Blood leukocyte microarrays to diagnose systemic onset juvenile idiopathic arthritis and follow the response to IL-1 blockade

Systemic onset juvenile idiopathic arthritis (SoJIA) represents up to 20% of juvenile idiopathic arthritis. We recently reported that interleukin (IL) 1 is an important mediator of this disease and that IL-1 blockade induces clinical remission. However, lack of specificity of the initial systemic manifestations leads to delays in diagnosis and initiation of therapy. To develop a specific diagnostic test, we analyzed leukocyte gene expression profiles of 44 pediatric SoJIA patients, 94 pediatric patients with acute viral and bacterial infections, 38 pediatric patients with systemic lupus erythematosus (SLE), 6 patients with PAPA syndrome, and 39 healthy children. Statistical group comparison and class prediction identified genes differentially expressed in SoJIA patients compared with healthy children. These genes, however, were also changed in patients with acute infections and SLE. An analysis of significance across all diagnostic groups identified 88 SoJIA-specific genes, 12 of which accurately classified an independent set of SoJIA patients with systemic disease. Transcripts that changed significantly in patients undergoing IL-1 blockade were also identified. Thus, leukocyte transcriptional signatures can be used to distinguish SoJIA from other febrile illnesses and to assess response to therapy. Availability of early diagnostic markers may allow prompt initiation of therapy and prevention of disabilities

Novel animal models of acetylcholine receptor antibody-related myasthenia gravis.

Experimental autoimmune myasthenia gravis (EAMG) in mice has been used to unravel the pathogenic mechanisms and to be used as a preclinical model of myasthenia gravis (MG). Induction of predominantly ocular EAMG in HLA-DQ8 transgenic mice immunized with acetylcholine receptor (AChR) subunits demonstrated the importance of nonconformationally expressed AChR subunits in extraocular muscle involvement. Wild-type (WT) and CD4(+) T cell knockout (KO) C57BL/6 mice developed EAMG upon immunization with AChR in incomplete Freund's adjuvant plus lipopolysaccharide. AChR-specific IgG2(+) B cell frequencies, estimated by Alexa-conjugated AChR, and AChR-reactive IgG2b levels significantly correlated with the clinical grades of EAMG in WT mice. CD4(+) T cell-deficient EAMG mice exhibited AChR antibodies mainly of the IgG2b isotype, emphasizing T helper independent B cell activation pathways in EAMG induction. These novel EAMG models have suggested that diverse immunopathological mechanisms might contribute to EAMG or MG pathogenesis

CD4 costimulation is not required in a novel LPS-enhanced model of myasthenia gravis

The potential of lipopolysaccharide (LPS) to induce antigen-specific B cell responses to acetylcholine receptor (AChR) in myasthenia gravis (MG) was evaluated in wild type (WT) and CD4-/- C57BL/6 mice. The WT mice immunized with AChR in LPS developed an MG-like disease (LPS-EAMG) similar to that induced by immunization with AChR in complete Freund's adjuvant (CFA-EAMG). CD4-/- mice were resistant to CFA-EAMG but susceptible to LPS-EAMG. LPS abrogated EAMG resistance in CD4-/- mice by increasing high-affinity anti-AChR IgG2b in sera and enhancing immune complex deposition in muscle. (C) 2012 Elsevier B.V. All rights reserved

MRL Strains Have a BAFFR Mutation without Functional Consequence

<div><p>It has been shown that B cell activating factor receptor (BAFFR) is critical for B cell development and survival. In this study, we sought to evaluate the expression and function of BAFFR across multiple stains of mice that vary in their potential to develop systemic autoimmune disease. The inability of a commercial antibody to bind to BAFFR in the autoimmune prone mouse strains, MRL and MRL/Lpr led to the discovery of a mutation in <i>TNFRSF13C</i> gene (encoding BAFFR) that resulted in a Pro44Ser substitution in the N-terminus near the BAFF binding site in these strains. To define the biological consequences of mutant BAFFR, we compared the expression and activity of BAFFR in MRL and MRL/Lpr mice to BALB/c, which express the consensus version of <i>TNFRSF13C</i>. B cells from MRL and MRL/Lpr mice expressed mutant BAFFR on surface and were capable of responding to BAFF as exhibited by BAFF-mediated reduction in apoptosis and NF-κB2 activation. Signaling through MAPK ERK1/2 was not significantly induced by BAFF in MRL/Lpr mice; however, MAPK ERK1/2 signaling was intact in MRL mice. The inability of MRL/Lpr B cells to significantly activate ERK1/2 in response to BAFF was due to the high basal activity of the signaling pathway in these cells. In fact, basal activity of ERK1/2 in B cells correlated with the degree of autoimmune susceptibility exhibited by each strain. In addition, aged MRL/Lpr mice with severe autoimmune disease had high BAFF levels, low surface BAFFR, and high basal NF-κB2 activation, a pattern which is attributed to the high frequency of antibody secreting cells. We conclude that P44S BAFFR mutation does not hinder BAFFR function or enhance B cell activity in MRL/Lpr and MRL mice and that other susceptibility loci on the MRL background contributed to the hyperactivity of these cells.</p></div

Evaluation of B cell survival in 2 month old mice.

