the human gastrointestinal mucosa constitutes the largest mucosal surface area in the human body interfacing the external environment .
a network of complementary regulatory interactions between different types of immune and nonimmune cells maintains mucosal homeostasis in the gut .
these regulatory interactions occur in the midst of a complex mixture of proteins , known as extracellular matrix ( ecm ) or stroma [ 1 , 2 ] , which together with soluble mediators such as cytokines and growth factors from mesenchymal cells , immune cells , and epithelial cells regulate cell activation and differentiation .
mesenchymal cells are actively involved in the inflammatory process in the gut and can perpetuate chronic gut inflammatory conditions like inflammatory bowel disease ( ibd ) [ 35 ] . transforming growth factor- ( tgf- )
 is one of the potential soluble mediators in homeostatic mechanisms in the gut that downregulates effector t - cell responses in the mucosa by reducing proliferation and interferon- ( ifn- ) production [ 4 , 6 ] .
however , tgf- together with il-6 and il-1 from the inflamed mucosa induces proinflammatory th17 cells , suggesting an innate regulatory function of the gut mucosal microenvironment [ 4 , 7 , 8 ] .
research is ongoing to elucidate the role of soluble factors and ecm in immunopathological mechanisms in ibd , but no such studies have so far been performed on microscopic colitis ( mc ) where the inflammation is subtler .
mc comprises two entities , collagenous colitis ( cc ) and lymphocytic colitis ( lc ) .
both conditions are characterized by chronic nonbloody , watery diarrhoea , often associated with abdominal pain and weight loss [ 911 ] .
the colonic mucosa is macroscopically normal or almost normal and the diagnosis relies on microscopic assessment of mucosal biopsies .
cc is presented with increased densities of lymphocytes and a thickened subepithelial collagen band ( 10  m ) adjacent to the basal membrane .
the pathophysiological data of cc are still limited , but it is postulated to be at least partially caused by disturbed immune responses to various luminal antigen(s ) , such as drugs , gluten , or infectious agents , in predisposed individuals .
nonsteroidal anti - inflammatory drugs ( nsaids ) , proton pump inhibitors , aspirin , and selective serotonin reuptake inhibitors have been associated with cc . in the majority of patients , however , no precipitating factor is found .
dysregulated myofibroblast function has also been implicated for collagen deposition in cc patients [ 10 , 13 ] .
recently , we reported on increased local activation of both cd4 and cd8 t cells in the lamina propria and epithelium of cc patients , demonstrated as increased expression of cd45ro and the proliferation marker ki67 , using flow cytometric analysis of freshly isolated lymphocytes from colonic biopsies .
in addition , mucosal transcript levels of ifn- , il-12 , il-1 , il-6 , il-17a , il-21 , il-22 , and il-23 are enhanced in the inflamed mucosa of cc patients compared to normal mucosa , together with elevated protein levels of il-6 , il-21 , and tnf . although the above findings suggest that the mucosal microenvironment is involved in cc immunopathology , the interplay between these factors and the proinflammatory activity of local mucosal t cells in cc patients has not been elucidated .
we therefore investigated the role of soluble factors from the intestinal mucosa of cc patients in the regulation of t cells using a novel in vitro model system with the aim of mimicking the in vivo exposure of newly recruited peripheral blood t cells to the soluble factors in the colonic milieu of inflamed cc and normal mucosa .
cc diagnosis was confirmed by clinical symptoms : 3 loose stools / day and/or abdominal pain and a macroscopically normal colonic mucosa with characteristic histopathological findings : increased numbers of lymphocytes in the epithelium and lamina propria with deposition of a 10  m thick subepithelial collagen layer .
patients with enteric infection , ischemic colitis , colonic cancer , or a previous history of crohn 's disease or ulcerative colitis were excluded .
we investigated colonic biopsies from 7 cc patients ( female ; n = 6 ) and 20 noninflamed controls ( female ; n = 11 ) without diarrhoeal symptoms , recruited among patients undergoing colonoscopy for examination of gastrointestinal bleeding or of abnormal radiological findings .
twelve biopsies from the hepatic flexure from each individual were obtained using standard biopsy forceps , placed in phosphate buffered saline ( pbs ) , and processed within 1 hour .
the colonoscopies were performed at the division of gastroenterology , rebro university hospital , sweden , between november 2012 and november 2013 . peripheral blood from healthy donors ( n = 6 ) was collected in heparin tubes for cd4 lymphocyte isolation as described below .
the study was approved by the regional ethical committee of rebro - uppsala county , sweden ( i d no .
