Sja-let-7 suppresses the development of liver fibrosis via Schistosoma japonicum extracellular vesicles

Schistosomiasis is a fatal zoonotic parasitic disease that also threatens human health. The main pathological features of schistosomiasis are granulomatous inflammation and subsequent liver fibrosis, which is a complex, chronic, and progressive disease. Extracellular vesicles (EVs) derived from schistosome eggs are broadly involved in host-parasite communication and act as important contributors to schistosome-induced liver fibrosis. However, it remains unclear whether substances secreted by the EVs of Schistosoma japonicum, a long-term parasitic “partner” in the hepatic portal vein of the host, also participate in liver fibrosis. Here, we report that EVs derived from S. japonicum worms attenuated liver fibrosis by delivering sja-let-7 into hepatic stellate cells (HSCs). Mechanistically, activation of HSCs was reduced by targeting collagen type I alpha 2 chain (Col1α2) and downregulation of the TGF-β/Smad signaling pathway both in vivo and in vitro. Overall, these results contribute to further understanding of the molecular mechanisms underlying host-parasite interactions and identified the sja-let-7/Col1α2/TGF-β/Smad axis as a potential target for treatment of schistosomiasis-related liver fibrosis.


Introduction
Schistosomiasis is a neglected tropical disease caused by infection with blood flukes of the genus Schistosoma that affects more than 250 million people each year mainly in tropical and subtropical regions of developing countries worldwide, accounting for an estimated 1.4-3.3 million disability-adjusted life years annually [1,2].The average survival of schistosomes in human hosts is 3-10 years and reportedly up to 40 years in extreme cases [3].Adult schistosomes typically exist in worm pairs.During mating, the male-female pairs migrate to the mesenteric vessels of the host, where the females produce eggs, which are responsible for the spread and primary pathology of schistosomiasis [4].Mature female S. japonicum and S. mansoni worms produce hundreds of eggs per day and most are deposited in the liver of the host, which leads to granulomatous inflammation and eventual fibrosis due to the host immune response.Schistosomiasis is particularly threatening to human health because of the lack of effective strategies for prevention and treatment [5].
Hepatic stellate cells (HSCs) are the principal collagen-producing cells in the liver and play a crucial role in schistosome-induced fibrogenesis [6].In response to liver damage, HSCs differentiate into myofibroblasts, which are characterized by synthesis of fibril-forming (type I and III) collagen and the extracellular matrix protein α-smooth muscle actin (α-SMA) [7].Therefore, it is particularly important to elucidate the roles and mechanisms of HSCs in schistosome-induced hepatic fibrosis.
The intricate relationship of S. japonicum with the human host enables the parasite to survive and evade host immunity.Extracellular vesicles (EVs) are small vesicles (diameter, ~100 nm) generated by all cells that transport various molecules, such as nucleic acids, proteins, lipids, and metabolites, and facilitate parasite-host crosstalk [8][9][10].Schistosome eggs, the central part of granulomas, mediate activation of HSCs through secretion of schistosome miRNAs, such as sja-miR-1 [11], sja-miR-71a [12], sja-miR-2162 [13] and a novel miRNA-33 [14].Each of these miRNAs plays a regulatory role through a unique mechanism and is involved in the dynamics of liver fibrosis.Notably, sja-miR-71a exhibits anti-fibrotic effects, suggesting that schistosomes secrete anti-pathological substances to prolong the life of the host as long as possible, resulting in chronic progressive disease, such as liver fibrosis.
However, comparisons of Sj-miRNAs in the EVs derived from the S. japonicum worm [15], egg [16] and primary HSCs of infected mice [13] identified four Sj-miRNAs transported to primary HSCs via worm-derived EVs, suggesting that in addition to the schistosome egg, the worm might also be involved in the process of liver fibrosis.However, the potential contributions of Sj-miRNAs from worm-derived EVs remain largely unknown.
The results of this study revealed that sja-let-7 released mostly by adult worm-derived vesicles reduced activation of HSCs by targeting Col1α2 and further attenuated progression of liver fibrosis by mediating the TGF-β/Smad signaling pathway.These findings highlight a worm-induced fibrotic regulatory process that expands current understanding of host-parasite interactions and also provides a potential target for treatment of liver fibrosis.

Comparisons of Sj-miRNAs from the EVs of S. japonicum worms and eggs with primary HSCs of infected mice
A previous study identified eight Sj-miRNAs from primary HSCs of infected mice by RNA sequencing [13], which are reportedly transported to the cytosol of HSCs via SjEVs [17].In the present study, Sj-miRNAs derived from the EVs of S. japonicum worms [15] and eggs [16] were compared to the eight Sj-miRNAs previously identified in the primary HSCs of infected mice (S1 Table ).A Venn diagram illustrated that four Sj-miRNAs (sja-bantam, sja-miR-2a, sja-miR-10, and sja-miR-2162) were likely transported to HSCs via egg-derived EVs, while the other four Sj-miRNAs (sja-let-7, sja-miR-1, sja-miR-125b, and sja-miR-190) might be transported to HSCs via worm-derived EVs (S1 Fig) .Comparisons of Sj-miRNAs from the EVs of S. japonicum worms and eggs with primary HSCs of infected mice suggest that in addition to schistosome eggs, worms may also be involved in the process of liver fibrosis with HSCs as the main effector cells.

