Probiotic Conditioned Media Research Articles

Abstract 2181: Endocrine-targeting therapies modify the breast microbiome to shift metabolism and reduce cancer risk

Abstract Studies indicate that breast tissue has a distinct modifiable microbiome population. However, whether endocrine-targeting therapies modify the non-cancerous breast microbiome to impact tumorigenesis is unknown. To examine this question, we performed 16S bacterial sequencing on DNA isolated from female C57BL/6 mouse mammary glands (MG) administered tamoxifen citrate for 16 weeks and breast tissue from ovariectomized non-human primates administered tamoxifen citrate (TAM) for 2.5 years. In both models, we show that TAM significantly shifted β-diversity and increased Firmicutes proportional abundance. TAM elevated the proportional abundance of Lactobacillus spp., Streptococcus luticea, and Staphylococcus sciuri. Female BALB/c mice received intra-nipple injections of Lactobacillus and MG were harvested at 1-, 3-, 7-, and 14-days post-injection to show bacteria colonization and modulation of metabolism-associated gene and protein expression. B6.MMTV-PyMT mice were injected with Lactobacillus bacteria into the MG. Elevating MG Lactobacillus reduced tumorigenesis with an associated decrease in tumor proliferation and shifts in non-cancerous MG tissue metabolism. To elucidate the effects of combining TAM and probiotic bacteria on breast cancer outcomes, 4T1.2ER+ tumor-bearing female BALB/c mice, fed either a control or Western diet were treated with TAM, Lactobacillus probiotic, or a combination of TAM + Lactobacillus. Results indicate that oral probiotic administration in combination with TAM reduced ER+ tumor growth. Plasma metabolomics revealed combination treatment elevated L-carnitine regardless of diet, suggesting drug-bug interactions modifying metabolite profiles. Cell viability assays on human MCF7 BC cells and non-cancerous mammary HMT-3522S1 cells treated with 1% probiotic-conditioned media (Pro-CM) revealed a significant decrease in cell viability in BC cells but not non-cancerous epithelial cells. Mitochondrial fuel oxidation Seahorse assay on murine 4T1.2ER+ cells treated with 1% Pro-CM, 1μm 4-hydroxytamoxifen (4-OHT) or combination revealed changes in metabolic substrate dependency, supporting role of microbial metabolites impacting metabolism. Tumor sections from patients with ER+ BC treated with aromatase inhibitors and/or Faslodex in the neoadjuvant setting were stained for Gram-positive bacteria, Gram-negative bacteria, and Ki67. Gram-positive bacteria negatively correlated with Ki67 immunoreactivity, suggesting endocrine-targeting therapies promote tumor Gram-positive bacteria associated with a decrease in proliferation. Taken together, our data indicates that endocrine-targeting treatments modify the breast microbiome associated with a shift in tissue metabolism to decrease proliferation and reduce ER+ BC risk. Citation Format: Alana Arnone, Yu-Ting Tsai, J Mark Cline, Adam S. Wilson, Brian Westwood, Meghan E. Seger, Akiko Chiba, Marissa Howard-McNatt, Edward A. Levine, Alexandra Thomas, David R. Soto-Pantoja, Katherine L. Cook. Endocrine-targeting therapies modify the breast microbiome to shift metabolism and reduce cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2181.

Probiotics Play a Role in Alleviating Bile Acid (BA)‐induced Apoptosis and Oxidative Stress in Human Colonic T84 Cells

