David Romanin

Down-regulation of intestinal epithelial innate response by probiotic yeasts isolated from kefir.

Kefir is obtained by milk fermentation with a complex microbial population included in a matrix of polysaccharide and proteins. Several health-promoting activities has been attributed to kefir consumption. The aim of this study was to select microorganisms from kefir able to down-regulate intestinal epithelial innate response and further characterize this activity. Caco-2 cells stably transfected with a human CCL20 promoter luciferase reporter were used to screen a collection of 24 yeast and 23 bacterial strains isolated from kefir. The Toll-like receptor 5 agonist, flagellin was used to activate the reporter cells, while pre-incubation with the selected strains was tested to identify strains with the capacity to inhibit cell activation. In this system, 21 yeast strains from the genera Saccharomyces, Kluyveromyces and Issatchenkia inhibited almost 100% of the flagellin-dependent activation, whereas only some lactobacilli strains showed a partial effect. K. marxianus CIDCA 8154 was selected for further characterization. Inhibitory activity was confirmed at transcriptional level on Caco-2/TC-7 and HT-29 cells upon flagellin stimulation. A similar effect was observed using other pro-inflammatory stimulation such as IL-1beta and TNF-alpha. Pre-incubation with yeasts induced a down-regulation of NF-kappaB signalling in epithelial cells in vitro, as well as expression of other pro-inflammatory chemokines such as CXCL8 and CXCL2. Furthermore, modulation of CCL20 mRNA expression upon flagellin stimulation was evidenced in vivo, in a mouse ligated intestinal loop model. Results indicate kefir contains microorganisms able to abolish the intestinal epithelial inflammatory response that could explain some of the properties attributed to this fermented milk.

Yeasts from kefir grains: isolation, identification, and probiotic characterization

Kefir—a traditional beverage whose consumption has been associated with health benefits—is a logical natural product to investigate for new probiotic strains. The aim of the present work was to isolate and identify kefir yeasts and select those with acid and bile tolerance to study their adhesion to epithelial cells and their transit through mouse gut. From 4 milky and 3 sugary kefir grains, 34 yeast strains were isolated and identified by means of classical microbiological and molecular-genetic methods (whole-cell protein pattern, internal-transcribed-spacer amplification, and analysis of restriction-fragment–length polymorphisms). We identified 4 species belonging to 3 genera—Saccharomyces cerevisiae (15 strains), Saccharomyces unisporus (6 strains), Issatchenkia occidentalis (4 strains), and Kluyveromyces marxianus (9 strains)—and selected 13 strains on the basis of resistance to low pH and bile salts. Among the strains selected, Kluyveromyces marxianus CIDCA 8154 and Saccharomyces cerevisiae CIDCA 8112 were further studied. Both strains evidenced the capacity to adhere to epithelial intestine-derived cells in vitro and to survive passage through the gastrointestinal tract of BALB/c mice. The investigation of the potential probiotic features of these kefir-yeast strains should be useful for the development of novel functional foods.

Down-regulation of NF-κB signaling by Gordonia bronchialis prevents the activation of gut epithelial cells

The immunomodulatory power of heat-killed Gordonia bronchialis was studied on gut epithelial cells activated with pro-inflammatory stimuli (flagellin, TNF-α or IL-1β). Light emission of luciferase-transfected epithelial cells and mRNA expression of IL-1β, TNF-α, IL-6, CCL20, IL-8 and MCP-1 were measured. NF-κB activation was assessed by immunofluorescence and immunoblotting, and induction of reactive oxygen species (ROS) was evaluated. In vivo inhibitory properties of G. bronchialis were studied with ligated intestinal loop assay and in a mouse model of food allergy. G. bronchialis promoted the down-regulation of the expression of CCL20 and IL-1β on activated epithelial cells in a dose-dependent manner. A concomitant blocking of nuclear p65 translocation with increased production of ROS was found. In vivo experiments confirmed the inhibition of CCL20 expression and the suppression of IgE sensitization and hypersensitivity symptoms in the food allergy mouse model. In conclusion, heat-killed G. bronchialis inhibited the activation of NF-κB pathway in human epithelial cells, and suppressed the expression of CCL20. These results indicate that G. bronchialis may be used to modulate the initial steps of innate immune activation, which further suppress the allergic sensitization. This approach may be exploited as a therapy for intestinal inflammation.

