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2.1 1.0 mg/ml, p<0.05) and IgG2a anti-chromatin autoantibodies (Fig 4B). autoimmune syndromes. == Introduction == Type I IFNs, particularly the IFN-s and IFN-, have received prominent attention for their role in the pathogenesis of systemic lupus erythematosus (SLE) and other autoimmune and inflammatory syndromes (1,2). By signaling through a common receptor (IFNAR), AC-42 these pleiotropic cytokines affect almost every aspect of innate and adaptive immune responses, including upregulation of MHC and costimulatory molecules, and production of B cell survival factors (BAFF, April) by antigen-presenting cells, culminating in the engagement and expansion of autoreactive T and B cells (1,2). Of particular relevance to lupus pathogenesis is the induction of type I IFNs under sterile conditions through the engagement of endosomal Toll-like receptors (TLRs) by self-nucleic acids (36). This systemic autoimmunity-inducing pathway has been well documented by studies showing reduced disease in predisposed mice lacking expression of endosomal TLRs (7), IFNAR (8,9), or Unc93b1 (10), a molecule that acts as a transporter of TLRs 3, 7 and 9 from ER to endolysosomes. These findings have AC-42 stimulated considerable interest in creating treatments based on blocking reagents against either the multiple IFN-s and the single IFN-, or their common receptor. The potential utility of these approaches would be considerably advanced by further defining the role of type I AC-42 IFNs in lupus mice with diverse genetic abnormalities, the potential difference in pathogenicity between the IFN- subtypes and IFN-, and the clinical stage where blockade of signaling by these cytokines is effective. Here, we address some of these issues and demonstrate that the disease-promoting effect of type I IFNs in lupus is primarily mediated by the IFN-s, type I IFN signaling significantly contributes to disease in BXSB mice but minimally in MRL-Faslprmice, treatment with an anti-IFNAR antibody has therapeutic efficacy even with partial IFNAR blockade, and effectiveness is most evident when treatment is initiated at early disease stages. These findings provide support for the potential utility of IFNAR blockade for the treatment of human SLE, but suggest that the type of patient and timing of treatment may be crucial factors in determining the outcome. == Materials and Methods == == Mice == BXSB.Yaa, MRL-Faslpr, and C57BL/6 (B6) mice were obtained from The Jackson Laboratory (Bar Harbor, ME) or The Scripps Research Institute Animal Facility. NZB mice deficient for IFNAR1 (Ifnar1/) have been reported (8), and marker-assisted congenic NZB mice deficient for IFN- (Ifnb/) were generated as described (8). Mice were housed under specific pathogen-free Rabbit polyclonal to SP1 conditions and all experimental protocols were performed according to the NIH Guide for the Care and Use of Laboratory Animals and approved by The Scripps Research Institute Animal Care and Use Committee. == Treatment with a monoclonal Anti-IFNAR Antibody == Male BXSB mice were treated with a monoclonal anti-IFNAR antibody of mouse origin (clone MAR1-5A3, Leinco Technologies) (11). Injections (i.p., 500 g for 3 consecutive days, followed by 250 g three times per week until experiment termination) were started either before (12 weeks of age) or after (17 weeks of age) appearance of disease manifestations, as suggested by detectable autoantibody titers and proteinuria. MRL-Faslprmice were similarly treated, but starting at 7 wks of age due to the expedited disease course in this strain. == Cell Preparations == Single cell suspensions were prepared from bone marrow (BM), blood, peritoneal cavity, spleen and lymph nodes (LN, inguinal, axillary, brachial, cervical), as described (12). B cells were purified from spleen or peritoneal cavity using magnetic beads (MACS, Miltenyi Biotec), while conventional DCs (cDCs) and plasmacytoid DCs (pDCs) were prepared by stimulating BM cells with recombinant mouse GM-CSF or Flt3 ligand (R&D Systems), respectively (13). == Flow Cytometry == Monoclonal antibodies to mouse CD4, CD8, B220, CD11b, CD11c, PDCA-1, IFNAR1, CD69, CD86, CD25, CD21, CD23, AA4.1, CD138, I-Ab, H2-Kb, and GR-1 were obtained from BD Pharmingen, Biolegend or eBioscience. For surface staining, cells were sequentially incubated with various combinations of antibodies or streptavidin (BD Pharmingen). Cell events were acquired on four-color FACSCalibur, and data analyzed using FlowJo software (Tree Star). == In Vitro Studies == Purified splenic B cells and BM-derived cDCs and pDCs were cultured in complete medium and stimulated or not with mouse IFN-11 (1000 U/ml, Miltenyi Biotec), the TLR7 ligand R848 (30 ng/ml, InvivoGen), or both, in.