Characteristics of IL-9 induced by Schistosoma japonicum infection in C57BL/6 mouse liver

Liver granulomatous inflammation and fibrosis were the primary pathological changes observed during Schistosoma japonicum (S. japonicum) infection. In the present study, the characteristics of IL-9 were investigated in the liver of S. japonicum infection C57BL/6 mice. Immunofluorescence, qRT-PCR, and ELISA results demonstrated that the expression of IL-9 significantly increased after infection (P < 0.01). FACS results indicated that the peak of IL-9+ Th9 cells in the liver mononuclear cells appeared at the early phase of infection (week 5), except that Th9 cells, CD8+ Tc cells, NKT and γδT cells could secrete IL-9 in this model. Although IL-9 neutralization has a limited effect on liver granulomatous inflammation, it could decrease the level of fibrosis-associated factor, PC-III, in the serum of infected mice (P < 0.05). Taken together, our results indicated that IL-9 was an important type of cytokine involved in the progression of S. japonicum infection-induced hepatic damage.

Schistosomiasis japonica is a chronic helminth infection of humans caused by S. japonicum. Although this disease has been controlled in many countries, it remains common in China, where over 50 million people are currently infected, as well as the Philippines and small pockets of Indonesia 1-3 . The primary pathological changes during S. japonicum infection are granulomatous inflammation and progressive fibrosis in liver. Additionally, the latter is a key reason for the induction of serious complications and death. The pathology of this disease is induced by CD4 + Th2 cell-driven response, with IL-4 and IL-13, as the dominant cytokines responsible, whereas IL-10, IFN-γ, IL-13Rα2 and regulatory T cells act to limit schistosome-induced pathology 4,5 . In recent studies, it was reported that increased Th17 cell populations were correlated with more severe pathology and IL-17 neutralization in an S. Japonicum infected mouse model could decrease granulomatous inflammation [6][7][8] .
IL-9 was discovered more than 20 years ago and initially described as a T cell and mast cell growth factor, produced by activated Th2 clones in vitro and during Th2-like T cell responses in vivo 9 . TGF-β interactions with IL-1 family members (IL-1α, IL-1β, IL-18, and IL-33) could trigger IL-4-independent IL-9 production by mouse CD4 + T cells 10 . IL-4 signalling promoted Th9 cell differentiation, in part, by suppressing the ability of TGF-β to induce the expression of the T regulatory (TReg) cell-associated transcription factor fork head box P3 (FOXP3) 11 . Recently, IL-9 was thought to be pleiotropic in function, and it can stimulate mast cell accumulation in tissues, promote ILC survival, enhance class switching to IgE in B cells, and alter haematopoietic progenitor cell activity, as its receptor is expressed on multiple cell types 12 . Except Th9 cells, IL-9 could be produced by mast cells, T C 9′ cells, natural killer T (NKT) cells, and other CD4 + T cells, including Th17 cells and Foxp3 + T regulatory (Treg) cells 12 .
As a prototypical type 2 cytokine, IL-9 has been implicated in many diseases, especially helminth infections. In individuals with lymphatic filariasis, antigen-specific Th9 cells were detected 13 . Licona-Limon et al. showed that Th9 cells are able to cause rapid Nippostrongylus brasiliensis worm expulsion in Rag2-deficient hosts, indicating a critical and non-redundant role for Th9 and IL-9 during parasitic worm infection 14 . In IL-9-deficient mice, after inducting pulmonary granuloma formation around the Schistosoma eggs, when compared with controls, the Isolation of hepatic lymphocytes. Mice were respectively sacrificed on week 4, 5, 6, 7, 8 and 9 after infection, and single hepatic lymphocyte suspensions were made for flow cytometry analysis. The precava was cut and then sterile saline was injected to remove blood from the livers. Livers were mechanically dissociated and processed through a 100 μm cell strainer (BD Falcon). Hepatic lymphocytes were isolated by lymphocyte separation medium. Isolated cells were washed twice in Hank's balanced salt solution and diluted to a final concentration of 2 × 10 6 cells/ml in complete RPMI-1640 medium, which contained 10% heat-inactivated foetal calf serum, 100 U/ ml penicillin and 100 μg/ml streptomycin.
