BACKGROUND
salmonella enterica is known to evade host defenses and induce persistent infections  <cit> .
salmonella sp.
persistence is also aided by a virulence mechanism that causes clonal deletion of high-avidity cd4+ t cells  <cit> .
furthermore, salmonella sp.
induces an inflammatory response and secretes a factor that blocks macrophage and dendritic cell migration  <cit> .
the intestinal microbiota can modulate the pathogenesis and host evasion mechanisms of s. enterica; for example, commensal e. coli improved the host response to salmonella sp.
infection by an immune system regulatory effect  <cit> .
the mechanisms of mucosal immune system regulation are only partially understood and the roles of microbes in this regulation are even less well understood.
addition of probiotic bacteria to the indigenous intestinal microbiota also affects mucosal immune regulation.
in a previous study, we observed that bacteria from a commercial probiotic product modified the host immune response to s. enterica by enhancing the proliferative response of spleen lymphocytes to s. enterica antigens.
we also observed that the t and b cell enriched splenocyte fractions had reduced activities of apoptosis-related cysteine peptidase or caspase   <dig> and casp <dig>  <cit> .
caspases are involved in the signaling pathway that directs programmed cell death  in activated immune cells.
these results were observed in mice that were not previously immunized to s. enterica, suggesting that the innate pathways of immune activation were involved.
t cells that have been activated, but lacking secondary signals, activate an intrinsic caspase pathway that is arrested by signals from b- cell cll/lymphoma  <dig>   <dig> and its related proteins.
the probiotic bacteria may be providing analogs to these secondary signals that maintain the survival of activated lymphocytes.
the present study sought to determine whether the secondary signals are mediated by intermediary antigen-presenting cells , such as dendritic cells and intestinal epithelial cells or by direct contact to lymphocytes.
apc would signal to lymphocytes with cytokines to affect a change in their activation and apoptotic status.
expression of these and other signaling molecules in mucosal tissues of probiotic-treated and untreated s. enterica-infected mice were compared.
a direct t cell response to microbial antigens would have to occur by a yet unrevealed mechanism or possibly by the same pathways that function in the apc.
intestinal epithelial cells are also involved in t cell activation by production of pro-inflammatory cytokines in the presence of commensal or probiotic bacteria  <cit> .
methods
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  <cit> .
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  agar  and bifidobacterium agars  incubated anaerobically for 48 h at 37 °c.
bacterial isolates were identified by their cellular fatty acid methyl ester compositions  and by their 16s rrna sequences with the microseq 16s rrna sequence assays .
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  <dig> × 106 cfu/mouse and administered to mice with a feeding tube, as previously described  <cit> .
salmonella enterica serotype cubana, originally isolated from poultry  <cit>  was grown on trypticase soy agar with 5% sheep blood  or in trypticase soy broth at 37 °c in an atmosphere of 5% co <dig> and air.
the salmonella isolate was originally characterized as serotype typhimurium and subsequently determined to be serotype g <dig>  by the arkansas regional laboratory of the food and drug administration , jefferson, ar.
the isolate is available upon reasonable request from the corresponding author.
mice
a total of  <dig> male and  <dig> female  <dig> week-old human microbiota-associated  <cit>  and  <dig> male and  <dig> female  <dig> week-old defined-microbiota balb/c mice  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- <dig> mouse chow  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 .
the cultures were incubated at 37 °c in 95% air, 5% co <dig> atmosphere overnight and the plates were assessed for bacterial growth.
the mice were fed a nih- <dig> diet sterilized in the transfer box as previously described  <cit> .
experimental design
control mice were human microbiota-associated in our laboratory, as previously described  <cit>  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  <dig> day later to culture for the presence of the probiotic bacteria.
at 7 days after probiotic treatment,  <dig> probiotic-treated mice and  <dig> untreated mice were each orally inoculated with  <dig> ×  <dig> colony forming units  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  <cit> .
total rna samples were extracted from the tissues and cdna were generated, from which quantitative real-time-polymerase chain reaction  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  <dig> 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, tlr <dig>  and v-rel avian reticuloendotheliosis viral oncogene homolog b  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  <cit> .
briefly, the entire volume of a 500 ml log phase broth culture of bacteria was centrifuged at  <dig> × 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  at  <dig>  lb./in <dig> to disrupt the bacteria.
the disrupted bacteria were centrifuged at  <dig> × g and the protein content of the supernatant was determined by the bicinchoninic acid protein assay  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  <cit> .
lymphocyte proliferation assays were performed with the celltiter aqueous  <dig> assay .
lymphocytes from the spleens of experimentally treated mice were prepared and incubated at a density of  <dig> ×  <dig> cells/well of a 96-well culture plate in rpmi medium  containing s. enterica antigens.
antigens were added to  <dig> 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 <dig> 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--5--2--2h–tetrazolium, inner salt  at 490 nm, which was measured with a plate reader .
the average of three wells per sample was used to determine the mean ± standard error of the mean  abs <dig> 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  <dig> and  <dig> .
each assay well of a nunc f <dig> black maxisorp  <dig> well fluorescent assay plate  contained 50 μl of cell suspension in roswell park memorial institute   <dig> medium at a cell concentration of  <dig> ×  <dig> cells/ml to which was added 10 μg antigen preparation per well.
after 56 h incubation at 37 °c in a humidified 5% co <dig> 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  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
rt <dig> profiler™ pcr arrays from qiagen bioscience were used to assess expression of mrna for  <dig> genes involved in the host response to bacteria in the mucosal immune tissues of the mouse gi tract.
arrays for genes involved in apoptosis , for genes involved in nf-κb activation , and for mouse t and b cell activation markers  were used according to the manufacturer’s instructions.
total cellular rna from peyer’s patches, mesenteric lymph nodes, and lamina propria from  <dig> mice were isolated using arraygrade total rna isolation kits .
the rna samples were treated with dnase- <dig>  reverse-transcribed with the rt <dig> pcr array first strand kit and the resulting cdna was analyzed by real-time pcr for detection in a biorad pci,q <dig> instrument.
the housekeeping genes used in the study were: beta glucuronidase, hypoxanthine phosphoribosyltransferase  <dig>  heat shock protein  <dig> alpha family class b member  <dig>  glyceraldehyde-3-phophate dehydrogenase, and beta actin.
results of the pcr array experiment were analyzed with the excel™  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  prepared frozen sections of the malt tissues from  <dig> 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 cd <dig>  cd <dig>  ptprc, tlr <dig>  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 .
analysis of data
evaluation of statistically significant differences between the results from treatment groups and control groups were determined with repeated measures analysis of variance and bonferroni’s post tests using prism v. <dig>  software .
numerical count data were log <dig> transformed prior to statistical analysis to make the data better fit a normal distribution.
statistical significance was defined at p <  <dig> .
