BACKGROUND
the human gastrointestinal tract  is one of the most complex ecosystems.
the interaction between git microbiota and the host is indispensable for the maturation of the immune system and for the regulation of intestinal physiology  <cit> .
firmicutes  and bacteroidetes are the two dominant phyla in the human gut, representing 90% of the microbiota, with actinobacteria being the third, comprising of only 3%  <cit> .
when the normal microbial ecosystem is disrupted, different non-predominant bacteria can bloom, constituting a potential trigger for disease.
the term dysbiosis refers to a type of microbial imbalance that may promote disease.
the strongest relationship between dysbiosis and pathological condition has been observed in inflammatory bowel diseases , where the proportion of firmicutes is lower than in healthy subjects  <cit> .
ibd, composed mainly of ulcerative colitis  and crohn’s disease , are characterized by an unusual activation of the git-immune system leading to a chronic inflammation of the gut with a mix of remission and exacerbation periods.
it has recently been suggested that the colonic microbiota is also abnormal in patients with irritable bowel syndrome , with a 2-fold increased ratio of the firmicutes to bacteroidetes  <cit> .
ibs is a symptom-based diagnosis characterized by chronic abdominal pain and altered bowel habits  <cit>  in the absence of detectable structural abnormalities  <cit> .
ibd and ibs are classically considered as dichotomous conditions.
however, increasing evidence, such as the involvement of brain-gut axis dysfunction  <cit> , as well as abnormal microbiota, supports some pathogenetic overlap between the two conditions  <cit> .
furthermore, the identification of low-grade inflammation  <cit> , histopathological observations and mucosal cytokine levels in the colon of a subset of ibs patients  <cit>  and the presence of ibs-like symptoms in quiescent ibd  <cit>  support this hypothesis.
low-grade inflammation can persist in many ibd patients who have otherwise obtained clinical remission  <cit> , as well as barrier alterations due to functional impairment of tight junction proteins  <cit> .
in fact, impaired gut barrier function appears to precede the development of ibd  <cit>  and increased epithelial permeability causes mucosal immune activation predisposing or enhancing disease progression  <cit> .
due to the importance of the microbiota in the development of these dysbiosis-related diseases, there is currently increasing interest in the development of microbiota-modulating therapies, including the use of probiotics  <cit> .
f. prausnitzii is a candidate probiotic bacterium that has been proposed as a sensor of intestinal health  <cit> .
changes in the abundance of this commensal anti-inflammatory bacterium  <cit>  and major member of the clostridium leptum group  <cit>  have been linked to dysbiosis in several human disorders  <cit> , such as ibd, where a low abundance was found in patients exhibiting endoscopic recurrence 6 months after surgery  <cit> .
furthermore, the recovery of f. prausnitzii counts after relapse is associated with maintenance of clinical remission in uc patients  <cit> .
besides, f. prausnitzii has shown protective effects in inflammation parameters in both acute  <cit>  and chronic  <cit>  models of chemical-induced active inflammation; however its beneficial role during low-grade inflammation and gut dysfunction has not been tested yet.
for these reasons, we hypothesized a possible protective role of f. pausnitzii on gut dysfunction mediated by low-grade inflammation.
therefore, we investigated the potential of f. prausnitzii as a probiotic for gut dysfunction prevention and therapy.
we tested the effects of f. prausnitzii in a murine model of chronic low-grade inflammation mimicking some of the most common gut dysfunction parameters found in ibd patients under remission.
RESULTS
validation of a low-grade inflammation model in dnbs-treated mice
the model of low-grade dnbs inflammation developed in this study involves the induction of a low grade, subclinical inflammatory status, followed by a recovery period and a reactivation period mimicking the relapsing nature in ibd .
the challenge with a sub-colitic dnbs dose  did not induce important changes in weight or colonic macroscopic or histological scores confirming the absence of a strong inflammation .
the degree of infiltration by polymorphonuclear neutrophils, as measured by tissue mpo activity, as well as lipocalin- <dig> , an early inflammation marker in inflammatory and autoimmune disorders, did not differ significantly between dnbs-treated and control animals .
these observations suggest that dnbs-treated mice tend to display a low-grade inflammatory status rather than a strong inflammation  thereby validating our model for further analysis of physiological changes induced by this chronic low-grade, subclinical inflammation.
the findings for all these markers thus suggest that the colons of dnbs-treated mice have a low-grade inflammatory status.
comparison of the levels of these markers with those previously published by our group following a colitic dose of dnbs that induced severe or moderate chronic inflammation  <cit>  provided further evidence that this protocol leads to low-grade inflammatory conditions .figure  <dig>
experimental protocols for the mouse model of chronic micro-inflammation and absence of inflammation.
a) colitis was induced by intrarectal administration of 100 mg/kg of dnbs in solution in 30% ethanol .
control mice  received only 30% etoh.
the effects of dnbs are highest during the first 3 days after its administration .
ten days after the end of the dnbs period, bacterial culture, pbs, or supernatant  were intragastrically administered daily for 10 days .
colitis was reactivated 21 days after the first dnbs injection with a second injection of 50 mg/kg of dnbs solution.
three days after reactivation mice were sacrificed.
