A genetic screen in macrophages identifies new regulators of IFNγ-inducible MHCII that contribute to T cell activation

Kiritsy, Michael C.; Ankley, Laurisa M; Trombley, Justin D.; Huizinga, Gabrielle P; Lord, Audrey E; Orning, Pontus; Elling, Roland; Fitzgerald, Katherine A.; Olive, Andrew J.

doi:10.7554/elife.65110

Cited by 18 publications

(40 citation statements)

References 78 publications

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“…Individual sgRNAs (2 per gene) from the Brie library or designed using Benchling (S3 Table ) were cloned into lentiGuide-Puro (Addgene #52963), lentiCRISPR v2-blast (Addgene #98293), or sgOpti (Addgene #85681) as previously described (22,(76)(77)(78). Briefly, sgRNAs were annealed, phosphorylated with T4 Polynucleotide Kinase, digested with BsmBI, and ligated into lentiGuide-Puro, lentiCRISPR v2-blast, or sgOpti.…”

Section: Construction Of Knockout Cell Lines and Crispr Screen Valida...mentioning

confidence: 99%

A genome-wide screen in macrophages identifies PTEN as required for myeloid restriction ofListeria monocytogenesinfection

Glover

Schwardt

Sanchez

et al. 2022

Preprint

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Listeria monocytogenes (Lm) is an intracellular foodborne pathogen which causes the severe disease listeriosis in immunocompromised individuals. Macrophages play a dual role during Lm infection by both promoting dissemination of Lm from the gastrointestinal tract and limiting bacterial growth upon immune activation. Despite the relevance of macrophages to Lm infection, the mechanisms underlying phagocytosis of Lm by macrophages are not well understood. To identify host factors important for Lm infection of macrophages, we performed an unbiased CRISPR/Cas9 screen which revealed pathways that are specific to phagocytosis of Lm and those that are required for internalization of bacteria generally. Specifically, we discovered the tumor suppressor PTEN promotes macrophage phagocytosis of Lm and L. ivanovii, but not other Gram-positive bacteria. Additionally, we found that PTEN enhances phagocytosis of Lm via its lipid phosphatase activity by promoting adherence to macrophages. Using conditional knockout mice lacking Pten in myeloid cells, we show that PTEN-dependent phagocytosis is important for host protection during oral Lm infection. Overall, this study provides a comprehensive identification of macrophage factors involved in regulating Lm uptake and characterizes the function of one factor, PTEN, during Lm infection in vitro and in vivo. Importantly, these results demonstrate a role for opsonin-independent phagocytosis in Lm pathogenesis and suggest that macrophages play a primarily protective role during foodborne listeriosis.

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Section: Construction Of Knockout Cell Lines and Crispr Screen Valida...mentioning

confidence: 99%

A genome-wide screen in macrophages identifies PTEN as required for myeloid restriction ofListeria monocytogenesinfection

Glover

Schwardt

Sanchez

et al. 2022

Preprint

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“…We previously generated a robust and reproducible pooled genome-wide loss-of-function library in Cas9+ iBMDMs (41). Knockouts in this pool were generated with sgRNAs from the Brie library which targets four independent single guide RNA molecules (sgRNAs) per coding gene and over 1,000 non-targeting controls (NTC) (42). To identify regulators of Mab uptake, we infected the loss-of-function library of macrophages with mEmerald Mab for four hours.…”

Section: Crispr-cas9 Loss-of-function (Lof) Screen Identifies Host Ge...mentioning

confidence: 99%

“…J2-virus immortalized murine bone marrow derived macrophages (iBMDMs) and Cas9+ iBMDMs were maintained in Dulbecco's Modified Eagle Medium (DMEM; Hyclone) supplemented with 10% heat inactivated fetal bovine serum (FBS; Seradigm) as previously described (42). All macrophage cell lines were incubated in 5% CO 2 at 37°C…”

Section: Cell Culturementioning

confidence: 99%

“…Mouse Brie CRISPR knockout pooled library was a gift from David Root and John Doench (Addgene #73633) (42) and infected with our smooth mEmerald-GFP Mab reporter strain at an MOI of five for four hours. Following the infection, macrophages were washed with PBS then fixed with 4% paraformaldehyde.…”