RESULTS
persistence of probiotic bacteria and salmonella sp.
in the murine gi tracts
in our previous study  <cit> , colonization of germfree mice by probiotic bacteria was easily verified by microbial culture from feces, but the mice in this study had conventional microbiota.
the presence of l. reuteri, which is not a component of the balb/c mouse colony microbiota  in the defined-microbiota mice in the present study was confirmed by culture on mrs agar and microbial identification of colonies from feces at 7 days after oral feeding of  <dig>  log <dig> cfu of the probiotic bacterial mixture.
infection of mice was confirmed by recovery of s. enterica from the mice 7 days after oral challenge .table  <dig> numbers of bacteria recovered from experimental mice
animala
amice 1– <dig> were non-treated with probiotic lactic acid bacteria.
mice 5– <dig> were orally challenged with 1 × 108 cfu probiotic bacteria mixture.
seven days later, all mice were orally challenged with 1 × 108 cfu/ml s. enterica.
bthe number of s. enterica isolated from feces of mice 7 days after challenge were enumerated on ss agar plates.
cthe numbers of lactic acid bacteria were enumerated on mrs agar plates and reported as cfu/g feces 7 days after s. enterica challenge
s. enterica was recovered from one of the probiotic-fed mice, and all of the control mice 7 days after oral salmonella challenge.
table  <dig> also shows numbers of lactic acid bacteria that were recovered from mice 7 days after oral s. enterica challenge and grown on mrs agar.
probiotic bacteria prevented immunosuppression of t cells in mice to s. enterica antigens
lymphocytes from the spleens of balb/c mice challenged with s. enterica only for 7 days did not proliferate in response to the b-cell mitogen lipopolysaccharide  or to the t cell mitogen concanavalin-a, nor did they respond to soluble antigens from s. enterica .
this lack of responsiveness was observed under similar conditions in our previous report  <cit> .
decreased lymphoproliferation of splenocytes in mice treated with probiotics was observed in the control treatment and lps treatment groups.
splenocytes from mice that were fed the mixture of probiotic bacteria prior to s. enterica challenge had significant proliferative responses to concanavalin-a and the s. enterica antigens , showing a preventative effect of the probiotic bacteria on s. enterica-induced immunosuppression.
the lymphoproliferative responses to s. enterica lps were not significantly changed by probiotic bacteria compared with the s. enterica-treated control mice.fig.
<dig> lymphocyte proliferation and apoptosis responses.
proliferation of splenocytes from uninfected mice , s. enterica-infected balb/c mice , or s. enterica-infected, probiotic-treated mice to lps , concanavalin-a , or soluble s. enterica antigens  was measured as change in absorbance of mts at 490 nm .
apoptosis was measured as activation of caspases  <dig> and  <dig> in splenocytes from uninfected mice , s. enterica-infected mice , or s. enterica-infected, probiotic-treated mice to lps , concanavalin-a , or soluble s. enterica antigens , detected as relative fluorescence intensity  of caspase substrate, were compared .
the asterisks indicate statistically significant differences between control  and treated groups by anova, p <  <dig>
the probiotic bacteria altered the activation of cellular apoptosis in lymphocytes, as reported previously  <cit> .
in the present study, activation of casp <dig> and casp <dig>  which mediate lymphocyte apoptosis, was significantly increased in s. enterica-infected balb/c mice, compared to the response to lps or concanavalin-a, but was strongly induced by s. enterica antigens .
the mice that were fed probiotic bacteria before s. enterica challenge did not have a significant increase in lymphocyte casp <dig> and casp <dig> activation by s. enterica antigens , which suggests that strong s. enterica induction of t cell apoptotic responses was suppressed by the probiotics.
probiotic bacteria altered mrna expression of genes involved in responses to s. enterica infection
the presence of probiotic bacteria induced changes in expression of immune response genes and cellular locations in the malt of mice infected with s. enterica.
there was a general increase in t cell function and a decrease in inflammatory responses, as evidenced by increased expression of t cell activation genes and apoptosis-related genes and decreased gene expression of nf-κb signal transduction pathway components and proinflammatory cytokines, respectively .
probiotic lactobacilli had significant effects on the expression of mrna from genes involved in b and t cell activation, signal transduction by the nf-κb pathway, and apoptosis, when the mice were challenged with s. enterica.
expression of the mrna for the lymphokines interleukin  - <dig> p <dig>  and il <dig> were reduced, while il <dig> mrna was significantly increased by the presence of probiotic lactobacilli .
other genes involved in lymphocyte activation were also affected by probiotic bacteria.
nearly 3-fold induction of mrna for cd <dig>  and ptprc genes  indicates activation and differentiation of b and t cells.
other components of the t and b cell activation signal-transduction pathways were also induced .
since activated splenocytes proliferated in response to concanavalin-a, but not salmonella lps , it appears that mostly t cell activation accounts for the increased expression of the lymphocyte activation genes listed in table  <dig>  this conclusion is supported by the result that expression of the mrna for tlr <dig>  the protein of which is a receptor for s. enterica lipopolysaccharides on apc, was not affected by probiotic bacteria .
decreased expression of tlr <dig> and increased expression of tlr <dig> in malt suggests that probiotic bacteria modified the spectrum of bacterial lipoproteins that could be detected by apc in the malt.
rna for cd <dig> was not detected in the s. enterica challenged mice that were not treated with probiotic bacteria.
it was present in samples of malt from the probiotic-treated mice, consistent with the possibility that probiotics prevented the loss of antigen-specific lymphocytes at sites of infection.table  <dig> gene expression effects of probiotic bacteria on s. enterica-challenged mice
b and t cell activation genesb
nf-κb signal transduction genesc
apoptosis genesd
athe mean  fold change in qrt-pcr threshold cycles  between tissue rna samples amplified from probiotic-treated versus untreated control mice were calculated as  <dig>  where Δct is the housekeeping gene-normalized average ct and ΔΔct is the Δct- Δct.
positive values indicate increased expression and negative values indicate reduced expression
bresults from qiagen pamm- <dig> mouse t and b cell activation array
cresults from qiagen pamm- <dig> mouse nf-κb array
dresults from qiagen pamm- <dig> mouse apoptosis array
additional effects of the probiotic lactobacilli were observed in genes that relate to the nf-κb signal transduction pathway that controls expression of cytokine genes in response to receptor detection of microbial antigens.
mrna expression of several signaling protein genes casp <dig> and fadd like apoptosis regulator , casp <dig>  receptor tnfrsf-interacting serine threonine kinase  <dig> , tnf receptor associated factor  <dig> , bcl <dig>  and bcl2) was slightly reduced, but not to the 2-fold change level of significance .
significant reduction  in mrna expression of relb, myeloid differentiation primary response gene  <dig> , ring finger protein  <dig> , inhibitor of light polypeptide gene enhancer in b cell kinase alpha , and map/erk kinase kinase  are indicative of a response that suppresses transduction of proinflammatory signals in lymphocytes .
the increased mrna expression of interleukin- <dig> receptor-associated kinase  <dig>  and v-jun sarcoma virus  <dig> oncogene homolog  induced by probiotic bacteria in table  <dig> suggest differential regulation of these genes that may function simultaneously in other signal transduction pathways.
activation of t and b lymphocytes to produce cytokines is linked to activation of programmed cell death responses in these cells.