the severity of the reactivated colitis was assessed from difference in body weight , macroscopic score , mpo activity , histological score   and lipocalin- <dig> levels   between control non-inflamed , control inflamed , bacteria-free culture medium , f. prausnitzii strain a2- <dig> , and f. prausnitzii a2- <dig> sn  groups.
experiments were performed at least in duplicate.
normal appearance of the colon of a control mouse with no inflammation  and of a mouse with micro-inflammation .
comparison of inflammation measures in severe and moderate colitis models
[
25
]
with the micro-inflammation model employed in this study.
severity of the colitis was assessed from differences in body weight , macroscopic score , histological score  and mpo activity  between the control non-inflamed , control inflamed severe colitis model )  <cit> , moderate colitis model )  <cit>  and micro-inflammation model ).
experiments were performed at least in duplicate.
comparisons involved the non-parametric kruskal-wallis test was used followed by a dunn’s multiple comparison test.
*p <  <dig>  vs. dnbs-pbs, +p <  <dig>  .
faecalibacterium prausnitzii a2- <dig> and its sn counteract dnbs-induced hyperpermeability in vivo
the gut of dnbs-treated mice showed high permeability to the paracellular tracer fitc-dextran in vivo  .
we tested whether f. prausnitzii modulates this dnbs-induced gut dysfunction.
treatment with either f. prausnitzii or its sn resulted in a significant reduction of intestinal permeability  .
to confirm that these findings corresponded to local paracellular translocation  of the fitc-dextran in vivo in this model, intestinal permeability was assessed in vitro in colonic samples mounted in ussing chambers extracted from mice at sacrifice .
the results were consistent with the pattern observed in vivo  confirming local translocation of the fitc-dextran.
we then tested f. prausnitzii sn  with samples from dnbs-challenged mice by ussing chamber analysis : the 51cr-edta flux, measured in % hot sample/hr/cm <dig>  was lower with sn than pbs  .
no significant differences were obtained in tissue conductance  .figure  <dig>
in vivo
and
in vitro
permeability measurements.
for in vivo measurements of gut permeability, animals were orally gavaged with fitc-dextran  .
for in vitro measures, sections of colon were mounted in ussing chambers and 51cr-edta flux   and tissue conductance   were measured .
control non-inflamed , control inflamed , bacteria-free culture medium , f. prausnitzii strain a2- <dig> , and f. prausnitzii a2- <dig> sn  groups.
experiments were performed at least in duplicate.
comparisons involved the non-parametric kruskal-wallis test was used followed by a dunn’s multiple comparison test.
*p <  <dig>  vs. dnbs-pbs, +p <  <dig> .
f. prausnitzii restores dnbs-induced apical junction proteins decrease
these effects on gut permeability prompted us to investigate the involvement of epithelial adherens junction  and tight junction  proteins, by assaying for relevant mrna levels.
the mrna levels for claudin- <dig> , e-cadherin , f11r  , occludin  and zo- <dig>  were all less abundant in dnbs-treated mice than in control mice  ; claudin- <dig>   <dig>   <dig> and  <dig> mrna levels were unaffected .
f. prausnitzii and its sn tended to normalize the amounts of all these mrna transcripts, the only statistically significant regulation was found for claudin- <dig> and f11r  .
claudin- <dig>  e-cadherin, f11r and occludin expression was also analyzed by immunofluorescence .
staining for the four proteins was reduced in dnbs + pbs mice compared to etoh + pbs group .
according to the qpcr results, f. prausnitzii tends to increase the expression of all the proteins.figure  <dig>
effect of
f. prausnitzii
on apical junction proteins mrnas in a dnbs-induced low-grade inflammation model.
apical junction protein expression levels were determined by real-time qpcr.
control non-inflamed , control inflamed , bacteria-free culture medium , f. prausnitzii strain a2- <dig> , and f. prausnitzii a2- <dig> sn  groups.
experiments were performed at least in duplicate.
comparisons involved the non-parametric kruskal-wallis test was used followed by a dunn’s multiple comparison test.
*p <  <dig>  vs. dnbs-pbs, +p <  <dig>  .
a systemic and mucosal reduction in pro-inflammatory cytokine production correlates with the protective effects of f. prausnitzii and its sn after dnbs challenge
we investigated the cytokines involved in this low-grade inflammation model and the mechanism by which f. prausnitzii and its sn protect the intestinal mucosa.
we assayed  <dig> cytokines  in both colonic and serum samples after the second dnbs injection .
the concentrations of il- <dig>  il-1α, il- <dig>  il- <dig>  il- <dig>  il- <dig>  il- <dig>  ifn-γ, and tnf-α in colon samples were higher in dnbs-treated mice than in controls , consistent with local inflammation.
similarly, cytokine concentrations in serum samples were consistent with the low-grade inflammatory status .
strikingly, the concentration of il- <dig>  ifn-γ and il- <dig> in colon samples and of il- <dig> and il- <dig> in serum was statistically significantly reduced by treatment with either f. prausnitzii or its sn  and there were clear tendencies for these treatments to reduce colonic il- <dig> and serum il- <dig> concentrations .figure  <dig>
cytokine concentrations in colon  and serum  in the dnbs micro-inflammation model.
control non-inflamed , control inflamed , bacteria-free culture medium , f. prausnitzii strain a2- <dig> , and f. prausnitzii a2- <dig> sn  groups.
experiments were performed at least in duplicate.
comparisons involved the non-parametric kruskal-wallis test was used followed by a dunn’s multiple comparison test.