we investigated the influence of two different preparations of the mucosa : one where the epithelium and intraepithelial cells were removed enzymatically , the denuded biopsies ( dnb ) , and one where collagenase was used to digest the lamina propria after the removal of the epithelium .
the dnb and the isolated mononuclear cells were cultured overnight , and the latter were termed lamina propria mononuclear cells ( lpmcs ) .
the cell populations are intact in the dnb fraction , whereas the lamina propria mononuclear cells ( lpmcs ) fraction is composed of a free leukocyte population as well as tissue , collagen , and cell debris .
twelve biopsies were thoroughly washed with pbs and incubated with prewarmed hank 's balanced salt solution ( hbss ) ( sigma aldrich , st .
louis , mo , usa ) containing 1  mm edta , 20  mm hepes , and 5% heat inactivated fetal bovine serum ( fbs ) at 37c , with constant stirring 4 times ( 15  min ) , to remove the epithelial layer .
six denuded biopsies were kept in serum - free rpmi-1640 containing 20  mm hepes , 100  g / ml streptomycin , 10  g / ml gentamycin , and 100 u / ml penicillin ( hereafter referred to as culture medium ) on ice until further use , and the remaining six biopsies were further digested with collagenase type viii and dnase i type iv ( sigma aldrich ) for 11.5  hrs to digest the collagen .
isolated lamina propria mononuclear cells were washed twice in pbs and resuspended in culture medium .
dnbs and lpmcs were cultured in culture medium overnight at 37c under 5% co2 - 95% air , to generate conditioned medium ( cm ) .
cm from the dnb and lpmc fractions from cc patients and noninflamed controls , respectively , were pooled .
endotoxin levels were quantified using the pierce lal chromogenic endotoxin quantitation kit ( pierce , rockford , il , usa ) according to the manufacturer 's protocol .
the maximum endotoxin level for dnb - cm and lpmc - cm was 4  eu / ml and 4.2  eu / ml , respectively .
total protein concentrations were determined using the bio - rad dc protein assay kit ( bio - rad , hercules , ca , usa ) .
dnb - cm was used at the total protein concentrations of 250 and 62.5  g / ml , whereas lpmc - cm was used at the total protein concentrations 125 and 62.5  g / ml , the highest possible concentrations of the respective conditioned media .
cm from the intestinal mucosa of noninflamed controls is referred to as ctrl - dnb - cm and ctrl - lpmc - cm , whereas cm derived from cc patients is referred to as cc - dnb - cm and cc - lpmc - cm .
cd4 peripheral blood lymphocytes were isolated from healthy donors using a human cd4 t - cell enrichment cocktail kit ( stemcell technologies , grenoble , france ) according to the manufacturer 's protocol .
the purity of cd4 t cells was 9095% as determined by flow cytometric analysis ( epics altra , beckman coulter , fullerton , ca , usa ) .
purified cd4 pbls were cultured in 96-well flat bottom assay plates ( sarstedt , newton , nc , usa ) precoated with 50  l of 5  g / ml anti - cd3 ( ucht1 , bd biosciences , san diego , ca , usa ) for 2  hrs at 37c , followed by two washes with pbs . 1  10 cd4 t cells were added to the washed wells together with 1  g / ml soluble anti - cd28 ( bd biosciences ) and were incubated in culture medium with the addition of 2  mm l - glutamine and 5% ab serum , with or without dnb - cm or lpmc - cm from noninflamed controls or cc patients in a total volume of 200  l . as controls , ctrl / cc - dnb - cm and ctrl / cc - lpmc - cm were cultured without cd4 t cells , and cd4 t cells alone were incubated without anti - cd3/anti - cd28 .
the assay was performed in duplicate wells for cytokine analysis and triplicate wells for the proliferation assay .
the cells were cultured at 37c under 5% co2 - 95% air for three days ; thereafter , supernatants were harvested and stored at 80c until determination of cytokine content , and t - cell proliferation was measured using the celltiter 96 aqueous one solution cell proliferation assay ( 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h - tetrazolium , promega , madison , wi , usa ) .
forty microliters of aqueous one solution reagent was added into each well and incubated at 37c for 4  hrs in a humidified , 5% co2 incubator followed by recording of the absorbance at 490  nm .
the pooled conditioned medium from inflamed cc mucosa and controls as well as supernatants from peripheral cd4 t cells were analyzed for il-1 , il-4 , il-6 , il-10 , il-17a , ifn- , and tnf using the xmap technology developed by luminex  ( austin , tx , usa ) , using two milliplex map kits ( cat .
