Interleukin‐9 blockage reduces early hepatic granuloma formation and fibrosis during Schistosoma japonicum infection in mice

Summary

Hepatic fibrosis induced by schistosomes is regulated by a complex network of cytokines. T helper type 9 (Th9) cells are a new type of effector T helper cells, which mainly secrete the specific cytokine interleukin‐9 (IL‐9). Interleukin‐9 has been shown to contribute to liver fibrosis in patients with chronic hepatitis B and in a mouse model due to carbon tetrachloride. However, the role of IL‐9 in schistosomiasis fibrosis remains unknown. In this study, we investigated the roles of IL‐9 in schistosomiasis through in vivo and in vitro studies. The in vivo studies found that neutralization of IL‐9 reduced liver granulomatous inflammation and collagen deposition around parasite eggs. The in vitro studies found that the treatment of primary hepatic stellate cells with IL‐9 induced a significant increase of collagen and α‐smooth‐muscle actin. Moreover, we also described the dynamics and relevance of IL‐9 and IL‐4 in mice infected with Schistosoma japonicum. We found that IL‐9 might appear more quickly and at higher levels than IL‐4. Hence, our findings indicated that IL‐9 might play a role in regulating hepatic fibrosis in early‐stage schistosomiasis and become a promising approach for regulating hepatic fibrosis caused by S. japonicum.

Introduction

Schistosomiasis is a tropical and subtropical disease infecting over 230 million people worldwide, causing between 500 000 and 800 000 deaths per year.1 Human schistosomiasis is caused by infection with one of six schistosome species: Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, Schistosoma intercalatum, Schistosoma malayensis and Schistosoma mekongi, with the former three species having the greatest clinical and socio‐economic significance. In China, only S. japonicum is endemic. The main pathogenesis of schistosomiasis caused by S. japonica is the occurrence of egg granulomas in the liver and intestine, which can result in severe liver fibrosis.2 Liver fibrosis is characterized by the deposition of excessive extracellular matrix, gradually causing permanent liver injury or chronic inflammation.3 In this repairing process, hepatic stellate cells (HSC) play an important role. Quiescent HSCs are usually activated by inflammatory stimuli, and then they undergo trans‐differentiation to myofibroblasts.4 The myofibroblasts can migrate to the necrotic tissue and the area of inflammation. Meanwhile, they have a powerful ability to produce α‐smooth‐muscle actin (α‐SMA) and collagens, the main components of extracellular matrix.5

CD4⁺ helper T (Th) cells play pivotal roles in both host humoral immunity and cellular immunity against parasitic infection and immunopathology in schistosomiasis by secreting a variety of cytokines and providing help to other cells. After infection, antigen secreted by schistosome eggs induces a continuous immune response of CD4⁺ T cells.6 During the course of the schistosome infection, there is an initial Th1‐derived pro‐inflammatory response marked by interferon‐γ.7, 8 As the disease progresses, the immune response undergoes a striking shift from the Th1 response to an anti‐inflammatory Th2‐dominated response, characterized by the rise of interleukin‐4 (IL‐4), IL‐5 and IL‐13, following the parasite oviposition around 5–6 weeks.9 Both IL‐4 and IL‐13 play a major role in egg granuloma formation, and the granulomas serve an important host‐protective role, sequestering the eggs away from surrounding tissues.10 However, IL‐13 is also important to the development of hepatic fibrosis.11, 12 In recent studies, it was reported that Th17/IL‐17 and follicular helper T cells (Tfh)/IL‐21 were correlated with severe pathology and subsequent fibrosis in schistosomiasis.13, 14 Down‐regulation of Th17/IL‐17 or Tfh/IL‐21 could decrease granulomatous inflammation and ameliorate liver fibrosis.15, 16, 17 During the chronic phase, the Th2 response is still predominant and modulated, but the granulomas are smaller than at earlier stages. The reason is that regulatory T cells play an important repressor role in the down‐regulation of pathological immune responses at this stage. Previous studies have shown that regulatory T cells and their functional factor IL‐10 could prevent the response of Th1, Th2 and Th17.18, 19 Hence, the schistosome‐induced hepatic pathology is regulated by multiple cytokines.