*p <  <dig>  vs. dnbs-pbs, +p <  <dig>  .
f. prausnitzii and its sn restore serotonin variations observed in dnbs-treated mice
serotonin  of the key neurotransmitters in the git, and affects motility, sensation and secretion  <cit> .
we therefore studied serotonin concentrations in colon tissue and content by elisa.
both colonic  and colon content  serotonin concentrations were higher in dnbs-treated mice than etoh-treated controls .
treatments with both f. prausnitzii and its sn counteracted these increases  .
serotonin reuptake transporter  mrna was assayed by qpcr in colon tissues: the value for inflamed mice was half than for control non-inflamed mice ; the value for mice treated with f. prausnitzii was the same as that for normal healthy mice .
similar positive effects were found in sn-treated mice  although the culture medium  itself had a positive effect .figure  <dig>
serotonin concentrations in colon  and colon contents  in the dnbs micro-inflammation model.
control non-inflamed , control inflamed , bacteria-free culture medium , f. prausnitzii strain a2- <dig> , and f. prausnitzii a2- <dig> sn  groups.
experiments were performed at least in duplicate.comparisons involved the non-parametric kruskal-wallis test was used followed by a dunn’s multiple comparison test.
*p <  <dig>  vs. dnbs-pbs, +p <  <dig>  .
discussion
there is growing evidence pointing at the role of the gut microbiota in the development of ibd  <cit> .
furthermore, at least 40% of ibd patients in clinical remission have ongoing histological evidence of inflammation  <cit>  and suffer from altered gut functions with the involvement of brain-gut axis dysfunction  <cit> .
interestingly, previous transient or chronic inflammation can lead to persistent gut dysfunction  <cit>  and it has been suggested that the host microbiota may affect the two-ways communication pathway between the gut and the brain  <cit> .
because the microbiota is a determinant of normal gut function and immunity  <cit> , and because there is evidence of dysbiosis in ibd, there is increasing interest in the development of new adjuvant therapies based on modulation of the microbiota  <cit> .
the traditional probiotic approach based on lactic acid bacteria  strains can be now extended to new candidate beneficial bacteria from the intestinal microbiota, including f. prausnitzii.
this metabolic active species is a member of the c. leptum group, and is one of the most prevalent commensal bacteria in the human gut  <cit> , involved in colonic epithelial physiology  <cit> , and a reduction in its abundance has been observed in ibd patients  <cit> .
the anti-inflammatory properties of the f. prausnitzii reference strain a2- <dig> have been demonstrated in vitro in various cellular models and validated in vivo in murine models of tnbs-induced acute colitis  <cit>  and dnbs-induced chronic severe and moderate colitis  <cit> .
we investigated the beneficial effects of both f. prausnitzii and its filtered sn  using a chronic low-grade inflammatory model, which mimics gut dysfunction related to colonic low-grade inflammation  <cit> .
we adjusted the dose of dnbs such that the challenge induced low-grade inflammation, as it has been described for tnbs  <cit> , a model that resembles dnbs  <cit> .
to mimic the relapsing character of ibd and reach a remission status, we used a chronic protocol with recovery and reactivation periods.
our results show that chronic, low-dose administration of dnbs did not induce severe colonic inflammation: there was no macro- or microscopic damages or granulocyte infiltration.
furthermore, no increase in the levels of lipocalin  <dig> , an early marker of inflammation, was observed  <cit> .
lcn- <dig> is an innate immune protein produced by a variety of cells, and its levels are increased during several pathologic conditions, including immune complex-mediated inflammatory or autoimmune disorders  <cit> .
these various results indicate the absence of any substantial active inflammatory tone in this dnbs-treated mouse model.
however, the presence of a low-grade inflammation was confirmed by the small increases in some pro-inflammatory cytokine titers .
the levels of most of the cytokines investigated, mainly in serum, were low, with no statistically significant difference between groups.
nevertheless, serum il- <dig>  colonic ifn-γ and il- <dig>  and both serum and colonic il- <dig> and il- <dig> were higher in dnbs treated mice.
ifn-γ is a th <dig> cytokine and an important activator of macrophages; it is involved in the regulation of inflammation and in barrier dysfunction through the redistribution and expression of tj proteins  <cit> .
the pro-inflammatory th <dig> cytokine il- <dig> modulates the excitability of submucosal neurons and increases colonic secretory function.
the cytokine il- <dig> regulates various processes linked to several autoimmune disorders  <cit>  exerting both antimicrobial and pro-inflammatory functions.
il- <dig> is a th- <dig> cytokine, canonical of il- <dig>  <cit> , with stimulatory effects on b- and t-cells  <cit> ; as well as involvement in dichotomous regulation of th cell differentiation, il- <dig> induces so-called alternative macrophage activation  <cit> .
