Host regulation of liver fibroproliferative pathology during experimental schistosomiasis via interleukin-4 receptor alpha

Interleukin-4 receptor (IL-4Rα) is critical for the initiation of type-2 immune responses and implicated in the pathogenesis of experimental schistosomiasis. IL-4Rα mediated type-2 responses are critical for the control of pathology during acute schistosomiasis. However, type-2 responses tightly associate with fibrogranulomatous inflammation that drives host pathology during chronic schistosomiasis. To address such controversy on the role of IL-4Rα, we generated a novel inducible IL-4Rα-deficient mouse model that allows for temporal knockdown of il-4rα gene after oral administration of Tamoxifen. Interrupting IL-4Rα mediated signaling during the acute phase impaired the development of protective type-2 immune responses, leading to rapid weight loss and premature death, confirming a protective role of IL-4Rα during acute schistosomiasis. Conversely, IL-4Rα removal at the chronic phase of schistosomiasis ameliorated the pathological fibro-granulomatous pathology and reversed liver scarification without affecting the host fitness. This amelioration of the morbidity was accompanied by a reduced Th2 response and increased frequencies of FoxP3+ Tregs and CD1dhiCD5+ Bregs. Collectively, these data demonstrate that IL-4Rα mediated signaling has two opposing functions during experimental schistosomiasis depending on the stage of advancement of the disease and indicate that interrupting IL-4Rα mediated signaling is a viable therapeutic strategy to ameliorate liver fibroproliferative pathology in diseases like chronic schistosomiasis.


Introduction
Schistosomiasis is a parasitic disease caused by blood-dwelling parasitic flatworms of the genus Schistosoma, mainly, Schistosoma mansoni (S. mansoni), S. japonicum and S. haematobium that are infective to humans and the most clinically relevant [1]. Schistosomiasis is estimated to affect more than 200 million people worldwide and causes up to 200,000 deaths per annum in developing countries [1]. The disease is caused by parasite eggs trapped in the microvasculature of the host organs (liver, intestine and bladder) that induce a vigorous inflammatory response [1]. The kinetics of the ensuing immune responses induced by S. mansoni infection are well defined and characterized [2,3]. Briefly, the outcomes of disease persistence and progression are organ enlargement, fibrosis, scarring, portal hypertension or hematuria (S. haematobium specifically) that drive host morbidity and eventually death in severe cases [1].
The immune response to schistosomiasis, similarly to that against other tissue-dwelling helminth infections [4][5][6], is highly polarized as it progresses, going from i) an early Th1 response to ii) a powerful Th2 response that culminates as the adult parasite-released eggs are trapped in the host tissues [2,3] and finally iii) a chronic regulatory phase with a minimized but still dominant Th2 response [3,7,8] with a more clinically relevant tissue fibroproliferative pathology. Our current understanding of schistosomiasis pathology heavily relies on the use of experimental murine models [2]. Studies aimed at uncovering factors that drive host protection or susceptibility to schistosomiasis have been conducted using gene-deficient mice. The disease associates with the formation of granulomas and excessive collagen deposition (fibrosis) around tissue-trapped eggs [3,7,8]. An important role was defined for the host immune effector responses in these pathognomonic processes as nude mice [9], T cell-depleted [10][11][12][13] or mice with severe combined immunodeficiency [14] failed to form proper fibrogranulomatous responses. Even though Th1, Th17 and Treg responses have been shown to play major roles in regulating schistosomiasis pathogenesis, type 2 immune responses, which are typically induced by the disease-mediating eggs of the parasite [15][16][17], have been ascribed a more dominant role [3,7,8,18].
