Omega-1 knockdown in Schistosoma mansoni eggs by lentivirus transduction reduces granuloma size in vivo

Hagen, Jana; Young, Neil D.; Every, Alison L.; Pagel, Charles N.; Schnoeller, Corinna; Scheerlinck, Jean-Pierre Y.; Gasser, Robin B.; Kalinna, Bernd H.

doi:10.1038/ncomms6375

Cited by 66 publications

(67 citation statements)

References 63 publications

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“…A long-established model of the schistosome egg granuloma employs tail vein injection of eggs into mice, which leads to formation of circumoval granuloma in the mouse lung (56-58). The latter approach has been extensively employed for immunopathogenesis-related studies of ω1 (16). Accordingly, to elicit circumoval granulomas, ∼3,000 WT or Δω1 LE were injected into the lateral vein of the tail of BALB/c mice.…”

Section: Resultsmentioning

confidence: 99%

Programmed genome editing of the omega-1 ribonuclease 1 of the blood fluke,Schistosoma mansoni

Ittiprasert

Mann

Karinshak

et al. 2018

Preprint

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34 35 CRISPR/Cas9 based genome editing has not yet been reported in schistosomes. Here, we tested 36 this approach by targeting omega-1 (ω1) of Schistosoma mansoni as a proof of principle. This 37 secreted ribonuclease is crucial for Th2 priming and granuloma formation, providing informative 38 immuno-pathological readouts for programmed genome editing. Schistosome eggs were either 39 exposed to recombinant Cas9 complexed with a synthetic guide RNA (sgRNA) complementary 40 to exon 6 of ω1 by electroporation or transduced with pseudotyped lentivirus encoding Cas9 and 41 the sgRNA. Some eggs were also transduced with a single stranded oligodeoxynucleotide donor 42 transgene that encoded six stop codons, flanked by 50 nt-long 5'-and 3'-microhomology arms 43 matching the predicted Cas9-catalyzed double stranded break (DSB) within ω1. CRISPResso 44 analysis of amplicons spanning the DSB revealed ~4.5% of the reads were mutated by insertions, 45deletions and/or substitutions, with an efficiency for homology directed repair of 0.19% insertion 46 of the donor transgene. Transcripts encoding ω1 were reduced >80%, and lysates of ω1-edited 47 eggs displayed diminished ribonuclease activity indicative that programmed editing mutated the 48 ω1 gene. Whereas soluble lysates of wild type eggs polarized Th2 cytokine responses including 49 IL-4 and IL-5 in human macrophage/T cell co-cultures, diminished levels of these cytokines 50followed the exposure to those of ω1-mutated schistosome eggs. Following injection of 51 schistosome eggs into the tail vein of BALB/c mice, the volume of pulmonary granulomas 52surrounding ω1-mutated eggs was 18-fold smaller than wild type eggs. Programmed genome 53 editing was active in schistosomes, Cas9-catalyzed chromosomal breakage was repaired by 54 homology directed repair and/or non-homologous end joining, and mutation of ω1 impeded the 55 capacity of schistosome eggs both to drive Th2 polarization and to provoke formation of 56 pulmonary circumoval granulomas. Knock-out of ω1 and the impaired immunological phenotype 57showcase the novel application of programmed gene editing in and functional genomics for 58 schistosomes. 59 60 61 Introduction 62 63Schistosomiasis is considered the most problematic of the human helminth diseases in terms of 64 morbidity and mortality [1][2][3][4]. The past decade has seen major advances in knowledge and 65 understanding of the pathophysiology, developmental biology, evolutionary relationships and 66 genome annotation of the human schistosomes [5][6][7][8][9][10][11][12][13][14][15][16]. Establishing CRISPR/Cas9 genome 67 editing in schistosomiasis would greatly enable effective functional genomics approaches. The 68 stable CRISPR/Cas9-based site-specific gene mutation and phenotyping will drive innovation 69 and a deeper understanding of schistosome pathogenesis, biology and evolution [17]. 70 71The schistosome egg plays a central role both in disease transmission and pathogenesis [1]. The 72 appearance of S. mansoni eggs in host tissues by six to seven week...

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Section: Resultsmentioning

confidence: 99%

Programmed genome editing of the omega-1 ribonuclease 1 of the blood fluke,Schistosoma mansoni

Ittiprasert

Mann

Karinshak

et al. 2018

Preprint

View full text Add to dashboard Cite

show abstract

“…# P < 0.05, vs the group of T2 RNase and TGF-β1 co-treatment (rT2 + TGF-β1+) potent Th2 polarization of CD4+ T cells during infection, due to its RNase activity and glycosylation (Everts et al 2009;Steinfelder et al 2009). Hagen et al (2014) have demonstrated that knockdown of Omega-1 in S. mansoni eggs could lead to a significant decrease in the size and cellular composition of granulomas. However, these findings have not established whether Omega-1 and other T2 RNase family members from schistosomes play a direct role in activation of HSCs.…”

Section: Discussionmentioning

confidence: 99%

Recombinant T2 RNase protein of Schistosoma japonicum inhibits expression of α-SMA in LX-2 cells

et al. 2016

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Recombinant T2 RNase glycoprotein, which showed a certain degree of homology to Omega-1 from Schistosoma mansoni eggs, was expressed in adult worms of Schistosoma japonicum, but not in eggs of S. japonicum. The direct biological role of the recombinant T2 RNase protein in activation of hepatic stellate cells (HSCs) remains unknown. In the present study, the immortalized human HSC line (LX-2 cells) was treated with the recombinant T2 RNase protein at indicated concentrations for various time points in vitro. The expression levels of α-smooth muscle actin (α-SMA) and Smad4 were detected by Western blot. The results showed that the recombinant T2 RNase protein significantly diminished the expression levels of α-SMA and Smad4 in LX-2 cells. The upregulated expression levels of α-SMA and Smad4 by TGF-β1 in LX-2 cells were both suppressed by the recombinant T2 RNase protein. These data suggest that the recombinant T2 RNase protein may be a potential target of therapeutic strategy for the treatment of hepatic fibrosis.

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“…Omega-1 can drive Th2 responses in dendritic cells and its cytotoxicity appears to control granuloma size [36]. Eggs from Trichuris suis (T. suis) have been tested as potential therapeutic agents against experimental and clinical inflammatory bowel disease (IBD) [37,38], but the responsible factors have not been elucdiated.…”

Section: Eggs and Soluble Egg Extracts (Sea)mentioning

confidence: 99%

Enteric nematodes and the path to up-regulation of type 2 cytokines IL-4 and IL-13

et al. 2015

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Omega-1 knockdown in Schistosoma mansoni eggs by lentivirus transduction reduces granuloma size in vivo

Cited by 66 publications

References 63 publications

Programmed genome editing of the omega-1 ribonuclease 1 of the blood fluke,Schistosoma mansoni

Programmed genome editing of the omega-1 ribonuclease 1 of the blood fluke,Schistosoma mansoni

Recombinant T2 RNase protein of Schistosoma japonicum inhibits expression of α-SMA in LX-2 cells

Enteric nematodes and the path to up-regulation of type 2 cytokines IL-4 and IL-13

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