Phenotypic, Metabolic, and Functional Characterization of Experimental Models of Foamy Macrophages: Toward Therapeutic Research in Atherosclerosis.

Different types of macrophages (Mφ) are involved in atherogenesis, including inflammatory Mφ and foamy Mφ (FM). Our previous study demonstrated that two-photon excited fluorescence (TPEF) imaging of NADH and FAD autofluorescence (AF) could distinguish experimental models that mimic the different atherosclerotic Mφ types. The present study assessed whether optical differences correlated with phenotypic and functional differences, potentially guiding diagnostic and therapeutic strategies. Phenotypic differences were investigated using three-dimensional principal component analysis and multi-color flow cytometry. Functional analyses focused on cytokine production, metabolic profiles, and cellular oxidative stress, in LDL dose-dependent assays, to understand the origin of AF in the FAD spectrum and assess FM ability to transition toward an immunoregulatory phenotype and function. Phenotypic studies revealed that FM models generated with acetylated LDL (Mac) were closer to immunoregulatory Mφ, while those generated with oxidized LDL (Mox) more closely resembled inflammatory Mφ. The metabolic analysis confirmed that inflammatory Mφ primarily used glycolysis, while immunoregulatory Mφ mainly depended on mitochondrial respiration. FM models employed both pathways; however, FM models generated with high doses of modified LDL showed reduced mitochondrial respiration, particularly Mox FM. Thus, the high AF in the FAD spectrum in Mox was not linked to increased mitochondrial respiration, but correlated with the dose of oxidized LDL, leading to increased production of reactive oxygen species (ROS) and lysosomal ceroid accumulation. High FAD-like AF, ROS, and ceroid accumulation were reduced by incubation with α-tocopherol. The cytokine profiles supported the phenotypic analysis, indicating that Mox FM exhibited greater inflammatory activity than Mac FM, although both could be redirected toward immunoregulatory functions, albeit to different degrees. In conclusion, in the context of immunoregulatory therapies for atherosclerosis, it is crucial to consider FM, given their prevalence in plaques and our results, as potential targets, regardless of their inflammatory status, alongside non-foamy inflammatory Mφ.

Engineering and Functional Evaluation of Neutralizing Antibody Fragments Against Congenital Toxoplasmosis.

Maternal-fetal transmission of Toxoplasma gondii tachyzoites acquired during pregnancy has potentially dramatic consequences for the fetus. Current reference-standard treatments are not specific to the parasite and can induce severe side effects. In order to provide treatments with a higher specificity against toxoplasmosis, we developed antibody fragments-single-chain fragment variable (scFv) and scFv fused with mouse immunoglobulin G2a crystallizable fragment (scFv-Fc)-directed against the major surface protein SAG1. After validating their capacity to inhibit T. gondii proliferation in vitro, the antibody fragments' biological activity was assessed in vivo using a congenital toxoplasmosis mouse model. Dams were treated by systemic administration of antibody fragments and with prevention of maternal-fetal transmission being used as the parameter of efficacy. We observed that both antibody fragments prevented T. gondii dissemination and protected neonates, with the scFv-Fc format having better efficacy. These data provide a proof of concept for the use of antibody fragments as effective and specific treatment against congenital toxoplasmosis and provide promising leads.

Cholesterol efflux pathways hinder KRAS-driven lung tumor progenitor cell expansion.

Cholesterol efflux pathways could be exploited in tumor biology to unravel cancer vulnerabilities. A mouse model of lung-tumor-bearing KRASG12D mutation with specific disruption of cholesterol efflux pathways in epithelial progenitor cells promoted tumor growth. Defective cholesterol efflux in epithelial progenitor cells governed their transcriptional landscape to support their expansion and create a pro-tolerogenic tumor microenvironment (TME). Overexpression of the apolipoprotein A-I, to raise HDL levels, protected these mice from tumor development and dire pathologic consequences. Mechanistically, HDL blunted a positive feedback loop between growth factor signaling pathways and cholesterol efflux pathways that cancer cells hijack to expand. Cholesterol removal therapy with cyclodextrin reduced tumor burden in progressing tumor by suppressing the proliferation and expansion of epithelial progenitor cells of tumor origin. Local and systemic perturbations of cholesterol efflux pathways were confirmed in human lung adenocarcinoma (LUAD). Our results position cholesterol removal therapy as a putative metabolic target in lung cancer progenitor cells.

Two-photon excited fluorescence (TPEF) may be useful to identify macrophage subsets based on their metabolic activity and cellular responses in atherosclerotic plaques.

BACKGROUND AND AIMS: Atherosclerosis is characterized by the formation of lipid plaques within the arterial wall. In such plaques, the massive and continuous recruitment of circulating monocyte-derived macrophages induces inflammation, leading to plaque destabilization and rupture. Plaque vulnerability is linked to the presence of (i) a large lipid core that contains necrotic, "foamy" macrophages (FMs), (ii) a thin fibrous cap that cannot limit the prothrombotic lipid core, and potentially (iii) an imbalance between inflammatory and immunoregulatory macrophages. These opposite macrophage functions rely on the use of different energy pathways (glycolysis and oxidative phosphorylation, respectively) that may lead to different levels of the auto-fluorescent cofactors nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). We hypothesized that high-resolution two-photon excited autofluorescence (TPEF) imaging of these cofactors may be used to monitor the metabolic activity and cellular responses of macrophages in atherosclerotic plaques. METHODS: Different models of human FMs were generated by exposure to acetylated or oxidized low-density lipoproteins (LDL), with/without human carotid extract (CE). Their phenotype and optical properties were compared with those of extremely polarized macrophages, inflammatory M1 (MLPS+IFNγ) and immunoregulatory M2 (MIL4+IL13). RESULTS: These FM models displayed an intermediate phenotype with low levels of M1 and M2 "specific" markers. Moreover, the NADH and FAD autofluorescence profiles of FMoxLDL ± CE cells were significantly distinct from those of M1 and M2 macrophages. CONCLUSIONS: TPEF imaging may be useful to follow the metabolic activity and cellular responses of the different macrophage subtypes present in atherosclerotic plaques in order to detect vulnerable areas.