Host liver-derived extracellular vesicles deliver miR-142a-3p induces neutrophil extracellular traps via targeting WASL to block the development of Schistosoma japonicum
Authors: Lifu Wang, Zifeng Zhu, Yao Liao, Lichao Zhang, Zilong Yu, Ruibing Yang, Ji Wu, Zhongdao Wu, Xi Sun
Journal: Molecular Therapy (2022)

Schistosomiasis is an important neglected tropical disease. Interactions between the host immune system and schistosomes are complex. Neutrophils contribute to clearance of large pathogens primarily by releasing neutrophil extracellular traps (NETs). However, the functional role of NETs in clearing schistosomes remains unclear. Herein, we report that extracellular vesicles (EVs) derived from the liver of Schistosoma japonicum-infected mice (IL-EVs) induce NET release by delivering miR-142a-3p to target WASL and block the development of S. japonicum. WASL knockout accelerated the formation of NETs that blocked further development of S. japonicum. miR-142a-3p and NETs upregulated the expression of CCL2, which recruits macrophages that block S. japonicum development. However, S. japonicum inhibited NET formation in wild-type mice by upregulating host interleukin-10 (IL-10) expression. In contrast, in WASL knockout mice, IL-10 expression was downregulated, and S. japonicum-mediated inhibition of NET formation was significantly reduced. IL-EV-mediated induction of NET formation is thus an anti-schistosome response that can be counteracted by S. japonicum. These findings suggest that IL-EV-mediated induction of NET formation plays a key role in schistosome infection and that WASL is a potential therapeutic target in schistosomiasis and other infectious diseases. Schistosomiasis is an important neglected tropical disease. Interactions between the host immune system and schistosomes are complex. Neutrophils contribute to clearance of large pathogens primarily by releasing neutrophil extracellular traps (NETs). However, the functional role of NETs in clearing schistosomes remains unclear. Herein, we report that extracellular vesicles (EVs) derived from the liver of Schistosoma japonicum-infected mice (IL-EVs) induce NET release by delivering miR-142a-3p to target WASL and block the development of S. japonicum. WASL knockout accelerated the formation of NETs that blocked further development of S. japonicum. miR-142a-3p and NETs upregulated the expression of CCL2, which recruits macrophages that block S. japonicum development. However, S. japonicum inhibited NET formation in wild-type mice by upregulating host interleukin-10 (IL-10) expression. In contrast, in WASL knockout mice, IL-10 expression was downregulated, and S. japonicum-mediated inhibition of NET formation was significantly reduced. IL-EV-mediated induction of NET formation is thus an anti-schistosome response that can be counteracted by S. japonicum. These findings suggest that IL-EV-mediated induction of NET formation plays a key role in schistosome infection and that WASL is a potential therapeutic target in schistosomiasis and other infectious diseases. IntroductionSchistosomiasis is a neglected tropical disease that affects 240 million people globally and causes 250,000 deaths annually.1Wendt G. Zhao L. Chen R. Liu C. O'Donoghue A.J. Caffrey C.R. Reese M.L. Collins J.J. A single-cell RNA-seq atlas of Schistosoma mansoni identifies a key regulator of blood feeding.Science. 2020; 369: 1644-1649https://doi.org/10.1126/science.abb7709Crossref PubMed Scopus (44) Google Scholar,2Liu S. Zhang X. Liu Y. Yuan X. Yang L. Zhang R. Wang X. Xu F. Zhu C. Early application of caffeine improves white matter development in very preterm infants.Respir. Physiol. Neurobiol. 2020; 281: 103495https://doi.org/10.1016/j.resp.2020.103495Crossref PubMed Scopus (10) Google Scholar Female Schistosoma japonicum worms live in the mesenteric veins of host organisms and produce numerous eggs, many of which are transported to the liver. Highly immunogenic substances released by these eggs elicit host immune responses, including granulomatous inflammation and fibrotic reactions. Schistosomes can flourish in the host despite the development of a pronounced immune response. Unfortunately, no schistosomiasis vaccines are available, and praziquantel is the only drug available for treatment. Therefore, a better understanding of how the immune system responds to schistosomes may facilitate the development of effective therapeutic strategies.In schistosomiasis, schistosomes evoke responses from many immune cells, including T helper cells, eosinophils, neutrophils, and macrophages, and induce chemokine release.3Chuah C. Jones M.K. Burke M.L. McManus D.P. 