Th9 cells, a new subset of T helper cells, are characterized by their specific cytokine IL‐9 and transcription factors PU.1 and IRF‐4.20, 21, 22 Interleukin‐9 was considered as a Th2‐specific cytokine more than 20 years ago. However, it has been recently shown that IL‐9 and IL‐4 were rarely produced by the same T cell, indicating that Th9 cells form a unique Th cell subset.23 The critical roles of IL‐9 in a broad range of autoimmune disorders, allergic inflammation and cancers, as well as parasitic infection, has aroused interest.24, 25, 26, 27, 28, 29, 30 However, studies on the relationship between IL‐9 and schistosomiasis are scarce. Our previous study investigated the dynamic changes of Th9/IL‐9 in S. japonicum‐infected mice and found that egg granulomas developed synchronously with the expression changes of IL‐9 and PU.1 in liver. Moreover, this difference was also reflected in the proportions of splenic IL‐9⁺ cells and levels of serum IL‐9.31 Though this is powerful evidence for IL‐9 being involved in the pathogenesis of schistosomiasis, the precise role of IL‐9 and its relationship with other cytokines remain unclear. Therefore, in the present study, we aimed to present the role of IL‐9 in hepatic fibrosis and compare it with levels of the Th2 cytokine IL‐4 during the course of schistosomiasis infection. This study may provide novel insights into the potential role of IL‐9 in the immunopathology of liver fibrosis in schistosomiasis.

Materials and methods

Ethics statement

The animal experiments using mice were performed in strict accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals (1988.11.1), and all efforts were made to minimize suffering. All animal procedures were approved by the Institutional Animal Care and Use Committee of Soochow University for the use of laboratory animals (Permit Number: 2007‐13).

Mice, parasites and infection

Female FVB/NJ mice aged 6–8 weeks with body weights of 18–20 g were purchased from the Centre of Comparative Medicine of Yangzhou University (Yangzhou, China). All mice were kept under specific pathogen‐free conditions at the laboratory animal research facility of Soochow University (Suzhou, China). Oncomelania hupensis snails harbouring S. japonicum cercariae (Chinese mainland strain) were purchased from the Jiangsu Institute for Schistosomiasis Control (Wuxi, China). Mice were infected with 15 ± 1 cercariae of S. japonicum per mouse through the abdominal skin. At 4, 7, 9 and 12 weeks post‐infection, five mice were randomly chosen from the infected mice and killed for further studies. Other infected mice were randomly divided into two groups (n = 6): infected mice treated with anti‐IL‐9 antibody (anti‐IL‐9 group) and treated with immunoglobulin G (IgG) isotype control (IgG group). The anti‐IL‐9 group was induced by intraperitoneal injection of 100 μg anti‐IL‐9 monoclonal antibody (BE0181; BioXCell, West Lebanon, NH) twice a week from the day of infection to the 9th week post‐infection. The IgG group was established through intraperitoneal injection with 100 μg IgG2a isotype control (BE0085; BioXCell). Non‐infected mice were used as a normal control group (NC group).

Histopathological study

The harvested liver specimens from the NC group, anti‐IL‐9 group and IgG group were fixed in 10% neutral buffered formalin, embedded in paraffin blocks and cut into 4‐μm‐thick serial sections. Liver sections were stained with haematoxylin & eosin or Masson Trichrome. To calculate the area of granulomas, we randomly captured images of single hepatic egg granulomas (five per mouse) and measured them using image‐pro plus 7·0 software (Media Cybernetics, Bethesda, MD). To assess the degree of hepatic fibrosis, images (five per mouse) were collected randomly. Masson‐Trichrome‐stained sections were collagen‐positive particles, and the ratio of the collagen area was measured by image‐pro plus 7·0 software. All images were photographed using a light microscope (Olympus, Tokyo, Japan).