Initiation and polarization of type 2 immune responses is orchestrated by interleukin-4 (IL-4) and IL-13 signaling via a common IL-4Rα chain [2,19]. Signaling via this receptor drives the activation of the transcription factor STAT6 in hematopoietic cells, the proliferation of T and B cells, the production of immunoglobulins by B cells, the priming and chemotaxis of mast cells and basophils [2,19]. In non-hematopoietic cells, this receptor plays a central role in inducing airway hyper-responsiveness by enhancing contractions and mucus secretion by gut epithelial cells [20] and has been shown to play a role in STAT6-dependent fibroblast activation leading to collagen deposition that define fibro-proliferative diseases [21,22]. Understandably, mice deficient in this receptor show impaired granuloma formation, enhanced liver damage and augmented gut inflammation that leads to endotoxemia and septic shock during acute schistosomiasis [23][24][25][26]. Moreover, studies conducted in our laboratory have refined the requirement of IL-4Rα to a cell-specific level showing that IL-4Rα-responsive macrophages [25], pan-T cells [27] and smooth muscles cells [28] are individually essential for driving host survival and limiting tissue pathology during acute schistosomiasis.
In all these studies employing mice constitutively deficient in IL-4Rα, a critical role for IL-4Rα mediated signaling during acute [25,26] and chronic schistosomiasis [25,29,30] is suggested. However, the constitutive lack of IL-4Rα led such transgenic mice to succumb prematurely to experimental schistosomiasis with high (acute model) as well as low (chronic model) infection doses i.e. chronic model of infections succumb during the acute phase in the absence of IL-4Rα [30] casting an equivoque on the reliability of using models of constitutive deletion of IL-4Rα to assess the role of this receptor during chronic schistosomiasis. Moreover, congenital IL-4Rα deletion has now been shown to affect the development of animals [31], challenging our current knowledge on the role of IL-4Rα throughout experimental schistosomiasis (acute and chronic) using mouse models of constitutive IL-4Rα deficiency.
In this study, the role of IL-4Rα during acute and chronic schistosomiasis was investigated using a novel murine model that allows for inducible deletion of il-4rα gene at any time point during S. mansoni infection. Our findings further confirmed a protective role played by IL-4Rα mediated signaling during acute schistosomiasis. Contrastingly, we showed for the first time that partial deletion of the il-4rα gene, specifically, at the chronic stage of schistosomiasis ameliorates the tissue pathology by reducing type-2 immune responses, improving immune balance between T helper cytokines and skewing the diminished immune response towards a more regulatory profile without affecting animal viability.
Host survival requires IL-4Rα mediated signaling during acute but not chronic schistosomiasis A protective role for IL-4Rα mediated signaling has been established during acute schistosomiasis, where IL-4Rα deficient mice but not wild-type (wt) mice died around 6 to 8 weeks after natural infection with S. mansoni [25]. To determine whether IL-4Rα mediated signaling is required throughout the course of experimental schistosomiasis, IL-4Rα was knocked down in S. mansoni-infected RosaCreER T2-/+ IL-4Rα -/lox mice at the early acute (Tamoxifen administration at 2 weeks post-infection termed Tam2), late acute (Tamoxifen administration at 6 weeks post-infection termed Tam6) and chronic phase (Tamoxifen administration at 16 weeks postinfection termed Tam16) (Fig 3A), as previously defined [3]. As expected, most of IL-4Rα deficient mice (70%) succumbed prematurely to infection with 35 S. mansoni cercariae as early as from 7 weeks post-infection ( Fig 3B). Similarly, the viability of Tam2-and Tam6-fed Rosa-CreER T2-/+ IL-4Rα -/lox mice declined rapidly (60 and 50% respectively at week 8 post-infection). From Tam-2-fed, Tam-6 fed or IL-4Rα deficient mice, no death was further reported as from 12 weeks post-infection, at the chronic phase of the disease. This indicated that IL-4Rα is necessary for host survival during acute schistosomiasis, but not required for host survival at the chronic phase of the disease. Indeed, removal of IL-4Rα in Tam16-fed S. mansoni-infected RosaCreER T2-/+ IL-4Rα -/lox mice failed to affect the morbidity (as indicated by serum levels of alanine transaminase as a marker of liver disease (S3 Fig) and the mortality ( Fig 3B) up to 24 weeks post-infection, further supporting a dispensable role of IL-4Rα mediated signaling during chronic schistosomiasis. Taken together our results suggest that IL-4Rα mediated signaling differentially regulates schistosomiasis disease depending on the stage of the infection.
