Contribution of tissue transglutaminase to the severity of hepatic fibrosis resulting from Schistosoma japonicum infection through the regulation of IL-33/ST2 expression

Background Tissue transglutaminase (tTG)-regulating IL-13 plays an important role in the pathogenesis of liver fibrosis resulting from Schistosoma japonicum (Sj) infection. IL-33 and its receptor ST2 are involved in Th2-biased immune responses through the release of IL-5 and IL-13 and subsequent hepatic granuloma pathology induced by Sj infection. However, the relationship between tTG, IL-33/ST2, and liver fibrosis during Schistosoma infection has not been established. Results This study investigated the link between tTG and IL-33/ST2 in the induction of liver fibrogenesis during Sj infection in mice. The extent of liver fibrosis coincided with an increase in tTG and IL-33/ST2 expression in the liver of infected mice between five to eight weeks, with a peak of correlation at six weeks after Sj infection. The inhibition of tTG activity through cystamine administration or gene knockout alleviated the level of TLR4, NF-κB pathway molecules, IL-33/ST2, and the severity of liver fibrosis resulting from Sj infection. Conclusions These results indicate that during Sj infection tTG may control liver fibrosis at least partially through TLR4, NF-κB pathway activation and then IL-33/ST2. tTG, IL-33 or ST2 might be promising drug targets against liver fibrosis induced by Sj infection.


Background
Schistosomiasis is a parasitic disease affecting more than 200 million people worldwide according to a report of the World Health Organization in 2016 [1]. The main pathogenic species causing schistosomiasis are Schistosoma japonicum (Sj), S. mansoni (Sm) and S. haematobium (Sh). In China, Sj is the only parasite causing this disease. Infection of Sj leads to severe liver granulomatous immunopathology and fibrosis induced by eggs trapped in the liver [2], which are the primary cause of morbidity and mortality. Th1 and Th2 responses play key roles in the immunity to infections. A robust Th2 response to Schistosoma egg antigens is a major factor leading to liver fibrosis, while the Th1-type response remains moderate [3]. Interleukin-33 (IL-33), a member of the IL-1 superfamily [4,5], can activate mast cells, Th2, group 2 innate lymphoid cells (ILC2) and eosinophils and leads to the release of IL-5 and IL-13, the major cytokines mediating the Th2-biased immune response [6][7][8][9][10]. The hepatocyte is a major source of IL-33 in mice liver fibrosis models induced by thioacetamide or carbon tetrachloride treatment [11]. Suppression of tumorigenicity 2 (ST2) is a high affinity receptor family member for IL-33

Open Access
Parasites & Vectors *Correspondence: D.Ferrandon@ibmc-cnrs.unistra.fr; lizi1002@gzhmu.edu.cn † Zhi-Yong Li, LinZhuo Xiao and GuiYing Lin contributed equally to this work 1 Sino-French Hoffmann Institute, Guangzhou Medical University, Guangzhou 511436, Guangdong Province, People's Republic of China Full list of author information is available at the end of the article [12]. Recently, it has been shown that IL-33 and ST2 are involved in Th2-biased immune responses by triggering on the one hand IL-5 and IL-13 release and on the other hand hepatic granuloma pathology induced by Sj infection [13,14]. However, the actual mechanism used by IL-33 to promote the formation of granuloma and fibrosis in the liver during Sj infection needs to be further delineated.
Tissue transglutaminase (tTG) is a distinctive member of the transglutaminase family, as it is ubiquitously expressed, catalyzes a number of different reactions and is involved in mediating numerous molecular processes, including cell death, signaling, cytoskeleton rearrangements, ECM stabilization, and fibrosis [15,16]. We recently reported that tTG-induced regulation of IL-13 plays an important role in the pathogenesis of liver fibrosis resulting from Sj infection [17,18].
Moreover, tTG reportedly induces IL-33 expression and a subsequent Th2 response in the allergen response of airway epithelial cells [19]. Given that both IL-33/ST2 and tTG have been implicated in fibrosis through the release of IL-13, this study was undertaken to genetically validate the role of tTG in liver fibrosis during Sj infection and the relationship between tTG and IL-33/ST2. Herein, we observed that the extent of liver fibrosis in Sj infection mice is consistent with the expression levels of tTG and IL-33/ST2. Importantly, we further demonstrated that tTG activity inhibition or tTG knockout leads to a decreased expression level of IL-33/ST2 and alleviated severity of liver fibrosis. These findings indicate that tTG contributes to the severity of hepatic fibrosis resulting from Sj infection by regulating IL-33 and ST2 expression.