Histochemistry. The experimental procedures used for routine histological examination of liver tissue were previously described 7 . Briefly, livers were removed and later fixed in 10% formalin, embedded in paraffin, and sectioned. The sections were then examined by light microscopy under 100 × or 200 × magnification after haematoxylin and eosin staining. The size of single hepatic egg granulomas was measured by computer-assisted morphometric analysis using DP-2BSW software (Olympus, Tokyo, Japan). Eighty to 100 granulomas were measured under a microscope for each group and only a visible central egg was analysed for accuracy. Granuloma size is expressed as the mean area ± SD.
For immunofluorescence detection of the Th9 cells in hepatic granuloma, fixed livers were embedded in optimal cutting temperature compound (OCT, Sakura Finetec Japan, Tokyo, Japan), frozen in liquid nitrogen and then sliced and stored at -70 °C before use. When ready for labelling, the sections of liver tissue were air-dried at room temperature for 1 h, roasted over flame 3 times, and then air-dried for another 10 min. Sections were then treated with blocking buffer (10% foetal calf serum in PBS) at room temperature for 1 h. After being washed Scientific RepoRts | 7: 2343 | DOI:10.1038/s41598-017-02422-8 with PBS three times, the sections were incubated with APC conjugated anti-mouse CD4 and PE conjugated anti-mouse IL-9 (BD, Inc., San Diego, CA) in a 1:50 dilution overnight in the dark at 4 °C. The next step, sections were washed with PBS three times. Then, the sections were treated with DAPI (10 µg/ml) for 10 min. Finally, the sections were washed with PBS three times and mounted with 50% glycerol/PBS. Fluorescent staining patterns were detected and acquired by confocal laser scanning microscope, CLSM (Leica, TCS SP8 X).
Cytokine measurement by Bio-Plex Multiplex System. Serum samples from mice infected at different points-in-time were collected to analyse the Th1/Th2 cytokines' profile using the Bio-Plex suspension array system (Bio-Rad Laboratories Inc., Hercules, CA, USA). The Bio-Plex Assay Kit, purchased from Bio-Rad, was applied to detect the Th1/Th2 cytokines including IL-2, IL-4, IL-5, IL-10, IL-12, IFN-γ, TNF-α, and GM-CSF in serum samples of infected mice and uninfected controls. The measurement method was performed according to the manufacturer's instructions. Briefly, a 12.5 µl sample was diluted 1:4 with sample diluent, incubated with antibody-coupled beads and washed. Next, the samples were incubated with biotinylated secondary antibodies, followed by incubation with streptavidin phycoerythrin. The beads readings were analysed using the Luminex System (Bio-plex 200, Bio-Rad), and the data were analysed by the Bio-Plex Manager Software (Bio-Rad).
ELISA detection. Levels of IL-9 in the supernatant of cultured cells were detected by ELISA according to the manufacturer's instructions (88-8092-22, eBioscience). The procollagen-III (PC-III) and hyaluronic acid (HA) in serum were analysed by ELISA according to the manufacturer's instructions (202124, Usbiological, abx052387 abbexa). Samples were read at 450 nm using a microplate reader (Moder ELX-800, BioTek).
Statistics. Data were analysed using SPSS (v11.0). Statistical evaluation of the difference between means was performed by unpaired, two-tailed Student's t-tests; P < 0.05 was considered to be significant.

