to investigate the immunologic events underlying the evolution of a chronic colitis we analyzed the chronic tnbs- colitis occurring in balb / c mice administered weekly doses of intra - rectal trinitrobenzene sulfonic acid ( tnbs ) ( 6 , 7 ) . mice treated in this way initially develop intense colitis associated with severe weight loss and considerable mortality . however , about three weeks after the initiation of this form of tnbs - colitis , the colitis moderates and , while the mice do not exhibit the weight gain of control mice , they regain their lost weight . importantly , the termination phase of this inflammatory cycle is accompanied by the development of fibrotic cycle . thus , four - five weeks after initiation of chronic tnbs - colitis , the mice develop steadily increasing fibrosis of the colonic lamina propria . as shown in figure 1 , this increase in intestinal fibrosis can be demonstrated by measuring the collagen content of the colon . to understand the immunologic basis of this complex series of events we determined the cytokines produced by lamina propria cells during the various stages of the inflammation . as established as an overview in figure 2 , we noted that the production of th1 cytokines , il-12p70 and ifn- by extracted cells were greatly elevated on day 7 after initiation of tnbs administration . however , surprisingly , the production of these cytokines gradually decreased over the next 2 weeks , returning to baseline levels by day 21 and then gradually declining to levels increasingly below that observed at baseline . interestingly , an increased production of an alternative set of pro - inflammatory cytokines , namely il-23 and il-17 , accompanied the decline in the production of il-12p70 and ifn-. increased il-23 production above baseline was noted first on day 21 , and thereafter increased further until it reached a plateau on day 42 . the expression levels of both cytokines increased further until they reached a plateau on day 42 . somewhat surprisingly , increased production of il-23 and il-17 coincided with increased production of cytokines usually considered a th2 cytokines , namely , il-25 and il-13 . previous studies have shown that il-25 , a member of the il-17 family of proteins ( and designated il-17e ) , is associated with expression of th2 cytokines , at least when it is over - expressed in transgenic mice ( 8) . on day 35 a massive increase in il-25 secretion appeared and thereafter il-25 secretion further increased , reaching its highest level on day 49 . at about the same time , the secretion of il-13 was initiated and the level of this cytokine also increased until it reached a plateau on day 49 . coming now to the molecular events during intestinal fibrogenesis , in previous studies we showed that il-13 induces tgf-1 production by signaling through cell - surface il-13r2 , an il-13 receptor formerly thought to function only as a soluble decoy receptor ( 912 ) . since il-13 is produced in the later phase of chronic tnbs - colitis we reasoned that this pathway might be involved in the development of fibrosis occurring during this phase . to explore this possibility we first determined the expression of both il-13r1 and il-13r2 in extracts of colonic lpmc during the course of chronic tnbs colitis . as shown in figure 3 , il-13r1 was constitutively expressed throughout the time course of the animal model . in contrast , il-13r2 was an induced receptor since it was not evident in nave mice or in the first few weeks of chronic tnbs - colitis , but could be detected initially at a low concentration on day 35 and later at a high concentration on days 42 and 49 after initiation of colitis . the above time course of il-13r2 expression correlated with that of il-13 and was thus consistent with the possibility that the latter was an inducing cytokine . to address this question more directly we next blocked il-13 activity by administration of a plasmid ( pci - sil-13r2-fc ) encoding a soluble il-13r2-fc fusion protein ( pci - sil-13r2-fc ) or a control plasmid ( pcl empty vector ) . the administration of the pcl - sil-13r2-fc plasmid by this route , but not the control plasmid , led to a reduction in lpmc production of il-13 . this effect is consistent with that noted previously in a study by chiaramonte et al where it was shown that mice with il-13r2 receptor deficiency have reduced il-13 production and is a possible result of the fact that autocrine il-13 signaling via this receptor is necessary for optimal il-13 production ( 9 ) . in addition , as shown in figure 4 , administration pci - sil-13r2-fc led to virtually complete loss of il-13r2 expression in extracts of colonic tissue obtained at the conclusion of the study ( day 49 ) . in contrast , neutralization of tgf-1 by administration of anti - tgf-1 had no effect on receptor expression or signaling ; this was expected since there is no evidence to suggest that tgf-1 induces up - regulation of il-13r2 . taken together , these results strongly suggest that il-13 production occurring in the late phase of chronic tnbs - colitis is indeed responsible for