il- <dig> and il- <dig> can decrease cellular trans-epithelial electric resistance  in vitro and in vivo  <cit> .
our findings for the cytokines are difficult to interpret as they seem to involve both th- <dig> and th- <dig> immune responses as well as effects in the th-17/th- <dig> subset.
this is not surprising because the particular inflammatory environment of our model could be the consequence of cross-regulation between all these possible pathways  <cit> .
although the profile of cytokines after chronic low dose dnbs challenge is not completely characterized, the altered cytokine profiles we document here in dnbs-treated mice and untreated control mice confirm mild immunological dysfunctions in the model.
furthermore, both f. prausnitzii and sn treatments had beneficial effects on indicators of immune status.
as ibd patients in remission often exhibit gut dysfunction  <cit> , we therefore chose to study colonic permeability and serotonin levels as functional markers in our model.
treatment with either f. prausnitzzi or its sn normalized in vivo permeability, suggesting that overall permeability was improved.
sn also improved permeability in vitro suggesting that the sn acts to protect the paracellular pathway in dnbs-challenged mice.
this result is consistent with the findings by carlssonn et al.
that f. prausnitzii sn counteracted the negative effects of dss challenge on colonic permeability  <cit> .
increased permeability after reactivation was accompanied by a reduction in the expression of apical junction proteins.
treatment with either f. prausnitzii or its sn restored the levels of claudin- <dig> and the junctional adhesion molecule f11r .
overall, our results suggest that the protective effect of f. prausnitzii and its sn may be the consequence, at least in part, of an effect on tight junctions mediated through certain tight junction proteins.
other common biomarkers in gut dysfunction are related to gut dysmotility  <cit> .
although serotonin is not a direct marker of motility, it stimulates peristalsis, secretion, vasodilatation and sensory signaling in the gut  <cit> .
it also activates the immune cells to produce pro-inflammatory mediators  <cit>  being a link between inflammation and functional symptoms.
enterochromaffin cells  containing serotonin transduce chemical and mechanical stimuli from the intestinal lumen by releasing the serotonin  <cit> .
serotonin selective reuptake transporter , expressed on intestinal enterocytes, terminates the action of 5-ht by eliminating it from the interstitial space  <cit> .
dysfunctional mucosal serotonin signaling has been implicated in visceral sensitivity and altered motility in ibd patients  <cit> .
colonic levels of 5-ht were high in dnbs-challenged mice, both in colon tissue and content.
this result is in agreement with previous studies reporting altered levels of this hormone in murine models of tnbs-induced inflammation and low grade inflammation  <cit> .
both f. prausnitzii and its sn were able to restore the serotonin level to normal.
in t-cell-mediated models of inflammation, an increase in ec and a decrease sert function have been described  <cit> .
the cytokines ifn-γ and il- <dig>  are involved in the metabolism of 5-ht, ifn-γ decreases sert expression in human epithelial caco- <dig> cells in vitro  <cit> , and il- <dig> contributes to increasing ec numbers  <cit> .
in agreement with what has been described for tnbs  <cit> , we found lower sert levels in dnbs-challenged mice.
moreover, f. prausnitzii and its sn restored these levels to normal.
however, it is possible that the abundance of sert reflects the general status of the epithelium, and it is not necessarily a direct consequence of the actions of f prausnitizii or its sn.
indeed, the lybhi medium may have an effect on sert.
this is not the first time that culture media  have been reported to show intrinsic anti-inflammatory properties in vivo and in vitro  <cit> ; this is presumably due to some of the components of the medium, as previously observed with mrs culture medium  <cit> .
CONCLUSIONS
chronic low-grade inflammation is common in patients with ibd in clinical remission as well as in ibs patients  <cit> .
patients with histological inflammation at baseline are at increased risk of clinical relapse, hospitalization, surgery, and colon cancer in observational longitudinal studies  <cit> .
it is widely recognized that microbiota can impact on gut homeostasis.
our study proposes that f. prausnitzii, one of the major members of the gut microbiota, protects from gut dysfunction-related symptoms in animal models.
in summary, we describe novel protective effects of both f. prausnitzii and its sn on chronic low-grade inflammation, in a murine model mimicking the relapsing low-grade inflammatory conditions present in some ibd patients under remission.
this bacterium exerts different beneficial effects in terms of cytokine levels, gut permeability and serotonin pathway.
furthermore, our results suggest that the protective effect of f. prausnitzii and its sn may be the consequence, at least in part, of barrier enhancement through certain tight junction proteins.
these results provide further evidence of the potential of f. prausnitzii as a novel probiotic for gut-dysfunction management.
future studies will need to be performed to validate the described beneficial effect in patients gut dysfunction and low grade inflammation.
also, it will be important to determine whether disease associated dysbiosis is corrected, or whether the effect is independent of a modulation of the gut microbiota.

