BACKGROUND
clostridium difficile is a gram-positive, spore-forming anaerobe that causes antibiotic-associated diarrhea, colitis, and pseudomembranous colitis in humans  <cit> .
c. difficile-associated disease  is acquired in association with the disruption and alteration of the gut microbiota  <cit> .
the frequency and severity of primary cdad are increasing; as well as recurrent cases and infections refractory to standard antibiotic therapy  <cit> .
c. difficile toxins, toxin a  and toxin b , are the main virulence factors contributing to intestinal tissue damage and severe inflammation  <cit> .
both toxins disrupt the actin cytoskeleton and tight junctions of intestinal epithelial cells  <cit> , and cause apoptotic and necrotic cell death  <cit> .
c. difficile toxins, tcda and tcdb, induce the release of chemokines, like il- <dig>  from intestinal epithelial cells  <cit> .
in addition to il- <dig>  tcda induces human epithelial cells to secrete other cxc chemokines, including growth-related oncogene -α and neutrophil activating protein- <dig> , along with the cc chemokine, monocyte chemoattractant protein - <dig>  <cit> .
the disruption of tight junctions is thought to enable tcda and tcdb to enter the laminar propria and submucosa to induce immune cells to secrete chemokines, proinflammatory cytokines, and mediators which promote proinflammatory and cytotoxic effects  <cit> .
proinflammatory cytokines, especially il-1β, also act on epithelial cells to increase il- <dig> secretion and upregulate intercellular adhesion molecule- <dig>  expression, leading to increased icam- <dig> and neutrophil cd11/cd <dig> receptor-dependent neutrophil adhesion  <cit> .
a prominent feature of cdad results from the release of il- <dig> from intestinal epithelial cells that causes a massive influx of neutrophils into the colonic mucosa  <cit> .
neutrophil-derived inflammatory mediators exert toxic effects on epithelial cells, causing congestion and edema of the mucosa and epithelial cell damage  <cit> .
il- <dig> is a major cxc chemokine that mediates neutrophil recruitment, activation, and adhesion.
il- <dig> potency relies on high binding affinity for neutrophil surface receptors, cxcr <dig> and cxcr2; which activate chemotaxis  <cit>  and robust effector functions  <cit> , and trigger the upregulation of adhesion molecules cd11/cd <dig> that facilitate transendothelial migration and subsequent tissue infiltration  <cit> .
cdad is most often associated with the disruption of a healthy intestinal microbiome after the administration of antibiotics  <cit> .
potential biotherapies for the treatment and prevention of cdad are probiotics.
in particular, candidate probiotics are capable of enhancing microbiome stability, interfering with pathogen activity, modulating the immune system, and are intrinsically resistant to broad-spectrum antibiotics  <cit> .
meta-analyses of randomized controlled trials  <cit>  support the efficacy of probiotic saccharomyces boulardii and lactobacillus rhamnosus gg in the prevention of cdad.
placebo-controlled clinical trials have shown that lyophilized s. boulardii decreased diarrhea associated with β-lactam antibiotics  <cit> , and when used in combination with metronidazole or vancomycin, s. boulardii significantly reduced recurrent episodes in patients with cdad  <cit> .
the probiotic effects of s. boulardii resulted from a secreted protease that interferes with receptor binding of c. difficile toxins a and b to brush borders of human colonic epithelial cells  <cit>  and the downstream modulation of host mitogen-activated protein kinase  signaling pathway  <cit> .
in vitro studies with probiotic bacteria show that lactobacillus delbrueckii ssp.
bulgaricus b- <dig> inhibits the cytotoxic effects and adhesion of c. difficile to caco- <dig> cells  <cit> , while specific strains of bifidobacterium spp.
and lactobacillus spp.
have antagonistic effects on the production of c. difficile toxins a and b  <cit> .
in the present study, we hypothesized that specific strains of lactobacillus species isolated from healthy hosts produce factors that suppress toxigenic c. difficile-induced il- <dig> production.
we investigated the probiotic effect of  <dig> lactobacillus infant-fecal isolates on the suppression of il- <dig> production from colonic epithelial cells stimulated by c. difficile.
conditioned media of two vancomycin-resistant isolates, l. rhamnosus l <dig> and l. casei l <dig>  suppressed il- <dig> production at the level of transcription without inhibiting c. difficile growth or toxin production.
large  heat-stable, soluble factors are suggested to be responsible for the observed il- <dig> suppression.
