The Microbial Metabolite Butyrate Stimulates Bone Formation via T Regulatory Cell-Mediated Regulation of WNT10B Expression.

Nutritional supplementation with probiotics can prevent pathologic bone loss. Here we examined the impact of supplementation with Lactobacillus rhamnosus GG (LGG) on bone homeostasis in eugonadic young mice. Micro-computed tomography revealed that LGG increased trabecular bone volume in mice, which was due to increased bone formation. Butyrate produced in the gut following LGG ingestion, or butyrate fed directly to germ-free mice, induced the expansion of intestinal and bone marrow (BM) regulatory T (Treg) cells. Interaction of BM CD8+ T cells with Treg cells resulted in increased secretion of Wnt10b, a bone anabolic Wnt ligand. Mechanistically, Treg cells promoted the assembly of a NFAT1-SMAD3 transcription complex in CD8+ cells, which drove expression of Wnt10b. Reducing Treg cell numbers, or reconstitution of TCRß-/- mice with CD8+ T cells from Wnt10b-/- mice, prevented butyrate-induced bone formation and bone mass acquisition. Thus, butyrate concentrations regulate bone anabolism via Treg cell-mediated regulation of CD8+ T cell Wnt10b production.

Autoinducer-2 promotes the colonization of Lactobacillus rhamnosus GG to improve the intestinal barrier function in a neonatal mouse model of antibiotic-induced intestinal dysbiosis.

BACKGROUND: Human health is seriously threatened by antibiotic-induced intestinal disorders. Herein, we aimed to determine the effects of Autoinducer-2 (AI-2) combined with Lactobacillus rhamnosus GG (LGG) on the intestinal barrier function of antibiotic-induced intestinal dysbiosis neonatal mice. METHODS: An antibiotic-induced intestinal dysbiosis neonatal mouse model was created using antibiotic cocktails, and the model mice were randomized into the control, AI-2, LGG, and LGG + AI-2 groups. Intestinal short-chain fatty acids and AI-2 concentrations were detected by mass spectrometry and chemiluminescence, respectively. The community composition of the gut microbiota was analyzed using 16S rDNA sequencing, and biofilm thickness and bacterial adhesion in the colon were assessed using scanning electron microscopy. Transcriptome RNA sequencing of intestinal tissues was performed, and the mRNA and protein levels of HCAR2 (hydroxycarboxylic acid receptor 2), claudin3, and claudin4 in intestinal tissues were determined using quantitative real-time reverse transcription PCR and western blotting. The levels of inflammatory factors in intestinal tissues were evaluated using enzyme-linked immunosorbent assays (ELISAs). D-ribose, an inhibitor of AI-2, was used to treat Caco-2 cells in vitro. RESULTS: Compared with the control, AI-2, and LGG groups, the LGG + AI-2 group showed increased levels of intestinal AI-2 and proportions of Firmicutes and Lacticaseibacillus, but a reduced fraction of Proteobacteria. Specifically, the LGG + AI-2 group had considerably more biofilms and LGG on the colon surface than those of other three groups. Meanwhile, the combination of AI-2 and LGG markedly increased the concentration of butyric acid and promoted Hcar2, claudin3 and claudin4 expression levels compared with supplementation with LGG or AI-2 alone. The ELISAs revealed a significantly higher tumor necrosis factor alpha (TNF-α) level in the control group than in the LGG and LGG + AI-2 groups, whereas the interleukin 10 (IL-10) level was significantly higher in the LGG + AI-2 group than in the other three groups. In vitro, D-ribose treatment dramatically suppressed the increased levels of Hcar2, claudin3, and claudin4 in Caco-2 cells induced by AI-2 + LGG. CONCLUSIONS: AI-2 promotes the colonization of LGG and biofilm formation to improve intestinal barrier function in an antibiotic-induced intestinal dysbiosis neonatal mouse model.

Lactobacillus rhamnosus GG ameliorates triptolide-induced liver injury through modulation of the bile acid-FXR axis.

Triptolide (TP) is the principal bioactive compound of Tripterygium wilfordii with significant anti-tumor, anti-inflammatory and immunosuppressive activities. However, its severe hepatotoxicity greatly limits its clinical use. The underlying mechanism of TP-induced liver damage is still poorly understood. Here, we estimate the role of the gut microbiota in TP hepatotoxicity and investigate the bile acid metabolism mechanisms involved. The results of the antibiotic cocktail (ABX) and fecal microbiota transplantation (FMT) experiment demonstrate the involvement of intestinal flora in TP hepatotoxicity. Moreover, TP treatment significantly perturbed gut microbial composition and reduced the relative abundances of Lactobacillus rhamnosus GG (LGG). Supplementation with LGG reversed TP-induced hepatotoxicity by increasing bile salt hydrolase (BSH) activity and reducing the increased conjugated bile acids (BA). LGG supplementation upregulates hepatic FXR expression and inhibits NLRP3 inflammasome activation in TP-treated mice. In summary, this study found that gut microbiota is involved in TP hepatotoxicity. LGG supplementation protects mice against TP-induced liver damage. The underlying mechanism was associated with the gut microbiota-BA-FXR axis. Therefore, LGG holds the potential to prevent and treat TP hepatotoxicity in the clinic.