BA‐malabsorption diarrhea (BAD) has been identified in one‐third of patients with inflammatory bowel disease (IBD) and affects >200 million people worldwide. Probiotic supplements are used to promote gut health, restoring microbiome and bile acid composition in these patients. We have previously reported the yin and yang in bile acid (BA) action, wherein the primary BA, chenodeoxycholic acid (CDCA; 500μM), altered tight junctions (TJ) and increased paracellular permeability in T84 cells, while its secondary BA, lithocholic acid (LCA; 50μM), did not. High colonic concentrations (>500μM) of CDCA have been reported in patients with IBD which can modify the gut microflora. Here, we studied the effect of a probiotic supplement on BA action in T84 cells and hypothesized that probiotics could ameliorate BA‐induced apoptosis, oxidative stress and barrier disruption.To study the crosstalk between BA and microflora, probiotic strains in Up & Up extra strength supplement (30 billion CFUs) were grown in T84 epithelial cell culture media ± BA under anaerobic conditions at 37°C. Bacteria grew well in control media (CFU x 108: 6 ± 1) after 48h with Lactobacillus paracasei identified as the predominant species by 16S rRNA sequencing. Further, microbial growth was modified by BAs. 500μM CDCA caused a significant reduction in the growth rate (CFU x 108: 0.07± 0.3; p<0.001, n=4) with L. paracasei remaining predominant. 50μM LCA alone did not alter growth but reversed the effect of CDCA on bacterial growth (CFU x 108: LCA: 6 ± 0.3; CDCA+LCA: 6 ± 1, n=4). Interestingly, LCA ± CDCA changed the bacterial composition with L. rhamnosus becoming the prevalent species. Culture media ± BA was sterile filtered to remove bacteria and the conditioned media (CM) obtained was used in subsequent experiments.T84 cells were grown to confluency in 6‐well plates and treated overnight with CM± BA. Apoptosis (Annexin V, flow cytometry, fluorescence microscopy), cell death (lactate dehydrogenase (LDH) assay, colorimetry) and mitochondrial ROS (mROS, CellRox, flow cytometry) were measured.Flow cytometry and LDH assay revealed that CM caused a dose‐dependent increase in T84 cell apoptosis with 100% CM causing significant (35‐fold) T84 cell death (n=3). The optimal CM dilution with minimal cell death was identified to be 1:8. As seen previously, CDCA (500μM) caused a small increase in apoptosis, while LCA (50μM) did not; CM± LCA reversed CDCA’s effect on cell death (% cell death, CTRL: 10±3 vs CM: 9±4; LCA: 9±3 vs CM+LCA: 10±2; CDCA: 19±4 vs CM+CDCA: 6±3; CDCA+LCA: 16±6 vs CM+CDCA+LCA: 8±3, p<0.05, n=5). Similarly, CDCA increased mROS and this was attenuated by CM (CellRox+ cells, in %, DMSO: 8±1; CDCA: 18±1; CM: 9±1; CM+CDCA: 9±2, n≥3). Though LCA alone did not have an effect, CM+LCA attenuated CDCA‐induced mROS release (LCA: 9±1; CDCA+LCA: 15±1 CM+LCA: 11±1, CM+CDCA+LCA: 9±2; n=4). We demonstrate that BA altered bacterial growth and probiotic CM±LCA reverses CDCA‐induced apoptosis and oxidative stress in T84 cells. We are currently studying the effect of CM on BA‐induced increases in paracellular permeability and TJ dysfunction. Delineating the role played by probiotics in alleviating BAD and understanding the mechanism of action could lead to novel therapeutic strategies for inflammation‐associated diarrheas.

The impact of indole-3-lactic acid on immature intestinal innate immunity and development: a transcriptomic analysis

An excessive intestinal inflammatory response may have a role in the pathogenesis of necrotizing enterocolitis (NEC) in very preterm infants. Indole-3-lactic acid (ILA) of breastmilk tryptophan was identified as the anti-inflammatory metabolite involved in probiotic conditioned media from Bifidobacteria longum subsp infantis. This study aimed to explore the molecular endocytic pathways involved in the protective ILA effect against inflammation. H4 cells, Caco-2 cells, C57BL/6 pup and adult mice were used to compare the anti-inflammatory mechanisms between immature and mature enterocytes in vitro and in vivo. The results show that ILA has pleiotropic protective effects on immature enterocytes including anti-inflammatory, anti-viral, and developmental regulatory potentials in a region-dependent and an age-dependent manner. Quantitative transcriptomic analysis revealed a new mechanistic model in which STAT1 pathways play an important role in IL-1β-induced inflammation and ILA has a regulatory effect on STAT1 pathways. These studies were validated by real-time RT-qPCR and STAT1 inhibitor experiments. Different protective reactions of ILA between immature and mature enterocytes indicated that ILA’s effects are developmentally regulated. These findings may be helpful in preventing NEC for premature infants.

