Indu Raman

Whole-genome transcription and DNA methylation analysis of peripheral blood mononuclear cells identified aberrant gene regulation pathways in systemic lupus erythematosus

BackgroundRecent achievement in genetics and epigenetics has led to the exploration of the pathogenesis of systemic lupus erythematosus (SLE). Identification of differentially expressed genes and their regulatory mechanism(s) at whole-genome level will provide a comprehensive understanding of the development of SLE and its devastating complications, lupus nephritis (LN).MethodsWe performed whole-genome transcription and DNA methylation analysis in PBMC of 30 SLE patients, including 15 with LN (SLE LN+) and 15 without LN (SLE LN−), and 25 normal controls (NC) using HumanHT-12 Beadchips and Illumina Human Methy450 chips. The serum proinflammatory cytokines were quantified using Bio-plex Human Cytokine 27-plex assay. Differentially expressed genes and differentially methylated CpG were analyzed with GenomeStudio, R, and SAM software. The association between DNA methylation and gene expression were tested. Gene interaction pathways of the differentially expressed genes were analyzed by IPA software.ResultsWe identified 552 upregulated genes and 550 downregulated genes in PBMC of SLE. Integration of DNA methylation and gene expression profiling showed that 334 upregulated genes were hypomethylated, and 479 downregulated genes were hypermethylated. Pathway analysis on the differential genes in SLE revealed significant enrichment in interferon (IFN) signaling and toll-like receptor (TLR) signaling pathways. Nine IFN- and seven TLR-related genes were identified and displayed step-wise increase in SLE LN− and SLE LN+. Hypomethylated CpG sites were detected on these genes. The gene expressions for MX1, GPR84, and E2F2 were increased in SLE LN+ as compared to SLE LN− patients. The serum levels of inflammatory cytokines, including IL17A, IP-10, bFGF, TNF-α, IL-6, IL-15, GM-CSF, IL-1RA, IL-5, and IL-12p70, were significantly elevated in SLE compared with NC. The levels of IL-15 and IL1RA correlated with their mRNA expression. The upregulation of IL-15 may be regulated by hypomethylated CpG sites in the promotor region of the gene.ConclusionsOur study has demonstrated that significant number of differential genes in SLE were involved in IFN, TLR signaling pathways, and inflammatory cytokines. The enrichment of differential genes has been associated with aberrant DNA methylation, which may be relevant to the pathogenesis of SLE. Our observations have laid the groundwork for further diagnostic and mechanistic studies of SLE and LN.

Kallikrein Transduced Mesenchymal Stem Cells Protect against Anti-GBM Disease and Lupus Nephritis by Ameliorating Inflammation and Oxidative Stress

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.

Glutathione S-transferase Mu 2-transduced mesenchymal stem cells ameliorated anti-glomerular basement membrane antibody-induced glomerulonephritis by inhibiting oxidation and inflammation.

IntroductionOxidative 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.MethodsThe 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.ResultsMSCs 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.ConclusionsOur 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.

Inducible expression of kallikrein in renal tubular cells protects mice against spontaneous lupus nephritis

OBJECTIVE To ascertain whether engineered expression of kallikreins within the kidneys, using an inducible Cre/loxP system, can ameliorate murine lupus nephritis. METHODS In mice with a lupus-prone genetic background, we engineered the expression of tamoxifen-inducible Cre recombinase under the control of a kidney-specific promoter whose activation initiates murine kallikrein-1 expression within the kidneys. These transgenic mice were injected with either tamoxifen or vehicle at age 2 months and then were monitored for 8 months for kallikrein expression and disease. RESULTS Elevated expression of kallikrein was detected in the kidney and urine of tamoxifen-injected mice but not in controls. At age 10 months, all vehicle-injected mice developed severe lupus nephritis, as evidenced by increased proteinuria (mean ± SD 13.43 ± 5.65 mg/24 hours), increased blood urea nitrogen (BUN) and serum creatinine levels (39.86 ± 13.45 mg/dl and 15.23 ± 6.89 mg/dl, respectively), and severe renal pathology. In contrast, the tamoxifen-injected mice showed significantly reduced proteinuria (6.6 ± 4.12 mg/24 hours), decreased BUN and serum creatinine levels (15.71 ± 8.17 mg/dl and 6.64 ± 3.39 mg/dl, respectively), and milder renal pathology. Tamoxifen-induced up-regulation of renal kallikrein expression increased nitric oxide production and dampened renal superoxide production and inflammatory cell infiltration, alluding to some of the pathways through which kallikreins may be operating within the kidneys. CONCLUSION Local expression of kallikreins within the kidney has the capacity to dampen lupus nephritis, possibly by modulating inflammation and oxidative stress.