Early IL-4Rα knockdown before egg production (week 2) exacerbates acute schistosomiasis The findings above demonstrated an impaired viability of S. mansoni-infected mice following IL-4Rα knockdown at 2 weeks post-infection (Tam2). Hence, the immune and histopathological response of Tam2-fed RosaCreER T2-/+ IL-4Rα -/lox mice (termed iCre -/+ IL-4Rα -/lox Tam2, Fig  4A), which might associate with the host premature death during experimental schistosomiasis was dissected. This impaired IL-4 production was confirmed within the supernatant of anti-CD3-stimulated MLN cells from S. mansoni-infected i Cre-/+ IL-4Rα -/lox Tam2 animals by ELISA where a greatly diminished production of other type-2 cytokines as well, i.e. IL-13, IL-5 and IL-10, amid rather minimally altered IFNγ responses (Fig 4E) was observed. Subsequently, Type 2 antibody responses (IgG1 and total IgE) appeared markedly reduced, whereas Type 1 antibodies (IgG2a) were similar to control mice (Fig 4F-4H). However, liver egg burden was similar between the different groups ( Fig 4I), ruling out a differential level of infection as the cause of the observed diminished type-2 responses in i Cre-/+ IL-4Rα -/lox Tam2 and IL-4Rα -/mice. Together, these results demonstrate that knocking down IL-4Rα at the early acute phase of experimental schistosomiasis considerably diminishes host ability to subsequently mount a type-2 immune response. Liver granuloma size (Fig 4J and 4K) and fibrosis (Fig 4L and 4M) were reduced in S. mansoni-infected i Cre-/+ IL-4Rα -/lox Tam2 similar to global IL-4Rα -/mice, translating into a significantly reduced level of hepato-( Fig 4N) and splenomegaly ( Fig 4O) compared with IL-4Rα-responsive control mice. As expected, from our previous mortality studies [25,27], body weights of S. mansoni-infected IL-4Rα -/and i Cre-/+ IL-4Rα -/lox Tam2 mice rapidly declined starting 6 weeks post-infection ( Fig 4P) that preceded the death of these animals ( Fig 4Q) when compared to IL-4Rα-responsive control mice. Bleeding was visible in the gut of the animals that rapidly succumbed to infection following removal of IL-4Rα. Taken together, these results suggest that IL-4Rα knockdown at the early acute phase of experimental schistosomiasis considerably diminishes the host ability to mount a protective fibro-granulomatous response around the S. mansoni eggs and this was associated with gut bleeding, rapid weight loss and premature death.
IL-4Rα knockdown after egg production (week 6) exacerbates acute schistosomiasis As impaired viability of S. mansoni-infected mice following IL-4Rα knockdown at 6 weeks post-infection (Tam6) was observed (Fig 3B), the associated immune and histopathological responses of Tam6-fed RosaCreER T2-/+ IL-4Rα -/lox mice (termed i Cre-/+ IL-4Rα -/lox Tam6, Fig  5A) (Fig 5B and 5C), whereas IFN-γ responses were similar compared to IL-4Rα -/lox and IL-4Rα +/+ control mice (Fig 5B and 5D). This suggests an impairment of type 2 immune responses in S. mansoni-infected i Cre-/+ IL-4Rα -/lox Tam6 animals as confirmed by the drastic reduction of IL-4-producing CD4 + T cell numbers in the MLN (~83%, S5E Fig), that paralleled a significant but less important reduction of IFNγ-producing CD4 + T cell numbers (~60%, S5F Fig). This reduction of IL-4 production was confirmed by anti-CD3-stimulated MLN cells from S. mansoni-infected i Cre-/+ IL-4Rα -/lox Tam6 mice and analysis of the released