Mikhail Olferiev

Targeting of type I interferon in systemic autoimmune diseases

Increased blood levels of type I interferon (IFN-I) and expression of a broad signature of gene transcripts that reflect induction by IFN-I are observed in many patients with systemic autoimmune diseases, and that pattern is most striking in systemic lupus erythematosus (SLE). Persistent production of IFN-α, the most abundant subtype measured in these patients, is an important feature of the immunopathogenesis of lupus and has stimulated current efforts to develop and test therapeutics that either block IFN-I or its receptor directly or target components of the IFN-I pathway involved in induction of or response to IFN-I. In this review data from animal models of chronic viral infection, examples of lupus-like syndromes associated with single-gene mutations that impact the IFN-I pathway, and longitudinal studies of patients with lupus are described and support the rationale for therapeutic targeting of the IFN-I pathway. However, the complexity of IFN-I regulation and the diversity of its effects on immune system function suggest that the definitive demonstration of that pathway as a valid and productive therapeutic target will only come from clinical trials of agents tested in patients with systemic autoimmune disease, with patients with lupus likely to be the most informative.

Inhibitory Fc Gamma Receptors: From Gene to Disease

Multiple lines of evidence have revealed a key role for inhibitory Fc gamma receptors class IIb (FcγRIIb) as negative modulators of innate and adaptive immune responses. Acquired and genetic factors regulate the expression of FcγRIIb receptors and modify their inhibitory potential. Recent advances have highlighted the importance of FcγRIIb receptors in influencing the development of cancer and autoimmunity. The association of increased FcγRIIb expression with tumor development is believed to operate at effector cell level resulting in inhibition of antitumor cytotoxicity. In autoimmune diseases, FcγRIIb receptors play a major role in controlling the amplitude of antibody- and immune complex-mediated reactions. Generally, FcγRIIb deficiency is associated with increased susceptibility and severity to organ-specific and systemic autoimmunity. This article discusses the proposed mechanisms for FcγRIIb deregulation associated with malignant and autoimmune pathology in animal models and human diseases.

The Role of Activating Protein 1 in the Transcriptional Regulation of the Human FCGR2B Promoter Mediated by the -343 G → C Polymorphism Associated with Systemic Lupus Erythematosus

The inhibitory receptor FcγRIIb is a negative regulator of antibody production and inflammatory responses. The -343 G → C polymorphism in the human FCGR2B promoter is associated with systemic lupus erythematosus. The -343 C mutant promoter has decreased transcriptional activity. In the present study, we show that the transcriptional change correlates with quantitative differences in the interaction of the activating protein 1 complex with the mutant FCGR2B promoter. Promoter pulldown and chromatin immunoprecipitation assays demonstrated binding of c-Jun to the FCGR2B promoter. Phosphorylation of c-Jun was accompanied by transactivation of both FCGR2B promoter variants, whereas dephosphorylation of c-Jun by an inhibitor of c-Jun N-terminal kinase, markedly decreased the promoter activities. The -343 G → C substitution enabled the specific interaction of the transcription factor Yin-Yang 1 with the mutant FCGR2B promoter. Yin-Yang 1 competed with activating protein 1 for binding at the -343 site, and contributed to the repression of the mutant FCGR2B promoter activity. This mechanism could be responsible for the decreased expression of FcγRIIb associated with the -343 C/C homozygous FCGR2B genotype in lupus patients. These findings provide a rationale for the transcriptional defect mediated by the -343 C/C FCGR2B promoter polymorphism associated with systemic lupus erythematosus, and add to our understanding of the complex transcriptional regulation of the human FCGR2B promoter.

Novel molecular signatures in mononuclear cell populations from patients with systemic lupus erythematosus

To gain novel insights into the immunopathogenesis of systemic lupus erythematosus we have analyzed gene expression data from isolated CD4+ T cells, CD8+ T cells, CD19+ B cells, and CD56+ NK-cell enriched peripheral blood cell fractions from patients and healthy donors. As predicted, type I interferon-inducible gene transcripts are overexpressed in all populations. Transcripts preferentially expressed in SLE CD4+ and CD8+ T cells include those associated with Tregulatory and Th17 effector cell programs, respectively, but in each case additional transcripts predicted to limit differentiation of those effector cells are detected. Evidence for involvement of the Wnt/β-catenin pathway was observed in both B and T cell fractions, and novel transcripts were identified in each cell population. These data point to disrupted T effector cell differentiation and the Wnt/β-catenin pathway as contributors to immune dysfunction in SLE while further supporting a central role for the type I interferon pathway in lupus.

Type I Interferons in Autoimmune Disease

Type I interferons, which make up the first cytokine family to be described and are the essential mediators of antivirus host defense, have emerged as central elements in the immunopathology of systemic autoimmune diseases, with systemic lupus erythematosus as the prototype. Lessons from investigation of interferon regulation following virus infection can be applied to lupus, with the conclusion that sustained production of type I interferon shifts nearly all components of the immune system toward pathologic functions that result in tissue damage and disease. We review recent data, mainly from studies of patients with systemic lupus erythematosus, that provide new insights into the mechanisms of induction and the immunologic consequences of chronic activation of the type I interferon pathway. Current concepts implicate endogenous nucleic acids, driving both cytosolic sensors and endosomal Toll-like receptors, in interferon pathway activation and suggest targets for development of novel therapeutics that may restore the immune system to health.