Lactobacillus rhamnosus GG cell-free supernatant as a novel anti-cancer adjuvant.

BACKGROUND: Gut microbiota modulation has been demonstrated to be effective in protecting patients against detrimental effects of anti-cancer therapies, as well as to improve the efficacy of certain anti-cancer treatments. Among the most characterized probiotics, Lactobacillus rhamnosus GG (LGG) is currently utilized in clinics to alleviate diarrhea, mucositis or intestinal damage which might be associated with several triggers, including Clostridium difficile infections, inflammatory gut diseases, antibiotic consumption, chemotherapy or radiation therapy. Here, we investigate whether LGG cell-free supernatant (LGG-SN) might exert anti-proliferative activity toward colon cancer and metastatic melanoma cells. Moreover, we assess the potential adjuvant effect of LGG-SN in combination with anti-cancer drugs. METHODS: LGG-SN alone or in combination with either 5-Fuorouracil and Irinotecan was used to treat human colon and human melanoma cancer cell lines. Dimethylimidazol-diphenyl tetrazolium bromide assay was employed to detect cellular viability. Trypan blue staining, anti-cleaved caspase-3 and anti-total versus anti-cleaved PARP western blots, and annexin V/propidium iodide flow cytometry analyses were used to assess cell death. Flow cytometry measurement of cellular DNA content (with propidium iodide staining) together with qPCR analysis of cyclins expression were used to assess cell cycle. RESULTS: We demonstrate that LGG-SN is able to selectively reduce the viability of cancer cells in a concentration-dependent way. While LGG-SN does not exert any anti-proliferative activity on control fibroblasts. In cancer cells, the reduction in viability is not associated with apoptosis induction, but with a mitotic arrest in the G2/M phase of cell cycle. Additionally, LGG-SN sensitizes cancer cells to both 5-Fluorouracil and Irinotecan, thereby showing a positive synergistic action. CONCLUSION: Overall, our results suggest that LGG-SN may contain one or more bioactive molecules with anti-cancer activity which sensitize cancer cells to chemotherapeutic drugs. Thus, LGG could be proposed as an ideal candidate for ground-breaking integrated approaches to be employed in oncology, to reduce chemotherapy-related side effects and overcome resistance or relapse issues, thus ameliorating the therapeutic response in cancer patients.

Lactobacillus rhamnosus GG treatment potentiates ethanol-induced behavioral changes through modulation of intestinal epithelium in Danio rerio.

The gut-brain axis directly regulates the brain homeostatic environment; an imbalance in gut microbial composition following ethanol exposure is maleficent. In this context, involvement of probiotics as prophylactic intervention against ethanol-induced neurotoxicity is elusive in the literature. Therefore, the present study was aimed to determine the impact of chronic ethanol exposure on the neurobehavioral response of zebrafish and possible neuroprotection through co-supplementation of probiotic Lactobacillus rhamnosus GG (LGG). Zebrafish were divided into naive, control, ethanol (0.01% v/v), LGG, and ethanol co-supplemented with LGG groups. Neurobehavioral assessment was performed after 7 days of chronic waterborne exposure to ethanol with LGG co-supplementation followed by histopathological studies. The findings indicated that there was a clear alteration in locomotor activity and habitat preference, with animals preferentially migrating toward altered zones on exposure to ethanol. However, co-supplementation of LGG showed restoration against ethanol-induced neurobehavioral and cognitive dysfunction. Brain tissue pyknosis and intestinal epithelial disruption were significantly mitigated on LGG co-supplementation against ethanol in zebrafish. The present study provides a novel approach toward supplementation of probiotics such as LGG in modulation of gut commensal microbiota influencing zebrafish behavior. Moreover, the findings delineate the possible role of probiotics as a curative administration to counter ethanol-persuaded neurological outcomes.

Effects of dietary Lactobacillus rhamnosus GG supplementation on the production performance, egg quality, eggshell ultrastructure, and lipid metabolism of late-phase laying hens.