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The presence of infiltrating neutrophils in S. japonicum-induced granulomas was observed in a transcriptomic study of hepatic granulomas.20Chuah C. Jones M.K. Burke M.L. Owen H.C. Anthony B.J. McManus D.P. Ramm G.A. Gobert G.N. Spatial and temporal transcriptomics of Schistosoma japonicum-induced hepatic granuloma formation reveals novel roles for neutrophils.J. Leukoc. Biol. 2013; 94: 353-365https://doi.org/10.1189/jlb.1212653Crossref PubMed Scopus (53) Google Scholar However, the role of neutrophils and subsequently formed NETs in schistosome infections remains poorly understood.Extracellular vesicles (EVs) are defined as heterogeneous plasma membrane vesicles that contain large quantities of proteins, lipids, and nucleic acids, and function in the long-distance exchange of biochemical information.21Lőrincz Á.M. Bartos B. Szombath D. Szeifert V. Timár C.I. Turiák L. Drahos L. Kittel A. Veres D.S. 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Schistosomal extracellular vesicle-enclosed miRNAs modulate host T helper cell differentiation.EMBO Rep. 2020; 21: e47882https://doi.org/10.15252/embr.201947882Crossref PubMed Scopus (38) Google Scholar We previously demonstrated that Sja-miR-71a in EVs derived from Schistosoma eggs inhibits liver fibrosis in S. japonicum infection.31Wang L. Liao Y. Yang R. Yu Z. Zhang L. Zhu Z. Wu X. Shen J. Liu J. Xu L. et al.Sja-miR-71a in Schistosome egg-derived extracellular vesicles suppresses liver fibrosis caused by schistosomiasis via targeting semaphorin 4D.J. Extracell. Vesicles. 2020; 9: 1785738https://doi.org/10.1080/20013078.2020.1785738Crossref PubMed Scopus (21) Google Scholar However, little is known about the role of host-derived EVs in infections with Schistosoma.In this study, we found that EVs derived from the liver of mice infected with S. japonicum (IL-EVs) induce the formation of NETs. The miRNA miR-142a-3p carried by IL-EVs plays a key role in IL-EV-mediated induction of NET formation via targeting of WASL. The resulting NETs block further development of S. japonicum in infected mice and attenuate the pathological progression of infection. However, S. japonicum is capable of inhibiting NET formation by upregulating the expression of interleukin-10 (IL-10).ResultsIL-EVs induced NET formationReleased EVs were isolated from liver tissues of S. japonicum-infected and uninfected mice at 56 days (Table S1). EVs were confirmed by negative-staining transmission electron microscopy (TEM) as having a diameter ranging from 50 to 150 nm and a characteristic cup-shaped morphology (Figure 1A , arrows). Western blotting analysis confirmed expression of the EV surface markers CD9, CD63, and CD81 (Figure 1B). The size distribution profile of the EVs was examined by nanoparticle tracking analysis (NTA), which revealed peak sizes of 84 nm (normal liver-derived EVs [NL-EVs]) and 75 nm (IL-EVs) (Figure 1C).A population of CD11b+Ly6G+ cells (neutrophils) surrounding the S. japonicum granuloma was observed (Figure 1D), and NL-EVs were internalized by neutrophils (Figure 1E). NETs, are released by neutrophils and neutralize and kill bacteria, fungi, viruses, and parasites.10Papayannopoulos V. Neutrophil extracellular traps in immunity and disease.Nat. Rev. Immunol. 2018; 18: 134-147https://doi.org/10.1038/nri.2017.105Crossref PubMed Scopus (1063) Google Scholar Interestingly, IL-EVs (but not NL-EVs) induced significant NET formation (Figure 1F). In addition, IL-EVs (but not NL-EVs) promoted phorbol-12-myristate-13-acetate (PMA)-induced NET formation (Figure 1G). Collectively, these data suggest that, during infection with S. japonicum, host liver tissue secretes EVs to induce the formation of NETs.IL-EVs induced the formation of NETs via delivery of miR-142a-3pmiRNAs are major components carried by EVs and play prominent roles in regulating gene expression.32Mori M.A. Ludwig R.G. Garcia-Martin R. Brandão B.B. Kahn C.R. Extracellular miRNAs: from biomarkers to mediators of physiology and disease.Cell Metab. 