Microorganisms

A blend of bacteria derived from a commercial probiotic product, which contained: Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus gasseri, Bifidobacterium thermophilus, Bifidobacterium longum, and Bifidobacterium adolescentis was used, as previously described [5]. These isolates are available from the corresponding author upon reasonable request. The products were cultured for isolation of component bacteria on de Man Rogosa Sharpe (MRS) agar (REMEL Laboratories) and Bifidobacterium agars (Anaerobe Systems) incubated anaerobically for 48 h at 37 °C. Bacterial isolates were identified by their cellular fatty acid methyl ester compositions (Microbial ID, Inc.) and by their 16S rRNA sequences with the MicroSeq 16S rRNA sequence assays (Applied Biosystems, Inc.). Rapid identifications were made with the Biolog Microbial Identification System. Serial dilution plate counts on MRS or Bifidobacterium agar were used to quantify the components of the probiotic products. Probiotic products were suspended in drinking water at a concentration of 5 × 106 CFU/mouse and administered to mice with a feeding tube, as previously described [5]. Salmonella enterica Serotype Cubana, originally isolated from poultry [5] was grown on Trypticase Soy agar with 5% sheep blood (REMEL) or in Trypticase Soy broth at 37 °C in an atmosphere of 5% CO 2 and air. The Salmonella isolate was originally characterized as Serotype Typhimurium and subsequently determined to be serotype G2 (serovar Cubana) by the Arkansas Regional Laboratory of the Food and Drug Administration (FDA), Jefferson, AR. The isolate is available upon reasonable request from the corresponding author.

Mice

A total of 4 male and 4 female 8 week-old human microbiota-associated [5] and 4 male and 4 female 8 week-old defined-microbiota BALB/c mice (Charles River) were used in this study with the approval of the Institutional Animal Care and Use Committee of the National Center for Toxicological Research. Sterile water and NIH-31 mouse chow (Purina) were supplied ad libitum to the mice. The probiotic bacteria were administered with a feeding tube to mice 7 days before oral challenge with S. enterica. Isolator sterility was assessed with weekly swab cultures on Trypticase soy blood agar plates (REMEL). The cultures were incubated at 37 °C in 95% air, 5% CO 2 atmosphere overnight and the plates were assessed for bacterial growth. The mice were fed a NIH-31 diet sterilized in the transfer box as previously described [5].

Experimental design

Control mice were human microbiota-associated in our laboratory, as previously described [5] or specific pathogen free from the vendor and fecal samples were cultured to assure their status. Treatment mice were colonized with the probiotic bacteria blend and fecal samples were collected from the mice 1 day later to culture for the presence of the probiotic bacteria. At 7 days after probiotic treatment, 4 probiotic-treated mice and 4 untreated mice were each orally inoculated with 2 × 108 colony forming units (CFU) of Salmonella enterica using a feeding tube. Seven days later, the mice were euthanized and MALT consisting of intestinal lamina propria, Peyer’s patches, and mesenteric lymph nodes were excised for analysis. Spleen cells were isolated and incubated with S. enterica antigens and mitogens to evaluate lymphoproliferative effects, as previously described [5]. Total RNA samples were extracted from the tissues and cDNA were generated, from which quantitative real-time-polymerase chain reaction (qRT-PCR) analyses were run to quantify mRNA expressed from genes of the mucosal immune system in probiotic-treated and untreated mice. The data were analyzed to identify cell type markers and intracellular signaling molecules involved in the host response to S. enterica that were affected by the probiotic bacteria. Control mice and probiotic-bacteria-treated BALB/c mice were orally challenged with S. enterica and pathway-focused gene expression profiles were generated from qRT-PCR expression arrays, as described below, to compare signal transduction in MALT from defined-microbiota mice treated with or without probiotic bacteria and orally challenged with S. enterica.

The experiment was repeated on a group of 8 specific pathogen free mice to obtain intestinal lamina propria, Peyer’s patches, and mesenteric lymph nodes for immunolocalization by an immunohistochemical method using antibodies from Santa Cruz Biotechnology. Immunolocalization of CD21+ B cells and dendritic cells, CD2+ T lymphocytes, and detection of PTPRC, TLR6, and v-rel avian reticuloendotheliosis viral oncogene homolog B (RELB) cellular expression was used to evaluate the probiotic effects that were observed on mRNA expression.

Antigen preparations

Antigens were prepared from crude lysates of S. enterica for in vitro activation and apoptosis assays of lymphocytes collected from the spleens of mice from the experiments, as previously described [5]. Briefly, the entire volume of a 500 ml log phase broth culture of bacteria was centrifuged at 2000 × g for 15 min. The bacterial pellet was washed three times with an equal volume of PBS and centrifuged again. The final bacterial pellet was suspended in 10 ml of PBS and passed through a French pressure cell (SLM/AMINCO) at 15,000 lb./in2 to disrupt the bacteria. The disrupted bacteria were centrifuged at 2000 × g and the protein content of the supernatant was determined by the bicinchoninic acid protein assay (Pierce Chemical Co.) to express antigen mass as mg protein, and used as the antigens for lymphocyte proliferation assays.

Lymphocyte proliferation assay

Lymphocytes from the spleens of mice treated with probiotics after S. enterica challenge were assayed for proliferative responses to S. enterica antigens, as previously described [5]. Lymphocyte proliferation assays were performed with the CellTiter Aqueous 96 assay (Promega, Corp.). Lymphocytes from the spleens of experimentally treated mice were prepared and incubated at a density of 5 × 105 cells/well of a 96-well culture plate in RPMI medium (Thermo Fisher Scientific, Inc.) containing S. enterica antigens. Antigens were added to 3 wells with spleen cells at a concentration of 10 μg whole cell lysate protein antigen preparation per well. Antigens were incubated with the cells 56 h at 37 °C in a humidified 5% CO 2 incubator before testing for lymphocyte proliferation. The formation of proliferating clonal clusters was also verified microscopically. The proliferation of lymphocytes in response to the antigens was measured as absorbance of reduced 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfonyl)-2H–tetrazolium, inner salt (MTS) at 490 nm, which was measured with a plate reader (Applied Biosystems). The average of three wells per sample was used to determine the mean ± standard error of the mean (SEM) Abs 490 for three mice per group. Proliferative responses of lymphocytes to antigens were compared as % increases in MTS absorbance as a result of the effects of probiotics.

Apoptosis assay

Lymphocytes from the spleens of the mice 7 days after probiotic treatment and S. enterica challenge were analyzed for activation of caspases 3 and 7 (Apo-ONE, Promega Corp.). Each assay well of a Nunc F16 black Maxisorp 96 well fluorescent assay plate (Thermo Fisher Scientific, Inc.) contained 50 μl of cell suspension in Roswell Park Memorial Institute (RPMI) 1640 medium at a cell concentration of 2 × 105 cells/ml to which was added 10 μg antigen preparation per well. After 56 h incubation at 37 °C in a humidified 5% CO 2 incubator, 100 μl of working substrate solution was added to each well. The plate was rotated at 300 rpm for 30 min at room temperature. Fluorescence intensities were measured in a plate reader (Applied Biosystems) set with filters for an excitation wavelength of 485 nm and an emission wavelength of 530 nm at 30 min intervals until the rates of increase reached a plateau state. Endpoint fluorescence intensities from each treatment group were compared as the % change in relative fluorescence intensities resulting from probiotic inhibition of CASP3/7activation.

qRT-PCR array profiling of signaling pathway and cytokine genes

RT2 Profiler™ PCR arrays from Qiagen Bioscience were used to assess expression of mRNA for 327 genes involved in the host response to bacteria in the mucosal immune tissues of the mouse GI tract. Arrays for genes involved in apoptosis (PAMM-012 Mouse Apoptosis Array), for genes involved in NF-κB activation (PAMM-025 Mouse NF-κB Array), and for mouse T and B cell activation markers (PAMM-053) were used according to the manufacturer’s instructions. Total cellular RNA from Peyer’s patches, mesenteric lymph nodes, and lamina propria from 8 mice were isolated using ArrayGrade total RNA isolation kits (Qiagen, Inc.). The RNA samples were treated with DNAse-1 (Thermo Fisher Scientific, Inc.) reverse-transcribed with the RT2 PCR Array first strand kit and the resulting cDNA was analyzed by real-time PCR for detection in a BioRad Pci,Q5 instrument. The housekeeping genes used in the study were: beta glucuronidase, hypoxanthine phosphoribosyltransferase 1, heat shock protein 90 alpha family class B member 1, glyceraldehyde-3-phophate dehydrogenase, and beta actin. Results of the PCR array experiment were analyzed with the Excel™ (Microsoft Corp.) template provided by Qiagen, Inc. to determine the key signal transduction pathways and immune system cells involved in the probiotic effects.

Immunolocalization of responses to probiotic bacteria in the murine MALT

The gene expression profiling was used to indicate specific mouse immune system genes that respond to the effect of probiotic bacteria in response to S. enterica. Two-color immunohistochemistry was used to determine the cell types involved with specific gene product markers and their tissue locations. Toxicologic Pathology Associates (Jefferson, Arkansas) prepared frozen sections of the MALT tissues from 8 mice and conducted the immunolocalization on those sections for cell markers and specific intracellular signal transduction gene products. Antibodies labelled with horseradish peroxidase specific for CD21, CD2, PTPRC, TLR6, and RELB were purchased from Santa Cruz Biologics. Densitometry of the stained areas in Peyer’s patches, lamina propria of intestinal villi, or the cortical and paracortical regions of mesenteric lymph nodes were measured by the Positive Pixel Count Algorithm from Aperio Technologies (Leica Biosystems).

Analysis of data