Recently, attention on the potential health benefits of probiotics has been focused on their production of soluble factors, defined as “postbiotics,” which are able to exert beneficial properties against pathogen-induced inflammation and related alteration of cytokine release.1 The identification of soluble factors may represent an opportunity to develop new, effective therapeutic strategies that would avoid the risks associated with the administration of live bacteria and problems related to bacterial vitality, as well as bacterial load linked with the correct use and effectiveness of probiotics.2
Postbiotic activity has been identified for several species of Bifidobacteri (breve, lactis, infantis),3–5Escherichia coli Nissle 1917,6,7 and Faecalibacterium prausnitzii.8,9 Postbiotic activity has also been reported for Lactobacilli whose soluble factors are able to modulate secretion of inflammatory mediators and activation of the nuclear factor kappa-light-chain-enhancer of activated B cells, reducing inflammation on several animal and human tissues.10,11 In particular, Lactobacillus rhamnosus GG (LGG) supernatants ameliorated alcohol-induced increase in intestinal permeability,12 and dose-dependently prevented cytokine-induced apoptosis.13
A previous study14 has shown the ability of LGG to protect human colonic muscle from damage by lipopolysaccharide (LPS) derived from the pathogen strain of E. coli (O111:B4). Protective effects have also been observed with the supernatants obtained from LGG culture. Soluble products of probiotics might easily reach deeper intestinal wall layers compared with luminal bacteria, providing a rational design for clinical studies in which the efficacy of probiotics and their by-products could ultimately be determined on bacterial-related gut motor disorders. However, the understanding of their fine mechanisms of action remains to be established to develop effective pharmacologic strategies finalized to integrate the action of treatments with live bacteria, and/or to replace it with postbiotic mediators.
The aim of our study was to evaluate whether supernatants harvested in different growth phases from LGG (ATCC53103 strain) culture protect human colonic smooth muscle cells from LPS-induced myogenic damage and to characterize their dose-dependent protective properties in terms of inhibition of LPS-induced biological effects and cytokine production.
MATERIALS AND METHODS
The following materials were used: Dulbecco modified Eagle’s medium (DMEM), de Man, Rogosa, Share (MRS) broth (Biolife, Italy), penicillin-streptomycin solution, gentamicin, anphotericin B, antimycin, fetal bovine serum (FBS; LONZA, Switzerland), collagenase type II (Worthington), highly purified LPS obtained from a pathogen strain of E. coli (O111:B4) (Vinci-Biochem, Italy), LGG strain ATCC53103, and enzyme-linked immunosorbent assay for dosage of the cytokine interleukin 6 (IL-6) (R&D System, Canada).
LGG (ATCC53103) was furnished as lyophilized by Dicofarm Spa. The freeze-dried bacterial cells where resuspended in MRS broth and grown under aerobiosis condition in MRS medium at 37°C. After the L. rhamnosus growth curve, samples were collected in different phases. Briefly, 20 mL of bacterial culture was harvested at middle exponential, late exponential, stationary, and overnight phases. To recover the supernatants, all samples collected were subjected to centrifugation at 4500 rpm for 30 minutes at 4°C. The recuperated supernatants were then filtered through a 0.20-μm filter. The filtered solutions were then aliquoted in 5 mL, and stored at −20°C until use.
Tissues were obtained from the circular layer of surgical specimens of disease-free human colon from 10 patients (7 men and 3 women; median age: 70 y, range, 62 to 78 y) submitted to colectomy for colorectal carcinoma. Segments (2 to 4 cm2) of normal colon were taken from the unobstructed macroscopically normal area, ∼7 to 8 cm from the neoplastic area, immediately after surgical removal of the tumor. All patients gave informed consent, and the study was approved by the Ethical Committee of Sapienza University of Rome.
Primary Culture of Human Colonic Smooth Muscle Cells
Primary colonic human smooth muscle cell (HSMCs) culture was prepared as previously described.15 Briefly, slices of circular muscle layer were incubated overnight in DMEM supplemented with penicillin-streptomycin solution (10,000 U/mL), gentamicin (1 mg/mL), amphotericin B (250 mg/mL), FBS, an adenosine triphosphate-regenerating system [adenosine triphosphate (3 mM), phosphocreatine (10 mM), creatine phosphokinase (10 U/mL)], antimycin (10 μM), and collagenase (150 U/mL). On the following day, digested colonic muscle was suspended in DMEM supplemented only with FBS and antibiotics for 20 minutes to allow spontaneous dissociation of HSMCs. The cells were then harvested and used immediately.
