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Abstract

Immune-mediated inflammatory diseases (IMIDs) are characterized by chronic non￾resolving inflammation leading to progressive tissue damage. IMIDs are commonly accompanied by diverse comorbidities and include a large set of diseases, such as systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis. Current therapies cannot cure IMIDs and primarily rely on suppressing autoimmune reactions and inflammation. The diseases commonly flare upon therapy cessation, indicating a need for lifelong anti￾inflammatory and immunosuppressive therapies. Thus, developing drugs that enhance the pro￾resolution in IMIDs could facilitate remission maintenance and boost tissue protection. Subsets of the tissue-resident macrophages (TRM) were shown to drive the pro-resolution and tissue￾protective programs during tissue inflammation. However, the inaccessibility of primary TRM from human tissues/organs affected by IMIDs, calls for the development of in vitro macrophage models that resemble the phenotypes and functions of protective TRM subsets. Thus, using a TRM in vitro model combined with CRISPR-Cas9 and tool molecules could help to identify molecular targets driving TRM differentiation/function Thereby accelerating the discovery of remission-promoting therapeutics in IMIDs. To study TRM differentiation, I implemented and further fine-tuned different iPSC-derived TRM-like models and an infiltrating macrophage model, including iMACs, iMicroglia and iMonocytes. I characterized these models for their yield, viability, target phenotypic features, functions and robustness. My experiments hinted that the iMonocyte protocol may be more dependent on iPSC lines used in differentiation. iMAC and iMicroglia protocols provided a good yield (3.5-9.5*106 and 3.5-4.7*106 per plate, respectively) and viability (92-96% and 94- 97%, respectively). Moreover, iMACs can be harvested multiple times from one differentiation run, while maintaining a similar phenotype. Furthermore, I assessed iMACs and iMicroglia for their amenability for a candidate drug screen and a pooled CRISPR-Cas9 knockout screen with a customized gRNA library of ~700 gene targets. I tested doxycycline-inducible and stably expressed Cas9. My experiments showed a difficulty in random Cas9 integration under antibiotic resistance and inferred an advantage in using Cas9 co-expressed with fluorescent protein. Moreover, iMAC model turned out to be difficult to use with the CRISPR-Cas9 system, but iMicroglia with shorter differentiation performed better. However, iMACs remained a highly reproducible and robust model that can be incorporated into TRM in vitro studies using tool molecules. Based on these results, I performed drug perturbation on iMACs and CRISPR experiments on iMicroglia. The differentiation CRISPR-Cas9 screen at the time of the thesis submission was submitted for sequencing and is yet to elucidate genes involved in TRM differentiation/function in iMicroglia. Additionally, I tested the effects of known (Cytochalasin D) and candidate (splicing inhibitors) drugs on iMAC surface marker phenotype and efferocytosis (apoptotic cell clearance). Splicing inhibitors Herboxidiene (GEX1A) and Pladienolide B (PladB) targeting SF3B1 subunit of the spliceosome, inhibited iMAC efferocytosis. Thus, iMAC platform combined with phenotypic and functional macrophage readouts can represent not only a drug screening system but also a new hypothesis-generating tool, facilitating new knowledge about macrophage biology and macrophage therapeutic targeting in IMIDs and possibly cancer. Finally, the pooled CRISPR screen is yet to define genes involved in TRM differentiation/function is ongoing in iMicroglia, which will be followed by validation studies of top hits (beyond my PhD thesis).