2019; 30: 656-673https://doi.org/10.1016/j.cmet.2019.07.011Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar We therefore assessed the expression profiles of miRNAs in IL-EVs and NL-EVs (Figures S1A and S1B). Compared with NL-EVs, miR-142a-3p levels were significantly higher in IL-EVs (Table S2; Figure 2A ). We then explored the potential role of miR-142a-3p in IL-EV-mediated induction of NET formation. Interestingly, miR-142a-3p induced significant NET formation (Figures 2B and 2C). In addition, miR-142a-3p promoted PMA-induced NET formation (Figure 2D). Furthermore, to assess the role of miR-142a-3p in IL-EV-induced NET formation, we treated cells with a miR-142a-3p inhibitor (chemically modified mature miR-142a-3p complementary single chain) to reduce miR-142a-3p levels in IL-EVs. IL-EV-mediated induction of NET formation decreased significantly after inhibition of miR-142a-3p (Figures 2E and 2F). Collectively, these results suggest that the induction of NET formation by IL-EVs is primarily mediated by the delivery of miR-142a-3p.Figure 2IL-EVs induced the formation of NETs via the delivery of miR-142a-3pShow full caption(A) miR-142a-3p levels in EVs were determined using quantitative real-time polymerase chain reaction (qRT-PCR). (B) Neutrophils were treated with PMA (500 nM, 4 h) and miR-142a-3p (50 nM, 24 h), and NETs were observed using SEM. (C) Neutrophils were treated with PMA (500 nM, 4 h) and miR-142a-3p (50 nM, 24 h), and NETs were detected based on H3cit and MPO co-localization. (D) Neutrophils were treated with PMA (500 nM, 4 h) and miR-142a-3p (50 nM, 24 h) + PMA (500 nM, 4 h), and NETs were observed using SEM. (E and F) Neutrophils were treated with PMA (500 nM, 4 h), IL-EVs (10 μg/mL, 24 h), and IL-EVs (10 μg/mL, 24 h) + miR-142a-3p inhibitor (100 nM, 24 h), and NETs were detected by immunohistochemistry and observed using SEM. (A) Unpaired two-sample t test. (C–F) One-way ANOVA with Dunnett's multiple comparison test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)miR-142a-3p induced NET formation and blocked further development of S. japonicum, thereby attenuating the pathological progression of S. japonicum infectionTo determine whether miR-142a-3p delivery by IL-EVs is involved in the progression of schistosomiasis, HBAAV2/9-miR-142a-3p recombinant adeno-associated virus (rAAV) were constructed. Mice were infected with S. japonicum and exposed to HBAAV2/9-miR-142a-3p rAAV (Figure 3A ). Liver sections reflected the highly hepatic tropism of HBAAV2/9-miR-142a-3p (Figure S2). As shown in Figure 3B, the number of CD11b+Ly6G+ neutrophils surrounding the S. japonicum granuloma significantly increased after HBAAV2/9-miR-142a-3p treatment. We then analyzed NET formation in the liver of infected mice. As expected, NETs were observed in S. japonicum granulomas (Figure S3), and a significant increase in NET formation was observed in mice treated with HBAAV2/9-miR-142a-3p (Figure 3C). Neutrophils were isolated from infected mice using Percoll density gradient centrifugation and flow cytometry (Figure S4) and then cultured in vitro for 24 h. NETs formed by these neutrophils increased markedly after HBAAV2/9-miR-142a-3p transfection (Figure 3D), and HBAAV2/9-miR-142a-3p promoted PMA-induced NET formation (Figure 3E).Figure 3miR-142a-3p in IL-EVs induced NET formationShow full caption(A) Time schedule for parasite infection and intravenous injection of rAAV vectors or PBS and sample examination. (B) Neutrophils in liver sections were detected based on CD11b and Ly6G co-localization (n = 6 mice per group). (C) NETs in liver sections were detected based on H3cit and MPO co-localization (n = 6 mice per group). (D) Neutrophils were isolated from infected mice using Percoll density gradient centrifugation and flow cytometry, and cultured in vitro for 24 h. NETs were then detected based on H3cit and MPO co-localization. (E) Neutrophils were isolated from infected mice and treated with PMA (500 nM) for 4 h and then detected based on H3cit and MPO co-localization. Differences were analyzed using one-way ANOVA with Dunnett's multiple comparison test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To obtain a better understanding of the effect of miR-142a-3p on IL-EV-induced NET formation in the progression of schistosomiasis, characteristics of the worms were examined. As shown in Figure 4A , development of both male and female worms was blocked by HBAAV2/9-miR-142a-3p treatment. Furthermore, worms collected from mice treated with HBAAV2/9-miR-142a-3p were significantly shorter in length than those obtained from mice treated with either PBS or HBAAV2/9 (Figure 4A). Furthermore, following exposure to HBAAV2/9-miR-142a-3p, a significant decrease in egg deposition in the liver was observed (Figure 4B). Hepatosplenomegaly was markedly attenuated after HBAAV2/9-miR-142a-3p