Many parasitic helminths induce highly polarized type 2 cytokine responses in their hosts and these responses control a variety of important functional activities including parasite expulsion (17
), eosinophil activation (57
), and development of fibrosis (11
). In schistosomiasis, the type 2 cytokine response is primarily directed at the parasite eggs (59
) and studies have shown that much of the egg-induced liver pathology is controlled by the relative dominance of type 1 versus type 2 cytokine expression (9
). Although it is clear that development of an egg-specific CD4+
Th2-type response is critical for normal granuloma formation (55
) and host survival after infection (60
), if these responses are too vigorous or persist for too long they can become detrimental and even lethal to the infected host (5
). Of the type 2 cytokines examined to date, IL-13 appears to be particularly important because studies show that mice deficient in this cytokine survive longer (12
) and fail to develop the fibrotic tissue pathology seen in chronic S. mansoni
). Therefore, to better understand disease pathogenesis, it is important to elucidate the mechanisms that control production of IL-13 and/or limit its various effector functions in vivo.
Although IL-4 and IL-13 share many functional activities, we hypothesized that the pathogenic effects of IL-13 are more marked than those of IL-4 because more IL-13 is produced during S. mansoni
). Indeed, our studies suggested that IL-13 production is nearly 100-fold greater than IL-4 at the site of granuloma formation. This may in part be explained by the kinetics of the response because IL-4 production is only transiently expressed after egg exposure whereas production of IL-13 is more rapid and sustained over a longer time period (42
). Thus, ligand availability is clearly one mechanism regulating the functional activities of these cytokines in vivo. Nevertheless, the pattern of IL-4/IL-13 receptor expression in the liver could be an equally important regulatory mechanism. Before this study, however, there was relatively little known about the IL-4–IL-13 receptor complex and whether expression of the four unique receptor subunits was regulated during infection.
Previous studies showed that IL-13 mRNA expression is induced rapidly after infection, with peak expression occurring in the liver on week 9 and remaining at relatively high levels through 16 wk after infection (7
). This work, examining the regulation of the IL-4Rα, γc, IL-13Rα1, and IL-13Rα2 genes, suggests a much more intriguing pattern of expression of the IL-4–IL-13 receptor complex in schistosomiasis. Although the γc and IL-13Rα1 chains showed little evidence of regulation, the signaling IL-4Rα chain and decoy IL-13Rα2 were highly regulated and displayed an opposite pattern of expression in the liver. IL-4Rα mRNA expression was high at early time points and showed little regulation before the onset of egg laying. However, by week 9, ~4 wk after egg laying commences, mRNA levels decreased markedly and remained at low but detectable levels through 16 wk after infection. In contrast, IL-13Rα2 mRNA was almost undetectable at early time points but was up-regulated after egg deposition. In situ hybridization for the three unique IL-4/IL-13–specific receptors showed significant expression of all three receptors throughout the liver of infected mice. IL-13Rα1 was expressed ubiquitously whereas the IL-4Rα chain was found at higher levels within the granuloma and the IL-13Rα2 localized at the periphery. Because IL-13Rα2 followed a pattern that closely mirrored the development of hepatic fibrosis, we examined both the regulation and functional activity of the decoy IL-13 receptor in more detail.
Induction of IL-13Rα2 expression during infection coincides with the emergence of the egg-specific type 2 cytokine response (59
). Therefore, we were initially interested in knowing whether type 1 or type 2 cytokines influence receptor up-regulation after infection. Experiments in several cytokine-deficient mice suggested that IL-10 and IL-13 are important mediators of IL-13Rα2 mRNA expression. The effects of IL-10, however, appear to be indirect because mice deficient in both IL-10 and IL-12 showed no reduction in IL-13Rα2 expression. This was in contrast to IL-10 and IL-10/IL-4–deficient mice, which displayed impaired receptor up-regulation. Because IL-10 and IL-10/IL-4–deficient mice develop strong IFN-γ responses, whereas IL-10/IL-12–deficient mice display polarized type 2 cytokine responses (39
), these data suggest that IL-10 regulates receptor expression by modulating the dominance of type 1/type 2 cytokine expression. This conclusion was strengthened by our immune deviation studies ( B) in which expression of the decoy receptor was clearly reduced in mice exposed to the Th1-inducing adjuvant IL-12. Moreover, immune deviation studies conducted in IFN-γ–deficient mice suggested that the suppressive effects of IL-12 were largely IFN-γ dependent.
