Identification of novel non-myelin biomarkers in multiple sclerosis using an improved phage-display approach.

Although the etiology of multiple sclerosis is not yet understood, it is accepted that its pathogenesis involves both autoimmune and neurodegenerative processes, in which the role of autoreactive T-cells has been elucidated. Instead, the contribution of humoral response is still unclear, even if the presence of intrathecal antibodies and B-cells follicle-like structures in meninges of patients has been demonstrated. Several myelin and non-myelin antigens have been identified, but none has been validated as humoral biomarker. In particular autoantibodies against myelin proteins have been found also in healthy individuals, whereas non-myelin antigens have been implicated in neurodegenerative phase of the disease. To provide further putative autoantigens of multiple sclerosis, we investigated the antigen specificity of immunoglobulins present both in sera and in cerebrospinal fluid of patients using phage display technology in a new improved format. A human brain cDNA phage display library was constructed and enriched for open-read-frame fragments. This library was selected against pooled and purified immunoglobulins from cerebrospinal fluid and sera of multiple sclerosis patients. The antigen library was also screened against an antibody scFv library obtained from RNA of B cells purified from the cerebrospinal fluid of two relapsing remitting patients. From all biopanning a complex of 14 antigens were identified; in particular, one of these antigens, corresponding to DDX24 protein, was present in all selections. The ability of more frequently isolated antigens to discriminate between sera from patients with multiple sclerosis or other neurological diseases was investigated. The more promising novel candidate autoantigens were DDX24 and TCERG1. Both are implicated in RNA modification and regulation which can be altered in neurodegenerative processes. Therefore, we propose that they could be a marker of a particular disease activity state.

B cell OX40L supports T follicular helper cell development and contributes to SLE pathogenesis.

OBJECTIVES: TNFSF4 (encodes OX40L) is a susceptibility locus for systemic lupus erythematosus (SLE). Risk alleles increase TNFSF4 expression in cell lines, but the mechanism linking this effect to disease is unclear, and the OX40L-expressing cell types mediating the risk are not clearly established. Blockade of OX40L has been demonstrated to reduce disease severity in several models of autoimmunity, but not in SLE. We sought to investigate its potential therapeutic role in lupus. METHODS: We used a conditional knockout mouse system to investigate the function of OX40L on B and T lymphocytes in systemic autoimmunity. RESULTS: Physiologically, OX40L on both B and T cells contributed to the humoral immune response, but B cell OX40L supported the secondary humoral response and antibody affinity maturation. Our data also indicated that loss of B cell OX40L impeded the generation of splenic T follicular helper cells. We further show that in two models of SLE-a spontaneous congenic model and the H2-IAbm12 graft-versus-host-induced model-loss of B cell OX40L ameliorates the autoimmune phenotype. This improvement was, in each case, accompanied by a decline in T follicular helper cell numbers. Importantly, the germline knockout did not exhibit a markedly different phenotype from the B cell knockout in these models. CONCLUSIONS: These findings contribute to a model in which genetically determined increased OX40L expression promotes human SLE by several mechanisms, contingent on its cellular expression. The improvement in pathology in two models of systemic autoimmunity indicates that OX40L is an excellent therapeutic target in SLE.

Dysregulated CD46 shedding interferes with Th1-contraction in systemic lupus erythematosus.

IFN-γ-producing T helper 1 (Th1) cell responses mediate protection against infections but uncontrolled Th1 activity also contributes to a broad range of autoimmune diseases. Autocrine complement activation has recently emerged as key in the induction and contraction of human Th1 immunity: activation of the complement regulator CD46 and the C3aR expressed by CD4+ T cells via autocrine generated ligands C3b and C3a, respectively, are critical to IFN-γ production. Further, CD46-mediated signals also induce co-expression of immunosuppressive IL-10 in Th1 cells and transition into a (self)-regulating and contracting phase. In consequence, C3 or CD46-deficient patients suffer from recurrent infections while dysregulation of CD46 signaling contributes to Th1 hyperactivity in rheumatoid arthritis and multiple sclerosis. Here, we report a defect in CD46-regulated Th1 contraction in patients with systemic lupus erythematosus (SLE). We observed that MMP-9-mediated increased shedding of soluble CD46 by Th1 cells was associated with this defect and that inhibition of MMP-9 activity normalized release of soluble CD46 and restored Th1 contraction in patients' T cells. These data may deliver the first mechanistic explanation for the increased serum CD46 levels observed in SLE patients and indicate that targeting CD46-cleaving proteases could be a novel avenue to modulate Th1 responses.

Mapping eQTLs with RNA-seq reveals novel susceptibility genes, non-coding RNAs and alternative-splicing events in systemic lupus erythematosus.

