Long-term effect of Toll-like receptor-2, -4, -5, -7 and NOD2 stimulation on Na+,K+-ATPase activity and expression in intestinal epithelial cells

Inappropriate activation of Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain receptors (NOD) is involved in many chronic disorders, including inflammatory bowel disease (IBD). Altered function and/or expression of Na+,K+-ATPase (NKA) and epithelial ion channels are the main cause of electrolyte absorption imbalance in patients with IBD, leading to diarrhea. We aimed to evaluate the effect of TLRs and NOD2 stimulation upon NKA activity and expression in human intestinal epithelial cells (IECs) using RT-qPCR, Western blot, and electrophysiology techniques. TLR2, TLR4, and TLR7 activation inhibited NKA activity [(means ± SE) -20.0 ± 1.2%, -34.0 ± 1.5%, and -24.5 ± 2.0% in T84 cells; and -21.6 ± 7.4%, -37.7 ± 3.5%, and -11.0 ± 2.3% in Caco-2 cells]. On the other hand, activation of TLR5 increased NKA activity (16.2 ± 2.9% in T84 and 36.8 ± 5.2% in Caco-2 cells) and β1-NKA mRNA levels (21.8 ± 7.8% in T84 cells). The TLR4 agonist synthetic monophosphoryl lipid A (MPLAs) reduced α1-NKA mRNA levels in both T84 and Caco-2 cells (-28.5 ± 3.6% and -18.7 ± 2.8%), and this was accompanied by a decrease in α1-NKA protein expression (-33.4 ± 11.8% and -39.4 ± 11.2%). NOD2 activation upregulated NKA activity (12.2 ± 5.1%) and α1-NKA mRNA levels (6.8 ± 1.6%) in Caco-2 cells. In summary, TLR2, TLR4, and TLR7 activation induce downregulation of NKA in IECs, whereas TLR5 and NOD2 activation has the opposite effect. A comprehensive understanding of the cross talk between TLRs, NOD2, and NKA is of utmost relevance for developing better IBD treatments.