Fig 2: Pro-inflammatory ß cell small EVs activate the CXCL10/CXCR3 axis to induce ß cell dysfunction(A) Proximity ligation assay (PLA) was used to determine CXCL10 (ligand):CXCR3(receptor) in situ binding. Shown are immunofluorescence images of dispersed mouse islets treated with CYTO, cytoEVs, cytoEV+CXCL10 neutralizing antibody (25 µg/mL), or cytoEV+AMG487 (1 µM, a CXCR3 receptor antagonist) for 48 h versus controls (-EV and –antibodies). Green punctatedots represent CXCL10/CXCR3 interactions, red represents insulin, and DAPI (blue) nuclear stain, imaged at 40× (scale bars, 10 µm).(B) Representative high-magnification images at 160× (scale bars, 50 µm) of CXCL10/CXCR3 interactions in single ß cells treated with cytoEVs or cytoEV+AMG487 for 48 h versus control (-EVs).(C) Average PLA particles/ß cell (CXCL10/CXCR3 interactions) quantified for each condition using ImageJ software (islets were isolated from 3 C57BL/6L mice, and 5–7 independent images containing ~11 ß cells/condition were examined).(D and E) GSIS assessed by static incubation at 4 mM and 16 mM glucose and corresponding insulin stimulation index (expressed as insulin release during 30 min at hyperglycemic 16 mM glucose over basal 4 mM glucose concentration) in isolated mouse islets. Treatments were for 48 h with cytoEVs or cytoEV+AMG487 versus UT (UT and cytoEV data were taken from Figure 2E for comparison; islets were isolated from 3 C57BL/6L mice, with 11 independent experiments for cytoEV+AMG487 treatment). Note restoration of (D) GSIS and (E) stimulation index (~2-fold, green bars).(F) mRNA expression assessed by qRT-PCR analysis of the ß cell identity genes Ins1, Glut2, and Pdx1 and the inflammatory genes Cxcl10, Nf?b1, and Stat1 in isolated islets treated with cytoEVs or cytoEV+AMG487 versus UT (islets were isolated from 8 C57BL/6L mice, with 2–7 independent experiments per given condition).All values are a mean ± SEM. Statistical significance among groups is indicated by *p < 0.05.

Fig 3: Schematic illustration of experimental design. a Labeling of genetically modified MSCs with TAT-CPNPs. Aiming to screen genes that promote the targeting migratory ability of MSCs, Cxcr3, whose ligand was up-regulated in the CHS animal model, was selected as a candidate gene and transduced into MSCs with lentivirus. To visualize gene modified MSCs via NIR-II PA imaging, TAT-CPNPs with high absorbance at 1064 nm were fabricated to label modified MSCs. b Longitudinal PA imaging of non-inflamed or inflamed ears in the CHS mouse model for assessing the targeting migratory ability of TAT-CPNPs labelled MSCs. TAT-CPNPs labeled MSCs were intravenously delivered to CHS mice, PA imaging at 1064 nm of non-inflamed or inflamed ears were performed at different time points after injection. The accumulation within targeted tissues and extravasation of MSCeGFP and MSCCxcr3 were analyzed

Fig 4: CXCR3 positive macrophages were located near the MES. (A) Representative immunofluorescent histochemical double staining for F4/80 (red) with CXCR3 (green) in HO-2 KO section (E16). Nuclear staining with DAPI (Blue). The mesenchyme demonstrates the presence of a F4/80 positive HO-2 KO macrophages, which also express CXCR3 (white arrows) (magnification: x200). One CXCR3 F4/80 positive HO-2 KO macrophage was located near the disintegrating MES (white arrow within the white square). (B) Magnification of a F4/80 positive HO-2 KO macrophage located near the MES (area between the dotted white lines) (magnification: x400). (C) Magnification of a F4/80 positive HO-2 KO macrophage located in the mesenchym of the palate shelf (magnification: x400). (D) Magnification of a F4/80 positive HO-2 KO macrophage located outside the palatal shelf (magnification: x400).

Fig 5: Palatal shelf surface measurement for determining the number of CXCL11, CXCR3 and HO-1 positive immunostained cells cells/mm2 within the mesenchyme of the palatal shelves. A square scale bar was drawn in the microscopic picture (magnification: x100) of the section of 1,000 × 1,000 µm (1 mm2) and the total number of pixels within the square was determined (e.g. 1 mm2 = 3,442,880 pixels). The contour of the mesenchyme of the shelves was drawn (yellow line). The number of pixels for this area was determined by the ImageJ (1.48 v) software (323,768 pixels). The number of positive immunostained cells within this mesenchymal area of the palatal shelves were counted by direct observation using the Zeiss microscope (e.g. 52 CXCL11 positive cells). The number of cells/mm2 was calculated (3,442,880/323,768 × 52 = 553 cells/mm2).