Siglec Genes Confer Resistance to Systemic Lupus Erythematosus in Humans and Mice

OPEN Cellular and Molecular Immunology (2019) 16, 154–164 www.nature.com/cmi ARTICLE Siglec genes confer resistance to systemic lupus erythematosus in humans and mice Rhonda Flores, Peng Zhang, Wei Wu, Xu Wang, Peiying Ye, Pan Zheng and Yang Liu A recent meta-analysis revealed the contribution of the SIGLEC6 locus to the risk of developing systemic lupus erythematosus (SLE). However, no specific Siglec (sialic acid-binding immunoglobulin-like lectin) genes (Siglecs) have been implicated in the pathogenesis of SLE. Here, we performed in silico analysis of the function of three major protective alleles in the locus and found that these alleles were expression quantitative trait loci that enhanced expression of the adjacent SIGLEC12 gene. These data suggest that SIGLEC12 may protect against the development of SLE in Asian populations. Consistent with human genetic data, we identified two missense mutations in lupus-prone B6.NZMSle1/Sle2/Sle3 (Sle1–3) mice in Siglece, which is the murine Siglec with the greatest homology to human SIGLEC12. Since the mutations resulted in reduced binding of Siglec E to splenic cells, we evaluated whether Siglece−/− mice had SLE phenotypes. We found that Siglece−/− mice showed increased autoantibody production, glomerular immune complex deposition and severe renal pathology reminiscent of human SLE nephropathy. Our data demonstrate that the Siglec genes confer resistance to SLE in mice and humans. Cellular and Molecular Immunology advance online publication, 5 March 2018; doi:10.1038/cmi.2017.160 Keywords: Siglecs; CD24; HMGB1; systemic lupus erythematosus INTRODUCTION damage. Although the etiology of SLE N-terminal V-set Ig-like domains that Systemic lupus erythematosus (SLE) is a remains elusive, genetic and environ- bind to sialoside-containing structures complex autoimmune inflammatory dis- mental factors, as well as a failure to with different specificities,aswellasa ease that predominately affects women of properly clear apoptotic cells leading to C2-set Ig-like domain that contains a childbearing age. The prevalence of SLE secondary necrosis and the release of variable number of C2-type repeats.10,16 ranges from 20 to 150 cases per 100 000 nuclear autoantigens, challenges immu- Many Siglecs have an intracellular individuals, with a 10-year survival rate nological tolerance, thereby exacerbating immune receptor tyrosine-based inhibi- of ~ 70%.1 Production of autoantibodies the risk of disease manifestation.3–5 tory motifs. These motifs are phosphory- against self-nucleic acids, such as double- Toll-like receptors (TLRs) recognize lated by tyrosine kinases and are stranded DNA (dsDNA), represents a both pathogen-associated molecular pat- subsequently bound by SHP-1 and serological hallmark of SLE.2 These auto- terns and danger-associated molecular SHP-2 tyrosine phosphatases17 and the antibodies contribute to the pathogenesis patterns (DAMPs) and induce the pro- E3 ligase Cbl,7 thereby dampening TLR of SLE by forming immune complex duction of inflammatory cytokines. TLRs cell signaling in response to DAMPs. We deposits in different parts of the body, play key roles in driving aberrant inflam- have recently reported that Siglec E leading to inflammation and organ mation in response to DAMPs in SLE is directly associated with TLRs and patients and SLE-prone mice.6–8 Sialic regulates TLR-mediated induction of acid-binding immunoglobulin-like lec- inflammatory responses, including Center for Cancer and Immunology Research, 18 Children’s National Medical Center, Washington, tins (Siglecs) are sialic acid-recognizing endotoxemia. DC 20010, USA cell surface receptors that are predomi- While the role for SIGLECS in SLE has Correspondence: Professor P Zheng or Professor nately expressed on immune and hema- not been systematically investigated, sev- Y Liu, Center for Cancer and Immunology topoietic cells.9–11 They comprise a eral lines of evidence suggest a potential Research, Children's National Medical Center, 111 Michigan Avenue NW, Washington, DC family of 14 receptors in humans and 9 role for Siglecs in the pathogenesis of 20010, USA. receptors in mice,12 and have been SLE. First, we and others have reported E-mail: [email protected] or yangl@on- shown to suppress TLR-mediated that CD24, which encodes the first coimmune.com inflammatory responses to DAMPs.13–15 known natural ligand for a Siglec,13 Received: 15 July 2017; Accepted: 30 Novem- 19–22 ber 2017 Siglecs have one or two extracellular affects the risk of developing SLE. Siglec genes and the risk of SLE R Flores et al 155 Sialylated CD24 has been shown to MATERIALS AND METHODS CCTCGCC, 5′-CTGGCGTGAGTATCG interact with Siglec-G and human Mice GCCTT; exon 6: 5′-TGGGTGTAAGGAC − − Siglec-10.23 This interaction attenuated Siglece / mice were generated by gene ACCAAGG, 5′-CAGTGTGCCTGTGCT proinflammatory TLR signaling in targeting from 129/Sv ES cells produced CAAGC; exon 7: 5′-GAAAGGAGAGAG response to a variety of DAMPs released by the Mutant Mouse Regional Resource TCAGAGAA, 5′-TGACCGTGGCTGGA by damaged