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1.  Autoimmune Diseases in the Bioinformatics Paradigm 
PMCID: PMC4610968  PMID: 26433001
2.  Regulatory polymorphisms modulate the expression of HLA class II molecules and promote autoimmunity 
eLife  null;5:e12089.
Targeted sequencing of sixteen SLE risk loci among 1349 Caucasian cases and controls produced a comprehensive dataset of the variations causing susceptibility to systemic lupus erythematosus (SLE). Two independent disease association signals in the HLA-D region identified two regulatory regions containing 3562 polymorphisms that modified thirty-seven transcription factor binding sites. These extensive functional variations are a new and potent facet of HLA polymorphism. Variations modifying the consensus binding motifs of IRF4 and CTCF in the XL9 regulatory complex modified the transcription of HLA-DRB1, HLA-DQA1 and HLA-DQB1 in a chromosome-specific manner, resulting in a 2.5-fold increase in the surface expression of HLA-DR and DQ molecules on dendritic cells with SLE risk genotypes, which increases to over 4-fold after stimulation. Similar analyses of fifteen other SLE risk loci identified 1206 functional variants tightly linked with disease-associated SNPs and demonstrated that common disease alleles contain multiple causal variants modulating multiple immune system genes.
eLife digest
The human immune system defends the body against microbes and other threats. However, if this process goes wrong the immune system can attack the body’s own healthy cells, which can lead to serious autoimmune diseases.
Systemic lupus erythematosus (SLE) is an autoimmune disease in which immune cells often attack internal organs – including the kidneys, nervous system and heart. Over the past decade, multiple genes have been linked with an increased risk of SLE. However, it is largely unknown how the sequences of these genes differ between individuals with SLE and healthy individuals, and the precise changes that lead to an increased risk of SLE are also not clear.
Now, Raj, Rai et al. have determined the genetic sequences of over 700 people with SLE and over 500 healthy individuals and looked for differences that influence susceptibility to the disease. The vast majority of differences were discovered in stretches of DNA that regulate the expression of nearby genes, rather than in DNA that encodes the structures of proteins. Notably, extensive differences were found in a region of the human genome that regulates the production of proteins called Human Leukocyte Antigen class II molecules; which are known to play a critical role in activating the immune system. Raj, Rai et al. found that slight changes to the regulatory DNA sequences resulted in an overabundance of these proteins, which led to a hyperactive immune system that is strongly associated with SLE.
Future studies could now ask if the changes to the regulatory DNA sequences highlighted by Raj, Rai et al. increase susceptibility to other autoimmune disorders as well. It may also be possible to use the increased understanding of how the immune system is regulated to develop new ways to minimize the rejection of organ transplants.
PMCID: PMC4811771  PMID: 26880555
targeted sequencing; HLA; SLE risk; haplotype; risk allele; LD; Human
3.  Delivering Oxidation Resistance-1 (OXR1) to Mouse Kidney by Genetic Modified Mesenchymal Stem Cells Exhibited Enhanced Protection against Nephrotoxic Serum Induced Renal Injury and Lupus Nephritis 
To elucidate the role of oxidation resistance 1 (OXR1) gene. Oxidative stress plays a pivotal role in pathogenesis of immune-mediated nephritis. Recently we identified oxidation resistance 1 (OXR1) is conventionally expressed in eukaryotes and has an ability to prevent oxidative damage caused by various oxidative stresses. However the protective effect of OXR1 in immune-associated inflammatory response and oxidative damage is not clear and will be investigated in this study.
We utilized mesenchymal stem cells (MSCs) as vehicles to carry OXR1 into the injured kidneys of nephritis model mice and investigated the influence of OXR1 on glomerulonephritis. Human OXR1 gene was integrated into genome of MSCs via lentiviral vector, and established hOXR1-MSC cell line which still maintains the differentiation property. 129/svj mice with anti-glomerular basement membrane (GBM) challenge and spontaneous lupus mice B6.Sle1.Sle2.Sle3 were injected with hOXR1-MSCs (i.v. injection) to evaluate the function of hOXR1. Immunohistochemistry was used to appraise the renal pathology and Tunel staining was applied to detect cell apoptosis.
Compared with control mice, hOXR1-MSCs administration showed significantly decreased blood urea nitrogen (BUN), proteinuria and ameliorated renal pathological damage. hOXR1-MSCs transplantation significantly reduced macrophage and T lymphocyte infiltration by inhibiting the expression of CCL2, CCL7, IL-1β, IL-6 and NFκB in mouse kidney. Moreover, hOXR1-MSCs prevented hydrogen peroxide (H2O2)-induced oxidative stress and its implantation reduced nitric oxide (NO) in mouse serum and urine to inhibit tubular cell apoptosis.
OXR1-MSCs transplantation may exert a certain protective effect on nephritis by suppressing inflammation and oxidative stress.
PMCID: PMC4435960  PMID: 25995969
Oxidation resistance 1; lupus nephritis; mesenchymal stem cell; oxidative stress; inflammation; apoptosis
5.  Insulin-Like Growth Factor Binding Protein-4 as a Marker of Chronic Lupus Nephritis 
PLoS ONE  2016;11(3):e0151491.
