Project description:Microglia, the brain’s resident macrophages, can be reconstituted by surrogate cells - a process termed “microglia replacement.” To expand the microglia replacement toolkit, we here introduce estrogen-regulated (ER) homeobox B8 (Hoxb8) conditionally immortalized macrophages, a cell model for generation of immune cells from murine bone marrow, as a versatile model for microglia replacement. We find that ER-Hoxb8 macrophages are highly comparable to primary bone marrow-derived (BMD) macrophages in vitro, and, when transplanted into a microglia-free brain, engraft the parenchyma and differentiate into microglia-like cells. Furthermore, ER-Hoxb8 progenitors are readily transducible by virus and easily stored as stable, genetically manipulated cell lines. As a demonstration of this system’s power for studying the effects of disease mutations on microglia in vivo, we created stable, Adar1-mutated ER-Hoxb8 lines using CRISPR-Cas9 to study the intrinsic contribution of macrophages to Aicardi-Goutières Syndrome (AGS), an inherited interferonopathy that primarily affects the brain and immune system. We find that Adar1 knockout elicited interferon secretion and impaired macrophage production in vitro, while preventing brain macrophage engraftment in vivo - phenotypes that can be rescued with concurrent mutation of Ifih1 (MDA5) in vitro, but not in vivo. Lastly, we extended these findings by generating ER-Hoxb8 progenitors from mice harboring a patient-specific Adar1 mutation (D1113H). We demonstrated the ability of microglia-specific D1113H mutation to drive interferon production in vivo, suggesting microglia drive AGS neuropathology. In sum, we introduce the ER-Hoxb8 approach to model microglia replacement and use it to clarify macrophage contributions to AGS.
Project description:The impact of LPS and LTA stimulation on differentiated bone marrow and Yolk sac Hoxb8 macrophages in comparison to untreated control cells was studied by global protein profiling using a bottom-up approach.
Project description:We recently identified CCDC134 as a novel regulator of TLR responses. To gain a comprehensive understanding of the effects of CCDC134 loss, we conducted total proteome analysis on control sgRen and sgCCDC134 Hoxb8 immortalized murine myeloid progenitors that were differentiated into macrophages. Interestingly, we observed that plasma membrane and endolysosomal TLRs, along with their associated chaperone Gp96, were among the most significantly downregulated proteins. Our findings further demonstrated that CCDC134 is essential for the proper folding and stability of Gp96. As a result, the deletion of CCDC134 in various cell lines and Hoxb8-derived macrophages results in altered TLR folding and trafficking.
Project description:Progenitor cells of yolk sac and bone marrow origin were transduced with an estrogen receptor Hoxb8 fusion protein to generate stable cell lines. Macrophages were differentiated with M-CSF in the absence of estrogen, and then stimulated with IL-4 or LPS. Hoxb8 progenitor cells and differentiated macrophages were analyzed by RNA sequencing.
Project description:Investigation of immune cell differentiation and function is limited by shortcomings of suitable and scalable experimental systems. Although forced expression of certain Hox genes allows immortalization of hematopoietic progenitor cells, their differentiation potential is limited to select myeloid lineages. Here we show that an estrogen-regulated form of Hoxb8 that is retrovirally delivered into bone marrow cells can be used along with FLT3 ligand to conditionally immortalize early hematopoietic progenitor cells (Hoxb8-FL). Hoxb8-FL cells have lost self-renewal capacity and the ability to adopt megakaryocyte/ erythroid lineage fates, but sustain myeloid and lymphoid differentiation potential. Hoxb8-FL cells differentiate in vitro and in vivo into different myeloid and lymphoid cell types, including macrophages, granulocytes, dendritic cells and B- and T-lymphocytes, which are phenotypically and functionally indistinguishable from their primary counterparts. Given the simplicity to generate Hoxb8-FL cells and their unlimited proliferative capacity, this system provides unique opportunities to investigate cell differentiation and immune cell functions. Hoxb8 expressing immortalized cells