 the human gastrointestinal tract is one of the most complex ecosystems. the interaction between git microbiota and the host is indispensable for the maturation of the immune system and for the regulation of intestinal physiology. firmicutes and bacteroidetes are the two dominant phyla in the human gut, representing 90% of the microbiota, with actinobacteria being the third, comprising of only 3%. when the normal microbial ecosystem is disrupted, different non-predominant bacteria can bloom, constituting a potential trigger for disease. the term dysbiosis refers to a type of microbial imbalance that may promote disease. the strongest relationship between dysbiosis and pathological condition has been observed in inflammatory bowel diseases, where the proportion of firmicutes is lower than in healthy subjects. ibd, composed mainly of ulcerative colitis and crohn’s disease, are characterized by an unusual activation of the git-immune system leading to a chronic inflammation of the gut with a mix of remission and exacerbation periods. it has recently been suggested that the colonic microbiota is also abnormal in patients with irritable bowel syndrome, with a 2-fold increased ratio of the firmicutes to bacteroidetes. ibs is a symptom-based diagnosis characterized by chronic abdominal pain and altered bowel habits in the absence of detectable structural abnormalities. ibd and ibs are classically considered as dichotomous conditions. however, increasing evidence, such as the involvement of brain-gut axis dysfunction, as well as abnormal microbiota, supports some pathogenetic overlap between the two conditions. furthermore, the identification of low-grade inflammation, histopathological observations and mucosal cytokine levels in the colon of a subset of ibs patients and the presence of ibs-like symptoms in quiescent ibd support this hypothesis. low-grade inflammation can persist in many ibd patients who have otherwise obtained clinical remission, as well as barrier alterations due to functional impairment of tight junction proteins. in fact, impaired gut barrier function appears to precede the development of ibd and increased epithelial permeability causes mucosal immune activation predisposing or enhancing disease progression. due to the importance of the microbiota in the development of these dysbiosis-related diseases, there is currently increasing interest in the development of microbiota-modulating therapies, including the use of probiotics. f. prausnitzii is a candidate probiotic bacterium that has been proposed as a sensor of intestinal health. changes in the abundance of this commensal anti-inflammatory bacterium and major member of the clostridium leptum group have been linked to dysbiosis in several human disorders, such as ibd, where a low abundance was found in patients exhibiting endoscopic recurrence 6 months after surgery. furthermore, the recovery of f. prausnitzii counts after relapse is associated with maintenance of clinical remission in uc patients. besides, f. prausnitzii has shown protective effects in inflammation parameters in both acute and chronic models of chemical-induced active inflammation; however its beneficial role during low-grade inflammation and gut dysfunction has not been tested yet. for these reasons, we hypothesized a possible protective role of f. pausnitzii on gut dysfunction mediated by low-grade inflammation. therefore, we investigated the potential of f. prausnitzii as a probiotic for gut dysfunction prevention and therapy. we tested the effects of f. prausnitzii in a murine model of chronic low-grade inflammation mimicking some of the most common gut dysfunction parameters found in ibd patients under remission.  validation of a low-grade inflammation model in dnbs-treated mice the model of low-grade dnbs inflammation developed in this study involves the induction of a low grade, subclinical inflammatory status, followed by a recovery period and a reactivation period mimicking the relapsing nature in ibd. the challenge with a sub-colitic dnbs dose did not induce important changes in weight or colonic macroscopic or histological scores confirming the absence of a strong inflammation. the degree of infiltration by polymorphonuclear neutrophils, as measured by tissue mpo activity, as well as lipocalin-, an early inflammation marker in inflammatory and autoimmune disorders, did not differ significantly between dnbs-treated and control animals. these observations suggest that dnbs-treated mice tend to display a low-grade inflammatory status rather than a strong inflammation thereby validating our model for further analysis of physiological changes induced by this chronic low-grade, subclinical inflammation. the findings for all these markers thus suggest that the colons of dnbs-treated mice have a low-grade inflammatory status. comparison of the levels of these markers with those previously published by our group following a colitic dose of dnbs that induced severe or moderate chronic inflammation provided further evidence that this protocol leads to low-grade inflammatory conditions.figure experimental protocols for the mouse model of chronic micro-inflammation and absence of inflammation. a) colitis was induced by intrarectal administration of 100 mg/kg of dnbs in solution in 30% ethanol. control mice received only 30% etoh. the effects of dnbs are highest during the first 3 days after its administration. ten days after the end of the dnbs period, bacterial culture, pbs, or supernatant were intragastrically administered daily for 10 days. colitis was reactivated 21 days after the first dnbs injection with a second injection of 50 mg/kg of dnbs solution. three days after reactivation mice were sacrificed. the severity of the reactivated colitis was assessed from difference in body weight, macroscopic score, mpo activity, histological score and lipocalin- levels between control non-inflamed, control inflamed, bacteria-free culture medium, f. prausnitzii strain a2-, and f. prausnitzii a2- sn groups. experiments were performed at least in duplicate. normal appearance of the colon of a control mouse with no inflammation and of a mouse with micro-inflammation. comparison of inflammation measures in severe and moderate colitis models [ 25 ] with the