Kidney biopsy remains the mainstay of Lupus Nephritis (LN) diagnosis and prognostication. The objective of this study is to identify non-invasive biomarkers that closely parallel renal pathology in LN. Previous reports have demonstrated that serum Insulin-like growth factor binding protein 4 (IGFBP-4) was increased in diabetic nephropathy in both animal models and patients. We proceeded to assess if IGFBP4 could be associated with LN. We performed ELISA using the serum of 86 patients with LN. Normal healthy adults (N = 23) and patients with other glomerular diseases (N = 20) served as controls. Compared to the healthy controls or other glomerular disease controls, serum IGFBP-4 levels were significantly higher in the patients with LN. Serum IGFBP-4 did not correlate well with systemic lupus erythematosus disease activity index (SLEDAI), renal SLEDAI or proteinuria, but it did correlate with estimated glomerular filtration rate (R = 0.609, P < 0.0001). Interestingly, in 18 patients with proliferative LN whose blood samples were obtained at the time of renal biopsy, serum IGFBP-4 levels correlated strongly with the chronicity index of renal pathology (R = 0.713, P < 0.001). IGFBP-4 emerges a potential marker of lupus nephritis, reflective of renal pathology chronicity changes.
PMCID: PMC4809566  PMID: 27019456
6.  AIM2 and endosomal TLRs differentially regulate arthritis and autoantibody production in DNaseII deficient mice 
Innate immune PRRs sense nucleic acids from microbes and orchestrate cytokine production to resolve infection. Inappropriate recognition of host nucleic acids also results in autoimmune disease. Here we utilize a model of inflammation resulting from accrual of self DNA (DNase II−/− Ifnar−/−) to understand the role of PRR sensing pathways in arthritis and autoantibody production. Using mice deficient in DNase II/Ifnar together with deficiency in either STING or AIM2 (TKO), we reveal central roles for the STING and AIM2 pathway in arthritis. AIM2 TKO mice show limited inflammasome activation and, like STING TKO mice, have reduced inflammation in joints. Surprisingly, autoantibody production is maintained in AIM2 and STING TKO mice, while DNase II−/− Ifnar−/− mice also deficient in Unc93b, a chaperone required for TLR7/9 endosomal localization, fail to produce autoantibodies to nucleic acids. Collectively, these data support distinct roles for cytosolic and endosomal nucleic acid sensing pathways in disease manifestations.
PMCID: PMC4299698  PMID: 25548216
7.  Cutting Edge: Inhibiting TBK1 by Compound II Ameliorates Autoimmune Disease in Mice 
The Journal of Immunology Author Choice  2015;195(10):4573-4577.
TANK-binding kinase 1 (TBK1) is a serine/threonine protein kinase that plays a crucial role in innate immunity. Enhanced TBK1 function is associated with autoimmune diseases and cancer, implicating the potential benefit of therapeutically targeting TBK1. In this article, we examined a recently identified TBK1 inhibitor Compound II on treating autoimmune diseases. We found that Compound II is a potent and specific inhibitor of TBK1-mediated IFN response. Compound II inhibited polyinosinic-polycytidylic acid–induced immune activation in vitro and in vivo. Compound II treatment also ameliorated autoimmune disease phenotypes of Trex1−/− mice, increased mouse survival, and dampened the IFN gene signature in TREX1 mutant patient lymphoblasts. In addition, we found that TBK1 gene expression is elevated in systemic lupus erythematosus patient cells, and systemic lupus erythematosus cells with high IFN signature responded well to Compound II treatment. Together, our findings provided critical experimental evidence for inhibiting TBK1 with Compound II as an effective treatment for TREX1-associated autoimmune diseases and potentially other interferonopathies.
PMCID: PMC4635567  PMID: 26432890
8.  Autoantigen Microarray for High-throughput Autoantibody Profiling in Systemic Lupus Erythematosus 
Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by the production of autoantibodies to a broad range of self-antigens. Profiling the autoantibody repertoire using array-based technology has emerged as a powerful tool for the identification of biomarkers in SLE and other autoimmune diseases. Proteomic microarray has the capacity to hold large number of self-antigens on a solid surface and serve as a high-throughput screening method for the determination of autoantibody specificities. The autoantigen arrays carrying a wide variety of self-antigens, such as cell nuclear components (nucleic acids and associated proteins), cytoplasmic proteins, phospholipid proteins, cell matrix proteins, mucosal/secreted proteins, glomeruli, and other tissue-specific proteins, have been used for screening of autoantibody specificities associated with different manifestations of SLE. Arrays containing synthetic peptides and molecular modified proteins are also being utilized for identification of autoantibodies targeting to special antigenic epitopes. Different isotypes of autoantibodies, including IgG, IgM, IgA, and IgE, as well as other Ig subtypes, can be detected simultaneously with multi-color labeled secondary antibodies. Serum and plasma are the most common biologic materials for autoantibody detection, but other body fluids such as cerebrospinal fluid, synovial fluid, and saliva can also be a source of autoantibody detection. Proteomic microarray as a multiplexed high-throughput screening platform is playing an increasingly-important role in autoantibody diagnostics. In this article, we highlight the use of autoantigen microarrays for autoantibody exploration in SLE.