treatment, and the liver and spleen indices declined significantly after HBAAV2/9-miR-142a-3p treatment (Figures 4C and 4D). In addition, thymus atrophy caused by S. japonicum infection diminished following HBAAV2/9-miR-142a-3p treatment (Figure 4E). Hematoxylin and eosin (H&E) staining of liver sections revealed that the percent egg granuloma area and area of a single granuloma declined significantly in the HBAAV2/9-miR-142a-3p-treated groups compared with the PBS- and HBAAV2/9-treated groups (Figures 4F and 4G). Masson's trichrome staining of liver sections showed significantly reduced collagen deposition in HBAAV2/9-miR-142a-3p-treated mice (Figure S5A). Similarly, significantly reduced expression of α-SMA and collagen I was observed in HBAAV2/9-Sja-miR-71a-treated mice (Figures S5B and S5C). The high expression of the inflammatory cytokines IL-1β, IL-23A, and IL-33, and tumor necrosis factor alpha (TNF-α) associated with S. japonicum infection decreased significantly after HBAAV2/9-miR-142a-3p treatment (Figures S6A–S6D).Figure 4miR-142a-3p induced NET formation to block further development of S. japonicum and attenuated the pathological progression of schistosomiasisShow full caption(A) Worms were stained with hydrochloric carmine red to assess development and length. (B) Liver egg burden was determined after digesting liver tissue in 4% KOH (n = 8–9 mice per group). (C–E) Macroscopic appearance of the liver, spleen, and thymus and related indices (n = 8–11 mice per group). (F) Hematoxylin and eosin (H&E) staining of liver sections. (G) Percent granulomatous area and area of a single granuloma were measured from H&E sections using Image Pro Plus 6.0 software (n = 9–11 mice per group). Differences were analyzed using one-way ANOVA with Dunnett's multiple comparison test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Taken together, these results suggest that IL-EVs induce NET formation by delivering miR-142a-3p, which blocks further development of S. japonicum and attenuates the pathological progression of schistosomiasis.miR-142a-3p induced NET formation via direct targeting of WASLTo further evaluate the molecular mechanisms involved in miR-142a-3p-mediated induction of NET formation, we identified genes directly targeted and thereby controlled by miR-142a-3p. TargetScan was used to predict potential miR-142a-3p targets. We found that expression of WASL in the liver of S. japonicum-infected mice declined after HBAAV2/9-miR-142a-3p treatment (Figure 5A ). Furthermore, as shown in Figure 5B, miR-142a-3p targeted four binding sites in the 3′ UTR of WASL. To confirm WASL as a direct target of miR-142a-3p, we generated a luciferase reporter plasmid containing the 3′ UTR of WASL and flanking putative miR-142a-3p-binding sites and a plasmid mutated miRNA binding sites. Dual-luciferase reporter assay results revealed that, compared with normal control mimic-transfected cells, cells transfected with miR-142a-3p mimic exhibited significantly reduced luciferase activity of the WASL construct (Figure 5B). In contrast, the miR-142a-3p mimic had no effect on the luciferase activity of the WASL construct with mutated miR-142a-3p-binding sites (Figure 5B).Figure 5miR-142a-3p induced NET formation via direct targeting of WASLShow full caption(A) WASL mRNA expression was analyzed by qRT-PCR (n = 7–10 mice per group). (B) Wild-type and mutated m-WASL-3′ untranslated regions (UTRs) were cloned into psi-CHECK-2, and four predicted binding sites of miR-142a-3p were identified in the 3′ UTR of the WASL gene; dual-luciferase reporter assay performed on HEK293T cells transfected with WASL UTR reporter plasmid together with miR-142a-3p mimic or control mimic. (C and D) Expression of N-WASP (translated from WASL) in mice livers was analyzed by western blotting and immunohistochemistry (n = 6 mice per group). (E) Neutrophils of infected mice were isolated, and the expression of N-WASP was analyzed by immunohistochemistry. (F and G) Neutrophils were treated with PMA (500 nM, 4 h) or Wiskostatin (N-WASP inhibitor, 20 μM, 24 h) + PMA (500 nM, 4 h), and NET formation was evaluated by SEM and immunohistochemistry. (A, C, and D–G) One-way ANOVA with Dunnett's multiple comparison test. (B) Unpaired two-sample t test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We analyzed the expression of neural Wiskott-Aldrich syndrome protein (N-WASP, translated from WASL) in mouse liver. As expected, N-WASP expression was markedly downregulated in the liver of mice treated with