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.

Objective 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. Methods 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. Results 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. Conclusion OXR1-MSCs transplantation may exert a certain protective effect on nephritis by suppressing inflammation and oxidative stress.

Clinical and Immunologic Profiles in Incomplete Lupus Erythematosus and Improvement with Hydroxychloroquine Treatment

Objective. The study goals were to evaluate performance of SLE classification criteria, to define patients with incomplete lupus erythematosus (ILE), and to probe for features in these patients that might be useful as indicators of disease status and hydroxychloroquine response. Methods. Patients with ILE (N = 70) and SLE (N = 32) defined by the 1997 American College of Rheumatology criteria were reclassified using the 2012 Systemic Lupus International Collaborating Clinics criteria. Disease activity, patient reported outcomes, and levels of Type I interferon- (IFN-) inducible genes, autoantibodies, and cytokines were measured. Subgroups treated with hydroxychloroquine (HCQ) were compared to patients not on this drug. Results. The classification sets were correlated (R2 = 0.87). ILE patients were older (P = 0.0043) with lower disease activity scores (P < 0.001) and greater dissatisfaction with health status (P = 0.034) than SLE patients. ILE was associated with lower levels of macrophage-derived cytokines and levels of expressed Type I IFN-inducible genes. Treatment of ILE with HCQ was associated with better self-reported health status scores and lower expression levels of Type I IFN-inducible genes than ILE patients not on HCQ. Conclusion. The 2012 SLICC SLE classification criteria will be useful to define ILE in trials. Patients with ILE have better health status and immune profiles when treated with HCQ.

Hyperactivated PI3Kδ promotes self and commensal reactivity at the expense of optimal humoral immunity

Gain-of-function mutations in the gene encoding the phosphatidylinositol-3-OH kinase catalytic subunit p110δ (PI3Kδ) result in a human primary immunodeficiency characterized by lymphoproliferation, respiratory infections and inefficient responses to vaccines. However, what promotes these immunological disturbances at the cellular and molecular level remains unknown. We generated a mouse model that recapitulated major features of this disease and used this model and patient samples to probe how hyperactive PI3Kδ fosters aberrant humoral immunity. We found that mutant PI3Kδ led to co-stimulatory receptor ICOS–independent increases in the abundance of follicular helper T cells (TFH cells) and germinal-center (GC) B cells, disorganized GCs and poor class-switched antigen-specific responses to immunization, associated with altered regulation of the transcription factor FOXO1 and pro-apoptotic and anti-apoptotic members of the BCL-2 family. Notably, aberrant responses were accompanied by increased reactivity to gut bacteria and a broad increase in autoantibodies that were dependent on stimulation by commensal microbes. Our findings suggest that proper regulation of PI3Kδ is critical for ensuring optimal host-protective humoral immunity despite tonic stimulation from the commensal microbiome.Patients who express a hyperactive mutant of the kinase PI3K exhibit defective humoral immunity. Preite et al. show that overactive PI3K leads to defective class-switched antigen-specific responses to immunization, despite augmented germinal-center formation and reactivity to commensal microbes and self antigens.