Probiotic Metabolites Promote Stem Cell Proliferation and Differentiation

Probiotic metabolites (PM) impart numerous health benefits in human and may have a potential to induce cell proliferation and differentiation. We tested the effects of PM released by probiotics found in yogurts on stem cell proliferation and differentiation. Five different brands of yogurts (Oikos, Stonyfield, Yoplait, Chobani and Activia) often sold in the supermarkets were tested in this study. Yogurt cultures analyzed by Gram‐staining and DNA sequencing of 16s rRNA gene identified that these yogurts are not equally supplemented and include different types and various amounts of probiotics. The probiotics were cultured and the condition media containing PMs were collected and used in our study. To evaluate the effect of PM on stem cells, human induced pluripotent stem cells (iPSC) were treated with probiotic conditioned media. We found that probiotic Lactobacillus reuteri conditioned media (LRCM) induced iPSC proliferation at lower doses but inhibited cell proliferation at higher doses. Additionally, expression of the proliferating cell nuclear antigen (PCNA) and pluripotent stem cell factors, OCT4, SOX2 and NANOG transcripts was upregulated in iPSCs treated with LRCM. iPS cells exposed to LRCM during in vitro differentiation led to enhanced expression of myocyte markers, Nkx2.5 and cTnT. Consistently, sodium butyrate, a defined bacterial metabolite (short chain fatty acid), had similar effect on iPSC proliferation and expression of pluripotent markers and myocyte markers. In summary, we have demonstrated that probiotics in different brands of yogurts are diverse in their numbers, viability and composition. Further, our results show that PMs can potentiate iPS proliferation and induce cardiomyocyte differentiation. Our findings have laid a foundation for further investigation of how probiotics and its metabolites from yogurt affect human health and disease.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Secretions of Bifidobacterium infantis and Lactobacillus acidophilus Protect Intestinal Epithelial Barrier Function.

The secreted metabolites of probiotics are cytoprotective to intestinal epithelium and have been shown to attenuate inflammation and reduce gut permeability. The present study was designed to determine the protective effects of probiotic conditioned media (PCM) from Bifidobacterium infantis (BCM) and Lactobacillus acidophilus (LCM) on interleukin (IL)-1β-induced intestinal barrier compromise. The epithelial barrier was determined by measuring the transepithelial electrical resistance (TER) across a Caco-2 cell monolayer using a Transwell model. The paracellular permeability was determined by fluorescein isothiocyanate-labeled dextran flux. The expression of tight junction (TJ) proteins and nuclear factor-kappa B (NF-κB) p65 were determined using Western blot and the distribution of NF-κB p65 was determined by immunofluorescence staining. BCM and LCM induced a dose-dependent increase in Caco-2 TER after 4 and 24 hours of incubation (P < 0.05). The maximal increase of Caco-2 TER occurred at 4 hours of treatment with a PCM concentration of 15%. Preincubation with BCM and LCM for 4 hours significantly prevented the decrease of Caco-2 TER induced by 24 hours of stimulation with 10 ng/mL IL-1β. BCM and LCM decreased paracellular permeability in both stimulated and unstimulated Caco-2 monolayers (P < 0.05). IL-1β stimulation decreased occludin expression and increased claudin-1 expression in Caco-2 cells (P < 0.05), which was prevented in cells treated with BCM or LCM. The changes of claudin-1 expression in H4 cells were similar to Caco-2 cells in response to PCM treatment and IL-1β stimulation; however, a similar response in occludin was not demonstrated. The IL-1β-induced nuclear translocation of NF-κB p65 in Caco-2 cells was prevented by pretreatment with both PCMs. BCM and LCM protected the intestinal barrier against IL-1β stimulation by normalizing the protein expression of occludin and claudin-1 and preventing IL-1β-induced NF-κB activation in Caco-2 cells, which may be partly responsible for the preservation of intestinal permeability.

Lactobacillus rhamnosus GR-1 Attenuates Induction of Hypertrophy in Cardiomyocytes but Not through Secreted Protein MSP-1 (p75).

Previous animal studies have shown that the administration of probiotic Lactobacillus rhamnosus can provide a protective effect against ischemia/reperfusion and necrotic injury to the intestine, liver, and heart, as well as a therapeutic effect to the outcome of ischemic injury to the heart, including cardiac hypertrophy and heart failure. We hypothesized that L. rhamnosus GR-1 major secreted protein 1 (MSP-1), also known as p75, plays a major role in this phenomenon. Experiments using neonatal rat ventricular cardiomyocytes showed that live and dead GR-1 bacteria, probiotic-conditioned media, and other probiotic species and strains inhibited the α1-adrenergic receptor agonist phenylephrine-induced hypertrophy as assessed by markers atrial natriuretic peptide and α-skeletal actin. However, using a mutant strain, we showed that this MSP-1 was not required for the inhibition. The ability of factors produced by lactobacilli to improve cardiac function warrants further study for the management of cardiac hypertrophy and heart failure.

