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Impaired Tricellular Tight Junctions Open the Barrier for Lipopolysaccharide Uptake
Author(s) -
Krug Susanne M.,
Lee In-Fah M.,
Weiß Franziska,
Hu Jia-Chen E.,
Fromm Michael,
Schulzke Jörg D.
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.03855
Subject(s) - paracellular transport , tight junction , lipopolysaccharide , microbiology and biotechnology , chemistry , transcellular , inflammation , phagocytosis , peptidoglycan , epithelium , immune system , intestinal epithelium , biology , immunology , permeability (electromagnetism) , biochemistry , cell wall , membrane , genetics
Lipopolysaccharides (LPS) are essential membrane components of most Gram‐negative bacteria and several pathologies have been linked to LPS‐induced inflammation. However, understanding the mechanisms of intestinal uptake of LPS is still at its beginning. Uptake is thought to happen either via toll‐like‐receptor‐4‐induced phagocytosis or via paracellular passage. In ulcerative colitis (UC), the paracellular barrier formed by tight junctions (TJ) is impaired and tricellulin, a protein of the tricellular TJ of importance for regulation of paracellular macromolecule passage, is downregulated. We hypothesize that a sole reduction in tricellulin expression leads to enhanced antigen uptake with the consequence that LPS is passing the intestinal epithelium via the tricellular TJ. For analyzing this, we generated clones of intestinal epithelial cell monolayers HT‐29/B6 at different tricellulin knockdown levels and measured the permeability for LPS. The passage of LPS through the tricellular TJ was visualized using a dedicated fluorescence microscopical technique. We then analyzed the capability of passaged LPS to stimulate immune cells and by that to induce inflammation. For this, we added LPS apically to the monolayers containing different levels of tricellulin and co‐cultured them with basolaterally located macrophages. Passing LPS should stimulate these after passage. The basolateral medium thus containing secreted cytokines was added to otherwise uninvolved HT‐29/B6 cells and the effect on transepithelial resistance (TER) was monitored. Using concentrations of LPS which were below to affect the epithelial barrier of HT‐29/B6 cells, at a decreased level of tricellulin of 50% the permeability for LPS was three times higher than in controls (shTric50: 2.09±0.56 × 10 −9 cm/s; control: 0.70±0.13 × 10 −9 cm/s; n=7, p=0.03). Although the permeability was quite low in absolute terms, LPS passage in shTric50 was sufficient to stimulate macrophages to secrete cytokines. This was not the case for cells with high tricellulin expression. TER of HT‐29/B6 cells treated with the supernatants of the macrophages of the co‐culture with LPS‐incubated controls did not change. We conclude that reduced tricellulin levels allow for paracellular passage of LPS via tricellular TJs and thereby may stimulate immune cells supporting inflammatory processes as happening in UC. This signifies a new pathomechanistic pathway for LPS‐induced inflammation. Support or Funding Information Supported by DFG GRK 2318 and DFG TRR 241