Of even more interest, studies conducted in IL-13–deficient mice demonstrated a critical role for the ligand of the IL-13Rα2 in receptor expression. In contrast to IL-10, these experiments suggest IL-13 acts more directly because exogenous administration of rIL-13 alone rapidly induced IL-13Rα2 mRNA expression in the liver ( B). Levels of IL-13Rα2 also increased in the serum after infection and expression was clearly dependent on IL-4/IL-13 and Stat6-dependent signaling pathways ( C), further emphasizing the importance of type 2 cytokine responses for decoy receptor production. The finding that receptor levels were markedly reduced in IL-13–deficient mice ( A) yet not significantly altered in IL-4–deficient animals ( A) also suggests IL-13 might be the primary type 2 cytokine stimulating IL-13Rα2 expression. Thus, although it is conceivable that rIL-4 and rIL-13 ( B) would both exhibit IL-13Rα2–inducing activity, IL-13 is likely the more important endogenous inducer simply because it is overproduced relative to IL-4 during infection (11
In a recent paper examining the regulation of the IL-13Rα2 in human monocytes, Daines and Hershey (62
) showed that IL-13Rα2 is rapidly mobilized from intracellular stores to the cell surface after treatment with IFN-γ. The authors hypothesized that induction of IL-13Rα2 expression by IFN-γ might represent a novel mechanism to regulate IL-13 responses. Surprisingly, our data, gathered mostly from in vivo studies, fails to support this hypothesis, which might be explained by the different models being investigated. Alternatively, it may simply indicate that different regulatory mechanisms are exhibited by whole tissues (this study) versus cells stimulated in vitro (62
). Our findings demonstrate that the decoy receptor is up-regulated to the greatest extent during polarized Th2 responses. IL-13Rα2 expression was highly IL-13/IL-4–, IL-4R–, and Stat6-dependent. Except for the small twofold decrease observed in the livers of infected IFN-γ–deficient mice, there was no evidence that IFN-γ was required for receptor up-regulation. On the contrary, mice polarized to a Th1-dominant response via treatment with rIL-12 showed decreased IL-13Rα2 expression. This IL-12–induced inhibition of IL-13Rα2 was largely dependent on IFN-γ because in the liver IL-12 failed to reduce receptor expression in IFN-γ KO mice, further highlighting the down-regulatory (rather than up-regulatory) role of IFN-γ in receptor expression. As such, our in vivo data suggest a different regulatory mechanism for the IL-13Rα2 than that proposed by Daines and Hershey (62
). Instead of a cross-regulatory system where IFN-γ drives IL-13Rα2 expression to attenuate IL-13 activity, our studies suggest that IL-13Rα2 is a negative feedback mechanism induced by the Th2 response itself. Interestingly, given that this system would act in an antigen-nonspecific way to attenuate IL-13 activity, this mechanism of helminth-induced IL-13Rα2 expression could, at least in part, also provide an explanation for the hygiene hypothesis, which suggests that the increase in allergic disease observed in westernized countries is due to decreased exposure to childhood infections (63
). Our findings demonstrate that schistosome infection provides a potent stimulus for decoy receptor expression, which presumably could act as a bystander mechanism to neutralize the allergy-inducing potential of IL-13.
Although expression of the IL-13Rα2 correlated with development of fibrosis ( B), the role of the endogenous IL-13Rα2 remained hypothetical until KO studies could be conducted. In previous work, we showed that administration of sIL-13Rα2-Fc fusion protein was highly efficacious for the prevention of fibrotic liver pathology (11
). Moreover, recent studies demonstrated that IL-13 antagonism could also be used to block the progression of established disease (7
). As such, these findings suggest that a primary function of the IL-13Rα2 is to inhibit the profibrotic activity of IL-13. To directly test this hypothesis and evaluate the function of the endogenous IL-13Rα2, we infected mice with a targeted deletion of IL-13Rα2 (38
). Strikingly, hepatic fibrosis increased significantly in IL-13Rα2–deficient mice. Importantly, these data were reproducible in KO mice generated on two different genetic backgrounds, providing one of the first in vivo examples of functional decoy activity for the endogenous IL-13Rα2.
The pattern of liver fibrosis in the IL-13Rα2–deficient animals was also quite intriguing. Although fibrosis in WT mice was primarily restricted to the areas surrounding the developing granulomas, collagen staining in the KO mice extended beyond the areas of lesion formation and appeared to spread throughout the liver parenchyma itself. These data suggest that the protective function of the decoy receptor might extend to areas not directly affected by the parasite eggs. Nevertheless, the exacerbated fibrotic response appeared to be restricted to the liver because hydroxyproline assays performed on kidney, lung, and brain of infected IL-13Rα2–deficient mice showed no evidence of excess collagen deposition in these nongranulomatous tissues (unpublished data). These findings are important because nanogram quantities of IL-13 are detectable in the serum of infected mice ( B) and the absence of decoy receptor could trigger fibrogenesis in tissues not directly affected by the parasite eggs. In some S. mansoni
–infected humans, pipe stem fibrosis along the hepatic portal tracks is not necessarily associated with the egg-induced granulomas (64
). It is intriguing to speculate that if the IL-13Rα2 exhibits similar protective activity in humans, it might represent an important mechanism to prevent fibrosis at distant sites within the liver.
