interleukin- ( il- ) 1 is a proinflammatory cytokine that drives an inflammatory response through il-1 receptor signaling . for example , il-1 is known to play an important role in the pathogenesis of metabolic inflammatory disorders . moreover il-1 induces expression of inflammatory cytokines , and il-1 signaling enhances differentiation into th17 cells [ 2 , 3 ] . thus , il-1 receptor antagonist ( il-1ra ) may be useful as an anti - inflammatory agent in inflammatory t cell - mediated diseases . additionally , il-1 is involved in the glycolysis pathway ; various studies have shown that il-1 is an important factor for upregulation of glucose uptake and glycolysis [ 4 , 5 ] . graft - versus - host disease ( gvhd ) , the leading cause of morbidity and mortality associated with an allogeneic hematopoietic cell transplant , is a complex illness involving dysregulation of inflammatory cytokine cascades and distortion of the donor 's cellular response to host alloantigens . activation of alloreactive donor t cells is initiated by host antigen - presenting cells ( apcs ) including dendritic cells . thus , t cells have been suggested as immunocompetent cells that cause gvhd , especially because th17 cells contribute to the development of gvhd . in addition , apcs play a significant role in the pathogenesis of gvhd ; evidence shows that inactivation of apcs alleviates gvhd [ 810 ] . th17 cells produce il-17 and can lead to an autoimmune disease by activating an inflammatory response and innate immunity . there is a general consensus that th17 cells control inflammation status and autoimmune diseases [ 11 , 12 ] . th17 cells are also involved in glucose and amino acid metabolism ; the latter processes require th17 cells , and hypoxia - induced factor-1-dependent glycolysis activates differentiation into th17 cells . blockade of il-1 signaling is an effective therapeutic strategy against inflammatory disorders ; it has been suggested that the inhibition of il-1 signaling suppresses inflammation , and il-1 antagonists are used as therapeutic agents in autoimmune diseases [ 1517 ] . nevertheless , there is a controversy regarding the therapeutic effects of il-1 antagonists in gvhd [ 18 , 19 ] . the aim of the present study was to determine the efficacy and mechanism of action of il-1ra treatment in acute gvhd . in this study , we performed in vivo and in vitro experiments to identify the effects and mechanisms of il-1ra activity during the development of acute gvhd in a mouse model . eight- to 10-week - old c57bl/6 ( h-2k , termed b6 ) and balb / c ( h-2k ) mice were purchased from orient bio ( sungnam , korea ) . the mice were maintained under specific pathogen - free ( spf ) conditions at an animal facility with controlled humidity ( 55  5% ) , light ( 12/12  h light / dark ) , and temperature ( 22  1c ) . the air at the facility was passed through a high - efficiency particulate arrestance ( hepa ) filter system designed to exclude bacteria and viruses . the protocols used in this study were approved by the animal care and use committee of the catholic university of korea . after lethal irradiation ( 800  cgy ) , recipient ( balb / c ) mice were injected intravenously ( i.v . ) with total bone marrow cells from donor mice . to induce acute gvhd , we isolated splenocytes from the donor mice , and then the splenocytes ( 1  10 ) from mhc major and minor antigen - disparate b6 donors were incubated with il-1ra ( anakinra , 50  ng / ml ) or with vehicle ( control ) for 2  h at 37c before adoptive transfer into the recipient mice . the clinical severity of gvhd was assessed twice a week using a scoring system consisting of five clinical parameters : weight loss , posture , activity , fur texture , and skin integrity . the mice were euthanized on day 14 after the bmt for blinded histopathological analysis of gvhd - affected organs ( skin and small intestine ) . the tissue slices were fixed in 10% buffered formalin and stained with hematoxylin and eosin ( h&e ) for histological examination . cd4 t cells were isolated from the spleen using cd4 t cell isolation kits according to the manufacturer 's instructions . these cells were stimulated with a plate - immobilized anti - cd3 antibody ( 0.5  g / ml ) and a soluble anti - cd28 antibody ( 1  g / ml ) for 72  h in 24-well plates . th17 cell development was induced by treatment with anti - interferon- ( ifn- )  ( 4  g / ml ) and anti - il-4 ( 4  g / ml ) antibodies , tgf- ( 2  ng / ml ) , and il-6 ( 20  ng / ml ) for 72  h. aliquots of 10 cd4 t cells ( responders ) were cultured with 10 irradiated ( 2500  cgy ) apcs in 96-well plates containing 200  l of the complete medium at 37c in a humidified atmosphere containing 5% co2 and were then pulsed with 1  ci of [ h]tdr for 18  h before harvesting and counted using an automated harvester . to analyze intracellular cytokines , we stained splenocytes with percp - conjugated anti - cd4 , apc - conjugated anti - cd25 , fitc - conjugated anti - il-17 , and pe - conjugated anti - foxp3 antibodies ( ebiosciences ) , followed by fixation and permeabilization using a foxp3 staining buffer kit ( bd bioscience ) . four hours before the staining , the cells were stimulated with phorbol myristate acetate ( 25  ng / ml ) and ionomycin ( 250  ng / ml ) ( all from sigma - aldrich ) and then treated with golgistop ( bd bioscience ) . all data were analyzed in the flowjo software ( tree star , ashland , or , usa ) . the mrna expression levels were estimated using a lightcycler 2.0 instrument ( roche diagnostic , mannheim , germany ) with version 4.0 software . all reactions were performed using the lightcyclerfaststart dna master sybr green i kit ( takara , shiga , japan ) . the concentrations of il-17 , ifn- , tgf- , and il-10 were measured using sandwich elisa ( r&d systems ) . serum levels of igg and igg1 antibodies were measured using a commercially available elisa kit ( bethyl laboratories , montgomery , tx , usa ) . the experimental data are presented as mean  sd of triplicate cell culture experiments and are representative of the three independent experiments ( not simultaneous ) . statistical significance was assessed using the mann - whitney u test or analysis of variance ( anova ) with bonferroni 's post hoc test using the graphpad prism software ( v.5.01 ) . total splenocytes from normal c57bl/6 mice were cultured with anti - cd28 and anti - cd3 antibodies in the presence or absence of il-1ra . this molecule inhibited differentiation into th17 cells in a dose - dependent manner ; il-17 concentration in the culture supernatant was significantly decreased by the il-1ra treatment ( figure 1(a ) ) . il-1ra also inhibited secretion of ifn- into the culture supernatant in the th0 condition . on the other hand , il-4 secretion into the culture medium il-1ra inhibited expression of il-17- and glycolysis - associated genes ( figure 1(b ) ) . on the other hand , il-1 treatment induced mrna expression of il-17 , rort , and genes involved in glycolysis such as hk2 ( figure 1(c ) ) . to determine whether il-1ra inhibits differentiation into th17 cells in vitro , we cultured the spleen cells under th17-polarizing conditions in the presence of il-1ra . differentiation into il-17-expressing cd4 t cells , mainly th17 cells , was suppressed significantly , whereas differentiation into cd4cd25foxp3treg cells was enhanced in a dose - dependent manner by il-1ra ( figure 1(d ) ) . in addition , il-1ra inhibited expression of the th17- and glycolysis - associated genes and of il-17 , il-21 , and rort genes ( figure 1(e ) ) . to determine whether blockade of il-1 signaling in t cells can inhibit differentiation into th17 cells , we cultured cd4 t cells ( that were isolated from the spleen ) under th17-polarizing conditions in the presence of il-1ra . il-1ra dose - dependently inhibited th17 cell development and promoted differentiation into treg cells in the culture of mouse splenocytes under th17 induction conditions ( figure 2(a ) ) . in addition , il-1ra inhibited the production of il-17 but enhanced tgf- production ( figure 2(b ) ) . the mrna levels of il-17 and glycolysis - associated genes were reduced dose - dependently by the il-1ra treatment ( figure 2(c ) ) . gene expression of glycolysis factors including triosephosphate isomerase ( tpi ) and lactate dehydrogenase a ( ldl ) was also decreased significantly by il-1ra treatment ( figure 2(d ) ) . these results suggested that the blockade of il-1 receptor drove the development of th17 cells and treg cells in opposite directions . to determine the impact of il-1 receptor blockade on the proliferative capacity of donor cd4 t cells in response to alloantigens , we measured t cell alloreactivity after treatment with il-1ra by means of [ h]thymidine incorporation . in contrast , treatment with il-1ra resulted in a potent dose - dependent inhibition of the proliferation of the alloreactive t cells ( figure 3(a ) ) . the elevation of ifn- and il-17 concentrations in the culture supernatant was also attenuated by the il-1ra treatment in a dose - dependent manner ( figure 3(b ) ) . in addition , the il-1ra treatment reduced the population of th1 cells and th17 cells in a dose - dependent manner ( figure 3(c ) ) . these data showed that il-1ra was an effective regulator of the alloreactive - cd4 t cell response . to test whether blockade of il-1 signaling has therapeutic effects on gvhd , a bmt was performed using splenocyte culture in the presence or absence of il-1ra . animals with acute gvhd who received a transplant of il-1ra - treated splenocytes showed a reduction of severity of acute gvhd and attenuation of weight loss , in comparison with the animals in the control gvhd group ( figure 4(a ) ) . in addition , we compared the histopathological features of gvhd - affected organs . in recipient mice that received a transplant of il-1ra - treated donor cells , the clinical severity of acute gvhd affecting the skin and small intestine was reduced . these findings were suggestive of decreased lymphocyte infiltration , inflammation , and fibrosis in comparison with control mice ( figure 4(b ) ) . to elucidate the in vivo mechanism of action of il-1ra in the murine model of acute gvhd , we used fluorescence - activated cell sorting ( facs ) to count the th1 , th2 , th17 , and treg cells in spleens isolated from each group of mice . the percentages of ifn--producing cd4 t cells and il-17-producing cd4 t cells in spleens were lower in il-1ra - treated gvhd animals than in the gvhd control group ( figure 5(a ) ) . in contrast , the numbers of il-4-producing cd4 t cells and cd4cd8foxp3treg cells were significantly higher in the il-1ra - treated group . to determine whether there was a change in b cell subpopulations after the il-1ra treatment , we analyzed splenocytes from the mice with acute gvhd . differentiation into mature b cells in gvhd was suppressed significantly in the il-1ra group compared to controls . on the other hand , differentiation into immature b cells was increased significantly in the gvhd il-1ra group compared to controls ( figure 5(c ) ) . concentrations of igg and igg1 decreased in the serum of il-1ra - treated animals compared to the gvhd control group ( figure 5(d ) ) . blockade of il-1 signaling can reduce inflammation , which makes il-1ra an important target of research in inflammatory diseases . nonetheless , the effects of il-1ra in gvhd are not clear [ 18 , 19 ] . however , il-1ra is involved in gvhd pathogenesis . indeed , il-1ra expression in saliva of gvhd patients recently , il-1 receptor deficiency in dendritic cells and t cells ameliorates acute gvhd enhancing survival . our study shows that il-1ra inhibits th17 cell development and the alloreactive t cell response through inhibition of the glycolysis pathway . additionally , we confirmed alleviation of severity and the immune response in gvhd by a transplant with il-1ra - treated splenocytes . this therapeutic effect and the apparent mechanism of the il-1ra treatment are the most substantial findings of our study . it is well known that th1 cells and th17 cells activate immunity and inflammation , whereas treg cells inhibit the development of th1 cells and th17 cells , thereby limiting redundant inflammatory responses . moreover , the th17/treg ratio plays an important role in gvhd . there is evidence that the th17/treg ratio in the peripheral blood of patients with gvhd is significantly higher in comparison with healthy controls , suggesting that the th17/treg ratio can be used as a sensitive and specific biomarker of gvhd . according to our data , il-1ra treatment reduces differentiation into th1 and th17 cells while inducing differentiation into treg cells . the th17 lineage has been recognized as the activator of proinflammatory responses ; in particular , th17 cells perform a pivotal function in inflammation in many autoimmune diseases including gvhd [ 26 , 27 ] . il-4 that is produced by th2 cells exerts a coordinated anti - inflammatory activity by inhibiting il-1 expression and by upregulating il-1ra [ 28 , 29 ] . in the present study , on the other hand , il-1ra inhibits differentiation into th1 cells and th17 cells while inducing