crohn 's disease ( cd ) is a form of inflammatory bowel disease ( ibd ) affecting the entire gastrointestinal ( gi ) tract [ 14 ] .
cd is characterized by transmural inflammation of gi wall in a noncontiguous pattern anywhere from the mouth to the anus [ 5 , 6 ] .
it is believed that , in genetically predisposed individuals , interaction between local microbiota and the intestinal mucosa can cause chronic inflammation and ulceration [ 79 ] .
a key pathological feature of cd is a granulomatous inflammatory response , characterized by the presence of epithelioid and multinucleated giant cells as well as macrophage infiltration [ 10 , 11 ] .
it is believed that the onset and development of the inflammatory reaction depend on persistence of an inciting agent , resulting in a chronic inflammatory milieu and perpetuating a complex immune reaction subsequent to the local granulomatous response .
persistent inflammation and immune activation are believed to be the leading causes of tissue necrosis and fibrosis .
newly diagnosed cd usually presents with diarrhea , abdominal pain , fever , fatigue , stomatitis , and weight loss . in more advanced cases ,
strictures and fistulas may develop , changing the clinical presentation to include severe abdominal pain , distension , bloating , vomiting , perianal fistulae , and abscesses .
cd can be diagnosed at any age ; however disease progression and clinical presentation frequently differ in young and adult patients .
cd in children and adolescents tends to have a more severe clinical manifestation often with development of strictures or fistulae [ 1 , 3 , 14 ] .
in contrast , later onset of cd is often characterized by a milder course with a lower frequency of complications [ 13 , 14 ] .
furthermore , it has been shown that the childhood - onset cd is more likely to require immunosuppressive therapy and has a higher frequency of surgery as compared to cd diagnosed in adulthood [ 4 , 13 ] .
additionally , juvenile and adolescent onset cd has more frequent involvement of the small bowel , while adult cd is generally more likely to be colon - associated [ 1519 ] .
an altered immune response , together with genetic and environmental factors , may govern the timing of cd onset .
there is a higher frequency of cd family history in patients diagnosed before the age of 20 relative to those diagnosed later in life , suggesting that genetic predisposition plays a role in early onset [ 13 , 20 ] .
for example , it has been shown in adult cd that higher levels of serum antibody reactivity towards increasing amount of microbiota in small intestine correlate with a greater frequency of complications [ 21 , 22 ] .
a similar observation has been made in a pediatric cohort [ 23 , 24 ] .
furthermore , cd patients have been shown to have increased numbers of circulating th17 lymphocytes and upregulation of il17 transcriptional activity in intestinal mucosa .
this indicates that the progression of cd is characterized by an exacerbated th17 response , which may explain the continuing nature of the disease .
immune mechanisms are believed to play a role in the pathogenesis of adult and juvenile cd , with cytokines being essential to establish and maintain the immune response .
however , little is known about differences in serum cytokine profiles between juvenile and adult cd cases .
we have found that all cd patients have upregulated serum cxcl10 levels , regardless of the age of diagnosis and the stage of the disease . also , we have demonstrated upregulation of il-1b and il-6 in juvenile cd , with no changes detected in levels of gm - csf and ifn-. in contrast , the adult cd serum profile was characterized by upregulation of gm - csf and ifn- , as well as il-6 and il-1b , when compared to controls .
it has been shown that gm - csf and ifn- promote a th1 immune response , while il-6 is essential for activation of th17 lymphocytes [ 2628 ] .
therefore , this suggests that the mechanisms of pathogenesis differ in adult and juvenile cd patients .
clinical surplus serum samples from 64 patients diagnosed with cd were utilized in this study ; 12 juvenile patients
( 6 male , 6 female ; average age 14.0  2.1 ) hospitalized in the volga region federal medical research center of the ministry of public health ( nizhny novgorod , russia ) and 52 adult patients ( 26 male , 26 female , average age 35.1  13 ) hospitalized in the department of gastroenterology of republican clinical hospital ( kazan , russia ) . diagnosis of cd was established in all patients based on clinical presentation and was verified by upper gi endoscopy .
additionally , a small number of samples ( 6 ) were collected from juvenile patients during their period of clinical improvement .
age and gender matched control samples were collected from 10 juvenile ( 5 male , 5 female ) as well as from 23 adult ( 13 male , 10 female ) controls . all serum samples were stored at 80c until use .
the study was approved by the ethics committee of kazan state medical university ( protocol 23 ) and the ethics committee of volga region federal medical research center of the ministry of public health nizhny novgorod ( protocol 4 ) .