PMCID: PMC4610965  PMID: 26415621
Systemic lupus erythematosus (SLE); Autoantibody profiling; Proteomic microarray; Biomarker; High-throughput assay
9.  CD11c-mediated Deletion of Flip Promotes Autoreactivity and Inflammatory Arthritis 
Nature communications  2015;6:7086.
Dendritic cells (DCs) are critical for immune homeostasis. To target DCs, we generated a mouse line with Flip deficiency in cells that express cre under the CD11c-promoter (CD11c-Flip-KO). CD11c-Flip-KO mice spontaneously develop erosive, inflammatory arthritis, resembling rheumatoid arthritis, which is dramatically reduced when these mice are crossed with Rag−/− mice. The CD8α+ DC subset is significantly reduced, along with alterations in NK cells and macrophages. Autoreactive CD4+ T cells and autoantibodies specific for joint tissue are present and arthritis severity correlates with the number of autoreactive CD4+ T cells and plasmablasts in the joint draining lymph nodes. Reduced T regulatory cells (Tregs) inversely correlate with arthritis severity, and the transfer of Tregs ameliorates arthritis. This KO line identifies a model that will permit in depth interrogation of the pathogenesis of rheumatoid arthritis, including the role of CD8α+ DCs and other cells of the immune system.
PMCID: PMC4429912  PMID: 25963626
10.  Opposing impact of B cell intrinsic TLR7 and TLR9 signals on autoantibody repertoire and systemic inflammation 
Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease characterized by autoantibodies targeting nucleic acid-associated antigens. The endosomal toll-like receptors TLR7 and TLR9 are critical for generation of Abs targeting RNA- or DNA-associated antigens, respectively. In murine lupus models, deletion of TLR7 limits autoimmune inflammation, while deletion of TLR9 exacerbates disease. Whether B cell or myeloid TLR7/TLR9 signaling is responsible for these effects has not been fully addressed. Here, we utilize a chimeric strategy to evaluate the effect of B cell-intrinsic deletion of TLR7 vs. TLR9 in parallel lupus models. We demonstrate that B cell-intrinsic TLR7 deletion prevents RNA-associated Ab formation, decreases production of class-switched Abs targeting non-nuclear antigens and limits systemic autoimmunity. In contrast, B cell-intrinsic TLR9 deletion results in decreased DNA-reactive Ab, but increased Abs targeting a broad range of systemic autoantigens. Further, we demonstrate that B-intrinsic TLR9 deletion results in increased systemic inflammation and immune-complex (IC) glomerulonephritis despite intact TLR signaling within the myeloid compartment. These data stress the critical importance of dysregulated B cell-intrinsic TLR signaling in the pathogenesis of SLE.
PMCID: PMC4041708  PMID: 24711620
11.  CD11c-mediated deletion of Flip promotes autoreactivity and inflammatory arthritis 
Nature Communications  2015;6:7086.
Dendritic cells (DCs) are critical for immune homeostasis. To target DCs, we generated a mouse line with Flip deficiency in cells that express cre under the CD11c promoter (CD11c-Flip-KO). CD11c-Flip-KO mice spontaneously develop erosive, inflammatory arthritis, resembling rheumatoid arthritis, which is dramatically reduced when these mice are crossed with Rag−/− mice. The CD8α+ DC subset is significantly reduced, along with alterations in NK cells and macrophages. Autoreactive CD4+ T cells and autoantibodies specific for joint tissue are present, and arthritis severity correlates with the number of autoreactive CD4+ T cells and plasmablasts in the joint-draining lymph nodes. Reduced T regulatory cells (Tregs) inversely correlate with arthritis severity, and the transfer of Tregs ameliorates arthritis. This KO line identifies a model that will permit in depth interrogation of the pathogenesis of rheumatoid arthritis, including the role of CD8α+ DCs and other cells of the immune system.
Dendritic cells are critical for initiation of immune responses and for induction of tolerance. Here the authors show that deletion of survival factor c-flip in CD11c-expressing cells subset perturbs CD8a+ dendritic cell, NK and macrophage pools, and leads to development of autoimmune arthritis.
PMCID: PMC4429912  PMID: 25963626
12.  A Locked Nucleic Acid (LNA)-Based Real-Time PCR Assay for the Rapid Detection of Multiple Bacterial Antibiotic Resistance Genes Directly from Positive Blood Culture 
PLoS ONE  2015;10(3):e0120464.
