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    • Enterocytes sample bacterial products such as lipopolysaccha

    2020-07-31

    Enterocytes sample bacterial products, such as lipopolysaccharide (LPS), with Toll-like receptors (TLRs). These receptors activate both the canonical pro-inflammatory transcriptional program mediated by nuclear factor kappa-light-chain-enhancer of activated C34 synthesis (NF-κB) [14], [15] and the UPR [16]. Inflammation stress can result in chaperone failure and subsequent accumulation of misfolded protein cargo in the ER, which is often a prelude to apoptosis [17]. ER stress under these conditions may initiate the UPR transcriptional program, which limits mRNA translation except for selected proteins (e.g., chaperones) to preserve the ER by clearing misfolded proteins [18]. At least three proteins are anchored in the ER membrane that sense stress and activate the UPR. These include (1) ATF6 (activating transcription factor 6), (2) PERK (protein kinase RNA-like endoplasmic reticulum kinase) and (3) IRE1a (inositol requiring enzyme 1a) [19]. The ultimate outcome of the UPR, whether it is cell preservation or the initiation of apoptosis to limit organ damage, is dependent upon the severity of the initiating pathological process. For example, the activation of PERK by severe, prolonged stress, may inactivate its substrate, the eukaryotic initiation translation factor 2a (eIF2a), but also activate the pro-apoptotic C/EBP homologous protein (CHOP) transcription factor [19]. On the other hand, a splice variant of the UPR, mediated by IRE1a and spliced X-box binding protein 1 (XBP1s), may constitutively maintain the quality of protein folding [20]. In this study we assessed the impact of OT-treatment upon UPR signaling molecules during or after exposure to LPS, which mimics bacterial endotoxin ingestion with breast milk, on enterocytic Caco2BB cells. Protein extracts of cells stimulated in vitro were assayed using automated immunocapillary electrophoresis. We show that OT activated select UPR mediators and inhibited and partially reversed LPS-induced inflammation. UPR induction by OT-rich milk may precondition enterocytes in the developing mammal to resist cellular stress associated with microbial colonization and may also impact enterocyte differentiation through effects on cellular metabolism [21]. OT may also protect other cell types, such as neurons, from stress-related complications during postnatal development.
    Materials and methods
    Results Our experimental conditions were optimized to coordinate the temporal parameters of NF-κB activation and the UPR to the time course of relevant intracellular signaling in response to OT that we have previously published [12], [13], [24]. This included physiological, and not necessarily pathological, LPS stimulation (e.g., lower dose and shorter duration of exposure vs. pathological conditions of 6–72h with a higher dose [27], [28], [29], [30]) so that we could assess signaling after 30min of exposure to OT and/or LPS. 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 cells [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 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.