cytokines by ELISA ( Fig 5E). Consistently, we observed a significant decrease in the production of type 2 cytokines, i.e. IL-4 and IL-10, but minimally altered IFN-γ responses ( Fig 5E). As a result of reduced IL-4, type 2 antibody responses (IgG1 and total IgE) were markedly reduced (Fig 5F and 5G), whereas type 1 antibodies (IgG2a) were similar to control mice ( Fig 5H). Liver egg burden was similar between the different groups ( Fig 5I), ruling out a differential level of infection as the cause of the observed diminished type 2 responses in i Cre-/+ IL-4Rα -/lox Tam6 and IL-4Rα -/mice. Together, these results demonstrate that knock down of IL-4Rα after egg deposition does diminish host ability to maintain the type 2 immune responses. Reduced type 2 responses decreases pathological features, including liver granuloma size (Fig 5J and 5K) and fibrosis (Fig 5L and 5M), hepato-( Fig 5N) and splenomegaly ( Fig 5O) compared with IL-4Rα-responsive control mice. However, the body weights of S. mansoni-infected i Cre-/+ IL-4Rα -/lox Tam6 mice rapidly declined following Tamoxifen-driven removal of IL-4Rα at 6 weeks post-infection similar to IL-4Rα -/mice ( Fig 5P) and culminated into the early death of these animals (Fig 5Q), when compared to IL-4Rα-responsive control mice. Bleeding was visible in the gut of the animals that rapidly succumbed to infection following removal of IL-4Rα. No premature mortality was reported with S. mansoni-infected Tam6-fed IL-4Rα +/+ (control for Tamoxifen side effects), Rosa-CreER T2-/+ IL-4Rα +/+ (control for activated CreER T2 ) and RosaCreER T2-/+ IL-4Rα -/lox (control for CreER T2 Tamoxifen-independent activity) mice when compared to S. mansoni-infected IL-4Rα +/+ (positive control) mice (S6 Fig) ruling out any non-specific effect(s) of Tamoxifen or CreERT2 as mediator(s) of the impaired survival of S. mansoni-infected i Cre-/+ IL-4Rα -/lox Tam6 mice. Taken together, these results show that IL-4Rα knockdown after egg deposition during the acute phase of experimental schistosomiasis considerably diminishes the host ability to maintain a type 2 immune response around the S. mansoni eggs which associates with gut bleeding, rapid weight loss and premature death.
IL-4Rα knockdown during chronic schistosomiasis (16 weeks) ameliorates disease S. mansoni-infected mice following IL-4Rα knockdown at 16 weeks post-infection, i.e. i Cre-/+ IL-4Rα -/lox Tam16 mice (Fig 6A) did not result in any weight loss (Fig 6B) or mortality (Fig 6C), for up to 24 weeks post infection. Liver egg burden was similar between the control IL-4Rα -/lox ( Fig  6D), ruling out a differential level of infection between both groups of mice. IL-4Rα knockdown considerably reduced liver ( Fig 6E) and spleen (Fig 6F) enlargement in chronically infected mice. Apparent scarification was visible on the liver lobes of control mice whereas IL-4Rα knockdown resulted in the removal/reversal/inhibition of liver scarification (Fig 6G). Moreover, IL-4Rα knockdown considerably reduced granuloma size (Fig 6H and 6I) and collagen levels (Fig 6J and  6K) in the livers of chronically infected mice. These data indicated that IL-4Rα knockdown ameliorate granulomatous inflammation, hepato-and splenomegaly and liver fibrosis during chronic schistosomiasis further consolidating the idea of a deleterious role for IL-4Rα signaling in mediating fibroproliferative pathology during chronic schistosomiasis.