Bacterial strains resistant to various antibiotic drugs are frequently encountered in clinical infections, and the rapid identification of drug-resistant strains is highly essential for clinical treatment. We developed a locked nucleic acid (LNA)-based quantitative real-time PCR (LNA-qPCR) method for the rapid detection of 13 antibiotic resistance genes and successfully used it to distinguish drug-resistant bacterial strains from positive blood culture samples. A sequence-specific primer-probe set was designed, and the specificity of the assays was assessed using 27 ATCC bacterial strains and 77 negative blood culture samples. No cross-reaction was identified among bacterial strains and in negative samples, indicating 100% specificity. The sensitivity of the assays was determined by spiking each bacterial strain into negative blood samples, and the detection limit was 1–10 colony forming units (CFU) per reaction. The LNA-qPCR assays were first applied to 72 clinical bacterial isolates for the identification of known drug resistance genes, and the results were verified by the direct sequencing of PCR products. Finally, the LNA-qPCR assays were used for the detection in 47 positive blood culture samples, 19 of which (40.4%) were positive for antibiotic resistance genes, showing 91.5% consistency with phenotypic susceptibility results. In conclusion, LNA-qPCR is a reliable method for the rapid detection of bacterial antibiotic resistance genes and can be used as a supplement to phenotypic susceptibility testing for the early detection of antimicrobial resistance to allow the selection of appropriate antimicrobial treatment and to prevent the spread of resistant isolates.
PMCID: PMC4361058  PMID: 25775001
13.  Allelic heterogeneity in NCF2 associated with systemic lupus erythematosus (SLE) susceptibility across four ethnic populations 
Human Molecular Genetics  2013;23(6):1656-1668.
Recent reports have associated NCF2, encoding a core component of the multi-protein NADPH oxidase (NADPHO), with systemic lupus erythematosus (SLE) susceptibility in individuals of European ancestry. To identify ethnicity-specific and -robust variants within NCF2, we assessed 145 SNPs in and around the NCF2 gene in 5325 cases and 21 866 controls of European-American (EA), African-American (AA), Hispanic (HS) and Korean (KR) ancestry. Subsequent imputation, conditional, haplotype and bioinformatic analyses identified seven potentially functional SLE-predisposing variants. Association with non-synonymous rs17849502, previously reported in EA, was detected in EA, HS and AA (PEA = 1.01 × 10−54, PHS = 3.68 × 10−10, PAA = 0.03); synonymous rs17849501 was similarly significant. These SNPs were monomorphic in KR. Novel associations were detected with coding variants at rs35937854 in AA (PAA = 1.49 × 10−9), and rs13306575 in HS and KR (PHS = 7.04 × 10−7, PKR = 3.30 × 10−3). In KR, a 3-SNP haplotype was significantly associated (P = 4.20 × 10−7), implying that SLE predisposing variants were tagged. Significant SNP–SNP interaction (P = 0.02) was detected between rs13306575 and rs17849502 in HS, and a dramatically increased risk (OR = 6.55) with a risk allele at each locus. Molecular modeling predicts that these non-synonymous mutations could disrupt NADPHO complex assembly. The risk allele of rs17849501, located in a conserved transcriptional regulatory region, increased reporter gene activity, suggesting in vivo enhancer function. Our results not only establish allelic heterogeneity within NCF2 associated with SLE, but also emphasize the utility of multi-ethnic cohorts to identify predisposing variants explaining additional phenotypic variance (‘missing heritability’) of complex diseases like SLE.
PMCID: PMC3929085  PMID: 24163247
14.  Discovery of Biomarkers for Systemic Lupus Erythematosus Using a Library of Synthetic Autoantigen Surrogates 
Journal of immunological methods  2013;402(0):23-34.
Antibodies to a wide range of self-antigens, including those directed against nucleic acids or nucleic acid-binding proteins are the essential biomarkers for diseases such as systemic lupus erythematosus (SLE). Highly complex libraries of nonamers consisting of N-substituted glycines (peptoids) were screened for compounds that bound IgG from patients with SLE and earlier, incomplete autoimmune syndromes. Peptoids were identified that could identify subjects with SLE and related syndromes with a high sensitivity (70%) and specificity (97.5%). Immobilized peptoids were used to isolate IgG from both healthy subjects and SLE patients that reacted with known RNA-binding proteins. In the case of SLE patients, the peptoid-purified IgG reacted with several autoantigens, suggesting that the peptoids are capable of interacting with multiple, structurally similar molecules. These results show that the measurement of IgG binding to peptoids can identify subjects with high levels of pathogenic autoantibodies.