Defining the Nonreturn Time for Intestinal Ischemia Reperfusion Injury in Mice

Among the abdominal organs, the intestine is probably the most sensitive to ischemia reperfusion injury (IRI), a phenomenon that occurs in many intestinal disorders. Few studies have reported in detail the impact of intestinal ischemia time in mice. We evaluated the effect of various warm intestinal ischemia times in an intestinal IRI model in mice. Adult male Balb/c mice were divided into 4 groups that differed in intestinal ischemia time: G1, 30; minutes; G2, 35 minutes; G3, 40 minutes; and G4, 45 minutes. Histological evaluation showed average Park scores as follows: G1 0.6 ± 0.55; G2 1.8 ± 0.45; G3 4.8 ± 2.25; and G4 5 ± 1.79. All animals from G1 survived 30 hours. G2 animals showed intermediate behavior with all succumbing between 18 and 30 hours postprocedure. G3 and G4 displayed similar survival results with animals succumbing before 6 hours after intestinal reperfusion. These data showed that Park index scores of 3 or higher were related to early death. We concluded that the 5 minutes between 35 and 40 minutes is the critical limit, after which all mice die after reperfusion. This result may represent a valuable tool for future research in mice.

Innate immune responses to Proteus mirabilis flagellin in the urinary tract

Flagella are bacterial virulence factors allowing microorganisms to move over surfaces. Flagellin, the structural component of flagella, is sensed by the host via Toll and NOD-like receptors and triggers pro-inflammatory responses. The use of Toll-like receptors agonists to modulate innate immune responses has aroused great interest as an alternative to improve the treatment of diverse infectious diseases. Proteus mirabilis is a Gram negative bacterium that causes urinary tract infections in humans. In the present work we used different approaches to study the ability of P. mirabilis flagellin to induce an innate immune response. We demonstrated that P. mirabilis flagellin has the ability to induce pro-inflammatory chemokines expression in T24 bladder cultures cells and in the mouse bladder after instillation. It was evidenced also that flagellin from different P. mirabilis strains differed in their capacity to induce an innate immune response in the CacoCCL20-Luc system. Also, flagellin elicited inflammation, with recruitment of leukocytes to the bladder epithelium. Flagellin instillation before an experimental P. mirabilis infection showed that the inflammatory response due to flagellin did not help to clear the infection but favored bacterial colonization. Thus, induction of inflammatory response in the bladder did not contribute to P. mirabilis infection neutralization.

Gut Permeability and Glucose Absorption Are Affected at Early Stages of Graft Rejection in a Small Bowel Transplant Rat Model

Background Intestinal transplantation (ITx) faces many challenges due to the complexity of surgery and to the multiple immunological reactions that lead to the necessity of rigorous follow-up for early detection of acute cellular rejection (ACR). Our aim was to determine the kinetics of ACR using an experimental ITx model, with emphasis in the characterization of the process using different approaches, including the use of functional assays of absorptive and barrier function. Methods ITx in rats conducting serial sampling was performed. Clinical monitoring, graft histology, proinflammatory gene expression, and nitrosative stress determination were performed. Also, glucose absorption, barrier function using ovalbumin translocation, and contractile function were analyzed. Results The model used reproduced the different stages of ACR. Allogeneic ITx recipients showed signs of rejection from postoperative day (POD) 5, with increasing severity until 12 POD. Histological evaluation showed mild rejection in early sampling and severe rejection at late stages, with alterations in all graft layers. IL-6, CXCL 10, IFNg, and nitrite plasmas levels showed behavior coincident with histopathology. Remarkably, allogeneic grafts showed a marked alteration of glucose absorptive capacity from POD 5 that was sustained until endpoint. Coincidently, barrier function alteration was evidenced by luminal ovalbumin translocation to serum. Contractile function was progressively impaired along ACR. Conclusions Glucose absorption and barrier function are altered at early stages of ACR when histological alterations or gene expression changes were much subtle. This observation may provide simple evaluation tools that could be eventually translated to the clinics to contribute to early ACR diagnosis.