Sja-let-7 derived from S. japonicum worms is involved in the activation of HSCs
Adult S. japonicum worms can survive in the portal vein of the host for decades [2].A transwell coculture system was used to simulate the environment of the portal vein to determine whether molecules of worm-derived EVs are involved in activation of HSCs.In the present study, adult paired worms from two naturally permissive hosts (BALB/c and Kunming (KM) mice) were used to strengthen the generality of the conclusions (Fig 1A).Following coculture for 48 h, the eight Sj-miRNAs mentioned above [13] were selected and measured by quantitative real-time polymerase chain reaction (qPCR) to investigate whether worms directly secrete substances into HSCs.The results showed that sja-let-7, along with six other Sj-miRNAs, were significantly increased in HSCs from both transwell systems, while sja-miR-2a was not detected (Fig 1B and  1C).In addition, qPCR analysis revealed that the mRNA levels of two markers of fibrosis (α-SMA and Col1α1) were significantly increased in HSCs from both transwell systems, while Col3α1 mRNA levels were only up-regulated in the transwell systems containing worms from BALB/c mice (Fig 1D and 1E).Since adult paired worms can also lay eggs in the upper wells of the transwell systems, to exclude the effects of unknown substances from eggs, mated male (MM) worms from BALB/c and KM mice were separated in vitro and cocultured with HSCs for 48 h in both transwell systems (Fig 1A).Then, 11 previously reported Sj-miRNAs, including sja-let-7, from MM derived-EVs were selected for further experiments [18].The presence of the 10 Sj-miRNAs out of 11 in HSCs, including sja-let-7, was verified by qPCR, while sja-miR-125b were undetectable in both transwell systems (Fig 1F and 1G).
To further explore whether sja-let-7 is involved in activation of HSCs, paired adult worms from BALB/c and KM mice were treated with sja-let-7 and negative control (NC) inhibitors, respectively.After coculture for 48 h, qPCR analysis was conducted to assess the uptake efficiency of the HSCs (Fig 1H -1O).The mRNA levels of two markers of fibrosis (α-SMA and Col1α1) were significantly increased in HSCs from both transwell systems, while there was no significant change in Col3α1 mRNA levels (Fig 1H -1O).These data suggest that sja-let-7 of S. japonicum worms is involved in activation of HSCs.

SjEVs transfer sja-let-7 into HSCs and reduced their activation
A previous transwell study reported that S.mansoni worms influenced the functions of T helper (Th) cells in the lower layer via EVs [19].The involvement of SjEVs in activation of
Next, the potential involvement of SjEVs on activation of HSCs was investigated using purified SjEVs labeled with a green fluorescent dye (PKH67) and EV-depleted ESPs (Excretorysecretory products), which was confirmed by showing no particles under TEM, was taken as a control (S2A Fig) .Following treatment with PKH67-labeled EVs for 2 h, uptake of SjEVs by HSCs cells was verified by fluorescence microscopy.The green fluorescence of PKH67-labeled SjEVs was observed in LX-2 cells, demonstrating uptake of SjEVs by HSCs (Fig 2C).This phenomenon could be blocked by the endocytosis inhibitors, dynasore (Fig 2C).Subsequent in vitro studies were conducted with SjEVs at a particle concentration of 4.3×10 10 / mL.Further qPCR analysis revealed that SjEVs treatment significantly increased mRNA levels of two markers of fibrosis (Col1α1, and Col3α1) (Fig 2D).Besides, the relative expression of sja-let-7 and other five Sj-miRNAs (sja-bantam, sja-miR-2162, sja-miR-10, sja-miR190 and sja-miR-125b) in HSCs was also up-regulated after SjEVs coculture (Figs 2E and S2B).In addition, the reduction of sja-let-7 abundance significantly increased the mRNA levels of all three markers of fibrosis as compared to the NC inhibitor group (Figs S2C and S2F).Collectively, these results indicate that EVs derived from S. japonicum worms were involved in general activation of HSC with reduced fibrotic functions in the presences of EV-derived sja-let-7.

Sja-let-7 reduces the activation of HSCs by targeting the Col1α2/TGF-β/ Smad axis in vitro
Transforming growth factor-β (TGF-β) is generally considered a potent activator of HSCs [20].To further clarify the potential impact of sja-let-7 on activation of HSCs, the inhibitory effects of sja-let-7 or NC mimics on TGF-β1-induced activation of HSCs (LX-2 cells) was investigated.After 48 h, the uptake efficiency was assessed by qPCR analysis.The results showed that the mRNA levels of all three markers of fibrosis (α-SMA, Col1α1, and Col3α1) were significantly decreased as compared to the NC group (Fig 3A -3D).
Next, target genes controlled by sja-let-7 were identified to clarify the mechanisms underlying sja-let-7-induced activation of HSCs.Two databases (miRanda [21] and RNAhybrid [22]) were used to identify potential sja-let-7 targets.A Venn diagram illustrated overlap of 1167 potential target genes between the two databases.Therefore, these genes were selected for gene ontology (GO) analysis (S3A Fig and S4 Table).Col1α2, an important component of type I collagen, was linked to the GO terms "collagen trimer (n = 2)", "extracellular matrix (n = 14)", and "extracellular matrix part (n = 8)" (S3B Fig and S5 Table ).Since let-7 is relatively conserved and there are multiple let-7 family members in the host, to avoid interference of identical seed sequences of let-7 on the screening process, the target genes of all let-7 family (n = 6).(F-G) Detection of 11 Sj-miRNAs in the LX-2 cells from the transwell system composed by MM worms coming from BALB/c mice (n = 3) and KM mice (n = 6).(H-K) Treatment efficiency analysis and detection of α-SMA, Col1α1 and Col3α1 mRNA expression towards LX-2 cells from the transwell system composed by worms coming from BALB/c mice (n = 3).(L-O) Treatment efficiency analysis and detection of α-SMA, Col1α1 and Col3α1 mRNA expression towards LX-2 cells from the transwell system composed by worms coming from KM mice (n = 3).All graph data are expressed as the mean ± SD of at least three biological replicates per group.*P< 0.05, **P< 0.01, ns, not significant.Abbreviation: Sj: S. japonicum; MM: mated male.Panel A was created with Biorender.com.https://doi.org/10.1371/journal.ppat.1012153.g001