RESULTS
human-derived lactobacillus spp.
produce factors that inhibit il- <dig> and gm-csf production by c. difficile-stimulated colonic epithelial cells
lactobacillus conditioned media  from thirty-four human-derived lactobacillus isolates  were screened for the ability to suppress pro-inflammatory cytokine production from c. difficile-stimulated colonic epithelial cells.
lcm from  <dig> of  <dig> lactobacillus isolates, l. rhamnosus l <dig> , l. rhamnosus l <dig> , and l. casei l <dig> , significantly suppressed il- <dig> production by approximately 50% or greater when compared to media control .
these three il-8-suppressing isolates did not stimulate il- <dig> production when co-cultured with ht- <dig> cells in the absence of c. difficile .
only a single isolate of l. casei was included in this study , and the third l. rhamnosus isolate in this collection, strain l <dig>  did not suppress il- <dig> in the same assay .
in a previous study  <cit> , we sequenced and compared the genomes of l. rhamnosus l <dig>  l <dig>  and l <dig>  due to the fact that lr-l <dig> and lr-l <dig> were isolated from the same host, had similar colony morphology, were nearly identical at the nucleotide level and similarly genetically distinct from strain lr-l <dig>  we determined that they were most likely independent isolates of the same strain.
therefore, the remaining experiments in this study were conducted excluding lr-l <dig>
lcm from lr-l <dig> and lc-l <dig> were screened for effects on fourteen additional cytokines in a multi-plex luminex assay.
in addition to il- <dig>  c. difficile stimulated gm-csf production, but affected no other cytokines tested .
gm-csf is an important pro-inflammatory cytokine that allows neutrophils to persist at sites of inflammation  <cit>  and enhances the chemotactic response of neutrophils to il- <dig>  <cit> .
in addition to il- <dig>  lcm from both anti-inflammatory lactobacilli strains significantly inhibited the production of gm-csf .
lcm from a different strain of a matched species of l. rhamnosus, strain l <dig>  not only had no effect on il- <dig> production, but also did not inhibit gm-csf production in the same assay .
further characterization of isolates lr-l <dig> and lc-l <dig> showed that they are resistant to both vancomycin and metronidazole , two drugs commonly used to treat c. difficile infection in humans  <cit> .
the anti-inflammatory effect of soluble factors produced by lr-l <dig> and lc-l <dig> is not unique to ht- <dig> cells as lcm from both strains also suppresses il- <dig> production greater than 50% from human colonocytes  stimulated by c. difficile .
trypan blue dye exclusion indicated that ht- <dig> and caco- <dig> cell viability  was not compromised by the presence of any lcm tested .
furthermore, neither viability of  nor toxin production by  c. difficile was negatively affected by the presence of lcm.
all further in vitro experimentation was carried out using the c. difficile-ht- <dig> cell co-culture model.
l. rhamnosus l <dig> and l. casei l <dig> affect il- <dig> gene transcription through decreased activity of nf-κb and c-jun
the effects of soluble factors produced by lr-l <dig> and lc-l <dig> on il- <dig> transcription were determined by il- <dig> gene-specific quantitative rt-pcr.
c. difficile-stimulated ht- <dig> cells were treated with lcm for 4 h prior to total rna isolation.
when compared to media control, treatment with lcm from lr-l <dig> or lc-l <dig> resulted in an approximate  <dig> -fold down-regulation  of il- <dig> mrna concentration relative to gapdh .