Characterization of Acute Cellular Rejection in the Different Layers of Rat Transplanted Intestines

Introduction Intestinal transplantation (IT) faces many challenges, among them, the necessity to understand and detect rejection processes. Rodent models of IT are used to provide evidence for intervention strategies as well as improve knowledge of IT biology. Our aim was to determine the kinetics of small bowel rejection with emphasis in the characterization of acute cellular rejection (ACR) in the different layers of the graft since most of the information coming from the clinics is on mucosal layer, the only one that is accessible by endoscopic biopsies. Methods Allogeneic (ALLO) heterotopic IT in rats was performed following standard procedure. ACR was diagnosed by H-E staining analysis. Also, real-time PCR from microdissected samples (epithelial, muscular and serosa layer) and whole graft to determine gene expression was performed at 5 and 10-12 postoperative days (POD). An Isogenic IT group was performed as a control. Results ACR was observed since 5 POD in ALLO group with mild rejection as the most characteristic grade. Severe ACR was diagnosed in all ALLO samples at 10-12 POD. Descriptive analysis showed a well-preserved architecture at 5 POD; confluent and loose apoptotic cells in the intestinal epithelium and perivascular infiltrate in all layers were evident. At 10-12 POD, significant cellular infiltrate, epithelial damage, ulcers and an increase of apoptotic cells were observed. Muscular and serosa layers showed inflammatory cell infiltrate and intercellular edema. At 5 POD, some markers were consistently increased in ALLO groups such as CXCL10 that showed a 120 ± 80-fold increase compared with nontransplanted tissue. Furthermore, IFNg and IDO showed a trend to be increased at these time points. Remarkably, other inflammation-related genes, such as CXCL1 and IL6 showed consistent increase in ALLO groups at 10-12 POD, when severe ACR was established. When a principal component analysis of the overall gene expression markers was performed, ALLO group at 10-12 POD clearly separated from the other conditions. The analysis of gene expression at different layers of the graft was coincident with whole tissue biopsies: higher levels of IL6 and CXCL1 were observed in ALLO groups at 10-12 POD with important activation of this response in serosa and muscular layer. Interestingly, IL22 expression was only measurable in epithelial layer in ALLO groups at 10-12 POD, indicating ACR-induced expression in this compartment. Serosa layer showed some of the highest relative increases in proinflammatory gene expression also in ALLO groups at 10-12 POD. Conclusion Although in the clinic mucosal rejection has been extensively characterized, in our animal model we could document that all graft layers are affected by ACR since the initial stages. Serosa and muscular layer show high expression of proinflammatory markers, with differential expression of IL22 in the epithelial compartment. This information could be useful in the search for early biomarkers of ACR.

Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) stimulates murine macrophages infected with Citrobacter rodentium

Citrobacter rodentium is a specific murine enteropathogen which causes diarrheal disease characterized by colonic hyperplasia and intestinal inflammation. Recruitment of neutrophils and macrophages constitute a key step to control the infection. Since modulation of the activity of professional phagocytic cells could contribute to improve host´s defences against C. rodentium, we investigated the effect of Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) on the interaction between murine macrophages (RAW 264.7) and C. rodentium. Phagocytosis, surface molecules and inducible nitric oxide synthase (iNOs) expression were determined by flow cytometry. Reactive oxygen species (ROS) were assessed by fluorescence microscopy. The presence of lactobacilli increased phagocytosis of C. rodentium whereas C. rodentium had no effect on lactobacilli internalization. Survival of internalized C. rodentium diminished when strain CIDCA 133 was present. CD-86, MHCII, iNOs expression and nitrite production were increased when C. rodentium and lactobacilli were present even though strain CIDCA 133 alone had no effect. Strain CIDCA 133 led to a strong induction of ROS activity which was not modified by C. rodentium. Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) is able to increase the activation of murine macrophages infected with C. rodentium. The sole presence of lactobacilli is enough to modify some stimulation markers (e.g. ROS induction) whereas other markers require the presence of both bacteria; thus, indicating a synergistic effect.Graphical Abstract