Whole-mount immunofluorescence imaging and isolation of mesothelium-bound immune cell aggregates during mouse peritoneal inflammation

Ferriz, Margarita; Vega-Pérez, Adrián; Gutiérrez-González, Alejandra; Alvarez-Ladrón, Natalia; Ardavı́n, Carlos

doi:10.1016/j.xpro.2023.102079

Cited by 2 publications

(2 citation statements)

References 6 publications

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“…Several recent studies have shown that these macrophages are primarily forming aggregates within the cavity or localizing to the omentum [ 30 ]. We adapted a recently published comprehensive methodology [ 30 , 31 ] to address MDHR in our studies by analyzing potential sites of LPM egress: floating and adherent cellular aggregates, homing to the omentum, and migration to draining lymphatics. We found that aggregated MHCII lo LPM and MHCII + SPM in RSM-treated mice expressed nearly no RELMα, similar to suspended lavage cells ( Fig 5F ).…”

Section: Resultsmentioning

confidence: 99%

“…For naïve in vivo experiments, peritoneal exudate cells (PECs) were recovered in a total of 6–8 mL of ice-cold PBS. For collection of peritoneal cellular aggregates, the inflated peritoneal cavity was vigorously massaged and cut open over a funnel [ 31 ]. Peritoneal aggregates were allowed to settle out of the lavage fluid for at least 20 min on ice and suspended PECs were aspirated with a plastic Pasteur pipette.…”

Section: Methodsmentioning

confidence: 99%

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Choline metabolism underpins macrophage IL-4 polarization and RELMα up-regulation in helminth infection

Ghorbani,

Kim,

Smith

et al. 2023

PLoS Pathog

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Type 2 cytokines like IL-4 are hallmarks of helminth infection and activate macrophages to limit immunopathology and mediate helminth clearance. In addition to cytokines, nutrients and metabolites critically influence macrophage polarization. Choline is an essential nutrient known to support normal macrophage responses to lipopolysaccharide; however, its function in macrophages polarized by type 2 cytokines is unknown. Using murine IL-4-polarized macrophages, targeted lipidomics revealed significantly elevated levels of phosphatidylcholine, with select changes to other choline-containing lipid species. These changes were supported by the coordinated upregulation of choline transport compared to naïve macrophages. Pharmacological inhibition of choline metabolism significantly suppressed several mitochondrial transcripts and dramatically inhibited select IL-4-responsive transcripts, most notably, Retnla. We further confirmed that blocking choline metabolism diminished IL-4-induced RELMα (encoded by Retnla) protein content and secretion and caused a dramatic reprogramming toward glycolytic metabolism. To better understand the physiological implications of these observations, naïve or mice infected with the intestinal helminth Heligmosomoides polygyrus were treated with the choline kinase α inhibitor, RSM-932A, to limit choline metabolism in vivo. Pharmacological inhibition of choline metabolism lowered RELMα expression across cell-types and tissues and led to the disappearance of peritoneal macrophages and B-1 lymphocytes and an influx of infiltrating monocytes. The impaired macrophage activation was associated with some loss in optimal immunity to H. polygyrus, with increased egg burden. Together, these data demonstrate that choline metabolism is required for macrophage RELMα induction, metabolic programming, and peritoneal immune homeostasis, which could have important implications in the context of other models of infection or cancer immunity.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Choline metabolism underpins macrophage IL-4 polarization and RELMα up-regulation in helminth infection

Ghorbani,

Kim,

Smith

et al. 2023

PLoS Pathog

View full text Add to dashboard Cite

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Choline metabolism underpins macrophage IL-4 polarization and RELMα up-regulation in helminth infection

Ghorbani

Kim

Smith

et al. 2022

Preprint

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Choline is an essential nutrient and in macrophages, mainly supports phosphatidylcholine (PC) synthesis. Cellular uptake and incorporation of choline into PC is critical for LPS-induced macrophage inflammation. Here, we examined choline metabolism in the context of IL-4 polarization of mouse macrophages in vitro and in vivo. Like LPS, IL-4 increased the levels of choline transporter-like protein 1, the rate of choline uptake, and incorporation into PC. Targeted lipidomics analysis revealed higher PC content in IL-4-polarized macrophages, with enrichment in low-saturated species. Pharmacological inhibition of choline metabolism significantly suppressed the transcription of certain hallmark IL-4 genes (Retnla) but not others (Chil3, Mrc1, Arg1). Blocking choline metabolism diminished the expression and secretion of RELMα protein (encoded by Retnla), while also limiting PD-L2 up-regulation and increasing PD-L1 expression. In vivo administration of RSM-932a, a choline kinase inhibitor, caused a dramatic shift in the peritoneal immune cell profile and up-regulated macrophage CD86 and PD-L1, while down-regulating CD206 and PD-L2. Strikingly, blocking choline metabolism lowered RELMα expression in multiple cell-types and tissues in naive mice as well as mice infected with the helminth pathogens Heligmosomoides polygyrus and Nippostrongylus brasiliensis. There were no changes in pathogen burden or clearance in the two separate helminth models. In contrast, in dextran sulfate sodium-induced colitis, loss of colon length as a marker of inflammation was mitigated by choline metabolism inhibition. These data demonstrate a critical link between choline and macrophage effector functions and suggest that targeting choline metabolism could be leveraged to fine-tune immunopathology.

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Whole-mount immunofluorescence imaging and isolation of mesothelium-bound immune cell aggregates during mouse peritoneal inflammation

Cited by 2 publications

References 6 publications

Choline metabolism underpins macrophage IL-4 polarization and RELMα up-regulation in helminth infection

Choline metabolism underpins macrophage IL-4 polarization and RELMα up-regulation in helminth infection

Choline metabolism underpins macrophage IL-4 polarization and RELMα up-regulation in helminth infection

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