• 2018-07
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  • 2020-07
  • We first confirmed LPS induction


    We first confirmed LPS-induction of markers known to induce NF-κB signaling in Caco2BB cells, as established for numerous other cell types [31]. We initially selected this approach to monitor NF-κB activation instead of directly monitoring p65 as it is technically straightforward and provides both upstream and downstream evidence for NF-κB activation. Cultures were treated for 30min with fresh growth medium (FGM; control), LPS (400ng/ml), OT (7.8nM) or both OT and LPS (7.8nM and 400ng/ml, respectively and for all other combined treatments unless otherwise indicated). We used 7.8nM OT based on our prior studies in Caco2BB GSK2606414 [12] showing that this concentration downregulated mRNA translation after 30min. We quantified phosphorylated IκΒ (pIκB; relative to Iκb) and total IκB (with reference to GAPDH (used as a loading control). The phosphorylation of IκB causes it to dissociate from NF-κB, which allows NF-κB to initiate transcriptional programs [15]. Compared with FGM, brief LPS stimulation for 30min significantly increased pIκB:IκB by 71% and significantly reduced total IκB by 48% (Fig. 1). Thus, LPS reduced NF-κB inhibition, as a result of lower levels of IκB-bound NF-κB. In contrast, OT treatment appeared to enhance NF-κB inhibition; relative to FGM, pIκB:IκB was unchanged and total IκB GSK2606414 was increased. Combined treatment with OT and LPS did not significantly alter pIκB:IκB and total IκB levels compared with OT. However, levels of pIκB:IκB and total IκB after combined treatment were significantly reduced when compared with LPS alone (p=0.02 and p=0.002, respectively), suggesting that OT treatment counters LPS-induced phosphorylation of IκB by enhancing total IκB, which would maintain NF-κB in an inactive state. These results confirm that, in Caco2BB cells, brief LPS treatment unleashes the inflammatory transcription factor NF-κB and that OT counteracts these effects. In more recent analyses we found that NF-κB p65, relative to total protein, was higher in OT treated cultures compared to controls (by 10.68 and 41.53%) while it was lower (by 29.59 and 74.99%) than controls in LPS treated cultures (Chi square=4, p=0.0455). This fits our results that show the increase in IkB by OT versus its decrease by LPS, respectively. We explored whether the OT-activated UPR [13] accounted for the differential impact of LPS and OT upon inflammatory signaling.
    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 expression 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.