Activation of inflammatory immune gene cascades by lipopolysaccharide (LPS) in the porcine colonic tissue ex-vivo model

Abstract

The technique of challenging postmortem tissue explants with inflammation inducer such as lipopolysaccharide (LPS) followed by gene expression analysis is used widely for evaluating the immune-suppressing effect of bioactives. Using porcine colonic tissue as an ex-vivo model of mammalian intestinal gut, this study evaluated the effect of incubation time on the integrity of gene transcripts and activation of inflammatory immune gene cascade by LPS treatment. Post-slaughter colon was removed surgically and explants were incubated for 0, 3, 6 and 12 h and the abundance of mRNA transcripts of a panel of 92 immune genes were evaluated using quantitative polymerase chain reaction (qPCR) arrays. The mRNA transcripts were highly intact after 0 and 3 h of incubation; however, after 6 h the degradation was clearly evident. Following 3 h incubation, 98·8% and 100% mRNA transcripts were detectable in the colonic tissue harvested from weaned and mature pigs, respectively. In the explants of weaned piglets, LPS treatment activated inflammatory signalling pathways [high mobility group B1 (HMGB1), dendritic cell maturation, interleukin (IL)-6, IL-8, IL-17F], while these pathways were inhibited by dexamethasone treatment. Activations of inflammatory genes were also evident in the explants collected from the mature pigs subjected to ex-vivo incubation for 3 h in the absence or presence of LPS. It is concluded that the colonic explant remains physiologically viable and responsive to immunological challenge for up to 3 h ex-vivo.

Analysis of the basal colonic innate immune response of pigs divergent in feed efficiency and following an ex vivo lipopolysaccharide challenge

“…LPS is a component of the outer membrane of gram-negative bacteria that binds the CD14/TLR4/MD2 receptor complex in many cell types including monocytes, dendritic cells, macrophages, and B cells, subsequently promoting the secretion of proinflammatory cytokines (17). The effects of LPS were evident across both LRFI and HRFI pigs in this study with increases in expression of the pro-and anti-inflammatory cytokines, inflammatory transcription factors, and cytokine regulators in agreement with previous studies (3,24,34). Following the LPS challenge, the expression of GPR43, NFAM1, JAK2, TLR1, and IL17 was upregulated in the colonic tissue of LRFI compared with the HRFI pigs.…”

Activation of ectopic olfactory receptor 544 induces GLP-1 secretion and regulates gut inflammation

“…The ex vivo cultures of colonic tissue explants was established according to the previously described methods with slight modifications. 64 Briefly, WT and Olfr544-KO mice were sacrificed, and an area of the proximal colon was dissected, and the tissue was rinsed with PBS, and a section of colonic explants was maintained in a humidified cell culture incubator with 5% CO 2 at 37°C for 4 h, in DMEM buffer with added 10 μg/mL of bacterial LPS (source: Escherichia coli strain B4). During the incubation period, tissues were treated with AzA (50 μM).…”

Fucaceae: A Source of Bioactive Phlorotannins

“…Notably, Toll-like receptor 4 (TLR-4) was identified in this study as an important target for the anti-inflammatory effect of this extract [ 128 ]. It is interesting to note that dexamethasone (as shown in pig or in rat colonic tissue models) does not seem to interfere with the expression of TLR-4 [ 129 , 130 ]. However, further investigations are still needed in order to understand whether the A. nodosum anti-inflammatory bioactivity mediated through inhibition of TLR-4 expression has any distinct advantage over the inflammatory immune diseases treatments based on dexamethasone.…”