The richly pure and homogeneous primary culture of colonic HSMCs that was obtained was exposed for 24 hours only to DMEM (control) or to highly purified LPS (1 μg/mL) obtained from a pathogen strain of E. coli (O111:B4) in the absence and presence of the harvested LGG supernatants collected during different phases of LGG culture growth. Different dilutions in DMEM broth (1:1, 1:2, and 1:4) of the collected supernatants were tested. At the end of the treatments, the cells were prepared for the following analyses.
Biological morphofunctional features (cell length and contractile response) were measured both in the untreated cells (control) and after LPS, in the presence or absence of LGG supernatants. For contractile response, 0.5 mL of cell suspension was added to the maximal dose of acetylcoline (1 μM) and the reaction terminated after 30 seconds, the time required for peak contraction, with 1% acrolein. For the cell length, the agent was omitted and an equivalent volume of medium was added. The length of 50 cells in sequential microscopic fields was measured by image-scanning micrometry both in the control state and upon addition of tested agents using a ProgRes camera with CapturePro 2.6 application software (Jenoptik Laser Optik, Jena, Germany) installed on a phase-contrast microscope, Leica 2500 (Leica Microsystems, Wetzlar, Germany). Contraction was expressed as percentage decrease in cell length from control taken as 100.
Analysis of Cytokine Secretion
IL-6 levels were determined in the control and in treated cells using sensitive enzyme-linked immunosorbent assay kits (R&D System, Minneapolis, MN). Briefly, supernatant aliquots of cell culture were removed at the end of treatments and assayed for IL-6 presence according to the manufacturer’s instructions.
Data and Statistical Analysis
Data are expressed as mean±SE of duplicate examinations of n experiments, “n” referring to the number of individual patients from whom the colonic specimens were obtained. Statistical analysis was performed by parametric analysis of variance test, corrected for multiple comparison by the Bonferroni procedure. P-values of <0.05 were considered significant (P<0.05).
HSMCs presented a resting cell length of 88.46±0.92 μm and a contraction of 25.65%±0.78% in response to a maximal concentration of acetylcholine (1 μM). The sole effects of MRS, the medium used for LGG culture, and of the single supernatants were tested on HSMC morphofunctional parameters. LGG supernatants have been separately collected from LGG culture corresponding to the middle exponential (5 h), late exponential (11 h), stationary (15.50 h), and overnight (29 h) phases of the LGG growth curve (Fig. 1).
The supernatants collected during the middle exponential phase induced a significant 16.33%±1.83% cell shortening (P<0.0001) that progressively disappeared with supernatants obtained in the later phases of LGG culture (Table 1). Concerning acethylcoline-induced contraction, an inhibition of response was observed in the presence of the sole supernatants, higher (32.94%±7.64%, P=0.0019) with supernatants collected during the stationary phase of LGG growth culture and lesser with supernatants collected in the overnight phase (26.93±9.28, P=0.0434).
LPS exposure of HSMCs for 24 hours caused a 20.00%±0.68% cell shortening, a 50.00%±0.82% inhibition of maximal contraction, and a 300.00%±2.75% increase in IL-6 levels. LGG supernatants were able to counteract all these LPS effects. The restoration of LPS-induced cell shortening and IL-6 production occurred in parallel to LGG culture growth, progressively significantly (P<0.0005) increasing with supernatants collected from the middle exponential phase to that overnight (Fig. 2). In terms of restoration of LPS-induced inhibition of cell contraction, a significant (P=0.0014) reestablishment of contractile response was observed only with supernatants collected in the overnight phase of LGG culture, with nonsignificant effects with supernatants collected up to 15.50 hours.
