus can contribute to mouse lupus by synergizing with the overlapping region of the rec1a and rec1d subloci, because the rec1d.lpr mice developed more severe autoimmune disease than rec1a.lpr mice (14).

Recently, we produced a novel recombinant rec1d1 from the rec1d sublocus (**Figure 1**) that narrowed down the location of the gene responsible for the severe autoimmune disease with striking lymphadenopathy in the rec1d1.lpr mice (15). The only gene in the rec1d1 interval that presented a non-synonymous mutation was Skint6, and this mutation resulted in a truncated secretory peptide (15). The Skint6 protein is mainly expressed in mouse skin, and we obtained evidence that non-hematopoietic cells expressing the rec1d1 Skint6 allele promoted T cell proliferation in vivo, suggesting that the Skint6 variant is the most likely causal gene in the rec1d1 sublocus.

The focus of this study was to analyze the phenotypes associated with the expression of rec1c, which is telomeric of rec1d1 (14, 15). When combined with lpr, rec1c showed a modest effect on autoimmune phenotypes, and greatly aggravated kidney and lung pathology. Exon sequencing of all the coding genes

present in the rec1c sublocus identified two non-synonymous mutations in the rec1c allele of the sNASP gene. The sNASP is the somatic isoform of the NASP protein, a histone-binding protein that controls H3.1 folding and regulates the pool of soluble H3- H4 histones available for DNA synthesis (16). NASP controls the progression through the cell cycle (17, 18) and regulates chromatin folding (19, 20). More recently, it has been shown that NASP regulates chromatin accessibility by maintaining a pool of H3K9me1 methylated histones (21), an epigenetic mark associated with active transcription sites (22). We show here that the rec1c allele of sNASP has a greater binding affinity for H4 histone or H3.1/H4 tetramers in vitro. Further, B6.lpr mice in which the mutated sNASP has been knocked-in displayed phenotypes similar to that of the rec1c.lpr mice, but also showed some extra autoimmune alterations. These results identify a gain of function allele of sNASP with the ability of increasing autoimmunity and aggravating inflammatory damage in end organs during lupus development. This discovery adds a new member to the list of pathogenic genes in murine lupus models and provides insights into new mechanisms of autoimmune diseases. It also identifies for the first time a natural variant of a histone-binding gene as a lupus susceptibility gene, potentially by regulating chromatin accessibility**.**

#### MATERIALS AND METHODS

#### Mice

B6.lpr mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). The B6.Sle2c1rec1c.lpr strain was previously described (7). All available polymorphic genetic markers were

**Abbreviations:** NASP, nuclear autoantigenic sperm protein; sNASP, somatic nuclear autoantigenic sperm protein; SLE, systemic lupus erythematosus; SNPs, single nucleotide polymorphisms; ES, embryonic stem cell; KI, knockin; IPTG, Isopropyl β-D-1-thiogalactopyranoside; APS, aminopropylsilane; GN, glomerulonephritis; BLI, biolayer interferometer.

used to refine the rec1c interval and define its ends. The B6.1sNASP mouse with the mutated bases of the rec1c sNASP allele introduced into the B6 genome was created by Cyagen Biosciences Inc. with the targeting strategy presented in **Figure 6A**. The mutated bases of the rec1c sNASP allele are in exon 12 of Nasp-001 ENSMUST00000030456. To construct the targeting vector, two homology arms were generated by PCR using BAC clone RP24-384F21 and RP24-72F14 from the C57BL/6J library as template. The CTGTACTCCATGAGC sequence in exon 12 of the NASP gene, corresponding to exon 10 of the sNASP isoform, was mutated to CT**A**TA**T**TCCATGAGC in the 5′ homology arm. In the targeting vector, a Neo cassette was flanked by Frt sites and DTA was used for negative selection. The constructed targeting vector was electroporated into C57BL/6 mouse embryonic stem (ES) cells, and then selected positive ES clones were microinjected into blastocysts. Chimeric mice were screened by genotyping and then bred with an Flp-deleter mouse to generate F1 mouse with constitutive knockin (KI) rec1c sNASP allele through Flp-mediated recombination. Finally, F1 mice were intercrossed to obtain a homozygous transgenic B6.1sNASP model. Following the previously described protocol (6), the lpr mutation was bred into the B6.1sNASP mouse to generate a B6.1sNASP.lpr strain. Both male and female mice were used in this study, without difference between genders. The protocols for mice used in this research were approved by the Institute Animal Care and Use Committees of the University of Florida, USA and Weifang Medical University, China.

#### DNA Sequencing and RT-PCR

Genomic DNA of the lupus-prone strains MRL/MpJ-Faslpr/J, NZM2410/J, BXSB/MpJ, NZB/B1NJ, and NZW/Lac/J was purchased from Jackson Laboratory. The Agilent SureSelect XT Mouse All Exon Capture Kit (Agilent Technologies, Inc., Santa Clara, CA, USA) used in this project has 50 Mb capture, covering the complete mouse exome and spanning over 221,784 exons and 24,306 genes. Mouse whole exome sequencing was performed by the Beijing Genomics Institute (Shenzhen, China), including DNA fragmentation, adapter ligation, hybridization with capture library, next-generation Illumina sequencing with an average 30x coverage, and bioinformatics analysis according to mouse genome assembly NCBIm37 (strain C57BL/6J). We selected the homozygous SNPs corresponding to nonsynonymous mutations, frameshifts, deletions, insertions, stop loss or gain in coding regions. Total RNA was purified from tissues using the Qiagen RNeasy kit (Qiagen, Valencia, CA, USA) and converted into cDNA by reverse transcription using the SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific, Waltham, MA). The Sanger method was used to sequence cDNA or specific exons. RT-PCR was utilized to semiquantitatively detect sNASP mRNA expression (Forward primer: 5 ′ ACAAGCCCATCTTAAACTTGGAG3′ ; Reverse primer: 5′ CTGAGATTCCTTTGCGTCTTCTA 3′ ).

#### Protein Expression, Purification, and Binding Kinetics of Protein Interaction

The full-length mouse sNASP cDNA (encoding 448 amino acids) was prepared from B6.lpr mouse using RT-PCR and then inserted into the pET30a expression vector to obtain a pET30a-WT sNASP protein expression vector. The mutated bases of the rec1c sNASP allele were introduced into the WT sNASP protein expression vector using the Q5 R Site-Directed Mutagenesis Kit (NEB, Ipswich, MA) to generate a pET30a-rec1c sNASP allele protein expression vector. All sNASP constructs were confirmed by DNA sequencing. WT and mutated expression vectors were transformed into E. coli BL21(DE3)and protein expression was induced by Isopropyl β-D-1-thiogalactopyranoside (IPTG). Ionexchange chromatography and size exclusion chromatography were used to purify proteins from the bacterial lysate. The protein purity was verified using SDS-PAGE electrophoresis. Western-blotting was used to identify mouse sNASP protein using anti-mouse NASP mAb (A-7, Santa Cruz Biotechnology, Inc., Dallas, TX).

Mouse histones H1a, H3.1, H4 were purchased from Lifespan Biosciences (Seattle, WA). The H3.1/H4 tetramer complex was prepared by incubating a mixture of mouse H3.1 and H4 overnight at room temperature followed by purification with size exclusion chromatography. The binding affinity of sNASP for histones was determined using biolayer interferometer Octet K2 system (Pall Fortebio Corp., Menlo Park, CA) at 30′C, following the instrument user guide. Briefly, aminopropylsilane (APS) biosensors were rinsed in assay buffer for 120 s to obtain an initial baseline. Next, mouse sNASP WT and mutant proteins were immobilized on the APS biosensors for 110 s to get a loading curve. Third, the sNASP-immobilized-APS biosensors were dipped into assay buffer for 120 s to acquire another baseline. Fourthly, the sNASP-immobilized-APS biosensors were exposed to various concentrations of histone H1a, H3.1, H4, and H3.1/H4 tetramer complex in assay buffer for 240 s to obtain association curves (Kon/M−1 s −1). Finally, the sNASPimmobilized-APS biosensors were again dipped into assay buffer without histones to get disassociation curves (Koff/s−1 ). The interaction of mouse sNASP and mouse histones was expressed as layer thickness (nm) over time (second). The binding affinity (KD) was calculated by dividing Kon by Koff. The protein expression, purification and measurements of binding kinetics were performed by Detai Biologics Company (Nanjing, China).

# IgG Autoantibody Detection

IgG anti-dsDNA and anti-chromatin IgG were measured by ELISA as previously described (6). Briefly, mBSA-coated plates were coated overnight with 50 mg/ml dsDNA for anti-dsDNA autoantibody detection. 10 mg/ml of histone H1, H2A, H2B, H3, and H4 were added to the dsDNA-coated plate for antichromatin autoantibody measurement. Test sera at 1:100 dilution was added to the plates and bound autoantibodies were detected using alkaline phosphatase-conjugated goat anti-mouse IgG and pNPP substrate. Raw optical densities were converted to units per milliliter, using a standard curve derived from pooled MRL/lpr serum, arbitrarily setting the reactivity of a 1:100 dilution of this serum to 100 U/ ml.

#### Flow Cytometry

Cell subsets and activation status in spleen and lymph nodes were determined by flow cytometry as previously described (8). In brief, single-cell suspensions were prepared and depleted of red blood cells with 0.83% NH4Cl Tris-buffer. Cells were blocked with saturating amounts of anti-CD16/CD32 (2.4G2) and stained with fluorochrome-conjugated antibodies against CD3e (145-2C11), CD4 (RM4-5), CD69 (H1.2F3), CD44 (IM7). All antibodies were purchased from BD Pharmingen (San Jose, CA, USA) or eBioscience (San Diego, CA, USA). At least 50,000 events were acquired per sample using a FACSCalibur cytometer (BD Biosciences, San Jose, CA, USA).

#### Kidney, Lung, and Liver Pathology

Tissues from 4 to 5-months-old mice were fixed and stained with hematoxylin and eosin (H&E). In addition, kidneys were also stained with periodic acid Schiff (PAS). Renal lesions were scored in a blinded manner following the previous report (7), and briefly speaking: grade 0, normal glomeruli, and evident capillary loops and unexpanded mesangium; grade 1, evident capillary loops, and widened mesangium with mild hypercellularity; grade 2, evident capillary loops, and expanded mesangium with more than moderate hypercellularity; grade 3, diminished capillary loops, swollen glomeruli, and more than 50% of all glomeruli with diffuse endocapillary proliferation; grade 4, no capillary loops, and basement membrane thickening and significant mesangial proliferation, more than 90% of all glomeruli with diffuse endocapillary proliferation. Lung pathology alterations were evaluated semi-quantitatively following the protocols in our publication (15), to briefly summarize: grade 0, normal lung architecture; grade 1, 1–10% of alveolar in lung has the pathological alterations of exudates, atelectasis and increased inflammatory cell number; grade 2, 10–25% of alveolar in lung shows the above pathological alterations and mild infiltrate of inflammatory cells around arteries and veins; grade 3, 25–50% alveolar in lung displays the above pathological alterations and moderate infiltrate of inflammatory cells around arteries and veins; grade 4, >50% alveolar in lung demonstrates the above pathological alterations and heavy infiltrate of inflammatory cells around arteries and veins.

The presence of immune complexes in the kidneys were evaluated on 5µm frozen sections stained with FITC-conjugated rat anti-mouse C3 (SC-58926, Santa Cruz Biotechnology, Dallas, TX) and IgGκ BP-CFL 488 (SC-516176, Santa Cruz Biotechnology, Dallas, TX). Staining intensity was evaluated by examining sections with Olympus BX53 fluorescence microscope and DP80 camera (Diagnostic Instruments). Average 20 glomeruli for each sample was recorded as semi quantitative 0–4 scale using Image J software (NIH).

#### Statistical Analysis

Data were analyzed with GraphPad Prism 5.0 software with the statistical tests indicated in the text. Non-parametric tests were used when data were not distributed normally.

# RESULTS

#### Fine-Mapping of the rec1c Sublocus

Since the rec1c interval is of NZB origin (8), we refined its map and defined its ends by genotyping all available markers that are polymorphic between the NZB and B6 genomes (**Figure 1**), including microsatellite Mit and singlenucleotide polymorphisms (SNPs) markers collected from the Mouse Genome Informatics (MGI), the National Center for Biotechnology Information (NCBI) or identified through our own genomic sequencing. The rec1c interval includes D4Mit278 at the centromeric end and rs27480282 at the telomeric end, but excludes the Novel5 marker and rs27513842, defining rec1c as a 1.39–2.99 Mb interval (**Figure 1**). The rec1c and rec1d1 subloci do not overlap, but together cover the entire rec1d sublocus. The rec1c is in a gene-rich region, which contains 44 protein-coding genes, including those in the intersection area of B6 and NZB genomes (**Figure 1**).