Anti-inflammatory effects of Bifidobacterium longum subsp infantis secretions on fetal human enterocytes are mediated by TLR-4 receptors.

The therapeutic and preventive application of probiotics for necrotizing enterocolitis (NEC) has been supported by more and more experimental and clinical evidence in which Toll-like receptor 4 (TLR-4) exerts a significant role. In immune cells, probiotics not only regulate the expression of TLR-4 but also use the TLR-4 to modulate the immune response. Probiotics may also use the TLR-4 in immature enterocytes for anti-inflammation. Here we demonstrate that probiotic conditioned media (PCM) from Bifidobacterium longum supp infantis but not isolated organisms attenuates interleukin-6 (IL-6) induction in response to IL-1β by using TLR-4 in a human fetal small intestinal epithelial cell line (H4 cells), human fetal small intestinal xenografts, mouse fetal small intestinal organ culture tissues, and primary NEC enterocytes. Furthermore, we show that PCM, using TLR-4, downregulates the mRNA expression of interleukin-1 receptor-associated kinase 2 (IRAK-2), a common adapter protein shared by IL-1β and TLR-4 signaling. PCM also reduces the phosphorylation of the activator-protein 1 (AP-1) transcription factors c-Jun and c-Fos in response to IL-1β stimulation in a TLR-4-dependent manner. This study suggests that PCM may use TLR-4 through IRAK-2 and via AP-1 to prevent IL-1β-induced IL-6 induction in immature enterocytes. Based on these observations, the combined use of probiotics and anti-TLR-4 therapy to prevent NEC may not be a good strategy.

Secreted Metabolites of Bifidobacterium infantis and Lactobacillus acidophilus Protect Immature Human Enterocytes from IL-1β-Induced Inflammation: A Transcription Profiling Analysis.

Combination regimens of Bifidobacterium infantis and Lactobacillus acidophilus have been demonstrated to prevent necrotizing enterocolitis (NEC) in clinical trials. However, the molecular mechanisms responsible for this protective effect are not well understood. Additionally, conditioned media from individual cultures of these two probiotics show strain specific modulation of inflammation using in vitro human intestinal NEC models. Here we report a transcription profiling analysis of gene expression in immature human fetal intestinal epithelial cells (H4 cells) pretreated with conditioned media from B. infantis (BCM) or L. acidophilus (LCM) prior to IL-1β stimulation. Compared with control media, the two probiotic-conditioned media (PCM) treatments altered the expression of hundreds of genes involved in the immune response, apoptosis and cell survival, cell adhesion, the cell cycle, development and angiogenesis. In IL-1β-stimulated cells, PCM treatment decreased the upregulation of genes in the NF-κB activation pathway and downregulated genes associated with extracellular matrix (ECM) remodeling. Compared with LCM, BCM showed more significant modulatory effects on ECM remodeling, reflected by a lower p value. IL-6 and IL-8 production was significantly reduced in IL-1β-stimulated cells pretreated with PCM (p<0.05), which was consistent with their altered gene expression. Western blot analysis showed that compared with IL-1β stimulation alone, PCM treatment attenuated the decrease of cytoplasmic IκBα and NF-κB p65 levels as well as the increase of nuclear NF-κB p65 levels in the stimulated cells (p<0.05). In conclusion, PCM treatment exerted anti-inflammatory effects in immature human fetal enterocytes primarily by modulating genes in the NF-κB signaling and ECM remodeling pathways. Additionally, some components of these signaling pathways, particularly the ECM remodeling pathway, were more profoundly affected by BCM than LCM.

