Marta Llopis

Reshaping the gut microbiome with bacterial transplantation and antibiotic intake

The intestinal microbiota consists of over 1000 species, which play key roles in gut physiology and homeostasis. Imbalances in the composition of this bacterial community can lead to transient intestinal dysfunctions and chronic disease states. Understanding how to manipulate this ecosystem is thus essential for treating many disorders. In this study, we took advantage of recently developed tools for deep sequencing and phylogenetic clustering to examine the long-term effects of exogenous microbiota transplantation combined with and without an antibiotic pretreatment. In our rat model, deep sequencing revealed an intestinal bacterial diversity exceeding that of the human gut by a factor of two to three. The transplantation produced a marked increase in the microbial diversity of the recipients, which stemmed from both capture of new phylotypes and increase in abundance of others. However, when transplantation was performed after antibiotic intake, the resulting state simply combined the reshaping effects of the individual treatments (including the reduced diversity from antibiotic treatment alone). Therefore, lowering the recipient bacterial load by antibiotic intake prior to transplantation did not increase establishment of the donor phylotypes, although some dominant lineages still transferred successfully. Remarkably, all of these effects were observed after 1 mo of treatment and persisted after 3 mo. Overall, our results indicate that the indigenous gut microbial composition is more plastic that previously anticipated. However, since antibiotic pretreatment counterintuitively interferes with the establishment of an exogenous community, such plasticity is likely conditioned more by the altered microbiome gut homeostasis caused by antibiotics than by the primary bacterial loss.

Effects of Nonpathogenic Bacteria on Cytokine Secretion by Human Intestinal Mucosa

OBJECTIVE:The human intestine harbors a complex microbial ecosystem, and the mucosa is the interface between the immune system and the luminal environment. The aim of this study was to elucidate whether host–bacteria interactions influence mucosal cytokine production.METHODS:Macroscopically normal colonic specimens were obtained at surgery from eight patients with neoplasm, and inflamed ileal specimens were obtained from two patients with Crohns disease. Mucosal explants were cultured for 24 h with either nonpathogenic Escherichia coli ECOR-26, Lactobacillus casei DN-114 001, L. casei DN-114 056, L. casei ATCC-334, or Lactobacillus bulgaricus LB-10. Each study included blank wells with no bacteria. Tissue and bacteria viability were confirmed by LDH release and culture. Concentration of tumor necrosis factor (TNF)α, transforming growth factor β1, interleukin (IL)-8, and IL-10 was measured in supernatants. In parallel experiments, neutralizing anti-TNFα antibody was added to the culture.RESULTS:Co-culture of mucosa with bacteria did not modify LDH release. Co-culture with L. casei strains significantly reduced TNFα release, whereas E. coli increased it. These effects were observed both in normal and inflamed mucosa. In combination studies, L. casei DN-114 001 prevented TNFα stimulation by E. coli. L. casei DN-114 001 also reduced IL-8 release via a TNFα-independent pathway. L. casei DN-114 056 or E. coli increased IL-10 release in the presence of neutralizing anti-TNFα.CONCLUSIONS:Nonpathogenic bacteria interact with human intestinal mucosa and can induce changes in cytokine production that are strain specific.

Lactobacillus casei downregulates commensals' inflammatory signals in Crohn's disease mucosa

Background: The interaction of commensal bacteria with the intestinal immune system is an essential factor in the development of inflammatory bowel disease (IBD). The study of isolated commensal bacterias effects on the mucosal immune response might be relevant for a better understanding of pathophysiological mechanisms in IBD. Methods: We investigated the immune responses to signals from the commensal Escherichia coli ATCC 35345 and the probiotic Lactobacillus casei DN‐114 001 in Crohns disease (CD) mucosa. Ileal specimens were obtained during surgery from CD patients. Mucosal explants were incubated with L. casei or its genomic DNA; TNF‐&agr;, IFN‐&ggr;, IL‐2, IL‐6, IL‐8, and CXCL1 were measured in the supernatant. Second, tissue expression of key proinflammatory cytokines (IL‐6, TGF‐&bgr;, IL‐23p19, IL‐12p35, IL‐17F), and chemokines (IL‐8, CXCL1, CXCL2) was evaluated after incubation with L. casei or E. coli. Finally, combination experiments were carried out by incubating both strains with mucosal explants at different timepoints. Results: Live L. casei significantly decreased secretion of TNF‐&agr;, IFN‐&ggr;, IL‐2, IL‐6, IL‐8, and CXCL1 by CD mucosa, but the effect was not reproduced by L. casei DNA. Second, live L. casei downregulated expression of IL‐8, IL‐6, and CXCL1 and did not modify expression of IL‐23p19, IL‐12p35, and IL‐17F. In contrast, E. coli significantly upregulated expression of all these cytokines. Interestingly, combination experiments revealed the ability of L. casei to prevent and counteract the proinflammatory effects of E. coli. Conclusions: Live L. casei can counteract the proinflammatory effects of E. coli on CD inflamed mucosa by specific downregulation of key proinflammatory mediators.

