Cas9+ conditionally-immortalized macrophages as a tool for bacterial pathogenesis and beyond

Abstract

Macrophages play critical roles in immunity, development, tissue repair, and cancer, but studies of their function have been hampered by poorly-differentiated tumor cell lines and genetically-intractable primary cells. Here we report a facile system for genome editing in non-transformed macrophages by differentiating ER-Hoxb8 myeloid progenitors from Cas9-expressing transgenic mice. These conditionally immortalized macrophages (CIMs) retain characteristics of primary macrophages derived from the bone marrow yet allow for easy genetic manipulation and a virtually unlimited supply of cells. We demonstrate the utility of this system for dissection of host genetics during intracellular bacterial infection using two important human pathogens: Listeria monocytogenes and Mycobacterium tuberculosis.

Genetic engineering of Hoxb8-immortalized hematopoietic progenitors – a potent tool to study macrophage tissue migration

“…Again, the phagocytic capacity of Hoxb8-macrophages was comparable to that of BMDMs, with similar percentages of phagocytic cells and similar number of ingested particles per cell being observed ( Fig. 2C), as recently described for engulfment of Mycobacterium tuberculosis and Listeria monocytogenes (Roberts et al, 2019).…”

“…Expansion of murine hematopoietic precursors that were transiently immortalized with a retrovirus-delivered and estrogeninducible form of the transcription factor Hoxb8 have been described (Wang et al, 2006) and validated for the study of hematopoietic cell biology (Cabron et al, 2018;Chu et al, 2019;Di Ceglie et al, 2017;Gurzeler et al, 2013;Hammerschmidt et al, 2018;Lee et al, 2017;Rosas et al, 2011;Wang et al, 2006;Witschi et al, 2010;Zach et al, 2015). The recent coupling of this long-term hematopoietic progenitor cell line to the CRISPR/Cas9 technology (Hammerschmidt et al, 2018;Roberts et al, 2019) has enabled the creation of new genetically modifiable cell models. During the last few years, expression of Hoxb8 has been used in several studies mainly focused on DC biology (Cabron et al, 2018;Grajkowska et al, 2017;Hammerschmidt et al, 2018;Leithner et al, 2018;Rosas et al, 2011), but only rare studies explored its use to generate surrogate macrophages (Cabron et al, 2018;Roberts et al, 2019;Wang et al, 2006).…”

“…The recent coupling of this long-term hematopoietic progenitor cell line to the CRISPR/Cas9 technology (Hammerschmidt et al, 2018;Roberts et al, 2019) has enabled the creation of new genetically modifiable cell models. During the last few years, expression of Hoxb8 has been used in several studies mainly focused on DC biology (Cabron et al, 2018;Grajkowska et al, 2017;Hammerschmidt et al, 2018;Leithner et al, 2018;Rosas et al, 2011), but only rare studies explored its use to generate surrogate macrophages (Cabron et al, 2018;Roberts et al, 2019;Wang et al, 2006). Only one of these studies directly compared macrophages derived from Hoxb8 progenitors to macrophages derived from bone marrow.…”

“…Only one of these studies directly compared macrophages derived from Hoxb8 progenitors to macrophages derived from bone marrow. This study describes the association of Hoxb8 conditional immortalization and CRISPR/Cas9 to specifically study macrophage biology in infectious settings (Roberts et al, 2019).…”

Why isListeria monocytogenessuch a potent inducer of CD8+ T‐cells?

“…Shortly after intravenous infection with L. monocytogenes, most of the bacteria are found within macrophages and dendritic cells (DCs) of the spleen and liver where the majority of bacteria are killed, but some of the bacteria escape from phagocytic vacuoles, replicate rapidly in the cytosol of infected cells and exploit host actin dynamics to spread to neighbouring cells (Portnoy, Auerbuch, & Glomski, 2002;Serbina, Shi, & Pamer, 2012;Waite et al, 2011). This infectious process occurs in most, if not all adherent cells, including primary or immortalised cultures of bone marrow-derived macrophages (BMMs) (Roberts et al, 2019) that are ideal cells for studies on innate immunity (see below) and can be activated with interferongamma (IFN-γ), to kill and degrade phagosomal bacteria (Herskovits, Auerbuch, & Portnoy, 2007).…”