Mouse primary bone marrow-derived macrophages (BMDMs) are an important tool for studying macrophage physiology ex vivo. They recapitulate the complex biology of macrophages more closely than immortalized macrophage cell lines and can often be derived from mice carrying mutations of interest. However, the genetic manipulation of primary macrophages has remained challenging. Here, we present a protocol for efficient genome editing in primary mouse BMDMs using sgRNA-Cas9 ribonucleoprotein particles (RNP) assembled in vitro, including the methods for designing and synthesizing sgRNAs and RNP complex delivery. We also provide a rapid protocol to evaluate editing efficiency using routine Sanger sequencing and a readily available online analysis tool. The protocol requires no plasmid construction, can be performed in under aweek, and results in up to 95% editing efficiency.

Mycobacterium tuberculosis (Mtb) is the etiological agent of Tuberculosis, a disease that has persisted over the past century. The success of this pathogen lies in its ability to avoid induction of the host immune response or subvert the consequences of induction altogether. Upon entering the lung of the host, Mtb is phagocytosed by resident alveolar macrophages, which serve as a replicative niche. Mtb escapes degradation by preventing phagosome maturation and permeabilizing the phagosome to secrete Mtb effectors and nucleic acids. Mtb-derived nucleic acids have been shown to induce the expression of type I IFN in host cells, which dampens host protective pathways and promotes Mtb growth, suggesting the importance of this pathway that needs further characterization. Our lab recently discovered that the E3 ubiquitin ligase CBL negatively regulated type I IFN during Mtb infection. However, the exact mechanism through which CBL represses type I IFN during Mtb infection is unknown. Here we show that CBL regulates type I IFN in both human and mouse macrophages and responds to other stimuli such as DNA and RNA, suggesting that CBL controls response to multiple stimuli. Consequently, along with the repression of type I IFN, CBL is also required to create a restrictive environment for Mtb growth in human macrophages. Transcriptomic analysis reveals that CBL operates by restricting innate immune and antiviral defense pathways, suggesting a novel role for CBL as an innate immune response regulator. Due to the nature of CBL being an E3 ubiquitin ligase, we tested the involvement of ubiquitination in the control Mtb growth and determined that ubiquitination is necessary to control bacteria burden. Upon identifying the importance of ubiquitination, we attempted to identify the CBL-dependent substrate and discovered ARL8B as a host factor that promotes Mtb growth. Further work is needed to determine the mechanism through which ARL8B creates a more permissive environment for Mtb growth. These studies reveal a new innate immune regulation module that helps to gain a better insight into how Mtb manipulates the host cell environment and how the host cell responds to manipulation tactics.

Objective

Design

Setting

Patients

Methods

Results

Conclusion