#### The rec1c Sublocus Promotes End Organ Inflammation in the rec1c.lpr Mouse

We analyzed the autoimmune pathology of the rec1c.lpr strain by comparing with control B6.lpr mice at the age of 4–6 months. The spleen sizes of rec1c.lpr mice were similar as that of B6.lpr mice, but the rec1c.lpr mice presented larger pooled lymph nodes (469 ± 46 mg), about 2 times larger than that of B6.lpr mice (292 ± 16 mg) (**Figure 2A**). The rec1c.lpr mice showed the same percentage of CD3+ T cells in spleen and lymph nodes (**Figure 2B**) and similar frequencies of CD4+ T cell expressing the early activation marker CD69 (**Figure 2C**) as B6.lpr mice. A small but significant increased frequency of CD44+CD4+ effector T cells was however observed in rec1c.lpr mice (**Figure 2D**).

The rec1c.lpr mice produced the same amount of serum antidsDNA and anti-chromatin IgG as the B6.lpr mice (**Figure 3A**). As the rec1c.lpr mice displayed a milder lymphadenopathy than rec1a.lpr or rec1d.lpr mice, the pathology of their kidneys and lungs was not examined in our previous report (7). Unexpectedly, we found that rec1c.lpr mice developed significantly more severe renal and lung inflammation than age-matched B6.lpr mice (**Figures 3B,C**). B6.lpr mice showed a mild mesangial expansion, but the rec1c.lpr mice displayed a markedly proliferative kidney pathology with glomerular cell proliferation and inflammatory cell infiltrates in addition to mesangial expansion (**Figure 3B**). Most of B6.lpr mice exhibited normal blood vessels in their lungs, thin inter-alveolar septum, or a low degree of inflammatory infiltrates. In contrast, the rec1c.lpr lungs showed obvious histopathological alterations, including the presence of numerous congested blood vessels, large peribronchiolar and perivascular inflammatory cell infiltrates (**Figure 3C**). We also examined liver tissues and skin appearance. Most of B6.lpr or rec1.lpr mice displayed normal liver histology, although a few of them had perivascular inflammatory cell infiltrations without a difference between strains. Neither rec1c.lpr nor B6.lpr mice develop skin disease. These results indicated that the rec1c sublocus contains some potential disease-causing allele(s), which promotes inflammation of end organs.

## A sNASP Variant Allele Was Identified in the rec1c Interval

To uncover potentially pathogenic genetic variants in the rec1c interval, we sequenced all exons of its 44 protein-coding genes using whole exome sequencing (WES). As a result, we identified a variant of somatic nuclear autoantigenic sperm protein gene

spleen and lymph nodes (A), and percentages of CD3+ T cells (B) as well as CD69+CD4+ (C) and CD44+CD4+ (D) T cell subsets in spleen and lymph nodes in B6.lpr and rec1c.lpr mice at age of 4–5 months. Statistical analysis was performed using two-tailed Mann-Whitney tests.

(sNASP) with two mutations in exon 10: Chr4:g116,276,661 G>A and Chr4:g116,276,664 C>T (NCBI m37 assembly), which correspond to 841G>A and 844C>T, respectively, in the sNASP cDNA sequence (**Figure 4A**). Consequently, the rec1c allele of the sNASP protein has a substitution of two consecutive amino acid residues, V281I and L282F, in its putative histone-binding motif (19). We therefore anticipate that the rec1c sNASP protein may have an altered binding to histones. Sequencing of sNASP exon 10 in the NZB, NZW, NZM2410, MRL/lpr, and BXSB lupus-prone mice showed the rec1c mutations in the NZB and NZM2410 genomes, as expected, also in the NZW and MRL/lpr strains, but not in the BXSB strain (**Figure 4B**). B6.lpr and rec1c.lpr mice produced comparable amount of sNASP mRNA in their skin, thymus, bone marrow, and spleen (**Figure 4C**). Overall,

these results identify the sNSAP allele as a candidate gene for the rec1c interval through its possibly altered binding to histones, and show that this allele is shared among several lupus-prone mouse genomes.

#### The rec1c sNASP Protein Is Dysfunctional in Binding Histones

We next investigated the histone-binding function of the rec1c sNASP protein function. WT sNASP and rec1c sNASP proteins were expressed in E. coli and purified by ion-exchange chromatography and size exclusion chromatography with more than 90% purity (**Figure 5A**). Quantitative binding studies of the sNASP protein interacting with mouse histones H1a, H3.1, H4, and the H3.1/H4 tetramer were measured using biolayer interferometry (BLI). A representative BLI assay graph in **Figure 5B** shows the interaction of WT sNASP binding H4 histone as expressed by layer thickness (nm) over time (second). **Table 1** lists the binding constants of the WT sNASP and rec1c sNASP proteins interacting with histones H1a, H3.1, H4, and

and spleen (Spl) was detected by RT-PCR. *Gapdh* was used as a control.

H3.1/H4 tetramer. Both WT and rec1c sNASP proteins showed a stronger binding affinity for H3.1 than for H1a histone, with almost a 40-fold difference. However, the WT and rec1c sNASP proteins did not show any different affinity in binding these two histones. sNASP also showed a strong binding affinity for H4 histone or H3.1/H4 tetramer in comparison with its binding to

mouse histones H1a, H3.1, H4, and H3.1/H4 tetramer. (B) A graph of a representative assay shows the interaction of WT sNASP binding histone 4 at the indicated concentrations from 62.5 to 1,000 nM. Each experiment is represented by an initial baseline (a), a sNASP immobilization curve (b), another baseline (c), an association curve (d), and a disassociation curve (e).

H1a histone (**Table 1**). The rec1c sNASP showed significantly lower Kd values for binding to H4 histone or H3.1/H4 tetramer than WT sNASP. This indicated that the rec1c sNASP protein has a significantly stronger affinity for binding H4 histone or H3.1/H4 tetramer than WT sNASP protein. These data demonstrate that the substitution of two consecutive amino acid residues in the rec1 sNASP protein leads to an increased affinity of binding mouse H4 histone or H3.1/H4 tetramer.

### The rec1c sNASP Allele Promotes Autoimmunity and Exacerbates End Organ Inflammation in a Transgenic 1sNASP.lpr Model

To test the hypothesis that the rec1c sNASP protein, which displays an increased affinity for H4 histone and H3.1/H4 tetramer, is involved in autoimmune diseases, the most reliable

TABLE 1 | Binding kinetics and affinities for the interactions of mouse WT sNASP and *rec1c* variant sNASP proteins with mouse histones H1a, H3.1, H4, and H3.1/H4 tetramer.

*Quantitative binding studies of the interactions of mouse WT sNASP and rec1c variant sNASP proteins with mouse histones H1a, H3.1, H4, and H3.1/H4 tetramer were measured using biolayer interferometer. The binding kinetic parameters were determined from four separate experiments (n* = *4). Values in the table indicate mean and SEM (standard error of mean). Calculated Kd* = *koff /kon.*

approach is to construct a transgenic model with the mutated bases of the rec1c sNASP allele on the B6 background. Following the targeting strategy shown in **Figure 6A**, we used DNA homologous recombination to substitute the guanine at 4:116276661 and cytosine at 4:116276664 in the B6 genome with the corresponding adenine and thymine present in the rec1c sNASP allele. This transgenic model was called B6.1sNASP. DNA sequencing confirmed that B6.1sNASP mouse has the mutated bases of the rec1c allele in sNASP cDNA sequence (**Figure 6B**), which indicates that the rec1c sNASP allele was correctly introduced into B6 genome. Western blotting revealed that both B6 and B6.1sNASP strains express similar amounts of sNASP protein in the skin, spleen, and thymus (**Figure 6C**), indicating that the sNASP protein expression was not affected in the B6.1sNASP model. Moreover, similar to B6.rec1c mouse, the B6.1sNASP mouse displayed a normal growth and procreation, and did not develop any detectable autoimmune phenotypes (data not shown). Adopting the same strategy as we used with B6.rec1c.lpr, we introduced the lpr mutation into the B6.1sNASP model to generate B6.1sNASP.lpr (1sNASP.lpr) mice.

We comprehensively evaluated the autoimmune phenotypes and organ pathology of the 1sNASP.lpr as compared to B6.lpr mice at the age of 4–6 months. The 1sNASP.lpr mice developed an enhanced lymphadenopathy with an average weight of the spleen or lymph nodes about twice and triple that of B6.lpr mice, respectively (**Figure 7A**). Total cell numbers in spleen and lymph node of 1sNASP.lpr mice significantly increased in comparison with B6.lpr mice (**Figure 7B**). The percentages of CD3+ T cells (**Figure 7C**) and CD19+ B cells (**Figure 7D**) in spleen and lymph nodes were comparable between B6.lpr and 1sNASP.lpr mice. However, 1sNASP.lpr mice have more absolute numbers of splenic and LN T cells (**Figure 7E**) as well as splenic B cells (**Figure 7F**) than B6.lpr mice. In addition, 1sNASP.lpr mice showed higher percentages of activated CD69+CD4+ T cells (**Figure 7G**) and effector CD44+CD4+ T cells (**Figure 7H**) than B6.lpr mice in spleen and lymph nodes.

The 1sNASP.lpr mice produced modestly elevated levels of serum anti-chromatin and anti-dsDNA IgG as compared with B6.lpr mice (**Figure 8A**). The immune complexes in kidney were detected using the indirect immunofluorescence technique. Although a small amount of mouse IgG was present in glomeruli, 1sNASP.lpr mice showed significantly more IgG deposits in glomeruli than B6.lpr mice (**Figure 8B**). The 1sNASP.lpr mice showed trace C3 deposit in glomeruli (**Figure 8C**). B6.lpr mice seemed to have less C3 accumulation in glumeruli than 1sNASP.lpr mice. However, there were no statistical difference for C3 deposit in glomeruli between 1sNASP.lpr and B6.lpr mice (**Figure 8C**). Pathological examination showed that the 1sNASP.lpr mice, in addition to mild mesangial expansion, develop an enhanced proliferative renal pathology with an increased glomerular cell number and an infiltration of inflammatory cells in comparison with B6.lpr mice (**Figure 8D**). The renal pathology scores of the 1sNASP.lpr mice were significantly higher than that of B6.lpr mice. However, both sNASP.lpr and B6.lpr mice at age of 4–6 months have trace proteinuria, without a significant difference between these two strains. Moreover, the 1sNASP.lpr mice also showed significantly a more severe lung inflammation than B6.lpr mice (**Figure 8E**). The lung pathological characteristics of 1sNASP.lpr mice were similar to what we observed in the rec1c.lpr mice. On the other hand, the 1sNASP.lpr mice did not develop liver inflammation and dermatitis. In summary, the 1sNASP.lpr mice not only reproduced all autoimmune phenotypes and organ pathology alterations of rec1c.lpr mice, but also developed additional autoimmune phenotypes, including increased sizes of spleen and lymph nodes, lymphocyte increase, expansion of activated or effector CD4+ T cells, IgG autoantibody elevation, and more IgG deposit in glomeruli. The characterization of the 1sNASP.lpr

B6.1sNASP mouse was verified to have the mutated bases (red arrows) of *rec1c sNASP* allele. (C) The sNASP protein expression was determined by Western blotting in tissues and compared between WT B6 and transgenic B6.1sNASP mice, and mouse β-actin was used as control.

model demonstrates that the sNASP allele is responsible for pathogenic contribution of the rec1c sublocus to mouse lupus.