BACKGROUND: Toward the late phase of laying, the production performance of laying hens decreases, egg quality deteriorates, lipid metabolism weakens, and hepatic lipid accumulation is exacerbated. Probiotics as an alternative to antimicrobials have been employed in poultry-related industries. Lactobacillus rhamnosus GG (LGG) is currently the most researched and clinically validated probiotic, showing promising effects in multiple application areas. However, few studies have been conducted on livestock (including poultry) production. RESULTS: Compared with the CON group, the feed conversion ratio (P < 0.01) declined significantly in the LGG group. Eggshell strength (P < 0.001) and eggshell thickness (P < 0.001) were significantly increased by supplementation with LGG in the diet. The height (P < 0.001) and proportion (P < 0.05) of the effective layer and the mammillary knob density (P < 0.01) in the eggshell ultrastructure of the LGG group increased significantly, while the mammillary layer (P < 0.05) and knob width (P < 0.01) decreased significantly. The LGG-treated hens had significantly lower serum concentrations of low-density lipoprotein (P < 0.05), free fatty acids (P < 0.01), and liver triglyceride (P < 0.05) levels than those in the CON group. CONCLUSIONS: LGG supplementation significantly decreases the feed conversion ratio, improves eggshell quality by altering the ultrastructure, and improves lipid metabolism in the late laying period.

Lactobacillus rhamnosus GG combined with inosine ameliorates alcohol-induced liver injury through regulation of intestinal barrier and Treg/Th1 cells.

BACKGROUND: Intestinal epithelial barrier disruption and bacterial translocation exacerbates the progression of alcoholic liver disease. Lactobacillus rhamnosus GG (LGG), a probiotic, has been shown benefits in chronic liver disease and in regulating gut dysbiosis. Previous studies showed the protective roles of LGG in ethanol-disrupted gut barrier functions and liver injury. Inosine, a metabolite produced by intestinal bacteria, has the anti-inflammatory and immunregulatory functions. In this study, the synergistic effect of LGG and inosine was investigated in a mouse model of alcohol-induced liver disease (ALD). METHODS: Male C57BL/6 mice were fed with a Lieber-DeCarli diet containing 5% alcohol for four weeks to establish a model of alcohol-induced liver injury. LGG or a combination of LGG and inosine were administrated orally to explore a new therapeutic method for alcohol-induced liver disease and to investigate the underlying mechanisms. Liver damage was evaluated by transaminases and pathological changes. Tight junction proteins, composition of the gut microbiome, cytokines, lipopolysaccharides (LPS), glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), F4/80+ macrophages, as well as p38, Jun N-terminal kinase (JNK), were determined by qRT-PCR, RNAseq, ELISA, IHC and western blot. Regulatory T (Treg) cells were characterized by positive staining of CD4, CD25 and Foxp3 using flow cytometry. IFN-γ-producing CD4+ T (Th1) cells were examined by intracellular cytokine staining. RESULTS: Alcohol consumption induced elevated liver enzymes, steatosis and inflammation, while LGG combined with inosine treatment was more significant to ameliorate these symptoms compared with LGG alone. When LGG combined with inosine were administered to ALD mice, intestinal microecology significantly improved reflected by intestinal villi and tight junction proteins recovery and the restoration of intestinal flora. Combined therapy inhibited phosphorylation of p38 and JNK to alleviate hepatic inflammation. Moreover, flow cytometry analysis showed that long-term excessive alcohol consumption reduced Tregs population while increased Th1 population, which was restored by a combination of LGG and inosine treatment. CONCLUSIONS: The findings from the study indicate that the combined LGG and inosine treatment ameliorates ALD by improving the gut ecosystem, intestinal barrier function, immune homeostasis and liver injury.

Lactobacillus rhamnosus GG colonization in early life regulates gut-brain axis and relieves anxiety-like behavior in adulthood.

Evidence reveals that gut dysbiosis is involved in bidirectional interactions in gut-brain axis and participates in the progress of multiple disorders like anxiety. Gut microbes in early life are crucial for establishment of host health. We aimed to investigate whether early life probiotics Lactobacillus rhamnosus GG (LGG) colonization could relieve anxiety in adulthood through regulation of gut-brain axis. Live or fixed LGG was gavaged to C57BL/6 female mice from day 18 of pregnancy until natural birth, and newborn mice from day 1 to day 5 respectively. In this study, we found that live LGG could be effectively colonized in the intestine of offspring. LGG colonization increased intestinal villus length and colonic crypt depth, accompanied with barrier function protection before weaning. Microbiota composition by 16S rRNA sequencing showed that some beneficial bacteria, such as Akkermansia and Bifidobacteria, were abundant in LGG colonization group. The protective effect of LGG on gut microbiota persisted from weaning to adulthood. Intriguingly, behavioral results assessed by elevated plus mazed test and open field test demonstrated relief of anxiety-like behavior in adult LGG-colonized offspring. Mechanically, LGG colonization activated epithelial growth factor receptor (EGFR) and enhanced serotonin transporter (SERT) expression and modulated serotonergic system in the intestine, and increased brain-derived neurotrophic factor and γ-aminobutyric acid receptor levels in the hippocampus and amygdala. Blocking EGFR blunted LGG-induced the increased SERT and zonula occludens-1 expression. Collectively, early life LGG colonization could protect intestinal barrier of offspring and modulate gut-brain axis in association with relief of anxiety-like behavior in adulthood.