To analyse the immune polarization and responses that are triggered by IL-4Rα knockdown during chronic schistosomiasis and associate with the amelioration of tissue disease, IL-4Rα was knockdown in mice chronically infected with S. mansoni at week 16 post infection and the immune response analyzed at week 18 post infection (Fig 7A).  Fig  7C) and ratios (Fig 7D) of cytokine-producing MLN CD4 + T cells ( S6 Fig and Fig 7B). Canonical transcription factor analysis ( Fig 7E) confirmed this conclusion with reduction of GATA3 but normal Tbet production in effector T cells (Fig 7E and 7F). Interestingly, the frequencies of Foxp3 + regulatory T cell responses were increased in the MLNs of S. mansoni-infected i Cre-/+ IL-4Rα -/lox Tam16 mice (Fig 7G and 7H), when compared with S. mansoni-infected IL-4Rα -/lox control mice. However, most likely as a result of total CD4 + T cell drop (S7A Fig), Treg cell numbers were reduced following Tam16 treatment in S. mansoni-infected i Cre-/+ IL-4Rα -/lox Tam16 animals when compared to their littermate controls (Fig 7I). Serum titers of type 2 antibodies (IgG1 and total IgE) were reduced (Fig 7J and 7K) but not type 1 antibodies (IgG2a, Fig 7L), supporting reduced type 2 responses in S. mansoni-infected i Cre-/+ IL-4Rα -/lox Tam16 mice. Of interest, regulatory B cell frequencies increased during infection and particularly in infected i Cre-/+ IL-4Rα -/lox Tam16 mice ( Fig  7M and 7N) amid a rather stable total count in S. mansoni-infected i Cre-/+ IL-4Rα -/lox Tam16 animals when compared to littermate controls (Fig 7O).
Innate type 2 immune effectors i.e. eosinophils [34,35], ILC2 [36] and macrophages [37] have been positively linked to liver fibrosis, the pathophysiological process that drives the host morbidity during chronic schistosomiasis. Conversely, arginase expression by macrophages has been shown to counter tissue inflammation and fibrosis [38]. The analysis of the MLN cells of S. mansoni-infected i Cre-/+ IL-4Rα -/lox Tam16 mice for these cell types by flow cytometry (S7D Fig-S7G Fig) revealed that the pro-fibrotic innate effectors i.e. eosinophils (Fig 7P, S8A  Fig and S8B Fig), ILC2 (Fig 7Q and S8C Fig) and macrophages (S8D Fig) were significantly diminished. Conversely, the mean arginase expression by macrophages (S8E Fig) was not affected in S. mansoni-infected i Cre-/+ IL-4Rα -/lox Tam16 mice, when compared to S. mansoniinfected IL-4Rα -/lox control mice. This suggests that IL-4Rα knockdown in chronically infected mice does skew the MLN response away from a pro-fibrotic response. Taken together, these results suggest that knocking down IL-4Rα at the chronic phase of experimental schistosomiasis considerably skews the host immune response away from the type 2 arm of the immune response, fosters a qualitatively more regulatory, anti-inflammatory and anti-fibrotic profile with no deleterious effect on host survival.

Discussion
Taking advantage of a newly established temporal inducible IL-4Rα deficient mouse model, we demonstrated that interrupting IL-4Rα mediated signaling prevents the onset and maintenance of egg-driven type 2 immune responses and its associated fibro-granulomatous inflammation during schistosomiasis. Whereas early knockdown of the receptor during the acute phase of the disease led to aggravated morbidity and mortality, late targeting at the chronic phase considerably ameliorated fibrogranulomatous inflammation and reduced hepato-and splenomegaly without impairing the animal viability. Amelioration of chronic schistosomiasis pathology was further associated with reduction of type 2 immune effector responses but expansion of regulatory T and B cells, suggesting that IL-4Rα mediated immune responses are detrimental in chronic schistosomiasis. Hence, therapeutic intervening of IL-4Rα mediated signaling to reduce type 2 responses might provide a strategy to ameliorate fibroproliferative pathology in diseases like chronic schistosomiasis.