# DISCUSSION

The rec1c.lpr mice exhibited a normal spleen size and a modest lymphadenopathy in comparison with control B6.lpr mice, but they developed more significant kidney and lung inflammation, two end-organ manifestations of SLE. These results suggest that the rec1c sublocus seemingly is not involved in systemic autoimmunity, but is rather aggravating its consequences. This is a contrast to the adjacent rec1d1 sublocus since the rec1d1.lpr mice showed 3-fold expanded spleen or even 10-fold enlarged lymph node relative to B6.lpr mice, and this expansion was largely accounted for T cells, suggesting that red1d1 contributes to lupus by targeting T cells (15). We have proposed a model for the genes involved in lupus pathogenesis with a first group of genes breaking tolerance, such as Ly108 in Sle1b (23), a second group amplifying/ polarizing autoimmune activation, such as Pbx1 in Sle1a (24), and a third group of genes modulating disease severity in target organs, such as the kallycrein gene family in Sle3 (25) in the NZM2410 lupus model (26). We propose that the Skint6 allele in rec1d1 belong to group 2 while sNASP variant in rec1c belongs to the third group. The detailed analysis of the Sle2c locus revealed a complex architecture with a total of four genes so far associated with lupus susceptibility: Cdkn2c, which regulates B1a cell expansion, the original selecting phenotype for Sle2c (8), a protective allele of Csf3r (11), a variant of Skint6 associated with T cell activation (15) and now a variant of sNASP that amplifies autoimmunity and aggravate tissue pathology. The presence of these two latter variants may explain why Sle2, which is not associated by itself to any end-organ pathology (27), was mapped in association with glomerulonephritis when it interacts with other NZM2410 loci (4) or with lpr (7).

Exon sequencing of the rec1c interval identified the substitution of two consecutive amino acid residues in the NASP gene. NASP contains two isoforms, a longer testis-specific tNASP and a shorter somatic sNASP. However, both isoforms often occur in transformed cell lines (28). It is well known that the sNASP functions as a histone chaperone to perform their vital role in genome maintenance by interacting with soluble histones, driving the accurate assembly and disassembly of nucleosomes (9). The substitution of two consecutive amino acid residues in the rec1c sNASP variant protein occurs in the histone-binding domain. We demonstrated that this variant has an increased binding affinity for histone H4 and the H3.1/H4 tetramer, suggesting that the amino acid substitutions alter its three-dimensional structure and dysfunction.

To test the functional significance of the rec1c sNASP variant, we introduced the corresponding two mutations into the B6 genome to generate a transgenic B6.1sNASP mouse and its derived 1sNASP.lpr strain. The B6.1sNASP mice did not develop any detectable autoimmunity. However, the 1sNASP.lpr mice produced more IgG autoantibodies, had bigger spleen and lymph nodes along with lymphocyte elevation, displayed mild increase of activated and effector CD4+ T cells in peripheral lymph organs, and more IgG deposit in glomeruli in comparison to B6.lpr mice. These phenotypes of 1sNASP.lpr mice are a sign of autoimmunity. The 1sNASP.lpr mice developed more severe kidney and lung inflammation than the control B6.lpr mice. Therefore, the 1sNASP.lpr mice reproduced most of the autoimmunepathological phenotypes of the rec1c.lpr mice. These findings establish that the sNASP mutant allele is responsible for the contribution of the rec1c interval to lupus pathogenesis. On

the other hand, as the 1sNASP.lpr mice did not develop significant proteinuria, their exacerbated kidney inflammation was not sufficient to result in renal dysfunction. As for the reason why 1sNASP.lpr mice presented some autoimmune phenotypes different that were not found in rec1c.lpr mice, we speculate it most likely due to unlinked NZM2410 genetic contamination carried over in the rec1c.lpr congenic genome that may interfere with the sNASP allele. Such contamination has been documented in other NZM2410-derived congenics [(24) and Morel unpublished].

The MRL/lpr strain develops a rapid onset of lupus due to the lpr mutation in the Fas gene on chromosome 19. A lpr modifier locus, Lprm1, has been mapped to chromosome 4 in a genomic location close to the rec1c sublocus (29). We found that the MRL/lpr genome shares the same sNASP allele with the rec1c NZM2410 allele, suggesting that it may be responsible for the Lprm1 phenotypes. Our study demonstrated that the sNASP mutant allele with higher binding affinity for histone interacts with the lpr mutation to modestly enhance lymphadenopathy and autoimmunity and greatly promote tissue inflammation in

performed using two-tailed Mann-Whitney tests.

the 1sNASP.lpr model. Therefore, it is reasonable to hypothesize that the interaction of the sNASP mutant allele and the lpr mutation represents an important contribution to autoimmune pathogenesis in the MRL/lpr model.

How the sNASP allele in the rec1c sublocus promotes inflammation needs to be elucidated in the future studies. We hypothesize that the increased histone-binding affinity of the sNASP allele may enhance the transcription of inflammatory cytokines, either by immune cells or local cells in target organs. A recent study has reported that sNASP maintains homeostasis of the innate immune response as a negative regulator of TLR signaling by binding TRAF6 and preventing its auto-ubiquitination in unstimulated macrophages (30). Following LPS stimulation, CK2 binds and phosphorylates sNASP protein at serine 158, allowing sNASP protein to dissociate from TRAF6. Free TRAF6 is then auto-ubiquitinated and participates in TLR signaling to trigger the transcription of inflammatory cytokines (30). We speculate that the sNASP variant protein in the rec1c sublocus may have a decreased binding affinity for TRAF6, or be more easily phosphorylated by CK2 in innate immune cells following TLR stimulation, leading to excessive TRAF6 autoubiquitination and inflammatory cytokine release. The rec1c sNASP allele may also enhance the production of inflammatory cytokines directly by facilitating access of transcriptional site or through long-range chromatin alterations. Indeed, NASP regulates chromatin accessibility by maintaining a pool of H3K9me1 methylated histones (21), an epigenetic mark associated with active transcription sites (22). Abnormal histone modification patterns have been reported in the CD4+ T cells of lupus patients (31). Epigenetic factors play a pivotal role in regulating cytokine expression, and hence effector functions, in lupus T cells (32). Specifically, CREMa increases IL-17A transcription (33) and the transcription factor RFX1 regulates the expression of CD11a and CD70 (34) through histone modifications in lupus CD4+ T cells. The critical and complex role of epigenetic regulations in lupus T cells was demonstrated by showing that specifically demethylating either CD4+ or CD8+ T cells had beneficial effects while systemic demethylation worsened disease in MRL/lpr lupus-prone mice (35). A complex pattern of DNA methylation profiles has been revealed in twins discordant for lupus with hypo-and hyper-methylation differences, including some that were cell-specific (36). Defining the mechanisms by which the histone-binding protein NASP variant contributes to lupus pathogenesis using the mouse models that we have generated, either through epigenetic alterations, or other processes such as TRAF6 activation will benefit our understanding of lupus and the regulation of inflammation in autoimmune diseases.

#### DATA AVAILABILITY

All datasets generated for this study are included in the manuscript and/or the supplementary files.

#### AUTHOR CONTRIBUTIONS

JJ supervised the construction, genotyping, and husbandry of the transgenic B6.1sNASP and B6.1sNASP.lpr mice,

two-tailed Mann-Whitney tests.

and performed some experiments on B6.1sNASP.lpr mice. JX performed multiple pathological examinations of kidney and lung tissues of all mouse strains. YZ performed some genotyping and experiments on B6.1sNASP.lpr mice. XF performed recombinant vector construction and some flow cytometry experiments on B6.1sNASP.lpr mice. LM participated in some experimental designs, interpreted data, and wrote the manuscript. ZX designed and supervised whole project, performed experiments on rec1c.lpr mouse, and was responsible for data analysis, interpretation, and manuscript writing.

#### REFERENCES

#### FUNDING

This project was supported by the National Natural Science Foundation of China (Grant No. 81373185) and National Institutes of Health Grant K01AR056725 to ZX, and the Natural Science Foundation of Shandong, China (ZR2018MH014) to JJ.

#### ACKNOWLEDGMENTS

We thank Jianye Zhang, Peng Xie, Shujuan Liang, Yuqing Liu, Wentong Li, Yuewen Li, Kai Zheng, and Gaohang Mu for their technical assistance and discussion.

prone mice. JCI Insight. (2018) 3:120880. doi: 10.1172/jci.insight.1 20880

36. Ulff-Møller CJ, Asmar F, Liu Y, Svendsen AJ, Busato F, Grønbæk K, et al. Twin DNA methylation profiling reveals flare-dependent interferon signature and B cell promoter hypermethylation in systemic lupus erythematosus. Arthr Rheumatol. (2018) 70:878–90. doi: 10.1002/art. 40422

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Ju, Xu, Zhu, Fu, Morel and Xu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Opposite Profiles of Complement in Antiphospholipid Syndrome (APS) and Systemic Lupus Erythematosus (SLE) Among Patients With Antiphospholipid Antibodies (aPL)

Stephanie L. Savelli 1,2,3 \*, Robert A. S. Roubey 4 \*, Kathryn J. Kitzmiller 1,3, Danlei Zhou1,3,5 , Haikady N. Nagaraja6 , Evan Mulvihill 3,5, Fatima Barbar-Smiley 3,5, Stacy P. Ardoin3,5 , Yee Ling Wu1,3,7 \* and Chack-Yung Yu1,3,5 \*

1 The Research Institute at Nationwide Children's Hospital, Columbus, OH, United States, 2 Division of Hematology/Oncology, Nationwide Children's Hospital, Columbus, OH, United States, 3 Department of Pediatrics, College of Medicine, The Ohio State University, Columbus, OH, United States, 4 Division of Rheumatology, Allergy and Immunology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, United States, 5 Division of Rheumatology, Nationwide Children's Hospital, Columbus, OH, United States, 6 Division of Biostatistics, College of Public Health, The Ohio State University, Columbus, OH, United States, 7 Department of Microbiology and Immunology, Loyola University Chicago, Maywood, IL, United States

APS is the association of antiphospholipid antibodies (aPL) with thromboses and/or recurrent pregnancy loss (RPL). Among patients with SLE, one-third have aPL and 10–15% have a manifestation of secondary APS. Animal studies suggested that complement activation plays an important role in the pathogenesis of thrombosis and pregnancy loss in APS. We performed a cross-sectional study on complement proteins and genes in 525 patients with aPL. Among them, 237 experienced thromboses and 293 had SLE; 111 had both SLE and thromboses, and 106 had neither SLE nor thrombosis. Complement protein levels were determined by radial immunodiffusion for C4, C3 and factor H; and by functional ELISA for mannan binding lectin (MBL). Total C4, C4A and C4B gene copy numbers (GCN) were measured by TaqMan-based realtime PCR. Two to six copies of C4 genes are frequently present in a diploid genome, and each copy may code for an acidic C4A or a basic C4B protein. We observed significantly (a) higher protein levels of total C4, C4A, C4B, C3, and anticardiolipin (ACLA) IgG, (b) increased frequencies of lupus anticoagulant and males, and (c) decreased levels of complement factor H, MBL and ACLA-IgM among patients with thrombosis than those without thrombosis (N = 288). We also observed significantly lower GCNs of total C4 and C4A among aPL-positive patients with both SLE and thrombosis than others. By contrast, aPL-positive subjects with SLE had significantly reduced protein levels of C3, total C4, C4A, C4B and ACLA-IgG, and higher frequency of females than those without SLE. Patients with thrombosis but without SLE (N = 126), and patients with SLE but without thrombosis (N = 182) had the greatest differences in mean protein levels of C3 (p = 2.6 × 10−6 ), C4 (p = 2.2 × 10−9 ) and ACLA-IgG (p = 1.2 × 10−5 ). RPL occurred in 23.7% of female patients and thrombotic SLE patients had the highest frequency of RPL (41.0%; p = 3.8 × 10−10). Compared with non-RPL females, RPL had significantly

#### *Edited by:*

José Carlos Crispín, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (INCMNSZ), Mexico

#### *Reviewed by:*

Kenji Oku, Hokkaido University, Japan Gabriela Hernandez-Molina, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (INCMNSZ), Mexico

#### *\*Correspondence:*

Stephanie L. Savelli<EMAIL_ADDRESS>Robert A. S. Roubey<EMAIL_ADDRESS>Yee Ling Wu<EMAIL_ADDRESS>Chack-Yung Yu chack-yung.yu@ nationwidechildrens.org

#### *Specialty section:*

This article was submitted to Autoimmune and Autoinflammatory Disorders, a section of the journal Frontiers in Immunology

> *Received:* 11 December 2018 *Accepted:* 05 April 2019 *Published:* 07 May 2019

#### *Citation:*

Savelli SL, Roubey RAS, Kitzmiller KJ, Zhou D, Nagaraja HN, Mulvihill E, Barbar-Smiley F, Ardoin SP, Wu YL and Yu C-Y (2019) Opposite Profiles of Complement in Antiphospholipid Syndrome (APS) and Systemic Lupus Erythematosus (SLE) Among Patients With Antiphospholipid Antibodies (aPL). Front. Immunol. 10:885. doi: 10.3389/fimmu.2019.00885 higher frequency of thrombosis and elevated C4 protein levels. Female patients with homozygous C4A deficiency all experienced RPL (p = 0.0001) but the opposite was true for patients with homozygous C4B deficiency (p = 0.017). These results provide new insights and biomarkers for diagnosis and management of APS and SLE.