Probiotics prevent necrotizing enterocolitis by modulating enterocyte genes that regulate innate immune-mediated inflammation

Necrotizing enterocolitis (NEC), an extensive intestinal inflammatory disease of premature infants, is caused, in part, by an excessive inflammatory response to initial bacterial colonization due to the immature expression of innate immune response genes. In a randomized placebo-controlled clinical trial, supplementation of very low birth weight infants with probiotics significantly reduced the incidence of NEC. The primary goal of this study was to determine whether secreted products of these two clinically effective probiotic strains, Bifidobacterium infantis and Lactobacillus acidophilus, prevented NEC by accelerating the maturation of intestinal innate immune response genes and whether both strains are required for this effect. After exposure to probiotic conditioned media (PCM), immature human enterocytes, immature human intestinal xenografts, and primary enterocyte cultures of NEC tissue (NEC-IEC) were assayed for an IL-8 and IL-6 response to inflammatory stimuli. The latter two models were also assayed for innate immune response gene expression. In the immature xenograft, PCM exposure significantly attenuated LPS and IL-1β-induced IL-8 and IL-6 expression, decreased TLR2 mRNA and TLR4 mRNA, and increased mRNA levels of specific negative regulators of inflammation, SIGIRR and Tollip. In NEC-IEC, PCM decreased TLR2-dependent IL-8 and IL-6 induction and increased SIGIRR and Tollip expression. The attenuated inflammatory response with PCM was reversed with Tollip siRNA-mediated knockdown. The anti-inflammatory secreted factor is a 5- to 10-kDa molecule resistant to DNase, RNase, protease, heat stress, and acid exposure. B. infantis-conditioned media showed superior anti-inflammatory properties to that of L. acidophilus in immature human enterocytes, suggesting a strain specificity to this effect. We conclude that PCM promotes maturation of innate immune response gene expression, potentially explaining the protective effects of probiotics in clinical NEC.

Probiotic Conditioned Media Induces an Anti-Inflammatory Macrophage Phenotype In Vivo and In Vitro

Diet in western societies is characterized by a high intake of fat and refined carbohydrates which can alter gut microflora. Gut bacteria can modulate immune function as well as behaviour through the gut-brain axis. Lactobacillus helveticus and Bifidobacterium infantis are probiotics used clinically with immunomodulatory effects. The aim of this study was to examine if host diet alters the ability of probiotics to modulate immune function and behaviour under normal and inflammatory conditions. Methods: At weaning, wild type (WT) and IL-10 deficient (IL-10-/-) 129/SvEv mice were placed on a standard mouse chow or a western style chow (fat 40%, carb 40%) ± Lactobacillus helveticus ROO52 (109 cfu/d) or Bifidobacterium infantis ROO33 (109 cfu/d) for 21 days. Animal weight and food eaten were monitored weekly. To examine mucosal immune function, large and small intestine were homogenized and analyzed for cytokine expression by MesoScale discovery platform. Spatial memory and anxiety-like behavior was assessed in a Barnes maze. Terminal Restriction Fragment Length Polymorphism (TRFLP) was used to analyze the faecal microflora in stool samples. Results:WT and IL-10-/mice on the western diet had a significantly higher weight gain per mass of food eaten. L. helveticus, but not B. infantis, reduced the western-diet induced weight gain. Mice on the western diet also had decreased microbial diversity and alterations in composition within the phyla Firmicutes. IL-10-/mice on normal chow developed colitis with high expression of pro-inflammatory cytokines. A western diet further increased expression of IL-1β and IL-12 in the small intestine, but had an immunosuppressive effect in the colon, with decreased expression of IFNγ, IL-1β, IL-8, and TNFα. L. helveticus prevented the western-diet induced alterations in cytokine expression in the small intestine, but not in the colon. L. Helveticus induced IL-1β, IL-2, IL-4, and IL-5 in WT mice on a chow, but not a western, diet. A western diet suppressed IFNγ, IL-1β, and IL-8 expression in the colon of WT mice, and this was not prevented by L. helveticus. IL-10-/mice had greater anxiety and reduced exploratory behavior in the Barnes maze as compared with WT mice. Mice on the western diet had decreased learning ability and enhanced anxiety. L. helveticus enhanced learning ability and increased exploratory behavior in IL-10-/mice on both diets. In WT mice, L. helveticus increased exploratory behaviour only in mice on the western diet. Conclusion: Both WT and IL-10-/mice on a western diet had increased weight gain, altered gut microflora, an impairment of learning behavior, and altered gut cytokine secretion. The ability of probiotic strains to modulate these factors was genotype, strainand diet-dependent. These findings have direct relevance to the clinical use of probiotics.