th2 cell development in our mouse model of gvhd . thus , il - ra may be used to inhibit t cell - related inflammation and to enhance an anti - inflammatory response . gvhd is characterized by weight loss and selective damage to several organs including the skin and gastrointestinal tract . in gvhd patients , moreover , a skin biopsy is necessary for diagnosis of gvhd after an intestinal transplant . we demonstrate here that il - ra treatment suppresses weight loss and decreases tissue damage in a mouse model of gvhd . it has been suggested that expansion and development of alloreactive t cells contribute to the development of gvhd . inhibition of differentiation into alloreactive t cells suppresses preexisting gvhd ; for example , an inhibitor of proliferation of alloreactive t cells ( inducing apoptosis ) slows down the development of gvhd . our study shows that il-1ra inhibits the alloreactive t cell response and the production of ifn- and il-17 in vitro . glycolysis is known to be involved in th17 cell development . because suitable energy precursors and synthetic precursors are necessary for activation of t cells , during this process , glucose uptake and glycolysis are enhanced , as are amino acid transport and glutaminolysis [ 3540 ] . specific metabolic pathways are needed to activate different t cell subsets in order to utilize their unique activities in immunity and an inflammatory response . for example , lipid oxidation enhances treg cell development and reduces the activity and endurance of th17 cells . in addition , glucose uptake and expression of genes that are involved in glycolysis ( such as glut1 ) are induced in th17 cells compared to treg cells . it is also known that aerobic glycolysis in response to hypoxia induces differentiation into th17 cells , thus regulating the th17/treg balance . although il-1 blockade revealed nontherapeutic effect in gvhd patients , il-1 exacerbated the severity of gvhd in murine model . thus , clinical trial in gvhd patients will be needed to confirm the therapeutic effect of il-1 blockade . in the present study , il-1 stimulates th17 cell development by upregulating the glycolysis pathway . on the other hand , il-1ra suppresses differentiation into th17 cells , while upregulating treg cells through inhibition of the glycolysis pathway . the therapeutic properties of il-1ra can be explained by downregulation of th17 cells via inhibition of glycolysis . the observations pointing to the anti - inflammatory effects of il-1ra open up new possibilities with respect to treatment of gvhd . we believe that il-1ra induces treg cell development and downregulates the th17 cells , thereby reducing an inflammatory response through inhibition of the glycolysis pathway in th17 cells . this observational evidence demonstrates that il-1ra is a strong candidate for a new therapeutic agent against gvhd .
t helper ( th ) 17 cells are a subset of th cells expressing interleukin- ( il- ) 17 and initiating an inflammatory response in autoimmune diseases . 
 graft - versus - host disease ( gvhd ) is an immune inflammatory disease caused by interactions between the adaptive immunity of donor and recipient . 
 the th17 lineage exhibits proinflammatory activity and is believed to be a central player in gvhd . 
 il-1 performs a key function in immune responses and induces development of th17 cells . here 
 , we show that blockade of il-1 signaling suppresses th17 cell differentiation and alleviates gvhd severity . 
 we hypothesized that the il-1 receptor antagonist ( il-1ra ) would suppress th17 cell differentiation in vitro via inhibition of glycolysis - related genes . 
 blockade of il-1 using il-1ra downregulated th17 cell differentiation , an alloreactive t cell response , and expression of genes of the glycolysis pathway . 
 severity of gvhd was reduced in mice with a transplant of il - ra - treated cells , in comparison with control mice . to clarify the mechanisms via 
 which il-1ra exerts the therapeutic effect , we demonstrated in vivo that il-1ra decreased the proportion of th17 cells , increased the proportion of foxp3-expressing t regulatory ( treg ) cells , and inhibited expression of glycolysis - related genes and suppressed th17 cell development and b - cell activation . 
 these results suggest that blockade of il-1 signaling ameliorates gvhd via suppression of excessive t cell - related inflammation .