Figure 1. STEMprep™ Mouse Lung Dissociation Kit Achieves High Cell Viability and Yield
Mouse lung tissue was processed into single-cell suspensions using the STEMprep™ Mouse Lung Dissociation Kit and the STEMprep™ Tissue Dissociator, an alternative automated system, or a manual dissociation method. (A) Viability of total nucleated cells. (B) Yield of viable cells per whole lung tissue (251 - 495 mg). (C and D) Proportion and yield of CD45+ immune, CD31+ endothelial cells, and EpCAM+ epithelial cells. Viability, yield, and subset composition were assessed by flow cytometry. Red blood cells were lysed with ammonium chloride solution before analysis. Data are presented as mean ± SD (n = 20), * p < 0.05, one-way ANOVA with Tukey's multiple comparisons test.
Figure 2. STEMprep™-Processed Mouse Lung Macrophages Are Phagocytic and Produce Cytokines upon Activation
Mouse lung tissue was processed into single-cell suspensions using the STEMprep™ Mouse Lung Dissociation Kit and the STEMprep™ Tissue Dissociator, an alternative automated system, or a manual dissociation method. (A) Lung F4/80+ macrophages were isolated from the single-cell suspensions using EasySep™ Mouse F4/80 Positive Selection Kit. (B) The isolated F4/80+ cells were incubated for 2 hours in the presence of pHrodo™ Green-conjugated E. coli BioParticles™ at 2 - 8°C (Cold) or 37°C. The fluorescence of phagocytosed BioParticles™ was measured by flow cytometry. (C) Intracellular flow cytometry staining of TNF-ɑ production by F4/80+ macrophages cultured overnight in the presence of 3 µg/mL Brefeldin A and treated with (+) or without (-) 100 ng/mL of lipopolysaccharides (LPS). Data are presented as mean ± SD (n = 4 - 6).
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Microbial dysbiosis sculpts a systemic ILC3/IL-17 axis governing lung inflammatory responses A Kabil et al. Mucosal Immunology 2025 July
Abstract
Advancements in vaccination and sanitation have significantly reduced the prevalence and burden of infectious diseases; however, these benefits have coincided with a marked rise in autoimmune and allergic disorders. Recent studies have investigated these linked trends through the lens of host-microbiome alterations, proposing these shifts as a potential explanatory mechanism. Previously, we demonstrated that vancomycin-induced depletion of short-chain fatty acid (SCFA)-producing bacteria results in hyperactivation of ILC2s and exacerbated allergic responses. Here we investigate the effects of low-dose streptomycin on innate and adaptive immune cell populations and their activation states. Although streptomycin-treated mice exhibit normal allergic responses, they display heightened susceptibility to Th1/Th17-mediated disease, specifically hypersensitivity pneumonitis (HP). This is characterized by a two-fold increase in ILC3s and Th17 cells in the lungs, alongside activation of antigen-presenting cells (APCs) at steady state-an effect that is further amplified upon exposure to HP-inducing agents. Shotgun metagenomic analysis revealed that streptomycin-induced dysbiosis reduces microbial diversity, depletes bile acid-metabolizing bacteria, and enriches for metabolic pathways involved in branched-chain amino acid biosynthesis, including leucine-a known activator of mTORC1. Strikingly, administration of the secondary bile acid metabolite isolithocholic acid (an inverse agonist of RORγt), or an IL-23 neutralizing antibody, reverses the enhanced susceptibility to HP. Inhibition of mTORC1 significantly reduced Th17/ILC3 responses and histopathology. Our findings underscore microbial equilibrium as a key determinant of susceptibility to HP and uncover a positive feedback loop between IL and 23-producing APCs and ILC3/Th17 cells that mechanistically links dysbiosis to sustained type 3 inflammation, and we identify a simple, actionable means of intervention.
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