in this study, expression of mrna for genes involved in apoptosis was reduced by the presence of probiotic bacteria .
multiple signaling pathways of apoptosis induction and suppression function simultaneously to regulate this important process.
we observed that probiotic bacteria were associated with suppression of caspase recruitment domain protein  <dig> , tumor necrosis factor alpha , and fas-associated death domain protein  mrna expression, which are involved in initial steps of the apoptosis activation cascade and also reduction of mrna expression of casp <dig> and casp <dig>  which are involved in the activation of enzymes involved in apoptosis .
some genes involved in suppression of apoptosis cascade activation were also suppressed, including: defender against cell death  <dig> , p21-activated kinase  <dig> , v-akt murine thymoma viral oncogene homolog  <dig> , bcl <dig>  and bcl2-associated agonist of cell death  .
these seemingly contradictory findings illustrate the precise balance needed in feedback regulation mechanisms for apoptosis in antigen-activated lymphocytes.
probiotic bacteria changed the tissue distribution of cellular activation markers
in addition to mrna expression effects of probiotic bacteria, tissue distributions of several proteins were investigated.
immunohistochemical staining of malt and mesenteric lymph node tissues was used to trace the tissue locations of cells with changes in production of cd <dig>  cd <dig>  ptprc, relb, and tlr <dig> proteins within the malt tissues.
immunolocalization helped to interpret the qpcr results in terms of probiotic effects on host responses to s. enterica and these results will be presented in the following narrative that includes the observations and discussion of their significance in the context of t cells and antigen-presenting cells responding to s. enterica in the malt and movement of these cells between malt and mln.
the cd <dig> protein is expressed on b lymphocytes and dendritic cells, which can be differentiated morphologically in malt and mln.
the germinal centers of peyer’s patches  and secondary follicles of mln  contained numerous cells with cd <dig> that have lymphocyte morphology.
densitometry of the peyer’s patches stained for cd <dig> suggested no change in the numbers of cells expressing this marker in the probiotic-treated group,  by anova .
the increased cd <dig> mrna expression in peyer’s patches of the mice with probiotic treatment  did not appear to change the numbers of cd21-producing cells in the peyer’s patches.fig.
<dig> immunohistochemical staining of cd21in peyer’s patches and mln.
the germinal centers of peyer’s patches   and mln  contained numerous cells with cd <dig>  that have lymphocyte morphology.
images 10× magnification.
there were no significant differences in optical density of stained cells in probiotic-treated peyer’s patches  and lamina propria  compared to control s. enterica-challenged peyer’s patches and lamina propria by anova, p <  <dig>
peyer’s patches of s. enterica-infected mice were highly reactive with heavy follicular cd2- staining .
this is indicative of a strong migration of activated t cells to the site.
there was reduced diffuse cd <dig> staining of cortical and paracortical lymph node tissue  in probiotic-treated s. enterica-infected mice, which was statistically significant between cortical and paracortical regions of the lymph nodes .
these results are consistent with reduced inflammatory response in the probiotic-treated mice.fig.
<dig> immunohistochemical staining of cd <dig> in peyer’s patches and mln.
peyer’s patches  of s. enterica-infected mice were highly reactive with heavy follicular cd2- staining , especially in probiotic-treated mice .
there were no significant differences in staining between control and treated peyer’s patches or lamina propria  .
cd <dig> staining of cortical and paracortical lymph node tissue  in probiotic-treated salmonella-infected mice were different than control mice.
images 10× magnification.
statistically significant differences in staining of cortical and paracortical regions of the lymph nodes between control and probiotic-treated mice are shown at the asterisk, p <  <dig>
the infiltration of ptprc-expressing cells in peyer’s patches  and mln  of probiotic treated and control s. enterica-infected mice in the two tissues appeared similar.
comparisons of staining densities of the replicates in these tissues of both treatment groups also showed no significant difference .fig.
<dig> ptprc production shown by immunohistochemistry.
peyer’s patches  and lamina propria   appeared to have the same amount of ptprc staining .
there were no significant differences measured by densitometry .
mln  of probiotic-treated and s. enterica-infected mice also showed similar amounts of ptprc staining in germinal centers.
no significant differences in ptprc staining between control and treated mice were confirmed by densitometry .
ptprc staining is shown at the arrows
expression of tlr <dig> was investigated immunohistochemically because its mrna expression was increased in the probiotic-treated mice.
tlr <dig> was not strongly stained in peyer’s patches from probiotic-treated s. enterica-infected mice .
the amount of tlr <dig> in cortical areas of probiotic-treated mln , was significantly reduced, compared with untreated s. enterica-challenged mice .fig.
<dig> immunohistochemical staining of tlr <dig>  cells were stained for tlr <dig>  in peyer’s patches  and mln  from s. enterica-infected and probiotic-treated s. enterica-infected mice.
the amount of tlr <dig> in cortical areas of probiotic-treated mln was significantly reduced, p <  <dig>  by anova, compared with untreated s. enterica-challenged mice
relb cortical staining was apparent in peyer’s patches of probiotic-treated s. enterica-infected mice .
staining was also seen in mln from the probiotic-treated mice  but was not significantly different between regions of the tissues or between treatments .fig.
<dig> immunohistochemical staining of relb.
the peyer’s patches and lamina propria  and mln of probiotic-treated s. enterica-infected mice  had relb expressed in cells  with lymphocyte and epithelial cell morphologies.
there were no significant differences in staining of lamina propria  of control or probiotic-treated mice .
staining in mln from the probiotic-treated mice was not significantly different from controls, p <  <dig>  by anova
discussion
the concept of t cell depletion by early induction of a strong inflammatory response by s. enterica has been previously described  <cit> .
in the present study, we observed recovery of t cell proliferative responses in splenocytes from mice treated with the probiotic bacterial blend prior to s. enterica infection, which is consistent with the concept that t cell depletion was averted.
in a previous report, an immunosuppressive effect of s. enterica infection was observed on splenic lymphoproliferative responses in gnotobiotic balb/c mice  <cit> .
treatment of the mice with probiotic bacteria prevented the immunosuppressive effects.
there are several immunosuppressive mechanisms used by virulent s. enterica to evade host responses, including elimination of high-avidity antigen-specific t cells  <cit> , activation of suppressive regulatory t cells  <cit> , and phagolysosomal escape in macrophages and dendritic cells  <cit> .
in the present study, the probiotic bacteria suppressed basal and lps-induced splenic lymphoproliferation.
a growing body of literature is reporting this characteristic of probiotic lactobacilli with inhibition of lps-stimulated lymphocyte proliferation  <cit> , inhibition of concanavalin-a-stimulated proliferation  <cit> , and anti-cd3ε-stimulated splenocyte proliferation  <cit> .
it is consistent with the concept that pathogen-induced lymphocyte clonal deletion can be blocked by bacterial induction of mechanisms that inhibit proliferation.
cell surface receptors for commensal or probiotic bacteria may activate pathways that prevent proliferation and apoptosis of lymphocytes.
bacterial activation of a pathway that inhibits lymphocyte activation and apoptotic caspase activity most likely occurs through the recognition of bacterial surface glycoproteins by tlr <dig>  tlr <dig>  or tlr <dig> on apc  <cit> .