Studies attempting to functionally interpret complex-disease susceptibility loci by GWAS and eQTL integration have predominantly employed microarrays to quantify gene-expression. RNA-Seq has the potential to discover a more comprehensive set of eQTLs and illuminate the underlying molecular consequence. We examine the functional outcome of 39 variants associated with Systemic Lupus Erythematosus (SLE) through the integration of GWAS and eQTL data from the TwinsUK microarray and RNA-Seq cohort in lymphoblastoid cell lines. We use conditional analysis and a Bayesian colocalisation method to provide evidence of a shared causal-variant, then compare the ability of each quantification type to detect disease relevant eQTLs and eGenes. We discovered the greatest frequency of candidate-causal eQTLs using exon-level RNA-Seq, and identified novel SLE susceptibility genes (e.g. NADSYN1 and TCF7) that were concealed using microarrays, including four non-coding RNAs. Many of these eQTLs were found to influence the expression of several genes, supporting the notion that risk haplotypes may harbour multiple functional effects. Novel SLE associated splicing events were identified in the T-reg restricted transcription factor, IKZF2, and other candidate genes (e.g. WDFY4) through asQTL mapping using the Geuvadis cohort. We have significantly increased our understanding of the genetic control of gene-expression in SLE by maximising the leverage of RNA-Seq and performing integrative GWAS-eQTL analysis against gene, exon, and splice-junction quantifications. We conclude that to better understand the true functional consequence of regulatory variants, quantification by RNA-Seq should be performed at the exon-level as a minimum, and run in parallel with gene and splice-junction level quantification.

Autophagy is activated in systemic lupus erythematosus and required for plasmablast development.

BACKGROUND: Autophagy has emerged as a critical homeostatic mechanism in T lymphocytes, influencing proliferation and differentiation. Autophagy in B cells has been less studied, but genetic deficiency causes impairment of early and late developmental stages OBJECTIVES: To explore the role of autophagy in the pathogenesis of human and murine lupus, a disease in which B cells are critical effectors of pathology. METHODS: Autophagy was assessed using multiple techniques in NZB/W and control mice, and in patients with systemic lupus erythematosus (SLE) compared to healthy controls. We evaluated the phenotype of the B cell compartment in Vav-Atg7(-/-) mice in vivo, and examined human and murine plasmablast formation following inhibition of autophagy. RESULTS: We found activation of autophagy in early developmental and transitional stages of B cell development in a lupus mouse model even before disease onset, and which progressively increased with age. In human disease, again autophagy was activated compared with healthy controls, principally in naïve B cells. B cells isolated from Vav-Atg7(F/F) mice failed to effectively differentiate into plasma cells following stimulation in vitro. Similarly, human B cells stimulated in the presence of autophagy inhibition did not differentiate into plasmablasts. CONCLUSIONS: Our data suggest activation of autophagy is a mechanism for survival of autoreactive B cells, and also demonstrate that it is required for plasmablast differentiation, processes that induce significant cellular stress. The implication of autophagy in two major pathogenic pathways in SLE suggests the potential to use inhibition of autophagy as a novel treatment target in this frequently severe autoimmune disease.

Phagocytosis is the main CR3-mediated function affected by the lupus-associated variant of CD11b in human myeloid cells.

The CD11b/CD18 integrin (complement receptor 3, CR3) is a surface receptor on monocytes, neutrophils, macrophages and dendritic cells that plays a crucial role in several immunological processes including leukocyte extravasation and phagocytosis. The minor allele of a non-synonymous CR3 polymorphism (rs1143679, conversation of arginine to histidine at position 77: R77H) represents one of the strongest genetic risk factor in human systemic lupus erythematosus, with heterozygosity (77R/H) being the most common disease associated genotype. Homozygosity for the 77H allele has been reported to reduce adhesion and phagocytosis in human monocytes and monocyte-derived macrophages, respectively, without affecting surface expression of CD11b. Herein we comprehensively assessed the influence of R77H on different CR3-mediated activities in monocytes, neutrophils, macrophages and dendritic cells. R77H did not alter surface expression of CD11b including its active form in any of these cell types. Using two different iC3b-coated targets we found that the uptake by heterozygous 77R/H macrophages, monocytes and neutrophils was significantly reduced compared to 77R/R cells. Allele-specific transduced immortalized macrophage cell lines demonstrated that the minor allele, 77H, was responsible for the impaired phagocytosis. R77H did not affect neutrophil adhesion, neutrophil transmigration in vivo or Toll-like receptor 7/8-mediated cytokine release by monocytes or dendritic cells with or without CR3 pre-engagement by iC3b-coated targets. Our findings demonstrate that the reduction in CR3-mediated phagocytosis associated with the 77H CD11b variant is not macrophage-restricted but demonstrable in other CR3-expressing professional phagocytic cells. The association between 77H and susceptibility to systemic lupus erythematosus most likely relates to impaired waste disposal, a key component of lupus pathogenesis.

