Introduction: Helicobacter pylori (H.pylori) is a Gram-negative bacterium that causes chronic gastritis, peptic ulcers and stomach cancer. The activity of immune cells, including monocytes, infiltrating gastric mucosa of H. pylori infected individuals depend on the ability to recognize and react to the bacterial antigens including lipopolysaccharide (LPS) H.pylori. The LPS of this bacterium has a unique structure of lipid A and possess Lewis (Le) determinants that allow bacteria to modulate host immune responses and promote chronic infection. However little is known about the role of Le determinants in H.pylori – host cell interactions and receptors involved in binding of H.pylori LPS. Aim: To evaluate TLR receptors involved in binding H.pylori LPS with or without Lewis antigens and to evaluate the consequences of this interaction: cell signaling, activity, morphology, ROS production and tumor necrosis factor (TNF-α) secretion. Material and Methods: THP-1XBlue™ cells were used as a model of human monocytes – a reporter cell line that when activated via TLR receptors induce NFk-B transcription factor and secrete embryonic alkaline phosphatase (SEAP). Cells were seeded in 96-well tissue culture plates (1×10⁵ cells/well) and stimulated for 24 h with LPS of: H.pylori Le^XY(+) or H.pylori Le^XY(-), or standard LPS E. coli O55:B5, in a final concentration of 100ng/ml. To evaluate the involvement of particular TLR receptors and signaling molecules in LPS binding 30 min. prior stimulation monoclonal antibodies: mAbα: TLR2, TLR4, TLR6 (Novus, CO, USA) or MyD88 inhibitor (Selleckchem) were introduced. After stimulation supernatants were used for SEAP (QuantiBlue reagent) and TNF-α quantification (ELISA, ElabSciences). ROS production was estimated with Total ROS Detection Kit (Enzo Life Sciences). Simultaneously, the cells were fixed for 10 min. with 99.8% methanol and stained with DAPI (1:2000) and DiOC (2: 1000) for 15 min. and 5 min. respectively. The microscopic evaluation was performed with Leica TCS SP8 (Leica-Microsystems, Wetzlar, Germany). Results: All LPS types have potential to activate monocytes, however each LPS with different intensity. The LPS of H.pylori Le^XY(-) (OD=0.870±0.07) as well as H.pylori Le^XY(+) (OD=0.677±0.05) exhibited significantly lower potential to activate monocytes in comparison to  the standard LPS of E. coli (OD=1.361±0,.44), p<0.001). Confocal microscopy analysis showed that the cells stimulated with LPS of E. coli lost their ability to adhere (78%) and the integrity of cellular membrane was disturbed (Figure 1), whereas the cells incubated in the presence of H.pylori LPS Le^XY(-) remained in a condition similar to untreated controls. The LPS of H.pylori Le^XY(+) did not affect the adhesive properties however we observed a significant drop of fluorescence signal from DAPI which suggest its influence on cellular nuclei. The experiments with mAbαTLR receptors revealed that the activation induced by standard LPS of E. coli solely depended on the presence of TLR4 receptor (or in complex with TLR2), whereas the H.pylori Le^XY(+)/Le^XY(-) – mediated monocyte activation was related with the presence of receptor complexes: TLR2/4 (p=0.0001) or TLR2/6 (p=0.003), whereas the blocking of TLR4 did not reduce the level of monocyte activation. The experiments with LPS evaluated by confocal microscopy were consistent with cellular culture study and showed that the lower activation of monocytes mediated by H.pylori LPS is accompanied by lower ROS production in comparison to that induced by E. coli LPS (3631±184). H. pylori LPS with Lewis antigens induced significantly higher ROS production than the H.pylori LPS without Lewis residues(1446±138 vs 853±62), which might suggest the involvement of sugar moieties in this phenomenon. Further studies showed that LPS E.coli-mediated stimulation was strongly correlated with the secretion of high doses of TNF-α by monocytes (150,5±9.4 pg/ml) whereas the level of this cytokine secreted in response to H.pylori Le^XY(+) or H.pylori Le ^XY(-) was notably lower (101.5±18.1 and 64.2±5.9, respectively, p<0.001). Additional experiments with the utility of Myd88 inhibitor used before stimulation showed that in all cases TNF-α production was Myd88 dependent and resulted with at least 70% inhibition of its release. Conclusion: Our results suggest that the LPS of H.pylori is recognized not solely by classic TLR4 receptor but rather by TLR4/2 or TLR2/6 receptor complexes and that the Lewis residues present in 80% of H.pylori isolates are involved in that process. The immunomodulatory activity of H.pylori is weaker in regard to ROS production and TNF-α which is associated with the level of monoCyte activation and might explain the chronic character of this infection.

Figures:

Figure 1. The morphological changes of THP1xBlueTM monocytes induced by standard lipopolysaccharide (LPS) of E. coli (b), H. pylori LPS LeXY(-) (c) and H. pylori LPS LeXY(+) (d) in comparison to untreated control (a). Cells were stimulated for 24h, fixed with methanol, stained with DAPI (blue) and DiOC (green) and observed under confocal microscope (20x – left column and 63x/1.20 right column).

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EMC Abstracts - https://emc-proceedings.com/abstract/the-activation-ros-induction-and-cytokine-secretion-in-human-monocytes-exposed-to-h-pylori-lipopolysaccharide-depend-on-the-availability-of-tlr42-and-tlr26-receptor-complexes/