tlr <dig>  and tlr <dig>  are also involved in the t cell activation response to s. enterica by apc  <cit> .
in our present study, gene expression profiling qrt-pcr panels and immunohistochemistry were used to observe changes in expression of genes associated with lymphocyte activation and apoptosis and signal transduction from toll-like receptors through the nf-κb and mapk signal transduction pathways that showed evidence for a t cell-sparing effect from the influence of probiotic bacteria.
activation and signaling responses by lymphocytes are regulated by the nf-κb transcription factor and signal transduction pathway  <cit> .
some of the intracellular molecules that activate nf-κb include: cflar  <cit> , casp <dig>  <cit> , conserved helix-loop-helix ubiquitous kinase   <cit> , ripk <dig>  <cit> , traf <dig>  <cit> , and bcl <dig>  <cit> .
here we show evidence for a probiotic mechanism that prevents s. enterica immune evasion by t cell deletion through a mechanism of reduced inflammatory response early during the infection in balb/c mice.
the probiotic bacteria modulated expression of genes related to inflammation and t cell apoptosis, including some of the toll-like receptors involved in s. enterica-induced inflammation.
in this present study, we observed increased tlr <dig> mrna in malt of probiotic-treated mice compared with s. enterica-treated mice.
this agrees with evidence that probiotic lactobacillus plantarum induced tlr <dig> mrna expression in intestinal epithelial cells of cyclophosphamide-suppressed mice  <cit> .
we also observed that there was not an increase in tlr <dig> expressing cells in peyer’s patches, suggesting a role for post-transcriptional regulation of tlr <dig> on the surface of lymphocytes in our mice.
it is possible that tlr <dig> and tlr <dig> heterodimer activation can be inhibited by probiotics through activation of peptide regulators, as shown with tlr <dig> transmembrane domain peptides in vitro  <cit> .
the latter paper suggested this inhibition is specific to tlr <dig>  but the work was in vitro and may not show how the process may affect tlr2/tlr <dig> heterodimer surface expression in cells in vivo.
another possibility for this in the present study is that the different microbiota used in the human microbiota-associated mice versus the conventional mice may not have supported the probiotic effect.
since we saw probiotic bacteria could reverse the suppression of splenic lymphocyte proliferation to s. enterica antigens, we looked at changes in expression of genes associated with t cell activation in mice treated with probiotic bacteria.
initial contacts by intestinal epithelial cells or professional phagocytes with s. enterica surface molecules that have pathogen-associated molecular patterns activate receptors that initiate signal transduction within the cell.
these signals initiate mrna transcription and protein synthesis of cytokines that are secreted into the extracellular environment and recruit inflammatory cells.
when dendritic cells encounter s. enterica antigens in the intestinal tissues, they process the antigens for presentation to lymphocytes and migrate to mln  <cit> .
in the mln, the dendritic cells activate antigen-specific t cells that are selected by stromal cells for expression of α4β <dig> integrin and ccr <dig>  which is essential for their migration along a gradient of ccl <dig> and other chemokines to the intestinal malt .
clues to the probiotic mechanism that blocks s. enterica-induced immunosuppression might be seen in changes of expression of genes involved in recruitment of immune responses to the malt and mln of mice protected by the bacteria from s. enterica infection.
mln are essential for resistance to s. enterica infections, but are also the sites of long term persistence of infections  <cit> .
the mln hold s. enterica-infected dendritic cells and limit the dissemination of the infection by arresting dc migration  <cit> .
still, some of the bacteria can escape the mln because host cell death can be induced by s. enterica, especially serovar typhimurium  <cit> .
some probiotic mechanisms do not involve immune and inflammatory cell migration.
for example, cell-free spent media from probiotic bifidobacterium bifidum inhibits growth of s. enterica  <cit> .
these authors suggest that bifidobacter-derived factors interfere with expression of s. enterica virulence genes encoded on the salmonella pathogenicity islands  <dig> and  <dig>  some effects of commensal and probiotic bacteria appear to occur at the mucosal surface in epithelial cells.
some surface layer proteins of lactobacillus acidophilus inhibit casp <dig> responses to salmonella sp.
in iec cell lines  <cit>  thus, reducing apoptotic death and loss of epithelial barrier functions to salmonella sp.
invasion.
clearly, evidence exists for the combination of microbial interactions for colonization resistance to s. enterica infections and influences on the innate and adaptive immune responses can be attributed to probiotic bacteria.
CONCLUSIONS
despite all the efforts of public health and agricultural programs to reduce acquisition of salmonella sp.
infections, they persist as a major health issue.
one reason for this is the asymptomatic carrier state of some individuals who have recovered from acute infections.
persistence of a carrier state is facilitated by immune system evasion mechanisms inherent to the genus.
strains of bacteria in the intestinal microbiota and found in probiotic products can antagonize salmonella sp.
infections, perhaps by inhibition of these inherent immune system evasion mechanisms.
in this study, the presence of probiotic bacteria in the intestinal tracts of mice during s. enterica infections prevented loss of splenocyte proliferation and decreased splenocyte apoptosis.
gene expression data showed changes that suggest lymphocyte activation, survival.
and immune cell homing functions were restored, this was related to an apparent ability of the probiotic bacteria to limit exhaustive lymphocyte proliferation and apoptosis.
the migration of lymphocytes and dendritic cells in intestinal mucosal tissues was restored by the presence of the probiotic bacteria.
these results are consistent with the theory that s. enterica induced clonal deletion of lymphocytes that was inhibited by the presence of probiotic bacteria.