micro-inflammation model employed in this study. severity of the colitis was assessed from differences in body weight, macroscopic score, histological score and mpo activity between the control non-inflamed, control inflamed severe colitis model ), moderate colitis model ) and micro-inflammation model ). experiments were performed at least in duplicate. comparisons involved the non-parametric kruskal-wallis test was used followed by a dunn’s multiple comparison test. *p <  vs. dnbs-pbs, +p < . faecalibacterium prausnitzii a2- and its sn counteract dnbs-induced hyperpermeability in vivo the gut of dnbs-treated mice showed high permeability to the paracellular tracer fitc-dextran in vivo. we tested whether f. prausnitzii modulates this dnbs-induced gut dysfunction. treatment with either f. prausnitzii or its sn resulted in a significant reduction of intestinal permeability. to confirm that these findings corresponded to local paracellular translocation of the fitc-dextran in vivo in this model, intestinal permeability was assessed in vitro in colonic samples mounted in ussing chambers extracted from mice at sacrifice. the results were consistent with the pattern observed in vivo confirming local translocation of the fitc-dextran. we then tested f. prausnitzii sn with samples from dnbs-challenged mice by ussing chamber analysis: the 51cr-edta flux, measured in % hot sample/hr/cm was lower with sn than pbs. no significant differences were obtained in tissue conductance.figure in vivo and in vitro permeability measurements. for in vivo measurements of gut permeability, animals were orally gavaged with fitc-dextran. for in vitro measures, sections of colon were mounted in ussing chambers and 51cr-edta flux and tissue conductance were measured. control non-inflamed, control inflamed, bacteria-free culture medium, f. prausnitzii strain a2-, and f. prausnitzii a2- sn groups. experiments were performed at least in duplicate. comparisons involved the non-parametric kruskal-wallis test was used followed by a dunn’s multiple comparison test. *p <  vs. dnbs-pbs, +p < . f. prausnitzii restores dnbs-induced apical junction proteins decrease these effects on gut permeability prompted us to investigate the involvement of epithelial adherens junction and tight junction proteins, by assaying for relevant mrna levels. the mrna levels for claudin-, e-cadherin, f11r, occludin and zo- were all less abundant in dnbs-treated mice than in control mice; claudin- and mrna levels were unaffected. f. prausnitzii and its sn tended to normalize the amounts of all these mrna transcripts, the only statistically significant regulation was found for claudin- and f11r. claudin- e-cadherin, f11r and occludin expression was also analyzed by immunofluorescence. staining for the four proteins was reduced in dnbs + pbs mice compared to etoh + pbs group. according to the qpcr results, f. prausnitzii tends to increase the expression of all the proteins.figure effect of f. prausnitzii on apical junction proteins mrnas in a dnbs-induced low-grade inflammation model. apical junction protein expression levels were determined by real-time qpcr. control non-inflamed, control inflamed, bacteria-free culture medium, f. prausnitzii strain a2-, and f. prausnitzii a2- sn groups. experiments were performed at least in duplicate. comparisons involved the non-parametric kruskal-wallis test was used followed by a dunn’s multiple comparison test. *p <  vs. dnbs-pbs, +p < . a systemic and mucosal reduction in pro-inflammatory cytokine production correlates with the protective effects of f. prausnitzii and its sn after dnbs challenge we investigated the cytokines involved in this low-grade inflammation model and the mechanism by which f. prausnitzii and its sn protect the intestinal mucosa. we assayed cytokines in both colonic and serum samples after the second dnbs injection. the concentrations of il- il-1α, il- il- il- il- il- ifn-γ, and tnf-α in colon samples were higher in dnbs-treated mice than in controls, consistent with local inflammation. similarly, cytokine concentrations in serum samples were consistent with the low-grade inflammatory status. strikingly, the concentration of il- ifn-γ and il- in colon samples and of il- and il- in serum was statistically significantly reduced by treatment with either f. prausnitzii or its sn and there were clear tendencies for these treatments to reduce colonic il- and serum il- concentrations.figure cytokine concentrations in colon and serum in the dnbs micro-inflammation model. control non-inflamed, control inflamed, bacteria-free culture medium, f. prausnitzii strain a2-, and f. prausnitzii a2- sn groups. experiments were performed at least in duplicate. comparisons involved the non-parametric kruskal-wallis test was used followed by a dunn’s multiple comparison test. *p <  vs. dnbs-pbs, +p < . f. prausnitzii and its sn restore serotonin variations observed in dnbs-treated mice serotonin of the key neurotransmitters in the git, and affects motility, sensation and secretion. we therefore studied serotonin concentrations in colon tissue and content by elisa. both colonic and colon content serotonin concentrations were higher in dnbs-treated mice than etoh-treated controls. treatments with both f. prausnitzii and its sn counteracted these increases. serotonin reuptake transporter mrna was assayed by qpcr in colon tissues: the value for inflamed mice was half than for control non-inflamed mice; the value for mice treated with f. prausnitzii was the same as that for normal healthy mice. similar positive effects were found in sn-treated mice although the culture medium itself had a positive effect.figure serotonin concentrations in colon and colon contents in the dnbs micro-inflammation model. control non-inflamed, control inflamed, bacteria-free culture medium, f. prausnitzii strain a2-, and f. prausnitzii a2- sn groups. experiments were performed at least in duplicate.comparisons involved the non-parametric kruskal-wallis test was used followed by a dunn’s multiple comparison test. *p <  vs. dnbs-pbs, +p < . discussion there is growing evidence pointing at the role of the gut microbiota in the development of ibd. furthermore, at least 40% of ibd patients in clinical remission have ongoing histological