Hepatic stellate cells: isolation and culture

Hepatic stellate cells were isolated from livers of FVB/NJ mice (non‐infected) according to a modified method described previously.13 In brief, after anaesthesia and sterilization, the abdominal cavity was opened. Preheating perfusion buffer 1 (RPMI‐1640; Sigma Corporation, St Louis, MO) was injected into the liver through the hepatic portal vein and drained via the inferior vena cava with a peristaltic pump. When the blood was washed clean, preheating perfusion buffer 2 [0·04% collagenase I (Gibco, Grand Island, NY) in RPMI‐1640, pH 7·3–7·4, sterile‐filtered, and stored at 37°] was pumped into liver for 6 min at a flow rate of 15 ml/min. Then the liver was ground and digested at 39° with 20 ml oscillating digestive buffer [0·08% pronase E (Solarbio, Beijing, China), 0·08% collagenase I and 5 U/ml DNase I (Solarbio) in RPMI‐1640, pH 7·3–7·4, sterile‐filtered and stored at 37°]. After 15 min, the digestion was terminated immediately by adding 20 ml RPMI‐1640 and filtered using 70‐μm nylon gauze. The homogenate was centrifuged at 400 g for 6 min at 4° to remove the hepatocytes and washed twice with RPMI‐1640 buffer. The cell pellets were re‐suspended in 5 ml of 15% OptiPrep™ (Axis‐Shield, Oslo, Norway) and loaded carefully with 5 ml of 11·5% OptiPrep and 2 ml RPMI‐1640. After centrifugation at 3000 g for 20 min without brake, HSCs in the 11·5% OptiPrep and RPMI‐1640 interphase were gently aspirated. The obtained objective cells were then washed twice using RPMI‐1640, and the final cell pellets were re‐suspended in Dulbecco's modified Eagle's medium containing 15% premium fetal bovine serum (Gibco), 100 U/ml penicillin and 100 μg/ml streptomycin. A density of 5 × 10⁵ cells per well was inoculated in 12‐well culture plates (Costar, Cambridge, UK), changing liquid for 24 hr. After 24 hr, HSCs were cultured with IL‐9 cytokines (20 ng/ml per well) (R&D Systems, Minneapolis, MN) for 48 and 72 hr at 37° in 5% CO₂ before the cells were harvested.

Western blot

Proteins extracted from HSCs using lysis buffer (BestBio, Shanghai, China) were separated by 5% or 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% non‐fat dry milk and incubated with primary antibodies of α‐SMA (Ab124964; Abcam, Cambridge, UK), collagen type I (Bs‐10423R; Bioss, Beijing, China) and collagen type III (22734‐1‐AP; Proteintech, Rosemont, IL) overnight at 4°. The levels of β‐tubulin (CW0098; CwBio, Taizhou, China) served as control. The membranes were incubated with horseradish peroxidase‐conjugated secondary goat anti‐rabbit antibodies and then visualized by the super‐sensitive chemiluminescence reagent (Meilun, Dalian, China). Finally, individual protein bands were quantified by densitometry with image J and normalized for β‐tubulin.

Flow cytometry

Spleens were freshly harvested from normal or infected mice (4, 7, 9, 12 weeks post‐infection). Single‐cell suspensions were prepared by mincing fresh spleen tissue in phosphate‐buffered saline containing 1% fetal bovine serum (Gibco) and followed by lysis of red blood cells using ACK lysis buffer. Approximately 10⁶ splenocytes per 100 μl were stimulated with 50 ng/ml phorbol 12‐myristate 13‐acetate (Sigma‐Aldrich, St Louis, MO) and 1 μg/ml ionomycin (Sigma‐Aldrich) in the presence of 10 μg/ml brefeldin A (BD Biosciences, San Jose, CA) for 5 hr at 37° in 5% CO₂. After incubation, the cells were surface stained with fluorescein isothiocyanate‐conjugated anti‐mouse CD4 antibody (BD Bioscience) for 30 min at 4° in the dark. Subsequently, the cells were fixed and permeabilized with Cytofix/Cytoperm buffer (BD Biosciences), and then intracellularly stained with Peridinin chlorophyll protein‐Cy5·5‐conjugated anti‐mouse IL‐9 (BD Bioscience) and phycoerythrin‐conjugated anti‐mouse IL‐4 (eBioscience, San Diego, CA). All of the stained cells were examined using a flow cytometer (Beckman Coulter, Brea, CA) and data were analysed with CXP software.