Mice, parasite infection and CTM, TAK242 treatment
Six to eight-week-old female C57BL/6 wild-type mice were fed with commercially available diet and housed in a controlled environment at 25 ± 2 °C. They were infected per-cutaneously through the abdomen with 20 ± 3 Sj cercariae of the Chinese mainland strain. The mice developed liver granuloma, then acute and advanced fibrosis at week 5, 6 and 8 post-infection (n = 6-10), respectively. The uninfected mice were used as normal controls (n = 6). According to our previous experiments, inhibition of tTG with CTM at the early stage of Sj infection strikingly decreases the extent of liver fibrosis [17,20]. We treated mice with CTM (10 −2 M, 100 µl per mouse, n = 4) in PBS by intraperitoneal injection once a day from day 3 to day 10, while the infection control group (n = 10) only received PBS. Two non-infected control mice groups were treated with CTM (n = 5) and PBS (n = 6) respectively. TAK242 treatment and the allocation of mice to each group were performed according to our previous study [20]. All mice were sacrificed at indicated time points and liver tissues were collected for further analysis.

Measurement of collagen deposition area in mice liver via Sirius red staining
Fresh hepatic tissues were fixed in 4% paraformaldehyde for 24 h, and then embedded with paraffin. The 4 µm liver sections were prepared and stained with Sirius red to evaluate the extent of collagen deposition, which mirrors the severity of liver fibrosis. The morphometric collagen area of Sj single egg granuloma as displayed in deep red was measured and analyzed by Image J software. Each stained section was evaluated in double-blind fashion by two independent researchers.

RNA isolation and quantitative PCR
Total RNA was extracted from fresh liver of mice using Trizol reagent. Genomic DNA was removed, and then cDNA was synthesized using PrimeScript ™ RT reagent Kit with gDNA Eraser. The relative RNA expression level of target genes was measured by real-time quantitative reverse transcription polymerase chain reaction (q-PCR) with SYBR ® Premix Ex Taq ™ II kit and the CFX96 ™

Western blotting
Fresh liver tissues of mice were placed on ice for grinding and crushing, and the total protein homogenate was prepared and then treated with protein extraction solution (RIPA:PMSF = 100:1). In order to remove nucleic acid, the protein homogenate was placed on ice for 1 h, and then centrifuged at high speed at 4 °C for 15 min. Protein concentrations were determined using the BCA Protein Assay Kit. Equal amounts of total proteins were resolved by 10% SDS-PAGE, and next transferred to PVDF. After blocking with 5% milk or bovine serum albumin (BSA), and incubating with the primary and secondary antibodies, the target protein bands were revealed by enhanced chemiluminescence (ECL) reagent. The primary antibodies used were as follows: anti-GAPDH, -tTG, -IL-33, -ST2 and anti-Col I.

Immunohistochemical (IHC) assay with semi-quantitative analysis
Endogenous peroxidase in mouse liver sections was blocked with 3% hydrogen peroxide (H 2 O 2 ). Immunohistochemical (IHC) staining assay was used to determine the expression level and location of IL-33 and ST2 in the mice liver tissue using anti-IL-33 (1:500) and anti-ST2 (1:500) primary antibodies, followed by HRP-conjugated secondary anti-rabbit or anti-mouse antibodies. The images were observed and captured with an optical microscope equipped with a camera (Olympus, Tokyo, Japan). The semi-quantitative analysis was determined by image J software and modified H-score [20].

Measurement and analysis of Sj egg load
Liver tissue sections were prepared and stained with Sirius red. The average total number of living eggs (with miracidium inside) in five fields (at a magnification of 200×) of each section was counted. Average number of eggs of each liver section was calculated and reported as the Sj egg load.

Statistical analysis
The results are given as the mean ± standard error of the mean (SEM) or standard deviation (SD) as noted of several replicates or experiments. SPSS22.0 software was used to analyze the statistical differences among multiple groups and the Studentʼs t-test, Mann-Whitney U-test were used to compare pairs of experiments. A P-value of < 0.05 was considered statistically significant.

