Gary S. Gilkeson

Genome-wide association scan in women with systemic lupus erythematosus identifies susceptibility variants in ITGAM , PXK , KIAA1542 and other loci

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.

Mycophenolate Mofetil versus Cyclophosphamide for Induction Treatment of Lupus Nephritis

Recent studies have suggested that mycophenolate mofetil (MMF) may offer advantages over intravenous cyclophosphamide (IVC) for the treatment of lupus nephritis, but these therapies have not been compared in an international randomized, controlled trial. Here, we report the comparison of MMF and IVC as induction treatment for active lupus nephritis in a multinational, two-phase (induction and maintenance) study. We randomly assigned 370 patients with classes III through V lupus nephritis to open-label MMF (target dosage 3 g/d) or IVC (0.5 to 1.0 g/m(2) in monthly pulses) in a 24-wk induction study. Both groups received prednisone, tapered from a maximum starting dosage of 60 mg/d. The primary end point was a prespecified decrease in urine protein/creatinine ratio and stabilization or improvement in serum creatinine. Secondary end points included complete renal remission, systemic disease activity and damage, and safety. Overall, we did not detect a significantly different response rate between the two groups: 104 (56.2%) of 185 patients responded to MMF compared with 98 (53.0%) of 185 to IVC. Secondary end points were also similar between treatment groups. There were nine deaths in the MMF group and five in the IVC group. We did not detect significant differences between the MMF and IVC groups with regard to rates of adverse events, serious adverse events, or infections. Although most patients in both treatment groups experienced clinical improvement, the study did not meet its primary objective of showing that MMF was superior to IVC as induction treatment for lupus nephritis.

A Pathogenetic Role for TNFα in the Syndrome of Cachexia, Arthritis, and Autoimmunity Resulting from Tristetraprolin (TTP) Deficiency

Tristetraprolin (TTP) is a widely expressed potential transcription factor that contains two unusual CCCH zinc fingers and is encoded by the immediate-early response gene, Zfp-36. Mice made deficient in TTP by gene targeting appeared normal at birth, but soon manifested marked medullary and extramedullary myeloid hyperplasia associated with cachexia, erosive arthritis, dermatitis, conjunctivitis, glomerular mesangial thickening, and high titers of anti-DNA and antinuclear antibodies. Myeloid progenitors from these mice showed no increase in sensitivity to growth factors. Treatment of young TTP-deficient mice with antibodies to tumor necrosis factor alpha (TNF alpha) prevented the development of essentially all aspects of the phenotype. These results indicate a role for TTP in regulating TNF alpha synthesis, secretion, turnover, or action. TTP-deficient mice may serve as useful models of the autoimmune inflammatory state resulting from chronic effective TNF alpha excess.

Histone deacetylase inhibitors modulate renal disease in the MRL-lpr/lpr mouse

Studies in human systemic lupus erythematosus (SLE) suggest a possible role for histone deacetylases (HDACs) in skewed gene expression and disease pathogenesis. We used the MRL-lpr/lpr murine model of lupus to demonstrate that HDACs play a key role in the heightened levels of both Th1 and Th2 cytokine expression that contribute to disease. The availability of specific HDAC inhibitors (HDIs) such as trichostatin A (TSA) and suberonylanilide hydroxamic acid (SAHA) permits the study of the role of HDACs in gene regulation. Our results indicate that HDIs downregulate IL-12, IFN-gamma, IL-6, and IL-10 mRNA and protein levels in MRL-lpr/lpr splenocytes. This effect on gene transcription is associated with an increased accumulation of acetylated histones H3 and H4 in total cellular chromatin. To elucidate the in vivo effects of TSA on lupuslike disease, we treated MRL-lpr/lpr mice with TSA (0.5 mg/kg/d) for 5 weeks. Compared with vehicle-treated control mice, TSA-treated mice exhibited a significant reduction in proteinuria, glomerulonephritis, and spleen weight. Taken together, these findings suggest that increased expression of HDACs leading to an altered state of histone acetylation may be of pathologic significance in MRL-lpr/lpr mice. In addition, TSA or other HDIs may have therapeutic benefit in the treatment of SLE.

