Fig 2: 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 3: Adhesion of THP-1 cells to human brain microvascular endothelial cells (HBMECs) stimulated with extracellular vesicles (EVs) is modulated by intercellular adhesion molecule 1 (ICAM-1). EVs were obtained from HBMEC grown in RPMI supplemented with extracellular vesicle-depleted fetal bovine serum (EVdS) using a differential centrifugation method. HBMECs were grown in 24-well plates and incubated with EVs isolated from cells cultured for 2 h (EV2h) or EV24h (100 ng/mL), TNF (10 ng/mL), or PBS for 24 h (A). Another group of cells was subsequently incubated for 1 h with specific antibodies against ICAM-1 (Ab-ICAM-1) and washed with PBS to remove unbound antibodies. THP-1 cells maintained in RPMI were labeled with CFSE (green) and cocultured with HBMEC at a 1:1 ratio for 1 h. The cells were then sequentially washed, fixed with 4% paraformaldehyde, stained with DAPI (blue), and evaluated under a fluorescence microscope (B). The 400× magnified image shows THP-1 cells (arrows) adhered to HBMEC (B). The adherend THP-1 cells in the absence (C) or presence (D) of Ab-ICAM-1 were counted, and the results are expressed graphically. The data were obtained from three biological replicates and five technical replicates and are expressed as the mean ± standard deviation [ANOVA/Tukey (C) t-test (D)]; *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001. The scale bar represents values of 100 μm (A) and 50 μm (B).

Fig 4: The expression of intercellular adhesion molecule 1 (ICAM-1) on human brain microvascular endothelial cells (HBMECs) is modulated by extracellular vesicles (EVs). EVs were obtained from HBMEC cultured in DMEM supplemented with EV-depleted FBS (EVdS) by differential centrifugation. HBMEC were grown in 24-well plates and incubated with EV2h, EV24h (100 ng/mL), TNF (10 ng/mL), or PBS for 24 h. A second experiment was conducted with cells incubated with extracellular vesicles isolated from cells cultured for 2 h (EV2h) or EV24h at concentrations of 0.01, 0.1, or 1.0 μg/mL. Following the incubation, both groups of cells were washed, fixed with 4% paraformaldehyde, and then incubated with a primary anti-ICAM-1 antibody. Subsequently, a secondary anti-mouse IgG antibody (H+L) conjugated to Alexa Fluor® 488 was applied (green), and the cells were stained with DAPI (blue). The cells were then evaluated under a fluorescence microscope (A). The obtained images were analyzed using specific software, and comparisons between EV2h and EV24h (B) and between different concentrations of EV2h (C) or EV24h (D) are expressed as the integrated density. The data were obtained from three biological replicates with five technical replicates each and are presented as the mean ± standard deviation (ANOVA/Tukey). Statistical significance was determined as follows: *: p-value < 0.05; **: p-value < 0.01; and ***: p-value < 0.001. Scale bar, 50 μm (A).