The level of tTG and IL-33/ST2 is consistent with the severity of hepatic fibrosis resulting from Sj infection
The severity of liver granuloma and fibrosis around Sj single egg was evaluated by measuring collagen area using Sirius red staining (in red, shown in Fig. 1a, left panel). At week (wk) 6 and 8 of Sj infection, the extent of hepatic fibrosis reached significantly high levels (t-test: wk 6 and wk 8 vs wk 5: t (116) = 6.233, P < 0.0001) (Fig. 1a), in agreement with our previous studies [17,20]. However, there were no significant differences between week 6 and week 8 (t-test: wk 8 vs wk 6: t (195) = 0.1558, P = 0.8763).
To evaluate the correlation between tTG, IL-33/ST2 expression and hepatic fibrosis, the expression level of tTG, IL-33 and ST2 in mice liver were measured using RT-qPCR ( IL-33, an alarmin, has been reported to be widely expressed in the cells of liver tissue, while ST2 expression is limited to ILC2 and macrophages [11,14]. Herein, IL-33, ST2 expression was found mainly in the cells surrounding the Sj eggs. IL-33 was located in cells at the outer rim of granulomas while ST2 was found relatively more in cells inside. The role of tTG in hepatic fibrosis has been reported in several studies [15][16][17]20]; however, genetic evidence of the involvement of this enzyme in Sj-induced liver fibrosis is lacking and its specific regulatory mechanisms remain incompletely understood. IL-33 and ST2 have been reported to be associated with liver fibrosis [11]. We tested whether tTG regulates IL-33 and ST2 expression during liver fibrosis induced by Sj infection either using tTG knock-out mice or by inhibiting tTGʼs enzymatic activity. Compared with wild type (WT) mice, the extent of liver fibrosis in tTG null mice after Sj infection was decreased significantly (Mann-Whitney U-test: WT wk 6 vs tTG-/-wk 6: U (139) = 1500.05, Z = -3.249, P = 0.0010; WT wk 8 vs tTG-/-wk 8: U (257) = 6941.5, Z = -1.968, P = 0.0492) (Fig. 2a). Western blotting revealed that the expression level of Collagen I in the liver of tTG null mice after Sj infection decreased (Fig. 2b), in agreement with the Sirius Red staining analysis. The Sj egg load showed no significant differences (t-test: WT wk 6 vs tTG-/-wk 6: t (11) = 0.526, P = 0.6093; WT wk 8 vs tTG-/wk 8: t (12) = 0.117, P = 0.9088. WT wk 6 vs WT wk 8: t (11) = 0.9406, P = 0.3671; tTG-/-wk 6 vs tTG-/-wk 8: The percentage of morphometric collagen areas around Sj single egg was calculated and is shown on the right panel (Mann-Whitney U-test, **P = 0.001, *P = 0.0492). b The protein expression levels of tTG and Col1 in mice liver homogenates detected by Western blotting are displayed on the left panel, and semi-quantitative analysis is shown on the right panel. GAPDH was used as loading control. c The average Sj egg numbers present in each liver section is shown. Data are presented as the mean ± SD from approximately three to six mice per group. All in vitro experiments were performed twice or thrice. Red arrows indicate the Sj eggs (t-test, ns) t (12) = 0.3315, P = 0.7460) in all Sj infected groups, whether expressing tTG or not (Fig. 2c). Thus, it can be ruled out that the lower extent of liver fibrosis in TG2 mutant mice was due to a lower number of Sj eggs being deposited in the liver Compared to WT mice, IL-33 and ST2 protein expression level in livers of tTG-knockout mice was decreased significantly at 6-week Sj infection as monitored