Umbilical Cord Mesenchymal Stem Cell Transplantation in Severe and Refractory Systemic Lupus Erythematosus

OBJECTIVE Umbilical cord (UC)-derived mesenchymal stem cells (MSCs) have shown marked therapeutic effects in a number of diseases in animal studies, based on their potential for self-renewal and differentiation. No data are available on the effectiveness of UC MSC transplantation (MSCT) in human autoimmune disease. This study was undertaken to assess the efficacy and safety of allogeneic UC MSCT in patients with severe and treatment-refractory systemic lupus erythematosus (SLE). METHODS We conducted a single-arm trial that involved 16 SLE patients whose disease was refractory to standard treatment or who had life-threatening visceral involvement. All of the patients gave consent and underwent UC MSCT. Clinical changes were evaluated before and after transplantation using the SLE Disease Activity Index (SLEDAI), measurement of serum antinuclear antibody (ANA), anti-double-stranded DNA (anti-dsDNA) antibody, serum complement C3 and C4, and albumin levels, and assessment of and renal function. Evaluation of potential mechanisms of MSCT effects focused on the percentage of peripheral blood Treg cells and serum levels of cytokines. RESULTS From April 2007 to July 2009, a total of 16 patients with active SLE were enrolled and underwent UC MSCT. The median followup time after MSCT was 8.25 months (range 3-28 months). Significant improvements in the SLEDAI score, levels of serum ANA, anti-dsDNA antibody, serum albumin, and complement C3, and renal function were observed. Clinical remission was accompanied by an increase in peripheral Treg cells and a re-established balance between Th1- and Th2-related cytokines. Significant reduction in disease activity was achieved in all patients, and there has been no recurrence to date and no treatment-related deaths. CONCLUSION Our findings indicate that UC MSCT results in amelioration of disease activity, serologic changes, and stabilization of proinflammatory cytokines. These data provide a foundation for conducting a randomized controlled trial of this new therapy for severe and treatment-refractory SLE.

Allogenic mesenchymal stem cells transplantation in refractory systemic lupus erythematosus: a pilot clinical study

Objective To determine the safety and efficacy of allogeneic mesenchymal stem cell transplantation (MSCT) in refractory systemic lupus erythematosus (SLE). Methods A total of 15 patients with persistently active SLE underwent MSCT. Outcome was evaluated by changes in the SLE disease activity index (SLEDAI), serological features (anti-nuclear antibodies and anti-double-stranded DNA (anti-dsDNA)), renal function and percentage of peripheral blood regulatory T cells. Results From 11 March 2007 to 4 November 2008, 15 patients with persistently active SLE were enrolled and underwent MSCT. The mean follow-up period was 17.2±9.5 months. A total of 13 patients have been followed for more than 12 months. All patients clinically improved following treatment with mesenchymal stem cells with a marked decrease in the SLEDAI score and 24 h proteinuria. At 12-month follow-up, SLEDAI scores decreased from 12.2±3.3 to 3.2±2.8 and proteinuria decreased from 2505.0±1323.9 to 858.0±800.7 mg/24 h (all p<0.05, by paired t test, n=12). At 1-year follow-up in 13 patients, 2 had a relapse of proteinuria, while the other 11 continue to have decreased disease activity on minimal treatment. Anti-dsDNA levels decreased. Improvement in glomerular filtration rate was noted in two patients in which formal testing was performed. Non-renal-related manifestations also improved significantly. No serious adverse events were reported. Conclusion Allogeneic MSCT in patients with refractory lupus resulted in amelioration of disease activity, improvement in serological markers and stabilisation of renal function. MSCT appears beneficial in treatment of patients with SLE refractory to conventional treatment options.

A nonsynonymous functional variant in integrin-|[alpha]|M (encoded by ITGAM) is associated with systemic lupus erythematosus

We identified and replicated an association between ITGAM (CD11b) at 16p11.2 and risk of systemic lupus erythematosus (SLE) in 3,818 individuals of European descent. The strongest association was at a nonsynonymous SNP, rs1143679 (P = 1.7 × 10−17, odds ratio = 1.78). We further replicated this association in two independent samples of individuals of African descent (P = 0.0002 and 0.003; overall meta-analysis P = 6.9 × 10−22). The genetic association between ITGAM and SLE implicates the αMβ2-integrin adhesion pathway in disease development.