BD-07 Gene transcripts expressed in peripheral blood of patients with lupus nephritis identify novel mechanisms and therapeutic targets

Background Lupus nephritis affects more than one-half of patients with SLE and is the most common serious manifestation of the disease. Lupus nephritis is more common in Hispanic and African-American patients than in those of European ancestry, and class III and IV nephritis progresses to end-stage renal disease in 10%–15% of patients within 15 years of diagnosis. Identification of markers and mechanisms of lupus nephritis could provide new approaches to predict and treat disease. Methods To identify blood cell transcriptome biomarkers that differentiate renal and non-renal disease we performed RNA sequencing on peripheral blood samples from 15 patients with lupus nephritis and 14 patients with non-nephritis manifestations of SLE (samples represented each patient during flaring and quiescent disease states) and from 5 healthy donors. To relate gene expression to activity of nephritis, 216 longitudinal samples from 30 patients with lupus nephritis covering a median time frame of 28 months were analyzed using the Illumina HT-V4 Bead array. Serum albumin levels were documented at the time of each visit. Results Principal component analysis of RNA sequencing data clearly differentiated SLE patients with nephritis from those without nephritis, and linear models for microarray (limma) analysis identified 153 gene transcripts differentially expressed between the two patient groups (fold change >1.5; p<0.05). U1 and U3 RNA transcripts were increased in lupus nephritis samples, and the most highly expressed transcript based on fold change was TREML4, encoding a protein previously identified as amplifying TLR7 signaling and promoting type I interferon production. Analysis of longitudinal microarray data in relation to serum albumin identified 120 transcripts. Those most significantly correlated with lupus nephritis activity were pituitary tumor-transforming gene 1 (PTTG1), recently identified as polymorphic and associated with SLE, uridine cytosine kinase 2 (UCK2), thioredoxin (TXN), and RNASE2. Expression of PTTG1 fluctuated over time, with elevated levels preceding the time of peak renal disease activity. Conclusions Spliceosome-associated RNAs and TREML4, a TLR7-associated gene product, may represent biomarkers of lupus nephritis, and PTTG1, the product of a lupus-associated gene reported to be involved in epithelial-mesenchymal transition, may be a novel therapeutic target associated with active nephritis. These studies provide a rich data set stimulating new understanding of mechanisms contributing to lupus nephritis. Acknowledgements This work was supported by the Lupus Research Alliance, Pfizer-Centers for Therapeutic Innovation, and the Emerald Foundation.

II-19 Elevated plasma cell-free mitochondrial DNA defines a subgroup of lupus patients with membranous lupus nephritis

Background Systemic lupus erythematosus (SLE) is an autoimmune disease with protean manifestations, characterised by production of antibodies against nucleic acids and upregulation of type I interferon-inducible genes in a majority of SLE patients. The principal drivers of this interferon signature are still not well understood. Recent work has shown that oxidised mitochondrial DNA released by neutrophils can stimulate plasmacytoid dendritic cells to produce interferon-α.1 We hypothesised that cell-free mitochondrial DNA might contribute to type I interferon production in SLE, and we sought to examine whether cell-free mtDNA levels were increased in SLE patients relative to controls, or during disease flares. Materials and methods A retrospective analysis was performed using banked plasma samples from 164 patients in our SLE cohort along with 57 banked plasma samples from healthy donors. DNA was isolated from the plasma samples and real-time quantitative PCR was performed, amplifying a target sequence in the mitochondrially-encoded gene NADH dehydrogenase I, as previously published.2 In-depth clinical phenotyping of SLE patients in the cohort was performed and used to define subgroups of SLE patients, as well as the specific disease manifestations present during flares. Results No significant difference was seen in cell-free mtDNA in plasma from SLE patients versus healthy donors (HD – 3060.3 copies/uL, N = 57, SLE - 3845.5 copies/uL, N = 164, p = 0.22). However, cell-free mtDNA levels were elevated in a subset of SLE patients with a history of membranous lupus nephritis, including those with a component of proliferative nephritis (WHO class V/III+V/IV+V), relative to patients with proliferative nephritis alone (WHO class III or class IV) (5313.9 copies/uL, N = 34 vs. 2062.5 copies/uL, N = 17, p = 0.02). A subset of 70 patients had multiple samples collected at visits before, during, and after flares of disease activity. Cell-free mtDNA levels rose at the peak of disease activity as assessed by SLEDAI score in 11/16 flares of class V/III+V/IV+V nephritis (p = 0.04), while it only did so in 4/11 of the remaining nephritis flares. In contrast, cell-free mtDNA rose at the peak of disease activity in only 4/20 flares where alopecia was present (p = 0.02). Conclusions Cell-free mtDNA levels are elevated in a subset of SLE patients with a history of membranous nephritis, and were more likely to rise during flares of membranous nephritis versus other types of disease flares. Acknowledgements Supported by a Lupus and Antiphospholipid Centre of Excellence Research Fund Award References Lood C, et al. Neutrophil extracellular traps enriched in oxidised mitochondrial DNA are interferogenic and contribute to lupus-like disease. Nat Med advance on, 2016 Nakahira K, et al. Circulating mitochondrial DNA in patients in the ICU as a marker of mortality: derivation and validation. PLoS Med 2013;10:e1001577; discussion e1001577