Journal: Frontiers in Immunology

Article Title: Lung epithelial and alveolar macrophage-like cell interactions significantly modify innate responses to bacterial endotoxin with the involvement of direct cellular contacts, TNF-α, ICAM1 and MCP-1

doi: 10.3389/fimmu.2025.1715943

Figure Lengend Snippet: MLE-12 cells produce LBP for enhanced co-culture LPS response and co-culture of lung epithelial cells and MPI macrophages increases the production of LBP independent TLR2 and allergen stimulated cytokines. LBP production in MLE-12 and MPI cultures, or co-cultures containing both cell types, when left unstimulated or after exposure to LPS (A) . Cells were stimulated with LPS and LBP was quantified at 24h post treatment via ELISA. Production of IL-6, MCP-1, and TNF-α in response to FSL-1 (Pam2CGDPKHPKSF) (B) or IL-6 production in response to Cockroach extract allergen (C) . Cells were stimulated and cytokines were quantified at 24h post treatment via ELISA. Statistical significance between mono-cultures and co-cultures were determined by Prism software using the One-Way ANOVA followed by Dunnett’s post hoc test. **p-value < 0.01; ***p-value < 0.001. ns was used to indicate when the differences were found to be non-significant.

Article Snippet: Response to FSL-1 (Invivogen) and Cockroach Extract (Greer Laboratories) was determined by exposing the cells to varying concentrations of these stimulants for 24h.

Techniques: Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: Nature

Article Title: Bidirectional CRISPR screens decode a GLIS3-dependent fibrotic cell circuit

doi: 10.1038/s41586-025-09907-x

Figure Lengend Snippet: (a) Secreted IL-11 after co-culture of fibroblast knockouts for TGFB1 - and IL1B -related ligands or receptors with TLR2/6-activated macrophages (top) or knockouts in activated macrophage knockouts with fibroblasts (bottom). n=3 cell lines per condition. (b) Z-score heatmap of IAF genes in TGFBR1/2 , IL1R1 CRISPRko fibroblasts co-cultured with TLR2/6-activated macrophages, normalized to HPRT. n=3 cell lines per condition. (c) qPCR of gene knockouts normalized to HPRT . Macrophages: IL-4/IL-13 (10 ng/mL); FSL-1 (10 ng/mL) + ATP (5 mM). Fibroblasts: media. n=2 cell lines per condition. (d) Secreted TGF-β and IL-1β after co-culture of primary human macrophages and fibroblasts (24 hours) ( Methods ). n=3 cell lines per condition. (e) Secreted IL-11 from primary colonic fibroblasts stimulated with TGF-β and/or IL-1β (10 ng/mL, 24 hours). Dashed lines: additive or synergistic response ( Methods ). One-way ANOVA with Tukey’s multiple-comparisons test. n=3 cell lines per condition. (f) Left: immunofluorescence of Il11 mNG colons after intraperitoneal injection with IgG control or dual anti-TGF-β and anti-IL-1β antibodies (100 μL in PBS, 100 μg/mouse). Right: IL-11 mNG cell percentage in all DAPI-imaged cells from two pooled independent experiments. Mice (co-housed, 13-20 weeks) were treated with chronic DSS (2.0%, 35 days). n=7 mice per condition. (g) qPCR quantification of Il11 from lysates from (f) normalized to Eef2 . Kruskal-Wallis test with Dunn’s multiple-comparison test. (h) Total colonic collagen percentage quantification from (f). (i) Quantification of colonic hydroxyproline normalized to total protein from lysates from (f). (j) Percent starting weight of mice from (f). Filled lines represent s.e.m. Linear mixed-effects analysis with Dunnett’s multiple comparison test. (k) Histopathological scoring ( Methods ) of H&E-stained tissues from (f). (l) Colon length measurements from (f). Unless otherwise stated, statistics are by a one-way ANOVA with Dunnet’s multiple comparison test on distinct biological replicates and error bars are the mean ± s.e.m. ns, not significant.

Article Snippet: Cells were then washed, trypsinized, and seeded onto the labeled macrophages along with the macrophage-activating ligand FSL-1 (1 ng/ml) (Invivogen, #tlrl-fsl) overnight.

Techniques: Co-Culture Assay, Cell Culture, Immunofluorescence, Injection, Control, Comparison, Staining