number hcyp2mag-62k ) , according to the manufacturer 's instructions ( millipore , ma , usa ) .
the assays were performed in duplicate and the levels of different cytokines were expressed as pg / ml , according to a standard curve with known amounts of each analyte ( millipore ) .
tgf- levels were determined by elisa , according to the manufacturer 's instructions ( bd biosciences , san diego , ca , usa ) .
cytokine amounts were calculated as follows : cytokine amounts released by peripheral t cells incubated with cm minus cytokine amounts in cm alone . for each blood donor , the cytokine amounts released by cd4 t cells incubated with cm from cc patients
were compared with the cytokine amounts released by cd4 t cells incubated with cm from controls . as the data obtained were not normally distributed , wilcoxon 's signed rank nonparametrical test was used for statistical comparison between groups .
to mimic in vitro the exposure of peripheral t lymphocytes that have newly arrived into the colonic mucosa and to determine whether the local intestinal milieu affects t - cell activation and differentiation , we investigated t - cell cytokine production by -cd3 plus -cd28 stimulated cd4 peripheral blood t cells in the presence of soluble factors from the intestinal mucosa .
there was significantly increased production of the proinflammatory cytokines ifn- , il-17a , il-6 , and il-1 in the presence of cm generated by culture of denuded biopsies ( dnb - cm ) from the colon of collagenous colitis patients , compared to dnb - cm from noninflamed controls ( figure 1 ) . this was evident with the lower protein concentration of cm tested for il-17a , il-6 , and il-1 and with the higher concentration of cm tested for ifn- production .
we also noted a significantly increased production of the anti - inflammatory cytokines il-4 and il-10 in the presence of dnb - cm from cc patients compared to noninflamed controls , with the lower protein concentration of cm ( figure 1 ) .
in contrast , no significant differences were noted for tgf- production by peripheral cd4 t cells in the presence of dnb - cm from cc patients compared to noninflamed controls ( data not shown ) . in general ,
lpmc - cm had less impact on peripheral cd4 t - cell activation and differentiation .
a trend towards increased production of il-17a and il-10 ( both p = 0.06 ) was noted in the presence of lpmc - cm from cc patients compared to noninflamed controls in the lower protein concentration ( figure 2 ) .
we next investigated the ability of soluble factors from the colonic mucosa to inhibit t - cell proliferation , as this has previously been demonstrated in vitro [ 4 , 16 , 17 ] .
a tendency towards reduced proliferation inhibition of peripheral cd4 t cells was noted in the presence of cm from culture of denuded biopsies ( dnb - cm ) ( p = 0.06 ) from inflamed cc patients compared to noninflamed controls ( figure 3 ) . this was evident with both total protein concentrations of dnb - cm tested for peripheral t - cell proliferation .
in contrast , no differences in proliferation inhibition were observed in the presence of lpmc - cm from noninflamed controls compared to cc patients ( figure 3 ) . as t - cell differentiation and function
are regulated by different cytokines , we next analysed eight cytokines in pooled conditioned medium from dnb and lpmc fractions derived from inflamed cc mucosa compared to controls .
we found more than twofold and eightfold increased levels of il-6 and il-1 , respectively , in dnb - cm from cc patients compared to controls , whereas no alterations were found in the levels of ifn- , il-17a , tnf , il-4 , and il-10 ( table 1 ) or tgf- ( data not shown ) .
similar trends of increased levels of il-6 and il-1 were noted in lpmc - cm from cc patients compared to controls , though it was investigated in pooled cm from only two cc patients ( data not shown ) .
we here report on a novel in vitro model for analysis of the impact of the soluble factors from the colonic mucosa of cc patients on peripheral t lymphocyte activation and differentiation .
we found that despite the subtle inflammation in the mucosa of collagenous colitis patients , not visible by the naked eye upon colonoscopy , soluble factors in the mucosa are sufficient to significantly enhance the production of ifn- , il-17a , il-6 , il-1 , il-4 , and il-10 by peripheral cd4 t cells exposed to them in vitro .
this is the first study to investigate the role of soluble factors from the intestinal mucosa of cc patients in the regulation of t cells , where this novel system reflects the impact of in vivo exposure of newly recruited peripheral blood t cells to soluble factors in the colonic milieu of inflamed mucosa from cc patients compared to normal mucosa .
cm from denuded biopsies from inflamed cc mucosa induced increased production of both pro- and anti - inflammatory cytokines by peripheral t cells . as microscopic colitis
is subtler compared to ulcerative colitis and crohn 's disease , this may suggest that the colonic microenvironment in cc promotes production of anti - inflammatory cytokines to counterbalance inflammatory responses .