The Gut Microbiota Predispose to the Pathophysiology of Acute Postradiotherapy Diarrhea

INTRODUCTION:Severe diarrhea may complicate pelvic radiotherapy and force interruption of treatment. As there is no current clinical or experimental information on the role of the gut microbiota in this pathogenesis, we conducted a pilot observational study on the fecal microbiota in patients receiving pelvic radiotherapy.METHODS:The study involved 10 patients who underwent 5 wk of radiotherapy for abdominal tumors and 5 controls. Four fecal samples were collected from each individual: before, during, at the end, and 2 wk after treatment. Following the amplification of the bacterial 16S rRNA gene from the samples, DNA fingerprinting and cloning-sequencing techniques were used to determine their microbial profile and composition, respectively.RESULTS:Six patients suffered acute postradiotherapy diarrhea and 4 did not. In patients without diarrhea, as well as in healthy volunteers, microbial diversity was stable over a period of 7 wk. However, patients exhibiting diarrhea showed a progressive modification in their microbial diversity. A radical drop in similarity index was observed at the end (P= 0.026) and still 2 wk after radiotherapy (P= 0.014). Interestingly, cluster analysis of the microbial profile in the first sample (S1) (collected before radiotherapy) displayed a dendogram where patients that presented diarrhea clustered separately from those that did not develop diarrhea after radiotherapy. Moreover, sequence analysis of dominant bacteria in the S1 sample confirmed differences between the diarrhea and nondiarrhea groups.DISCUSSION: In this set of patients, susceptibility or protection against diarrhea after radiotherapy could be linked to different initial microbial colonization.

Modulation of apoptosis in intestinal lymphocytes by a probiotic bacteria in Crohn’s disease

Apoptosis of active T lymphocytes constitutes a maor control mechanism of immune homeostasis and tolerance. In Crohns disease, abnormal activation of mucosal T lymphocytes against enteric bacteria is the key event triggering intestinal inflammation. Resistance of lymphocytes to apoptosis has been proposed as the pathogenetic defect. We examined the influence of bacteria‐mucosa interactions on apoptosis of mucosal T lymphocytes. Ileal specimens were obtained at surgery from 12 patients with Crohns disease. Mucosal explants from each specimen were cultured with nonpathogenic Escherichia coli ATCC 35345, Lactobacillus casei DN‐114 001, or no bacteria. Cytokine release was measured in supernatant, and mononuclear cells were isolated for phenotypic characterization and Bcl‐2 family protein expression. Coculture of inflamed tissue with L. casei significantly reduced the release of interleukin (IL)‐6 and tumor necrosis factor α (P<0.05). In addition, coculture with L. casei significantly reduced the number of T cells displaying the IL‐2 receptor in the lamina propria. Expression of the antiapoptotic protein Bcl‐2 in lamina propria lymphocytes was also reduced after coculture with L. casei, and the percentage of deoxyuridine triphosphate nick‐end labeling positive lymphocytes increased. The nonpathogenic E. coli strain had no significant effect. In conclusion, L. casei reduces the number of activated T lymphocytes in the lamina propria of Crohns disease mucosa. A balanced, local microecology may restore immune homeostasis.

Adhesion properties of Lactobacillus casei strains to resected intestinal fragments and components of the extracellular matrix.

Adhesion to intestinal epithelium is an outcome property for the selection of probiotic lactic acid bacteria strains. We have analyzed the adhesion properties of a collection of Lactobacillus casei strains from different origins, ranging from cheese isolates to commercial probiotics. Analysis of the surface characteristics of the strains by measuring adhesion to solvents (MATS test) showed that most of the strains have a basic and hydrophobic surface. The strains were able to bind ex vivo to human colon fragments at different levels and, in most cases, this adhesion correlated with the ability to in vitro binding of mucin. Attachment to this later substrate was not enhanced by growing the cells in the presence of mucin and was independent of proteinaceous factors. On the contrary, adhesion to other extracellular matrix components, such as collagen, fibronectin, or fibrinogen was partially or totally dependent on the presence of surface proteins. These results show that most of L. casei strains have in their surfaces factors that promote binding to intestinal epithelium, however, no clear correlation appears to exist between the origin of the strains and their adhesion capacities.