transcriptional regulation of il- <dig> is mediated via nf-κb and ap- <dig> by the upstream phosphorylation of subunits p <dig> and c-jun, which result in the respective activation of nf-κb and ap- <dig> and subsequent downstream transcription of il- <dig>  <cit> .
to determine whether or not lr-l <dig> or lc-l <dig> produce factors that affect activation of nf-κb or c-jun, c. difficile-stimulated ht- <dig> cells were treated with lcm from either lr-l <dig> or lc-l <dig> for  <dig> and 30 min, then were assayed for effects on phosphorylated nf-κb  and c-jun  concentrations by western blot.
lcm from lr-l <dig> decreased p-nf-κb by  <dig> % at 30 min only , and did not affect p-c-jun at either time point .
a 15 min treatment with lc-l <dig> lcm resulted in decreased concentrations of p-nf-κb  and p-c-jun  while a 30 min treatment resulted in a  <dig> % decrease in p-nf-κb .
the anti-inflammatory factor produced by l. rhamnosus l <dig> and l. casei l <dig> is heat-stable and greater than 100 kda in size
heat tolerance and size prediction of anti-inflammatory factors in lcm from lr-l <dig> and lc-l <dig> were characterized as follows.
lcm from lr-l <dig> and lc-l <dig> were heated to 100°c for 15 min, 30 min, and 1 h and then tested for the ability to suppress c. difficile-induced il- <dig> production in ht- <dig> cells.
the ability to suppress il- <dig> production in this assay was retained at all time points .
size fractionation of lr-l <dig> and lc-l <dig> lcm was performed using 30 kda and  <dig> kda amicon® centrifugal filters and filtrates containing factors <30 kda and <100 kda did not suppress il- <dig> activity, while fractions containing factors >100 kda retained the anti-il- <dig> activity in each case .
discussion
standard therapy for treating cdad with metronidazole and vancomycin is effective, but the association of these drugs with high relapse rates represents a major health problem  <cit> .
recent reports estimate the rate of recurrence after an initial episode of cdad to be 13-38%  <cit> .
standard therapy for recurrent cdad has been modified to include probiotics like l.rhamnosus gg, l. plantarum 299v, or s. boulardii which reduce the rate of recurrence around 20-30%  <cit> .
health promoting effects of lactobacilli include the stabilization of indigenous microbial populations, protection against intestinal infection, modulation of the immune system, and effects on gene expression in the human mucosa  <cit> .
in addition, a recent systematic review and meta-analysis indicated that probiotics given concurrently with antibiotics reduced the risk of antibiotic-associated diarrhea and c. difficile infection  <cit> .
a key characteristic of the pathophysiology of cdad is an inflammatory response with a marked neutrophil accumulation resulting from secreted il- <dig>  <cit> .
lactobacilli are known to inhibit il- <dig> production by intestinal epithelial cells stimulated with bacterium-derived lps  <cit>  but have not yet been associated with the modulation of c. difficile-induced il- <dig> production.
we identified two lactobacillus isolates from a library of infant feces-derived lactobacillus spp.
that can significantly suppress il- <dig> production by c. difficile-stimulated ht- <dig> cells.
il- <dig> suppression by lr-l <dig> and lc-l <dig> did not result from negative effects on c. difficile viability as colony counts from the co-culture supernatants of either c. difficile alone, or in combination with mrs, or lcm from lr-l <dig> or lc-l <dig>  were not significantly different .
trejo et al.
<cit>  demonstrated that spent culture supernatants of b. bifidum  <dig> and l. plantarum  <dig> diminished the production of toxin a and toxin b by c. difficile atcc  <dig> and clinical isolate  <dig>  il- <dig> suppression by lr-l <dig> and lc-l <dig> seems not to result from attenuated toxin production and endocytosis by ht- <dig> cells.
no significant difference of intracellular toxin concentrations in c. difficile and ht- <dig> cells was seen by lcm treatment as compared to medium control .
however, we cannot exclude the possibility that lcm of lr-l <dig> and lc-l <dig> interfere with toxin self-cleavage in the endosome prior to entering the cytosol which induces several downstream inflammatory consequences  <cit> .
it is likely that c. difficile-secreted toxins were endocytosed by ht- <dig> cells as a very small amount of toxin was present in tissue culture medium at 24 h of the co-culture assay.
lr-l <dig> and lc-l <dig> suppressed il- <dig> gene transcription at 4 h after co-culture with ht- <dig> cells which is in agreement with a report of imaoka et al.