Lactobacillus rhamnosus GG protects against atherosclerosis by improving ketone body synthesis.

Atherosclerosis (AS) is a major cause of death and morbidity worldwide. There is an increasing amount of evidence that the gut microbiota plays an important role in disorders associated with lipid metabolism, such as AS, and alterations in the composition of the gut microbiota and its metabolic potential have been identified as contributing factors in the development of AS. Recently, probiotics have attracted great interest for their excellent cholesterol-lowering ability, their capacity to improve vascular endothelial function, and their participation in the remodeling of the intestinal flora to prevent AS. The incidental findings of our other study suggest that probiotic Lactobacillus rhamnosus GG may be associated with slowing the progression of AS. Thus, we delivered strain GG into mice by oral feeding and found that strain GG could effectively inhibit AS plaque generation. We analyzed the differences in gut microbiota composition and the peripheral blood metabolome in mice after oral feeding of strain GG by 16S DNA sequencing and untargeted metabolomics, respectively. The results showed that strain GG changed the composition of the gut microbiota in mice fed a high-fat diet; elevated the abundance of beneficial bacteria, such as Bilophila and Alistipes, and decreased the abundance of harmful bacteria, such as Deltaproteobacteria. The results of enrichment analysis of the gut microbiota and the peripheral blood metabolome both indicated that the antiatherosclerotic effect of strain GG might be associated with the biosynthesis pathway of ketone bodies. In addition, strain GG attenuated endothelial injury and elevated peripheral blood ketone body content in mice but did not significantly affect low-density lipoprotein cholesterol (LDL-C) and total cholesterol (TC) content. In conclusion, our study provides new evidence that strain GG slows the progression of AS, which may be associated with its improvement of the gut microbiome and peripheral blood metabolome, its ability to increase the abundance of beneficial bacteria, and its participation in unsaturated fatty acid and ketone body synthesis and degradation. KEY POINTS: • L. rhamnosus GG attenuated endothelial injury and atherosclerotic plaque formation • L. rhamnosus GG elevated the abundance of beneficial bacteria • L. rhamnosus GG elevated peripheral blood ketone body content in mice.

Gut Bifidobacteria enrichment following oral Lactobacillus-supplementation is associated with clinical improvements in children with cystic fibrosis.

BACKGROUND: Relationships between gut microbiomes and airway immunity have been established in murine and human studies of allergy and asthma. Early life Lactobacillus supplementation alters the composition and metabolic productivity of the gut microbiome. However, little is known of how Lactobacillus supplementation impacts the gut microbiota in children with cystic fibrosis (CF) and whether specific microbiota states that arise following gut microbiome manipulation relate to pulmonary outcomes. METHODS: Stool samples were collected from CF patients enrolled in a multi-center, double-blind, randomized placebo-controlled trial of daily Lactobacillus rhamnosus strain GG (LGG) probiotic supplementation over a 12-month period. Fecal 16S rRNA biomarker sequencing was used to profile fecal bacterial microbiota and analyses were performed in QiiME. RESULTS: Bifidobacteria-dominated fecal microbiota were more likely to arise in LGG-treated children with CF (P = 0.04). Children with Bifidobacteria-dominated gut microbiota had a reduced rate of pulmonary exacerbations (IRR = 0.55; 95% CI 0.25 to 0.82; P = 0.01), improved pulmonary function (+ 20.00% of predicted value FEV1; 95% CI 8.05 to 31.92; P = 0.001), lower intestinal inflammation (Calprotectin; Coef = - 16.53 µg g-1 feces; 95% CI - 26.80 to - 6.26; P = 0.002) and required fewer antibiotics (IRR = 0.43; 95% CI 0.22 to 0.69; P = 0.04) compared to children with Bacteroides-dominated microbiota who were less likely to have received LGG. CONCLUSIONS: The majority of pediatric CF patients in this study possessed a Bacteroides- or Bifidobacteria-dominated gut microbiota. Bifidobacteria-dominated gut microbiota were more likely to be associated with LGG-supplementation and with better clinical outcomes.

Cytokine-specific autoantibodies shape the gut microbiome in autoimmune polyendocrine syndrome type 1.

