Fig 1: A conserved TNFA-CXCL8 axis in β cells under ER stress(A) Representative RNAscope images of insulin, mpeg, and cxcl8a expression at 64 and 66 h zMIR fish. A yellow line in each image outlines the islet, and a red line outlines macrophages. Scale bars, 20 μm.(A′) Quantification of cxcl8a signals in the islet area (outlined in yellow) from RNAscope images at 64 and 66 h in zMIR fish. Unpaired t test; bar graphs represent mean ± SEM; n = 5/group, ***p < 0.001.(B) Representative RNAscope images of insulin, mpeg, and cxcl8a expression at 66 h in tnfa−/−, zMIR and control zMIR fish. Scale bars, 20 μm.(B′) Quantification of cxcl8a signals in the islet area (outlined in yellow) from RNAscope images at 66 h in tnfa−/−, zMIR, and control zMIR fish. Unpaired t test; bar graphs represent mean ± SEM; n = 5/group; ***p < 0.001.(C) Islet qRT-PCR analysis of tnfa at 66 hpt in irf8−/−, zMIR, and control zMIR fish. Data are mean ± SEM; n = 3/group; two-way ANOVA followed by Tukey’s multiple comparisons test; ***p < 0.001.(D) Islet qRT-PCR analysis of islet cxcl8a expression at 66 hpt in irf8−/−, zMIR, and control zMIR fish. Data are mean ± SEM. n = 3/group, two-way ANOVA followed by Tukey’s multiple comparisons test; ***p < 0.001.(E) Effect of TNFA treatment on CXCL8 expression in EndoC-H1 cells cultured under high-palmitate (0.5 mM) and glucose (25 mM) conditions (pal&glu). Data are mean ± SEM. n = 6/group, one-way ANOVA followed by Tukey’s multiple comparisons test; ***p < 0.001.(F) Effect of TNFA treatment on expression of Cxcl1, Cxcl2, Cxcl5, and Cxcl15 in MIN6 cells under pal&glu conditions. Data are mean ± SEM. n = 4/group, one-way ANOVA followed by Tukey’s multiple comparisons test; **p < 0.01, ***p < 0.001.(G) Effect of the TNFA-neutralizing antibody on Cxcl15 expression in MIN6 cells cultured in Raw264.7 conditioned medium. The schematic shows generation of Raw264.6 conditioned medium for MIN6 cells. Neutralized TNFA significantly decreases induction of cxcl15 expression under conditioned medium in MIN6 cells. Data are mean ± SEM. n = 4/group, multiple t tests; *p < 0.05.
Fig 2: Macrophage-derived Tnfa is necessary for β cell loss in zMIR fish(A) Intra-islet macrophage number in zMIR fish from 64–72 h based on 30 s/frame time-lapse videos hourly. Data represent means ± SEM, n ≥ 10 per group, multiple t tests; *p < 0.05, ***p < 0.001.(B) Macrophage surveillance of the principal islet during 65–66 hpt in zMIR and control fish, showing that macrophage-contacted β cells remained intact. Live imaging videos were recorded at 30-s intervals. Yellow arrows indicate a macrophage-contacted β cell. Scale bars, 20 μm.(C) Representative RNAscope images of insulin, mpeg1.1, tnfa expression at 64 h in control and zMIR fish. Scale bars, 20 μm.(D) Islet images of TgBAC(tnfa:GFP), Tg(ins:H2B-mCherry), zMIR fish at 64 and 66 h. The GFP signal was detected by immunofluorescence. Scale bars, 20 μm.(E) Quantification of GFP signals in the islet area (outline in D). Data are mean ± SEM, n = 10/group, unpaired t test; bar graphs represent mean ± SEM; ***p < 0.001.(F) The effect of tnfa re-expression in macrophages on β cell loss in zMIR fish. Data are mean ± SEM, n > 15/group, two-way ANOVA followed by Tukey’s multiple comparisons test; ***p < 0.001.(G) Representative images showing increased intra-islet macrophages in zMIR fish with different tnfa genotypes (+/+, −/−, and −/− with macrophage rescue). Red marks β cells, and white labels macrophages. Scale bars, 10 μm.(H) Quantification of intra-islet macrophages in zMIR fish with different tnfa genotypes (+/+, −/−, and −/− with macrophage rescue) at 66 hpt. Data are mean ± SEM, n = 10 per group, two-way ANOVA followed by Tukey’s multiple comparisons test; ***p < 0.001.
Supplier Page from Thermo Fisher Scientific for Recombinant Mouse TNF-alpha (ELISA Standard) Protein