IL-4 receptor drives liver fibrosis
The group of Cheever et al. (1994) were the first to show that abrogation of type 2 immune responses in S. mansoni-infected mice resulted in impaired granulomatous inflammation around the trapped eggs in tissue. A subsequent study by Chiaramonte et al. [21] showed the importance of IL-13-mediated signaling in fibrogenesis through the blockade of IL-13. Moreover, this group further reported a critical role for IL-13 in granuloma formation induced by S. mansoni eggs [39]. An independent group further reported on the achievement of significantly reduced tissue fibrosis by blocking type 2 responses in S. mansoni-infected mice with anti-IL-4 antibody treatment [40]. This indicated that IL-4-orchestrated type 2 responses, as well as IL-13-driven responses, are all causally linked to fibrogranulomatous pathology. More recently, using Schistosomiasis-infected IL-4Rα deficient mice, we and others demonstrated reduced IL-4 receptor drives liver fibrosis fibrogranulomatous inflammation. However, these mice died during acute schistosomiasis due to cachexia [25]. Using inducible IL-4Rα deficient mice in the present study, we now dissected the role of IL-4/IL-13-mediated type 2 responses during acute and chronic murine schistosomiasis. IL-4Rα removal (knockdown) early during schistosomiasis infection led to impaired type 2 responses with reduced fibrogranulomatous inflammation around the trapped eggs of the parasites, as demonstrated before. This resulted in exacerbated morbidity and premature death of the animals, as demonstrated previously in IL-4Rα deficient mice [25]. Thus, IL-4Rα-elicited type 2 immune effector responses like granuloma formation and fibrosis are important for the host survival during acute schistosomiasis. This concept has been previously established where a tissue protective role of these responses against the toxic secretions of the parasite eggs was suggested [41]. Interestingly, liver integrity was not affected after acute knockdown of IL-4Rα in S. mansoni-infected animals. This finding argues against liver toxicity being the pathological event that drives death in these animals. A more likely explanation for their premature death would be the intensive gut bleeding reported in our previous study on IL-4Rα deficient mice [25], and similarly observed in this study. As a result of the compromised gut integrity, bacteria would translocate to the blood stream and death by septic shock would ensue, as previously demonstrated [25].
Tamoxifen-induced knockdown of the IL-4Rα after egg deposition during the chronic phase (16 weeks post-infection) uncovered a hitherto unappreciated facet of the IL-4Rα mediated type 2 responses. Indeed, IL-4Rα knockdown during chronic schistosomiasis did not lead to gut bleeding and did not affect animal viability but ameliorated liver pathology with reduced granuloma size and fibrosis in the liver and no visible scarification and reduced level of liver and spleen enlargement. This clearly suggests that IL-4Rα mediated type 2 responses are detrimental during chronic schistosomiasis and the cause for fibroproliferative liver pathology. Of interest, regulatory T and B cell compartments were significantly increased following IL-4Rα removal during chronic schistosomiasis. It is tempting to associate the beneficial effect of IL-4Rα blockade on tissue pathology during chronic schistosomiasis to the enhanced regulatory response observed. In fact, previous studies have reported an amelioration of the fibrogranulomatous inflammation during chronic schistosomiasis by Foxp3 + regulatory T cells [42,43]. Whether IL-4Rα mediated signaling causally dictates the anti-inflammatory and anti-fibrotic activities of these regulatory cells during chronic schistosomiasis is not known.
As of now, a role for IL-4Rα signaling in the development of immune hyporesponsiveness after chronic exposure of host immune cells to schistosomal antigens has been demonstrated [44]. This further re-emphasizes the potential of IL-4Rα in modulating the dynamics of the host regulatory responses during chronic diseases such as schistosomiasis. Such a potential has already been widely reported with a negative regulation of Foxp3 + Tregs and a loss of their suppressive capacity suggested to occur when the IL-4Rα signaling was solicited [45][46][47]. Alternatively, however, the remnant IL-4Rα mediated signaling in Tam16 mice argues against the absence of Th2 responses as the sole driver of the ameliorated pathology observed. A rather noticeable finding is the upregulation of other cytokines i.e. IL-10 and IFNγ resulting in a better balanced cytokine profile between T cells producing IL-4, IFN-γ and/or IL-10. Consequently, the impairment of IL-4Rα mediated signaling during chronic schistosomiasis by inducing a more equilibrated and mixed Th profile might prevent untoward immune polarization and tissue immunopathology. This hypothesis is strongly supported by the recently demonstrated role for immune balance rather than strong immune polarization in controlling fibrogranulomatous pathology during experimental schistosomiasis [48]. Further experiments are now required to empirically disentangle these hypotheses. What remains clear and worth focus at present is the fact that targeting IL-4Rα mediated signaling for the management of non-communicable type 2-mediated diseases in humans is in advanced clinical trials [49][50][51].
