Self-replicating murine ex vivo cultured alveolar macrophages as a model for toxicological studies of particle-induced inflammation

Kendall, Rebekah L.; Ray, Jessica L.; Hamilton, Raymond F.; Holian, Andrij

doi:10.1016/j.taap.2023.116400

Cited by 5 publications

(4 citation statements)

References 65 publications

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“…In addition, FLAMs expressed high levels of resident macrophage-associated surface markers, including SiglecF, Siglec1, Marco, CD200, TLR2, MRC1, Itgal, and Itgax, which were expressed at low levels or not expressed in iBMDMs. In line with there being functional similarities between AMs and FLAMs, we observed high expression of genes associated with lipid and cholesterol metabolism in FLAMs ( 26 ). Interestingly, when we examined genes that modulate T cell activation, we observed high expression of the coinhibitory markers PDL1 and PDL2 on FLAMs ( Fig.…”

Section: Resultssupporting

confidence: 81%

GSK3α/β Restrain IFN-γ–Inducible Costimulatory Molecule Expression in Alveolar Macrophages, Limiting CD4+ T Cell Activation

Ankley,

Conner,

Vielma

et al. 2024

ImmunoHorizons

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Macrophages play a crucial role in eliminating respiratory pathogens. Both pulmonary resident alveolar macrophages (AMs) and recruited macrophages contribute to detecting, responding to, and resolving infections in the lungs. Despite their distinct functions, it remains unclear how these macrophage subsets regulate their responses to infection, including how activation by the cytokine IFN-γ is regulated. This shortcoming prevents the development of therapeutics that effectively target distinct lung macrophage populations without exacerbating inflammation. We aimed to better understand the transcriptional regulation of resting and IFN-γ–activated cells using a new ex vivo model of AMs from mice, fetal liver–derived alveolar-like macrophages (FLAMs), and immortalized bone marrow-derived macrophages. Our findings reveal that IFN-γ robustly activates both macrophage types; however, the profile of activated IFN-γ–stimulated genes varies greatly between these cell types. Notably, FLAMs show limited expression of costimulatory markers essential for T cell activation upon stimulation with only IFN-γ. To understand cell type–specific differences, we examined how the inhibition of the regulatory kinases GSK3α/β alters the IFN-γ response. GSK3α/β controlled distinct IFN-γ responses, and in AM-like cells, we found that GSK3α/β restrained the induction of type I IFN and TNF, thus preventing the robust expression of costimulatory molecules and limiting CD4+ T cell activation. Together, these data suggest that the capacity of AMs to respond to IFN-γ is restricted in a GSK3α/β-dependent manner and that IFN-γ responses differ across distinct macrophage populations. These findings lay the groundwork to identify new therapeutic targets that activate protective pulmonary responses without driving deleterious inflammation.

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Section: Resultssupporting

confidence: 81%

GSK3α/β Restrain IFN-γ–Inducible Costimulatory Molecule Expression in Alveolar Macrophages, Limiting CD4+ T Cell Activation

Ankley,

Conner,

Vielma

et al. 2024

ImmunoHorizons

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“…Macrophages are central cells present in various tissues that are involved in the regulation of inflammatory responses [1][2][3][4]. The mexAM cell model used in this study represented primary AMs phenotypically, transcriptionally, and morphologically [21,22]. Furthermore, when engrafted into AM-depleted mice, the mexAMs were able to restore lung function in an alveolar proteinosis disease model, illustrating the in vivo translatability of these AMs [21].…”

Section: Discussionmentioning

confidence: 96%

“…To aid in the detachment from the flask bottom, the mexAMs were incubated with a local anesthetic lidocaine (20 mM, cat # J63035.14, Thermo Fisher Scientific) for 4 min at 37 • C and then gently scraped from the flask bottom. The usage of lidocaine for the detachment of mexAMs and other cell types has been used previously by our laboratory and others and has been shown to preserve the cell surface proteins that are otherwise lost through trypsinization or scraping alone [21][22][23]. For the drug treatments, the mexAMs were incubated at a cell density of 10 6 cells/mL in a complete media without growth factors, with either imipramine (25 µM, cat # 113-52-0, Cayman Chemical, Ann Arbor, MI, USA) or U18666A (1 µg/mL, cat # 3039-71-2, Cayman Chemical) for 24 h prior to the lysosome isolations.…”

Section: Cell Culturementioning

confidence: 99%

Imipramine Treatment Alters Sphingomyelin, Cholesterol, and Glycerophospholipid Metabolism in Isolated Macrophage Lysosomes

