Project Description TNFα/TNFR has received the greatest attention because of its position at the apex of the pro-inflammatory cy- tokine cascade, and its dominance in the pathogenesis of inflammation. TNFR1 primarily mediates inflammato- ry activity of TNFα, whereas TNFR2 plays a protective and anti-inflammatory role in various diseases. Our ge- netic screen for the binding partners of progranulin (PGRN) growth factor led to the isolation of TNFR2 as the PGRN-binding receptor (Tang, et al, Science, 2011). Remarkably, PGRN exhibits an approximately 600-fold higher binding affinity to TNFR2 than does TNFα. During the initial funding period, we have successfully identi- fied in a proteomics screen 14-3-3ε, an important intracellular signaling molecule, as a novel component of TNFR2 complexes in response to PGRN stimulation. PGRN deficiency promoted inflammatory classically- activated macrophage (CAM) but inhibited anti-inflammatory alternatively-activated macrophage (AAM) polari- zation, whereas recombinant PGRN inhibited AAM-CAM but stimulated CAM-AAM switching. In addition, knockout of 14-3-3ε abolished PGRN’s regulation of macrophage. More excitingly, our direct protein-protein interaction screen of nine central members of the complement system identified C5a as a novel binding partner of PGRN with high binding affinity, and PGRN inhibited the binding of C5a to C5aR1. Thus, the scientific prem- ise of this competitive continued application is based on 1) the identification of 14-3-3ε as a novel component of PGRN/TNFR2 pathway, and 2) the isolation of C5a as a novel PGRN-binding factor. The central hypothesis is that PGRN regulates macrophage polarization and inflammatory arthritis through a) recruitment of 14-3-3ε to TNFR2 and activation of the PGRN/TNFR2 anti-inflammatory pathway; and b) interplay with C5a and inhibition of C5a/C5aR1 inflammatory pathway. The Specific Aims are: (1) To elucidate 14-3-3ε’s role in PGRN/TNFR2- mediated macrophage polarization in the course of inflammatory arthritis. We will determine the dependence on 14-3-3ε of PGRN's regulation of macrophage polarization (SA#1.1); the signaling, target genes and co- factor(s) of 14-3-3ε that mediate PGRN action in macrophage polarization (SA#1.2); and the importance of PGRN regulation of macrophage phenotypic switch in the context of inflammatory arthritis and its dependence on 14-3-3ε (SA#1.3). (2) To define the importance of PGRN/C5a interplay in the pathogenesis of inflammatory arthritis. We will characterize and dissect the PGRN/C5a interaction and to identify PGRN-derived minimal fragment/peptide that retains C5a binding activity (SA#2.1); elucidate the interplay between PGRN and C5a in regulating macrophage polarization, as well as the signaling pathways involved (SA#2.2); and determine the interplay between PGRN and C5a/C5aR1 signaling and the contribution of inhibition of C5a/C5aR1 signaling by PGRN to PGRN's anti-inflammatory action in inflammatory arthritis (SA#2.3). Th...