Mucosal adhesion and anti-inflammatory effects of Lactobacillus rhamnosus GG in the human colonic mucosa: A proof-of-concept study

Cristiano Pagnini, Vito Domenico Corleto, Michela Martorelli, Claudio Lanini, Giancarlo D'Ambra, Emilio Di Giulio, Gianfranco Delle Fave, Cristiano Pagnini, Vito Domenico Corleto, Michela Martorelli, Claudio Lanini, Giancarlo D'Ambra, Emilio Di Giulio, Gianfranco Delle Fave

Abstract

Aim: To investigate the adhesion and anti-inflammatory effects of Lactobacillus rhamnosus GG (LGG) in the colonic mucosa of healthy and ulcerative colitis (UC) patients, both in vivo and ex vivo in an organ culture model.

Methods: For the ex vivo experiment, a total of 98 patients (68 UC patients and 30 normal subjects) were included. Endoscopic biopsies were collected and incubated with and without LGG or LGG-conditioned media to evaluate the mucosal adhesion and anti-inflammatory effects [reduction of tumor necrosis factor alpha (TNFα) and interleukin (IL)-17 expression] of the bacteria, and extraction of DNA and RNA for quantification by real-time (RT)-PCR occurred after the incubation. A dose-response study was performed by incubating biopsies at "regular", double and 5 times higher doses of LGG. For the in vivo experiment, a total of 42 patients (20 UC patients and 22 normal controls) were included. Biopsies were taken from the colons of normal subjects who consumed a commercial formulation of LGG for 7 d prior to the colonoscopy, and the adhesion of the bacteria to the colonic mucosa was evaluated by RT-PCR and compared with that of control biopsies from patients who did not consume the formulation. LGG adhesion and TNFα and IL-17 expression were compared between UC patients who consumed a regular or double dose of LGG supplementation prior to colonoscopy.

Results: In the ex vivo experiment, LGG showed consistent adhesion to the distal and proximal colon in normal subjects and UC patients, with a trend towards higher concentrations in the distal colon, and in UC patients, adhesion was similar in biopsies with active and quiescent inflammation. In addition, bioptic samples from UC patients incubated with LGG conditioned media (CM) showed reduced expression of TNFα and IL-17 compared with the corresponding expression in controls (P < 0.05). Incubation with a double dose of LGG increased mucosal adhesion and the anti-inflammatory effects (P < 0.05). In the in vivo experiment, LGG was detectable only in the colon of patients who consumed the LGG formulation, and bowel cleansing did not affect LGG adhesion. UC patients who consumed the double LGG dose had increased mucosal concentrations of the bacteria and reduced TNFα and IL-17 expression compared with patients who consumed the regular dose (48% and 40% reduction, respectively, P < 0.05).

Conclusion: In an ex vivo organ culture model, LGG showed consistent adhesion and anti-inflammatory effects. Colonization by LGG after consumption for a week was demonstrated in vivo in the human colon. Increasing the administered dose increased the adhesion and effectiveness of the bacteria. For the first time, we demonstrated that LGG effectively adheres to the colonic mucosa and exerts anti-inflammatory effects, both ex vivo and in vivo.

Keywords: Adhesion; Cytokines; Lactobacillus rhamnosus GG; Probiotic; Ulcerative colitis.