abbreviations
akt1v-akt murine thymoma viral oncogene homolog 1
apcantigen presenting cell
badbcl2-associated agonist of cell death
bcl2b- cell cll/lymphoma 2
card10caspase recruitment domain protein 10
casp2apoptosis-related cysteine peptidase 2
cd2clusters of differentiation antigen 2
cflarcasp <dig> and fadd like apoptosis regulator
cfucolony forming unit
dad1defender against cell death 1
faddfas-associated death domain protein
il4interleukin-4
irak2interleukin- <dig> receptor-associated kinase 2
iκκbinhibitor of light polypeptide gene enhancer in b cell kinase alpha
junv-jun sarcoma virus  <dig> oncogene homolog
lpslipopolysaccharide
maltmucosa associated lymphoid tissue
mekkmap/erk kinase kinase
mlnmesenteric lymph node
mrsde man rogosa sharpe
mts3--5--2--2h–tetrazolium, inner salt
myd88myeloid differentiation primary response gene 88
nf-κbnuclear factor of kappa light chains enhancer in b cells
pak7p21-activated kinase 7
ptprcprotein tyrosine phosphatase receptor type c
qrt-pcrquantitative real-time polymerase chain reaction
relbv-rel avian reticuloendotheliosis viral oncogene homolog b
ripk1receptor tnfrsf-interacting serine threonine kinase 1
rnf7ring finger protein 7
rpmi 1640roswell park memorial institute 1640
semstandard error of the mean
tlr6toll-like receptor 6
tnftumor necrosis factor alpha
traf2tnf receptor associated factor 2

 salmonella enterica is known to evade host defenses and induce persistent infections. salmonella sp. persistence is also aided by a virulence mechanism that causes clonal deletion of high-avidity cd4+ t cells. furthermore, salmonella sp. induces an inflammatory response and secretes a factor that blocks macrophage and dendritic cell migration. the intestinal microbiota can modulate the pathogenesis and host evasion mechanisms of s. enterica; for example, commensal e. coli improved the host response to salmonella sp. infection by an immune system regulatory effect. the mechanisms of mucosal immune system regulation are only partially understood and the roles of microbes in this regulation are even less well understood. addition of probiotic bacteria to the indigenous intestinal microbiota also affects mucosal immune regulation. in a previous study, we observed that bacteria from a commercial probiotic product modified the host immune response to s. enterica by enhancing the proliferative response of spleen lymphocytes to s. enterica antigens. we also observed that the t and b cell enriched splenocyte fractions had reduced activities of apoptosis-related cysteine peptidase or caspase and casp. caspases are involved in the signaling pathway that directs programmed cell death in activated immune cells. these results were observed in mice that were not previously immunized to s. enterica, suggesting that the innate pathways of immune activation were involved. t cells that have been activated, but lacking secondary signals, activate an intrinsic caspase pathway that is arrested by signals from b- cell cll/lymphoma and its related proteins. the probiotic bacteria may be providing analogs to these secondary signals that maintain the survival of activated lymphocytes. the present study sought to determine whether the secondary signals are mediated by intermediary antigen-presenting cells, such as dendritic cells and intestinal epithelial cells or by direct contact to lymphocytes. apc would signal to lymphocytes with cytokines to affect a change in their activation and apoptotic status. expression of these and other signaling molecules in mucosal tissues of probiotic-treated and untreated s. enterica-infected mice were compared. a direct t cell response to microbial antigens would have to occur by a yet unrevealed mechanism or possibly by the same pathways that function in the apc. intestinal epithelial cells are also involved in t cell activation by production of pro-inflammatory cytokines in the presence of commensal or probiotic bacteria. methods 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. 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 agar and bifidobacterium agars incubated anaerobically for 48 h at 37 °c. bacterial isolates were identified by their cellular fatty acid methyl ester compositions and by their 16s rrna sequences with the microseq 16s rrna sequence assays. 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 × 106 cfu/mouse and administered to mice with a feeding tube, as previously described. salmonella enterica serotype cubana, originally isolated from poultry was grown on trypticase soy agar with 5% sheep blood or in trypticase soy broth at 37 °c in an atmosphere of 5% co and air. the salmonella isolate was originally characterized as serotype typhimurium and subsequently determined to be serotype g by the arkansas regional laboratory of the food and drug administration, jefferson, ar. the isolate is available upon reasonable request from the corresponding author. mice a total of male and female week-old human microbiota-associated and male and female week-old defined-microbiota balb/c mice 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- mouse chow 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. the cultures were incubated at 37 °c in 95% air, 5% co atmosphere overnight and the plates were assessed for bacterial growth. the mice were fed a nih- diet sterilized in the transfer box as previously described. experimental design control mice were human microbiota-associated in our laboratory, as previously described 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 day later to culture for the presence of the probiotic bacteria. at 7 days after probiotic treatment, probiotic-treated mice and untreated mice were each orally inoculated with × colony forming units 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. total rna samples were extracted from the tissues and cdna were generated, from which quantitative real-time-polymerase chain reaction 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 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, tlr and v-rel avian reticuloendotheliosis viral oncogene homolog b 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. briefly, the entire volume of a 500 ml log phase broth culture of bacteria was centrifuged at × 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 at  lb./in to disrupt the bacteria. the disrupted bacteria were centrifuged at × g and the protein content of the supernatant was determined by the bicinchoninic acid protein assay 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. lymphocyte proliferation assays were performed with the celltiter aqueous assay. lymphocytes from the spleens of experimentally treated mice were prepared and incubated at a density of × cells/well of a 96-well culture plate in rpmi medium containing s. enterica antigens. antigens were added to 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 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--5--2--2h–tetrazolium, inner salt at 490 nm, which was measured with a plate reader. the average of three wells per sample was used to determine the mean ± standard error of the mean abs 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 and. each assay well of a nunc f black maxisorp well fluorescent assay plate contained 50 μl of cell suspension in roswell park memorial institute medium at a cell concentration of × cells/ml to which was added 10 μg antigen preparation per well. after 56 h incubation at 37 °c in a humidified 5% co 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 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 rt profiler™ pcr arrays from qiagen bioscience were used to assess expression of mrna for genes involved in the host response to bacteria in the mucosal immune tissues of the mouse gi tract. arrays for genes involved in apoptosis, for genes involved in nf-κb activation, and for mouse t and b cell activation markers were used according to the manufacturer’s instructions. total cellular rna from peyer’s patches, mesenteric lymph nodes, and lamina propria from mice were isolated using arraygrade total rna isolation kits. the rna samples were treated with dnase- reverse-transcribed with the rt pcr array first strand kit and the resulting cdna was analyzed by real-time pcr for detection in a biorad pci,q instrument. the housekeeping genes used in the study were: beta glucuronidase, hypoxanthine phosphoribosyltransferase heat shock protein alpha family class b member glyceraldehyde-3-phophate dehydrogenase, and beta actin. results of the pcr array experiment were analyzed with the excel™ 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 prepared frozen sections of the malt tissues from 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 cd cd ptprc, tlr 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. analysis of data evaluation of statistically significant differences between the results from treatment groups and control groups were determined with repeated measures analysis of variance and bonferroni’s post tests using prism v. software. numerical count data were log transformed prior to statistical analysis to make the data better fit a normal distribution. statistical significance was defined at p < .  