through Western blotting (Fig. 3a) and IHC assay [t-test: IL-33 (WT wk 6 vs tTG-/-wk 6: t (147) = 2.117, P = 0.0360); ST2 (WT wk 6 vs tTG-/-wk 6: t (78) = 4.361, P < 0.0001) (Fig. 3b, c). IL-33-and ST2-positive staining cells were mostly located on the outside rim and inside of granulomas, respectively. Fig. 3 tTG knockout down-regulated the expression level of IL-33/ST2. a The protein level of IL-33 and ST2 in mice liver homogenates detected by Western blotting is shown on the left panel, and semi-quantitative analysis is displayed on the right panel. GAPDH was used as a loading control. b, c The protein expression levels and localization of IL-33 (b) and ST2 (c) in mice liver tissue were determined by IHC assay (200× and 400× (insets); scale-bar: 50 μm), and the quantification of the signal using modified H score analysis is shown on the right. Data are presented as the mean ± SEM from 3-6 mice per group. All in vitro experiments were performed three times. Red arrows indicate the Sj eggs (t-test, *P = 0.0360, ****P < 0.0001) tTG activity, TLR4 and NF-κB pathway activation are involved in tTG-regulated IL-33/ST2 expression Cystamine (CTM), an inhibitor of tTG, selectively inhibits the activity of tTG by modifying a cysteine with disulfide interchange at the active site of tTG. To verify the effect of tTG enzyme deficiency on hepatic fibrosis in C57BL/6 mice, we treated Sj-infected mice with CTM. Compared to mock-treated mice, the severity of hepatic fibrosis in Sj-infected mice for 6 and 8 weeks with CTM treatment was obviously alleviated as shown by Sirius red staining (Fig. 4a) and ColI Western blotting analysis ( Fig. 4b) (t-test: WT wk 6 vs CTM wk 6: t (175) = 6.85, P < 0.0001; WT wk 8 vs CTM wk 8: t (213) = 3.661, P = 0.0003). As for the knock-out mice, CTM treatment did not change the Sj egg load (Fig. 4c) (t-test: WT wk 6 vs CTM wk 6: t (15)  The protein expression levels of IL-33 and ST2 in Sjinfected mice liver with CTM treatment, were reduced as shown using Western blotting (Fig. 5a) and IHC assay [t-test: IL-33 (WT wk 6 vs CTM wk 6: t (160) = 3.248, P = 0.0014); ST2 (WT wk 6 vs CTM wk 6: t (134) = 9.354, P < 0.0001)] (Fig. 5b, c). tTG has previously been reported to regulate TLR4 [20] and NF-κB pathway activation [16]. The protein expression levels of TLR4 (Fig. 6a), of some NF-κB pathway molecules including phosphorylated p65 (p-p65), p-IKKα, p-IKKβ, and IKKγ (Fig. 6b) were lowered in tTG-null mice compared to wild type (WT) mice after Sj infection. RNA expression levels of IL-33 and ST2 in mice liver induced by Sj infection were strongly reduced when TLR4 signaling was inhibited by TAK242 GAPDH was used as a loading control. b, c The protein expression levels and location of IL-33 (B) and ST2 (C) in mice liver tissue were determined by IHC assay (200× and 400× left, scale-bar: 50 μm), and the result of semi-quantitative analysis of the protein signal using modified H score is shown on the right panel. Data are presented as the mean ± SD. All experiments were performed twice or thrice. Red arrows indicate the Sj eggs (t-test, **P = 0.0014, ****P < 0.0001) drug treatment [t-test: IL-33 (WT wk 8 vs TAK wk 8: t (4) = 5.107, P = 0.0069); ST2 (WT wk 8 vs TAK wk 8: t (4) = 5.406, P = 0.0057)] (Fig. 6c).
Taken together, these results suggest that tTG activity, TLR4, and NF-κB pathway activation are at least partially involved in regulating IL-33/ST2 induction.