PLOS PATHOGENS
Sja-let-7 suppresses liver fibrosis via Schistosoma japonicum extracellular vesicles members were predicted and cross-referenced with the 1167 target genes of sja-let-7.The results found that Col1α2, which was not identified as a target gene of any let-7 family member, was the only target gene of sja-let-7 (S3C and S3D Fig, S6 and S7 Tables).
Based on the binding site of sja-let-7 on the 3 0 -untranslated region (UTR) of Mus musculus Col1α2, which is homologue to Homo sapiens Col1α2, a luciferase reporter plasmid was generated containing the 3 0 -UTR of M. musculus Col1α2 flanking the putative sja-let-7 binding sites (Fig 3E).The dual-luciferase reporter assay revealed that as compared to NC mimic-treated cells, sjalet-7 significantly reduced luciferase activity of the Col1α2 construct (Fig 3F).In addition, TGF-β1-mediated upregulation of Col1α2 in HSCs was downregulated by treatment with sja-let-7 mimics (Fig 3G).Furthermore, treatment with a sja-let-7 inhibitor in both transwell systems or in vitro treatment with SjEVs (described above) significantly upregulated expression of Col1α2 (S3E-S3J Fig) .These results suggest that sja-let-7 inhibited Col1α2 expression via the 3 0 -UTR.
GO analysis was conducted to further characterize the role of Col1α2.The GO terms associated with to Col1α2 included "transforming growth factor beta receptor signaling pathway", "response to transforming growth factor beta", "cellular response to transforming growth factor beta stimulus" and "SMAD binding" (S8 Table ).These findings suggest that the Col1α2/ TGF-β/Smad axis regulates sja-let-7-induced suppression of HSC activation.To test this hypothesis, components of the TGF-β/Smad signaling pathways were targeted in further experiments.The results of qPCR analysis found that the expression levels of Smad2 and Smad3, which are key components of the TGF-β/Smad signaling pathway, were significantly reduced after treatment with sja-let-7 mimics, while expression of the antagonist Smad7 was increased (Fig 3H -3J).Collectively, these results suggest that sja-let-7 reduces activation of HSCs by targeting the Col1α2/TGF-β/Smad axis.

Establishment of a BALB/c mouse model of schistosome-induced liver fibrosis
Pathological changes vary among the naturally permissive hosts of S. japonicum.Therefore, the stages of schistosome-induced liver fibrosis in mice were investigated as a foundation for subsequent analysis.Mice were percutaneously infected with 20 ± 2 cercariae and sacrificed at 2, 4, 6, 8, 10, and 12 weeks post infection (wpi).From 2 to 4 wpi, there were no significant differences in the liver and spleen indices, hematological index, or hydroxyproline content in the liver tissues of mice in the uninfected group, but all indices increased significantly from 6 wpi (S4A-S4F Fig).Histological analysis found no eggs in the liver tissues at 2 wpi.In contrast, by 4 wpi, after maturation and mating of the schistosomes, eggs were found in the liver tissues, although no granulomas were present (S5A Fig) To further evaluate the extent of liver fibrosis, the expression profiles of three markers of fibrosis (α-SMA, Col1α1, and Col3α1) were detected by qPCR analysis.The results showed that from 2 to 4 wpi, there was no significant change to the expression levels of the three markers as compared to the uninfected group, while at 6 wpi, the expression levels of all three markers were significantly increased, but then decreased to different degrees at 8-12 wpi (S5B-S5D Fig) .Besides, the expression levels of sjalet-7 and its target gene Col1α2 were also detected.Similar to the above indices, the expression levels of both sja-let-7 and Col1α2 started to increase at 6 wpi (S5E-S5F Fig) .These results indicate that 6 wpi is the initiation phase of liver fibrosis in BALB/c mice infected with 20 ± 2 cercariae and that the functionality of sja-let-7 may be initiated at this time point.
Analysis of transcriptomic data retrieved from the Gene Expression Omnibus (GEO) database showed that Col1α2 was significantly upregulated in the liver tissues of C57BL/6J mice at PLOS PATHOGENS Sja-let-7 suppresses liver fibrosis via Schistosoma japonicum extracellular vesicles the liver fibrosis stage (6 wpi and 7 wpi), suggesting that Col1α2 is a target gene of sja-let-7 and plays a pivotal role in liver fibrosis (S6 Fig).
To further verify the correlation between sja-let-7 and its target gene Col1α2 in vivo, fluorescence in situ hybridization (FISH) of mouse liver slices was conducted at 6 wpi.As shown in Fig 4, sja-let-7 and Col1α2 were widely expressed around the granuloma and co-localized with the nuclei, thereby confirming a targeting relationship.
Collectively, these results suggest that 6 wpi is the starting point of schistosome-induced liver fibrosis of the BALB/c mice model in the present study and sja-let-7 and its target gene Col1α2 are closely involved in this process.