Enzyme‐linked immunosorbent assay

Mice were killed and sera were harvested at 0, 4, 7, 9 and 12 weeks post‐infection. Levels of IL‐9 and IL‐4 in serum were examined by enzyme‐linked immunosorbent assay (ELISA) Ready‐SET‐Go kits (Multi sciences, Hangzhou, China) according to the manufacturer's instructions. Samples were read at 450 nm using an ELISA reader and the concentrations were calculated using standard curves.

Immunohistochemistry

Livers were freshly harvested from normal or infected mice (4, 7, 9 and 12 weeks post‐infection) and fixed in 10% buffered formalin. Sections (4 μm) of liver were embedded in paraffin. Immunostaining for IL‐9 and IL‐4 were performed using monoclonal anti‐IL‐9 primary antibody (Ab203386; Abcam) and monoclonal anti‐IL‐4 primary antibody (Ab9811; Abcam), respectively. The immunohistochemical technique used a two‐step method (peroxidase‐conjugated polymer). Three per cent H₂O₂ was used to block the endogenous peroxidase activity, and the non‐specific binding was inhibited with 10% normal goat serum. All sections were incubated with primary antibodies at 37° for 1 hr and then at 4° overnight. The sections were further incubated with the secondary antibodies (ChemMate Envision/HRP, rabbit/mouse detection immunohistochemistry kit; Gene‐tech, Shanghai, China) at 37° for 1 hr. Immunoreactivity was visualized using diaminobenzidine as the chromogen. Five high‐power fields (×400) per mouse liver were randomly photographed with a light microscope (Olympus). The percentages of positively staining cells were measured by image‐pro plus 7·0 software (Media Cybernetics).

Statistical analysis

All statistical analyses were carried out with spss 21·0 data editor (SPSS Inc., Chicago, IL). Data were shown as the mean ± standard deviation (SD). More than two groups were performed using one‐way analysis of variance (anova). Two group comparisons were performed by unpaired, two‐tailed Student's t‐tests.

Results

Treatment with anti‐IL‐9 antibody ameliorated granulomatous inflammation

To determine whether IL‐9 is involved in the egg‐induced granulomatous response, we treated mice with anti‐IL‐9 antibody or IgG twice a week. Nine weeks after infection, haematoxylin & eosin staining showed that the structure of hepatic lobules was normal and the hepatic cells were arranged well in the NC group; whereas obvious egg granulomas, cell degeneration and inflammatory cell infiltration appeared in the livers of the IgG group and the anti‐IL‐9 group. Significantly, treatment with the anti‐IL‐9 antibody moderately attenuated hepatic egg granulomas and reduced inflammatory cell infiltration (Fig. 1a). As shown in Fig. 1(b), the data suggested that the anti‐IL‐9 group had smaller egg granulomas than the IgG‐treated group (t = 2·63, P < 0·05).

Figure 1.

Haematoxylin & eosin staining of mouse livers after treatment with anti‐interleukin‐9 (IL‐9) antibody or IgG. (a) Representative images of hepatic egg granuloma from Schistosoma japonicum‐infected mice in the normal control (NC) group, IgG group and anti‐IL‐9 group (original magnification: ×100). (b) Comparison of the areas of hepatic egg granuloma between the IgG group and anti‐IL‐9 group. Data are presented as mean ± SD. *P < 0·05.