Understandably, building on the present study, the translatability of targeting IL-4Rα mediated signaling during fibroproliferative diseases like chronic schistosomiasis is further supported.
What do we add to the current knowledge on the control of fibroproliferative disease? It should be recalled that our present report builds on the previous observations made during IL-13 blockade experiments where a key role for this cytokine, and the indication of the potential of the IL-4Rα signaling axis in driving fibroproliferative responses during experimental schistosomiasis was defined [21]. In as much as an efficient anti-fibrotic strategy already transpired from the sole blockade of IL-13 [21], the noticeable and independent pro-fibrotic effect of IL-4 [21,40] altogether argues for the higher anti-fibrotic potential of dually targeting IL-4 and IL-13 by blocking IL-4Rα rather than IL-13 alone. The picture might not be that straightforward, however, as caution should also be exerted in dually targeting IL-4 and IL-13 via IL-4Rα given that IL-4 unlike IL-13 is critical for type 2 immune responsiveness. A state of immune deficiency might therefore arise from IL-4Rα targeting as opposed to IL-13 targeting where Th2 responses are optimally elicited [21]. Also, consistent with the observation that IL-13 targeting was not toxic for the host [21], our present report shows that IL-4R targeting does not impair animal fitness. This strongly argues for the safety of our approach. Conclusively, as of yet, one could therefore speculate on an added value of targeting IL-4Rα rather than just IL-13 given the different profibrotic potentials of IL-13 [21,39,52,53] and IL-4 [40,[54][55][56][57][58] as both cytokines signal through IL-4Rα. Clearly, such a conclusion would still need to be experimentally validated.
In summary, we provide evidence on the role of IL-4Rα during experimental schistosomiasis whereby early signaling helps the host survive the acute phase of the disease whereas signaling at the late chronic phase mediate the morbidity. Targeting IL-4Rα might therefore represent a novel therapeutic strategy against the fibroproliferative pathology that drives the morbidity of fibrotic diseases like chronic schistosomiasis.

Materials and methods Mice
IL-4Rα -/-, IL-4Rα -/lox and CreER T2 mice on a C57/BL6 background were previously described [2,25,33]. We generated a novel inducible IL-4Rα deleting mouse strain (RosaCreER T2-/+ IL-4Rα -/lox ) by intercrossing transgenic RosaCreER T2-/+ mice with IL-4Rα -/and IL-4Rα Lox/Lox mice. CreER T2 transgenic negative littermates (IL-4Rα -/lox ) expressing functional IL-4Rα were used as controls in all experiments. Mice were maintained in the University of Cape Town specific pathogen-free animal facility in accordance with the guidelines established by the Animal Research Ethics committee of the Faculty of Health Science of the University of Cape Town and the South African Veterinary Council (SAVC).

Parasite infection and Tamoxifen administration
Mice were infected percutaneously via the abdomen with 35, 80 or 100 cercariae, as indicated, with a Puerto Rican strain of Schistosoma mansoni obtained from infected Biomphalaria glabrata (a generous gift from Adrian Mountford, York, UK). Eggs were purified from digested sections of liver or ileum from infected animals and counted at 40× magnification as previously described [40]. To activate il-4rα gene excision by CreER T2 , Tamoxifen (Sigma, Deisenhofen, Germany) solubilized in vegetable oil was administered by oral gavage to mice for four consecutive days (2.5mg/day).