Conflict of interest statement

Conflict-of-interest statement: The authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1
Lactobacillus rhamnosus GG adhesion in the normal colon and ulcerative colitis patients’ colon was evaluated by the ex vivo organ culture experimental model. A: Mucosal Lactobacillus rhamnosus GG (LGG) quantification from total bioptic samples showed consistent adhesion in mucosal biopsies, with a trend toward higher LGG concentrations in distal specimens compared with proximal specimens (n = 20 per group); B: Mucosal LGG quantification from bioptic samples from ulcerative colitis patients confirmed consistent bacterial adhesion after incubation (n = 15 per group). The bars represent the mean values. LGG: Lactobacillus rhamnosus GG; UC: Ulcerative colitis.
Figure 2
Figure 2
Lactobacillus rhamnosus GG adhesion in bioptic samples from the rectum of ulcerative colitis patients was evaluated by the ex vivo organ culture experimental model. Similar concentrations of adherent Lactobacillus rhamnosus GG were found in the mucosa from ulcerative colitis patients without and with active endoscopic inflammation, as defined by Mayo Endoscopic Scores of ≤ 1 and 2, respectively (n = 15 per group). Mean ± standard error is represented. LGG: Lactobacillus rhamnosus GG; UC: Ulcerative colitis.
Figure 3
Figure 3
Effect of Lactobacillus rhamnosus GG on cytokine expression in the colon was evaluated by the ex vivo organ culture experimental model. A: Tumor necrosis factor alpha (TNFα) mRNA quantification in proximal (n = 15) and distal (n = 20) colon biopsies from ulcerative colitis (UC) patients incubated with Lactobacillus rhamnosus GG (LGG) conditioned media (CM) and control biopsies; B: IL-17 mRNA quantification in proximal and distal colon biopsies from UC patients incubated with LGG CM and control biopsies; C: TNFα mRNA quantification in proximal and distal colon biopsies (n = 8 per group) from normal subjects incubated with LGG CM and control biopsies. aP < 0.05. TNFα: Tumor necrosis factor alpha; IL: Interleukin; LGG: Lactobacillus rhamnosus GG; UC: Ulcerative colitis.
Figure 4
Figure 4
Lactobacillus rhamnosus GG dose-finding study in the ex vivo organ culture experimental model. A: Mucosal Lactobacillus rhamnosus GG (LGG) quantification in distal colon bioptic samples after incubation with a regular (6 × 106 CFU), double or 5-times dose of LGG. aP < 0.05 vs regular dose (n = 8 per group); B: TNFα mRNA quantification in distal colon bioptic samples incubated with a regular (1:10), double (2:10) or 5-times (5:10) dose of LGG conditioned media (CM) (n = 10 per group); C: IL-17 mRNA quantification in distal colon bioptic samples incubated with a regular (1:10), double (2:10) or 5-times (5:10) dose of LGG CM. Mean ± standard error is represented. aP < 0.05 vs regular dose. TNFα: Tumor necrosis factor alpha; IL: Interleukin; LGG: Lactobacillus rhamnosus GG; CM: Conditioned media.
Figure 5
Figure 5
Lactobacillus rhamnosus GG adhesion in the normal colon in vivo after 7 d of Lactobacillus rhamnosus GG administration. A: Lactobacillus rhamnosus GG (LGG) detection in the colon of normal subjects who did (n = 12) and did not (n = 10) consume the probiotic formulation. Relative value bars and visualization on an agarose gel are shown. Mean ± standard error is represented; B: Relative segmental adhesion of LGG in the proximal and distal colon biopsies (n = 12 per group), with the bars representing the mean values. ND: Not detectable; LGG: Lactobacillus rhamnosus GG.
Figure 6
Figure 6
Lactobacillus rhamnosus GG adhesion and mucosal effect in the colon of ulcerative colitis patients. A: Mucosal Lactobacillus rhamnosus GG (LGG) quantification in colon biopsies of ulcerative colitis (UC) patients who consumed a regular (12 × 109 UFC/die) or a double (24 × 109 UFC/die) dose of LGG supplement for 7 d (n = 10 per group); B: Tumor necrosis factor alpha mRNA quantification in colon biopsies from UC patients who consumed a regular or a double dose of LGG supplement for 7 d. Mean ± standard error is represented; C: Interleukin-17 mRNA quantification in colon biopsies from UC patients who consumed a regular or a double dose of LGG supplement for 7 d. aP < 0.05. TNFα: Tumor necrosis factor alpha; IL: Interleukin; LGG: Lactobacillus rhamnosus GG; UC: Ulcerative colitis.

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