Dynamic changes of granulomatous inflammation during S. japonicum infection. S. japonicum
infection can result in hepatic granulomatous inflammation and fibrosis. To investigate the relationship between granulomatous inflammation and infection period, the dynamic changes of the single egg granuloma area in mice livers were analysed. Haematoxylin and eosin (HE) staining of liver sections revealed the normal cellular organization of uninfected hepatic lobules, with the typical actinomorphic distribution of hepatic cord centred around central veins. In infected livers, a few small and mixed inflammatory cell foci appeared on week 3, and Schistosome eggs without granulomatous reaction could be observed on week 4. On week 5, egg granulomas appeared (mean cross-sectional area: 12.7 ± 3.5 × 10 −3 mm 2 ). With the extension of the infection period, more and larger egg granulomas and many more inflammatory cells gradually appeared in mice livers (Fig. 1A). The single egg granuloma area reached a maximum on weeks 6, seven and 8 (week 6, 66.6 ± 13.1 × 10 −3 mm 2 ; week 7, 70.8 ± 10.8 × 10 −3 mm 2 ; week 8, 67.3 ± 12.4 × 10 −3 mm 2 ). However, on week 9, the area began to decrease (week 9, 53.1 ± 9.1 × 10 −3 mm 2 ; week 10, 53.5 ± 7.3 × 10 −3 mm 2 , Fig. 1B). At the same time, serum from each group of mice was collected and levels of Th1/Th2 cytokines were detected by Bio-Plex Multiplex System, as described in Materials and Methods. The results (Fig. 1C) showed that compared to others the changes of TNF-, Il-12, IL-5, and GM-CFS were significant, and the level of these cytokines in serum increased on week 5 after infection, peaked on week 6, and then began to decrease. The serum level of IL-5 had a significant rebound on week 8.

Detection of IL-9 in S. japonicum-infected mouse liver.
To detect the existence of IL-9 in S. japonicum-infected mouse liver, C57BL/6 mice were sacrificed at 5-6 weeks after S. japonicum infection. Livers were picked out, and ice-slices were made. PE-labelled anti-mouse IL-9, APC-labelled anti-mouse CD4, and DAPI were used to do immunofluorescence. Results showed that some IL-9 + cells (red) and, seldom, some IL-9 + CD4 + Th cells (yellow) resided around Schistosome eggs ( Fig. 2A). Moreover, hepatic lymphocytes from normal and 5-6 weeks-infected mice were prepared and incubated for 5 hours with PMA and ionomycin or not, respectively. RNA was collected, cDNA were transcribed, and the level of IL-9 mRNA was detected by qRT-PCR. As shown in Fig. 2B, the IL-9 gene stripe of stimulated, infected hepatic lymphocytes was brighter than others. The qRT-PCR results indicated that the expression of IL-9 mRNA significantly increased after S. japonicum infection (Fig. 2C). Additionally, hepatic lymphocytes from normal and infected mice were isolated and cultured with or without stimulation of CD3 plus CD28 for 72 h. The cell supernatants were collected and the level of IL-9 was detected by ELISA. As shown in Fig. 2D, the concentration of IL-9 of stimulated hepatic lymphocytes was 123.395 ± 3.765 pg/ml (normal mice), and increased to 215 ± 2.89 pg/ml (infected mice, P < 0.01).

Kinetic analysis of hepatic Th9 cells during S. japonicum infection. To observe the changes in
Th9 cells during S. japonicum infection, lymphocytes were isolated from blood-free livers at 4, 5, 6, 7, 8 and 9 weeks after infection (normal control was set), and stimulated with PMA plus ionomycin for 5 hours, and intracellular cytokines staining (ICS) was done. Cells were gated on CD3 + CD4 + cells first, and the expression of IL-9 and IFN-γ was detected. The results (Fig. 3) showed that the percentage of Th9 cells increased on week 4 (0.65 ± 0.04%), reached the maximum on week 5 (1.53 ± 0.20%), decreased on week 6 to 0.81 ± 0.21%, which was close to the value on week 7 (0.81 ± 0.08%), and then continued to decrease to 0.41 ± 0.13% and 0.36 ± 0.07% on week 8 and week 9, respectively. Changes of IFN-γ + Th1 cells were similar to Th9 cells.
Source of IL-9 in S. japonicum-infected mouse liver. To detect the source of IL-9 in S. japonicum-infected mouse liver, single hepatic lymphocyte suspensions of infected mice were stimulated with PMA and ionomycin. Next, cells were stained with different fluorescent-conjugated antibodies for FACS analysis. First, CD3 + T cells were gated. Then, the proportion of IL-9 + cells in CD4-, CD8-, NK1.1-, and γδTCR-positive cells was detected, respectively. As shown in Fig. 4, CD4 + Th cells, CD8 + Tc cells, NKT and γδT cells could secrete IL-9 after non-specific stimulation during S. japonicum infection. The percentage of IL-9 + CD4 + T cells detected by FACS analysis was 1.11%, which was close to the proportion of IL-9 + NKT cells (1.23%), and higher than IL-9 + CD8 + Tc cells (0.23%) and γδT cells (0.07%).

Roles of IL-9 in S. japonicum infection-induced hepatic granulomatous and fibrosing inflammation.
To explore the role of IL-9 in hepatic granulomatous inflammation, we divided mice into three groups: control, infected and infected/anti-IL-9 mAb-treated mice. These mice were sacrificed 8 weeks after infection and livers were isolated. First, we compared the gross appearance of livers from different groups (Fig. 5A). Normal liver was light red with smooth surface. In contrast, the infected liver was dark red with many small white spots on its surface, indicating that infected mice were experiencing severe inflammation and egg pyogenic granulomas were formed in their liver. Additionally, livers from infected anti-IL-9 mAb-treated mice seemed a little shallower than the infected ones, but there were still many white spots on the surface (Fig. 5A). The sections of livers were detected by haematoxylin and eosin staining (Fig. 5B). Normal livers, showed the regular actinomorphic distribution of hepatic cord, centring around central veins. However, there were many Schistosome eggs in livers from infected mice and infected/anti-IL-9 mAb-treated mice. Around these eggs, there were a variety of inflammatory cells, which formed the granulomatous inflammation. We compared the size of a single egg granuloma in mice livers (Fig. 5C) and found that the mean size of a single granuloma from infected/anti-IL-9 mAb-treated livers was 55.04 ± 18.59 × 10 −3 mm 2 , slightly smaller than the infected group (63.34 ± 20.65 × 10 −3 mm 2 ). However, there was no significant difference between the two groups.
The level of PC-III and HA were detected by ELISA (Fig. 5D). Serum of control, infected and infected/ anti-IL-9 mAb-treated mice were collected on week 8 post infection. The level of PC-III in infected mice was significantly higher than control and anti-IL-9 groups (infected: 893.06 ± 23.42 pg/ml; Normal: 29.64 ± 19.42 pg/ml; anti-IL-9: 122.71 ± 8.25 pg/ml, Fig. 5D). In contrast, HA level in normal control mice was significantly higher than The results are expressed as mean ± SD (n = 5). (C) The levels of Th1/Th2 cytokines in mice serum were determined by using Bio-Plex multiplex system; *P < 0.05. Three independent experiments were performed, and representative results are shown.
in infected mice and anti-IL-9-treated mice (Normal: 16044.5 ± 2413.21 pg/ml; infected: 7540.27 ± 930.73 pg/ml; anti-IL-9: 10840.8 ± 3064.02 pg/ml, Fig. 5D). HA level in anti-IL-9-treated mice was higher than the infected mice, however, there was no significant difference between these two groups. Hence, the results indicate that IL-9 might influence the hepatic fibrosis by promoting the synthesis and accumulation of PC-III, whereas it moderately regulates HA expression during S. japonicum infection.

Discussion
Liver granuloma response is considered to be a specific mark of Schistosome infection 17 , and this inflammatory process is the key pathological change of the disease. Most of the time, severity and progression of disease are determined by the size of granuloma and its component. In our model of S. japonicum infection, we used praziquantel intragastric injection to establish the status of chronic S. japonicum infection in mice. As shown in Fig. 1A/B, the size of a single egg granuloma increased slowly from three weeks post-infection to five weeks post-infection, but then increased very rapidly between five and six weeks post-infection and remained at peak between six and eight weeks post-infection. These results indicated that the granulomatous inflammation is extraordinarily active during this period, as reported in many previous studies, and further implied that 6 weeks after S. japonicum infection is a good time point to observe the induced granulomatous inflammation.
It has been reported that both type 1 cytokines, such as IL-2, IL-12, IFN-γ, and type 2 cytokines, such as IL-4, IL-5, IL-10, IL-13, are involved in the liver egg granulomas in S. Mansoni infection [18][19][20] . TNF-α is a kind of   multifunctional cytokine secreted by microphage. The excessive secretion of TNF-α can induce the hyperplasia of granulocytes and fibroblasts, injure endothelial cells and promote the generation of IL-1, leading to shock, tissue injury, fibrosis, etc. In S. japonicum-infected BALB/c mice, IL-12 is considered to play an impeditive role in granuloma formation and fibrosis 21 . Therefore, we detected the levels of Th1/Th2 cytokines in serum during this process. The results showed that the expressions of TNF-α, IL-12, IL-5, IL-10 and GM-CSF in serum increased from five weeks post-infection and to peak levels at week 6 post-infection; however, as typical type 1 cytokines, the levels of IL-2 and IFN-γ in serum did not change during the key period. The similar situation appears with IL-4, a typical type 2 cytokine (Fig. 1C). These results are consistent with the granulomatous formation results (Fig. 1A/B), which demonstrated that week 6 is a good time point to observe S. japonicum infection-induced inflammatory response in mouse liver. The small change of typical Th1/2 cytokines in mouse serum is related to the low level of these cytokines in serum, as detected in previous clinical research 22,23 , and also suggested that these typical Th1/2 cytokines primarily affect the site of local inflammation.

Figure 5.
Roles of anti-IL-9 in S. japonicum infection-induced hepatic granulomatous and fibrosing inflammation. Female C57BL/6 mice were randomly divided into three groups: normal group, infected group and anti-IL-9 group. The infected group and anti-IL-9 group were infected with 40 ± 5 S. japonicum cercariae per mouse. From week 5 post-infection, the control IgG mAb or anti-IL-9 mAb were injected intraperitoneally into the infected group and anti-IL-9 group mice every 3 days, for a total of 6 times, respectively. These mice were sacrificed at 8 weeks post-infection. Mice livers were fixed in paraformaldehyde, embedded in paraffin, and then sliced and examined by haematoxylin and eosin staining. (A) The gross appearance of livers from the three groups. (B) Representative photomicrographs are shown at x100 magnification for upper panels and ×200 for lower panels. (C) Sizes of single egg granuloma were measured by computer-assisted morphometric analysis. Only granulomas with a visible central egg were analysed for accuracy. The results are expressed as mean ± SD (6 mice per group); n.s, P > 0.05. (D) Levels of pro-collagen type III (PC-III, left) and hyaluronic acid (HA, right) in the serum of mice were detected by ELISA. The results are expressed as mean ± SD; *P < 0.05, **P < 0.01. One representative result of 3 to 5 independent results is shown.
Scientific RepoRts | 7: 2343 | DOI:10.1038/s41598-017-02422-8 As mentioned before, IL-9 was a prototypical type 2 cytokine, which has been implicated in many diseases, especially helminth infections. The existence of IL-9 in S. japonicum-infected mouse liver was detected in this study. Immunofluorescent results ( Fig. 2A), PCR results ( Fig. 2B/C), and ELISA results (Fig. 2D) indicated that S. japonicum infection could induce IL-9 in mouse liver, as detected in Schistosoma mansoni-infected mice 16 . Moreover, the percentage of IL-9-secreted CD4 + Th9 cells was kinetically observed in S. japonicum-infected mouse liver by ICS. As shown in Fig. 3, the percentage of Th9 cells increased on week 4 and reached the maximum on week 5. The time of IL-9 + Th9 cells' peak in infected mouse liver was one week before the peak of the granuloma area in liver and cytokine level in serum and implied that IL-9 could mediate the pathogenic changes induced by S. japonicum.
