Probiotic Lactobacillus and Bifidobacterium Strains Counteract Adherent-Invasive Escherichia coli (AIEC) Virulence and Hamper IL-23/Th17 Axis in Ulcerative Colitis, but Not in Crohn's Disease

Abstract

Hypersecretion of proinflammatory cytokines and dysregulated activation of the IL-23/Th17 axis in response to intestinal microbiota dysbiosis are key factors in the pathogenesis of inflammatory bowel diseases (IBD). In this work, we studied how Lactobacillus and Bifidobacterium strains affect AIEC-LF82 virulence mechanisms and the consequent inflammatory response linked to the CCR6-CCL20 and IL-23/Th17 axes in Crohn's disease (CD) and ulcerative colitis (UC) patients. All Lactobacillus and Bifidobacterium strains significantly reduced the LF82 adhesion and persistence within HT29 intestinal epithelial cells, inhibiting IL-8 secretion while not affecting the CCR6-CCL20 axis. Moreover, they significantly reduced LF82 survival within macrophages and dendritic cells, reducing the secretion of polarizing cytokines related to the IL-23/Th17 axis, both in healthy donors (HD) and UC patients. In CD patients, however, only B. breve Bbr8 strain was able to slightly reduce the LF82 persistence within dendritic cells, thus hampering the IL-23/Th17 axis. In addition, probiotic strains were able to modulate the AIEC-induced inflammation in HD, reducing TNF-α and increasing IL-10 secretion by macrophages, but failed to do so in IBD patients. Interestingly, the probiotic strains studied in this work were all able to interfere with the IL-23/Th17 axis in UC patients, but not in CD patients. The different interaction mechanisms of probiotic strains with innate immune cells from UC and CD patients compared to HD suggest that testing on CD-derived immune cells may be pivotal for the identification of novel probiotic strains that could be effective also for CD patients.

Keywords: AIEC; Crohn’s disease; IL-23/Th17; intestinal inflammation; pro- and anti-inflammatory mechanisms; probiotics; ulcerative colitis.

Figure 1

Effects of probiotic strains on adhesion and invasion of intestinal epithelial cells (IECs) by AIEC-LF82 and the relative inflammatory response. (A) Adhesion to HT29 cell monolayers of AIEC-LF82 alone (taken as 100%) or in the presence of Lactobacillus acidophilus LA1 (+L₁), Lactobacillus paracasei 101/37 (+L₂), Bifidobacterium animalis spp. lactis Bi1 (+B₁), Bifidobacterium breve Bbr8 (+B₂), S. epidermidis ATCC-155 (+S.e.) at 1:1 ratio or in the presence of 6-mercaptopurine (+6MP, 2 µg/mL) was quantified after a 3 h incubation period; (B) invasion of AIEC-LF82 alone (taken as 100%) or in the presence of probiotic strains or S. epidermidis (+S.e.) at 1:1 ratio or in the presence of 6MP was quantified after a 3 h or 7 h incubation period. Results are expressed as the percentage of cell-associated LF82 (adherent plus intracellular LF82 cells) or intracellular LF82 relative to those obtained in monoinfection with LF82 alone, taken as 100%; (C) IL-8 and CCL20 secretion by HT29 cells after 7 h of infection with AIEC-LF82 alone or in the presence of probiotic strains or S. epidermidis (+S.e.) at 1:1 ratio or in the presence of 6MP was quantified by ELISA. Dot lines (---) represent the median values for cytokine secretion of HT29 cells infected with LF82 alone. Each experiment was performed in triplicate, and data are represented with box-plots showing the median, range and upper and lower quartiles of at least three independent experiments. Means are represented as black cross (+), median as horizontal lines, and outliers are marked as dots. Statistical significance for each condition compared to HT29 infected with LF82 alone was reported (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 2

Effects of probiotic strains on phagocytosis and intracellular survival of AIEC-LF82 within human monocyte-derived macrophages (MDM). Percentage of internalized AIEC-LF82 cells within MDM derived from HD, UC or CD patients after 1 h (A) or 8 h of infection (C) with LF82 alone (taken as 100%) or in coinfection with Lactobacillus acidophilus LA1 (+L₁) or Lactobacillus paracasei 101/37 (+L₂) or Bifidobacterium animalis spp. lactis Bi1 (+B₁) or Bifidobacterium breve Bbr8 (+B₂) or S. epidermidis ATCC-155 (+S.e.), at 1:1 ratio or in the presence of 6MP (2 µg/mL). Each experiment was performed in triplicate and data are represented as described in Figure 1. Statistical significance for each condition compared to MDM infected with LF82 alone was reported (* p < 0.05, ** p < 0.01, *** p < 0.001); (B) confocal microscopic examination of HD-derived MDM infected with AIEC-LF82 alone or in coinfection with L. acidophilus LA1 (+L₁) at 1 h post infection. Cell nuclei (stained by DAPI) are shown in blue and AIEC-LF82 in green. Larger images: magnification 200×, scale bars: 50 µm. Smaller images: magnification 630×, scale bars: 20 µm.

Figure 3

Effects of probiotic strains on cytokine secretion by MDM derived from HD, UC and CD patients infected with AIEC-LF82 strain. (A) TNF-α and (B) IL-10 secretion by MDM derived from HD, UC patients or CD patients after 8 h of infection with AIEC-LF82 alone or in the presence of Lactobacillus strains (+L₁, +L₂) or Bifidobacterium strains (+B₁, +B₂) or S. epidermidis ATCC-155 (+S.e.), at 1:1 ratio or in the presence of 6MP (2 µg/mL) was quantified by ELISA. Dot lines (---) represent the median values for cytokine secretion of MDM infected with LF82 alone. Data are represented as explained in Figure 1. Statistical significance for each condition compared to MDM infected with LF82 alone was reported (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 4

Effects of probiotic strains on phagocytosis and intracellular survival of AIEC-LF82 within human monocyte-derived dendritic cells (MoDC). Percentage of internalized AIEC-LF82 cells within MoDC derived from HD, UC or CD patients after 1 h (A), 8 and 24 h of infection. (B) MoDC were infected with LF82 alone (taken as 100%) or in the presence of Lactobacillus strains (+L₁, +L₂) or Bifidobacterium strains (+B₁, +B₂) or S. epidermidis ATCC-155 (+S.e.), at 1:1 ratio or in the presence of 6MP (2 µg/mL). Each experiment was performed in triplicate and data are represented as described in Figure 1. Statistical significance for each condition compared to MoDC infected with LF82 alone was reported (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 5

Effects of probiotic strains on cytokine secretion by MoDC derived from HD, UC and CD patients infected with AIEC-LF82 strain. Secretion of IL-1β (A), IL-23 (B), IL-12 (C) and IL-10 (D) in the supernatants of MoDC derived from HD, UC patients or CD patients after 24 h of infection with AIEC-LF82 alone or in the presence of either Lactobacillus strains (+L₁ or +L₂) or Bifidobacterium strains (+B₁ or +B₂) or S. epidermidis ATCC-155 (+S.e.), at 1:1 ratio or in the presence of 6MP (2 µg/mL), was quantified by ELISA. Dot lines (---) represent the median values for cytokine secretion of MoDC infected with LF82 alone. Data are represented as described in Figure 1. Statistical significance for each condition compared to MoDC infected with LF82 alone was reported (* p < 0.05, ** p < 0.01, *** p < 0.001).