serum cytokine levels were analyzed using single - plex sets for il-1b , il-2 , il-5 , il-4 , il-6 , il-8 , il-10 , il-12p40 , gm - csf , ifn- , cxcl10 , and tnf- ( bio - rad , hercules , ca , usa ) following the manufacturer 's instructions .
serum aliquots ( 50  l ) were used for analysis , with a minimum of 50 beads acquired per analyte .
data collected was analyzed with masterplex ct control software and masterplex qt analysis software ( hitachi software , san bruno , ca , usa ) .
statistical analysis was performed using the statistica 7.0 software package ( statsoft , tulsa , ok , usa ) .
levels of 12 cytokines were analyzed in juvenile cd serum collected during both the acute stage and remission .
acute serum was characterized by upregulation of il-1b , il-6 , cxcl10 , and tnf- levels compared to controls ( table 1 ) .
in contrast , no changes in il-2 , il-5 , il-4 , il-10 , il-12p40 , gm - csf , and ifn- were found in acute juvenile patients compared to controls .
median values of il-8 in acute serum were upregulated ( 1415.30 [ 997.12654.4 ] versus 2777.00 [ 611.07382.5 ] ) ; however , differences were not statistically significant from controls . during remission , serum levels of cxcl10 ( p < 0.05 ) and il-1b (
p < 0.05 ) remained significantly upregulated in juvenile cd patients compared to controls ( table 1 ) .
the median value of il-1b during remission was higher than that in acute patients ( 832.75 [ 481.91016.0 ] versus 550.00 [ 192.0784.3 ] ) , suggesting an upward trend in cytokine production ( p = 0.067 ) . il-6 and tnf- serum levels
were significantly higher during the acute stage but then declined in remission and remained comparable to controls .
similarly , in acute stage patients , median values of il-8 were upregulated during remission ( 1415.30 [ 997.12654.4 ] versus 4  194.0 [ 411.97  220.9 ] ) ; however , differences were not statistically significant from controls .
several leukocyte surface markers were examined including cd3 ( lymphocytes ) , cd4 ( t - helper ( th ) lymphocytes ) , cd8 ( cytotoxic t lymphocytes ( ctl ) ) , cd16 ( natural killer ( nk ) cells ) , cd19 ( b lymphocytes ) , and cd56 ( nk cells ) ( table 2 ) . increased numbers of cd16+cd56
+ leukocytes were characteristic for acute stage juvenile cd compared to remission ( 0.26  0.08 versus 0.14  0.02  10/l ; p < 0.02 ) ( table 2 ) .
when the cd4/cd8 ratio was analyzed , two groups of acute stage patients could be identified having a ratio higher or lower than 2.0 . since cytokines play a central role in differentiation and proliferation of leukocyte cd4 and cd8 subsets , we analyzed serum cytokines in the cd4/cd8 subgroups of juvenile cd patients ( table 3 ) .
acute stage patients with a cd4/cd8 ratio above 2.0 were characterized by upregulated serum levels of il-1b , cxcl10 , tnf- , and il-10 . additionally , high cd4/cd8 ratio patients had a greater il-8 median value compared to those with a cd4/cd8 ratio below 2.0 ( 4161.50 [ 2956.08372.8 ] versus 1760.00 [ 257.46014.1 ] ) ; however , these differences were not significant .
interestingly , cxcl10 levels were significantly higher in juvenile cd patients with a cd4/cd8 ratio below 2.0 than those with a cd4/cd8 ratio above 2.0 .
serum cytokine profiles in adult cd patients depended on the stage ( acute or remission ) of the disease .
for example , a subset of serum cytokines , including il-1b , il-6 , gm - csf , ifn- , and cxcl10 , were upregulated in the acute stage of cd relative to controls ( table 4 ) and remained upregulated as in remission . however , serum levels of tnf- and il-10 , unchanged in acute stage the disease , were downregulated during remission ( table 4 ) .
it should be noted that the il-12 median value was 20-fold higher in adult cd patients in either acute stage or remission than controls , though not statistically significant .
although these differences were not significant , there was a strong trend towards upregulation in adult serum , both during the acute stage and the remission .
interestingly , in contrast , the serum level of il-12 in juveniles in both the acute stage and the remission did not differ from controls .
further , in serum of acute stage , adults with cd both gm - csf and ifn- were upregulated , whereas these cytokines remained unchanged in acute juvenile cd patients .
this strongly suggests that several cytokines , including gm - csf , ifn- , and il-12 , play a role in the pathogenesis of cd in adult patients .
cd is a chronic inflammatory bowel disease affecting the integrity and function of the gi tract .
the disease has a bimodal age distribution , with 2025% of cases diagnosed early in life ( childhood and adolescence ) , while 70% of cases are diagnosed in adulthood .
clinical symptoms of cd include abdominal pain , diarrhea , weight loss , and fever .