BACKGROUND: Gastrointestinal dysfunction is a frequent and disabling manifestation of autoimmune polyendocrine syndrome type 1 (APS-1), a rare monogenic multiorgan autoimmune disease caused by the loss of central AIRE-controlled immune tolerance. OBJECTIVES: This study aimed to understand the role of the gut microbiome in APS-1 symptoms and potentially alleviate common gastrointestinal symptoms by probiotic intervention. METHODS: This study characterized the fecal microbiomes of 28 patients with APS-1 and searched for associations with gastrointestinal symptoms, circulating anti-cytokine autoantibodies, and tryptophan-related metabolites. Additionally, daily doses of the probiotic Lactobacillus rhamnosus GG were administered for 3 months. RESULTS: Of 581 metagenomic operational taxonomic units (mOTUs) characterized in total, 14 were significantly associated with patients with APS-1 compared with healthy controls, with 6 mOTUs depleted and 8 enriched in patients with APS-1. Four overabundant mOTUs were significantly associated with severity of constipation. Phylogenetically conserved microbial associations with autoantibodies against cytokines were observed. After the 3-month intervention with the probiotic L rhamnosus GG, a subset of gastrointestinal symptoms were alleviated. L rhamnosus GG abundance was increased postintervention and corresponded with decreased abundances of Alistipes onderdonkii and Collinsella aerofaciens, 2 species positively associated with severity of diarrhea in patients with APS-1. CONCLUSIONS: The APS-1 microbiome correlates with several APS-1 symptoms, some of which are alleviated after a 3-month L rhamnosus GG intervention. Autoantibodies against cytokines appear to shape the gut microbiome by positively correlating with a taxonomically consistent group of bacteria.

Label-free quantitative proteomic analysis of the inhibition effect of Lactobacillus rhamnosus GG on Escherichia coli biofilm formation in co-culture.

BACKGROUND: Escherichia coli (E. coli) is the principal pathogen that causes biofilm formation. Biofilms are associated with infectious diseases and antibiotic resistance. This study employed proteomic analysis to identify differentially expressed proteins after coculture of E. coli with Lactobacillus rhamnosus GG (LGG) microcapsules. METHODS: To explore the relevant protein abundance changes after E. coli and LGG coculture, label-free quantitative proteomic analysis and qRT-PCR were applied to E. coli and LGG microcapsule groups before and after coculture, respectively. RESULTS: The proteomic analysis characterised a total of 1655 proteins in E. coli K12MG1655 and 1431 proteins in the LGG. After coculture treatment, there were 262 differentially expressed proteins in E. coli and 291 in LGG. Gene ontology analysis showed that the differentially expressed proteins were mainly related to cellular metabolism, the stress response, transcription and the cell membrane. A protein interaction network and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis indicated that the differentiated proteins were mainly involved in the protein ubiquitination pathway and mitochondrial dysfunction. CONCLUSIONS: These findings indicated that LGG microcapsules may inhibit E. coli biofilm formation by disrupting metabolic processes, particularly in relation to energy metabolism and stimulus responses, both of which are critical for the growth of LGG. Together, these findings increase our understanding of the interactions between bacteria under coculture conditions.

Growth and tolerance of healthy, term infants fed lower protein extensively hydrolyzed or amino acid-based formula: double-blind, randomized, controlled trial.

BACKGROUND: Optimal protein level in hypoallergenic infant formulas is an area of ongoing investigation. The aim was to evaluate growth of healthy term infants who received extensively hydrolyzed (EH) or amino acid (AA)-based formulas with reduced protein. METHODS: In this prospective, multi-center, double-blind, controlled, parallel group study, infants were randomized to receive a marketed EH casein infant formula at 2.8 g protein/100 kcal (Control) or one of two investigational formulas: EH casein formula at 2.4 g protein/100 kcal (EHF) or AA-based formula at 2.4 g total protein equivalents/100 kcal (AAF). Control and EHF each had 2 × 107 CFU Lactobacillus rhamnosus GG/100 kcal. Anthropometrics were measured and recall of formula intake, tolerance, and stool characteristics was collected at 14, 30, 60, 90, 120 days of age. Primary outcome was weight growth rate (g/day) between 14 and 120 days of age (analyzed by ANOVA). Medically confirmed adverse events were recorded throughout the study. RESULTS: No group differences in weight or length growth rate from 14 to 120 days were detected. With the exception of significant differences at several study time points for males, no group differences were detected in mean head circumference growth rates. However, mean achieved weight, length, and head circumference demonstrated normal growth throughout the study period. No group differences in achieved weight or length (males and females) and head circumference (females) were detected and means were within the WHO growth 25th and 75th percentiles from 14 to 120 days of age. With the exception of Day 90, there were no statistically significant group differences in achieved head circumference for males; means remained between the WHO 50th and 75th percentiles for growth at Days 14, 30, and 60 and continued along the 75th percentile through Day 120. No differences in study discontinuation due to formula were detected. The number of participants for whom at least one adverse event was reported was similar among groups. CONCLUSIONS: This study demonstrated hypoallergenic infant formulas at 2.4 g protein/100 kcal were safe, well-tolerated, and associated with appropriate growth in healthy term infants from 14 to 120 days of age. TRIAL REGISTRATION: ClinicalTrials.gov, ClinicalTrials.gov Identifier: NCT01354366 . Registered 13 May 2011.

N, O-codoped hierarchical porous graphitic carbon for electrochemical immunosensing of Lactobacillus rhamnosus GG.

An ultrasensitive label-free electrochemical immunosensor was fabricated for quantitative detection of Lactobacillus rhamnosus GG (LGG). The N/O co-doped three-dimensional hierarchical porous graphitic (THPG) carbon was synthesized by a one-step synthesis of polyaniline hydrogel, and followed by simple carbonization and chemical activation procedures. Because of the unique structure design, the obtained THPG carbon networks possess an ultra-large specific surface area of 4859 m2 g-1 along with a class of highly graphitic carbons. The results offer an enormous surface area and excellent electrical conductivity for label-free electrochemical immunosensing of probiotic L. rhamnosus strain. Under optimal conditions, the immunosensor showed a good linear relationship between peak current and concentration of LGG (R2 = 0.9976), with a detection limit of 2 CFU mL-1. Furthermore, this label-free immunosensor also shows good specificity, long-term stability, and reliability, and could be applied to detect probiotic LGG in dairy products and drinks with satisfactory results. The present protocol was shown to be quite promising for practical screening and functional evaluation of probiotic products containing LGG. A ultrasensitive label-free electrochemical immunosensor based on THPG carbon was fabricated for detection of Lactobacillus rhamnosus GG.

Lactobacillus rhamnosus GG attenuates the pathology of Chlamydial muridarium in the upper genital tract in mice. / å£æœé¼ æŽç³–ä¹³æ†èŒGGå‡è½»é¼ è¡£åŽŸä½“æ„ŸæŸ“å¼•èµ·çš„ä¸Šç”Ÿæ®–é“ç— å˜.

OBJECTIVES: Chlamydia trachomatis is a pathogen which can cause hydrosalpinx and tubal fibrosis when infecting the urogenital tract. However, the mechanism is still not clear. There is growing evidence that the gut microbiota is associated with the pathogenesis of both intestinal and extra-intestinal disorders, such as cardiovascular disease, hepatocirrhosis, allergy, respiratory tract infection, polycystic ovary syndrome, endometriosis, and bacterial vaginitis. Lactobacillus rhamnosus GG (LGG) is one of the most extensively studied and widely used probiotic bacteria, the benefits of LGG including the treatment in gastrointestinal disorders and immunomodulation are well demonstrated, and it can also alleviate hypersensitivity reaction and diarrhoea, inhibit a variety of respiratory and urogenital diseases. Chlamydia muridarium (Cm) infection is a good model for the study on human Chlamydia pathogenicity in genitourinary tract. The mice infected with Cm were used as animal models to preliminarily explore the mechanism for the effect of LGG on upper reproductive tract infection in the mice, and to provide experimental basis for the pathogenesis of Chlamydia trachomatis genitourinary tract infection and the new idea for the treatment of Chlamydia trachomatis infection. METHODS: Five to six weeks-old C57BL/6J mice were divided into 2 groups: An experimental group and a control group. The experimental group were administrated with 5×108 colony forming units (CFU) LGG for 19 consecutive days, while the control group were feed PBS. The mice in the 2 group were subcutaneously injected with 2.5 mg progesterone on Day 9 and infected with 1×105 inclusion body forming unit of Cm via the vaginal tract on Day 14. Vaginal and rectal swabs were taken every 7 days to infect HeLa cells for 24 hours, then the indirect immunofluorescence assay was used and the number of inclusion bodies of Chlamydia were calculated. Mice were euthanized on Day 14 and Day 63 after Cm inoculation, the vaginal tracts were dissected, and the tissue homogenates were prepared to culture the pathogens for 24 hours. The Cm bearing capacity in the bilateral uterine horn, tubal ovary, and cervical vaginal tissues in the 2 groups were calculated. The spleen cells were harvested to assay the intracellular IFN-γ, IL-5, and IL-17 by flow cytometry. On Day 63 after the Chlamydia infection, the pathology injury in the bilateral uterine horn and oviduct was observed, and the pathological sections and HE staining in the various part of genital tract were performed. The inflammatory cell infiltration and lumen dilatation was assessed. The specific IgM and IgG in sera were detected by indirect ELISA on Day 14 and 63 after infection. RESULTS: There was no effect of LGG on the clearing of Cm from the urogenital tract, the Chlamydia ascending to fallopian tube or the uterine horn, and the organism dissemination and colonization to the gastrointestinal tract (all P>0.05). On Day 14 after Cm infection via the vagina, the IL-17 expression level in the experimental group was significant decreased than that in the control group (t=2.486, P<0.05), but there was no significant difference between the 2 groups in the CD4+ T rate in spleen and IgM and IgG levels in serum after Cm intravaginal infection (all P>0.05). On Day 63 after Cm infection, there was no difference in the severity of inflammation in the uterine horns and fallopian tubes between the 2 groups (P>0.05), but the dilation of the fallopian tubes and hydrosalpinx was attenuated in the experimental group compared with the control group (P<0.05). CONCLUSIONS: Oral administration of LGG has no effect on inhibiting Cm ascending to upper genital tract and preventing the dissemination and colonization of Cm to the gastrointestinal tract, which also cannot affect the secretion of specific IgM and IgG in sera. Oral administration of LGG can suppress the production of IL-17 in the spleen cells and attenuate hydrosalpinx development when following Cm intravaginal infection in mice.