Additionally, it was reported that IL-9 could be produced by mast cells, T C 9′ cells, natural killer T (NKT) cells, and other CD4 + T cells, including Th17 cells and Foxp3 + T regulatory (Treg) cells 12 . Even innate lymphoid cells are a significant source of IL-9 24 . In this study, the expression of IL-9 was compared in CD4 + T cells, CD8 + T cells, NKT cells and γδT cells in r S. Japonicum-infected C57BL/6 mice liver. As shown in Fig. 4, the percentage of IL-9 + CD4 + T cells was close to the proportion of IL-9 + NKT cells (1.23%) and higher than IL-9 + CD8 + Tc cells (0.23%) and γδT cells (0.07%). A similar study, showed that peripheral iNKT cells can adopt IL-9 producing NKT cell phenotype, which was able to mediate pro-inflammatory effects in vivo 25 , and suggested that NKT cell was a main source of IL-9 in S. Japonicum-infected C57BL/6 mice liver. For NKT cells, it is a kind of innate lymphocyte, implying that NKT cells might be producing IL-9 sooner than Th9 cells in response to invading S. Japonicum. Although, it was reported that IL-9 + γδT cells increased in asthmatic mice, demonstrating that γδT cells may promote inflammation by secreting IL-9 26 . The percentage of IL-9 + γδT cells in infected mouse liver is 0.44% in CD3 + T cells, which means that γδT cells are not a main source of IL-9 in S. Japonicum-infected mouse liver. However, this figure is approximately 7% in γδT cells, which is higher than other cell populations (P < 0.05). It is suggested that γδT cells are apt to secrete IL-9, and might play some function in S. Japonicum-infected induced liver damage.
Granuloma formation is the result of a host adaptive immune response mediated by CD4 + T cells specific for Schistosome egg Ags (SEA), which damaged hepatocytes and destroyed the normal histological structure of the liver. Although livers from infected anti-IL-9 mAb-treated mice appeared slightly shallower than the infected ones (Fig. 5A), no obvious difference in the size of a single egg granuloma in mice liver was detected between the two (P > 0.05), and these results differ from our findings in IL-17 blockage mice 7 ; which, indicated that the effect of IL-9 to induce inflammation in S. Japonicum-infected mouse liver is limited, and may be related to the powerful function of IL-7 in recruiting inflammatory cells 27 . The lower percentage of IL-9 + cells (Fig. 3) and lower concentration of IL-9 induced (Fig. 2D) in the infected mice liver lymphocytes may be another reason. Additionally, hyaluronic acid (HA), type III pre-collagen (PC III), collagen IV (C IV) and laminin (LN), as serum markers, were widely used in the diagnosis of liver fibrosis in patients with chronic viral infections or alcoholic liver diseases 28 . Results (Fig. 5D) demonstrated that IL-9 antibody could obviously reduce the level of PC-III in mouse serum (P < 0.05), and suggested that IL-9 could influence hepatic fibrosis by promoting the synthesis and accumulation of PC-III in the course of S. japonicum infection. Similarly, aberrant expression of cytokine interleukin 9 was found along with interleukin 4 and interferon γ in connective tissue disease-associated interstitial lung disease (association with severity of pulmonary fibrosis) 29 . Serum levels of IL-9 cytokine showed no significant differences (p > 0.05) between chronic clinical forms of Schistosoma mansoni infection disease, and in comparisons with the different classifications of periportal fibrosis 30 .

Conclusions
We demonstrated that the IL-9 + lymphocyte population significantly expands in the liver of C57BL/6 mice during infection by S. japonicum. IL-9-producing cells might mediate the immune response induced by S. japonicum in mouse liver. Anti-IL-9 mAb therapy may be helpful in preventing inflammation-mediated liver damage caused by schistosomiasis.