persistence of probiotic bacteria and salmonella sp. in the murine gi tracts in our previous study, colonization of germfree mice by probiotic bacteria was easily verified by microbial culture from feces, but the mice in this study had conventional microbiota. the presence of l. reuteri, which is not a component of the balb/c mouse colony microbiota in the defined-microbiota mice in the present study was confirmed by culture on mrs agar and microbial identification of colonies from feces at 7 days after oral feeding of log cfu of the probiotic bacterial mixture. infection of mice was confirmed by recovery of s. enterica from the mice 7 days after oral challenge.table numbers of bacteria recovered from experimental mice animala amice 1– were non-treated with probiotic lactic acid bacteria. mice 5– were orally challenged with 1 × 108 cfu probiotic bacteria mixture. seven days later, all mice were orally challenged with 1 × 108 cfu/ml s. enterica. bthe number of s. enterica isolated from feces of mice 7 days after challenge were enumerated on ss agar plates. cthe numbers of lactic acid bacteria were enumerated on mrs agar plates and reported as cfu/g feces 7 days after s. enterica challenge s. enterica was recovered from one of the probiotic-fed mice, and all of the control mice 7 days after oral salmonella challenge. table also shows numbers of lactic acid bacteria that were recovered from mice 7 days after oral s. enterica challenge and grown on mrs agar. probiotic bacteria prevented immunosuppression of t cells in mice to s. enterica antigens lymphocytes from the spleens of balb/c mice challenged with s. enterica only for 7 days did not proliferate in response to the b-cell mitogen lipopolysaccharide or to the t cell mitogen concanavalin-a, nor did they respond to soluble antigens from s. enterica. this lack of responsiveness was observed under similar conditions in our previous report. decreased lymphoproliferation of splenocytes in mice treated with probiotics was observed in the control treatment and lps treatment groups. splenocytes from mice that were fed the mixture of probiotic bacteria prior to s. enterica challenge had significant proliferative responses to concanavalin-a and the s. enterica antigens, showing a preventative effect of the probiotic bacteria on s. enterica-induced immunosuppression. the lymphoproliferative responses to s. enterica lps were not significantly changed by probiotic bacteria compared with the s. enterica-treated control mice.fig. lymphocyte proliferation and apoptosis responses. proliferation of splenocytes from uninfected mice, s. enterica-infected balb/c mice, or s. enterica-infected, probiotic-treated mice to lps, concanavalin-a, or soluble s. enterica antigens was measured as change in absorbance of mts at 490 nm. apoptosis was measured as activation of caspases and in splenocytes from uninfected mice, s. enterica-infected mice, or s. enterica-infected, probiotic-treated mice to lps, concanavalin-a, or soluble s. enterica antigens, detected as relative fluorescence intensity of caspase substrate, were compared. the asterisks indicate statistically significant differences between control and treated groups by anova, p <  the probiotic bacteria altered the activation of cellular apoptosis in lymphocytes, as reported previously. in the present study, activation of casp and casp which mediate lymphocyte apoptosis, was significantly increased in s. enterica-infected balb/c mice, compared to the response to lps or concanavalin-a, but was strongly induced by s. enterica antigens. the mice that were fed probiotic bacteria before s. enterica challenge did not have a significant increase in lymphocyte casp and casp activation by s. enterica antigens, which suggests that strong s. enterica induction of t cell apoptotic responses was suppressed by the probiotics. probiotic bacteria altered mrna expression of genes involved in responses to s. enterica infection the presence of probiotic bacteria induced changes in expression of immune response genes and cellular locations in the malt of mice infected with s. enterica. there was a general increase in t cell function and a decrease in inflammatory responses, as evidenced by increased expression of t cell activation genes and apoptosis-related genes and decreased gene expression of nf-κb signal transduction pathway components and proinflammatory cytokines, respectively. probiotic lactobacilli had significant effects on the expression of mrna from genes involved in b and t cell activation, signal transduction by the nf-κb pathway, and apoptosis, when the mice were challenged with s. enterica. expression of the mrna for the lymphokines interleukin - p and il were reduced, while il mrna was significantly increased by the presence of probiotic lactobacilli. other genes involved in lymphocyte activation were also affected by probiotic bacteria. nearly 3-fold induction of mrna for cd and ptprc genes indicates activation and differentiation of b and t cells. other components of the t and b cell activation signal-transduction pathways were also induced. since activated splenocytes proliferated in response to concanavalin-a, but not salmonella lps, it appears that mostly t cell activation accounts for the increased expression of the lymphocyte activation genes listed in table this conclusion is supported by the result that expression of the mrna for tlr the protein of which is a receptor for s. enterica lipopolysaccharides on apc, was not affected by probiotic bacteria. decreased expression of tlr and increased expression of tlr in malt suggests that probiotic bacteria modified the spectrum of bacterial lipoproteins that could be detected by apc in the malt. rna for cd was not detected in the s. enterica challenged mice that were not treated with probiotic bacteria. it was present in samples of malt from the probiotic-treated mice, consistent with the possibility that probiotics prevented the loss of antigen-specific lymphocytes at sites of infection.table gene expression effects of probiotic bacteria on s. enterica-challenged mice b and t cell activation genesb                                     nf-κb signal transduction genesc                                   apoptosis genesd                   athe mean fold change in qrt-pcr threshold cycles between tissue rna samples amplified from probiotic-treated versus untreated control mice were calculated as where Δct is the housekeeping gene-normalized average ct and ΔΔct is the Δct- Δct. positive values indicate increased expression and negative values indicate reduced expression bresults from qiagen pamm- mouse t and b cell activation array cresults from qiagen pamm- mouse nf-κb array dresults from qiagen pamm- mouse apoptosis array additional effects of the probiotic lactobacilli were observed in genes that relate to the nf-κb signal transduction pathway that controls expression of cytokine genes in response to receptor detection of microbial antigens. mrna expression of several signaling protein genes casp and fadd like apoptosis regulator, casp receptor tnfrsf-interacting serine threonine kinase, tnf receptor associated factor, bcl and bcl2) was slightly reduced, but not to the 2-fold change level of significance. significant reduction in mrna expression of relb, myeloid differentiation primary response gene, ring finger protein, inhibitor of light polypeptide gene enhancer in b cell kinase alpha, and map/erk kinase kinase are indicative of a response that suppresses transduction of proinflammatory signals in lymphocytes. the increased mrna expression of interleukin- receptor-associated kinase and v-jun sarcoma virus oncogene homolog induced by probiotic bacteria in table suggest differential regulation of these genes that may function simultaneously in other signal transduction pathways. activation of t and b lymphocytes to produce cytokines is linked to activation of programmed cell death responses in these cells. in this study, expression of mrna for genes involved in apoptosis was reduced by the presence of probiotic bacteria. multiple signaling pathways of apoptosis induction and suppression function simultaneously to regulate this important process. we observed that probiotic bacteria were associated with suppression of caspase recruitment domain protein, tumor necrosis factor alpha, and fas-associated death domain protein mrna expression, which are involved in initial steps of the apoptosis activation cascade and also reduction of mrna expression of casp and casp which are involved in the activation of enzymes involved in apoptosis. some genes involved in suppression of apoptosis cascade activation were also suppressed, including: defender against cell death, p21-activated kinase, v-akt murine thymoma viral oncogene homolog, bcl and bcl2-associated agonist of cell death. these seemingly contradictory findings illustrate the precise balance needed in feedback regulation mechanisms for apoptosis in antigen-activated lymphocytes. probiotic bacteria changed the tissue distribution of cellular activation markers in addition to mrna expression effects of probiotic bacteria, tissue distributions of several proteins were investigated. immunohistochemical staining of malt and mesenteric lymph node tissues was used to trace the tissue locations of cells with changes in production of cd cd ptprc, relb, and tlr proteins within the malt tissues. immunolocalization helped to interpret the qpcr results in terms of probiotic effects on host responses to s. enterica and these results will be presented in the following narrative that includes the observations and discussion of their significance in the context of t cells and antigen-presenting cells responding to s. enterica in the malt and movement of these cells between malt and mln. the cd protein is expressed on b lymphocytes and dendritic cells, which can be differentiated morphologically in malt and mln. the germinal centers of peyer’s patches and secondary follicles of mln contained numerous cells with cd that have lymphocyte morphology. densitometry of the peyer’s patches stained for cd suggested no change in the numbers of cells expressing this marker in the probiotic-treated group, by anova. the increased cd mrna expression in peyer’s patches of the mice with probiotic treatment did not appear to change the numbers of cd21-producing cells in the peyer’s patches.fig. immunohistochemical staining of cd21in peyer’s patches and mln. the germinal centers of peyer’s patches and mln contained numerous cells with cd that have lymphocyte morphology. images 10× magnification. there were no significant differences in optical density of stained cells in probiotic-treated peyer’s patches and lamina propria compared to control s. enterica-challenged peyer’s patches and lamina propria by anova, p <  peyer’s patches of s. enterica-infected mice were highly reactive with heavy follicular cd2- staining. this is indicative of a strong migration of activated t cells to the site. there was reduced diffuse cd staining of cortical and paracortical lymph node tissue in probiotic-treated s. enterica-infected mice, which was statistically significant between cortical and paracortical regions of the lymph nodes. these results are consistent with reduced inflammatory response in the probiotic-treated mice.fig. immunohistochemical staining of cd in peyer’s patches and mln. peyer’s patches of s. enterica-infected mice were highly reactive with heavy follicular cd2- staining, especially in probiotic-treated mice. there were no significant differences in staining between control and treated peyer’s patches or lamina propria. cd staining of cortical and paracortical lymph node tissue in probiotic-treated salmonella-infected mice were different than control mice. images 10× magnification. statistically significant differences in staining of cortical and paracortical regions of the lymph nodes between control and probiotic-treated mice are shown at the asterisk, p <  the infiltration of ptprc-expressing cells in peyer’s patches and mln of probiotic treated and control s. enterica-infected mice in the two tissues appeared similar. comparisons of staining densities of the replicates in these tissues of both treatment groups also showed no significant difference.fig. ptprc production shown by immunohistochemistry. peyer’s patches and lamina propria appeared to have the same amount of ptprc staining. there were no significant differences measured by densitometry. mln of probiotic-treated and s. enterica-infected mice also showed similar amounts of ptprc staining in germinal centers. no significant differences in ptprc staining between control and treated mice were confirmed by densitometry. ptprc staining is shown at the arrows expression of tlr was investigated immunohistochemically because its mrna expression was increased in the probiotic-treated mice. tlr was not strongly stained in peyer’s patches from probiotic-treated s. enterica-infected mice. the amount of tlr in cortical areas of probiotic-treated mln, was significantly reduced, compared with untreated s. enterica-challenged mice.fig. immunohistochemical staining of tlr cells were stained for tlr in peyer’s patches and mln from s. enterica-infected and probiotic-treated s. enterica-infected mice. the amount of tlr in cortical areas of probiotic-treated mln was significantly reduced, p <  by anova, compared with untreated s. enterica-challenged mice relb cortical staining was apparent in peyer’s patches of probiotic-treated s. enterica-infected mice. staining was also seen in mln from the probiotic-treated mice but was not significantly different between regions of the tissues or between treatments.fig. immunohistochemical staining of relb. the peyer’s patches and lamina propria and mln of probiotic-treated s. enterica-infected mice had relb expressed in cells with lymphocyte and epithelial cell morphologies. there were no significant differences in staining of lamina propria of control or probiotic-treated mice. staining in mln from the probiotic-treated mice was not significantly different from controls, p <  by anova discussion the concept of t cell depletion by early induction of a strong inflammatory response by s. enterica has been previously described. in the present study, we observed recovery of t cell proliferative responses in splenocytes from mice treated with the probiotic bacterial blend prior to s. enterica infection, which is consistent with the concept that t cell depletion was averted. in a previous report, an immunosuppressive effect of s. enterica infection was observed on splenic lymphoproliferative responses in gnotobiotic balb/c mice. treatment of the mice with probiotic bacteria prevented the immunosuppressive effects. there are several immunosuppressive mechanisms used by virulent s. enterica to evade host responses, including elimination of high-avidity antigen-specific t cells, activation of suppressive regulatory t cells, and phagolysosomal escape in macrophages and dendritic cells. in the present study, the probiotic bacteria suppressed basal and lps-induced splenic lymphoproliferation. a growing body of literature is reporting this characteristic of probiotic lactobacilli with inhibition of lps-stimulated lymphocyte proliferation, inhibition of concanavalin-a-stimulated proliferation, and anti-cd3ε-stimulated splenocyte proliferation. it is consistent with the concept that pathogen-induced lymphocyte clonal deletion can be blocked by bacterial induction of mechanisms that inhibit proliferation. cell surface receptors for commensal or probiotic bacteria may activate pathways that prevent proliferation and apoptosis of lymphocytes. bacterial activation of a pathway that inhibits lymphocyte activation and apoptotic caspase activity most likely occurs through the recognition of bacterial surface glycoproteins by tlr tlr or tlr on apc. tlr and tlr are also involved in the t cell activation response to s. enterica by apc. in our present study, gene expression profiling qrt-pcr panels and immunohistochemistry were used to observe changes in expression of genes associated with lymphocyte activation and apoptosis and signal transduction from toll-like receptors through the nf-κb and mapk signal transduction pathways that showed evidence for a t cell-sparing effect from the influence of probiotic bacteria. activation and signaling responses by lymphocytes are regulated by the nf-κb transcription factor and signal transduction pathway. some of the intracellular molecules that activate nf-κb include: cflar, casp, conserved helix-loop-helix ubiquitous kinase, ripk, traf, and bcl. here we show evidence for a probiotic mechanism that prevents s. enterica immune evasion by t cell deletion through a mechanism of reduced inflammatory response early during the infection in balb/c mice. the probiotic bacteria modulated expression of genes related to inflammation and t cell apoptosis, including some of the toll-like receptors involved in s. enterica-induced inflammation. in this present study, we observed increased tlr mrna in malt of probiotic-treated mice compared with s. enterica-treated mice. this agrees with evidence that probiotic lactobacillus plantarum induced tlr mrna expression in intestinal epithelial cells of cyclophosphamide-suppressed mice. we also observed that there was not an increase in tlr expressing cells in peyer’s patches, suggesting a role for post-transcriptional regulation of tlr on the surface of lymphocytes in our mice. it is possible that tlr and tlr heterodimer activation can be inhibited by probiotics through activation of peptide