Crystal structure of lactobacillar SpaC reveals an atypical five-domain pilus tip adhesin: Exposing its substrate-binding and assembly in SpaCBA pili.

Adhesion to cell surfaces is an essential and early prerequisite for successful host colonization by bacteria, and in most instances involves the specificities of various adhesins. Among bacterial Gram-positives, some genera and species mediate attachment to host cells by using long non-flagellar appendages called sortase-dependent pili. A case in point is the beneficial Lactobacillus rhamnosus GG gut-adapted strain that produces the so-called SpaCBA pilus, a structure noted for its promiscuous binding to intestinal mucus and collagen. Structurally, SpaCBA pili are heteropolymers of three different pilin-protein subunits, each with its own location and function in the pilus: backbone SpaA for length, basal SpaB for anchoring, and tip SpaC for adhesion. Previously, we solved the SpaA tertiary structure by X-ray crystallography and also reported on the crystallization of SpaB and SpaC. Here, we reveal the full-length high-resolution (1.9 Å) crystal structure of SpaC, a first for a sortase-dependent pilus-bearing commensal. The SpaC structure, unlike the representative four-domain architecture of other Gram-positive tip pilins, espouses an atypically longer five-domain arrangement that includes N-terminal 'binding' and C-terminal 'stalk' regions of two and three domains, respectively. With the prospect of establishing new mechanistic insights, we provide a structural basis for the multi-substrate binding nature of SpaC, as well as a structural model that reconciles its exclusive localization at the SpaCBA pilus tip.

Lactobacillus rhamnosus GG Genomic and Phenotypic Stability in an Industrial Production Process.

Lactobacillus rhamnosus GG is one of the most widely marketed and studied probiotic strains. In L. rhamnosus GG, the spaCBA-srtC1 gene cluster encodes pili, which are important for some of the probiotic properties of the strain. A previous study showed that the DNA sequence of the spaCBA-srtC1 gene cluster was not present in some L. rhamnosus GG variants isolated from liquid dairy products. To examine the stability of the L. rhamnosus GG genome in an industrial production process, we sequenced the genome of samples of L. rhamnosus GG (DSM 33156) collected at specific steps of the industrial production process, including the culture collection stock, intermediate fermentations, and final freeze-dried products. We found that the L. rhamnosus GG genome sequence was unchanged throughout the production process. Consequently, the spaCBA-srtC1 gene locus was intact and fully conserved in all 31 samples examined. In addition, different production batches of L. rhamnosus GG exhibited consistent phenotypes, including the presence of pili in final freeze-dried products, and consistent characteristics in in vitro assays of probiotic properties. Our data show that L. rhamnosus GG is highly stable in this industrial production process.IMPORTANCELactobacillus rhamnosus GG is one of the best-studied probiotic strains. One of the well-characterized features of the strain is the pili encoded by the spaCBA-srtC1 gene cluster. These pili are involved in persistence in the gastrointestinal tract and are important for the probiotic properties of L. rhamnosus GG. Previous studies demonstrated that the L. rhamnosus GG genome can be unstable under certain conditions and can lose the spaCBA-srtC1 gene cluster. Since in vitro studies have shown that the loss of the spaCBA-srtC1 gene cluster decreases certain L. rhamnosus GG probiotic properties, we assessed both the genomic stability and phenotypic properties of L. rhamnosus GG throughout an industrial production process. We found that neither genomic nor phenotypic changes occurred in the samples. Therefore, we demonstrate that L. rhamnosus GG retains the spaCBA-srtC1 cluster and exhibits excellent genomic and phenotypic stability in the specific industrial process examined here.

Lactobacillus rhamnosus GG Attenuates Lipopolysaccharide-Induced Inflammation and Barrier Dysfunction by Regulating MAPK/NF-κB Signaling and Modulating Metabolome in the Piglet Intestine.

