Epidermal growth factor receptor tyrosine kinase inhibitors (EGFr TKIs) are first‐line therapies for various cancers, and cause dose‐limiting severe diarrhea in many patients. We hypothesized that diarrhea caused by EGFr TKIs might reflect actions on epithelial transport, barrier function, or both, which we tested using cell cultures including murine and human enteroid‐derived monolayers (EDMs), analyzed using electrophysiological and other relevant methods. EGFr TKIs (such as afatinib, erlotinib, and osimertinib) reversed the acute inhibitory effect of EGF on chloride secretion induced by carbachol (CCh) across T84 human colonic epithelial cells, which correlated with the diarrhea‐inducing effect of each agent clinically. EGFr TKIs also reduced transepithelial electrical resistance (TEER), whereas co‐treatment with CCh delayed the decrease in TEER compared with that of cells co‐treated with EGF. Furthermore, afatinib and erlotinib prevented EGF‐ or CCh‐induced EGFr phosphorylation. EGFr TKIs also suppressed phosphorylation of extracellular signal‐regulated kinase (Erk)1/2 in response to EGF, whereas they had weaker effects on CCh‐induced Erk1/2 phosphorylation. In human EDMs, EGF potentiated ion transport induced by CCh, whereas afatinib reversed this effect. The ability of EGFr TKIs to reverse the effects of EGF on calcium‐dependent chloride secretion could contribute to the diarrheal side effects of these agents, and their disruption of epithelial barrier dysfunction is likely also pathophysiologically significant. CCh‐activated Erk1/2 phosphorylation was relatively insensitive to EGFr TKIs and delayed the deleterious effects of EGFr TKIs on barrier function. These findings confirm and extend those of other authors, and may be relevant to designing strategies to overcome the diarrheal side effects of EGFr TKIs.
Aims
At conception, the infant gut barrier is immature, gradually developing with regular intake of maternal milk. This study addressed whether the barrier-strengthening effect of breast feeding might be attributable, at least in part, to autochthonous beneficial human milk bacteria.
Methods and results
Twelve bacterial strains from the breast milk of Pakistani mothers who underwent cesarean delivery (NPL-88, NPL-157, NPL-179, NPL-181, NPL-388 (Limosilactobacillus reuteri), NPL-76, NPL-495, NPL-504 (Limosilactobacillus fermentum), NPL-415 (Lactobacillus pentosus), NPL-412, NPL-416 (Lactiplantibacilllus plantarum) and NPL-374 (Bifidobacterium longum) were shortlisted based on their tolerance to acidic pH (2.8–4.2) and bile (0.1–0.3%). The effect of these bacteria on gut barrier function in the presence and absence of pathogens was assessed as changes in transepithelial electrical resistance (TEER) in the human T84 colonic epithelial cell line and in murine enteroid-derived monolayers (EDMs). The TEER of T84 cells monolayers rose in the presence of most of the human milk strains, being most pronounced in case of L. reuteri NPL-88 (34% within five h), exceeding the effect of the well-known probiotic L. acidophilus (20%). qRT-PCR, western blot and immunofluorescent staining associated the increase in TEER with enhanced expression of tight junction proteins. Pretreatment of murine EDMs with NPL-88 also largely prevented the ability of the pathogen, Salmonella, to decrease TEER (87 ± 1.50%; P < 0.0001, n = 4).
Conclusions
Human milk lactic acid bacteria are potential probiotics that can strengthen gut barrier function and protect breastfed neonates against enteric infections.
Salmonella enterica serovar Typhimurium is one of the most burdensome foodborne diarrheal pathogens worldwide. However, despite its prevalence, the mechanism by which Salmonella causes diarrhea is not entirely known. We have shown in mice that Salmonella infection decreases expression of SLC26A3 (Down‐Regulated in Adenoma; DRA), a chloride/bicarbonate exchanger. In order to understand the mechanism of this effect, we employed an enteroid model from murine colon. Enteroid‐derived monolayers (EDM) that recapitulate both the absorptive and secretory cell lineages of the native intestinal epithelium (enterocytes vs. enteroendocrine, goblet, and Paneth cells, respectively) were infected apically with Salmonella and relevant protein expression was studied using qRT‐PCR and western blot. Infection significantly reduced expression of DRA (40%, p = 0.0124) and Hes1 (45%, p = 0.0004) while upregulating expression of ATOH1 (3‐fold, p = 0.0292) and mucin 2 (Muc2) (2‐fold, p = 0.0134). Hes1 is a Notch pathway downstream signaling molecule and thus a precursor to the absorptive epithelial lineage that expresses DRA. ATOH1, on the other hand, is a Wnt pathway downstream signaling molecule and a precursor to secretory lineages, including goblet cells that express Muc2. Infection of EDMs also reduced levels of the Notch intracellular domain. The involvement of Notch was further investigated by inhibiting Notch signaling using a γ‐secretase inhibitor, which reproduced the downregulation in Hes1 and DRA and upregulation in ATOH1 and Muc2 as seen with infection. Because the balance of Wnt and Notch signaling determines relative intestinal epithelial differentiation, our findings suggest that the diarrheal pathogenesis of Salmonella may reflect Notch inhibition and an accompanying shift in epithelial differentiation from absorptive to secretory cell types, a decreased capacity for absorption, and thus the accumulation of diarrheal fluid.
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