Markedly, the increase in hepatic fibrosis in IL-13Rα2–deficient mice on week 9 was not accompanied by any obvious change in the size nor cellular composition of the granulomas. Similar findings were also reported in infected IL-13Rα2-Fc–treated (11
) and IL-13–deficient mice (12
). In each of these studies, granulomatous inflammation was regulated by the combined actions of IL-4 and IL-13 whereas fibrosis was regulated primarily by IL-13. Thus, the primary function of IL-13 and its receptors appears to be the regulation of fibrosis. Hepatic fibrosis in schistosomiasis is also tightly controlled by the relative dominance of IFN-γ versus IL-13 produced locally in the liver (7
). Indeed, studies showed that mice that produce high levels of IL-13 and low levels of IFN-γ develop much more severe fibrosis than animals that have a high IFN-γ/IL-13 ratio (5
). Therefore, we were interested in knowing whether changes in type 1/type 2 cytokine dominance were contributing to the exacerbated fibrotic response. Strikingly, however, our findings suggested that type 2 cytokine expression was actually decreasing in the livers of the KO animals (). This conclusion was strengthened by analyzing serum levels of IL-13, which were also markedly reduced in the IL-13Rα2–deficient mice (). Finally, decreasing levels of egg-specific IgG1 Abs (the dominant Ab isotype expressed during infection) provided additional evidence of a general decline in type 2 cytokine expression (). Thus, fibrosis increases in the IL-13Rα2 KO mice despite the significant decrease in IL-13 levels. Importantly, expression of IL-13Rα1 and IL-4Rα mRNA chains did not differ in the WT and IL-13Rα2–deficient mice, which may explain why IL-13 remains active in both animals. These results suggest that even reduced levels of IL-13 are sufficient to generate significant fibrosis when IL-13Rα2 expression is low or absent. As such, these findings add additional weight to the importance of the IL-13Rα2 in the regulation of type 2 immune responses because they clearly demonstrate an enhancement of IL-13 activity in the absence of the decoy receptor. The results also suggest the decoy receptor has a much greater influence on the development of fibrosis than the relative level of IL-13.
The decrease in IL-13 expression in the KO mice was particularly surprising because it is clear from both blocking and KO studies that IL-13 is required for the development of hepatic fibrosis in schistosomiasis (11
). These findings suggest that a sensitive and highly regulated mechanism is in place to control production of IL-13, IL-13Rα2, and development of tissue fibrosis. This elegant system for controlling tissue pathology was best exemplified in the last series of experiments () in which IL-13Rα2 activity was restored in the KO mice by treating them with a sIL-13Rα2-Fc construct. Fibrosis more than doubled by 12 wk after infection in the IL-13Rα2–deficient mice whereas the fibrotic response was largely prevented in the group receiving sIL-13Rα2-Fc. In fact, the sIL-13Rα2-Fc construct reduced fibrosis in the KO animals by >70%, formally demonstrating that their exacerbated pattern of fibrosis was entirely due to the absence of IL-13Rα2 expression and heightened IL-13 activity. We observed that the impaired serum IL-13 response in the KO animals was restored and even increased after administration of sIL-13Rα2-Fc (). As such, these data suggest that as fibrosis increases in IL-13Rα2–deficient mice, IL-13 levels drop, perhaps to compensate for the worsening tissue response. Similarly, when fibrosis is reduced in the KO mice after sIL-13Rα-Fc treatment, levels of the profibrotic cytokine IL-13 increase in the serum. Alternatively, IL-13 may simply act as a negative regulator of its own production. It is also possible that serum levels of IL-13 increase after sIL-13Rα2-Fc treatment because the receptor is acting as a depot or carrier for the cytokine, essentially extending the in vivo half-life of IL-13 via a mechanism analogous to cytokine–anti–cytokine antibody complexes (38
). Regardless of the exact mechanisms involved, this reciprocal pattern of regulation suggests that a tight balance between IL-13 and IL-13Rα2 expression might be required for the establishment of an optimal healing response.
Th2-mediated inflammation plays a central role in the pathogenesis of a variety of other fibrotic disorders including asthma (29
), idiopathic pulmonary fibrosis (67
), chronic graft rejection (69
), bleomycin-induced pulmonary fibrosis (70
), progressive systemic sclerosis (71
), radiation-induced pulmonary fibrosis (72
), and hepatic fibrosis (9
). The studies presented here document the important protective function of the endogenous IL-13Rα2 and its role in the inhibition of IL-13–mediated tissue fibrosis. Moreover, these studies demonstrate that the decoy IL-13Rα2 can also regulate the magnitude of Th2-type cytokine production in vivo. As such, our findings illustrate a novel and previously unappreciated mechanism for limiting the pathogenesis of chronic Th2-mediated inflammatory responses.