Albright,

Sydor,

Shannahan

et al. 2023

Biomolecules

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Lysosomes are degradative organelles that facilitate the removal and recycling of potentially cytotoxic materials and mediate a variety of other cellular processes, such as nutrient sensing, intracellular signaling, and lipid metabolism. Due to these central roles, lysosome dysfunction can lead to deleterious outcomes, including the accumulation of cytotoxic material, inflammation, and cell death. We previously reported that cationic amphiphilic drugs, such as imipramine, alter pH and lipid metabolism within macrophage lysosomes. Therefore, the ability for imipramine to induce changes to the lipid content of isolated macrophage lysosomes was investigated, focusing on sphingomyelin, cholesterol, and glycerophospholipid metabolism as these lipid classes have important roles in inflammation and disease. The lysosomes were isolated from control and imipramine-treated macrophages using density gradient ultracentrifugation, and mass spectrometry was used to measure the changes in their lipid composition. An unsupervised hierarchical cluster analysis revealed a clear differentiation between the imipramine-treated and control lysosomes. There was a significant overall increase in the abundance of specific lipids mostly composed of cholesterol esters, sphingomyelins, and phosphatidylcholines, while lysophosphatidylcholines and ceramides were overall decreased. These results support the conclusion that imipramine’s ability to change the lysosomal pH inhibits multiple pH-sensitive enzymes in macrophage lysosomes.

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“…It is important to understand how these lung-resident cells uniquely respond to inflammatory signals compared to other macrophages to develop lung-specific therapies that protect against infection while maintaining pulmonary function. To address this gap, several groups developed approaches to culture AM-like cells ex vivo that maintain AM functions (21,(23)(24)(25). While the details of these approaches differ, they all leverage lung-specific cytokine cues from GM-CSF and TGFβ that are required to maintain AM populations in the lung environment.…”

Section: Introductionmentioning

confidence: 99%

GSK3α/β restrains IFNγ-inducible costimulatory molecule expression in alveolar macrophages, limiting CD4⁺T cell activation

Ankley,

Conner,

Vielma

et al. 2023

Preprint

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Macrophages play a crucial role in eliminating respiratory pathogens. Both pulmonary resident alveolar macrophages (AMs) and recruited macrophages contribute to detecting, responding to, and resolving infections in the lungs. Despite their distinct functions, it remains unclear how these macrophage subsets regulate their responses to infection, including how activation by the cytokine IFNγ is regulated. This shortcoming prevents the development of therapeutics that effectively target distinct lung macrophage populations without exacerbating inflammation. We aimed to better understand the transcriptional regulation of resting and IFNγ-activated cells using a new ex vivo model of AMs from mice, fetal liver-derived alveolar-like macrophages (FLAMs), and immortalized bone marrow-derived macrophages (iBMDMs). Our findings reveal that IFNγ robustly activates both macrophage types; however, the profile of activated IFNγ-stimulated genes varies greatly between these cell types. Notably, FLAMs show limited expression of costimulatory markers essential for T cell activation upon stimulation with only IFNγ. To understand cell type-specific differences, we examined how the inhibition of the regulatory kinases GSK3α/β alters the IFNγ response. GSK3α/β controlled distinct IFNγ responses, and in AM-like cells, we found GSK3α/β restrained the induction of type I IFN and TNF, thus preventing the robust expression of costimulatory molecules and limiting CD4+ T cell activation. Together, these data suggest that the capacity of AMs to respond to IFNγ is restricted in a GSK3α/β-dependent manner and that IFNγ responses differ across distinct macrophage populations. These findings lay the groundwork to identify new therapeutic targets that activate protective pulmonary responses without driving deleterious inflammation.

show abstract

Self-replicating murine ex vivo cultured alveolar macrophages as a model for toxicological studies of particle-induced inflammation

Cited by 5 publications

References 65 publications

GSK3α/β Restrain IFN-γ–Inducible Costimulatory Molecule Expression in Alveolar Macrophages, Limiting CD4+ T Cell Activation

GSK3α/β Restrain IFN-γ–Inducible Costimulatory Molecule Expression in Alveolar Macrophages, Limiting CD4+ T Cell Activation

Imipramine Treatment Alters Sphingomyelin, Cholesterol, and Glycerophospholipid Metabolism in Isolated Macrophage Lysosomes

GSK3α/β restrains IFNγ-inducible costimulatory molecule expression in alveolar macrophages, limiting CD4⁺T cell activation

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Self-replicating murine ex vivo cultured alveolar macrophages as a model for toxicological studies of particle-induced inflammation

Cited by 5 publications

References 65 publications

GSK3α/β Restrain IFN-γ–Inducible Costimulatory Molecule Expression in Alveolar Macrophages, Limiting CD4+ T Cell Activation

GSK3α/β Restrain IFN-γ–Inducible Costimulatory Molecule Expression in Alveolar Macrophages, Limiting CD4+ T Cell Activation

Imipramine Treatment Alters Sphingomyelin, Cholesterol, and Glycerophospholipid Metabolism in Isolated Macrophage Lysosomes

GSK3α/β restrains IFNγ-inducible costimulatory molecule expression in alveolar macrophages, limiting CD4+T cell activation

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GSK3α/β restrains IFNγ-inducible costimulatory molecule expression in alveolar macrophages, limiting CD4⁺T cell activation