regulators, as shown with tlr transmembrane domain peptides in vitro. the latter paper suggested this inhibition is specific to tlr but the work was in vitro and may not show how the process may affect tlr2/tlr heterodimer surface expression in cells in vivo. another possibility for this in the present study is that the different microbiota used in the human microbiota-associated mice versus the conventional mice may not have supported the probiotic effect. since we saw probiotic bacteria could reverse the suppression of splenic lymphocyte proliferation to s. enterica antigens, we looked at changes in expression of genes associated with t cell activation in mice treated with probiotic bacteria. initial contacts by intestinal epithelial cells or professional phagocytes with s. enterica surface molecules that have pathogen-associated molecular patterns activate receptors that initiate signal transduction within the cell. these signals initiate mrna transcription and protein synthesis of cytokines that are secreted into the extracellular environment and recruit inflammatory cells. when dendritic cells encounter s. enterica antigens in the intestinal tissues, they process the antigens for presentation to lymphocytes and migrate to mln. in the mln, the dendritic cells activate antigen-specific t cells that are selected by stromal cells for expression of α4β integrin and ccr which is essential for their migration along a gradient of ccl and other chemokines to the intestinal malt. clues to the probiotic mechanism that blocks s. enterica-induced immunosuppression might be seen in changes of expression of genes involved in recruitment of immune responses to the malt and mln of mice protected by the bacteria from s. enterica infection. mln are essential for resistance to s. enterica infections, but are also the sites of long term persistence of infections. the mln hold s. enterica-infected dendritic cells and limit the dissemination of the infection by arresting dc migration. still, some of the bacteria can escape the mln because host cell death can be induced by s. enterica, especially serovar typhimurium. some probiotic mechanisms do not involve immune and inflammatory cell migration. for example, cell-free spent media from probiotic bifidobacterium bifidum inhibits growth of s. enterica. these authors suggest that bifidobacter-derived factors interfere with expression of s. enterica virulence genes encoded on the salmonella pathogenicity islands and some effects of commensal and probiotic bacteria appear to occur at the mucosal surface in epithelial cells. some surface layer proteins of lactobacillus acidophilus inhibit casp responses to salmonella sp. in iec cell lines thus, reducing apoptotic death and loss of epithelial barrier functions to salmonella sp. invasion. clearly, evidence exists for the combination of microbial interactions for colonization resistance to s. enterica infections and influences on the innate and adaptive immune responses can be attributed to probiotic bacteria.  despite all the efforts of public health and agricultural programs to reduce acquisition of salmonella sp. infections, they persist as a major health issue. one reason for this is the asymptomatic carrier state of some individuals who have recovered from acute infections. persistence of a carrier state is facilitated by immune system evasion mechanisms inherent to the genus. strains of bacteria in the intestinal microbiota and found in probiotic products can antagonize salmonella sp. infections, perhaps by inhibition of these inherent immune system evasion mechanisms. in this study, the presence of probiotic bacteria in the intestinal tracts of mice during s. enterica infections prevented loss of splenocyte proliferation and decreased splenocyte apoptosis. gene expression data showed changes that suggest lymphocyte activation, survival. and immune cell homing functions were restored, this was related to an apparent ability of the probiotic bacteria to limit exhaustive lymphocyte proliferation and apoptosis. the migration of lymphocytes and dendritic cells in intestinal mucosal tissues was restored by the presence of the probiotic bacteria. these results are consistent with the theory that s. enterica induced clonal deletion of lymphocytes that was inhibited by the presence of probiotic bacteria. abbreviations akt1v-akt murine thymoma viral oncogene homolog 1 apcantigen presenting cell badbcl2-associated agonist of cell death bcl2b- cell cll/lymphoma 2 card10caspase recruitment domain protein 10 casp2apoptosis-related cysteine peptidase 2 cd2clusters of differentiation antigen 2 cflarcasp and fadd like apoptosis regulator cfucolony forming unit dad1defender against cell death 1 faddfas-associated death domain protein il4interleukin-4 irak2interleukin- receptor-associated kinase 2 iκκbinhibitor of light polypeptide gene enhancer in b cell kinase alpha junv-jun sarcoma virus oncogene homolog lpslipopolysaccharide maltmucosa associated lymphoid tissue mekkmap/erk kinase kinase mlnmesenteric lymph node mrsde man rogosa sharpe mts3--5--2--2h–tetrazolium, inner salt myd88myeloid differentiation primary response gene 88 nf-κbnuclear factor of kappa light chains enhancer in b cells pak7p21-activated kinase 7 ptprcprotein tyrosine phosphatase receptor type c qrt-pcrquantitative real-time polymerase chain reaction relbv-rel avian reticuloendotheliosis viral oncogene homolog b ripk1receptor tnfrsf-interacting serine threonine kinase 1 rnf7ring finger protein 7 rpmi 1640roswell park memorial institute 1640 semstandard error of the mean tlr6toll-like receptor 6 tnftumor necrosis factor alpha traf2tnf receptor associated factor 2 
we previously observed that probiotic bacteria could prevent inhibition of lymphoproliferation and apoptosis responses of t cells associated with s. enterica infections in orally challenged mice. in this study, changes in expression of genes related to lymphocyte activation in mucosa-associated lymphoid tissues of mice orally infected with s. enterica with and without treatment with probiotic bacteria were evaluated. probiotic bacteria increased expression of mrna for clusters of differentiation antigen, protein tyrosine phosphatase receptor type c, and toll-like receptor genes related to t and b cell activation in mouse intestinal tissue. the probiotic bacteria were also associated with reduced mrna expression of a group of genes related to nuclear factor of kappa light chains enhancer in b cells signal transduction pathway-regulated cytokine responses. probiotic bacteria were also associated with reduced mrna expression of apoptotic genes that suggest high avidity lymphocyte sparing. the results of this study are consistent with prevention of s. enterica-induced deletion of lymphocytes by the influence of probiotic bacteria in mucosal lymphoid tissues of mice. reduced cd immunostaining in mesenteric lymph nodes was suggestive of reduced lymphocyte activation in probiotic-treated mice. reduced immunostaining of tlr in malt of probiotic-treated, s. enterica-infected mice suggests that diminished innate immune sensitivity to s. enterica antigens is associated with preventing lymphocyte deletion.
 salmonella enterica infections often exhibit a form of immune evasion. we previously observed that probiotic bacteria could prevent inhibition of lymphoproliferation and apoptosis responses of t cells associated with s. enterica infections in orally challenged mice.  in this study, changes in expression of genes related to lymphocyte activation in mucosa-associated lymphoid tissues of mice orally infected with s. enterica with and without treatment with probiotic bacteria were evaluated. probiotic bacteria increased expression of mrna for clusters of differentiation antigen, protein tyrosine phosphatase receptor type c, and toll-like receptor genes related to t and b cell activation in mouse intestinal tissue. the probiotic bacteria were also associated with reduced mrna expression of a group of genes related to nuclear factor of kappa light chains enhancer in b cells signal transduction pathway-regulated cytokine responses. probiotic bacteria were also associated with reduced mrna expression of apoptotic genes that suggest high avidity lymphocyte sparing. reduced cd immunostaining in mesenteric lymph nodes was suggestive of reduced lymphocyte activation in probiotic-treated mice. reduced immunostaining of tlr in malt of probiotic-treated, s. enterica-infected mice suggests that diminished innate immune sensitivity to s. enterica antigens is associated with preventing lymphocyte deletion.  the results of this study are consistent with prevention of s. enterica-induced deletion of lymphocytes by the influence of probiotic bacteria in mucosal lymphoid tissues of mice. keywords probioticgene expressionsignal transduction genessalmonellaapoptosishttp://dx.doi.org/ /100007197u.s. public health serviceissue-copyright-statement© the author 2017 