<cit>  for b. bifidum strain yakult.
the il- <dig> gene promoter contains binding sites for transcription factors such as nf-κb and ap- <dig>  <cit> , which control the transcription of il- <dig> in c. difficile-stimulated ht- <dig> colonic epithelial cells  <cit> .
lcm from lr-l <dig> decreased p-nf-κb, and did not affect p-c-jun while lc-l <dig> lcm decreased both of p-nf-κb and p-c-jun, with greater effects on pnf-κb.
modulation of pro-inflammatory signaling pathways by probiotic bacteria and yeasts have been demonstrated previously.
ma et al.
<cit>  reported that l. reuteri inhibited tnf-induced il- <dig> production in both t <dig> and ht- <dig> intestinal epithelial cells by inhibiting nuclear translocation of nf-κb.
s. boulardii modifies host cell pro-inflammatory signaling pathways during bacterial infection by blocking the activation of nf-κb and mapk  <cit> .
our data showed that c. difficile stimulated gm-csf production in addition to il- <dig> and conditioned media generated from lr-l <dig> and lc-l <dig> can also suppress gm-csf production.
the ability to suppress gm-csf of these lactobacilli potentially enhances their anti-inflammatory effects on c. difficile infection.
probiotic factors can vary from organism to organism in regards to activity, size, and stability.
sougioultzis et al.
<cit>  reported that s. boulardii probiotic yeast produce a heat stable, <1 kda soluble, anti-inflammatory compound that blocks nf-κb activation and nf-κb-mediated il- <dig> gene expression in both ht- <dig> colonic epithelial cells and thp- <dig> monocytes.
l. rhamnosus gg secretes p <dig>  and p <dig>  proteins that activate egfr and downstream pi3k/akt and pkc signaling that modulate intestinal epithelial cell survival and growth  <cit> .
castagliuolo et al.
<cit>  reported that a 54-kda protease of s. boulardii can inhibit the effect of c. difficile toxins a and b in ht- <dig> cells.
l. reuteri atcc pta  <dig> produces the small molecule, histamine, which inhibits tnf production in thp- <dig> monocytes via pka and erk signaling  <cit> .
active substances in lcm of lr-l <dig> and lc-l <dig> were found to be heat-stable and >100 kda.
although lr-l <dig> and lc-l <dig> also inhibit tnf production by thp- <dig> monocytes, they do not produce histamine .
therefore the tnf-suppressive mechanism of action of these lactobacilli must be different from l. reuteri, and further investigation is needed to characterize the soluble factors produced by these specific lactobacillus isolates.
the discovery of vancomycin-resistant lr-l <dig> and lc-l <dig> with il-8-suppressing ability in this study offers us potential probiotic strains for combating cdad.
these strains may harbor other probiotic properties, including the ability to replenish normal microbiota, a key factor for treating cdad as shown by the recent successes with intestinal microbiome transplant therapies  <cit> .
CONCLUSIONS
we have demonstrated the probiotic effect of two vancomycin-resistant strains, l. rhamnosus l <dig> and l. casei l <dig>  on the suppression of il- <dig> production from c. difficile-stimulated colonic epithelial cells.
these strains suppressed il- <dig> production by inhibiting activation of transcription factors for il- <dig> gene expression without inhibiting c. difficile growth or toxin production.
our data also suggest that heat-stable, >100 kda factors are responsible for il- <dig> suppression.
these vancomycin-resistant lactobacillus strains are potential probiotics for treating or preventing cdad.

 clostridium difficile is a gram-positive, spore-forming anaerobe that causes antibiotic-associated diarrhea, colitis, and pseudomembranous colitis in humans. c. difficile-associated disease is acquired in association with the disruption and alteration of the gut microbiota. the frequency and severity of primary cdad are increasing; as well as recurrent cases and infections refractory to standard antibiotic therapy. c. difficile toxins, toxin a and toxin b, are the main virulence factors contributing to intestinal tissue damage and severe inflammation. both toxins disrupt the actin cytoskeleton and tight junctions of intestinal epithelial cells, and cause apoptotic and necrotic cell death. c. difficile toxins, tcda and tcdb, induce the release of chemokines, like il- from intestinal epithelial cells. in addition to il- tcda induces human epithelial cells to secrete other cxc chemokines, including growth-related oncogene -α and neutrophil activating protein-, along with the cc chemokine, monocyte chemoattractant protein -. the disruption of tight junctions is thought to enable tcda and tcdb to enter the laminar propria and submucosa to induce immune cells to secrete chemokines, proinflammatory cytokines, and mediators which promote proinflammatory and cytotoxic effects. proinflammatory cytokines, especially il-1β, also act on epithelial cells to increase il- secretion and upregulate intercellular adhesion molecule- expression, leading to increased icam- and neutrophil cd11/cd receptor-dependent neutrophil adhesion. a prominent feature of cdad results from the release of il- from intestinal epithelial cells that causes a massive influx of neutrophils into the colonic mucosa. neutrophil-derived inflammatory mediators exert toxic effects on epithelial cells, causing congestion and edema of the mucosa and epithelial cell damage. il- is a major cxc chemokine that mediates neutrophil recruitment, activation, and adhesion. il- potency relies on high binding affinity for neutrophil surface receptors, cxcr and cxcr2; which activate chemotaxis and robust effector functions, and trigger the upregulation of adhesion molecules cd11/cd that facilitate transendothelial migration and subsequent tissue infiltration. cdad is most often associated with the disruption of a healthy intestinal microbiome after the administration of antibiotics. potential biotherapies for the treatment and prevention of cdad are probiotics. in particular, candidate probiotics are capable of enhancing microbiome stability, interfering with pathogen activity, modulating the immune system, and are intrinsically resistant to broad-spectrum antibiotics. meta-analyses of randomized controlled trials support the efficacy of probiotic saccharomyces boulardii and lactobacillus rhamnosus gg in the prevention of cdad. placebo-controlled clinical trials have shown that lyophilized s. boulardii decreased diarrhea associated with β-lactam antibiotics, and when used in combination with metronidazole or vancomycin, s. boulardii significantly reduced recurrent episodes in patients with cdad. the probiotic effects of s. boulardii resulted from a secreted protease that interferes with receptor binding of c. difficile toxins a and b to brush borders of human colonic epithelial cells and the downstream modulation of host mitogen-activated protein kinase signaling pathway. in vitro studies with probiotic bacteria show that lactobacillus delbrueckii ssp. bulgaricus b- inhibits the cytotoxic effects and adhesion of c. difficile to caco- cells, while specific strains of bifidobacterium spp. and lactobacillus spp. have antagonistic effects on the production of c. difficile toxins a and b. in the present study, we hypothesized that specific strains of lactobacillus species isolated from healthy hosts produce factors that suppress toxigenic c. difficile-induced il- production. we investigated the probiotic effect of lactobacillus infant-fecal isolates on the suppression of il- production from colonic epithelial cells stimulated by c. difficile. conditioned media of two vancomycin-resistant isolates, l. rhamnosus l and l. casei l suppressed il- production at the level of transcription without inhibiting c. difficile growth or toxin production. large heat-stable, soluble factors are suggested to be responsible for the observed il- suppression.  