PMCID: PMC3946867  PMID: 24269750
15.  Overexpression of Membrane-bound Fas Ligand (CD95L) Exacerbates Autoimmune Disease and Renal Pathology in Pristane-induced Lupus 
Loss of function mutations in the Fas death receptor or its ligand result in a lymphoproliferative syndrome and exacerbate clinical disease in most lupus-prone strains of mice. One exception is mice injected with 2,6,10,14-Tetramethylpentadecane (TMPD), a hydrocarbon oil commonly known as pristane, which induces SLE-like disease. While Fas/FasL interactions have been strongly implicated in the activation induced cell death of both lymphocytes and other antigen presenting cells, FasL can also trigger the production of pro-inflammatory cytokines. FasL is a transmembrane protein with a matrix metalloproteinase (MMP) cleavage site in the ectodomain. MMP cleavage inactivates membrane-bound FasL (mFasL) and releases a soluble form, sFasL, reported to have both antagonist and agonist activity. To better understand the impact of FasL cleavage on both the pro-apoptotic and proinflammatory activity of FasL, its cleavage site was deleted through targeted mutation, to produce the ΔCS mouse line. ΔCS mice express higher levels of mFasL than WT mice and fail to release sFasL. To determine to what extent FasL promotes inflammation in lupus mice, TMPD-injected FasL-deficient and ΔCS BALB/c mice were compared to control TMPD-injected BALB/c mice. We found that FasL-deficiency significantly reduced the early inflammatory exudate induced by TMPD injection. By contrast, ΔCS mice developed a markedly exacerbated disease profile associated with a higher frequency of splenic neutrophils and macrophages, a profound change in ANA specificity, and markedly increased proteinuria and kidney pathology, compared to controls. These results demonstrate that FasL promotes inflammation in TMPD-induced autoimmunity, and its cleavage limits FasL pro-inflammatory activity.
PMCID: PMC4219920  PMID: 23918976
16.  IL-21 promotes the production of anti-DNA IgG but is dispensable for kidney damage in lyn−/− mice 
European journal of immunology  2012;43(2):382-393.
The autoimmune disease systemic lupus erythematosus (SLE) is characterized by loss of tolerance to nuclear antigens and a heightened inflammatory environment, which together result in end organ damage. Lyn-deficient mice, a model of SLE, lack an inhibitor of B- and myeloid cell activation. This results in B-cell hyperresponsiveness, plasma cell (PC) accumulation, autoantibodies, and glomerulonephritis (GN). IL-21 is associated with autoimmunity in mice and humans and promotes B-cell differentiation and class switching. Here, we explore the role of IL-21 in the autoimmune phenotypes of lyn−/− mice. We find that IL-21 mRNA is reduced in the spleens of lyn−/−IL-6−/− and lyn−/−Btklo mice, neither of which produce pathogenic autoantibodies or develop significant GN. While IL-21 is dispensable for PC accumulation and IgM autoantibodies in lyn−/− mice, it is required for anti-DNA IgG antibodies and some aspects of T cell activation. Surprisingly, GN still develops in lyn−/−IL-21−/− mice. This likely results from the presence of IgG autoantibodies against a limited set of non-DNA antigens. These studies identify a specific role for IL-21 in the class switching of anti-DNA B-cells and demonstrate that neither IL-21 nor anti-DNA IgG is required for kidney damage in lyn−/− mice.
PMCID: PMC3768150  PMID: 23169140
autoimmunity; Lyn; IL-21; IL-6; autoantibody; glomerulonephritis
17.  Glutathione S-transferase Mu 2-transduced mesenchymal stem cells ameliorated anti-glomerular basement membrane antibody-induced glomerulonephritis by inhibiting oxidation and inflammation 
Oxidative stress is implicated in tissue inflammation, and plays an important role in the pathogenesis of immune-mediated nephritis. Using the anti-glomerular basement membrane antibody-induced glomerulonephritis (anti-GBM-GN) mouse model, we found that increased expression of glutathione S-transferase Mu 2 (GSTM2) was related to reduced renal damage caused by anti-GBM antibodies. Furthermore, mesenchymal stem cell (MSC)-based therapy has shed light on the treatment of immune-mediated kidney diseases. The aim of this study was to investigate if MSCs could be utilized as vehicles to deliver the GSTM2 gene product into the kidney and to evaluate its potential therapeutic effect on anti-GBM-GN.
The human GSTM2 gene (hGSTM2) was transduced into mouse bone marrow-derived MSCs via a lentivirus vector to create a stable cell line (hGSTM2-MSC). The cultured hGSTM2-MSCs were treated with 0.5mM H2O2, and apoptotic cells were measured by terminal dUTP nick-end labeling (TUNEL) assay. The 129/svj mice, which were challenged with anti-GBM antibodies, were injected with 106 hGSTM2-MSCs via the tail vein. Expression of hGSTM2 and inflammatory cytokines in the kidney was assayed by quantitative PCR and western blotting. Renal function of mice was evaluated by monitoring proteinuria and levels of blood urea nitrogen (BUN), and renal pathological changes were analyzed by histochemistry. Immunohistochemical analysis was performed to measure inflammatory cell infiltration and renal cell apoptosis.
MSCs transduced with hGSTM2 exhibited similar growth and differentiation properties to MSCs. hGSTM2-MSCs persistently expressed hGSTM2 and resisted H2O2-induced apoptosis. Upon injection into 129/svj mice, hGSTM2-MSCs migrated to the kidney and expressed hGSTM2. The anti-GBM-GN mice treated with hGSTM2-MSCs exhibited reduced proteinuria and BUN (58% and 59% reduction, respectively) and ameliorated renal pathological damage, compared with control mice. Mice injected with hGSTM2-MSCs showed alleviated renal inflammatory cell infiltration and reduced expression of chemokine (C-C motif) ligand 2 (CCL2), interleukin (IL)-1β and IL-6 (53%, 46% and 52% reduction, respectively), compared with controls. Moreover, hGSTM2-MSCs increased expression of renal superoxide dismutase and catalase, which may associate with detoxifying reactive oxygen species to prevent oxidative renal damage.