Sja-let-7 suppresses schistosome-induced liver fibrosis in vivo
As mentioned above, 6 wpi is the starting point of schistosome-induced liver fibrosis in BALB/ c mice.Therefore, in this experiment, mice were infected with S. japonicum and administered sja-let-7 agomir, experimentally mimicing the endogenous parasite miRNA function, through the tail vein once per week for six weeks (Fig 5A).Analysis of serum and liver tissues from mice injected with sja-let-7 agomir for 6 weeks showed that the miRNAs in both samples were significantly upregulated (Fig 5B ), indicating that sja-let-7 is processed and matured in mice.Administration of sja-let-7 agomir markedly ameliorated hepatosplenomegaly, as indicated by the reductions in the liver (aspartate transaminase (AST), alanine transaminase (ALT), and hydroxyproline), spleen, and hematological indices (S7A-S7J Fig) .Besides, hematoxylin and eosin (H&E) staining of liver sections showed that the Ishak score of liver fibrosis and areas with a single egg granuloma were significantly reduced in the sja-let-7 agomir group as compared to the control group (Fig 5C -5F).Furthermore, Masson and Sirius red staining of liver sections showed significantly reduced collagen deposition in the sja-let-7 agomir-treated mice (Fig 5G -5J).
To determine the role of sja-let-7 on liver fibrosis, qPCR analysis was conducted of the liver tissues of mice from all groups.The results showed that the mRNA levels of all three markers of fibrosis (α-SMA, Col1α1, and Col3α1) were significantly reduced (Fig 6A -6C).Next, both the detection of the 3 fibrotic markers separately using immunohistochemistry (IHC) and the combined observation of the 3 fibrotic markers using immunofluorescence showed a significant decrease of positive area in the sja-let-7 agomir group compared to the infected group (Figs 6D-6E and S7K-S7P).In addition, enzyme linked immunosorbent assay (ELISA) revealed that the serum levels of α-SMA were also significantly reduced in the sja-let-7 agomir group (S7Q Fig).The process of liver fibrosis is accompanied by the secretion of large amounts of inflammatory cytokines [23].So, several inflammatory cytokines (IL-1β, IL-6, TNF-α, and HMGB1) were selected for qPCR analysis.The results revealed that the expression levels of all indices were significantly reduced in the sja-let-7 agomir-treated mice (S7R-S7U Fig) .Taken together, these findings indicate that sja-let-7 attenuated progression of schistosome-induced liver fibrosis and decreased liver inflammation in vivo.

Sja-let-7 suppression of schistosome-induced liver fibrosis is mediated by the Col1α2/TGF-β/Smad axis
To determine whether sja-let-7 also regulates liver fibrosis via the Col1α2/TGF-β/Smad axis in vivo, the expression levels of Col1α2 and components of the TGF-β/Smad signaling pathway were determined.First, the contents of type I and type III collagen fibers were quantified by polarization microscopy.As shown in Fig 7A and 7B, the content of type I collagen fibers, as indicated by bright red or yellow staining, was significantly reduced in the liver tissues of mice in the sja-let-7 agomir group.As an important component of type I collagen, the expression level of Col1α2 was measured by qPCR and immunohistochemical analyses.The results revealed that Col1α2 expression was significantly reduced at both the mRNA and protein levels, indicating that uptake of sja-let-7 miRNA agomir inhibits the sja-let-7 target gene, Col1α2 (Fig 7C -8E).Next, sja-let-7-induced suppression of liver fibrosis through the TGF-β/Smad signaling pathway was verified.Notably, sja-let-7 agomir treatment significantly reduced the expression levels of TGF-β1, Smad2, Smad3, and p-Smad2/3, while on the TGFβ-antagonist Smad7 there was an opposite effect (Fig 7F -7M).

Discussion
The present study provides evidence of a functional role of sja-let-7 in S. japonicum wormderived EVs in schistosome-induced liver fibrosis.Comparisons of Sj-miRNAs from S. japonicum worm-and egg-derived EVs and primary HSCs of infected mice found that in addition to