Discussion
In this study, we observed that tTG plays an important role in regulating IL-33/ST2 expression and alleviating the severity of liver fibrosis following Sj challenge. We rigorously established the role of tTG in the pathogenesis of liver fibrosis by using in parallel a genetic approach, tTG knockout mice, and a biochemical approach, the injection of cystamine, a selective inhibitor of tissue transglutaminase activity. Lowered IL-33 and ST2 expression following Sj infection was observed after implementing both strategies. Our results open the possibility that the fibrosis induced by Sj through the activation of tTG is mediated via the IL-33/ST2 axis.
In schistosomiasis, the immune response against Schistosoma eggs results in granuloma formation in liver and initiates the Th2-biased immunity that prevents acute mortality of the host but ultimately causes liver fibrosis [21]. IL-33 being a strong inducer of the Th2 immune response, the role of IL-33 in schistosomiasis has also been intensively studied. Mchedlidze et al. [11] reported that high levels of IL-33 in sera were critical for inducing the development of IL-13-dependent hepatic fibrosis in a murine model of Sm infection. Higher IL-33 expression also occurs in murine models of Sj [13,14] and Sh infection [22]. Furthermore, a recent study showed that IL-33 needs to synergize with two other cytokine alarmins, thymic stromal lymphopoietin (TSLP) and IL-25, to induce IL-4/IL-13-dependent inflammation and fibrosis after Sm infection [23]. Our previous results showed the association of IL-13 and tTG for the development of liver fibrosis after Sj infection [17]. We did not use IL-13 or IL-33 knockout mice or neutralizing antibody to verify that IL-13 or IL-33 play a role in liver fibrosis during Sj infection as several publications have already reported that IL-13 [24] and IL-33 [11] mediate liver fibrosis during Schistosoma infection. Herein, the extent of liver fibrosis following Sj infection correlates with the expression level of IL-33 and ST2.
The roles of tTG in the pathogenesis of organ-specific and systemic inflammatory responses including fibrosis have been documented [25]. Although an important role for tTG in liver fibrosis has been suggested by several reports [15-17, 20, 25, 26], how tTG alleviates fibrosis is not thoroughly understood. In our previous studies, we have provided evidence that tTG regulates TGF-β1 of Sj origin and IL-13 of the host and documented the importance of tTG in liver fibrosis during Sj infection [17,26]. The positive feedback regulation between tTG and Toll-Like Receptor 4 signaling in hepatic stellate cells correlates with liver fibrosis resulting from Sj infection [20]. In this study, our results utilizing tTG-/-mice and tTG inhibitor validated the importance of tTG during the pathogenesis of liver fibrosis induced by Sj infection and indicated that tTG is a putative novel disease target of parasite-induced liver fibrosis. tTG-null mice displayed reduced IL-33 expression following induction of allergic asthma compared to those in the WT control [19], which was also found in tTG-/-mice and tTG inhibitor-treated mice in our study of Sj parasitism. Our results taken in the light of this previous work suggests that tTG-IL-33/ ST2 regulation may commonly happen in the pathogenesis of diseases displaying a Th2-biased immune response.
In mouse and human corneal epithelial cells, short ragweed pollen-stimulated IL-33 production was blocked by TLR4 antibody and a NF-κB inhibitor [27]. As shown in [27] and previous studies [16,20], tTG is involved in TLR4 signaling [20] and NF-κB pathway activation [16]. Furthermore, the full induction of IL-33 and ST2 by Sj infection appears to be dependent on NF-κB signaling. How tTG regulates TLR4 or IL-33/ST2 will require further investigations, including the identification of the cell types involved in the process and the determination of whether the regulation is direct or indirect, given tTG's multiple functions.
Herein, we evaluated collagen deposition area around Sj single egg but not multiple neighboring eggs included in one granuloma using Sirius red staining, which provides solid evidence of the extent of liver fibrosis. Here, the extent of liver fibrosis of C57BL/6 mice between week 6 and 8 post-infection showed no significant difference, whereas it was not the case in Sj-infected Balb/C mice [17,20,26]. However, in these previous studies, we analyzed collagen area around single or multiple Sj eggs in a granuloma. Higher expression level of tTG at week 6 in comparison with week 5 and 8 post-infection in both C57BL/6 and Balb/C [17] mice liver led us to evaluate its regulation relationship with IL-33/ST2 at week 6 postinfection. Our IHC results showed that IL-33 was mostly expressed in the cells of the outer rim of the granulomas, whereas ST2 was essentially expressed inside granulomas where macrophages are extensively present [28], in keeping with the report of Peng et al. [14] that ST2-expressing cells in the liver were mainly F4/80+ macrophages. As reported, IL-33 was critical for hepatic accumulation of ILC2 with high expression of ST2, and ILC2 mediated hepatic fibrosis resulting from Sm eggs challenge [11]. The role and mechanisms of ILC2 in liver fibrosis during Sj infection also deserve further study.

Conclusions
In summary, we have validated the importance of tTG in liver fibrosis during Sj infection through knockout mice and inhibitor treatment. Our data suggest that tTG may function at least partially through the indirect induction of IL-33 and ST2 expression. The modulation of tTG activity with a consequent diminution of fibrosis in the liver may represent a potential therapeutic approach to hepatic granuloma pathology caused by S. japonicum, in as much as the absence of tTG does not appear to be required to control Sj egg numbers in infected livers.