cells, such as nuclear protein Center (MMRRC) at UC Davis (Davis, GAAAGC). PCR was performed using high mobility group box 1 (HMGB1) and CA, USA), as described here (https:// GoTaq Green Master Mix (Promega, heat-shock proteins HSP70 and www.taconic.com/knockout-mouse/ Madison, WI, USA) for 40 cycles at 96 °C HSP90.14,15 Previous studies have siglece-targeted). These mice were back- for10s,57°Cfor30s and72°Cfor60s. demonstrated that, during cell damage crossed to C57BL/6 mice for five gen- and death, molecules such as HMGB1, erations. B6.NZMSle1/Sle2/Sle3 (Sle1–3) Immunofluorescence analysis HSPs and possibly HMGB1-containing mice were purchased from the Jackson For antinuclear antibody (ANA) level nucleosomes induce the production of Laboratory. All mice used were between measurement, HeLa cells were seeded on inflammatory cytokines in a TLR2- 12 and 14 months of age. All mice were coverslips and fixed with 4% paraformal- dependent manner, as well as the pro- bred and maintained under specific dehyde (Sigma, St Louis, MO, USA) in duction of anti-dsDNA antibodies in pathogen-free conditions at the Chil- phosphate-buffered saline (PBS) for BALB/c mice.6,24 Therefore, disruption dren’s National Medical Center. All pro- 20minatroomtemperature,followed of Siglecs and their sialylated ligands cedures were approved by the Animal by permeabilization with 0.1% Triton may promote autoimmunity. Second, a Care and Use Committee of the Chil- X-100 (Sigma) for an additional 10 min. recent study showed that loss of Siglec-G dren’s National Medical Center. After washing with PBS, cells were blocked expression in the SLE-prone MRL/lpr with 10% fetal bovine serum in DMEM mouse strain moderately contributed to Genetic analysis and stained with serum from 6-month- disease severity.25 Likewise, Siglecg single Genomic DNA was extracted from three old mice diluted 1:1000. Alexa Fluor knockout mice show a massive increase C57BL/6 and three B6.NZMSle1/Sle2/Sle3 488-conjugated goat anti-mouse immuno- in B1 B cells,26,27 and mice deficient for mice. Tail digestion was performed over- globulin G (IgG) (Invitrogen, Carlsbad, both Siglec-G and CD22 have an exacer- night at 55 °C in STE buffer (100 mM CA, USA) was used to detect ANA. bation of this phenotype and develop Tris, 5 mM EDTA, 0.2% sodium dodecyl For evaluation of glomerular IgG, IgM systemic autoimmunity with limited fea- sulfate, 200 mM NaCl, pH 8.5) and and C3 deposition, kidneys were snap tures of SLE.28 Third, mutations in sialic proteinase K. Genomic DNA was then frozen in OCT medium directly after acid acetylesterase, the enzyme involved purified using phenol–chloroform extrac- dissection. After sectioning using a cryo- in modifying sialylated Siglec-G ligands, tion. Amplification and sequencing pri- stat, the 8-μm-thick frozen sections were led to autoimmunity in mice and was mers were designed for each of the seven fixed with 4% paraformaldehyde in PBS associated with autoimmune diseases in exons in Siglece (amplification primers— for 30 min at room temperature, fol- humans.29 Nevertheless, no genetic poly- exon 1: 5′-TAAAACTGTCTCTCCAGGCT lowed by permeabilization with 0.3% morphisms of either Siglecg or SIGLEC10 ,5′-CTGGGAGCAGCTGGGTTT; exon 2: Triton X-100 at room temperature for genes have been reported to be associated 5′-CAGCTCCTCCCCTGAGC, 5′-TAAG 15 min. After washing, the tissue sections with an increased risk of developing SLE. GGTGCTTGTCAGGATG; exon 3: 5′-CT were blocked with 3% normal goat Most recently, an association study GAACTTACTTTCCGCCTT, 5′-TACCT serum (Sigma) and then stained with including 4478 SLE cases and 12 656 GACCTTGAGTCCAGG; exon 4: 5′-AGT Alexa Fluor 488-conjugated goat anti- controls from six East Asian cohorts AGGGAGCAAAGGACAGG, 5′-TCCCT mouse IgG (Invitrogen), goat anti-mouse identified SIGLEC6 as a major SLE risk ATTAGCCTTGTTAGCT; exon 5: 5′-CT IgM (Santa Cruz Technology, Dallas, TX, locus among Asian populations.30 Our in GAACTTACTTTCCGCCTT, 5′-GATGG USA) and rat anti-mouse C3 (Abcam, silico analysis in this study suggests that TGAGGGACCAGCCTG; exon 6: 5′-AC Cambridge, Cambridgeshire, UK). Depos- both predisposing and protective alleles CCTCTGCTTGCAGTTAAG, 5′-GCCTG its in the glomeruli were scored in a can be found within this region. Surpris- GACTCCTCCCCTGAGA; exon 7: 5′-TG blinded manner on a scale of 0–4 ingly, all protective alleles were found to TAGGGGTATATACACATAA, 5′-GTTG (0 = negative, 1 = weak, 2 = moderate, be associated with enhanced expression ACATGTGATACACAGGG; sequencing 3 = strong, 4 = maximal fluorescence) in of a SIGLEC12 gene. We also show that primers—exon 1: 5′-GCATGTCCAGC five different fields for each kidney sec- mice with significantly enhanced devel- TAAAACTGT, 5′-CCATGGGTTGGGAG tion. All images were acquired at the opment of SLE due to expression of the CAGT;exon2:5′-AATGGAGCATCAGG same exposure time to allow comparison alleles Sle1–3 have two mutations in the ATGGGA, 5′-CCTGTTTTTCTAGTACA among samples with an Olympus X51 IgV-like domain of Siglec E, the closest AAG; exon 3: 5′-GAATGCTAAGAAACC microscope (Temple Hills, ML, USA).

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