Digestive proteases in villous CD68⁺ LP macrophages locally degrade transferrin in cre/+ mice. (A) Representative image of immunofluorescent staining for CD68 (macrophage activation marker) on mouse duodenum cryosections obtained from control (+/+) and cre/+ mice. Scale bar, 100 μm. Quantification of CD68 mean fluorescence intensity in the LP (∗P < .05). (B) Representative image of immunohistochemistry staining for pS6 (phosphorylated S6 ribosomal protein [Ser240/244]), mTORC1 signal activation marker) on FFPE sections obtained from +/+ and cre/+ mice, and its quantification in the LP. Scale bar, 25 μm (∗∗∗∗P < .0001). Arrows indicate LP region with macrophages. (C-F) Single-cell RNA-seq analysis of CD68⁺ cells: Umap plots of gene expression levels in CD68⁺ cell clusters (C) and differentially expressed genes between cre/+ and +/+ mice (D). Significantly enriched pathways in GO terms, category cellular components with differentially expressed genes from cluster 1 (E). Heatmap of identified genes in enriched GO pathways (F). (G) Representative image of immunofluorescent staining for ATP6V0D2 (vATPases) on mouse duodenum cryosections obtained from control (+/+) and cre/+ mice. Scale bar, 100 μm. (H) Flow cytometry quantitative analysis of CD71 (Trf1, transferrin receptor) mean fluorescence intensity of LP macrophages (CD64⁺ Ly6C⁻ CD11b⁺ CD11c^int) (I) and of pH-sensitive pHrodo-Red Tf uptake in LP macrophages from +/+ and cre/+ mice. Histogram of pHrodo-Red Tf⁺ LP macrophages compared between +/+ and cre/+ mice with or without lysosomal pH-disrupting drug hydrochloroquine. (J) Representative images of chymotrypsin enzyme activity on duodenum cryosections with an activity-based probe (ABP; fluorophore-labeled covalent inhibitor). Scale bar, 100 μm. Quantification of enzyme activity based on mean fluorescence intensity in the duodenal villi. (K) Representative image of immunofluorescent staining for Tf in LP of control (+/+) and cre/+ mice after protease inhibitor nafamostat (FUT-175) treatment for 1 week. Quantification of Tf mean fluorescence intensity in the LP (∗P < .05; ∗∗∗P < .001). Scale bar, 50 μm. (L) Tf saturation was determined in serum of mice treated with protease inhibitor nafamostat (FUT-175) for 1 week (∗P < .05). Control animals were treated with 1× PBS.