PLOS PATHOGENS
Sja-let-7 suppresses liver fibrosis via Schistosoma japonicum extracellular vesicles the eggs, which are the central part of a granuloma, the worm may also deliver informative substances through EVs, such as sja-let-7, to regulate activation of the host HSCs as a form of long-term symbiosis and the anti-fibrotic effect of sja-let-7 is mediated via the Col1α2/TGF-β/ Smad axis (Fig 8).
Accumulating evidence shows that Sj-miRNAs in the schistosome EVs play pivotal roles in the host-parasite interaction [24].Liu et al. [15] reported that EVs released by paired adult S. japonicum worms are primarily absorbed by mouse peripheral blood monocytes, resulting in increased production of TNF-α, which is essential for the survival and egg production of S. japonicum.Overexpression of sja-miR-125b and sja-bantam has been implicated as the main contributor to this phenomenon.With the use of a transwell system with Th cells in the lower well, Meningher et al. [19] showed that adult S. mansoni worms in the upper well differentially restricted Th2 polarization by interfering with Th2-specific transcription, which was partially mediated by sma-miR-10 in the S. mansoni EVs.Another study found that activation of the NF-kappa B target gene Map3k7, which is a critical transcription factor for Th2 differentiation, was inhibited by sma-miR-10 [25].In the present study, a similar transwell system was established with adult S. japonicum worms in the upper wells and HSCs in the lower wells to verify the hypothesis that substances carried by EVs and other soluble secreted factors of adult schistosome worms can directly regulate activation of HSCs.The presence of four Sj-miRNAs in both primary HSCs of infected mice and S. japonicum worm-derived EVs was confirmed.The use of recent methodologies to effectively isolate schistosome EVs further revealed that EVs derived from S. japonicum worms could be absorbed by HSCs, indicating the miRNA-loaded schistosomal EVs could transfer from the upper well to the lower well of the transwell system [26,27].In addition, a transwell system with adult mated male worms in the upper wells confirmed the presence of Sj-miRNAs from MM-derived EVs in HSCs [18].Although the function of these Sj-miRNAs in activation of HSCs remains unclear, the results of this study provide evidence that single-sex or bisexual infected male and female schistosome worms have different effects on liver fibrosis [28][29][30].
Among the four Sj-miRNAs from both primary HSCs of infected mice and S. japonicum worm-derived EVs, sja-let-7 was selected for further study, which has not shown high expression in schistosome egg-derived EVs based on current available studies [12,14,16].Members of the miRNA let-7 family, which were originally discovered in Caenorhabditis elegans, control the timing of stem-cell division and differentiation of nematodes [31].Other biological functions of let-7 family members include neuromusculature development and adult behaviors in flies [32], limb development in chicken and mice [33], and cell proliferation and differentiation [34].Other studies have reported the involvement of sja-let-7 in the transformation from a miracidium to sporocyst in the snail as an intermediate host and higher expression in singlesex infected than bisexual female worms, suggesting potential functions in regulation of the sexual status of female worms [35,36].In mammals, many let-7-family members function as tumor suppressors in a variety of cancers [37,38].Recent studies have revealed that some let-7-family members act as anti-fibrotic factors in various fibrotic diseases.Let-7a-5p, which is highly expressed in human bone marrow mesenchymal stem cell-derived EVs, was reported to inhibit TGF-β-induced fibroblast activation and collagen secretion in vitro and extenuate capsular stiffness in vivo by targeting TGFβRI [39].Let-7b is also reported to inhibit S. japonicum recombinant P40 protein-induced activation of HSCs by direct targeting of Col1α1 [40].In another study, transfection with a lentivirus encoding let-7b significantly reduced liver fibrosis in mice infected with S. japonicum [41].These findings suggest that some members of the let-7-family have the potential to negatively regulate fibrotic diseases.However, to our best knowledge, there have been no reports of sja-let-7 in schistosome-induced liver fibrosis.Based on the same seed sequence and highly conserved functions across species, sja-let-7 mimics and agomir were synthesized and applied in in vitro and in vivo to verify the hypothesis that substances carried by EVs of adult schistosome worms can directly regulate activation of HSCs.The results of the present study showed that sja-let-7 significantly inhibited activation of HSCs in vitro and attenuated progression of liver fibrosis of mice infected with S. japonicum.A previous study by our group found that the increase of sja-let-7 by injecting sja-let-7 mimics to the infected mice had no impact on egg burden, indicating the anti-fibrotic effect was not caused by decreased parasite reproduction, but rather the direct activities of sja-let-7 in the liver [42].In contrast, an inhibitor of sja-let-7 induced stronger activation of HSCs in vitro.
To further elucidate the molecular mechanisms of sja-let-7 in liver fibrosis, FISH analysis and a dual-luciferase reporter assay were conducted to identify direct target genes that might be controlled by sja-let-7 rather than other let-7-family members.The results confirmed that Col1α2 is a direct target of sja-let-7.A previous study also confirmed the relationship between let-7 carried by the EVs of S. mansoni and Col1α2 of the host [43].Schistosome-induced liver damage resulted in fibroblast activation and extracellular matrix accumulation, especially type I collagen, which is composed of one Col1α2 and two Col1α1 chains [44].In the present study, the upregulation of sja-let-7 down-regulated Col1α2 expression both in vitro and in vivo.In addition, upregulation of sja-let-7 inhibited expression of key components of the TGF-β/ SMAD signaling pathway, which promotes liver fibrosis by activation of HSCs and production of type I collagen [45,46].Thus, sja-let-7 suppresses liver fibrosis by inhibiting the Col1α2/ TGF-β/Smad axis, as verified by bioinformatics analysis.Furthermore, after treatment of sjalet-7 agomir, the expression of inflammatory cytokines (IL-1β, IL-6, TNF-α, and HMGB1) were also reduced, suggested alleviation of liver inflammation.
Overall, the results of this study demonstrate that sja-let-7 carried by the EVs of S. japonicum worms reduced activation of HSCs by targeting Col1α2 and further inhibited progression of liver fibrosis by mediating the TGF-β/Smad signaling pathway.These findings expand current understanding of host-parasite interactions during schistosomiasis and identified a promising target for treatment of schistosomiasis.

Ethics statement
All animal experiments were performed in accordance with the guidelines of the Committee for the Care and Use of Laboratory Animals of the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Shanghai, China, permit no.SYXK-20160010).The study protocol was approved by the Ethics and Animal Welfare Committee of the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Shanghai, China, experiment no.SV-20230505-03).

Laboratory animals, parasites and infection
Specific-pathogen-free (SPF) male BALB/c mice (6-8 weeks old; body weight 18 ± 2 g), KM mice (6-8 weeks old; body weight 20 ± 2 g) and New Zealand rabbits (7-8 weeks old; body weight 2 kg) were purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd.(Shanghai, China) and housed in SPF-grade animal rooms at the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Shanghai, China).Animals were randomly allocated to certain groups before the start of the study.
S. japonicum cercariae were obtained from the National Reference Laboratory for Animal Schistosomiasis, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Shanghai, China).BALB/c mice, KM mice or New Zealand rabbits were percutaneously infected by applying cercariae (the number of cercariae varies according to the PLOS PATHOGENS experiments and will be described in the following method sections) to the shaved skin of the abdomen.At a particular point in time, depending on different experiments, animals were euthanized and paired adult worms were collected through hepatic-portal perfusion as previously described [47].The mated male (MM) worms were carefully separated from the paired worms under a microscope, then collected.
For transfection, when cells had reached a density of 1×10 6 cells/well in the 6-well plate (or 5×10 5 cells/well in the 12-well plate), they were transfected with 100 pmol/well (or 40 pmol/ well in the 12-well plate) Sja-let-7 mimics/inhibitors (GenePharma, China) or corresponding negative control (NC) mimic/inhibitors with Lipofectamine 3000 (Invitrogen, USA) for 48 h according to the manufacturer's instructions.An additional group was named as Mock group with liposomal transfection reagent and phosphate buffer solution (PBS, Corning, USA) only.The detailed sequences of miRNA mimics/inhibitors are shown in S9 Table .For stimulation, when cells had reached a density of 1×10 6 cells/well in the 6-well plate, they were challenged with 2.5 ng/mL/well recombinant TGF-β1 for 12 h followed by correspondent transfection with Sja-let-7 or NC mimic or with PBS only.Or the cells were stimulated with SjEVs at a final particle concentration of 4.3×10 10 /mL for 2 h.
Each group of cells had at least three technical replicates.Depending on different experiments, cells were harvested for qPCR analyses at a particular point in time.