<p>(A) Flow cytometry sorted B cells were incubated for 2 days in culture with increasing concentrations of BAFF (0, 15, 75, or 375 ng/ml). Cell apoptosis was measured in flow cytometry by staining with propidium iodide (PI) and annexin V. Annexin V⁺PI^- cells are in early apoptosis, annexin V⁺PI⁺ are in late apoptosis, and annexin V^-PI^- are live cells. (A) Shown bi-exponential plots of PI vs annexin V are data from one representative experiment out of three. (B) Mean live cell frequency ± SD from three experiments are plotted. ** p < 0.01 and *** p < 0.001 indicate statistically significant differences between MRL/Lpr or MRL vs BALB/c, while # p < 0.05 and ### p < 0.001, indicates statistical differences within a strain induced by BAFF treatment.</p

Absolute B cell and B cell subset numbers in 5 month old mice.

<p>(A) Mean ± SD spleen weight for each strain is plotted (n = six mice per strain). (B) Graph of mean number ± SD of total splenocytes and (C) splenic B cells (CD19⁺) determined by flow cytometry (n = 6 mice per strain). (D) Splenic B cells (CD19⁺) were gated for B cell subsets T1, T2, T3, MZ, FO and AEC as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154518#pone.0154518.g002" target="_blank">Fig 2</a>. * p < 0.05, ** p < 0.01, and *** p < 0.001 indicate statistically significant differences between MRL/Lpr or MRL vs BALB/c, while †† p < 0.01, and ††† p < 0.001 indicate statistical differences between MRL and MRL/Lpr strains.</p

Absolute B cells numbers and the characterization of BAFFR expression on B cell subsets in 2 month old mice.

<p>(A) Graph of mean number ± SD of splenic B cells (CD19⁺) determined by flow cytometry (n = 6 mice per strain). (B) Concentrations of total IgM and IgG were determined by ELISA. Mean ± SD of sera IgM and IgG concentrations from 11 to15 female mice are shown. (C-D) B cells (CD19⁺) were gated for splenic B cell subsets based on CD93, IgM and IgD expression, using the previously described gating strategy published by Uslu and colleagues [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154518#pone.0154518.ref034" target="_blank">34</a>]. Transitional B cell populations are defined as T1 (CD93⁺IgM⁺IgD^-), T2 (CD93⁺IgM⁺IgD⁺), and T3 (CD93⁺IgM^-IgD^-). Mature B cell populations are defined as MZ (CD93^-IgM⁺IgD^-), FO (CD93^-IgM⁺IgD⁺), and AEC (CD93^-IgM^-IgD^-). (C) Graphs of mean number ± SD of splenic B subset from 6 mice per strain as determined by flow cytometry are shown. (D) MFI ± SD of BAFFR on B cells subsets was determined by flow cytometry. Data shown are from 6 mice per group. * p < 0.05, ** p < 0.01, and *** p < 0.001 indicate statistically significant differences between MRL/Lpr or MRL vs BALB/c, while † p < 0.05, †† p < 0.01, and ††† p < 0.001 indicate statistical differences between MRL and MRL/Lpr strains.</p

MRL/Lpr mice with SLE have elevated sera BAFF, elevated anti-dsDNA antibodies and kidney disease.

<p>(A) Mean ± SD of serum BAFF levels determined by ELISA. The data shown are from 5 to13 female mice per group. (B) Titers of anti-dsDNA IgG in serum are plotted. Sera from 2 and 5 month old mice were tested for anti-dsDNA IgG levels by ELISA. Titer is defined by the serum dilution giving an OD reading 2 times higher than background. Mean titers ± SEM from 3 to11 mice in each group were plotted. (C) Mean ± SD of proteinuria score is plotted. Approximate levels of protein levels in urine: score 0 = 0- trace, score 1 = 30 mg/dL, score 2 = 100 mg/dL, score 3 = 300 mg/dL, and score 4 = > 2000 mg/dL. The data shown are from 5 to13 female mice per group. * p < 0.05, ** p < 0.01, and *** p < 0.001 indicate statistically significant differences between MRL/Lpr or MRL vs BALB/c, while † p < 0.05, †† p < 0.01, and ††† p < 0.001 indicates statistical differences between MRL and MRL/Lpr strains.</p