Molecular mechanisms regulating pathogenic pathways in B cells and plasmacytoid dendritic cells in lupus

Systemic lupus erythematosus (SLE) is a prototype autoimmune disease characterized by autoantibody production, inflammation and end organ damage resulting from an overactivation of the immune system through mechanisms that are still not completely understood. One critical component of this pernicious cycle is a loss of tolerance to self-antigen that precedes disease manifestations. Importantly, there has only been one FDA approved treatment for SLE in the last 60 years, though an additional treatment has recently been approved for lupus nephritis. Therefore, there is a need to understand how pathogenic pathways are regulated in the context of SLE. This thesis focused on not only further understanding how interferon regulatory factor 5 (IRF5) drives pathogenicity in a mouse of model of lupus but also discerning the molecular pathways important for blood dendritic cell antigen 2 (BDCA2) inhibition of type I interferon (IFN-I) release from plasmacytoid dendritic cells (pDCs). Gain-of-function polymorphisms in the transcription factor IRF5 are associated with an increased risk of developing systemic lupus erythematosus. However, the IRF5-expressing cell type(s) responsible for lupus pathogenesis in vivo is not known. This work shows that monoallelic IRF5 deficiency in B cells markedly reduces disease in the FcγRIIB−/−Yaa mouse model of lupus. Mechanistically, B cell receptor and TLR7 signaling synergize to promote IRF5 phosphorylation through IRAK4, and also increase IRF5 protein expression, with these processes being independently regulated. This synergy increases B cell-intrinsic IL-6 and TNF- production, both key requirements for germinal center responses, with IL-6 and TNF- production in vitro and in vivo being substantially lower with loss of one allele of IRF5. Moreover, this thesis further strengthens the notion of IRF5 as a target for the treatment of SLE by therapeutically targeting IRF5 in the FcγRIIB−/−Yaa mouse lupus model. This work shows that tamoxifen driven deletion of IRF5 after disease associated manifestations have already developed confers protection in the FcγRIIB−/−Yaa mouse model. IFN-I is a family of pleotropic cytokines that are thought to be pathogenic in the context of SLE. PDCs are a major source of IFN-I. As such, they have been shown to play a major role in anti-viral immunity and are thought to be pathogenic in context of SLE. I used the anti-BDCA2 antibody 24F4A to ligate the receptor in a human pDC cell line (Gen 2.2) and applied a variety of proteomics methods, including profiling of post-translational modifications, to evaluate signaling downstream of BDCA2. I found that phosphorylation of phosphoinositide 3-kinase adapter protein 1 (PIK3AP1)/ B cell adaptor protein (BCAP) was increased after BDCA2 engagement by 24F4A. Deletion of BCAP from Gen2.2 cells reversed BDCA2 mediated AKT phosphorylation and IFN inhibition, suggesting that PI3 kinase (PI3K) may be important for BDCA2 mediated IFN inhibition. Treatment of human pDCs with PI3kinase inhibitor followed by TLR9 stimulation confirmed that PI3K activity is critical for BDCA2 mediated IFN inhibition. This work describes a critical threshold of IRF5 expression in B cells necessary for the development of the lupus like disease in the FcγRIIB−/−Yaa mice and that systemic deletion of IRF5 after disease onset reduces the disease manifestations in the same mouse model. Additionally, this work demonstrated that BCAP and PI3K are important pathways to mediate the IFN inhibition observed with engagement of the BDCA2 pathway. This work supports IRF5 as a therapeutic target in SLE and furthermore, describes a molecular pathway important for BDCA2 mediated IFN-I inhibition from pDCs