Transwell system
BALB/c or KM mice were percutaneously infected with approximately 100 cercariae and worms were collected from the infected mice at 28 days post infection (dpi).Parasites were thoroughly and gently washed three times with 20 mL PBS and then maintained in preheated DMEM.In each transwell system of 12-well plate (PET membrane, pore size 0.4 μm) (Corning, USA), LX-2 cells (5×10 5 cells/well) were placed in the lower well with 1.5 mL DMEM containing 10% FBS and 1% penicillin-streptomycin. 5 pair adult worms or 10 MM worms (28-day) coming from BALB/c or KM mice were transferred from the previous DMEM culture and placed in the upper well with 900 μL fresh DMEM containing 10% FBS and 1% penicillinstreptomycin.A transwell insert with only an unused schistosomal medium was utilized as a control.Each transwell system had at least three technical replicates and were cultured in a humidified incubator at 37˚C with 5% CO 2 .The protocols involving transfection in the transwell system are shown in S8 Fig.Cells were harvested for qRT-PCR 48 h after co-incubation was established.

Isolation and purification of SjEVs
The isolation of SjEVs was performed as previous described with modification [26,27].Briefly, New Zealand rabbits were percutaneously infected with approximately 3,000 cercariae and worms were collected through hepatic-portal perfusion at 28 dpi.Worms were gently washed 3-5 times with 50 mL PBS and were microscopically examined to ensure the teguments were intact, the dead or fragmentary ones were discarded.The remaining worms were then maintained in preheated RPMI-1640 culture medium (Corning, USA) containing 1% penicillin-streptomycin at 37˚C under 5% CO 2 at a density of ~15 worm pairs / mL for 2 h.After 2 h incubation, the supernatant was collected and fresh culture medium was added for the next collection (the whole collecting procedure could last for 3-4 days until the worms were less active).The pellets in the collected supernatant were discarded by centrifugation at 2,000× g and 14,000× g for 30 min each at 4˚C, respectively.Then, the supernatant was collected and dialyzed in PBS for 24 h at 4˚C followed by centrifugal ultrafiltration through a 3K Omega membrane (Pall, USA).The supernatant was then filtered using a 0.22 μm syringe filter (Pall, USA) and a total EV isolation kit (Thermo Fisher Scientific, USA) was used according to the manufacturer's instructions.The EV pellet was resuspended in 200 μL of PBS and then stored at -80˚C until further analysis.The supernatant after pelleting the SjEVs was collected and named as "SjEV-depleted ESPs".The workflow of SjEVs isolation and purification protocols are shown in S9 Fig.The detailed information for SjEVs collection based on "roadmap of EVs from parasitic helminths" [48] is listed in S1 Data.

Electron microscopy and NTA
SjEVs or SjEV-depleted ESPs suspension was adsorbed onto 200 mesh formvar-coated grids (Agar Scientific, UK) for 2 min at room temperature (25˚C).The grids were then stained with 2% phosphotungstic acid (Solarbio, China) for 2 min and examined under a TEM (FEI, Netherlands).The size distributions of EVs were determined by NTA using the ZetaView system (Particle Metrix, Germany).

Enzymatic digestion of SjEVs protein, mass spectrometry and data analysis
The purified EVs were dissolved in PBS.To reduce the proteins, dithiothreitol was then added at a final concentration of 10 mM followed by incubation at 37˚C for 1.5 h.Iodoacetamine was then added at a final concentration 50 mM to alkylate the proteins followed by incubation at room temperature (RT) in the dark for 40 min.Trypsin was then added at a trypsin to protein ration of 1:50 (w/w) for overnight digestion at 37˚C after 4-fold dilution in 25mM NH 4 HCO 3 buffer to achieve a final urea concentration of less than 2M.Trypsin digestion was stopped by adding trifluoroacetic acid to a final concentration of 1%.The peptides of each sample were desalted on C18 cartridges, concentrated by vacuum centrifugation and reconstituted in 40 μL of 0.1% (v/v) formic acid.
For total protein identification, LC-MS/MS, Thermo Scientific, USA) analysis was performed using a Q Exactive mass spectrometer coupled to an Easy nLC system (Thermo Scientific).Trypsin-digested peptides (~5 μg) were trapped and desalted on Zorbax 300SB-C18 peptide traps (Agilent Technologies, USA) and separated on a C18-reversed phase column (0.15 mm × 150 mm, Column Technology, USA).The Easy nLC system (Thermo Scientific, USA) was used to deliver mobile phases A (0.1% formic acid in HPLC-grade water) and B (0.1% Formic acid in 84% acetonitrile) with a linear gradient of 4-50% B (0-50 min), 50-100% B (50-54 min), and then 100% B (54-60 min) at a flow rate of 250 nL/min.The nanoliter liquid phase separation end was directly connected to the mass spectrometer.
To acquire the MS data, a data-dependent top ten method was used, in which the ten most abundant precursor ions were selected for HCD fragmentation.For survey scans (m/z 300-1800), the target value was determined based on predictive Automatic Gain Control at a resolution of 70,000 at m/z 200 and dynamic exclusion duration of 25 s.Resolution for HCD spectra was set to 17,500 at m/z 200.Normalized collision energy was 27 eV and the under fill ratio, which specifies the minimum percentage of the target value likely to be reached at maximum fill time, was defined as 0.1%.