BACKGROUND: Probiotic Lactobacillius rhamnosus GG (LGG) shows beneficial immunomodulation on cultured cell lines in vitro and in mouse models. OBJECTIVE: The aim was to investigate the effects of LGG on intestinal injury and the underlying mechanisms by elucidating inflammatory signaling pathways and metabolomic response to LPS stimulation in the piglet intestine. METHODS: Piglets (Duroc × Landrace × Large White, including males and female; 8.6 ± 1.1 kg) aged 28 d were assigned to 3 groups (n = 6/group): oral inoculation with PBS for 2 wk before intraperitoneal injection of physiological saline [control (CON)] or LPS (25 µg/kg body weight; LPS) or oral inoculation with LGG for 2 wk before intraperitoneal injection of LPS (LGG+LPS). Piglets were killed 4 h after LPS injection. Systemic inflammation, intestinal integrity, inflammation signals, and metabolomic characteristics in the intestine were determined. RESULTS: Compared with CON, LPS stimulation significantly decreased ileal zonula occludens 1 (ZO-1; 44%), claudin-3 (44%), and occludin (41%) expression; increased serum diamineoxidase (73%), D-xylose (19%), TNF-α (43%), and IL-6 (55%) concentrations; induced p38 mitogen-activated protein kinase (p38 MAPK; 85%), extracellular signal-regulated kinase (ERK; 96%), and NF-κB p65 phosphorylation (37%) (P < 0.05). Compared with LPS stimulation alone, LGG pretreatment significantly enhanced the intestinal barrier by upregulating expressions of tight junction proteins (ZO-1, 73%; claudin-3, 55%; occludin, 67%), thereby decreasing serum diamineoxidase (26%) and D-xylose (28%) concentrations, and also reduced serum TNF-α expression (16%) and ileal p38 MAPK (79%), ERK (43%) and NF-κB p65 (37%) phosphorylation levels (P < 0.05). Metabolomic analysis showed clear separation between each group. The concentrations of caprylic acid [fold-change (FC) = 2.39], 1-mono-olein (FC = 2.68), erythritol (FC = 4.62), and ethanolamine (FC = 4.47) significantly increased in the intestine of LGG + LPS piglets compared with the LPS group (P < 0.05). CONCLUSIONS: These data suggest that LGG alleviates gut inflammation, improves intestinal barrier function, and modulates the metabolite profile of piglets challenged with LPS. This trial was registered at the Zhejiang University (http://www.lac.zju.edu.cn) as ZJU20170529.

Hepatoprotective Effect of Probiotic Lactobacillus rhamnosus GG Through the Modulation of Gut Permeability and Inflammasomes in a Model of Alcoholic Liver Disease in Zebrafish.

Objective: Alcoholic liver disease (ALD) is among the leading causes of death from liver disease. Among the factors involved in its pathogenesis are inflammation and increased intestinal permeability. The aim of this study was to assess the effect of Lactobacillus rhamnosus GG (LGG) on hepatic lipid accumulation, activation of inflammasomes, and gut permeability markers in experimental model of ALD with zebrafish.Methods: An experiment was conducted to assess the effective LGG dose capable of promoting intestinal colonization. Animals were divided into three groups (n = 64/group): ethanol group (E), ethanol + probiotic group (EP), and control group (C). Groups E and EP were exposed to 0.5% ethanol concentration for 28 days. At the end of this period, animals were euthanized, and livers were collected for Oil Red staining and assessment of the inflammasome system. Intestines were collected for evaluation of gut permeability markers.Results: The dose of 1.55 × 106 UFC LGG/fish/d promoted intestinal colonization. Group EP presented lower hepatic lipid accumulation, lower il-1ß expression, and higher cldn15a expression when compared to group E.Conclusions: Supplementation with LGG was protective for hepatic steatosis in ALD model. In addition, LGG influenced the modulation of the inflammatory response and markers of gut permeability, improving the gut barrier structure.

Antibiotics can cause weight loss by impairing gut microbiota in mice and the potent benefits of lactobacilli.

This study assessed whether antibiotics could alter gut microbiota to affect host growth and the possibility of alleviation by lactobacilli. We divided four-week-old BABL/c mice into control (Ctrl), antibiotic exposure (Abx), Lactobacillus plantarum PC-170 (PC), and Lactobacillus rhamnosus GG (LGG) group and the Abx, LGG, and PC group received an one-week antibiotic/antibiotic + probiotic treatment. The fecal microbiota and the expression of splenic cytokines were determined. Following the ceftriaxone treatment, the body weight gain of Abx was delayed compared with others. The ceftriaxone treatment significantly decreased the alpha-diversity of the fecal microbiota and altered the fecal microbiota but LGG and PC can partly alleviate the effect. At the end of the study, the microbial community of LGG and PC group were more similar to Ctrl compared with Abx group. The results indicated that ceftriaxone could significantly alter intestinal microbiota. Lactobacilli might alleviate the side effects of antibiotics by stabilizing the intestinal microbiota.