Induction of colonic transmural inflammation by bacteroides fragilis. Implication of Matrix Metalloproteinases

Background: Commensal bacteria are implicated in the pathophysiology of intestinal inflammation, but the precise pathogenetic mechanisms are not known. We hypothesized that Bacteroides fragilis‐produced metalloproteinases (MMPs) are responsible for bacterial migration through the intestinal wall and transmural inflammation. Aim: To investigate the role of bacterial‐MMP activity in an experimental model of colitis induced by the intramural injection of bacteria. Methods: Suspensions of viable B. fragilis or Escherichia coli were injected into the colonic wall, and the effect of the MMP inhibitor (phenantroline) on histologic lesion scores was tested. MMP activity in bacterial suspensions was measured by azocoll assay. Results: The inoculation with B. fragilis induced chronic inflammatory lesions that were preferentially located in the subserosa, whereas inoculation with E. coli induced acute‐type inflammatory reactions, evenly distributed in both the submucosa and subserosa. Treatment with phenantroline significantly decreased subserosal lesion scores in rats inoculated with B. fragilis, but not in rats inoculated with E. coli. Bacterial suspensions of B. fragilis showed MMP activity, but E. coli suspensions did not. Sonication of B. fragilis reduced MMP activity and virulence to induce serosal lesions. Conclusion: Our data suggest that bacterial MMPs may be implicated in the serosal migration of B. fragilis and in the induction of transmural inflammation.

Lactobacillus paracasei and Lactobacillus plantarum strains downregulate proinflammatory genes in an ex vivo system of cultured human colonic mucosa

Significant health benefits have been demonstrated for certain probiotic strains through intervention studies; however, there is a shortage of experimental evidence relative to the mechanisms of action. Here, noninvasive experimental procedure based on a colon organ culture system has been used that, in contrast to most experimental in vitro models reported, can preserve natural immunohistochemical features of the human mucosa. This system has been used to test whether commensal lactobacilli (Lactobacillus paracasei BL23, Lactobacillus plantarum 299v and L. plantarum 299v (A−)) were able to hinder inflammation-like signals induced by phorbol 12-myristate 13-acetate (PMA)/ionomycin (IO). Whole genome microarrays have been applied to analyze expression differences, from which mRNA markers could be inferred to monitor the effect of putative probiotic strains under such conditions. Regarding the gene expression, PMA/IO treatment induced not only interleukin (IL)-2 and interferon gamma (IFN-γ), as expected, but also other relevant genes related to immune response and inflammation, such as IL-17A, chemokine (C-X-C motif) ligand (CXCL) 9 and CXCL11. The ex vivo culturing did not modify the pattern of expression of those genes or others related to inflammation. Interestingly, this study demonstrated that lactobacilli downregulated those genes and triggered a global change of the transcriptional profile that indicated a clear homeostasis restoring effect and a decrease in signals produced by activated T cells.

Transportome Profiling Identifies Profound Alterations in Crohn’s Disease Partially Restored by Commensal Bacteria

BACKGROUND AND AIMS Several transport alterations have been described in intestinal inflammatory diseases. This is relevant because the primary function of the intestine is nutrient and mineral absorption. However, analysis of the transportome as a whole and the effect of commensal bacteria on it have not been addressed so far. METHODS Five healthy and 6 Crohns disease (CD) samples were hybridized to human HT-12 V4 Illumina GeneChip. Results were validated by reverse transcription-polymerase chain reaction (RT-PCR) analysis and with additional array data. Organ culture assays were performed from mucosa ileal wall specimens collected at surgery. Samples were incubated with or without commensal bacteria for 4 hours. Finally, RNA was isolated for microarray processing. RESULTS The analysis of CD versus healthy ileal mucosa demonstrated upregulation of previously described genes involved in immunity and the inflammatory response in this disease. Interestingly, whole transcriptional analysis revealed profound alterations in the transportome profile. Sixty-two solute carrier (SLC) transporters displayed different expression patterns, most of them being downregulated. Changes were confirmed by RT-PCR in a randomly chosen subset of SLCs. A large number of amino acid transporters and most members of the enteric purinome were found to be altered. Most of these proteins were found at the apical membrane of the enterocyte, which could impair both amino acid absorption and purinergic signalling. Treatment of ileum specimen explants with commensal bacteria restored almost all CD transportome alterations. CONCLUSIONS These results describe the altered transportome profile in CD and open the possibility of restoring transportome complications with commensal bacteria.

Original contributionEffects of nonpathogenic bacteria on cytokine secretion by human intestinal mucosa

Objective The human intestine harbors a complex microbial ecosystem, and the mucosa is the interface between the immune system and the luminal environment. The aim of this study was to elucidate whether host–bacteria interactions influence mucosal cytokine production.