Effects of the pro-inflammatory cytokine TNF on functional and molecular barrier properties of porcine intestinal epithelial models | Auswirkungen des proinflammatorischen Zytokins TNF auf die funktionellen und molekularen Barriereeigenschaften von porzinen intestinalen epithelialen Modellen

One of the major proteins involved in the pathogenesis of inflammatory diseases of the intestine is tumor necrosis factor (TNF), a pro-inflammatory cytokine that can disturb epithelial barrier function by altering the composition of tight junction (TJ) proteins, such as claudins (Mullin and Snock, 1990). A disbalance of sealing and pore-forming claudins in impaired intestinal barrier function attributable to TNF is often characterized by an uncontrolled permeability to toxins and bacteria, resulting in diarrhea. Nevertheless, the detailed functional and molecular mechanisms of TNF-induced barrier disturbance remain the focus of current research in order to provide improved preventive and therapeutical approaches in the future. Because of the morphological and nutritional similarities between the gastrointestinal tract of pigs and humans, porcine models are suitable for analyzing both pig and human intestinal diseases. However, to date, only limited information regarding TNF effects on porcine epithelial barrier function is available. Therefore, the objective of the present work has been to analyze the functional and molecular impact of the pro-inflammatory cytokine on epithelial barrier function in porcine models. Subsequent to experiments, the effects of TNF on barrier function have been examined more closely, by paying special attention to TJ proteins and TNF signaling. These aspects have been assessed by using two distinct models as explained in the following. 1. The non-transformed cell line IPEC-J2 During the first study described in the present thesis, barrier-disturbing effects of TNF on the porcine intestinal epithelium were examined in vitro by using IPEC-J2 cells. The IPEC-J2 cell line, which originated from the jejunal epithelium of a neonatal piglet, constitutes a well-established model for the analysis of gastrointestinal barrier function (Schierack et al., 2006). Incubation with TNF led to a considerable disturbance of the epithelial barrier, represented by a decreased transepithelial resistance (TER) and an increased paracellular permeability to 3H-D-Mannitol. This barrier damage was accompanied by a significant reduction of the sealing TJ proteins claudin-1, -3, and occludin. Furthermore, incubation of IPEC-J2 cells with TNF led to an increase in the expression of specific TNF receptor-1 (TNFR-1), verifying the self-enhancing inflammatory effect under TNF treatment. Use of ML-7, a specific blocker of the myosin light chain kinase (MLCK), significantly prevented effects on TER and alterations in claudin-3 and TNFR-1 expression. Moreover, cells recovered from TNF-induced barrier loss, as 48 h after removal of the cytokine the TJ proteins claudin-1 and occludin were integrated once again at the functional lateral membrane of the cells. In addition, no significant changes in an ApoTox-Glo assay were observed, excluding an induction of apoptosis by TNF in IPEC-J2 cells. The obtained data from this study explain (i) the barrier-disturbing effect of TNF on TJ composition, (ii) the involvement of MLCK in TNF-induced signaling in IPEC-J2, and (iii) the up-regulation of the specific TNFR-1, underlining the exponential nature of inflammation. 2. The follicle-associated epithelium of porcine Peyer’s patches The second part of the present work examined the effects of TNF on porcine intestinal tissue samples ex vivo by using the Ussing chamber technique. The experiments were conducted by using two different types of tissue, one being the immunological active Peyer’s patch (PP), and the other one being the neighboring villus epithelium (VE), both obtained from adult pigs at a slaughterhouse. The PP is covered by the follicle-associated epithelium (FAE), whose primary purpose is to limit the uptake of antigens from the intestine, and which therefore requires an efficient epithelial barrier (Jung et al., 2010). As one major restriction of the Ussing chamber technique is the experimental time limitation (Pearce et al., 2018), an improved setup was established to analyze the effects of TNF on PP and VE for up to 10 h. An incubation of PP with TNF led to a significant decrease in TER after 8 h, whereas the paracellular flux to 3H-D-Mannitol was unaltered. Western blot analysis and immunostaining revealed a decrease of sealing claudin-1 and -4, whereas the pore-forming claudin-2 was significantly up-regulated and functionally integrated into the TJ structure. In contrast to in vitro experiments, a significant increase of TNFR-2 could be observed in PP FAE after addition of TNF. In adjacent VE, neither TER alterations, nor effects on TJ proteins or TNF receptors could be observed after incubation with the cytokine. This highlights the important role of FAE in intestinal mucosal defense.