Our data suggest that the enhanced protective effect of GSTM2-transduced MSCs against anti-GBM-GN might be associated with inhibition of oxidative stress-induced renal cell apoptosis and inflammation, through over-expression of hGSTM2 in mouse kidneys.
PMCID: PMC4055015  PMID: 24480247
18.  SNPs in VKORC1 are Risk Factors for Systemic Lupus Erythematosus in Asians 
Arthritis and rheumatism  2013;65(1):211-215.
The increased risk of thrombosis in systemic lupus erythematosus (SLE) may be partially explained by interrelated genetic pathways for thrombosis and SLE. In a case-control analysis, we investigated whether 33 established and novel single nucleotide polymorphisms (SNP) in 20 genes involved in hemostasis pathways that have been associated with deep venous thrombosis in the general population were risk factors for SLE development among Asians.
Patients in the discovery cohort were enrolled in one of two North American SLE cohorts. Patients in the replication cohort were enrolled in one of four Asian or two North American cohorts. SLE cases met American College of Rheumatology classification criteria. We first genotyped 263 Asian SLE and 357 healthy Asian control individuals for 33 SNPs using Luminex multiplex technology in the discovery phase, and then used Taqman and Immunochip assays to examine 5 SNPs in up to an additional 1496 cases and 993 controls in the Replication phase. SLE patients were compared to healthy controls for association with minor alleles in allelic models. Principal components analysis was used to control for intra-Asian ancestry in an analysis of the replication cohort.
Two genetic variants in the gene VKORC1, rs9934438 and rs9923231, were highly significant in both the discovery and replication cohorts: OR(disc) = 2.45 (p=2×10−9), OR(rep) = 1.53 (p=5×10−6) and OR(disc) = 2.40 (p=6×10−9), OR(rep) = 1.53 (p=5×10−6), respectively. These associations were significant in the replication cohort after adjustment for intra-Asian ancestry: rs9934438 OR(adj) = 1.34 (p=0.0029) and rs9923231 OR(adj) = 1.34 (p=0.0032).
Genetic variants in VKORC1, involved in vitamin K reduction and associated with DVT, are associated with SLE development in Asians. These results suggest intersecting genetic pathways for the development of SLE and thrombosis.
PMCID: PMC3670944  PMID: 23124848
systemic lupus erythematosus; single nucleotide polymorphisms; genetic risk factors
19.  The constant region contributes to the antigenic specificity and renal pathogenicity of murine anti-DNA antibodies 
Journal of autoimmunity  2012;39(4):398-411.
Affinity for DNA and cross-reactivity with renal antigens are associated with enhanced renal pathogenicity of lupus autoantibodies. In addition, certain IgG subclasses are enriched in nephritic kidneys, suggesting that isotype may determine the outcome of antibody binding to renal antigens. To investigate if the isotype of DNA antibodies affects renal pathogenicity by influencing antigen binding, we derived IgM, IgG1, IgG2b and IgG2a forms of the PL9–11 antibody (IgG3 anti-DNA) by in vitro class switching or PCR cloning. The affinity and specificity of PL9–11 antibodies for nuclear and renal antigens were analyzed using ELISA, Western blotting, surface plasmon resonance (SPR), binding to mesangial cells, and glomerular proteome arrays. Renal deposition and pathogenicity were assayed in mice injected with PL9–11 hybridomas. We found that PL9–11 and its isotype-switched variants had differential binding to DNA and chromatin (IgG3 > IgG2a > IgG1 > IgG2b > IgM) by direct and competition ELISA, and SPR. In contrast, in binding to laminin and collagen IV the IgG2a isotype actually had the highest affinity. Differences in affinity of PL9–11 antibodies for renal antigens were mirrored in analysis of specificity for glomeruli, and were associated with significant differences in renal pathogenicity in vivo and survival. Our novel findings indicate that the constant region plays an important role in the nephritogenicity of antibodies to DNA by affecting immunoglobulin affinity and specificity. Increased binding to multiple glomerular and/or nuclear antigens may contribute to the renal pathogenicity of anti-DNA antibodies of the IgG2a and IgG3 isotype. Finally, class switch recombination may be another mechanism by which B cell autoreactivity is generated.
PMCID: PMC3656415  PMID: 22841793
Systemic lupus erythematosus (SLE); Lupus nephritis; Anti-DNA antibodies; Isotype switching
20.  Molecular Basis of 9G4 B Cell Autoreactivity in Human Systemic Lupus Erythematosus 
The Journal of Immunology Author Choice  2013;191(10):4926-4939.
