Stromal Cells in the Small Intestine: Physiology, Localization, Function, and Alterations Induced by Chronic Arsenic Exposure

Arsenic is a prevalent toxicant found to affect around 94 to 240 million individuals in over fifty countries who are exposed to levels above the EPA and WHO standard of 10 ppb. As one of WHO’s top ten chemicals of public health concern, it is linked to several chronic diseases including diabetes, obesity, cancers, and inflammatory bowel diseases. Arsenic increases inflammation and disrupts the intestinal barrier in the small intestine and colon. Further, previous studies found that arsenic decreased secretory cell lineage and stromal cell gene expression markers in the small intestine. Thus, our goal was to investigate arsenic’s effect on Pdgfrα stromal cell numbers, localization and signaling, while also observing how stromal cells change between the duodenum, jejunum, and ileum. First, thirty-six Sox9 tm2Crm-EGFP (strain B6:129S4-Sox9/J) mice were exposed to 0, 33, and 100 ppb arsenic for 13 weeks, which are environmentally relevant concentrations. Sox9 fluorescence was used as a marker for enteroendocrine cells (EECs; secretory cell lineage), transit amplifying cells (TA; immature absorptive and secretory cells), and intestinal stem cells (ISCs). A fluorescein isothiocyanate-dextran 4000 (FD-4) gavage was also conducted to assess intestinal barrier damage. From this study, duodenal tissue was collected for flow cytometry, immunohistochemistry, and RT qPCR. Flow cytometry analysis revealed an overall population decrease in Sox9 expressing cells (EECs, TA cells, and ISCs), while no changes in barrier function were observed (FD-4). qPCR revealed a significant decrease in the trophocyte marker Cd81 by 10- and 9.0-fold in males and females, respectively. IHC data discovered sex-dependent differences in response to arsenic toxicity (at 100 ppb) with telocytes (PdgfrαHi) increasing in female mice. Trophocytes (PdgfrαLo) and Igfbp5+ fibroblasts (PdgfrαLo), with their signaling protein Grem1, increased in males. PCA validated these sex-dependent changes, which also revealed sex differences may also be present between male and female controls. The results of this study led to a second exposure with thirty-eight (B6129SF2/J) mice exposed to 0, 100, and 500 ppb for 13 weeks. The goal of this study was to expand our tissue collection to include all three regions of the small intestine - the duodenum, jejunum, and ileum. We also examined expression of Igfbp5+ and Fgfr2+ fibroblasts, which are recently discovered stromal cell types that were not analyzed in the first exposure. Tissue was collected and processed for RT-qPCR, IHC, and immunoblotting from the duodenum, jejunum, and ileum. Markers used to assess changes between small intestinal regions and arsenic exposure were Pdgfrα, Cd201, Bmp4, Bmp5, Cd81, Grem1, Igfbp5+, Fgfr2+, and Olfm4. Physiologically, telocyte (PdgfrαHi expressing) numbers and Cd201 levels in males and females were reduced in the ileum compared to the duodenum; meanwhile, only male Bmp4, a telocyte signaling protein, was reduced. Gene expression of Igfbp5+ markers decreased from the duodenum to jejunum, while Fgfr2+ fibroblasts increased. Finally, IHC revealed Igfbp5 fibroblast numbers were higher in male mice in all three regions of the small intestine. As a result of arsenic exposure, arsenic seemed to target ileal stromal cells the greatest. Pdgfrα, Cd201, Cd81, Grem1, Igfbp5, and Fgfr2 gene expression changes were observed in female mice; however, these changes were not seen in males. In both males and females, telocyte (PdgfrαHi) markers were significantly increased by 6.3-fold in males and 2.3-fold in females at 500 ppb. Overall, the findings suggest arsenic affects Pdgfrα stromal cells as an attempt to maintain the ISC niche resulting in the preservation of small intestine homeostasis.