Keywords: C3 and C4, C4A and C4B, Copy number variation, Factor H, Lupus anticoagulant, Mannan binding lectin, Recurrent pregnancy loss, Thrombosis

#### INTRODUCTION

Antiphospholipid syndrome (APS) is characterized by vascular thrombosis and/or pregnancy morbidity such as recurrent fetal loss in the persistent presence of antiphospholipid antibodies (aPL) (1–7). aPL are a heterogeneous group of autoantibodies that include antibodies against phospholipid binding protein β2-glycoprotein I (β2GPI), anticardiolipin antibodies (ACLA), and lupus anticoagulant (LAC) (8). Human subjects with triple positivity for all three groups of aPL appeared to be at high risk to experience recurrent thromboembolic events (9). A majority of clinical tests for aPL detects antibodies against β2GPI. β2GPI is a plasma protein consisting of five structural domains known as short consensus repeats that are characteristic features of controlling proteins for the complement system (10–12).

SLE is a common autoimmune disease associated with APS. SLE features the generation of autoantibodies against nuclear antigens including double-stranded DNA (13, 14). In a study of European APS patients, over 40% were found to have SLE or a lupus-like disease (15). Among the general SLE population, between 30 and 40% have aPL; and 10– 15% of patients with SLE also have clinical manifestations of APS (15–20). In addition to the presence of autoantibodies, hypocomplementemia is another hallmark of human SLE (21– 25). Low serum complement levels for C4 and C3 in patients with SLE can be triggered by a combination of heritable and acquired factors: genetic deficiencies, low copy number of complement C4 genes, robust consumption caused by immune complexmediated complement activation, or the presence of inhibitors that inactivate or prevent accessibility. A complete genetic deficiency in any one of the early components specific for the classical complement activation pathway almost always lead to pathogenesis of human SLE, inferring that an intact classical pathway of the complement system is essential for the protection against systemic autoimmunity (26–28).

Activations of complement C3 and C5 in the presence of antigen-antibody complexes occur via the formation of the C1 complex (C1q-C1r2-C1s2), followed by the activations of C4 and C2 to form C4b and C2a, respectively (29). C4b and C2a are subunits of the C3 and C5 convertases, essential for the classical and lectin activation pathways (26). There are two isotypes of native C4 proteins. C4A is the acidic isotype believed to play an essential role in immune clearance and immunotolerance. C4B is the basic isotype that is capable of rapid propagation of complement activation (30–34). In a diploid genome, complement C4 gene copy number varies among different individuals. Two to eight copies of C4 genes are generally present in a diploid genome among most human subjects (35, 36). Each C4 gene either codes for a C4A or a C4B protein. Such gene copy number variation contributes to quantitative and qualitative diversities in C4 protein levels and function, and therefore different intrinsic strengths for effector functions of innate and adaptive immune responses (25, 34, 36– 40). Among European and East-Asian subjects, low copy number of total C4 or C4A is a risk factor for SLE, while high copy number of total C4 or C4A is protective against susceptibility to SLE (22, 38, 41, 42).

An injection of human aPL into animal models including wild-type mice induced an increase in thrombus size (43, 44). An injection of human aPL into pregnant mice resulted in fetal resorption. (45, 46). Mice deficient in complement C3 or C5, as well as mice injected with a monoclonal antibody against C5, did not exhibit an increase in thrombus size in the presence of aPL. Blockade of complement activation by genetic deletion of C3 or C4, or with transgenic insertion of complement regulatory protein Crry-Ig, a soluble inhibitor of mouse C3 convertase, protected mice, rats or hamsters from pregnancy complications induced by injections of human aPL (45, 47–56). These phenomena suggest that complement proteins or their activated products are engaged in the pathogenesis of APS, as they probably provide immune effectors for aPLmediated thromboses, tissue injury and/or fetal loss in mouse models. The generation of immune complexes between aPL and ligands (such as β2GPI binding to phospholipids) leads to activation of the complement classical pathway, release of C5a and C3a anaphylatoxins (50, 57, 58), which may attract neutrophils and other granulocytes to the site of complement activation, increase vascular permeability, and elicit inflammatory response that contributes to tissue injuries including pregnancy morbidity. Culmination of complement activation pathways leads to the assembly of the membrane attack complex (C5b-9) and provides the "second-hit" to trigger vascular thrombosis (53). Consistent with this notion, it was shown that C3, C5, or C6-deficient rodents were protected from aPL induced thrombosis (56, 59). Such protective effects of complement deficiency in APS-associated disorders observed in

**Abbreviations:** ANOVA, analysis of variance; aPL, antiphospholipid antibodies; APS, antiphospholipid syndrome; CNV, copy number variation; GCN, gene copy number; LAC, lupus anticoagulant; MBL, mannan binding lectin; NS, no SLE; NT, no thrombosis; NTS, no thrombosis and no SLE; RMSE, root mean square error; RPL, recurrent pregnancy loss; S, SLE; So, SLE without thrombosis; SLE, systemic lupus erythematosus; T, thrombosis, To, thrombosis without SLE; TS, thrombosis and SLE.

animal models are opposite to the causal effects of deficiencies in early components for the classical complement pathway in human lupus (26, 60). Among human patients with APS, elevated levels of complement activation products (C4a, C3a, C5a, C5b-9) have been demonstrated (55, 61, 62). However, systematic and meticulous studies on how complement proteins and genes contribute to the pathology of human APS (recurrent vascular thrombosis or pregnancy morbidity) and the concurrence of SLE and APS were scarce or limited by small sample size.

Here we performed a cross-sectional study on 525 human subjects with aPL from the Antiphospholipid Syndrome Collaborative Registry. Based on clinical presentations of thrombosis and SLE, these subjects were categorized to four groups: patients with thrombosis only (To), with thrombosis and SLE (TS), with SLE only (So), and without thrombosis and without SLE (NTS). Plasma protein concentrations for complement total C4, C4A, C4B, C3, factor H, and functional mannan binding lectin (MBL) were measured. Total C4, C4A and C4B gene copy numbers were elucidated. The results reveal substantial phenotypic differences for complement protein concentrations among patients with thromboses or recurrent pregnancy loss, and SLE. There was also a significant difference in C4 gene copy number variations between patients with both thrombosis and SLE, and patients without SLE and thrombosis.

## PATIENTS AND METHODS

#### Study Population

This study was approved by the Institutional Review Board at Nationwide Children's Hospital. Peripheral blood plasma and matched genomic DNA samples without personal identifiers from 525 patients with aPL and clinical status were provided by the APS Core at University of North Carolina (8, 63). These aPL-positive patients were recruited with written informed consent. Of these patients, 444 (84.57%) were female and 81 (15.43%) were male. The mean age (±SD) was 45.01 ± 12.97 years old. Among these aPL-positive patients, 184 (35.05%) met the Sapporo criteria for definite APS (1); an additional 175 subjects (33.33%) met the extended definition of APS, and 166 asymptomatic subjects (31.6%) who had aPL but no manifestations of thrombosis or pregnancy morbidity. Patients with definite APS as defined by the preliminary or modified Sapporo criteria (1, 6) must have one or more clinical episodes of vascular thrombosis and/or pregnancy morbidity as well as ACLA, anti-β2GPI IgG and/or IgM or LAC present on two or more occasions at least 6 weeks apart. The expanded APS group was defined by institutions participating in APSCORE and include those patients with one or more clinical manifestations characteristic of APS but not fulfilling the strict definition and either the Sapporo laboratory criteria or one of a group of APS-associated autoantibodies. Asymptomatic patients fulfill the Sapporo laboratory criteria but have no clinical manifestations related to APS.

To perform refined analyses based on clinical presentations, we segregated the aPL subjects based on the presence and absence of thrombosis, SLE and recurrent pregnancy loss. Among the study cohort, 237 subjects had a history of thrombosis and 288 subjects did not have thrombosis. A total of 293 subjects were diagnosed with SLE according to the American College of Rheumatology criteria (64) and 232 subjects did not have a diagnosis of SLE at the time of recruitment. Of the 444 female subjects with aPL, 106 (23.87%) experienced recurrent pregnancy loss.

## Quantifications of Total *C4*, *C4A*, and *C4B* Genes by Real-Time PCR

A series of real-time PCR assays was applied to determine the copy number variations of total C4, C4A, and C4B genes (35). All real time PCR assays used the TaqMan MGB probes (ABI). The target probes (C4, C4A, and C4B) were VIC-labeled. The endogenous control probe, which targeted an invariant exon 4 of the RP1 gene, was FAM-labeled. Each reaction consisted of 0.5 to 1µM of both forward and reverse primers for the target and control amplicons, 100 nM of the target and endogenous control probes, 25 ng of sample DNA and TaqMan Universal PCR master mix (ABI, PN 4323018). All assays were performed in triplicates using the ABI 7500 RT-PCR system per manufacturer's recommendations. The relative standard curve method was utilized to calculate the copy number of each target gene. The accuracy of C4A and C4B gene copy number assignments for each sample was cross-confirmed as the gene copy number of total C4 equals the sum of C4A and C4B.

#### Complement C3, C4, Factor H (CFH) and Mannan Binding Lectin (MBL) Protein Concentrations in Citrate-Plasma

Platelet poor plasma samples were processed with a consistent protocol. Briefly, blood samples in citrate tubes were centrifuged at 1,500 g for 10 min, at 4–8◦C. Plasma samples were transferred to microcentrifuge tubes and spun again at 2,000 g for 5 min. Aliquots were kept frozen at −80◦C. Plasma protein concentrations of complement C3 and C4 were determined by single radial immunodiffusion assays using commercial kits from The Binding Site (Birmingham, United Kingdom). A comparison of C4 protein concentrations of (a) SLE patients without thrombosis from this study and (b) an independent cross-sectional study of Ohio SLE (38) revealed that protein concentrations of complement C4 assayed from plateletpoor citrate plasma, which were subjected to two rounds of centrifugation, were ∼14.5% lower than that of EDTA-plasma C4 harvested after a single round of centrifugation.

Complement factor H plasma protein concentrations were measured using homemade RID plates according to a standard protocol (65). Plasma concentrations for MBL were determined using a functional assay kit from the Antibody Shop (Denmark).

# Complement C4 Protein Allotyping

Plasma C4A and C4B protein allotypes were determined by immunofixation and immunoblot techniques, as described previously (66–68). The relative band intensities of C4A and C4B allotypes from each sample were quantified by ImageQuant Software. The corresponding plasma C4A and C4B protein concentrations were calculated from the total C4 protein concentrations.

r, coefficient of correlation.

\*p values obtained by χ 2 analyses for categorical data, or by ANOVA (analysis of variance) for continuous data; kothers: other racial and multi-racial groups; p- <0.05 are in bold fonts.

#### Statistical Analyses

Descriptive statistics, including means, standard deviations (SD), and 95% confidence intervals (95% CI) were computed for numeric data, and frequency distributions were determined for categorical variables, using statistical software JMP13 (SAS Institute) and GraphPad Prism6 software. Two group comparisons were based on t-tests that accounted for unequal variances if appropriate. Specifically, Tukey HSD test with an alpha set at 0.05 was applied, and was followed by pairwise Student's t-tests that yielded pvalues. Dunnett's test with an alpha of 0.05 was applied for comparing study groups to controls. Categorical data were compared by χ 2 analyses and odds ratios were calculated whenever appropriate.

To allow a standardized comparison of all continuous parameters contributing to thrombosis without SLE, SLE without thrombosis, SLE with thrombosis, and no thrombosis and no SLE, we determined the root mean square error (RMSE) of each parameter in these four groups by analysis of variance (ANOVA). The difference in the mean protein levels for each protein between any two groups divided by its RMSE to give the effect size index (69). The mean values of parameters in the NTS group were used as references and the effect size indices for To, So, and TS groups were graphically plotted. This enabled a uniform depiction on effects of all continuous parameters under different clinical conditions.