9G4+ IgG Abs expand in systemic lupus erythematosus (SLE) in a disease-specific fashion and react with different lupus Ags including B cell Ags and apoptotic cells. Their shared use of VH4-34 represents a unique system to understand the molecular basis of lupus autoreactivity. In this study, a large panel of recombinant 9G4+ mAbs from single naive and memory cells was generated and tested against B cells, apoptotic cells, and other Ags. Mutagenesis eliminated the framework-1 hydrophobic patch (HP) responsible for the 9G4 idiotype. The expression of the HP in unselected VH4-34 cells was assessed by deep sequencing. We found that 9G4 Abs recognize several Ags following two distinct structural patterns. B cell binding is dependent on the HP, whereas anti-nuclear Abs, apoptotic cells, and dsDNA binding are HP independent and correlate with positively charged H chain third CDR. The majority of mutated VH4-34 memory cells retain the HP, thereby suggesting selection by Ags that require this germline structure. Our findings show that the germline-encoded HP is compulsory for the anti–B cell reactivity largely associated with 9G4 Abs in SLE but is not required for reactivity against apoptotic cells, dsDNA, chromatin, anti-nuclear Abs, or cardiolipin. Given that the lupus memory compartment contains a majority of HP+ VH4-34 cells but decreased B cell reactivity, additional HP-dependent Ags must participate in the selection of this compartment. This study represents the first analysis, to our knowledge, of VH-restricted autoreactive B cells specifically expanded in SLE and provides the foundation to understand the antigenic forces at play in this disease.
PMCID: PMC3816606  PMID: 24108696
21.  Enoyl Coenzyme A Hydratase Domain–Containing 2, a Potential Novel Regulator of Myocardial Ischemia Injury 
We reported previously that Brown Norway (BN) rats are more resistant to myocardial ischemia/reperfusion (I/R) injury than are Dahl S (SS) rats. To identify the unique genes differentially expressed in the hearts of these rats, we used DNA microarray analysis and observed that enoyl coenzyme A hydratase–containing domain 2 (ECHDC2) is highly expressed (≈18‐fold) in the SS hearts compared with the BN hearts.
Methods and Results
RT‐PCR, Western blot, and immunohistochemistry analyses verified that ECHDC2 was highly expressed in SS hearts compared with the BN hearts. ECHDC2 gene locates at chromosome 5 of rat and is expressed in mitochondria of the heart, mainly in cardiomyocytes but not in cardiofibroblasts. Overexpression of ECHDC2 in cells increased susceptibility to I/R injury while knockdown of ECHDC2 enhanced resistance to I/R injury. Furthermore, we observed that left anterior descending coronary artery ligation–induced myocardial infarction was more severe in the SS hearts than in the BN hearts or SSBN5 hearts, which was built on SS rats but had the substitution of chromosome 5 from BN rats. We also demonstrated that ECHDC2 did not alter mitochondrial O2 consumption, metabolic intermediates and ATP production. By gas chromatography–mass spectrometry, we found that ECHDC2 overexpression increased the levels of the cellular branched chain amino acids leucine and valine.
ECHDC2, a mitochondrial protein, may be involved in regulating cell death and myocardial injury. Its deficiency in BN rats contributes to their increased resistance to myocardial I/R compared with SS rats. ECHDC2 increases branched chain amino acid metabolism and appears to be a novel regulator linking cell metabolism with cardiovascular disease.
PMCID: PMC3835224  PMID: 24108764
branched amino acid metabolism; cell death; ECHDC2; ischemia/reperfusion injury; myocardial infarction
22.  Kallikrein Transduced Mesenchymal Stem Cells Protect against Anti-GBM Disease and Lupus Nephritis by Ameliorating Inflammation and Oxidative Stress 
PLoS ONE  2013;8(7):e67790.
Previously we have shown that kallikreins (klks) play a renoprotective role in nephrotoxic serum induced nephritis. In this study, we have used mesenchymal stem cells (MSCs) as vehicles to deliver klks into the injured kidneys and have measured their therapeutic effect on experimental antibody induced nephritis and lupus nephritis. Human KLK-1 (hKLK1) gene was transduced into murine MSCs using a retroviral vector to generate a stable cell line, hKLK1-MSC, expressing high levels of hKLK1. 129/svj mice subjected to anti-GBM induced nephritis were transplanted with 106 hKLK1-MSCs and hKLK1 expression was confirmed in the kidneys. Compared with vector-MSCs injected mice, the hKLK1-MSCs treated mice showed significantly reduced proteinuria, blood urea nitrogen (BUN) and ameliorated renal pathology. Using the same strategy, we treated lupus-prone B6.Sle1.Sle3 bicongenic mice with hKLK1-MSCs and demonstrated that hKLK1-MSCs delivery also attenuated lupus nephritis. Mechanistically, hKLK1-MSCs reduced macrophage and T-lymphocyte infiltration into the kidney by suppressing the expression of inflammation cytokines. Moreover, hKLK1 transduced MSCs were more resistant to oxidative stress-induced apoptosis. These findings advance genetically modified MSCs as potential gene delivery tools for targeting therapeutic agents to the kidneys in order to modulate inflammation and oxidative stress in lupus nephritis.
PMCID: PMC3720854  PMID: 23935844
23.  Genome-wide association scan in women with systemic lupus erythematosus identifies susceptibility variants in ITGAM, PXK, KIAA1542 and other loci 
Nature genetics  2008;40(2):204-210.