PLOS PATHOGENS
Sja-let-7 suppresses liver fibrosis via Schistosoma japonicum extracellular vesicles Data interpretation and protein identification were performed with the MS/MS spectra data sets using the MaxQuant software (v 1.5.5.1, Max Planck Institutes, Germany) (http:// www.maxquant.org)[49] against the UniProtKB Schistosoma japonicum database (download on March 06, 2023).The search parameters were trypsin enzyme, two missed cleavages, fixed modifications of carbamidomethyl, variable modifications of oxidation, a fragment ion mass tolerance of 0.10 Da, and peptide tolerance of 20 ppm.Only proteins with at least two peptides (filtered by an ion score � 20 and false discovery rate of <0.01) uniquely assigned to the respective sequence were considered as identified.

SjEVs uptake experiment
Isolated SjEVs were labeled with PKH67 using a Green Fluorescent Labeling Kit (Sigma Aldrich, USA), and the procedures were performed according to the manufacturer's protocol.Briefly, SjEVs at a particle concentration of 4.3×10 10 /mL were stained with PKH67 dye in 500 μL of Diluent C fluid for 5 min at RT. Next, 1 mL 1% bull serum albumin (BSA, Yeasen, China) was added to stop the labeling process.Then, the labeled SjEVs were re-purified via ultracentrifugation at 100,000× g with PBS rinsing for 90 min.As a control for non-specific labeling of cells, SjEV-depleted ESPs was PKH67-stained, washed, and added to the cells as a parallel experiment.The whole procedure was conducted at 4˚C.Then, the PKH67-labeled SjEVs were co-incubated with LX-2 for 2 h in a humidified incubator at 37˚C with 5% CO 2 .For the inhibitor treatment, the cells were pretreated with 150 μM dynasore (Sigma Aldrich, USA) for 30 min at 37˚C and then incubated with PKH67-labeled SjEVs for 2 h as described above in the presence of inhibitors.Afterward, the culture medium was discarded, and then the cells were washed in PBS three times, fixed with 4% formaldehyde solution (Servicebio, China) for 15 min and washed twice more with PBS.After this, cells were then incubated with TRITC phalloidin (Yeasen, China) for 30 min and nuclei were stained with 4 0 ,6-diamidino-2-phenylindole (DAPI, Sigma Aldrich, USA) for 3 min.After washed in PBS three times to remove the remaining DAPI, the cells were observed using a fluorescence microscopy (Olympus, Japan).

Establishment of Sj-induced liver fibrosis model
To determine the initiation time of Sj-induced liver fibrosis in BALB/c mice with the infection dose of 20±2 cercariae, the overall course of pathology from 0−12 wpi were observed at first.21 mice were percutaneously infected with 20±2 cercariae to the shaved skin of the abdomen and sacrificed at 2, 4, 6, 8, 10 and 12 weeks post infection (wpi) (n = 3).Another 3 uninfected mice were placed as the uninfected group.Liver, spleen tissues and blood sample of each mouse were collected for further experiments.The distribution of samples for different experiments is shown in S10 Fig.
In the subsequent in vitro RNAi experiments, 18 BALB/c mice which were percutaneously infected with 20±2 cercariae for establishment of liver fibrosis mouse model were divided into Sj, Sj+NC agomir, Sj+Sja-let-7 agomir groups (n = 6).In the Sj+NC agomir and Sj+Sja-let-7 agomir groups, mice were injected with 120 μL of 1 OD (optical density) NC or Sja-let-7 agomir (GenePharma, China) via the tail vein once a week for 6 weeks, respectively.In another normal saline groups (control) group, mice were uninfected with cercariae and were injected with 120 μL PBS via the tail vein once a week for 6 weeks (n = 6).All mice were sacrificed at 6 wpi, liver, spleen tissues and blood sample of each mouse were collected for further experiments S10 secondary antibody at RT for 50 min in dark condition.The procedure was repeated twice with the second and the third set of primary and secondary antibodies.In order to remove the primary antibodies and secondary antibodies combined with tissue, microwave treatments were conducted between each set of antibodies.Afterward, nuclei were stained DAPI for 10 min.After washed in PBS three times to remove the remaining DAPI, the slides were observed using a fluorescence microscopy (Olympus, Japan).The percentage of positive areas was quantified by ImageJ software.
The detailed information of antibodies used in IHC and immunofluorescence experiment is presented in S10 Table.

Dual-luciferase reporter assay
To confirm Col1α2 was a target of sja-let-7, wild-type or mutant 3'UTRs of Col1α2 were chemically synthesized (GenePharma, China) and then cloned into the pmirGLO luciferase plasmid (Promega, USA).The HEK293T cells were seeded in a 24-well plate (3 × 10 5 cells/ well).When the cells density reached up to 70%, 25 pmol sja-let-7 or NC mimics, together with 500 ng wild-type Col1α2 3'UTR plasmid or mutant Col1α2 3'UTR plasmid, were transfected into the HEK293T cells using Lipofectamine 3000.Subsequently, the cells were cultured for 48 h and then collected.A Dual-luciferase Reporter Assay Kit (Promega, USA) was used to detect the effect of sja-let-7 on the luciferase activity of the Col1α2 3'UTR plasmid.