Analysis of IgM antibody production and repertoire in a mouse model of Sjögren’s syndrome

This study tested the hypothesis that B cells from salivary tissue are distinct in terms of proliferative capacity, immunoglobulin M secretion, repertoire, and autoantibody enrichment in Sjögrens syndrome. We sorted purified B cells from the spleen, cervical lymph nodes, and submandibular glands of a primary Sjögrens syndrome mouse model (Id3−/−). Enzyme‐linked immunospot and proliferation assays were performed with stimulated B cells. We single‐cell sorted B cells from the spleen, cervical lymph nodes, and submandibular gland tissue from Sjögrens syndrome mice and sequenced immunoglobulin M heavy‐chain variable regions. Finally, autoantigen arrays were performed using immunoglobulin M derived from sera, cervical lymph nodes, spleens, and submandibular gland tissue of Id3−/− animals. Results suggest B cells from salivary tissue of Sjögrens syndrome mice are similar to those from secondary immune sites in terms of proliferative and secretory capacity. However, differences in repertoire usage, heavy chain complementarity‐determining region 3 length, mutational frequency, and N region addition were observed among B cells derived from submandibular gland, cervical lymph node, and spleen tissue. Moreover, autoantigen array data show immunoglobulin M from salivary B cells have enriched specificity for Ro (Sjögrens syndrome A) and La (Sjögrens syndrome B). All together, these data suggest salivary B cells have unique repertoire characteristics that likely influence autoantigen binding and contribute to Sjögrens syndrome disease in a tissue‐specific manner.

CE-12 Comparison of classification criteria, self-assessments and immunologic profiles in patients with incomplete and systemic lupus erythematosus

Background The syndrome of incomplete lupus erythematosus (ILE) likely includes individuals at risk for development of systemic lupus erythematosus (SLE). Studies of interventions to lower risk or prevent further disease in ILE are of interest. Design of such trials will require methods to classify patients and to assess risk. The goals of the present study were to evaluate performance of updated SLE classification criteria to define ILE and to probe for other features in these patients that might be useful as indicators of disease status. A long term goal is to develop prognostic multifaceted risk profiles that would have clinical applications. Materials and methods Patients with ILE (N = 70) and SLE (N = 32) defined by the 1997 American College of Rheumatology (ACR) criteria were then reclassified using the 2012 Systemic Lupus International Collaborating Clinics (SLICC) criteria. Disease activity, patient self-assessments and levels of autoantibodies, soluble mediators and expressed Type I interferon (IFN) genes also were measured in the ILE and SLE patients and compared to healthy control (HC) individuals. Results The two sets of classification criteria were highly correlated (Figure 1; R2 = 0.87). ILE patients were older (P = 0.0043), with lower SLEDAI scores (P = 0.023) and greater dissatisfaction with their health status (P = 0.034) than SLE patients. Anti-C1q and sCD27 levels were correlated with ACR criteria and SLE Disease Activity Index (SLEDAI) scores (P ≤ 0.0004). Three cytokines, IL-7, IL12p70 and IL-13, were lower in both ILE and SLE than in HCs. Two IFN-related cytokines IP = 10 and MCP-1, were higher in SLE than in ILE. Of three IFN genes measured, IFI27 showed the greatest difference between ILE and SLE. Conclusions The 2012 SLICC SLE classification criteria likely can be used to define ILE in future research trials. Patients with ILE are somewhat dissatisfied with their condition, possibly related to anxiety about the lack of a clear diagnosis. Further patient-reported outcome studies in this population would be of interest. Reliable assessment of lupus risk will likely include demographic, clinical and immunologic features. Some of the latter may suggest novel approaches to early treatment. Abstract CE-12 Figure 1 Correlation between two SLE classification criteria, the 1997 ACR and 2012 SLICC sets, in 102 patients with either ILE or SLE. Values on each axis correspond to numbers of criteria in each of the sets. Significance determined using Pearson’s R. Acknowledgements This project was funded, in part, with a grant from the Pennsylvania Department of Health using Tobacco CURE Funds. The Department specifically disclaims responsibility for any analyses, interpretations or conclusions. It was also?supported in part by the National Institutes of Health, NIAMS U34 AR067392. The data entry assistance of Fan He is appreciated.