Single-sex infection with female Schistosoma mansoni cercariae mitigates hepatic fibrosis after secondary infection

Koslowski, Nicole; Sombetzki, Martina; Loebermann, Micha; Engelmann, Robby; Grabow, Niels; Österreicher, Christoph H.; Trauner, Michael; Mueller-Hilke, Brigitte; Reisinger, Emil C.

doi:10.1371/journal.pntd.0005595

Cited by 23 publications

(40 citation statements)

References 37 publications

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“…The level of IL-4 was increased only in mice injected with X5-KI eggs complementing the significantly increased level of IL-4 observed in the lung cells and splenocytes. The expression of Ctla4, which inhibits the Th2 response [65], was increased in mice injected with AChE-X7 KI eggs, and this was reflected by the increased level of TNF (typifying a Th1 response) observed in splenocytes isolated from mice injected with AChE-X7 KI eggs. RetnlA is known to be involved in the down modulation of Th2 responses [66] and is expressed by alternatively activated macrophages [66].…”

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9-mediated genome editing ofSchistosoma mansoniacetylcholinesterase

You

Mayer

Johnston

et al. 2020

Preprint

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AbstractCRISPR/Cas9-mediated genome editing shows cogent potential for the genetic modification of helminth parasites. Here we report successful gene knock-in (KI) into the genome of the egg of Schistosoma mansoni by combining CRISPR/Cas9 with single-stranded oligodeoxynucleotides (ssODNs). We edited the acetylcholinesterase (AChE) gene of S. mansoni targeting two guide RNAs (gRNAs), X5 and X7, located on exon 5 and exon 7 of Smp_154600, respectively. A CRISPR/Cas9-vector encoding gRNA X5 or X7 was introduced by electroporation into eggs recovered from livers of experimentally infected mice. Simultaneously, eggs were transfected with a ssODN donor encoding a stop codon in all six frames, flanked by 50 nt-long 5’- and 3’-homology arms matching the predicted Cas9-catalyzed double stranded break at X5 or X7. Next generation sequencing analysis of reads of amplicon libraries spanning targeted regions revealed that the major modifications induced by CRISPR/Cas9 in the eggs were generated by homology directed repair (HDR). Furthermore, soluble egg antigen from AChE-edited eggs exhibited markedly reduced AChE activity, indicative that programmed Cas9 cleavage mutated the AChE gene. Following injection of AChE-edited schistosome eggs into the tail veins of mice, a significant decrease in circumoval granuloma size was observed in the lungs of the mice. Notably, there was an enhanced Th2 response involving IL-4, −5, −10, and-13 induced by lung cells and splenocytes in mice injected with X5-KI eggs in comparison to control mice injected with unmutated eggs. A Th2-predominant response, with increased levels of IL-4, −13 and GATA3, also was induced by X5 KI eggs in small intestine-draining mesenteric lymph node cells when the gene-edited eggs were introduced into the subserosa of the ileum of the mice. These findings confirmed the potential and the utility of CRISPR/Cas9-mediated genome editing for functional genomics in schistosomes.Author SummarySchistosomiasis is the most devastating of the parasitic helminth diseases. Currently, no vaccines are available for human use and praziquantel is the only available treatment raising considerable concern that drug resistance will develop. A major challenge faced by the schistosomiasis research community is the lack of suitable tools to effectively characterise schistosome gene products as potential new drug and/or vaccine targets. We introduced CRISPR/Cas9 mediated editing into S. mansoni eggs targeting the gene encoding acetylcholinesterase (AChE), a recognized anthelminthic drug target. We found that the major modifications induced by CRISPR/Cas9 in the eggs were generated by homology directed repair (HDR). This platform provides a unique opportunity to generate precise loss-of-function insertions into the schistosome genome. We pre-screened the activity of two guide RNAs of the AChE gene and compared/validated the mutation efficacy using next-generation sequencing analysis at the genomic level and phenotypic modifications at the protein level. That resulted in reduced AChE activity observed in AChE-edited eggs, and decreased lung circumoval granuloma size in mice injected with those edited eggs. The CRISPR/Cas9-genome editing system we established in this study provides a pivotal platform for gene functional studies to identify and test new anti-schistosome intervention targets, which can be extended to the other human schistosome species and other important parasitic helminths.

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Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9-mediated genome editing ofSchistosoma mansoniacetylcholinesterase

You

Mayer

Johnston

et al. 2020

Preprint

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“…e current study was performed as a proof-of-principle study. We previously demonstrated that the antifibrotic effect of single-sex infection with female schistosomes is associated with increased CTLA-4 expression in livers of these mice [15]. Based on these data, it was obvious for us to investigate the potential direct antifibrotic effect of CTLA-4 on S. mansoni-induced hepatic fibrosis by mimicking the effect of female worms.…”

Section: Discussionmentioning

confidence: 97%

“…With respect to murine S. mansoni infections, blocking of CTLA-4 during acute infection was associated with significant weight loss and altered type 2 cytokine responses indicating the crucial importance of this regulator during S. mansoni infection [14]. Moreover, we recently reported that single-sex infection with female S. mansoni cercariae mitigates hepatic fibrosis after secondary infection, which was associated with an increased expression of CTLA-4 in these mice [15,16].…”

