br Discussion We have demonstrated that ER stress induced se

Discussion We have demonstrated that ER stress-induced sensors and mediators of the UPR are stimulated by OT-alone or in combination with LPS, whereas many of these same markers are inhibited by LPS alone, as summarized in Table 1. The dynamic upregulation of OTR Lithocholic Acid mg in the gut during the milk suckling period [5] may protect enterocytes by opposing LPS-induced UPR silencing and also play a fundamental role in enterocyte differentiation secondary to changes in cellular metabolism [21], which may account for the gut phenotype in OTR deficient mice [6]. The experimental system is meant to model the exposure of rat newborn enterocytes to the normal physiological intestinal intake of microbial flora and mother\'s milk. Upon vaginal birth, the neonate is exposed to bacteria derived from both the birth canal and large intestine, which include Gram negative species that specifically supported by glycans in breast milk and are rich in both the breast milk and the neonatal gut [36]. This represents a main source of LPS exposure of developing enterocytes in vivo and supports the germane nature of our study of LPS, as opposed to other bacterial triggers. Milk contains oligosaccharides that inhibit LPS-induced inflammation [37], and we show that OT (as one component of milk) can physiologically inhibit the inflammatory signals of LPS physiologically encountered by newborn-type enterocytes. However, we cannot estimate the quantity of LPS encountered by these newborn type enterocytes, which is a limitation of our interpretation. LPS contains lipopeptide traces that can target TLR2, according to our LPS supplier, InvivoGen. Therefore, with low TLR4 expression, TLR2 may substitute for the opsonizing complex that consists of LPS binding protein and myeloid differentiation protein-2+TLR4 in a non-enterocyte membrane [14], [15]. In our experiments TLR2/4 signaling is mostly likely responsible for LPS-elicited increases in pIκB [14], [15], rather than IKK (inhibitor of IκB kinase) secondary to IRE1a activation [38], as LPS reduced pIRE1a levels. Furthermore, we also ruled out IκB degradation and NF-κB activation by IKK secondary to p-eIF2a signaling (via PERK activation) and/or pIRE1a-mediated, concerted activation of TRAF2, JNK and IKK [38] because LPS alone did not induce markers of ER stress with our experimental parameters, whereas OT induced ER stress sensors, apart from pPERK. Our results indicate an OT-specific activation of PKR that inactivates eIF2a and may, by this means, reduce protein translation by utilizing only select sensors and mediators of the UPR (i.e., avoiding significant PERK activation). Interferon is a PKR activator [32] that temporarily subdues cellular and viral mRNA translation without killing the host cell [33]. Interferon transcription can be induced by XBP1s [39]; Interferon may thus allow for eIF2a inactivation via PKR after OT treatment. Our observation of minimal PERK activation agrees with Martinon et al. [39] who demonstrated that LPS-TLR2/4 stimulation of macrophages repressed PERK and IRE1a activation in combination with tunicamycin, which by itself activated these ER-sensors and who suggest that bacterial endotoxin-induced ER-stress specifically utilizes IRE1a and XBP1s, but not ATF6 and PERK. However, this study only detected XBP1 splicing with 3h LPS stimulation (vs. our 30min).