in addition to the common gi symptoms , juvenile cd is often associated with failure to thrive that can include retarded growth , malnutrition , pubertal delay , and bone demineralization [ 31 , 32 ] .
studies using animal models have shown that cd pathogenesis is closely associated with a th1 cytokine profile , while activation of the th2 cytokines is more prevalent in the pathogenesis of ulcerative colitis [ 33 , 34 ] .
activation of t cell - mediated immunity is linked to the development of epithelioid granulomas , which are pathognomonic to cd .
the serum cytokine profile of cd patients is also indicative of activation of a th1 and a th17 type immune response .
for example , upregulation of il-17a , il-17f , il-21 , il-22 , il-26 , and ccl20 has been demonstrated in cd cases pointing to activation of th17 type immunity .
increased serum levels of tnf- are a hallmark of cd suggesting a fundamental role for this cytokine in disease pathogenesis [ 3739 ] .
the role of cytokines in cd was supported by prehn et al . who demonstrated enhanced production of ifn- , the key cytokine for activation of a th1 type immune response , by mucosal t lymphocytes upon stimulation with tnf- .
therefore , these data suggest that cytokines play an important role in activating and maintaining the th1 and th17 type immune response , which is indicative of cd . although the role of cytokines in cd pathogenesis is well established , little is known about age differences in cytokine activation in cd patients .
juvenile cd is usually characterized by severe clinical manifestations , often including development of strictures or fistulae requiring surgical intervention , whereas adults frequently have a milder clinical presentation with a lower likelihood of developing complications [ 4 , 13 ] .
therefore , we sought to determine whether the cytokine profiles in juvenile and adult cd differed .
serum levels of 12 cytokines were evaluated during both the acute stage and the remission in juvenile and adult cd patients .
an interesting observation was that there was upregulation of cxcl10 cytokine in all patients regardless of age and stage of the disease .
although increased cxcl10 serum levels and enhanced expression of cxcl10 in inflammatory bowel tissue has previously been shown in adult cd , here we present the first evidence for upregulation of this chemokine in the serum from juvenile cd cases .
cxcl10 is a chemokine known to exclusively recruit activated t lymphocytes and nk cells [ 4143 ] .
additionally , it has been demonstrated that cxcl10 targets th1 lymphocytes [ 42 , 43 ] .
the essential role of cxcl10 in the gut lymphocyte infiltration and inflammation has been established by grip and janciauskiene .
these authors demonstrated that reduced plasma levels of cxcl10 correlate with decreased level of the prominent inflammation marker , c reactive protein . in another study , hyun et al . demonstrated that a reduction in cxcl10 serum levels impairs activation and reduces recruitment of th1 lymphocytes into the gut mucosa and lymphoid tissue
therefore , our data support the notion that upregulation of cxcl10 can facilitate th1 lymphocyte infiltration and perpetuate epithelial inflammation in the gut . furthermore , increased serum levels of cxcl10 were found in all patients regardless of the stage of the disease , suggesting chronic th1 lymphocyte activation , which is ongoing even during clinical remission .
an interesting observation regarding serum il-8 levels in juvenile and adult cd patients was that , although not significant , the serum level of il-8 was higher ( 1.9 and 2.9 times ) in acute and remission juvenile cd compared to controls . in contrast , serum il8 levels in adult cd remained similar to age - matched controls . il-8 is a pleotropic cytokine , known as the prototype neutrophil attractant , which also activates neutrophils by upregulating phagocytosis and respiratory burst .
since inflamed tissue is often infiltrated by activated neutrophils , this suggests a contribution of il-8 to both local and systemic inflammation . although high circulating il-8 is characteristic of inflammation , a lack of change in serum il-8 in some cases of gastroduodenitis has been documented .
further investigation demonstrated that although il-8 levels in serum were unchanged , there were increased levels of il-8 transcripts within intestinal tissue [ 50 , 51 ] , suggesting local production of il-8 .
therefore , this suggests that lack of changes in il-8 in serum may not always reflect ongoing in situ inflammation .
therefore , we propose that in juvenile cd that high serum il-8 may reflect systemic inflammation , whereas , in adult cd , the lack of changes in circulating il-8 level indicates that inflammation could be restricted to bowel tissue .
another finding was that the serum level of tnf- was significantly upregulated in juvenile cd compared to controls , while it remained unchanged in adult cases .
these data suggest that juvenile cd , relative to adult , is characterized by a stronger inflammatory milieu in the gut . additionally , increased serum il-6 was detected all cd cases , regardless of patient 's age , relative to controls .
il-6 is a b cell differentiating factor   that , when combined with tgf , promotes the differentiation of th17 lymphocytes .