evidence of inflammation and suffer from altered gut functions with the involvement of brain-gut axis dysfunction. interestingly, previous transient or chronic inflammation can lead to persistent gut dysfunction and it has been suggested that the host microbiota may affect the two-ways communication pathway between the gut and the brain. because the microbiota is a determinant of normal gut function and immunity, and because there is evidence of dysbiosis in ibd, there is increasing interest in the development of new adjuvant therapies based on modulation of the microbiota. the traditional probiotic approach based on lactic acid bacteria strains can be now extended to new candidate beneficial bacteria from the intestinal microbiota, including f. prausnitzii. this metabolic active species is a member of the c. leptum group, and is one of the most prevalent commensal bacteria in the human gut, involved in colonic epithelial physiology, and a reduction in its abundance has been observed in ibd patients. the anti-inflammatory properties of the f. prausnitzii reference strain a2- have been demonstrated in vitro in various cellular models and validated in vivo in murine models of tnbs-induced acute colitis and dnbs-induced chronic severe and moderate colitis. we investigated the beneficial effects of both f. prausnitzii and its filtered sn using a chronic low-grade inflammatory model, which mimics gut dysfunction related to colonic low-grade inflammation. we adjusted the dose of dnbs such that the challenge induced low-grade inflammation, as it has been described for tnbs, a model that resembles dnbs. to mimic the relapsing character of ibd and reach a remission status, we used a chronic protocol with recovery and reactivation periods. our results show that chronic, low-dose administration of dnbs did not induce severe colonic inflammation: there was no macro- or microscopic damages or granulocyte infiltration. furthermore, no increase in the levels of lipocalin, an early marker of inflammation, was observed. lcn- is an innate immune protein produced by a variety of cells, and its levels are increased during several pathologic conditions, including immune complex-mediated inflammatory or autoimmune disorders. these various results indicate the absence of any substantial active inflammatory tone in this dnbs-treated mouse model. however, the presence of a low-grade inflammation was confirmed by the small increases in some pro-inflammatory cytokine titers. the levels of most of the cytokines investigated, mainly in serum, were low, with no statistically significant difference between groups. nevertheless, serum il- colonic ifn-γ and il- and both serum and colonic il- and il- were higher in dnbs treated mice. ifn-γ is a th cytokine and an important activator of macrophages; it is involved in the regulation of inflammation and in barrier dysfunction through the redistribution and expression of tj proteins. the pro-inflammatory th cytokine il- modulates the excitability of submucosal neurons and increases colonic secretory function. the cytokine il- regulates various processes linked to several autoimmune disorders exerting both antimicrobial and pro-inflammatory functions. il- is a th- cytokine, canonical of il-, with stimulatory effects on b- and t-cells; as well as involvement in dichotomous regulation of th cell differentiation, il- induces so-called alternative macrophage activation. il- and il- can decrease cellular trans-epithelial electric resistance in vitro and in vivo. our findings for the cytokines are difficult to interpret as they seem to involve both th- and th- immune responses as well as effects in the th-17/th- subset. this is not surprising because the particular inflammatory environment of our model could be the consequence of cross-regulation between all these possible pathways. although the profile of cytokines after chronic low dose dnbs challenge is not completely characterized, the altered cytokine profiles we document here in dnbs-treated mice and untreated control mice confirm mild immunological dysfunctions in the model. furthermore, both f. prausnitzii and sn treatments had beneficial effects on indicators of immune status. as ibd patients in remission often exhibit gut dysfunction, we therefore chose to study colonic permeability and serotonin levels as functional markers in our model. treatment with either f. prausnitzzi or its sn normalized in vivo permeability, suggesting that overall permeability was improved. sn also improved permeability in vitro suggesting that the sn acts to protect the paracellular pathway in dnbs-challenged mice. this result is consistent with the findings by carlssonn et al. that f. prausnitzii sn counteracted the negative effects of dss challenge on colonic permeability. increased permeability after reactivation was accompanied by a reduction in the expression of apical junction proteins. treatment with either f. prausnitzii or its sn restored the levels of claudin- and the junctional adhesion molecule f11r. overall, our results suggest that the protective effect of f. prausnitzii and its sn may be the consequence, at least in part, of an effect on tight junctions mediated through certain tight junction proteins. other common biomarkers in gut dysfunction are related to gut dysmotility. although serotonin is not a direct marker of motility, it stimulates peristalsis, secretion, vasodilatation and sensory signaling in the gut. it also activates the immune cells to produce pro-inflammatory mediators being a link between inflammation and functional symptoms. enterochromaffin cells containing serotonin transduce chemical and mechanical stimuli from the intestinal lumen by releasing the serotonin. serotonin selective reuptake transporter, expressed on intestinal enterocytes, terminates the action of 5-ht by eliminating it from the interstitial space. dysfunctional mucosal serotonin signaling has been implicated in visceral sensitivity and altered motility in ibd patients. colonic levels of 5-ht were high in dnbs-challenged mice, both in colon tissue and content. this result is in agreement with previous studies reporting altered levels of this hormone in murine models of tnbs-induced inflammation and low grade inflammation. both f. prausnitzii and its sn were able to restore the serotonin level to normal. in t-cell-mediated models of inflammation, an increase in ec and a decrease sert function have been described. the cytokines ifn-γ and il- are involved in the metabolism of 5-ht, ifn-γ decreases sert expression in human epithelial caco- cells in vitro, and il- contributes to increasing ec numbers. in agreement with what has been described for tnbs, we found lower sert levels in dnbs-challenged mice. moreover, f. prausnitzii and its sn restored these levels to normal. however, it is possible that the abundance of sert reflects the general status of the epithelium, and it is not necessarily a direct consequence of the actions of f prausnitizii or its sn. indeed, the lybhi medium may have an effect on sert. this is not the first time that culture media have been reported to show intrinsic anti-inflammatory properties in vivo and in vitro; this is presumably due to some of the components of the medium, as previously observed with mrs culture medium.  chronic low-grade inflammation is common in patients with ibd in clinical remission as well as in ibs patients. patients with histological inflammation at baseline are at increased risk of clinical relapse, hospitalization, surgery, and colon cancer in observational longitudinal studies. it is widely recognized that microbiota can impact on gut homeostasis. our study proposes that f. prausnitzii, one of the major members of the gut microbiota, protects from gut dysfunction-related symptoms in animal models. in summary, we describe novel protective effects of both f. prausnitzii and its sn on chronic low-grade inflammation, in a murine model mimicking the relapsing low-grade inflammatory conditions present in some ibd patients under remission. this bacterium exerts different beneficial effects in terms of cytokine levels, gut permeability and serotonin pathway. furthermore, our results suggest that the protective effect of f. prausnitzii and its sn may be the consequence, at least in part, of barrier enhancement through certain tight junction proteins. these results provide further evidence of the potential of f. prausnitzii as a novel probiotic for gut-dysfunction management. future studies will need to be performed to validate the described beneficial effect in patients gut dysfunction and low grade inflammation. also, it will be important to determine whether disease associated dysbiosis is corrected, or whether the effect is independent of a modulation of the gut microbiota. 
however, gut permeability, colonic serotonin levels and the colonic levels of the cytokines il- inf-γ, il- and il- were higher in dnbs-treated than in untreated mice. importantly, mice treated with either f. prausnitzii or its sn exhibited significant decreases in intestinal permeability, tissue cytokines and serotonin levels. the importance of this intestinal microbiota is highlighted when a disruption of the intestinal ecosystem equilibrium appears leading to an illness status, such as inflammatory bowel diseases. here, we report the beneficial effects of f. prausnitzii in the physiological changes induced by a chronic low-grade inflammation in a murine model. chronic low-grade inflammation and gut dysfunction were induced in mice by two episodes of dinitro-benzene sulfonic acid instillations. our results show that f. prausnitzii and its sn had beneficial effects on intestinal epithelial barrier impairment in a chronic low-grade inflammation model. these observations confirm the potential of this bacterium as a novel probiotic treatment in the management of gut dysfunction and low-grade inflammation. the effects of f. prausnitzii strain a2- and its culture supernatant were then investigated. no significant differences were observed in classical inflammation markers confirming that inflammation was subclinical. markers of inflammation, gut permeability, colonic serotonin and cytokine levels were studied. indeed, the reduction of the commensal bacterium faecalibacterium prausnitzii has been correlated with several diseases, including ibd, and most importantly, it has been shown that this bacterium has anti-inflammatory and protective effects in pre-clinical models of colitis.
 the human gut houses one of the most complex and abundant ecosystems composed of up to 1013- microorganisms. the importance of this intestinal microbiota is highlighted when a disruption of the intestinal ecosystem equilibrium appears leading to an illness status, such as inflammatory bowel diseases. indeed, the reduction of the commensal bacterium faecalibacterium prausnitzii has been correlated with several diseases, including ibd, and most importantly, it has been shown that this bacterium has anti-inflammatory and protective effects in pre-clinical models of colitis. some dysbiosis disorders are characterized by functional and physiological alterations. here, we report the beneficial effects of f. prausnitzii in the physiological changes induced by a chronic low-grade inflammation in a murine model. chronic low-grade inflammation and gut dysfunction were induced in mice by two episodes of dinitro-benzene sulfonic acid instillations. markers of inflammation, gut permeability, colonic serotonin and cytokine levels were studied. the effects of f. prausnitzii strain a2- and its culture supernatant were then investigated.  no significant differences were observed in classical inflammation markers confirming that inflammation was subclinical. however, gut permeability, colonic serotonin levels and the colonic levels of the cytokines il- inf-γ, il- and il- were higher in dnbs-treated than in untreated mice. importantly, mice treated with either f. prausnitzii or its sn exhibited significant decreases in intestinal permeability, tissue cytokines and serotonin levels.  our results show that f. prausnitzii and its sn had beneficial effects on intestinal epithelial barrier impairment in a chronic low-grade inflammation model. these observations confirm the potential of this bacterium as a novel probiotic treatment in the management of gut dysfunction and low-grade inflammation. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords microbiotadysbiosisibd-remissionlow-grade inflammationprobioticsissue-copyright-statement© the author 2015 