Section: Introductionmentioning

confidence: 94%

“…Design. Eight-week-old female C57BL/6 mice were percutaneously infected with 50 cercariae of S. mansoni (Belo Horizonte strain) obtained from our in-house cycle of infected Biomphalaria glabrata snails (Brazilian strain) as previously described [15]. For treatment, belatacept (Nulojix, Bristol-Myers Squibb, Germany) and appropriate control antibodies (MP Biomedicals/Fisher scientific, Germany) were diluted in PBS (100 μg/ml).…”

Section: Mice Infection and Studymentioning

confidence: 99%

“…In consideration to S. mansoni infection in mice, it has been shown that blocking of CTLA-4 by anti-CTLA-4 antibodies leads to significant weight loss and altered 2 response in these mice when administered during the acute stage of infection [14]. Using a single-sex infection model of S. mansoni, we have recently shown that an initial infection with female S. mansoni reduces hepatic fibrosis of a bisexual challenge infection [15]. We concluded that this inhibited 2 response and the antifibrotic effect was due to a significant increase of CTLA-4 expression in the livers of these mice.…”

Section: Biomed Research Internationalmentioning

confidence: 99%

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Preventive CTLA-4-Ig Treatment Reduces Hepatic Egg Load and Hepatic Fibrosis inSchistosoma mansoni-Infected Mice

Sombetzki

Rabes

Bischofsberger

et al. 2019

BioMed Research International

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Background. Hepatic fibrosis and granuloma formation as a consequence of tissue entrapped eggs produced by female schistosomes characterize the pathology of Schistosoma mansoni infection. We have previously shown that single-sex infection with female schistosomes mitigates hepatic fibrosis after secondary infection. This was associated with an increased expression of cytotoxic T-lymphocyte-associated protein-4 (CTLA-4), known as a negative regulator of T cell activation. Based on these findings, we hypothesized that administration of agonistic CTLA-4-Ig (Belatacept) is capable to prevent and/or treat hepatic fibrosis during schistosomiasis. Methods. Mice were infected with 50 S. mansoni cercariae and CTLA-4-Ig, or appropriated control-Ig was administered for 4 weeks. Preventive treatment started 4 weeks after infection, before onset of egg production, and therapeutic treatment started 8 weeks after infection when hepatic fibrosis was already established. Results. When given early after infection, livers of CTLA-4-Ig-treated mice showed significantly reduced collagen deposition and decreased expression of profibrotic genes in comparison to controls. In addition, administration of CTLA-4-Ig suppressed the inflammatory T cell response in infected mice. If therapy was started at a later time point when fibrogenesis was initiated, CTLA-4-Ig had no impact on hepatic fibrosis. Conclusion. We could demonstrate that an early preventive administration of CTLA-4-Ig suppresses effector T cell function and therefore ameliorates liver fibrosis. CTLA-4-Ig administration after onset of egg production fails to treat hepatic fibrosis.

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Human serum activates the tegument of female schistosomes and supports recovery from Praziquantel

et al. 2020

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Schistosomiasis is one of the most devastating parasitic disease in the world. Schistosoma spp. survive for decades within the vasculature of their human hosts. They have evolved a vast array of mechanisms to avoid the immune reaction of the host. Due to their sexual dimorphism, with the female worm lying within the gynecophoric canal of the male worm, it is the male that is exposed to the immediate environment and the soluble parts of the host’s immune response. To understand how the worms are so successful in fending off the immune attacks of the host, comparative analyses of both worm sexes in human serum (with or without Praziquantel) were performed using scanning electron microscopy, transmission electron microscopy, and immunohistochemistry. Further, gene expression analyses of tegument-specific genes were performed. Following the incubation in human serum, males and females out of pairs show morphological changes such as an altered structure of the pits below the surface and an increased number of pits per area. In addition, female schistosomes presented a marked tuft-like repulsion of their opsonized surface. The observed resistance of females to Praziquantel seemed to depend on active proteins in the human serum. Moreover, different expression profiles of tegument-specific genes indicate different functions of female_single and male_single teguments in response to human serum. Our results indicate that female schistosomes developed different evasion strategies toward the host’s immune system in comparison to males that might lead to more robustness and has to be taken into account for the development of new anti-schistosomal drugs.

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Single-sex infection with female Schistosoma mansoni cercariae mitigates hepatic fibrosis after secondary infection

Cited by 23 publications

References 37 publications

CRISPR/Cas9-mediated genome editing ofSchistosoma mansoniacetylcholinesterase

CRISPR/Cas9-mediated genome editing ofSchistosoma mansoniacetylcholinesterase

Preventive CTLA-4-Ig Treatment Reduces Hepatic Egg Load and Hepatic Fibrosis inSchistosoma mansoni-Infected Mice

Human serum activates the tegument of female schistosomes and supports recovery from Praziquantel

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