IL-33 Involved in the Progression of Liver Fibrosis Through Innate Immunity Cells (Mφ) Regulated by ICOS/ICOSL Signaling in Early Stage of Mice Schistosomiasis

Schistosomiasis liver fibrosis is characterized by the foci of a classical type 2 granulomatous inflammation in response to egg soluble antigen. The inducible costimulator (ICOS)/ICOSL signaling can mediate Th2 polarization and regulate the disease process of chronic schistosomiasis by adaptive immune response. ICOS/ICOSL signaling could regulate innate immune cells, including B cells and macrophage (Mφ), and participate in pulmonary fibrosis. The purpose of the study was to identify whether ICOS/ICOSL signaling could regulate the polarization of Mφ, and to explore the potential role of IL-33 in ICOSL knockout (ICOSL-KO) mice with schistosomiasis. Firstly, the expression level of CD86, CD206 and IL-33 was checked respectively in ICOSL-KO and WT mice infected with Schistosoma japonicum (S. japonicum). Then recombinant IL-33 (rIL-33) was injected into ICOSL-KO mice infected with S. japonicum. Our experiments showed that the liver Mφ was successfully polarized toward to classical activated Mφ (M1) and the expression of IL-33 was inhibited in ICOSL-KO mice. Furthermore, the injection of rIL-33 successfully aggravated liver fibrosis in ICOSL-KO mice, increased the numbers of lymphocyte antigen 6C (Ly6C)hi, enhanced the expression of C-C chemokine ligand (CCL)2,, CCL5 and C-X-C motif chemokine 2 (CXCL2), and promoted polarization of T helper (Th) cells to Th2 cells, as well as induced the autophagy and apoptosis of hepatic stellate cells (HSCs). Overall, the liver fibrosis was alleviated in ICOSL-KO mice along with the reduced expression of IL-33, which could skew the polarization of Mφ toward to M1, induce Th cells activation, HSCs apoptosis and autophagy through Smad2/3 and TGF-β signaling pathway, thus participated in the homeostasis of liver fibrosis of schistosomiasis.


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Liver fibrosis is a chronic liver injury caused by a variety of factors, including viral infection, 89 mice compared with that of WT mice ( Supplementary Fig 1a, b). Histological examination was done to 90 examine the area of granulomas and to evaluate the content of fibrosis. Compared with WT mice, the size

ICOSL-KO mice upregulated the expression of M1
100 ICOSL is mainly expressed in B cells and Mφ, and the polarization of Mφ is related to liver fibrosis.

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Given the prior evidence, we hypothesized that ICOS/ICOSL signaling could also regulate the week 4 to week 8 after infection in ICOSL-KO mice compared with that of WT mice. But M2 108 decreased from week 4 to week 8 after infection in ICOSL-KO mice compared with that of WT mice 109 (Fig 1a-c). RT-PCR analysis showed that the expression of iNOS increased, while that of Arg1 110 decreased in ICOSL-KO mice, P<0.05 compared to that of WT mice (Fig 1d, e).

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These immune responses are associated with liver fibrosis in schistosomiasis [17,18]. Along with shrunken 114 size of granulomas and content of fibrosis in the liver of ICOSL-KO mice, the expression of IL-33 and ST2 115 significantly declined in murine schistosomiasis ( Supplementary Fig 2a-f). The above data proved that 116 ICOSL-KO mice have protective effect against liver fibrosis by inhibiting the expression of IL-33.

IL-33 aggravated the liver fibrosis in ICOSL-KO mice
118 To investigate if IL-33 take part in the progression of S. japonicum hepatic pathology in ICOSL-KO 119 mice, we injected exogenous IL-33 or recombinant IL-33, 1 μg/injection, once every week, totally five 120 times, to ICOSL-KO mice after S. japonicum infection [19,20]. Samples were harvested at the indicated 121 times (Fig 2a). We found that the size of the hepatic granulomas and the degree of liver fibrosis were 122 greater in rIL-33 injected mice than in control mice (P<0.05) (Fig 2b-e). Similarly, rIL-33 not only 123 enhanced collagen deposition, but also increased hydroxyproline levels in 6 weeks, 8 weeks and 10 weeks 124 after infection compared with those in control mice (P<0.05) (Fig 2f). This manifested that rIL-33 125 injection aggravated hepatic fibrosis in ICOSL-KO mice with S. japonicum. In addition, HA levels in 126 serum were significantly higher in rIL-33 injected mice than that of control mice (P<0.05) (Fig 2g).

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Taken together, these data revealed an important role of IL-33 in aggravating S. japonicum egg-induced 128 granuloma, hepatic injury, and hepatic fibrosis in ICOSL-KO mice.

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The expression of F4/80 and Ly6C increased in the liver of rIL-33 injection mice   156

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To unearth the function of IL-33 on HSCs, we examined the apoptosis rate of JS1. Our data showed that 158 the apoptosis rate of activated HSCs significantly decreased in rIL-33 group than in control group (Fig 5a,   159 b). Additionally, we analyzed the role of IL-33 in the autophagy of HSCs. As expected, the autophagy rate

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Our findings above are well in accordance with this.

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As the second signaling, ICOS/ICOSL costimulatory signaling is necessary for the homeostasis and

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Through binding to its receptor ST2, IL-33 shows crucial roles in regulating type-2 immune response 199 [31]. The schistosomal liver fibrosis was also reported to associated with Th2 polarization, caused by Th2-

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Since liver fibrosis in schistosomiasis involves strong immune components, we applied this model to 208 ICOSL-KO mice to investigate the roles of these molecules in modulating innate and adaptive immunity.

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IL-33 has been considered to be an important cytokine to adjust the immunity effect of Th2/M2, and it 210 contributes to HSCs activation and collagen deposition in liver fibrosis [35]. We premised that the Mφ macrophage. Plasma IL-33 level was suggested to negatively correlate with schistosomiasis infection [37].

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We observed that IL-33 and ST2 expressed less in Mφ of ICOSL-KO mice than of WT mice. Accordingly,

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we speculated that there are some correlations between Mφ and IL-33 in ICOSL-KO model of

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In addition, IL-33 also induces Mφ to release CCL2 that could recruit monocytes from blood into liver and  Fig 3).

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The study was supported by grants from National Natural Science Foundation of China (No. 82172294),

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the Priority Academic Program Development of Jiangsu Higher Education Institutions (YX13400214).

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The authors declare that they have no competing interests.

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Animal experiments were carried out in strict accordance with the Regulations for the Administration of

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Availability of data and material

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The data and material that support the findings of this study are available from the corresponding author 290 on reasonable request.

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All the study subjects provided informed consent to participate in this study.

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Flow cytometry analysis was performed using the FCM Calibur (BD FACSVerse system, CA).

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We thank the study participants as well as the staff involved in the collection of blood samples. We

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(a-c) The liver macrophages were collected from S. japonicum infected mice by density gradient 575 centrifugation and purified according to surface marker of F4/80 and CD11b by flow cytometry (FCM).

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The expression of CD86 and CD206 in the liver macrophage were determined by FCM (from two 577 experiments with 4 mice per group). (d, e) The expression of iNOS and Arg1 in the liver macrophage were 578 determined by qRT-PCR (from two experiments with 4 mice per group). Data are presented as the 579 mean ± SD from multi-group experiments. Statistical significance was calculated using independent t-test.