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Summary: Microglial inflammation driven by the NACHT, LRR and PYD domain‐containing protein 3 (NLRP3) inflammasome contributes to brain disease and is a therapeutic target. Most mechanistic studies on NLRP3 activation use two‐dimensional pure microglial cell culture systems. Here we studied the activation of the NLRP3 inflammasome in organotypic hippocampal slices, which allowed us to investigate microglial NLRP3 activation in a three‐dimensional, complex tissue architecture. Toll‐like receptor 2 and 4 activation primed microglial inflammasome responses in hippocampal slices by increasing NLRP3 and interleukin‐1β expression. Nigericin‐induced NLRP3 inflammasome activation was dynamically visualized in microglia through ASC speck formation. Downstream caspase‐1 activation, gasdermin D cleavage, pyroptotic cell death and interleukin‐1β release were also detected, and these findings were consistent when using different NLRP3 stimuli such as ATP and imiquimod. NLRP3 inflammasome pathway inhibitors were effective in organotypic hippocampal slices. Hence, we have highlighted organotypic hippocampal slice culture as a valuable ex vivo tool to allow the future study of NLRP3 inflammasomes in a representative tissue section, aiding the discovery of further mechanistic insights and drug development. [ABSTRACT FROM AUTHOR]

Summary: Monocyte‐derived macrophages (MDMs) generated from peripheral blood monocytes are widely used to model human macrophages for in vitro studies. However, the possible impact of different isolation methods on the resulting MDM phenotype is poorly described. We aimed to investigate the effects of three commonly used monocyte isolation techniques on the resulting MDM phenotype. Plastic adhesion, negative selection, and CD14pos selection were compared. Monocyte‐derived macrophages were generated by 5‐day culture with macrophage and granulocyte–macrophage colony‐stimulating factors. We investigated monocyte and MDM yields, purity, viability, and cell phenotype. CD14pos selection resulted in highest monocyte yield (19·8 × 106 cells, equivalent to 70% of total) and purity (98·7%), compared with negative selection (17·7 × 106 cells, 61% of total, 85·0% purity), and plastic adhesion (6·1 × 106 cells, 12·9% of total, 44·2% purity). Negatively selected monocytes were highly contaminated with platelets. Expression of CD163 and CD14 were significantly lower on CD14pos selection and plastic adhesion monocytes, compared with untouched peripheral blood mononuclear cells. After maturation, CD14pos selection also resulted in the highest MDM purity (98·2%) compared with negative selection (94·5%) and plastic adhesion (66·1%). Furthermore, MDMs from plastic adhesion were M1‐skewed (CD80high HLA‐DRhigh CD163low), whereas negative selection MDMs were M2‐skewed (CD80low HLA‐DRlow CD163high). Choice of monocyte isolation method not only significantly affects yield and purity, but also impacts resulting phenotype of cultured MDMs. These differences may partly be explained by the presence of contaminating cells when using plastic adherence or negative selection. Careful considerations of monocyte isolation methods are important for designing in vitro assays on MDMs. [ABSTRACT FROM AUTHOR]

Mast cells are proposed to be one of the targets for mucosal vaccine adjuvants. We previously demonstrated that mucosal adjuvants containing IgG immune complexes could activate connective tissue mast cells enhancing immune responses. Here we suggest that mucosal mast cells (MMC) may also contribute to augmentation of antigen-specific immune responses following treatment with antigens complexed with IgG. We demonstrated that both bone marrow-derived cultured MMC and tissue resident MMC incorporated ovalbumin (OVA) at a greater level in the presence of anti-OVA IgG. Co-culture of OVA/IgG-pulsed bone marrowderived MMC with splenocytes from OT-II mice promoted OVA-specific activation and proliferation of T cells, a process known as cross-presentation. Furthermore, bone marrow-derived cultured MMC underwent apoptosis following treatment with IgG immune complexes, a feature that has been described as favouring phagocytosis of mast cells by professional antigen-presenting cells. [ABSTRACT FROM AUTHOR]