the expression of il-13r2 . in previous studies we have shown that intra - rectal administration of nf-b decoy oligonucleotides on days 35 and 42 abrogates the inflammation and the accompanying cytokine response characteristic of the chronic inflammation at this stage of the inflammation , including the high levels of il-23 , il-17 and il-13 ( 6 ) . in addition , such treatment prevented the production of tgf-1 and the development of intestinal tissue fibrosis . however , while these data established that the il-13 and tgf-1 responses as well as the collagen deposition were an outgrowth of the inflammation , they did not address the question of whether or not il-13 and/or tgf-1 were the cause of such deposition . to address the question of whether il-13 and tgf-1 are responsible for the collagen deposition , in the present study we determined the effect of in vivo inhibition of either il-13 signaling or tgf-1 activity on the generation of tissue fibrosis . in these studies we inhibited il-13 signaling in two ways : 1 ) by intra - nasal administration of the plasmid described above encoding a soluble il-13r2-fc fusion protein ( pci - sil-13r2-fc ) or a control plasmid ( pcl empty vector ) to block the receptor with the use of a decoy ; and 2 ) by intra - rectal administration of il-13r2-specific sirna encapsulated in hvj - e or a control sirna to down - regulate the receptor by gene silencing . both the sil-13r2-fc plasmid and the sirna were administered every other day starting on day 35 after the initiation of the chronic inflammation . on the other hand , for study of the effects of inhibition of tgf-1 we administered anti - tgf--antibody or isotype control ig by an intra - peritoneal route over the same time period . the effects of these treatments on il-13 and tgf-1 levels were subsequently determined at the termination of the study on day 49 . for this purpose , lpmc extracted from the colons of sacrificed mice were cultured for 48h with anti - cd3/anti - cd28 and their secretion of il-13 and tgf-1 into the culture supernatant measured by elisa . neutralization of il-13 signaling by fusion protein or gene - silencing of il-13r2 by sirna as well as neutralization of tgf-1 by antibody was accompanied by only a small , non - significant increase in body weight and no measurable change in colitis score . thus , lpmc extracted from the colons of sacrificed mice on day 49 and evaluated for secretion of cytokines secreted equal amounts of il-12p70 , ifn- , tnf- , il-23 , il-17 , il-25 and il-4 whether or not they were subjected to neutralization of il-13 signaling or tgf-1 . in contrast , a very different picture was obtained with respect to tgf-1 secretion . in this case , the cells extracted from the colons of mice subjected to neutralization of il-13 signaling by fusion protein or il-13r2 downregulation by sirna exhibited greatly reduced production of tgf-1 . in addition , as shown in figure 5 , those mice subjected to neutralization of il-13 signaling as well as those subjected to tgf-1 neutralization displayed basal collagen levels seen in nave mice whereas those mice not subjected to such neutralization displayed undiminished collagen levels as compared to other mice with chronic tnbs - colitis . overall , these data show quite clearly that neutralization of il-13 signaling via il-13r2 does not alter the course of the inflammation of chronic tnbs - colitis but does block the development of fibrosis ; in addition , they show that such signaling results in fibrosis via its effect on the induction of tgf-1 . in summary , these findings suggest that chronic inflammation is orchestrated by a succession of cytokines that ultimately result in il-13 and tgf-1 production . in addition , blockade of this cytokine interaction can prevent intestinal fibrosis by selectively interfering with the pro - fibrotic program .
to investigate the immunopathogenesis of inflammation - associated fibrosis we analyzed the chronic colitis and late - developing fibrosis occurring in balb / c mice administered weekly doses of intra - rectal trinitrobenzene sulfonic acid ( tnbs ) . 
 we showed first in this model that an initial th1 response involving il-12p70 and ifn- subsides after three weeks to be supplanted by an il-23/il-25 response beginning after 45 weeks . 
 this evolution is followed by gradually increasing production of il-17 and cytokines ordinarily seen in a th2 response , particularly il-13 , which reaches a plateau at 89 weeks . 
 we then show that il-13 production results in the induction of an il-13 receptor formerly thought to function only as a decoy receptor , il-13r2 , and this receptor is critical to the production of tgf-1 and the onset of fibrosis . 
 thus , if il-13 signaling through this receptor is blocked by administration of soluble il-13r2-fc , or by administration of il-13r2specific sirna , tgf-1 is not produced and fibrosis does not occur . 
 these studies show that in chronic tnbs colitis , fibrosis is dependent on the development of an il-13 response that acts through a novel cell - surface - expressed il-13 receptor to induce tgf-1 .