Stepwise multiple logistic regression analyses were used to identify independent parameters significant for clinical outcomes: thrombosis, arterial thrombosis, venous thrombosis, pregnancy loss, and SLE. Such analyses allowed controlled studies for continuous and categorical parameters. For analyses of a clinical presentation as a response, we first put all parameters studied [i.e., C3 or C4, factor H, MBL, ACLA-IgM, ACLA-IgG, BMI, age, gender (F/M), LAC (presence or absence), SLE (presence or absence)] into the regression model. Those that did not give a p-value smaller than 0.1 were removed from the subsequent analyses. The last best model with parameters represented by p-values smaller than 0.05 was maintained and presented. Unit Odds Ratio (OR) and range OR were computed. Parameters that could not coexist in the regression model because of strong correlation (e.g., C3 and C4) were put into the regression models separately and the stronger parameter was kept. When C4 was identified as a significant parameter in a model, we further asked whether C4A or C4B or both C4 isotypes were playing a major role.

#### RESULTS

The study population consisted of 525 human subjects with antiphospholipid antibodies (aPL), recruited through the APSCORE. The mean age of subjects at the time of recruitment was 45.0 ± 13.0 (mean ± SD) years old. These study subjects were initially segregated into three groups: definite APS, extended APS, and non-APS based on clinical manifestations associated with APS, which included vascular thromboses and pregnancy morbidity. Results for an initial characterization for these three groups of patients are shown in **Table 1**. One remarkable feature emerged was the steady and highly significant increase in the mean protein concentration of complement C4 from non-APS (16.6 ± 8.8 mg/dL), to extended APS (20.0 ± 9.2 mg/dL), and definitive APS (22.9 ± 10.0 mg/dL) (p = 2.6 × 10−8 ). These three groups of patients had different frequencies of SLE, thromboses and pregnancy morbidities. Thus, we set to examine quantitative variations of plasma complement proteins among aPL subjects with different clinical manifestations for thrombosis and/or SLE in both female and male patients, and recurrent pregnancy loss among female patients. The demographic and clinical features for these aPL-positive subjects are shown in **Table 2**.

#### Variations of Plasma Complement Protein and ACLA Levels in aPL-Positive Subjects With and Without Thromboses

Among the aPL-positive subjects, 45.1% had a past history of thrombosis, and 55.8% were diagnosed with SLE at the time of recruitment. When the mean plasma complement TABLE 2 | Demographic data and disease status of aPL-positive subjects.

Key: BMI, body mass index; NS, no SLE; NT, no thrombosis; NTS, no thrombosis and no SLE; S, SLE; So, SLE without thrombosis; T, thrombosis; To, thrombosis without SLE; TS, thrombosis and SLE; frequency in each group is shown in parenthesis. p-values of statistical significance are in bold fonts.

protein concentrations and aPL between thrombotic and nonthrombotic groups were compared, highly significant phenotype differences for total C4, C4A, C4B, MBL, ACLA-IgM, and ACLA-IgG were observed (**Table 3**). The mean protein level (and 95% confidence interval) for total C4 was 22.7 (21.3–24.0) mg/dL in the thrombotic group, and 17.7 (16.6–18.7) mg/dL in the non-thrombotic group, which represented a difference of 28.2% (p = 1.3 × 10−8 ).

Parallel increases in complement C4A and C4B were observed in the thrombotic group. Mean concentration of plasma C4A in

CI, confidence interval. Odds ratios were calculated for T vs. NT. p-values of statistical significance are in bold fonts.

the thrombotic group was 12.1 (11.2–12.9) mg/dL, and 9.6 (8.9– 10.3) mg/dL in the non-thrombotic group (p = 8.2 × 10−6 ). Plasma C4B mean protein concentration in the thrombotic group was 10.5 (9.8–11.3) mg/dL, and 8.3 (7.8–8.9) mg/dL in the non-thrombotic group (p = 6.7 × 10−6 ). For complement C3, moderately higher mean protein level was observed in the thrombotic group than the non-thrombotic group (T: 128.1 mg/dL; NT: 120.2 mg/dL; p = 0.013).

By contrast, mean plasma protein level for functional MBL among thrombotic subjects was significantly lower than that of non-thrombotic subjects, which were 0.117 (0.100–0.135) mg/dL and 0.167 (0.145–0.189) mg/dL, respectively (p = 0.0007). Slightly lower levels of factor H protein were also observed in the thrombotic group (T: 49.9 mg/dL; NT: 52.3 mg/dL; p = 0.057).

The mean values of anticardiolipn antibodies among thrombotic subjects were 19.5 (14.9–24.1) units for ACLA-IgM and 45.1 (35.7–54.5) units for ACLA-IgG. In non-thrombotic subjects, the corresponding values were 29.9 (25.3–34.6) and 29.6 (23.9–35.4) units (p = 0.0022 for ACLA-IgM; p = 0.0043 for ACLA-IgG). Thrombotic subjects had lower levels of ACLA-IgM but higher levels of ACLA-IgG. Three-quarters (74.9%) of thrombotic subjects tested positive for the presence of lupus anticoagulant (LAC), compared to slightly over one-half (53.3%) among non-thrombotic subjects (p = 1.7 × 10−6 ).

Thrombotic subjects had significantly lower female to male ratio (3.23 to 1) when compared with non-thrombotic subjects (10.0 to 1; p = 7.2 × 10−6 ).

#### Plasma Complement Protein and ACLA Levels in aPL-Positive Subjects With and Without SLE

Quantitative variations of complement and ACLA plasma protein were compared between the aPL-positive patients with and without SLE (**Table 4**). The mean C3 concentrations were 118.3 (114.2–122.4) mg/dL in SLE and 130.8 (126.2– 135.4) mg/dL in non-SLE, which represented a reduction

LAC, lupus anticoagulant. Odds ratios were calculated for S vs. NS. p-values of statistical significance are in bold fonts.

of 9.6% of mean C3 level in SLE (p = 8 × 10−5 ). The mean total C4 concentrations were 18.6 (17.4–19.8) mg/dL in SLE and 21.6 (20.4–22.9) mg/dL in non-SLE, which corresponded to a reduction of 13.9% of total C4 in SLE (p = 0.0006). The mean C4A and C4B concentrations were 10.0 (9.2–10.8) mg/dL and 8.7 (8.1–9.4) mg/dL, respectively, in the SLE group; and were 11.6 (10.9–12.4) mg/dL and 10.1 (9.4–10.8) mg/dL, respectively, in the non-SLE group (p = 0.0038 for C4A; p = 0.005 for C4B). Thus, lower plasma levels of complement C3 and C4 were conspicuous in the SLE group.

The mean plasma protein levels of MBL between the SLE and non-SLE groups were not significantly different [S: 0.151 (0.130– 0.172) mg/dL, NS: 0.135 (0.116–0.154) mg/dL; p = 0.29]. The mean plasma protein level of factor H was slightly lower in the SLE group [49.9 (48.3–51.5) mg/dL] than that in the non-SLE group [52.9 (50.9–54.9) mg/dL; p = 0.02].

The mean ACLA-IgG level was significantly lower in the SLE than non-SLE [S: 28.2 (22.7–33.7) GPL, NS: 48.8 (38.7–59.0); p = 0.0002]. By contrast, the mean ACLA-IgM levels and the frequency for the presence of LAC were similar between the SLE and the non-SLE groups.

Among the aPL subjects, SLE patients had a higher female to male ratio (7.62 to 1) than non-SLE patients (3.81 to 1; p = 0.0031).

## Differential Plasma Protein Levels of Complement and ACLA in aPL-Positive Subjects With Thrombosis, SLE, Both Thrombosis and SLE, and Neither Thrombosis Nor SLE

Our study results revealed higher levels of mean plasma complement C4, C3, and ACLA-IgG in aPL-positive subjects

with a history of thrombosis, but lower levels of complement C4, C3, and ACLA-IgG among aPL-positive subjects with SLE. A proportion (37.9%) of aPL-positive subjects diagnosed with SLE also experienced thromboses. To distinguish the roles of complement proteins and ACLA in thromboses and SLE, we segregated the study subjects according to their thrombosis and SLE status: thrombosis only (To), thrombotic SLE (TS), SLE only (So), and no thrombosis and no SLE (NTS) (**Figure 1**, **Table 5**). Among these four groups, significantly higher mean protein levels were observed for patients with thrombosis only than with SLE only for plasma protein levels of total C4 (p = 2.2 × 10−9 ), C4A (p = 1.9 × 10−6 ), C4B (p = 1.3 × 10−6 ), C3, (p = 2.6 × 10−5 ), ACLA-IgG (p = 1.2 × 10−5 ), and in female to male ratio sex ratio (p = 1.6 × 10−6 ).

For functional MBL and the presence of LAC, main differences were observed between the thrombotic groups and the nonthrombotic groups. Mean MBL protein levels were significantly lower in To and TS than in So (To vs. So, p = 0.0041; TS vs. So, p = 0.0046). The mean level of MBL in aPL-positive subjects without thrombosis and SLE (NTS) was slightly higher than those of To and TS (p = 0.078 and 0.076), and similar to that of So (p = 0.416). For lupus anticoagulant, 73.5% of patients in To and 76.3% of patients in TS were tested positive, compared to 51.2% in So (To vs. So, p = 0.0002; TS vs. So, p = 0.000038) and 56.8% in NTS (To vs. NTS, p = 0.015; TS vs. NTS, p = 0.0048).

#### Gene Copy Number Variations of Total *C4*, *C4A*, and *C4B* in Patients With aPL

Gene copy numbers (GCN) for total C4, C4A, and C4B from 472 aPL-positive subjects were elucidated by TaqMan based real-time PCR using genomic DNA (35). The copy number of total C4 genes in this study cohort varied from 2 to 6; C4A from 0 to 5; and C4B from 0 to 4. The distribution of total C4, C4A, and C4B gene copy number variations among To, TS, So, and NTS are shown in **Table 6**.

The distribution of GCN groups was analyzed first as categorical data. The distribution of C4A genes was statistically different among To, TS, So, and NTS (p = 0.034, χ 2 analysis). Variations of GCNs for total C4 (p = 0.088) and C4B (p = 0.13) TABLE 5 | Mean plasma protein levels of complement and ACLA in aPL-positive subjects segregated by thrombosis and SLE status.

p-values of statistical significance are in bold fonts.

TABLE 6 | Gene copy number variations (CNVs) of total C4, C4A and C4B among aPL-positive subjects.

B. Mean gene copy numbers (±SD) for total C4, C4A and C4B among aPL-positive subjects.

To, thrombosis without SLE; TS, thrombotic SLE; So, SLE without thrombosis; NTS, non-thrombosis and non-SLE; f, frequency. GCN, gene copy number. The reference values for mean GCNs of total C4, C4A, and C4B are 3.82 ± 0.75, 2.09 ± 0.79, and 1.74 ± 0.63, respectively, for healthy subjects; and 3.56 ± 0.77, 1.81± 0.89, and 1.76 ± 0.58, respectively, for SLE subjects (38). p values <0.05 were in bold fonts.

had not reached statistical significance. The median GCN groups for total C4 is 4, and for C4A and C4B are both 2. Low and high copy number groups are defined as those below and above median GCN groups, respectively. Variations in frequencies were observed for the low and high GCN groups of C4 genes. For example, 41.0% of the TS group had 2 or 3 copies of total C4 genes (low GCN), compared to 27.5% in To and 31.6% in NTS. By contrast, only 2.0% of the TS group had 5 or 6 copies of total C4 (high GCN), compared to 10.2% in the NTS group. A similar pattern was observed for C4A genes. There was an increase in the frequency of low C4A GCN (27.0% in TS, 17.3% in NTS), and a decrease in the frequency of high C4A GCN in the TS group (12.0% in TS, 24.5% in NTS).

The GCN values were analyzed as continuous data to compare means by Student's t-test. The means for total C4, C4A, and C4B were 3.67, 1.87, and 1.80, respectively, for the thrombotic subjects; and were 3.74, 1.99, and 1.75, respectively, for the nonthrombotic subjects. Lower mean C4A gene copy number was observed in the thrombotic group (p = 0.049) (**Table 6**).

The mean GCN for total C4, C4A, and C4B were 3.65, 1.87, and 1.78, respectively, for the SLE patients; and were 3.78, 2.01, and 1.77, respectively, for non-SLE subjects. Significantly lower mean GCNs for total C4 and C4A were present in the SLE group (p = 0.035 for total C4; p = 0.022 for C4A) (**Table 6B**). The mean C4B gene copy numbers were almost identical between patients with and without SLE.

When the aPL subjects were segregated and compared based on both thrombosis and SLE status, it revealed that the NTS group without thrombosis and SLE had the highest mean GCNs for total C4 and C4A (3.78 and 2.12, respectively), but the lowest C4B mean GCN (1.66). The thrombotic SLE group (TS) had the lowest values of total C4 at 3.55 and C4A at 1.81.

The mean GCN of total C4 for NTS was significantly higher than that of TS (p = 0.012); of C4A for NTS was significantly higher than those of To (p = 0.027), So (p = 0.012) and TS (p = 0.001); and of C4B was significantly lower than that of To (p = 0.015).

For TS, the mean GCN of total C4 was significantly lower than those of To (p = 0.011), So (p = 0.042), and NTS (p = 0.012); and of C4A was significantly lower than that of NTS (p = 0.001).

#### Plasma C4 Protein Concentrations Per *C4* Gene Copy in Thrombosis and SLE

Both C4 gene copy number variation and clinical conditions of aPL subjects are important determining factors for C4 plasma protein concentrations. To examine the respective roles of genetic variants and clinical status on plasma protein levels of complement C4, we calculated the C4 protein per gene copy in each study subject by dividing the C4 plasma protein concentration with the C4 gene copy number. The mean C4 protein concentrations per gene dose among To, TS, So and NTS were 6.42, 6.21, 4.72, and 5.11 mg/dL, respectively. Thus, the C4 plasma protein yield per gene copy was the highest in aPL subjects with thrombosis only, and the lowest in aPL subjects with SLE only. Highly significant differences were present between the two thrombotic groups (To and TS) and the two non-thrombotic groups (So and NTS) (**Figure 2**).

## Differential Complement and ACLA Plasma Protein Levels and C4 Genetic Deficiencies in aPL-Positive Subjects With and Without Recurrent Pregnancy Loss (RPL)

Of the 444 female aPL-positive subjects, 106 experienced recurrent pregnancy loss (RPL). Thromboses occurred in 63.8% of the RPL patients, compared to 33.7% in non-RPL patients (p =

5.1 × 10−8 ). SLE were diagnosed in 54.3% of the RPL patients and 59.8% in non-RPL patients (p = 0.32). Strikingly, RPL had the highest frequency in patients with both thrombosis and SLE (TS, 41.0%) but lowest in patients with SLE only (So, 13.3%); patients of the NTS and To groups each had a frequency of 22.9%. The frequencies of RPL are significantly different among aPL-positive patients when segregated into To, So, TS, and NTS (**Table 6**; χ 2 = 46.2, degree of freedom = 3, p = 3.8 × 10−10). TS patients had an odds ratio (95% confidence interval) of 8.63 (4.37–17.0) over So patients to experience recurrent pregnancy loss (p = 1.6 × 10−11).

Lupus anticoagulant was present in 67.1% of patients with RPL and 58.4% of non-RPL female patients (p = 0.057). Mean total C4 and ACLA-IgG protein levels were significantly increased, while mean CFH level was reduced (**Table 7**) among RPL patients. The mean total C4 level was 21.8 (19.9 ± 23.7) mg/dL in RPL and 19.1 (18.1 ± 20.2) mg/dL in non-RPL (p = 0.015). The ACLA-IgG was 44.2 (35.1 ± 53.3) g/l in RPL and 30.0 (24.2 ± 35.8) g/l in non-RPL (p = 0.01). The CFH mean concentrations in RPL were 48.8 (46.6 ± 51.0) mg/dL and 52.1 (50.5 ± 53.7) mg/dL in non-RPL (p = 0.019).

Homozygous C4A deficiency (GCN of C4A = 0) was present in five female aPL patients and all of these five subjects experienced RPL (p = 0.0001). On the contrary, homozygous C4B deficiency (GCN of C4B = 0) was present in 11 female

among patients.

TABLE 7 | Plasma complement and ACLA levels and risk factors for recurrent pregnancy loss (RPL) in female aPL-positive subjects.

f, frequency; N-RPL, no recurrent pregnancy loss; RPL, with recurrent pregnancy loss. p-values of statistical significance are in bold fonts.

aPL patients and none of them experienced RPL (p = 0.017). Thus, homozygous C4A deficiency was a strong risk factor for, and homozygous C4B deficiency was a strong protective factor against, recurrent pregnancy loss.

## Standardized Comparison of Numeric Parameters Associated With Thrombosis and SLE

To allow a standardized comparison of complement and ACLA protein variations in thrombosis and SLE, we determined the effect size index (69) of each parameter in To, So, and TS, using the mean protein concentrations or antibody levels of the group without thrombosis and SLE (NTS) as a reference. The difference of mean protein levels for each protein at To, So, or TS from NTS was divided by its root mean square error (RMSE), which was computed by ANOVA, to yield the effect size index. The effect size indices for complement and ACLA proteins with quantitative variations are depicted in **Figure 3**. In descending order, the greatest intergroup effect size indices are: C4, To vs. So: 0.709; C4B, To vs. So: 0.570; C4A, To vs. So: 0.566; ACLA-IgG, To vs. So: 0.554; complement factor H, TS vs. NTS: 0.483; ACLA-IgM, To vs. NTS: 0.373; and MBL, TS vs. So: 0.367.

FIGURE 3 | (Upper) Categorization of aPL-positive subjects according to their thrombosis and SLE status. (Lower) Standardized differences (effect size index) of mean complement protein levels and ACLA levels among aPL-positive subjects with thrombosis without SLE (To), thrombosis and SLE (TS), SLE without thrombosis (So) when compared to the non-thrombotic and non-SLE (NTS) group. Root mean square error (RMSE) value for each protein was derived from Oneway ANOVA. Differences of mean protein levels for To, TS and So from NTS were each divided by their associated RMSE and charted to derive the effect size index. The effect size indices allow a standardized comparison of different parameters under different clinical conditions.

# DISCUSSION

This is a cross-sectional study of complement protein profiles and copy number variations of C4 genes in a relatively large cohort of human subjects with aPL antibodies. These aPL-positive subjects had a variety of clinical presentations: thrombosis, SLE, thrombosis and SLE, no thrombosis and no SLE. Many female aPL-positive subjects also experienced pregnancy morbidity such as recurrent pregnancy losses. Extensive analyses of gene copy number variations for total C4, C4A, and C4B, plasma protein levels of total C4, C4A, C4B, C3, factor H and MBL, and antiphospholipid antibodies revealed distinct patterns of diversity that can be relevant and effective quantitative biomarkers for thrombosis, SLE and recurrent pregnancy loss.

An intriguing aspect of complement C4 genetics is the frequent gene copy number variations (30, 38, 40). Genetic deficiency (60, 70) or low gene copy numbers of total C4 or C4A has been shown to be a prevalent risk factor for SLE in European and East-Asian Americans (22, 38, 41, 71–73). C4 gene copy number variations in the aPL-positive subjects were determined and validated by quantitative real-time PCR (35). When SLE and non-SLE subjects were compared, lower total C4 and C4A mean gene copy numbers were found among the SLE subjects, suggesting that aPL-positive subjects with low total C4 or C4A gene copy numbers carried a greater risk of developing SLE, as we reported in earlier studies (22, 38). Among the NTS subjects who were not afflicted with thrombosis and SLE, there were higher mean GCN of C4A, which would be protective against SLE; and low GCN of C4B that would lead to lower C4B protein levels and thereby reducing the risk of thrombosis.

Using thrombosis as a response, multiple logistic regression analyses suggested that higher plasma C4 protein levels and the presence of lupus anticoagulant (LAC) were among the strongest independent biomarkers associated with thrombosis (C4, p = 6.2 × 10−9 ; LAC, p = 6.9 × 10−5 ) (**Supplementary Table**). Other relevant parameters for increased risk of thrombosis included male sex and a reduction of complement factor H level, which was also observed by Nakamura and colleagues (74). Higher plasma C4 protein levels and the presence of LAC were the two most prominent risk factors for arterial thrombosis. The higher level of C4 protein in arterial thrombosis was mainly attributable to higher C4B (p = 2.1 × 10−5 ). The risk factors identified for venous thrombosis also included increased protein level of total C4 (p = 0.01) and the presence of LAC (p = 0.012). Reduced protein level of functional MBL (p = 0.0012) and male gender also had prominent effects.

While the presence of LAC and elevated ACLA-IgG levels have long been recognized for their connections with thrombosis and recurrent pregnancy loss (75), this report provides a firm documentation on the significance of higher C4 plasma protein levels among aPL-positive subjects with APS-related clinical manifestations. The presence of LAC and elevated protein level of complement C4 together are predictors for increased risk of thrombosis with values of sensitivity at 0.707 and specificity at 0.664. This study also reveals lower complement factor H protein levels among subjects with SLE and thrombosis. Deficiency, mutation or autoantibody of complement factor H have been linked to atypical hemolytic uremic syndrome that is characterized by thrombotic microangiopathy (76, 77). Along with observations that plasma protein levels of MBL were decreased, evidence for an involvement of complement proteins in human thrombosis or pregnancy loss are compelling and deserve clinical attention (78). High levels of plasma C4 among patients with thrombosis could result in a procoagulation or thromboinflammatory state, which provide large quantities of reagents to fuel the complement cascades, leading to greater extent of complement-mediated tissue injuries. The abundance of the fast-reacting C4B could aggravate the pathogenic process in arterial thrombosis.

Using SLE as a response, multiple logistic regression analysis of plasma protein data suggested that reduced levels of C3, C4, and ACLA-IgG, and female gender were strong risk factors for SLE C3 is downstream of C4 in the classical and the MBL activation pathways, the activation and consumption of C3 are amplified by a positive feedback mechanism (79–81). In other words, moderate activation of C4 can lead to large consumption of C3. Thus, fluctuations of serum C3 levels tend to be a more sensitive biomarker for SLE disease activity than C4 does.

Among the female aPL-positive subjects, patients with thrombosis and particularly, thrombotic SLE, had high frequencies of recurrent pregnancy loss. RPL patients had elevated levels of complement C4 and ACLA-IgG, and decreased concentration of factor H. Remarkably, female aPL-positive subjects with homozygous C4B deficiency were all protected from RPL, which is consistent with observations in mouse models that complement C4 deficiency or C3 deficiency were protective from RPL induced by injection of human aPL (16). It is also of interest to note that aPL-positive female (human) patients with homozygous C4A deficiency all experienced RPL, which underlies the importance of C4A protein in achieving tolerance or defense against autoimmunity and fetal rejections.

It is important to recognize that the direction of changes for plasma protein levels of complement C3, C4, and ACLA-IgG among aPL-positive patients with SLE and with thrombosis or pregnancy morbidity are mostly opposite to each other. The highest mean protein levels for these proteins were present in the To group (thrombosis without SLE), and the lowest in the So group (SLE without thrombosis). Thus, the inter-group differences of these proteins were highly significant (To vs. So: p = 2.6 × 10−5 for C3; p = 2.2 × 10−9 for C4; p = 1.2 × 10−5 for ACLA-IgG). The resultant effects for these two opposing forces, SLE and thrombosis, are shown in the thrombotic SLE group TS, by which the mean plasma protein levels of C3, C4, C4A, C4B, and ACLA-IgG all fell between those of To and So groups, and their values were closer to those present in the NTS group. When standardized by the gene copy numbers, highly significant differences for mean C4 protein concentrations per gene-copy were observed between the thrombotic subjects and non-thrombotic subjects, and the greatest difference remained between To and So (6.42 mg/dL/gene for To, 4.72 mg/dL/gene for So; p = 3.5 × 10−8 ). The mean C4 protein per gene-copy in TS (6.21 mg/dL/gene) was only slightly lower than that of To. This implies the presence of trans-acting factor(s) among patients with thrombosis that upregulates C4 protein biosynthesis, and/or reduces its turnover that would have decreased the protein levels. While complement activation is a noted feature for clinical manifestations of APS, such activation likely occurs locally that may not result in systemic and parallel decline of plasma protein levels for C4 and C3, a phenomenon analogous to what we observed in many patients with juvenile dermatomyositis (82).

The target sites for most aPL appear to be located at the domain D1 or complement controlling protein repeat on β2GPI. Recombinant antibody recognizing this domain D1 induced fetal loss and coagulation in animal models (83). Interestingly, an engineered β2GPI antibody without the IgG heavy chain CH2 domain, which was devoid of the C1q binding site and unable to fix or activate complement, was shown to compete and control the coagulation and abortive effects in animals burgeoned by injection of human aPL (83). Biochemical studies revealed that β2GPI in its linear conformation can serve as a regulator for the classical and alternative pathway C3 convertases, as it diminished the activation of C3 (to form C3a) and the assembly of C5b-9 in a dose-dependent manner. Active β2GPI also enhanced the degradation of C3b in the presence of factor I and factor H (84, 85). The effects of aPL on the functional activities of β2GPI and plasma complement protein concentrations and activities remain to be elucidated.

The relationships among MBL deficiency, SLE and thrombosis were complex and it was not clear whether MBL deficiency was a risk factor for SLE (86, 87). In a study of 91 SLE patients, Garred et al. demonstrated a near doubling of thrombosis in individuals homozygous for MBL protein structural variants (B,D,C) that led to functional deficiencies of MBL (88). Subsequently, an association was made between MBL deficiency and arterial thrombosis (89). In a study of structural variants and promoter alleles for high and low expression of MBL2 gene in 114 SLE patients, Font et al. observed that low MBL expression genotypes were associated with venous thrombosis (90). Data from our study further clarifies the role of MBL in SLE and thrombosis: reduced plasma protein concentrations of functional MBL were present among aPL-positive patients with thrombosis, regardless of SLE status. Therefore, the link between MBL-deficiency (or low expression of MBL) and SLE could be secondary to low functional MBL in SLE patients with APS. As a lectin binding protein that binds to simple carbohydrate (mannose) components on cell membranes, it is possible that MBL could compete with aPL for binding to phospholipids or phospholipidbinding proteins to reduce the risk of aPL on initiating thrombotic events.

Our study population includes multiple racial and ethnic backgrounds but the majority were of Northern European ancestry. When we analyzed the complement and ACLA data on this specific ethnic group, similar conclusions on the contrasting patterns of complement C4 and ACLA in thromboses and SLE can be reached. Results on three clinical studies on stroke or recurrent pregnancy loss were in accord with our observations that high C4 and/or high C3 plasma protein levels are associated with thrombosis or recurrent pregnancy loss (91–93).

Our observations are consistent with a parallel and independent study that revealed that pediatric SLE patients undergoing a clinical trial (94) with a history of hypertension had persistently higher serum levels of complement C4 and C3 and higher gene copy number of C4B (Mulvihill et al, submitted). Here, we further show that patients with both SLE and thrombosis had the lowest mean GCNs for total C4 (3.55) and C4A (1.81), which underscores the importance of C4A deficiency as a genetic risk factor for systemic autoimmune disease.  SLE-associated disorders such as lupus nephritis, hemolytic anemia, high titers of anti-dsDNA, and lupus disease flares are notably marked by low serum complement levels due to massive consumption of C3 and C4 (21–23, 25). Systemic and concurrent consumptions of C4 and C3 can be reflected by higher coefficients of correlation (r or r 2 ) between these two proteins, which are conspicuous among aPL-positive patients in the So and NTS groups (**Table 2**) (25). Detailed diagnostic disorders of SLE (64) and triple positivity of aPL autoantibodies (9) were not available for this study to examine the extent of hypocomplementemia in various organ involvement and tissue damage, but these would be relevant topics for future investigations.

This cross-sectional study represents a snapshot of complement and aPL in a population of human subjects with antiphospholipid antibodies. The relatively large study population provided an informative dataset to examine specific patterns of complement and aPL among patients with thrombosis, SLE and recurrent pregnancy loss. Along time courses of patients with chronic, systemic autoimmune disease, plasma or serum complement C3 and C4 levels and their cell-bound products would fluctuate with disease activities. The status of SLE/APS disease activities including flare and remission for each patient at the time of sample collection was not available and therefore not accounted for in our data analyses. The lack of data from longitudinal studies, and blood samples from healthy subjects with and without aPL being processed in parallel with similar methodologies are other limitations of this study. Further studies with large sample size of patients for measurements of complement component protein levels under defined genetic backgrounds, plus determination of activation products C3a, C4a, and C5a, cell-bound and fluid phase levels of C4d and C3d, and membrane attack complexes may provide important insights into mechanism(s) on how complement modulate aPL associated clinical manifestations and disease activities of SLE and APS (55, 58, 61, 62). In addition, effects of complement-mediated tissue damage and thromboses would be more readily demonstrated by immunohistochemical methods.

In conclusion, our results can serve as a foundation for further studies of SLE and APS disease mechanisms, more sensitive disease diagnosis, and possibly better prognosis of disease course and profile. It would be desirable to elucidate the C4 gene copy numbers among aPL-positive subjects for a prevention purpose, as those with low total C4 or C4A gene copy number would have a higher risk to develop SLE, and high C4B GCN would have greater risk for complement-mediated complications such as thrombosis, recurrent pregnancy loss in females, and tissue injuries.

#### AUTHOR CONTRIBUTIONS

C-YY, SS, YW, and RR designed the research. RR contributed patient samples and clinical data. SS, KK, DZ, YW, and C-YY performed experiments. HN, YW, SS, and C-YY performed statistical analyses. SS, YW, DZ, EM, FB-S, SA, RR, and C-YY analyzed and interpreted data. SS, YW, KK, DZ, EM, FB-S, SA, RR, HN, and C-YY wrote the paper.

#### ACKNOWLEDGMENTS

We thank Gail Buxton and Bi Zhou for their assistance. This work was supported by grants 1R01 AR050078, 1R21 AR070905, 1R01 AR073311 from the National Institute of Arthritis, Musculoskeletal and Skin Diseases of the NIH (C-YY), a grant from the General Clinical Research Centers Program of the Division of Research Resources, National Institutes of Health, M01 RR00034 (OSU) and RR00046 (UNC).

#### REFERENCES

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.00885/full#supplementary-material

50 consanguineous subjects with defined HLA genotypes. J Immunol. (2007) 179:3012–25. doi: 10.4049/jimmunol.179.5.3012

injury in complement receptor 2/complement receptor 1-deficient mice. J Immunol. (2004) 173:7055–61. doi: 10.4049/jimmunol.173.11.7055

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Savelli, Roubey, Kitzmiller, Zhou, Nagaraja, Mulvihill, Barbar-Smiley, Ardoin, Wu and Yu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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# Mechanistic Characterization of RASGRP1 Variants Identifies an hnRNP-K-Regulated Transcriptional Enhancer Contributing to SLE Susceptibility

#### Julio E. Molineros 1†, Bhupinder Singh1†, Chikashi Terao2,3, Yukinori Okada4 Jakub Kaplan1 , Barbara McDaniel 1 , Shuji Akizuki 3 , Celi Sun1 , Carol F. Webb5 Loren L. Looger 6 and Swapan K. Nath1 \*

 Arthritis and Clinical Immunology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, United States, 2 Laboratory for Statistical Analysis, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan, Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, Kyoto University, Kyoto, Japan, Department of Statistical Genetics, Osaka University Graduate School of Medicine, Osaka, Japan, 5 Departments of Medicine, Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma, OK, United States, Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, VA, United States

Systemic lupus erythematosus (SLE) is an autoimmune disease with a strong genetic component. We recently identified a novel SLE susceptibility locus near RASGRP1, which governs the ERK/MAPK kinase cascade and B-/T-cell differentiation and development. However, precise causal RASGRP1 functional variant(s) and their mechanisms of action in SLE pathogenesis remain undefined. Our goal was to fine-map this locus, prioritize genetic variants likely to be functional, experimentally validate their biochemical mechanisms, and determine the contribution of these SNPs to SLE risk. We performed a meta-analysis across six Asian and European cohorts (9,529 cases; 22,462 controls), followed by in silico bioinformatic and epigenetic analyses to prioritize potentially functional SNPs. We experimentally validated the functional significance and mechanism of action of three SNPs in cultured T-cells. Meta-analysis identified 18 genomewide significant (p < 5 × 10−8 ) SNPs, mostly concentrated in two haplotype blocks, one intronic and the other intergenic. Epigenetic fine-mapping, allelic, eQTL, and imbalance analyses predicted three transcriptional regulatory regions with four SNPs (rs7170151, rs11631591-rs7173565, and rs9920715) prioritized for functional validation. Luciferase reporter assays indicated significant allele-specific enhancer activity for intronic rs7170151 and rs11631591-rs7173565 in T-lymphoid (Jurkat) cells, but not in HEK293 cells. Following up with EMSA, mass spectrometry, and ChIPqPCR, we detected allele-dependent interactions between heterogeneous nuclear ribonucleoprotein K (hnRNP-K) and rs11631591. Furthermore, inhibition of hnRNP-K in Jurkat and primary T-cells downregulated RASGRP1 and ERK/MAPK signaling. Comprehensive association, bioinformatics, and epigenetic analyses yielded putative functional variants of RASGRP1, which were experimentally validated. Notably, intronic

#### Edited by:

José Carlos Crispín, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (INCMNSZ), Mexico

#### Reviewed by:

Shinsuke Yasuda, Hokkaido University, Japan Sergio Iván Valdés-Ferrer, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (INCMNSZ), Mexico

#### \*Correspondence:

Swapan K. Nath<EMAIL_ADDRESS>
†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Autoimmune and Autoinflammatory Disorders, a section of the journal Frontiers in Immunology

> Received: 02 November 2018 Accepted: 25 April 2019 Published: 20 May 2019

#### Citation:

Molineros JE, Singh B, Terao C, Okada Y, Kaplan J, McDaniel B, Akizuki S, Sun C, Webb CF, Looger LL and Nath SK (2019) Mechanistic Characterization of RASGRP1 Variants Identifies an hnRNP-K-Regulated Transcriptional Enhancer Contributing to SLE Susceptibility. Front. Immunol. 10:1066. doi: 10.3389/fimmu.2019.01066 variant (rs11631591) is located in a cell type-specific enhancer sequence, where its risk allele binds to the hnRNP-K protein and modulates RASGRP1 expression in Jurkat and primary T-cells. As risk allele dosage of rs11631591 correlates with increased RASGRP1 expression and ERK activity, we suggest that this SNP may underlie SLE risk at this locus.

Keywords: RASGRP1, homology, ERK (extracellular-signal-regulated kinase), genetic variant, luciferase, ChIPqPCR, EMSA (electrophoretic mobility shift assay)

#### INTRODUCTION

Systemic lupus erythematosus (SLE) is a complex autoimmune disease that disproportionately affects people of Asian, African, and Hispanic ethnicities and women, in particular, with higher incidence and disease severity (1). Much of SLE etiology remains mysterious. It has been proposed that complex interactions amongst numerous genes and their products with pathogens and other environmental factors promotes dysregulation of both the innate and adaptive immune responses in SLE. Over 80 SLE susceptibility loci have been identified so far across multiple ethnic groups by genome-wide association studies (GWAS) and candidate gene studies (2, 3). However, the precise underlying variants and functional mechanisms associated with disease are largely unidentified for the vast majority of these SLE-associated signals. Understanding SLE pathogenesis requires identification of true causal variants and the target genes and mechanisms by which they contribute to disease.

Previously, we reported a novel SLE susceptibility signal near the RAS guanyl-releasing protein 1 (RASGRP1) in Asians (4). We identified several associated variants, the most significant being an intergenic variant (rs12900339) between RASGRP1 and C15orf53 (4). However, the actual predisposing variants, target genes, and underlying mechanisms of action for this region are largely unknown. RASGRP1 belongs to a family of RAS guanyl nucleotide-releasing proteins (RASGRPs) comprising four members (RASGRP1 through RASGRP4), all with a diacylglycerol (DAG)-binding C1 catalytic domain. Upon antigen stimulation, DAG binding and phospholipase C (PLC) signaling drive RASGRPs to the membrane, where they play important roles in RAS activation (5, 6). RASGRP1, originally cloned from the brain (7), was later found highly expressed in T-lymphocytes (8); small amounts of RASGRP1 expression can also occur in B-lymphocytes, neutrophils, mast cells, and natural killer cells (9–11). RASGRP1 has been shown to be involved in B-cell development, activation and tolerance, in both mice and humans (12, 13). RASGRP1−/− mice have been reported for marked deficiency in thymocyte and lymphocyte development, which was associated with impaired proliferation in response to TCR stimulation (14). Deficiency in RASGRP1 in mice has been associated with CD4+ and CD8+ T cell lymphopenia (8). However, humans deficient in RASGRP1 show a decrease in CD4+T concurrent with a relative increase in CD8+T cells (15). RASGRP1 inhibition impairs T-cell expansion and increases susceptibility to Epstein-Barr virus infection, as well as suppressing proliferation of activated T-cells occurring in autoimmune conditions (16). A recent study reported a heterozygous mutation in RASGRP1 correlated with autoimmune lymphoproliferative syndrome (ALPS)-like disease (17). RASGRP1 expression in T-cells also correlated negatively with rheumatoid arthritis disease activity (18). Dysregulated expression of RASGRP1 has been observed in human SLE. The ratio of normal RASGRP1 isoforms to isoforms missing exon-11 could be linked to defective poly[ADP-ribose] polymerase 1 (PARP1) expression and reduced lymphocyte survival in SLE patients (19, 20). Aberrant splice variants accumulate in SLE patients and adversely affect Tcell function (21). There are conflicting reports of the effect of RASGRP1 on ERK signaling. On one hand, deficiency in RASGRP1 expression reportedly decreases ERK phosphorylation in B- and T-cells (15). Hydralazine, a drug that causes druginduced lupus erythematosus, is reported to inhibit ERK signaling, inducing autoimmunity and the production of antidsDNA autoantibodies in mice (22). However, some reports found significantly higher levels of pERK and pJNK in SLE patients with active disease vs. controls and inactive SLE patients (23–25), contradicting earlier reports. In spite of these conflicting reports, the consensus is that RASGRP1 dysfunction is mechanistically associated with autoimmune phenotypes including SLE.

Here, we fine-mapped an SLE locus near RASGRP1 that we previously identified (4). Using trans-ethnic metaanalysis across six Asian and European cohorts followed by bioinformatic analyses and experimental validation, we identified potential SLE predisposing variants and defined mechanisms by which these functional variants contribute to SLE pathogenicity.

#### MATERIALS AND METHODS

#### Patients and Data

We used all associated SNP data at this locus from six cohorts reported previously (**Table 1**). We began with our published Asian cohort report [see Supplementary Table 5 in Sun et al. (4)] and augmented this with two publicly available sets of GWAS summary statistics (26, 27) and a partially published Japanese cohort (28). Our original report contained three Asian cohorts (3AS: Korean, Han Chinese, and Malaya Chinese). Japanese samples included samples (456 cases and 1,102 controls) collected under support of the Autoimmune Disease Study Group of Research in Intractable Diseases, Japanese Ministry of Health, Labor and Welfare, and the BioBank Japan Project (28), and added samples obtained at Kyoto University, Japan. SLE classification followed the

We utilized samples from our previous report (4) (3AS: Korean, Han Chinese and Malaya Chinese) for RASGRP1 SLE association. We added a Han Chinese (HC) and a European (EU) cohort from (26) and a Japanese cohort containing the patients from Okada et al. (28) and additional Japanese samples (JAP).

American College of Rheumatology criteria (29). All sample collections were approved by the Institutional Review Board of the Oklahoma Medical Research Foundation as well as by the collaborating institutions.

## Quality Control

SNP quality control for our initial Asian cohort has been described elsewhere (4). Quality control for European, Han Chinese 2, and Japanese samples was described in the original publications (26–28). All SNPs in the study were in Hardy-Weinberg equilibrium (P > 1 × 10−6 ) and had minor allele frequency >0.5%. Genotypic missingness was <10%. In order to match risk alleles between cohorts, we compared their allele frequencies to the parent populations from the 1,000 Genomes Project. We used the SNP reference dbSNP142 as the SNPnaming convention in common for all variants. SNP imputation for all cohorts was described in their original publications. For this study, SNPs with r 2 and imputation quality information <0.7 were dropped.

#### Study Design

In order to identify RASGRP1 functional variants and their mechanisms of action, our analysis followed the workflow presented in **Figure 1**. We first extracted all summary GWAS information in and around RASGRP1 (118 SNPs) from **Supplementary Table 5** in our previous study of Asian SLE (4). We combined these results with a European (27), an Asian (26), and a partially published Japanese cohort (28), to perform meta-analysis. SNPs that passed the genome-wide significant association threshold (p = 5 × 10−8 ) were further annotated with functional information. A series of bioinformatics and epigenomic analyses was conducted for each of the candidate SNPs including their effects on gene expression (expression quantitative trait loci, eQTLs), transcription factor binding, promoter/enhancer activities, and chromatin interaction sites. Together, we prioritized and nominated SNPs with stronger association signals and with higher annotated likelihood of being functional (**Supplementary Tables 1, 2**). Finally, we experimentally validated predicted functions of the nominated SNPs in Jurkat and HEK293 cell lines. Following SNP prioritization, we performed electrophoretic mobility shift assays (EMSAs), followed by mass spectrometry, chromatin immuno-precipitation quantitative PCR (ChIP-qPCR), and inhibition-based expression assays.

# Association Analysis and Trans-Ethnic Meta-Analysis

Association analysis for all cohorts was performed using PLINK (30) and SNPTEST. Meta-analysis for all cohorts was performed in METAL (31) using cohort sample size correction and standard error correction to estimate the 95% confidence interval for odds ratios. Heterogeneity of odds ratios was estimated and informed the use of Pmeta values in the study. Variants with Pmeta < 5 × 10−3 were selected for further study.

#### Bioinformatic Analysis

Given that candidate SNPs were located in non-coding regions of the genome, we performed a thorough epigenetic annotation of the variants. Initial annotation of epigenetic features was performed in Haploreg (32). Each SNP in the region was collocated with active and regulatory histone marks including H3K27ac, H3K4me1, and H3K4me3, and DNase hypersensitivity sites (DHS) in GM12878, and CD4+ and CD8+ T cells (**Supplementary Figure 1**). Histone modifications and DHS data were obtained from the ENCODE project (33) and the BLUEPRINT epigenome project (34).

### SNP Prioritization

We used a prioritization algorithm to narrow down the large list of SNPs for further validation. Our strategy consisted of two Bayesian algorithms to score each SNP [3dSNP (35) and RegulomeDB (36)], as well as additional expression, epigenetic, and preferential allele-specific information about each SNP. First, we used the 3dSNP (35) tool to assign functional weights based on the presence of enhancers, promoters, experimentally determined (ChIP-seq) transcription factor binding sites (TFBSs), TFBS motif matching, evolutionary conservation, and presence of 3D chromatin interactions. We assigned a 3dSNP weight of 2 to SNPs >2 standard deviations above the mean, a weight of 1 for scores above the mean, and a weight of 0 for the rest. RegulomeDB (36) scores were also assigned for each candidate SNP and converted to an associated weight. Each functional category, i.e., eQTL, enhancer/superenhancer, rSNP (37), capture Hi-C, TFBS, and allele-specific expression/binding, was assigned a weight of 1 if the SNP had this feature. Finally, we summed all weights for each SNP and nominated the top SNPs for further experimental validation.

# Expression Quantitative Trait Loci (eQTLs)

All the candidate SNPs were annotated for the presence of eQTLs changing expression of RASGRP1 and its surrounding genes in multiple tissues. We used expression databases for whole blood (38, 39), immune cell lines (40), and multiple tissues (41) (GTEx Analysis Release V6p). In order to identify quantitative changes in methylation in blood cell lines, we used the WP10 database from the Blueprint epigenome project (42).

dimensions.

# Transcription Factor Binding Sites (TFBSs)

In order to identify allele-specific effects on transcription factor binding (TFBSs), we used the motifBreakr (43) algorithm implemented in R, as well as the PERFECTOS-APE algorithm that identifies fold-changes in binding affinity of SNPs against HOCOMOCO10, HOMER, JASPER, Swiss Regulon, and HT-Selex motif databases. We selected only TFBSs that had at least 5-fold change in affinity.

### Assessing SNP Effects on Enhancer/Promoter Sequences

We assessed whether each SNP was located within regulatory (enhancer/promoter) regions across multiple cell lines using active histone marks (H3K27ac, H3K4me1, and H3K4me3) collocation implemented in the 3dSNP application (35). Superenhancers were annotated using the dbSuper (44), Prestige (45), and EnhancerAtlas (46) databases.

#### Chromatin Interactions

Chromatin looping was identified using capture Hi-C assays obtained from 3D Genome (47), 3DSNP (35) and CHiCP (48); as well as from Promoter-capture Hi-C (49–52) experiments.

# Allele-Specific Binding

Candidate SNPs within the association peaks were further targeted to assess allele-specific binding (ASB) of histone marks H3K4me1 and H3K4me3 in and around them. ASB was calculated using seven heterozygous cell lines (GM10847, GM12890, GM18951, GM19239, GM19240, GM2610, and SNYDER). ASB was implemented in SNPhood (53).

## Luciferase Reporter Assays

To test candidate SNP-containing regions for allele-specific enhancer activity, we cloned all three SNPs (rs1163159, rs7173565-rs7173565, and rs9920715) individually into the enhancer reporter plasmid pGL4.26[luc2/minP/Hygro] (Promega, USA). In brief, genomic DNA from the Coriell cell line having different genotypes for the SNP tested (obtained from NIGMS Human Genetic Cell Repository at the Coriell Institute for Medical Research) was amplified using specific primers containing KpnI and HindIII sites (**Supplementary Table 3**). These amplified PCR products surrounding rs11631591 (481 bp), rs7170151 (579 bp), and rs9920715 (455 bp) were digested with KpnI and HindIII restriction enzymes and ligated into the pGL4.26 plasmid. After cloning and transformation, the plasmids generated for each genotype were confirmed by direct Sanger DNA sequencing. To study cell type-dependence, we used two different cell types: human embryonic kidney HEK293 and T-lymphoid Jurkat cell lines. HEK293 cells were seeded in 24-well sterile plastic culture plates at a density of 1x105 cells per well with complete growth medium. The cells were transfected with 500 ng of pGL4.26 (with or without insert) along with 50 ng Renilla plasmid as control vector to control for differences in transfection efficiency. LipofectAMINE 3000 (Invitrogen, USA) was used for transfection into HEK293 cells, according to the manufacturer's protocol. For Jurkat transfections, we used the Neon Transfection System (Thermo Fisher Scientific). A total of 5 × 105 Jurkat cells was electroporated with a Neon Transfection System (Invitrogen) under the following conditions: voltage (1,050 V), width (30 ms), pulses (Two), 10-µl tip, and Buffer R. For transfection, we used 2 µg of each plasmid

containing the insert with risk or non-risk allele, along with 50 ng Renilla plasmid. Firefly and Renilla luciferase activities were measured consecutively at 24 h after transfection using Dualluciferase assays (Promega), according to the manufacturer's instructions. Luciferase activity was analyzed with Student's t-test implemented in GraphPad Prism7. Differences between relative luciferase activity levels were considered significant if Student's t-test P-value < 0.05.

#### Identification of DNA-Binding Proteins Electrophoretic Mobility Shift Assays (EMSAs) and DNA Pulldown Assays

Jurkat cell lines were obtained from ATCC and maintained in RPMI 1640 medium with 2 mm L-glutamine, 100µg/ml each of streptomycin and penicillin, and 10% fetal bovine serum at 37◦C with 5% CO2. Cells were harvested at a density of 8 × 105 cells/ml, and nuclear extracts were prepared using the NER nuclear extraction kit (Invitrogen) with complete protease inhibitors (Roche Diagnostics). Protein concentrations were measured using a BCA reagent. Biotinylated DNA sequence surrounding the candidate SNPs (rs7170151 and rs11631591) was prepared using a synthetic single-stranded DNA sequence (Integrated DNA Technologies, USA) (**Supplementary Table 3**). Biotinylated DNA sequence with a 5-bp deletion at the SNP region served as a control for the assay. Twenty-five pmol of each DNA product was bound to 1 mg Dynabeads R M-280 Streptavidin (Invitrogen, USA), as per the manufacturer's recommendations. Dynabeads M-280 Streptavidin (Dynal, Inc., Lake Success, NY, USA) were prepared by washing three times in phosphate-buffered saline (pH 7.4) containing 0.1% bovine serum albumin and two times with Tris-EDTA containing 1 M NaCl. Between each wash, beads were pulled down with a Dynal magnetic particle concentrator. Double-stranded, biotinylated oligonucleotides were added to the washed beads, and the mix was rotated for 20–30 min at 21 ◦C. Equal cpm of proteins translated in vitro were diluted to 1× with binding buffer and mixed with ∼100 µg of Dynabeads containing 10 pmol of the individual oligonucleotide probe in a final volume of 250 µl. The mixture was rotated at room temperature for 20 min. Proteins bound to the beads were separated from unbound proteins by successive washes, three times with 0.5× binding buffer and once with 1× binding buffer. Higher stringency washes included two washes with 2× binding buffer. Beads and bound proteins were pulled down with a magnetic concentrator, suspended in 1× sample buffer, boiled for 5 min, and resolved on SDS-PAGE gels, followed by peptide mass fingerprint MALDI-MS analysis of single bands.

#### Mass Spectrometry Analysis