Systemic lupus erythematosus (SLE) is a common systemic autoimmune disease with complex etiology but strong clustering in families (λS = ~30). We performed a genome-wide association scan using 317,501 SNPs in 720 women of European ancestry with SLE and in 2,337 controls, and we genotyped consistently associated SNPs in two additional independent sample sets totaling 1,846 affected women and 1,825 controls. Aside from the expected strong association between SLE and the HLA region on chromosome 6p21 and the previously confirmed non-HLA locus IRF5 on chromosome 7q32, we found evidence of association with replication (1.1 × 10−7 < Poverall < 1.6 × 10−23; odds ratio 0.82–1.62)in four regions: 16p11.2 (ITGAM), 11p15.5 (KIAA1542), 3p14.3 (PXK) and 1q25.1 (rs10798269). We also found evidence for association (P < 1 × 10−5) at FCGR2A, PTPN22 and STAT4, regions previously associated with SLE and other autoimmune diseases, as well as at ≥9 other loci (P < 2 × 10−7). Our results show that numerous genes, some with known immune-related functions, predispose to SLE.
PMCID: PMC3712260  PMID: 18204446
24.  SLE Peripheral Blood B Cell, T Cell and Myeloid Cell Transcriptomes Display Unique Profiles and Each Subset Contributes to the Interferon Signature 
PLoS ONE  2013;8(6):e67003.
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that is characterized by defective immune tolerance combined with immune cell hyperactivity resulting in the production of pathogenic autoantibodies. Previous gene expression studies employing whole blood or peripheral blood mononuclear cells (PBMC) have demonstrated that a majority of patients with active disease have increased expression of type I interferon (IFN) inducible transcripts known as the IFN signature. The goal of the current study was to assess the gene expression profiles of isolated leukocyte subsets obtained from SLE patients. Subsets including CD19+ B lymphocytes, CD3+CD4+ T lymphocytes and CD33+ myeloid cells were simultaneously sorted from PBMC. The SLE transcriptomes were assessed for differentially expressed genes as compared to healthy controls. SLE CD33+ myeloid cells exhibited the greatest number of differentially expressed genes at 208 transcripts, SLE B cells expressed 174 transcripts and SLE CD3+CD4+ T cells expressed 92 transcripts. Only 4.4% (21) of the 474 total transcripts, many associated with the IFN signature, were shared by all three subsets. Transcriptional profiles translated into increased protein expression for CD38, CD63, CD107a and CD169. Moreover, these studies demonstrated that both SLE lymphoid and myeloid subsets expressed elevated transcripts for cytosolic RNA and DNA sensors and downstream effectors mediating IFN and cytokine production. Prolonged upregulation of nucleic acid sensing pathways could modulate immune effector functions and initiate or contribute to the systemic inflammation observed in SLE.
PMCID: PMC3691135  PMID: 23826184
25.  Complete Genome Analysis of Three Acinetobacter baumannii Clinical Isolates in China for Insight into the Diversification of Drug Resistance Elements 
PLoS ONE  2013;8(6):e66584.
The emergence and rapid spreading of multidrug-resistant Acinetobacter baumannii strains has become a major health threat worldwide. To better understand the genetic recombination related with the acquisition of drug-resistant elements during bacterial infection, we performed complete genome analysis on three newly isolated multidrug-resistant A. baumannii strains from Beijing using next-generation sequencing technology.
Methodologies/Principal Findings
Whole genome comparison revealed that all 3 strains share some common drug resistant elements including carbapenem-resistant blaOXA-23 and tetracycline (tet) resistance islands, but the genome structures are diversified among strains. Various genomic islands intersperse on the genome with transposons and insertions, reflecting the recombination flexibility during the acquisition of the resistant elements. The blood-isolated BJAB07104 and ascites-isolated BJAB0868 exhibit high similarity on their genome structure with most of the global clone II strains, suggesting these two strains belong to the dominant outbreak strains prevalent worldwide. A large resistance island (RI) of about 121-kb, carrying a cluster of resistance-related genes, was inserted into the ATPase gene on BJAB07104 and BJAB0868 genomes. A 78-kb insertion element carrying tra-locus and blaOXA-23 island, can be either inserted into one of the tniB gene in the 121-kb RI on the chromosome, or transformed to conjugative plasmid in the two BJAB strains. The third strains of this study, BJAB0715, which was isolated from spinal fluid, exhibit much more divergence compared with above two strains. It harbors multiple drug-resistance elements including a truncated AbaR-22-like RI on its genome. One of the unique features of this strain is that it carries both blaOXA-23 and blaOXA-58 genes on its genome. Besides, an Acinetobacter lwoffii adeABC efflux element was found inserted into the ATPase position in BJAB0715.
Our comparative analysis on currently completed Acinetobacter baumannii genomes revealed extensive and dynamic genome organizations, which may facilitate the bacteria to acquire drug-resistance elements into their genomes.
PMCID: PMC3691203  PMID: 23826102

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