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Human iNKT Cells Modulate Macrophage Survival and Phenotype.

ABSTRACT: CD1d-restricted invariant Natural Killer T (iNKT) cells are unconventional innate-like T cells whose functions highly depend on the interactions they establish with other immune cells. Although extensive studies have been reported on the communication between iNKT cells and macrophages in mice, less data is available regarding the relevance of this crosstalk in humans. Here, we dove into the human macrophage-iNKT cell axis by exploring how iNKT cells impact the survival and polarization of pro-inflammatory M1-like and anti-inflammatory M2-like monocyte-derived macrophages. By performing in vitro iNKT cell-macrophage co-cultures followed by flow cytometry analysis, we demonstrated that antigen-stimulated iNKT cells induce a generalized activated state on all macrophage subsets, leading to upregulation of CD40 and CD86 expression. CD40L blocking with a specific monoclonal antibody prior to co-cultures abrogated CD40 and CD86 upregulation, thus indicating that iNKT cells required CD40-CD40L co-stimulation to trigger macrophage activation. In addition, activated iNKT cells were cytotoxic towards macrophages in a CD1d-dependent manner, killing M1-like macrophages more efficiently than their naïve M0 or anti-inflammatory M2-like counterparts. Hence, this work highlighted the role of human iNKT cells as modulators of macrophage survival and phenotype, untangling key features of the human macrophage-iNKT cell axis and opening perspectives for future therapeutic modulation.

SUBMITTER: Loureiro JP

PROVIDER: S-EPMC9313099 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Extracellular vesicles (EVs) mediate cell-to-cell communication by horizontally transferring biological materials from host cells to target cells. During exposure to pathogens, pathogen-associated molecular patterns (e.g., lipopolysaccharide, LPS) get in contact with endothelial cells and stimulate the secretion of endothelial cell-derived EVs (E-EVs). The triggered EVs secretion is known to have a modulating influence on the EVs-receiving cells. Macrophages, a major component of innate immunity, are polarized upon receiving external inflammatory stimuli, in which toll-like receptor4 (TLR4)-nuclear factor kappa B (NFκB) pathway plays a key role. However, the functions of LPS-induced E-EVs (ELPS-EVs) in modulating macrophage phenotype and activation remain elusive. We collected the EVs from quiescent endothelial cells (ENor-EVs) and ELPS-EVs to detect their stimulatory role on NR8383 macrophages. Isolated EVs were characterized by transmission electron microscopy (TEM), western blot assay, and nanoparticle tracking analysis (NTA). NR8383 macrophages were stimulated with ELPS-EVs, ENor-EVs, or PBS for 24 h. Hereafter, the uptake of EVs by the macrophages was investigated. Upon EVs stimulation, cellular viability was determined by MTT assay, while macrophage phenotype was analyzed by flow cytometry and immunofluorescence analysis. Furthermore, a western blot assay was conducted to evaluate the potentially involved TLR4-NFκB pathway. Interestingly, upon exposure to LPS, endothelial cells secreted significantly higher amounts of EVs (i.e., ELPS-EVs) when compared to quiescent cells or cells in PBS. The ELPS-EVs were also better internalized by NR8383 macrophages than ENor-EVs. The cellular viability of ELPS-EVs-treated macrophages was 1.2 times higher than those in the ENor-EVs and PBS groups. In addition, ELPS-EVs modulated NR8383 macrophages towards a proinflammatory macrophage M1-like phenotype. This was indicated by the significantly upregulated expressions of proinflammatory macrophage biomarkers CD86 and inducible nitric oxide synthase (iNOS) observed in ELPS-EVs-treated macrophages. The TLR4-NFκB signaling pathway was substantially activated in ELPS-EVs-treated macrophages, indicated by the elevated expressions of makers TLR4 and phosphorylated form of nuclear factor kappa B p65 subunit (p-NFκBp65). Overall, our results indicate that E-EVs play a crucial role in macrophage phenotype modulation under inflammatory conditions.

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Human iNKT Cells Modulate Macrophage Survival and Phenotype.

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