a study on the effects of stroma conditioned medium from crohn 's patients mucosa on cytokine production by t cells demonstrated increased ifn- and il-17 production but provided no data on the effect of anti - inflammatory cytokine production .
no differences were noted in tgf- production by peripheral cd4 t cells in the presence of cm from cc patients compared to controls . whereas tgf- in the normal mucosa likely suppresses t - cell function , the significantly increased amounts of il-6 and il-1 in the inflamed cc mucosa instead
likely promote differentiation of proinflammatory th17 cells [ 7 , 18 ] producing large amounts of il-17a .
the colonic milieu from cc patients might also promote differentiation of peripheral cd4 t cells into il-17/ifn- double producing th17/th1 cells that have been suggested to mediate gut inflammatory processes [ 1921 ] , corroborating our findings of enhanced levels of both il-17a and ifn-. we found a trend towards reduced inhibition of t - cell proliferation by soluble factors from denuded biopsies from the colonic mucosa of cc patients compared to controls .
older studies have demonstrated that the mucosal microenvironment reduces the proliferative responses of lamina propria lymphocytes to antigen receptor stimulation [ 17 , 22 ] but they are still active in their helper and cytolytic functions [ 22 , 23 ] .
the present study together with the study by huff et al .   indicates that these effects are at least partly imprinted in the t lymphocytes by the local milieu , rather than an intrinsic characteristic .
in contrast to cm from denuded biopsies , lamina propria mononuclear cell- ( lpmc- ) cm from cc patients did not affect t - cell proliferation compared to lpmc - cm from controls .
these different effects of the two types of cm are unclear and further experiments need to be performed to elucidate the differences in the composition of the cms . despite our observed enhanced production of il-10 by peripheral t cells in the presence of cm from cc patients , known to inhibit both t - cell proliferation and cytokine production , we found neither reduced proliferation of peripheral t cells nor production of proinflammatory cytokines .
this indicates that other immunoregulatory molecules drive synthesis of these proinflammatory cytokines in collagenous colitis .
the production of cytokines did not increase with higher total protein concentrations in the cm .
one explanation could be the presence of inhibiting and/or toxic factors in the cm limiting the t - cell responses .
in addition , various molecules have different optimal concentrations for their function and high concentrations can limit their activity . to further elucidate this and explain the differences observed on cd4 t - cell differentiation we want to investigate a larger panel of cytokines and compare the protein profile between cm from cc patients and that from noninflamed controls and between dnb and lpmc fractions by proteomics . in conclusion
, we have set up an in vitro model for analysis of the impact of the soluble factors from the colonic mucosa of cc patients on peripheral t lymphocyte activation and cytokine production . despite the subtle inflammation in cc ,
our data demonstrate significant alterations in cytokine production by peripheral cd4 t cells in the presence of mucosa - derived soluble factors from cc patients compared to controls .
one of our future goals is to test this in vitro model on differentiation of cd8 t cells , as we have previously reported on their increased numbers in the colonic mucosa of cc patients .
we also want to evaluate its use in evaluating the effect of drugs , including those in present use , on the colonic mucosal milieu and the lymphocytes there within , thereby facilitating the decision on optimum molecules as well as doses required for suppression of t - cell inflammatory responses .
 soluble factors from intestinal mucosal cells contribute to immune homeostasis in the gut . 
 we have established an in vitro model to investigate the regulatory role of soluble factors from inflamed intestinal mucosa of collagenous colitis ( cc ) patients in the differentiation of t cells . 
 peripheral blood cd4 + t cells from healthy donors were polyclonally activated in the presence of conditioned medium ( cm ) generated from denuded biopsies ( dnb ) or isolated lamina propria mononuclear cells ( lpmcs ) from mucosal biopsies from cc patients compared to noninflamed controls , to determine proliferation and secretion of cytokines involved in t - cell differentiation . compared to controls , we observed significantly increased production of the proinflammatory cytokines ifn- , il-17a , il-6 , and il-1 and the anti - inflammatory cytokines il-4 and il-10 in the presence of cc - dnb - cm . the most pronounced effect of cc - lpmc - cm on peripheral cd4 + t cells was a trend towards increased production of il-17a and il-10 . 
 a trend towards reduced inhibition of t - cell proliferation was noted in the presence of cc - dnb - cm . in conclusion , 
 our in vitro model reveals implications of soluble factors from cc colonic mucosa on peripheral t cells , enhancing their production of both pro- and anti - inflammatory cytokines . 