human-derived lactobacillus spp. produce factors that inhibit il- and gm-csf production by c. difficile-stimulated colonic epithelial cells lactobacillus conditioned media from thirty-four human-derived lactobacillus isolates were screened for the ability to suppress pro-inflammatory cytokine production from c. difficile-stimulated colonic epithelial cells. lcm from of lactobacillus isolates, l. rhamnosus l, l. rhamnosus l, and l. casei l, significantly suppressed il- production by approximately 50% or greater when compared to media control. these three il-8-suppressing isolates did not stimulate il- production when co-cultured with ht- cells in the absence of c. difficile. only a single isolate of l. casei was included in this study, and the third l. rhamnosus isolate in this collection, strain l did not suppress il- in the same assay. in a previous study, we sequenced and compared the genomes of l. rhamnosus l l and l due to the fact that lr-l and lr-l were isolated from the same host, had similar colony morphology, were nearly identical at the nucleotide level and similarly genetically distinct from strain lr-l we determined that they were most likely independent isolates of the same strain. therefore, the remaining experiments in this study were conducted excluding lr-l lcm from lr-l and lc-l were screened for effects on fourteen additional cytokines in a multi-plex luminex assay. in addition to il- c. difficile stimulated gm-csf production, but affected no other cytokines tested. gm-csf is an important pro-inflammatory cytokine that allows neutrophils to persist at sites of inflammation and enhances the chemotactic response of neutrophils to il-. in addition to il- lcm from both anti-inflammatory lactobacilli strains significantly inhibited the production of gm-csf. lcm from a different strain of a matched species of l. rhamnosus, strain l not only had no effect on il- production, but also did not inhibit gm-csf production in the same assay. further characterization of isolates lr-l and lc-l showed that they are resistant to both vancomycin and metronidazole, two drugs commonly used to treat c. difficile infection in humans. the anti-inflammatory effect of soluble factors produced by lr-l and lc-l is not unique to ht- cells as lcm from both strains also suppresses il- production greater than 50% from human colonocytes stimulated by c. difficile. trypan blue dye exclusion indicated that ht- and caco- cell viability was not compromised by the presence of any lcm tested. furthermore, neither viability of nor toxin production by c. difficile was negatively affected by the presence of lcm. all further in vitro experimentation was carried out using the c. difficile-ht- cell co-culture model. l. rhamnosus l and l. casei l affect il- gene transcription through decreased activity of nf-κb and c-jun the effects of soluble factors produced by lr-l and lc-l on il- transcription were determined by il- gene-specific quantitative rt-pcr. c. difficile-stimulated ht- cells were treated with lcm for 4 h prior to total rna isolation. when compared to media control, treatment with lcm from lr-l or lc-l resulted in an approximate -fold down-regulation of il- mrna concentration relative to gapdh. transcriptional regulation of il- is mediated via nf-κb and ap- by the upstream phosphorylation of subunits p and c-jun, which result in the respective activation of nf-κb and ap- and subsequent downstream transcription of il-. to determine whether or not lr-l or lc-l produce factors that affect activation of nf-κb or c-jun, c. difficile-stimulated ht- cells were treated with lcm from either lr-l or lc-l for and 30 min, then were assayed for effects on phosphorylated nf-κb and c-jun concentrations by western blot. lcm from lr-l decreased p-nf-κb by % at 30 min only, and did not affect p-c-jun at either time point. a 15 min treatment with lc-l lcm resulted in decreased concentrations of p-nf-κb and p-c-jun while a 30 min treatment resulted in a % decrease in p-nf-κb. the anti-inflammatory factor produced by l. rhamnosus l and l. casei l is heat-stable and greater than 100 kda in size heat tolerance and size prediction of anti-inflammatory factors in lcm from lr-l and lc-l were characterized as follows. lcm from lr-l and lc-l were heated to 100°c for 15 min, 30 min, and 1 h and then tested for the ability to suppress c. difficile-induced il- production in ht- cells. the ability to suppress il- production in this assay was retained at all time points. size fractionation of lr-l and lc-l lcm was performed using 30 kda and kda amicon® centrifugal filters and filtrates containing factors <30 kda and <100 kda did not suppress il- activity, while fractions containing factors >100 kda retained the anti-il- activity in each case. discussion standard therapy for treating cdad with metronidazole and vancomycin is effective, but the association of these drugs with high relapse rates represents a major health problem. recent reports estimate the rate of recurrence after an initial episode of cdad to be 13-38%. standard therapy for recurrent cdad has been modified to include probiotics like l.rhamnosus gg, l. plantarum 299v, or s. boulardii which reduce the rate of recurrence around 20-30%. health promoting effects of lactobacilli include the stabilization of indigenous microbial populations, protection against intestinal infection, modulation of the immune system, and effects on gene expression in the human mucosa. in addition, a recent systematic review and meta-analysis indicated that probiotics given concurrently with antibiotics reduced the risk of antibiotic-associated diarrhea and c. difficile infection. a key characteristic of the pathophysiology of cdad is an inflammatory response with a marked neutrophil accumulation resulting from secreted il-. lactobacilli are known to inhibit il- production by intestinal epithelial cells stimulated with bacterium-derived lps but have not yet been associated with the modulation of c. difficile-induced il- production. we identified two lactobacillus isolates from a library of infant feces-derived lactobacillus spp. that can significantly suppress il- production by c. difficile-stimulated ht- cells. il- suppression by lr-l and lc-l did not result from negative effects on c. difficile viability as colony counts from the co-culture supernatants of either c. difficile alone, or in combination with mrs, or lcm from lr-l or lc-l were not significantly different. trejo et al. demonstrated that spent culture supernatants of b. bifidum and l. plantarum diminished the production of toxin a and toxin b by c. difficile atcc and clinical isolate il- suppression by lr-l and lc-l seems not to result from attenuated toxin production and endocytosis by ht- cells. no significant difference of intracellular toxin concentrations in c. difficile and ht- cells was seen by lcm treatment as compared to medium control. however, we cannot exclude the possibility that lcm of lr-l and lc-l interfere with toxin self-cleavage in the endosome prior to entering the cytosol which induces several downstream inflammatory consequences. it is likely that c. difficile-secreted toxins were endocytosed by ht- cells as a very small amount of toxin was present in tissue culture medium at 24 h of the co-culture assay. lr-l and lc-l suppressed il- gene transcription at 4 h after co-culture with ht- cells which is in agreement with a report of imaoka et al. for b. bifidum strain yakult. the il- gene promoter contains binding sites for transcription factors such as nf-κb and ap-, which control the transcription of il- in c. difficile-stimulated ht- colonic epithelial cells. lcm from lr-l decreased p-nf-κb, and did not affect p-c-jun while lc-l lcm decreased both of p-nf-κb and p-c-jun, with greater effects on pnf-κb. modulation of pro-inflammatory signaling pathways by probiotic bacteria and yeasts have been demonstrated previously. ma et al. reported that l. reuteri inhibited tnf-induced il- production in both t and ht- intestinal epithelial cells by inhibiting nuclear translocation of nf-κb. s. boulardii modifies host cell pro-inflammatory signaling pathways during bacterial infection by blocking the activation of nf-κb and mapk. our data showed that c. difficile stimulated gm-csf production in addition to il- and conditioned media generated from lr-l and lc-l can also suppress gm-csf production. the ability to suppress gm-csf of these lactobacilli potentially enhances their anti-inflammatory effects on c. difficile infection. probiotic factors can vary from organism to organism in regards to activity, size, and stability. sougioultzis et al. reported that s. boulardii probiotic yeast produce a heat stable, <1 kda soluble, anti-inflammatory compound that blocks nf-κb activation and nf-κb-mediated il- gene expression in both ht- colonic epithelial cells and thp- monocytes. l. rhamnosus gg secretes p and p proteins that activate egfr and downstream pi3k/akt and pkc signaling that modulate intestinal epithelial cell survival and growth. castagliuolo et al. reported that a 54-kda protease of s. boulardii