´╗┐Proceedings of the National Academy of Sciences of the United States of America 113, 2218C2222, doi:10

´╗┐Proceedings of the National Academy of Sciences of the United States of America 113, 2218C2222, doi:10.1073/pnas.1600511113 (2016). would worsen inflammatory arthritis. Surprisingly, associated with arthritis display increased migratory capacity, whereas knockdown reduces human neutrophil migration to chemokines linked to arthritis. These data identify non-canonical roles for ELMO1 as an important cytoplasmic regulator of specific neutrophil receptors and promoter of arthritis. Rheumatoid arthritis (RA) affects millions of people worldwide with reduced quality of life and economic costs. RA is characterized by chronic inflammation and progressive joint destruction, with debilitating consequences1. A hallmark of human RA and mouse models of arthritis is the leukocyte influx into the joint synovium, with neutrophils being the most abundant2. Activated neutrophils promote chronic inflammation along with matrix and cartilage degradation2. Although effective therapies have been introduced, a significant fraction of RA patients are refractory to existing therapies3. Genome-wide association studies (GWAS) have identified many genetic loci; however, most of them are single nucleotide polymorphisms (SNPs) in non-coding genetic regions4 with no obvious causality. Therefore, a better understanding of causative and disease contributing factors is needed. AC-5216 (Emapunil) Cell death via apoptosis occurs during homeostasis and tissue inflammation5. While apoptotic cells have been detected in Keratin 18 (phospho-Ser33) antibody the synovial joints of RA patients6, resistance to apoptosis has also been implied as a contributory factor to chronic disease; therefore, induction of apoptosis has been proposed as a therapeutic avenue7. For these approaches, however, apoptotic cell clearance pathways also need to be considered. Inefficient clearance of apoptotic cells can result in secondary necrosis, and exposure of self-antigens, and cell clearance defects are linked to chronic inflammation and autoimmunity8. Apoptotic cells expose eat me signals on their surface that are recognized by specific receptors on phagocytes9C11. Binding of apoptotic cells to phagocyte recognition receptors results in activation of the engulfment machinery, dynamic changes of the actin cytoskeleton, and corpse uptake9C11. Phagocyte receptors can bind phosphatidylserine exposed on the apoptotic cell surface directly (such as TIM-412 and BAI113) or indirectly, through bridging molecules (such as MerTK14), or recognize cell surface modifications or opsonins bound to apoptotic cells15. The receptor redundancy and the specific signaling pathways downstream of these engulfment receptors are unclear16. One of the better characterized cytoplasmic signaling relays (in both professional and non-professional phagocytes) is the ELMO-DOCK-Rac signaling pathway16. In this mode of signaling, the ELMO-DOCK protein complex acts as a guanine nucleotide exchange factor (GEF) to activate the small GTPase Rac, leading to cytoskeletal rearrangements needed for engulfment17. In this work, we examined how components of a specific engulfment pathway may link to inflammatory arthritis. AC-5216 (Emapunil) AC-5216 (Emapunil) Surprisingly, loss of the engulfment signaling protein ELMO1 alleviated disease severity in mouse models of arthritis through ELMO1 regulation of neutrophil recruitment to inflamed joints. Via proteomic and transcriptomic approaches, we uncover an ELMO1-dependent signature in neutrophils and identify a requirement for ELMO1 in signaling downstream of the receptors for arthritis-associated molecules C5a and LTB4. These data suggest a neutrophil-specific ELMO1-dependent signaling nexus that controls different aspects of arthritis. RESULTS Engulfment protein ELMO1 is associated with arthritis To test whether specific engulfment machinery components are associated with human rheumatoid arthritis, we searched publicly available databases for SNPs. We found multiple SNP-Disease associations with human rheumatoid arthritis in and genes (see Methods; Fig. 1a and Supplementary Table 1). In a meta-analysis for common SNPs or gene linkages to both RA and celiac disease (CD), a SNP in human (rs11984075) was discovered18. A previous approach assessing the methylation status of arthritis associated genes also reported that locus was hypomethylated in fibroblast-like synoviocytes (FLS) that line the synovium of the joints19. ELMO1 functions at the interface between the phagocytic receptors and their downstream cytoplasmic signaling activity, leading to corpse internalization13,17,20,21. As apoptotic cell clearance is an anti-inflammatory process, we hypothesized that disruption of ELMO1 might lead to greater joint inflammation. Open in a separate window Fig. 1. Engulfment protein ELMO1 AC-5216 (Emapunil) contributes to inflammatory arthritis.a) Disease SNPs in Rheumatoid Arthritis discovered via search of the GWASdb SNP-disease association database. The data are plotted using AC-5216 (Emapunil) a standardized value. b) Expression of in total paw extracts from K/BxN mice.

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