Treatment of experimental arthritis by targeting synovial endothelium with a neutralizing recombinant antibody to C5.

OBJECTIVE: To show that a new recombinant protein (MT07) obtained by fusing a synovial-homing peptide to a neutralizing antibody to C5 can be selectively delivered to inflamed synovium and can effectively control joint inflammation in experimental models of arthritis. METHODS: Binding of MT07 to human, rat, and mouse synovial tissue was evaluated in vitro by immunofluorescence, and selective localization in the inflamed joints of rats was documented in vivo using time-domain optical imaging. The antiinflammatory effect of MT07 was tested in a rat model of antigen-induced arthritis (AIA) and in a mouse model of collagen antibody-induced arthritis (CAIA). RESULTS: MT07 was able to bind to samples of inflamed synovium from humans, mice, and rats while failing to recognize uninflamed synovium as well as inflamed mouse lung or rat kidney. In vivo analysis of the biodistribution of MT07 confirmed its preferential homing to inflamed joints, with negligible inhibition of circulating C5 levels. MT07 prevented and resolved established inflammation in a rat model of AIA, as demonstrated by changes in joint swelling, polymorphonuclear cell counts in synovial washes, release of interleukin-6 and tumor necrosis factor α, and tissue damage. A similar therapeutic effect was obtained testing MT07 in a CAIA model. CONCLUSION: Our findings show that the novel recombinant molecule MT07 has the unique ability to selectively target inflamed joints and to exert local control of the inflammatory process by neutralizing the complement system without interfering with circulating C5 levels. We believe that this approach can be extended to other antiinflammatory drugs currently used to treat patients with rheumatoid arthritis.

In vivo functional analysis and genetic modification of in vitro-derived mouse neutrophils.

Mature neutrophils are notoriously short-lived immune cells that cannot be genetically manipulated. Analysis of gene function therefore requires genetically modified animals, which is expensive, time-consuming, and costly in animal life. Analysis of gene function in neutrophils in a physiologically relevant context thus represents a significant problem in the field. We sought to overcome this obstruction in the field by developing a strategy for the analysis of gene function in neutrophils in a physiologically relevant context. Here, we demonstrate the functional relevance of in vitro conditional-Hoxb8 immortalized precursor-derived neutrophils. In vitro-derived neutrophils functionally resembled primary neutrophils, but critically, neutrophils generated in this way can be adoptively transferred into live animals and tracked during inflammatory responses using single-cell analysis to define functional attributes. We have validated this approach using CD11b-deficient neutrophils and replicated the key findings observed in gene-targeted animals and in naturally CD11b-deficient humans. Furthermore, we show that by retroviral transduction, one can generate stable alterations in the precursor cell lines and thus a continuous supply of functionally altered neutrophils. This novel technological advance offers for the first time the possibility of applying higher-throughput genetic modification and in vivo functional analysis to the neutrophil-lineage.

The alternative pathway is critical for pathogenic complement activation in endotoxin- and diet-induced atherosclerosis in low-density lipoprotein receptor-deficient mice.

BACKGROUND: The early components of the classical and lectin complement pathways have been shown to protect low-density lipoprotein receptor-deficient mice (Ldlr(-/-)) from early atherogenesis. However, the role of the alternative pathway remained unknown, and that was investigated in this study. METHODS AND RESULTS: Mice lacking factor B (Bf(-/-)), the initiator of the alternative pathway, were crossed with Ldlr(-/-) mice and studied under different proatherogenic conditions. There was no statistically significant difference in lipid profiles or atherosclerotic lesion development between Bf(-/-)/Ldlr(-/-) and Ldlr(-/-) mice fed a low-fat diet. However, in these groups, administration of bacterial lipopolysaccharide led to a significant increase in atherosclerosis only in Ldlr(-/-) and not in Bf(-/-)/Ldlr(-/-) mice, indicating that the alternative pathway is necessary for endotoxin-mediated atherogenesis. Bf(-/-)/Ldlr(-/-) mice also had significantly decreased cross-sectional aortic root lesion fraction area and reduced lesion complexity compared with Ldlr(-/-) animals after a 12-week period of high-fat diet, although this was also accompanied by reduced levels of serum cholesterol. Under both experimental conditions, the atherosclerotic changes in the Bf(-/-)/Ldlr(-/-) mice were accompanied by a marked reduction in complement activation in the circulation and in atherosclerotic plaques, with no statistically significant differences in immunoglobulin G deposition or in the serum antibody response to oxidized low-density lipoprotein. CONCLUSIONS: These data demonstrate that amplification of complement activation by the alternative pathway in response to lipopolysaccharide or high-fat diet plays a proatherogenic role.