Neutralization of IL‐9 attenuated the severity of hepatic fibrosis

Masson Trichrome staining was used to observe the hyperplastic state of collagen fibre, which indicates the severity of liver fibrosis. To compare the degree of liver fibrosis before and after treatment with anti‐IL‐9 antibody, liver tissues were stained with Masson Trichrome in the NC group, IgG group and anti‐IL‐9 group (Fig. 2a). The results showed that there was little collagen in the livers in the NC group; whereas, a large amount of collagen was deposited around the egg granuloma in the livers in the IgG group and anti‐IL‐9 group. We calculated the percentages of collagen area around single‐egg granulomas. As shown in Fig. 2(b), mice treated with anti‐IL‐9 antibody had a lower degree of liver fibrosis than the IgG‐treated group (t = 3·05, P < 0·01).

Figure 2.

Masson Trichrome staining of mouse livers after treatment with anti‐interleukin‐9 (IL‐9) antibody. (a) Representative images of collagen deposition from Schistosoma japonicum‐infected mice in the normal control (NC) group, IgG group and anti‐IL‐9 group (original magnification: ×100); the blue area indicates collagen particles. (b) Comparison of the degree of hepatic fibrosis between the phosphate‐buffered saline group and anti‐IL‐9 group. Data are presented as mean ± SD. **P < 0·01.

IL‐9 induced HSC activation and drove HSCs to secrete collagen‐I and collagen‐III

HSCs were isolated from livers and identified their purity (Fig. S1). To study the functional change of HSCs with or without stimulation of IL‐9, we examined the expression of α‐SMA, collagen‐I and collagen‐III. α‐SMA is one of the characteristics of HSC activation. Collagen‐I and collagen‐III are primary components of the extracellular matrix. The results showed that co‐culture of HSCs with IL‐9 could up‐regulate the expression of α‐SMA, collagen‐I and collagen‐III (Fig. 3a). Western blot analysis showed a significant increase of these three proteins from HSCs after stimulation of IL‐9 for 48 and 72 hr compared with controls at the same time‐points (P < 0·01 for all comparisons) (Fig. 3b).

Figure 3.

Expression of α‐smooth‐muscle actin (α‐SMA), collagen‐I and collagen‐III after stimulation of interleukin‐9 (IL‐9). (a) Representative graphs of protein expression, which were measured by Western blot. (b) Comparison of the protein expression between IL‐9 stimulation for 48 and 72 hr. Data are presented as mean ± SD. **P < 0·01.

Polarization of IL‐9⁺ cells in front of IL‐4⁺ cells

To investigate the different immune response characteristics between IL‐9 and IL‐4, we examined the proportions of IL‐9⁺ cells and IL‐4⁺ cells in splenic CD4⁺ T cells by flow cytometry (Fig. 4a). The statistical results (Fig. 4b) showed that after infection, the proportions of IL‐9⁺ cells increased rapidly and peaked at 7 weeks, then decreased gradually (anova: F _(4,20) = 59·15, P < 0·0001). The proportions of IL‐4⁺ cells were remarkably up‐regulated from 4 weeks and reached a peak at 12 weeks (anova: F _(4,20) = 107·97, P < 0·0001).

Figure 4.

Percentages of interleukin‐9‐positive (IL‐9⁺) cells and IL‐4⁺ cells in total CD4⁺ T cells from spleens of mice with schistosomiasis. (a) Representative dot plots of IL‐9⁺ cells and IL‐4⁺ cells by flow cytometry in Schistosoma japonicum‐infected mice at 0, 4, 7, 9 and 12 weeks. All of the values were gated on CD4⁺ cells. The percentages of IL‐9⁺ cells in CD4⁺ cells are indicated in the upper left of each chart, and the percentages of IL‐4⁺ cells in CD4⁺ cells are indicated in the lower right of each chart. (b) Quantitative changes of IL‐9⁺ cells and IL‐4⁺ cells in splenic CD4⁺ T cells at different time‐points. Data are presented as mean ± SD. **P < 0·01 compared with the 0 week; ^## P < 0·01 compared with the peak.

Peak of serum IL‐9 came before IL‐4

To confirm the dynamics of IL‐9 and IL‐4, we also measured the two cytokines in the serum from the same mouse groups using ELISA (Fig. 5). Consistent with the dynamics of IL‐9⁺ cells, the level of IL‐9 increased very quickly in the first 4 weeks after infection, and reached a peak at 7 weeks. Then the level of IL‐9 decreased gradually (anova: F _(4,20) = 48·72, P < 0·0001). The level of IL‐4, in parallel with the dynamics of IL‐4⁺ cells, grew very slowly during the first 4 weeks after infection, but rose rapidly thereafter until reaching a peak at 12 weeks (anova: F _(4,20) = 45·84, P < 0·0001).

Figure 5.

Level of serum interleukin‐9 (IL‐9) and IL‐4. Kinetics of serum IL‐9 and IL‐4. Enzyme‐linked immunosorbent assays (ELISA) were used to measured IL‐9 and IL‐4 from Schistosoma japonicum‐infected mice at 0, 4, 7, 9 and 12 weeks. Data are presented as mean ± SD. **P < 0·01 compared with the normal control (NC) group; ^## P < 0·01 compared with the peak.

IL‐9 appeared more quickly and at higher levels than IL‐4 in liver

To further elucidate the relationship between IL‐9 and IL‐4 in the development of liver pathology in mice infected with S. japonicum, the expressions of IL‐9 and IL‐4 in liver egg granulomas were examined by immunohistochemistry (Fig. 6a). As shown in Fig. 6(b), both IL‐9⁺ cells and IL‐4⁺ cells were expanded. After infection, the percentage of cells staining positive for IL‐9 increased and reached its peak at 7 weeks, then decreased slowly (anova: F _(4,20) = 140·22, P < 0·0001). The percentage of cells staining positive for IL‐4 did not increase until 4 weeks post‐infection, afterwards rising steadily and peaking at 12 weeks (anova: F _(4,20) = 246·53, P < 0·0001).

Figure 6.

Immunohistochemistry of interleukin‐9 (IL‐9) and IL‐4 in mouse livers. (a) Representative images of hepatic expression and distribution of IL‐9 and IL‐4 from Schistosoma japonicum‐infected mice at 0, 4, 7, 9 and 12 weeks (original magnification: ×200). (b) Comparison of the percentage of positive cells around granulomas among different time groups. Data are presented as mean ± SD. **P < 0·01 compared with the normal control (NC) group; ^# P < 0·05, ^## P < 0·01 compared with the peak.

Discussion

Increasing evidence suggests that several Th subsets and their specific cytokines, such as Th2/IL‐13, Th17/IL‐17 and Tfh/IL‐21, play important roles in the pathogenesis of liver fibrosis caused by schistosomiasis.13, 32, 33 Our previous study has shown that Th9/IL‐9 took part in the development of schistosomiasis.31 However, whether IL‐9 contributes to the pathology of this disease is unknown.

In this study, we established a mouse model of schistosomiasis and treated the mouse model with anti‐IL‐9 antibody or IgG by intraperitoneal injection for 9 weeks. Unlike the results of a previous study by Li et al.,34 we observed that the volumes of hepatic egg granulomas were smaller in the anti‐IL‐9 group than in the IgG group. These indicated that IL‐9 might contribute to aggravate liver granulomatous inflammation. It is well known that a granulomatous reaction appears at the period of oviposition around 5–6 weeks after infection. A large number of eosinophils, macrophages, lymphocytes, mast cells, mononuclear cells and neutrophils gather around schistosome eggs to protect the host against excessive pathology. Extensive studies have indicated that IL‐9 might be related to the infiltration and maturation of these inflammatory cells. For example, IL‐9 may promote influx and local maturation of eosinophils in a mouse model of peritoneal inflammation induced by carbon thioglycolate.35 Another study showed that IL‐9 might accumulate mast cells and neutralization of IL‐9 could reduce mast cell infiltration in experimental autoimmune encephalomyelitis.36 Other studies found that IL‐9 could induce the migration of Th17 cells to promote inflammation.37, 38 In addition, IL‐9 potentiates the production of immunoglobulin by B lymphocytes.39 A recent study also demonstrated that IL‐9 might facilitate the survival of neutrophils in rheumatoid arthritis.40 Therefore, in our model it is possible that anti‐IL‐9 effectively reduced egg granulomatous inflammation and decreased inflammatory cell infiltration around the trapped eggs through mechanisms similar to those mentioned above.