The efficacy of supernatants to revert LPS-induced cell shortening and inhibition of acetylcholine-induced contraction prompts the study of the dose-dependency of their effects. On both morphofunctional cell parameters, supernatant effects were concentration dependent. Lower the concentration of supernatant, lesser the inhibition of LPS-induced effects (Fig. 3). Undiluted supernatant (1:1) caused a 58.79%±21.69% inhibition of LPS-induced cell shortening, which decreased to 40.34%±13.75% with dilution 1:2 and to 14.64%±10.38% with dilution 1:4. Similarly, undiluted supernatant (1:1) caused a 49.05%±9.31% inhibition of LPS-induced inhibition of maximal contraction that decreased to 22.22%±9.02% with supernatant dilution 1:2 and 10.98%±11.01% with dilution 1:4.
The present study demonstrates that LGG might protect human smooth muscle from LPS-induced damage through the secretion of bioproducts. A progressive increase in postbiotic activity, able to reverse LPS effects, results in function of the LGG culture growth phase. The dose dependency of supernatant effects further suggests that LGG bioproducts directly interact with human smooth muscle. Similar effects have been observed on postinfective muscle hypercontractility with Lactobacillus paracasei or its heat-labile metabolite.16
Lactobacillus is a genus of Gram-positive facultative anaerobic bacteria belonging to the phylum of Firmicutes that has been shown to modulate gut innate immunity and to promote intestinal epithelial cell survival and barrier function.1,17 The effect of probiotics on gastrointestinal motility has been reported from different studies carried out in animal models18 highlighting the gut motor-neural apparatus as a potential target for probiotics in gut postinfective disorders. Lactobacillus species specifically regulate jejunal motility19 and colonic neuron excitability20 and alleviate visceral hypersensitivity.21 On human smooth muscle, LGG is able to counteract the inflammatory burst induced by LPS through the activation of membrane Toll-like receptor 2.14 On both mouse cardiac cells22 and rabbit colonic smooth muscle,23 Toll-like receptor 2 activation hampers the inflammatory the nuclear factor kappa-light-chain-enhancer of activated B cells pathway, through anti-inflammatory phosphatidylinositol-3′-kinase/protein kinase B signaling. This latter signaling pathway is responsible for the antiapoptotic effects reported for LGG culture supernatants.24
The inhibition of LPS-induced IL-6 secretion from HSMCs by LGG supernatants suggests that LGG bioproducts exert an anti-inflammatory effect on human smooth muscle as well. Downregulation by Lactobacilli of proinflammatory cytokine release has been previously reported in human intestinal dendritic cells challenged with Salmonella25 and in human peripheral blood monocyte–derived macrophages primed by LPS.12 Similar downregulation of inflammatory response has been observed with Lactobacilli metabolic products.26–28 Purification of LGG supernatants has highlighted mostly the presence of 2 proteins, 1 with a molecular mass of around 40 kDa and the other of 75 kDa—namely, p40 and p75, respectively—whose effects have been tested on both murine and human cell lines and cultured colon explants.11 These 2 proteins prevent tumor necrosis factor-induced intestinal epithelial cells and organ culture damage, inhibit apoptosis, and stimulate proliferative epithelial cell responses. p40 has relevant immunoregulatory functions, acting on macrophages and lymphocytes to directly downregulate proinflammatory cytokine production and modulate intestinal epithelial homeostasis.13,29
Interesting is the observation of the different effects of LGG supernatants on cell contraction depending on the time of collection from the broth of the culture. Supernatants collected in the middle exponential phase of growth induced a significant contraction of HSMCs and possessed only a slight protective effect on LPS-induced impairment of contractile response. Vice versa, supernatants collected in the overnight phase of growth were able to restore contraction after LPS exposure and had by themselves only light contractile activity. This apparently contradictory result suggests the presence/absence or loss of activity of different postbiotic mediators in the different collected samples that could exert different potential effects on human cells.
In conclusion, results obtained in the present study fortify the therapeutic value of the probiotic strain LGG in contrasting LPS tissue damages and emphasize the role of LGG posbiotic mediators. Further efforts will be necessary to investigate the postbiotic mediators involved. Such studies will emphasize new potentials in probiotic use, allowing the avoidance of existing issues linked to the use of live cells of probiotic bacterial strains such as bacterial viability, bacterial load, and vitality maintained in the gastrointestinal tract, and the problematic linked to the antibiotic resistance spread.
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