The canine isolate Lactobacillus acidophilus LAB20 adheres to intestinal epithelium and attenuates LPS-induced IL-8 secretion of enterocytes in vitro

Background

For a good probiotic candidate, the abilities to adhere to intestinal epithelium and to fortify barrier function are considered to be crucial for colonization and functionality of the strain. The strain Lactobacillus acidophilus LAB20 was isolated from the jejunum of a healthy dog, where it was found to be the most pre-dominant lactobacilli. In this study, the adhesion ability of LAB20 to intestinal epithelial cell (IECs) lines, IECs isolated from canine intestinal biopsies, and to canine, porcine and human intestinal mucus was investigated. Further, we studied the ability of LAB20 to fortify the epithelial cell monolayer and to reduce LPS-induced interleukin (IL-8) release from enterocytes.

Results

We found that LAB20 presented higher adhesion to canine colonic mucus as compared to mucus isolated from porcine colon. LAB20 showed adhesion to HT-29 and Caco-2 cell lines, and importantly also to canine IECs isolated from canine intestinal biopsies. In addition, LAB20 increased the transepithelial electrical resistance (TER) of enterocyte monolayers and thus strengthened the intestinal barrier function. The strain showed also anti-inflammatory capacity in being able to attenuate the LPS-induced IL-8 production of HT-29 cells.

Conclusion

In conclusion, canine indigenous strain LAB20 is a potential probiotic candidate for dogs adhering to the host epithelium and showing intestinal barrier fortifying and anti-inflammatory effects.

Electronic supplementary material

The online version of this article (doi:10.1186/s12866-014-0337-9) contains supplementary material, which is available to authorized users.     

Immunomodulatory effect of oxygen and pressure

The immunomodulatory effect of hyperbaric oxygen, involving altered cytokine release by macrophages, is well described. Importantly, however, it is not known what the relative contribution is of the hyperbaric environment of the cells vs. increased oxygen tension on these hyperbaric oxygen-dependent effects. We compared, therefore, cytokine release by murine macrophages under hyperbaric oxygen, hyperpressure of normal air and normobaric conditions. We observed that hyperbaric oxygen enhanced cytokine release of both unstimulated as well as lipopolysaccharide (LPS)-challenged macrophages. Hyperpressure of normal air, however, enhanced LPS-induced cytokine production but did not elicit cytokine release in unstimulated macrophages. To further investigate the molecular details underlying the effects of hyperbaric oxygen, we investigated the effect of the p42/p44 mitogen-activated protein (MAP) kinase inhibitor PD98059 and the p38 MAP kinase inhibitor SB203580. Neither inhibitor, however, had a significant effect on the modulatory effects of hyperbaric oxygen on cytokine release. We concluded that the immunomodulatory effect of hyperbaric oxygen contains a component for which hyperpressure is sufficient and a component that apart from hyperpressure also requires hyperoxygenation.     

Nucleocytoplasmic distribution of the ovalbumin serpin PI-9 requires a nonconventional nuclear import pathway and the export factor Crm1.

Proteinase inhibitor 9 (PI-9) is a human serpin present in the cytoplasm of cytotoxic lymphocytes and epithelial cells. It inhibits the cytotoxic lymphocyte granule proteinase granzyme B (graB) and is thought to protect cytotoxic lymphocytes and bystander cells from graB-mediated apoptosis. Following uptake into cells, graB promotes DNA degradation, rapidly translocating to the nucleus, where it binds a nuclear component. PI-9 should therefore be found in cytotoxic lymphocyte and bystander cell nuclei to ensure complete protection against graB. Here we demonstrate by microscopy and subcellular fractionation experiments that PI-9 is present in the nuclei of human cytotoxic cells, endothelial cells, and epithelial cells. We also show that the related serpins, PI-6, monocyte neutrophil elastase inhibitor (MNEI), PI-8, plasminogen activator inhibitor 2 (PAI-2), and the viral serpin CrmA exhibit similar nucleocytoplasmic distributions. Because these serpins lack classical nuclear localization signals and are small enough to diffuse through nuclear pores, we investigated whether import occurs actively or passively. Large (approximately 70 kDa) chimeric proteins comprising PI-9, PI-6, PI-8, MNEI, or PAI-2 fused to green fluorescent protein (GFP) show similar nucleocytoplasmic distributions to the parent proteins, indicating that nuclear import is active. By contrast, CrmA-GFP is excluded from nuclei, indicating that CrmA is not actively imported. In vitro nuclear transport assays show that PI-9 accumulates at a rate above that of passive diffusion, that it requires cytosolic factors but not ATP, and that it does not bind an intranuclear component. Furthermore, PI-9 is exported from nuclei via a leptomycin B-sensitive pathway, implying involvement of the export factor Crm1p. We conclude that the nucleocytoplasmic distribution of PI-9 and related serpins involves a nonconventional nuclear import pathway and Crm1p.     

Human endotoxemia activates p38 MAP kinase and p42/44 MAP kinase, but not c-Jun N-terminal kinase.

BACKGROUND: All three major members of the MAPK family (i.e., p38 MAPK, p42/p44 MAPK, and c-Jun N terminal kinase (JNK)) have been shown to control cellular responses to inflammation in vitro. Therefore these kinases have been designated suitable targets for anti-inflammatory therapy. However, the extent to which these kinases are actually activated during inflammation in humans in vivo has not been investigated. We employed experimental human endotoxemia, a model of systemic inflammation, to address this question. MATERIALS AND METHODS: Male volunteers were intravenously infused with 4 ng/kg bw lipopolysaccharide (LPS). Directly before LPS infusion and up to 24 h thereafter, activation of p38 MAPK, p42/p44 MAPK and JNK was assessed in peripheral blood, using Western blot and in vitro kinase assays. RESULTS: We observed that LPS induced a strong but transient phosphorylation and activation of p38 MAPK and p42/p44 MAPK, maximal activity being reached after 1 hr of LPS infusion. Strikingly, no JNK phosphorylation or activation was detected under these circumstances. CONCLUSIONS: These results suggest that both inhibitors of p38 MAPK and p42/p44 MAPK but not JNK are potentially useful for anti-inflammatory therapy.