can inhibit the effect of c. difficile toxins a and b in ht- cells. l. reuteri atcc pta produces the small molecule, histamine, which inhibits tnf production in thp- monocytes via pka and erk signaling. active substances in lcm of lr-l and lc-l were found to be heat-stable and >100 kda. although lr-l and lc-l also inhibit tnf production by thp- monocytes, they do not produce histamine. therefore the tnf-suppressive mechanism of action of these lactobacilli must be different from l. reuteri, and further investigation is needed to characterize the soluble factors produced by these specific lactobacillus isolates. the discovery of vancomycin-resistant lr-l and lc-l with il-8-suppressing ability in this study offers us potential probiotic strains for combating cdad. these strains may harbor other probiotic properties, including the ability to replenish normal microbiota, a key factor for treating cdad as shown by the recent successes with intestinal microbiome transplant therapies.  we have demonstrated the probiotic effect of two vancomycin-resistant strains, l. rhamnosus l and l. casei l on the suppression of il- production from c. difficile-stimulated colonic epithelial cells. these strains suppressed il- production by inhibiting activation of transcription factors for il- gene expression without inhibiting c. difficile growth or toxin production. our data also suggest that heat-stable, >100 kda factors are responsible for il- suppression. these vancomycin-resistant lactobacillus strains are potential probiotics for treating or preventing cdad. 
clostridium difficile is the main cause of hospital-acquired diarrhea and colitis known as c. difficile-associated disease.with increased severity and failure of treatment in cdad, new approaches for prevention and treatment, such as the use of probiotics, are needed. on the suppression of il- production in response to c. difficile infection. we screened lactobacillus conditioned media from infant fecal isolates for the ability to suppress c. difficile-induced il- production from ht- cells. factors produced by two vancomycin-resistant lactobacilli, l. rhamnosus l and l.casei l, suppressed the secretion and transcription of il- without inhibiting c. difficile viability or toxin production. conditioned media from lr-l and lc-l also decreased the production of c. difficile-induced gm-csf in ht- cells. our results suggest that l. rhamnosus l and l. casei l each produce factors capable of modulating inflammation stimulated by c. difficile. these vancomycin-resistant lactobacillus strains are potential probiotics for treating or preventing cdad. since the pathogenesis of cdad involves an inflammatory response with a massive influx of neutrophils recruited by interleukin - this study aimed to investigate the probiotic effects of lactobacillus spp.
 clostridium difficile is the main cause of hospital-acquired diarrhea and colitis known as c. difficile-associated disease.with increased severity and failure of treatment in cdad, new approaches for prevention and treatment, such as the use of probiotics, are needed. since the pathogenesis of cdad involves an inflammatory response with a massive influx of neutrophils recruited by interleukin - this study aimed to investigate the probiotic effects of lactobacillus spp. on the suppression of il- production in response to c. difficile infection.  we screened lactobacillus conditioned media from infant fecal isolates for the ability to suppress c. difficile-induced il- production from ht- cells. factors produced by two vancomycin-resistant lactobacilli, l. rhamnosus l and l.casei l, suppressed the secretion and transcription of il- without inhibiting c. difficile viability or toxin production. conditioned media from lr-l suppressed the activation of phospho-nf-κb with no effect on phospho-c-jun. however, lc-l conditioned media suppressed the activation of both phospho-nf-κb and phospho-c-jun. conditioned media from lr-l and lc-l also decreased the production of c. difficile-induced gm-csf in ht- cells. immunomodulatory factors present in the conditioned media of both lr-l and lc-l are heat-stable up to 100°c and > 100 kda in size.  our results suggest that l. rhamnosus l and l. casei l each produce factors capable of modulating inflammation stimulated by c. difficile. these vancomycin-resistant lactobacillus strains are potential probiotics for treating or preventing cdad. lactobacillusclostridium difficileprobioticil-8anti-inflammatory 