More importantly, we found that the degree of liver fibrosis was also alleviated after neutralization of IL‐9. This result was consistent with the findings of Li et al.,34 but the mechanisms of IL‐9 pro‐fibrosis remain unknown. As the granuloma matures, fibroblasts, which lead to production of extracellular matrix and collagen fibres, were recruited at the outer zone of the granuloma, resulting in fibrosis.41 Activation of HSCs is a critical process to drive liver fibrogenesis because of their production of a large amount of collagen. It is known that α‐SMA is a key indicator of HSC activation. Hence, we isolated HSCs from normal mice and cultured them in the presence of IL‐9 for 48 and 72 hr. Our results showed that IL‐9 could increase the expression of α‐SMA and promote the secretion of collagen‐I and collagen‐III from HSCs. Thus, IL‐9 plays a pro‐fibrogenic role through up‐regulating α‐SMA, collagen‐I and collagen‐III.

Interleukin‐9‐producing T cells were initially associated with Th2 cells and it was therefore categorized as a member of the Th2 cytokines. Recently, the production of IL‐9 cells has been linked to a unique subset of Th cells, termed Th9 cells.42 Studies have shown that the differentiation of Th9 cells requires transforming growth factor‐β and IL‐4. However, IL‐4 alone leads to Th2 differentiation. Moreover, Th9 cells can be generated by culturing Th2 cells in medium supplied with transforming growth factor‐β, without IL‐4.43 Hence, there is a close relationship between IL‐9 and IL‐4. We examined the percentages of IL‐9⁺ cells and IL‐4⁺ cells in spleen, levels of IL‐9 and IL‐4 in serum, and expression of IL‐9 and IL‐4 in liver during the process of schistosomiasis. Interestingly, the expression of IL‐9 was higher and faster than that of IL‐4. Th2/IL‐4 are widely regarded as a primary promoter in the development of liver fibrosis caused by schistosomiasis. In this study, we confirmed that IL‐9 not only promoted liver granulomatous inflammation and fibrosis, but also appeared earlier than IL‐4. So our study contributes to a better understanding of the immunopathogenesis of schistosomiasis and indicated that the IL‐9 might play a role in early regulation of hepatic fibrosis in schistosomiasis.

In conclusion, our data showed the role of IL‐9 in the pathogenesis of liver fibrosis. We found that neutralization of IL‐9 might ameliorate granulomatous inflammation and fibrosis in liver. The cause may be that IL‐9 could chemotactically accumulate inflammatory cells and stimulate the activation of HSCs. Recent studies have shown that percentages of Th9 cells and IL‐9, in the plasma of individuals with chronic hepatitis B who have hepatic fibrosis and individuals with hepatitis B virus‐associated liver cirrhosis were significantly higher than those in healthy control individuals.44 Further investigation found that the percentages of splenic IL‐9⁺ cells increased quickly and peaked at the 6th week in an experimental mouse model of liver fibrosis due to carbon tetrachloride, and treatment with anti‐IL‐9 antibody effectively weakened hepatic inflammation, necrosis and fibrosis.45 Therefore, IL‐9 neutralization might be potentially beneficial to human patients with chronic hepatitis and liver fibrosis.

Disclosures

The authors declare no conflicts of interest.

Supporting information

Figure S1. Identification of hepatic stellate cell (HSC) purity. The freshly isolated primary HSCs reached approximative concentrations at (2·34 × 10⁶ (± 0·05 × 10⁶) cells/ml per mouse, the viability had a good approximation at 91%, and the purity reached a reasonable approximation at 90%. According to the specific expression of glial fibrillary acidic protein (GFAP) in HSCs, the primary HSCs of mice were investigated for GFAP immunocytochemical staining. The presence of red fluorescence in HSC cytoplasm represents GFAP‐positive cells. Exhibited immunological characteristics under excitation (a), under light microscopy (b);merge (a) and (b) (original magnification: ×200).