Abstract
The intestine fulfills roles in the uptake of nutrients and water regulation and acts as a gatekeeper for the intestinal microbiome. For the latter, the intestinal gut barrier system is able to respond to a broad range of bacterial antigens, generally through Toll-like receptor (TLR) signaling pathways. To test the capacity of various in vitro intestinal models, we studied IL-8 secretion, as a marker of pro-inflammatory response through the TLR pathway, in a Caco-2 monoculture, Caco-2/HT29-MTX di-culture, Caco-2/HT29-MTX/HMVEC-d tri-culture and in a HT29-p monoculture in response to exposure to various TLR agonists. Twenty-one-day-old differentiated cells in Transwells were exposed to Pam3CSK4 (TLR1/2), lipopolysaccharide (TLR4), single-stranded RNA (TLR7/8), Poly(i:C) (TLR3) and flagellin (TLR5) for 24 h. In all systems IL-8 secretion was increased in response to flagellin exposure, with HT29-p cells also responding to Poly(I:C) exposure. All other agonists did not induce an IL-8 response in the tested in vitro models, indicating that the specific TLRs are either not present or not functional in these models. This highlights the need for careful selection of in vitro models when studying intestinal immune responses and the need for improved in vitro models that better recapitulate intestinal immune responses.
Highlights
The intestine fulfills many roles such as digestion of food, nutrient uptake, water regulation and it acts as a gatekeeper for the intestinal microbiome (Silverthorn et al, 2016)
In the Caco-2 mono culture, following exposure to the Toll-like receptor (TLR) agonist flagellin, we observed a significant increase in the apical IL-8 concentration (p < 0.01) (Fig. 3)
We aimed to evaluate the potential of in vitro intestinal models with increasing cellular complexity to study immunomodulation induced by exogenous factors by measuring their IL-8 responses to a broad range of bacterial antigens
Summary
The intestine fulfills many roles such as digestion of food, nutrient uptake, water regulation and it acts as a gatekeeper for the intestinal microbiome (Silverthorn et al, 2016). Monolayers of differen tiated Caco-2 cells are commonly used as a model to assess the potential transport of compounds from the intestine into the systemic blood cir culation. This Caco-2 cell-layer model has shown a high predictability towards intestinal transport in vivo for certain compounds (Artursson et al, 2001). This model has been used to study direct effects of chemicals on the intestinal epithelium and to study in teractions between the epithelium and antigens originating from bacteria residing in the human intestine (Sadabad et al, 2015; Tang et al, 1993)
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