Genotypic characterization
Polymerase chain reaction was used to confirm the genotype of RosaCreER T2-/+ IL-4Rα -/lox mice. PCR conditions were 94˚C for 2 minute, 94˚C for 20 seconds, 45˚C for 30 seconds, and 72˚C for 20 seconds for 40 cycles. To quantify the efficiency of deletion, real-time PCR was performed on genomic DNA from liver and spleen cells using primers specific for IL-4Rα exon 5 (control) and exon 8 (deleted by CreER T2 activation) as described previously [25].

In vitro stimulation
Stimulation with cytokines. Splenocytes or MLN cells were seeded at a density of 5 x 10 6 cells/ml and stimulated with 1 ng/ml of rIL-4 for 48h. After incubation, the cells were harvested and washed in PBS, 3% FCS and 0.1% NaN 3 before labeling of IL-4Rα for flow cytometry. To avoid nonspecific binding, cells were incubated with a mixture containing rat sera and unlabeled anti-CD32/CD16 Antibody (2.4G2). In another series of stimulation, cultures incubated for 48h with different amounts of rIL-2 then incubated with CellTiter-blue assay reagent (Promega, Germany) for 4h before OD measurement at 570/620 to measure the cell viability.
Ex vivo restimulation of MLN. Single cell suspensions from MLN cells of S. mansoniinfected animals were prepared by pressing the MLN through 70μm cell-strainers. Cells were resuspended in complete IMDM (Gibco) supplemented with 10% FCS (Gibco) and Penicillin and Streptomycin (100 U/ml and 100 μg/ml, Gibco). The cells were cultured at 1×10 6 cells/ml in 96-well plates coated with α-CD3 (20 μg/ml) or supplemented with SEA (20 μg/ml) and incubated at 37˚C in a humidified atmosphere containing 5% CO 2 . Supernatants were collected after 72 h and cytokines were measured by sandwich ELISA as previously described [33].

Serum analyses
Antibody titers. S. mansoni antigen-specific serum antibody isotypes and total IgE titers from infected mice were determined as follows. Blood was collected in serum separator tubes (BD Bioscience, San Diego, CA) and centrifuged at 8 000×g for 10 min at 4˚C to separate serum. The flat-bottom 96-well plates were coated with 10 μg/ml SEA, blocked with 2% (w/v) milk powder for 2 h at 37˚C and samples were loaded and incubated overnight at 4˚C. Alkaline phosphatase labeled secondary antibody was added and incubated for 2 h at 37˚C. The plates were developed by addition of 4-nitrophenyl substrate (Sigma). The absorbance was read at 405 nm using VersaMax microplate spectrophotometer (Molecular Devices, Germany).
Liver enzymes. Hepatocellular damage was assessed by measuring the serum levels of alanine transaminase at the National Health Laboratory Service of South Africa (Cape Town).

Histology and hydroxyproline quantification
Tissue samples were fixed in neutral buffered formalin, processed, and 5-7 μm sections stained with hematoxylin and eosin (H & E). Granuloma diameter of 20-50 granulomas per animal was determined using an ocular micrometer (Nikon NIS-Elements, Nikon Corporation, Tokyo, Japan). For fibrosis assessment, tissue sections were stained with chromotrope 2R and analine blue solution (CAB) and counterstained with Wegert's hematoxylin for collagen staining. Complementarily, a modified protocol of tissue hydroxyproline quantification was used [59]. In brief, weighed liver samples were hydrolyzed and the supernatant was neutralized with 1% phenolphthalein and titrated against 10 M NaOH. An aliquot was mixed with isopropanol and added to a chloramine-T/citrate buffer solution (pH 6.0) (Sigma). Ehrlich's reagent solution was added and measured at 570 nm. Hydroxyproline levels were calculated by using 4-hydroxy-L-proline (Calbiochem) as standard, and results were expressed as μg hydroxyproline per weight of liver tissue that contained 10 4 eggs.