FISH
To demonstrate the relationship between Col1α2 and sja-let-7 on liver sections, FISH was performed.Briefly, according to the tissue fixation time, the slices are boiled in the retrieval solution for 10-15 min and naturally cooled.Add 20 μg/mL proteinase K (Servicebio, China) working solution to cover objectives and incubate at 37˚C for 15 min.Wash in pure water, then wash three times in PBS on a rocker device, 5 min each.Pre-hybridization solution was added to each section and incubate for 1 h at 37˚C.Then, remove the pre-hybridization solution, add the sja-let-7 probe hybridization solution with concentration of 500nM, and incubate the section in a humidity chamber and hybridize overnight at 40˚C.Then, remove the hybridization solution.Wash sections in 2×SSC (Servicebio, China) for 10 min at 37˚C, in 1×SSC two times for 5 min each at 37˚C, and wash in 0.5×SSC for 10 min at RT. Discard the solution, add the Col1α2 probe hybridization solution with concentration of 500nM, and incubate the section in a humidity chamber and hybridize overnight at 40˚C.Remove the hybridization solution.Sections were washed with 2×SSC, 1×SSC, 0.5×SSC for 5 min each at 37˚C,

PLOS PATHOGENS
Sja-let-7 suppresses liver fibrosis via Schistosoma japonicum extracellular vesicles respectively.Afterward, nuclei were stained DAPI for 10 min.The slides were observed using a fluorescence microscopy (Olympus, Japan).
The detailed information of probes used in FISH experiment is presented in S11 Table.

GEO database analysis
To investigate the differential gene expression in the liver tissues between S. japonicum infected and uninfected mice, the gene expression profiles of GSE14367 (6 wpi vs uninfected C57BL/6J mice, 7 wpi vs uninfected C57BL/6J mice) and GSE59276 (6 wpi vs uninfected C57BL/6J mice) were acquired and then assessed from the GEO database, an open database that documents high-throughput microarray empirical data [56].P-value <0.01 and |log fold change (FC)| > 2 were used as the cut-off criteria to select the significant differentially expressed genes.The genes with logFC > 2 were thought to be upregulated genes, and those with logFC < −2 were regarded as downregulated genes.

RNA extraction and mRNA/miRNA quantification
To evaluate the level of mRNAs and miRNAs in LX-2 cells and liver tissues, total RNA was extracted from LX-2 cells and liver tissues by TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions [57] and quantified by Nanodrop (Thermo Scientific USA).
For mRNAs, reverse-transcription was performed using a Hifair III 1st Strand cDNA Synthesis SuperMix for qPCR kit (Yeasen, China).The resulting cDNA was used as template for qPCR with Hieff qPCR SYBR Green Master Mix (Yeasen, China).The relative mRNA expression levels of genes were quantified with Gapdh served as an endogenous control.The Light-Cycler 96 system (Roche, China) was used for qPCR analysis.The cycling conditions were as follows: preincubation, 95˚C for 60 s; 2 step amplification, 95˚C for 5 s, and 60˚C for 30 s, for 40 cycles; melting, 95˚C for 10 s, 65˚C for 60 s, 97˚C for 1 s.For miRNAs, the first-strand cDNA was reverse-transcribed using the miRNA First Strand cDNA Synthesis kit (Stem-loop Method) (Sangon biotech, China) with a stem-loop RT primer designed by each miRNA.The resulting miRNA cDNA was used as template for qPCR with MicroRNAs qPCR Kit (SYBR Green Method) (Sangon biotech, China).The relative expression levels of miRNAs were quantified with U6 served as an endogenous control.The LightCycler 96 system (Roche, China) was used for qPCR analysis.The cycling conditions were as follows: preincubation, 95˚C for 60 s; 2 step amplification, 95˚C for 5 s, and 62˚C for 30 s, for 40 cycles; melting, 95˚C for 10 s, 65˚C for 60 s, 97˚C for 1 s.All samples were assessed in triplicate.
To evaluate the level of miRNAs in the serum, all miRNAs in serum from BALB/c mice were extracted using miRcute Serum/Plasma miRNA Isolation Kit (TIANGEN, China) according to the manufacturer's recommendations.The procedures of first-strand cDNA reverse-transcription and qPCR for miRNAs were the same as above.
The 2 -ΔΔCt method [58] was used to calculate the fold change in the expression of all the mRNAs and miRNAs and all samples were assessed in triplicate.The primers used in this study are listed in S12 Table.

Statistical analysis
Data were analyzed with SPSS 25.0 software (SPSS Inc., USA) and expressed as mean ± standard deviation (SD) of three independent biological replicates.Data were statistically analyzed with Student's t-tests.A P-value of <0.05 was considered statistically significant in statistical analysis.

Fig 1 .
Fig 1. Sja-let-7 derived from S. japonicum worms involves in the activation of HSCs.(A) Workflow of transwell systems establishment.(B-C) Detection of 8 Sj-miRNAs in the LX-2 cells from the transwell system composed by worms coming from BALB/c mice (n = 3) and KM mice (n = 6) (D-E) Detection of α-SMA, Col1α1 and Col3α1 mRNA expression of the LX-2 cells from the transwell system composed by worms coming from BALB/c mice (n = 3) and KM mice

Fig 8 .
Fig 8. Graphical abstract of the mechanism.The S. japonicum worms dwelling in the host portal vein release SjEVs that contain sja-let-7 to suppress the schistosome-induced liver fibrosis via Col1α2/TGF-β/Smad axis.This figure was created with Biorender.com.https://doi.org/10.1371/journal.ppat.1012153.g008 Fig. The workflow of in vitro RNAi experiments of Sj-induced liver fibrosis model is shown in Fig 5A.The detailed sequences of miRNA agomirs are shown in S9 Table.
Sja-let-7 suppresses liver fibrosis via Schistosoma japonicum extracellular vesicles S1 Data.Details for SjEVs collection and function analysis.(DOCX) S2 Data.Excel spreadsheet containing, in separate sheets, the underlying numerical data and statistical analysis for Figures.(XLSX)