Fig. 6: Cellular dosimetry of fgTiO₂ across timepoints and Salmonella-induced migration.

a, b, k Confocal reflectance micrographs showing the subepithelial dome (SED) tissue region (n = 3 animals per group). After the 16-week fgTiO₂ feeding period, all animals were switched to a normal diet (without fgTiO₂ supplementation) and half were orally dosed with ΔaroA-Salmonella before tissue harvest at +3 or +28-day timepoints. a, b In the animals fed the fgTiO₂-supplemented diet without Salmonella exposure (WT +TiO₂), fgTiO₂-loaded cells were present in the SEDs at both +3 and +28 timepoints. c–j To gain insights into cellular dosimetry changes across the two timepoints, image analysis of Peyer’s patch transverse sections was used to measure the number of reflectant foci per-cell (i.e., the fgTiO₂-loaded vesicle (TLV) count per-cell) and the amount of reflectance per foci (i.e., equivalent to the fgTiO₂ dose per-vesicle). d, e At the +28 timepoint, the TLV count per-cell was elevated with a new population of cells with TLV counts >13 forming in the mid-to-base regions of the follicle. f, g Randomly sampling and montaging the reflectance (RL) information in cells from each of the two timepoints visually demonstrates this, showing (g) more heavily-loaded cells at the +28 timepoint. h Moving from cell to vesicle information, in contrast, the amount of reflected light per individual foci remained near-identical across the two timepoints and this was again borne out visually when (i, j) individual TLVs were randomly montaged. Collectively, the results suggest that once fgTiO₂-loaded cells move below the SED, there is a process of vesicular inheritance by certain cells over time, eventually yielding heavily-loaded pigment cells at the follicle base–as is well-described in humans (Fig. 1). k Challenge with ΔaroA-Salmonella (WT +TiO₂ +Sal) markedly changed this picture (n = 4 animals per group). At the +28 timepoint, SEDs were almost completely devoid of both fgTiO₂ and autofluorescence-positive cells. Out of four SEDs imaged, only one reflectant foci was detectable (indicated by arrow) showing marked migration of fgTiO₂ from the SED tissue compartment after ΔaroA-Salmonella exposure. Scale bars: a, b, k = 50 μm, c, d = 250 μm, f, g, i, j = 10 μm.