Section: Crispr Screen and Analysismentioning

confidence: 99%

“…Genomic DNA was isolated from each sorted population using Qiagen DNeasy kits after reversing DNA crosslinks following a 55°C incubation overnight. Amplification of sgRNAs by PCR was performed as previously described using Illumina compatible primers from IDT (42) and amplicons were sequenced on an Illumina NextSeq 500 at the Genomics Core at Michigan State University.…”

Section: Crispr Screen and Analysismentioning

confidence: 99%

See 2 more Smart Citations

A genome-wide screen in macrophages defines host genes regulating the uptake ofMycobacterium abscessus

Gilliland

Beckman

Olive

2022

Preprint

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The interactions between a host cell and a pathogen can dictate disease outcomes and are important targets for host-directed therapies.Mycobacterium abscessus(Mab) is a highly antibiotic resistant, rapidly growing non-tuberculous mycobacterium that infects patients with chronic lung diseases. Mab can infect host immune cells, such as macrophages, which contribute to its pathogenesis. However, our understanding of initial host-Mab interactions remains unclear. Here, we developed a functional genetic approach to define these host-Mab interactions by coupling a Mab fluorescent reporter with a genome-wide knockout library in murine macrophages. We used this approach to conduct a forward genetic screen to define host genes that contribute to the uptake of Mab by macrophages. We identified known regulators of phagocytosis, such as the integrin ITGB2, and uncovered a key requirement for glycosaminoglycan (sGAG) synthesis for macrophages to efficiently take up Mab. CRISPR-Cas9 targeting of three key sGAG biosynthesis regulators, Ugdh, B3gat3 and B4galt7 resulted in reduced uptake of both smooth and rough Mab variants by macrophages. Mechanistic studies suggest that sGAGs function upstream of pathogen engulfment and are required for the uptake of Mab, but notEscherichia colior latex beads. Further investigation found that the loss of sGAGs reduced the surface expression, but not the mRNA expression, of key integrins suggesting an important role for sGAGs in modulating surface receptor availability. Together, these studies globally define and characterize important regulators of macrophage-Mab interactions and are a first step to understanding host genes that contribute to Mab pathogenesis and disease.

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Combination of novel oncolytic herpesvirus with paclitaxel as an efficient strategy for breast cancer therapy

Deng

Shen

et al. 2023

Journal of Medical Virology

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Background: New strategies are needed to improve the treatment of patients with breast cancer (BC). Oncolytic virotherapy is a promising new tool for cancer treatment but still has a limited overall durable antitumor response. A novel replicable recombinant oncolytic herpes simplex virus type 1 called VG161 has been developed and has demonstrated antitumor effects in several cancers. Here, we explored the efficacy and the antitumor immune response of VG161 cotreatment with paclitaxel (PTX) which as a novel oncolytic viral immunotherapy for BC. Methods: The antitumor effect of VG161 and PTX was confirmed in a BC xenograft mouse model. The immunostimulatory pathways were tested by RNA-seq and the remodeling of tumor microenvironment was detected by Flow cytometry analysis or Immunohistochemistry. Pulmonary lesions were analyzed by the EMT6-Luc BC model. Results:In this report, we demonstrate that VG161 can significantly represses BC growth and elicit a robust antitumor immune response in a mouse model. The effect is amplified when combined with PTX treatment. The antitumor effect is associated with the infiltration of lymphoid cells, including CD4 + T cells, CD8 + T cells, and NK

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A genetic screen in macrophages identifies new regulators of IFNγ-inducible MHCII that contribute to T cell activation

Cited by 18 publications

References 78 publications

A genome-wide screen in macrophages identifies PTEN as required for myeloid restriction ofListeria monocytogenesinfection

A genome-wide screen in macrophages identifies PTEN as required for myeloid restriction ofListeria monocytogenesinfection

A genome-wide screen in macrophages defines host genes regulating the uptake ofMycobacterium abscessus

Combination of novel oncolytic herpesvirus with paclitaxel as an efficient strategy for breast cancer therapy

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