Lack of a Functional Alternative Complement Pathway Ameliorates Ischemic Acute Renal Failure in Mice

Ischemia/reperfusion (I/R) injury of the kidney is a common cause of acute renal failure (ARF) and is associated with high morbidity and mortality in the intensive care unit. The mechanisms underlying I/R injury are complex. Studies have shown that complement activation contributes to the pathogenesis of I/R injury in the kidney, but the exact mechanisms of complement activation have not been defined. We hypothesized that complement activation in this setting occurs via the alternative pathway and that mice deficient in complement factor B, an essential component of the alternative pathway, would be protected from ischemic ARF. Wild-type mice suffered from a decline in renal function and had significant tubular injury, particularly in the outer medulla, after I/R. We found that factor B-deficient mice (fB−/−) developed substantially less functional and morphologic renal injury after I/R. Furthermore, control wild-type mice had an increase in tubulointerstitial complement C3 deposition and neutrophil infiltration in the outer medulla after I/R, whereas fB−/− mice demonstrated virtually no C3 deposition or neutrophil infiltration. Our results demonstrate that complement activation in the kidney after I/R occurs exclusively via the alternative pathway, and that selective inhibition of this pathway provides protection to the kidneys from ischemic ARF.

Identification of IRAK1 as a risk gene with critical role in the pathogenesis of systemic lupus erythematosus

A combined forward and reverse genetic approach was undertaken to test the candidacy of IRAK1 (interleukin-1 receptor associated kinase-1) as an X chromosome-encoded risk factor for systemic lupus erythematosus (SLE). In studying ≈5,000 subjects and healthy controls, 5 SNPs spanning the IRAK1 gene showed disease association (P values reaching 10−10, odds ratio >1.5) in both adult- and childhood-onset SLE, in 4 different ethnic groups, with a 4 SNP haplotype (GGGG) being strongly associated with the disease. The functional role of IRAK1 was next examined by using congenic mouse models bearing the disease loci: Sle1 or Sle3. IRAK1 deficiency abrogated all lupus-associated phenotypes, including IgM and IgG autoantibodies, lymphocytic activation, and renal disease in both models. In addition, the absence of IRAK1 reversed the dendritic cell “hyperactivity” associated with Sle3. Collectively, the forward genetic studies in human SLE and the mechanistic studies in mouse models establish IRAK1 as a disease gene in lupus, capable of modulating at least 2 key checkpoints in disease development. This demonstration of an X chromosome gene as a disease susceptibility factor in human SLE raises the possibility that the gender difference in SLE may in part be attributed to sex chromosome genes.

Induction of cross-reactive anti-dsDNA antibodies in preautoimmune NZB/NZW mice by immunization with bacterial DNA.

To investigate the role of antigen drive in anti-double-stranded (ds) DNA production, the antibody response induced in lupus-prone NZB/NZW mice by E. coli (EC) dsDNA was evaluated. Preautoimmune NZB/NZW female mice were immunized with complexes of EC dsDNA with methylated bovine serum albumin (mBSA) in complete Freunds adjuvant; control mice received either mBSA complexes with calf thymus (CT) dsDNA or mBSA alone in adjuvant. IgG antibody responses were assessed by ELISA. Similar to normal mice, immunized NZB/NZW mice produced significant levels of anti-dsDNA when measured with EC dsDNA as antigen. Whereas normal mice produce antibodies which are specific for the immunizing bacterial DNA, NZB/NZW mice produced antibodies that bound crossreactively to CT dsDNA by ELISA. Furthermore, the induced antibodies resembled lupus anti-DNA in their fine specificity for polynucleotide antigens and reactivity with Crithidia luciliae DNA. Despite their response to EC dsDNA, NZB/NZW mice immunized with CT dsDNA failed to generate significant anti-dsDNA responses. These results provide further evidence for the enhanced immunogenicity of bacterial DNA and suggest that immune cell abnormalities in NZB/NZW mice promote the generation of crossreactive autoantibody responses when confronted with a foreign DNA.