In Peripheral Blood Mononuclear Cells Helicobacter pylori Induces the Secretion of Soluble and Exosomal Cytokines Related to Carcinogenesis

Abstract

Helicobacter pylori promotes the secretion of cytokines that regulate inflammation and carcinogenesis. Immune cells secrete cytokines into the extracellular medium or packaged in exosomes. The objective of this study was to analyze the profile of soluble and exosomal cytokines that were secreted by human peripheral blood mononuclear cells (PBMCs) that were infected with H. pylori and to build a network of interaction between cytokines and cellular proteins. PBMCs were obtained by density gradient centrifugation and infected with H. pylori for 24 h. The infection was verified by immunofluorescence and Western blot for CagA. The exosomes were obtained from culture supernatant by ultracentrifugation and characterized by transmission electron microscopy, particle size analysis, and Western blot for CD9 and CD81. Cytokines were quantified using a multiplex immunoassay in the culture supernatant, intact exosomes, and lysed exosomes. H. pylori adheres to lymphocytes and translocates CagA. In PBMCs, H. pylori induces an increase in the soluble and exosomal IL-1β, IL-6, TNF-α, IL-10, IL-17A, IL-21, and IL-22. The protein-protein interaction (PPI) network shows that soluble and exosomal cytokines interact with proteins that participate in signaling pathways such as NF-κB, MAPK, PI3K-Akt, Jak-STAT, FoxO, and mTOR, that are related to carcinogenesis; moreover, TNF-α had the highest number of interactions. Cytokine-loaded exosomes represent another means of intercellular communication that is activated by H. pylori to stimulate inflammation, carcinogenesis, or cancer progression. Cytokine-loaded exosomes are likely to be associated with extragastrointestinal diseases of inflammatory origin.

Keywords: Helicobacter pylori; PBMCs; carcinogenesis; cytokines; exosomes; inflammation.

Figure 1

H. pylori adheres to PBMCs and translocates CagA. PBMCs were infected with H. pylori strain 26695 at a MOI of 1:50, for 24 h. (a) H. pylori infection of PBMCs was documented by immunofluorescence. White arrows indicate bacteria that were adhered to the lymphocyte membrane (green), nuclei are seen in blue, and F-actin in red. Uninfected PBMCs were used as a negative control. The images were observed under an epifluorescence microscope at 100×. (b) For CagA detection, 50 µg of total protein from H. pylori-PBMCs or Control-PBMCs were subjected to Western blot. As a positive control for CagA, 15 µg of total proteins from H. pylori strain 26695 were used. GAPDH was used as an internal control. All the experiments were performed in triplicate.

Figure 2

H. pylori-infected and -uninfected PBMCs secrete exosomes. Exosomes were isolated by ultracentrifugation. (a) Representative photomicrographs of exosomes that were secreted by Control-PBMCs and H. pylori-PBMCs at a MOI 1:100, observed in a TEM. The scale bars represent 100 nm. (b) Exosome particle size that was determined using the Zetasizer instrument. In the graphs, the x axis represents the particle size in nm and the y axis the percentage of particles. Each curve represents the size and percentage of exosomes. (c) Tetraspanins CD9 and CD81 were detected by Western blot in 30 µg of total proteins from exosomes that were secreted by Control-PBMCs and H. pylori-PBMCs. As a negative control, 30 μg of BSA were used and as a positive control 30 μg of proteins from exosomes that were isolated from the breast cancer cell line MDA-MB-231 were used, which secrete abundant exosomes. All experiments were performed in triplicate.

Figure 3

H. pylori induces the secretion of soluble and exosomal cytokines in PBMCs. The concentration of IL-1β, IL-6, TNF-α, IFN-γ, IL-10, IL-31, IL-33, IL-17A, IL-17F, IL-21, IL-22, and IL-25 was determined in supernatant, intact exosomes, and lysed exosomes that were secreted by the Control-PBMCs and H. pylori-PBMCs by a bead-based multiplex immunoassay. The graphs show the mean ± SEM. Comparison between the groups was performed using the Mann–Whitney U test, and differences were considered statistically significant when * p < 0.05. All the experiments were performed in triplicate.

Figure 4

Cytokines increased in the supernatant and exosomes that were secreted by H. pylori-PBMCs participate in the regulation of signaling pathways that are involved in carcinogenesis. (a) The PPI network represents physical and functional interactions of IL-1β, IL-6, TNF-α, IL-10, IL-17A, IL-21, and IL-22 with cellular proteins that participate in signaling pathways that are related to carcinogenesis. (b) The PPI network constructed with TNF-α and cellular proteins. Each node represents a protein and the lines represent the interactions between the proteins (edges); pink lines denote experimentally tested interactions, and the blue lines denote the interactions with proteins from curated databases. Colored nodes represent KEGG pathways that are involved in carcinogenesis and white nodes symbolize proteins that are not involved in KEGG pathways. All interactions were statistically significant (p = 1.0 × 10⁻¹⁶).

Figure 5

H. pylori promotes the secretion of exosomal and soluble cytokines that are associated with inflammation and gastric carcinogenesis. H. pylori adheres PBMCs and translocates CagA to the cell cytoplasm. In infected PBMCs, H. pylori promotes the secretion of IL-1β, IL-6, TNF-α, IL-10, IL-17A, IL-21, and IL-22. TNF-α could play an important role in the activation of signaling pathways that are associated with inflammation, gastric carcinogenesis, or tumor progression.