moreover , il-6 is considered an essential cytokine for th17 differentiation of naive t cells .
th17 lymphocytes are believed to be pathogenic and are often associated with the severe tissue damage and development of autoimmunity .
the mechanisms of th17-associated tissue damage are linked to a breach in the integrity of the intestinal epithelium due to increased production of inflammatory cytokines and matrix metalloproteinases , as well as inhibition of proliferation of human gut epithelial cells [ 56 , 57 ] .
interestingly , juvenile cd is characterized by upregulation of the il-6 , while levels of ifn- were unaffected .
however , the opposite pattern of cytokine activation was found in adult cd , where only ifn- levels were significantly upregulated .
ifn- is secreted by ctl and often used as a marker of the th1 type immune response .
the th1 ( il12-ifn- ) and th17 ( il-6-il-17 ) pathways seem to be mutually exclusive , with ifn- and il-17 acting as reciprocal inhibitory cytokines .
however , a subpopulation of th17 lymphocytes producing il-17 and ifn- was identified in cd gut tissue by annunziato et al . .
the authors suggested that il-17/ifn- positive th17 are important for cd pathogenesis , as these cells were able to support b cell proliferation , had low cytotoxic activity , and were poorly responsive to autologous regulatory t cells .
therefore , we hypothesize that the th17 response is characteristic for juvenile and adult cd .
however , upregulation of il-17 and ifn- in adult cd suggests that there are more complex mechanisms of pathogenesis relative to juvenile cd .
we propose that the th17 ( il-17/ifn- ) , or a combination of th17 and th1 immune responses , play a leading role in pathogenesis of adult cd .
studies using tissue biopsies from juvenile and adult cd will help to better understand the role of these th subsets in cd pathogenesis .
it has been suggested that gm - csf can alter the course of intestinal inflammation .
have shown that gm - csf activated macrophages protect mice from t cell - induced colitis .
these authors suggested that gm - csf - activated macrophages facilitate differentiation of th2 lymphocytes by upregulating production of il-4 , il-10 , and il-13 in lamina propria resident leukocytes , which may decrease local inflammation , improve bacterial clearance , and promote healing . in another study ,
have shown that the presence of anti - gm - csf antibody can serve as marker for the severity of cd .
further , these authors demonstrated that elevated anti - gm - csf antibody levels were associated with intestinal stricture , penetration , and resection in cd patients .
a similar observation was published by gathungu et al . , where cd patients with elevated anti - gm - csf antibody exhibited increased bowel permeability as compared to those with lower levels of this antibody .
it has been suggested that gm - csf neutralization promotes ileac disease by modulating expression of ccl25 by intestinal epithelium and ccr9 by t lymphocytes via nod2-dependent and -independent pathways .
therefore , increased gm - csf levels in adult cd may contribute to inhibition of bowel inflammation and prevention of tissue damage .
adult cd was characterized by upregulation of serum gm - csf and ifn- , a combination with strong anti - inflammatory potential .
for example , it has been shown that treatment of an lps challenge with gm - csf and ifn- inhibited an inflammatory response by lowering neutrophil counts and increasing the number of suppressive neutrophils ( cd16 ( bright)/cd62l ( dim ) )   in healthy volunteers .
the gm - csf / ifn- combination may have a profound effect on leukocyte polarization .
both cytokines play a key role in developing m1 type macrophages , which are associated with th1 type immune response .
. therefore , increased serum gm - csf and ifn- may reflect activation of th1 type immune response in adult cd .
our data suggest that juvenile and adult cd are characterized by unique serum cytokine profiles , which may reflect age - dependent difference in disease pathogenesis .
 crohn 's disease ( cd ) is a chronic inflammatory bowel disease that can be diagnosed at any age . 
 there are two major patient groups based on diagnosis of this disease , before or after the age of 20 ( juvenile / adolescent or adult ) , with disease progression in adults usually milder than in juvenile cd patients . 
 immune mechanisms have been suggested to play an important role in cd pathogenesis , with cytokines governing the development of the immune response . 
 upregulation of inflammatory cytokines in serum of juvenile and adult cd patients has been documented ; still little is known about age - dependent differences in serum cytokine profiles of cd patients . 
 we applied multiplex technology to analyze serum levels of 12 cytokines in juveniles and adults . 
 we show that during the acute stage of the disease all cd patients have high serum levels of cxcl10 , which remains upregulated during remission . increased serum levels of tnf- and il-6 during the acute stage was characteristic of juvenile cd patients , whereas adult cd patients had upregulated levels of gm - csf and ifn-. taken together 
 , these results demonstrate age - dependent differences in cytokine profiles , which may affect the pathogenesis of cd in patients at different ages of disease onset . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion