Transcriptional profiling of Microtus fortis responses to S. japonicum: New sight into Mf‐Hsp90α resistance mechanism

Abstract Aims Schistosomiasis is a parasitic disease with a chronic debilitating character caused by parasitic flatworms of the genus Schistosoma. The main disease‐causing species of Schistosoma in China is S. japonicum. M fortis has been proved to be a nonpermissive host of S. japonicum. Mf‐HSP90α (Microtus fortis heat shock protein 90alpha), the homologue of HSP90α, display anti‐schistosome effect in vitro and in vivo. In the current study, in order to investigate the mechanism of anti‐schistosome effect of Mf‐HSP90α, we conducted RNA‐Seq to obtain the transcriptome profile of M. fortis liver infected with S. japonicum at different time points. Methods and Results By mapping the differential expressed genes (DEGs) to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), we found that the JAK2/STAT1 pathway was highly enriched with an elevated level of IL‐10 and HSP90α. We then checked the IL‐10‐JAK2/STAT1‐HSP90α pathway, and found that this pathway was activated in the infected mice with S. japonicum. The expression of the molecules in this pathway was elevated on the 10th day after infection and gradually decreased on the 20th day. Conclusions The IL‐10‐JAK2/STAT1‐HSP90α axis was associated with the anti‐schistosome effect of Mf‐HSP90α, and targeting IL‐10‐JAK2/STAT1‐HSP90α axis might be a novel therapeutic strategy for developing resistance to S. japonicum infection.

effective treatment needs to be carried out in order to eliminate the threat of Schistosoma infection on human health.
Microtus fortis is one of the rodents living on the shores of the Dongting Lake, Hunan province, China, where S. japonicum is highly epidemic. M. fortis has been proved to be a nonpermissive host of S. japonicum. 6 S. japonicum starts to grow slowly, become atrophied and finally died at 3-4 weeks after infection in M. fortis. The sera of M. fortis are found to have anti-schistosome effects in vivo by passive immunization. 7,8 Moreover, many studies have been conducted using M. fortis to investigate therapeutic strategies against schistosomiasis caused by S. japonicum. [9][10][11][12][13][14] We have conducted a series of studies to unveil the mechanism of extermination effect of M. fortis. Firstly, we found that the extermination effect on schistosomula of the sera of M. fortis was stronger than that of mice, but no significant difference in antischistosomula effect was found in vitro between tissue and organ of M. fortis. 15 Then, we compared killing effect of different fractional proteins from M. fortis serum to S. japonicum juveniles, and we found the serum albumin and Karyopherin alpha 2 (KPNA2) of. M. fortis have natural resistance to S. japonicum infection. [16][17][18] Moreover, we screened a M. fortis marrow cDNA library by expression cloning, the conditioned medium of full-length of Mf-HSP90α showed antischistosome function in vitro. The mice transferred with Mf-HSP90α displayed higher reduction in worm burden and liver eggs, indicating anti-schistosome ability of Mf-HSP90α in vivo. 19 However, the exact mechanism of anti-schistosome effect of Mf-HSP90α was still unclear. Therefore, this study was designed to further investigate the mechanism of anti-schistosome effect of Mf-HSP90α.

| Animals and parasites
Sexually mature closed colonies of M. fortis, provided by Department of Laboratory Animal, Central South University, Changsha, China, were used in this study. All animals were acclimatized for a week prior to the experiment. The infected Oncomelania hupensis snails were obtained from Hunan institute of Parasite Disease, Yueyang, China. We carried out all the animal experiments in strict accordance with the Laboratory Animal Regulation (1988.11.1) and made every effort to minimize suffering. All the procedures related to the use of experimental animals had been approved by the Ethical Committee of School of Life Science, Central South University (License No.2017-2-5). We monitored the health of the animals daily. Liver issues were harvested after animals were euthanized by isoflurane exposure, frozen in liquid nitrogen, and stored at −80°C for the RNA and protein extraction. These animals were housed in groups of three per cage with free access to food and water on a 12 h light/dark cycle. Every effort was made to minimize the suffering of animals.

| Animal infection and sample collection
The infected Oncomelania hupensis snails were placed in dechlo- were used as uninfected controls. We randomly selected three per group for liver transcriptome sequencing (three biological duplications).

| Bioimformatics analysis of transcriptome
Differential expression analysis of two samples was performed using the DEGseq (2010) R package. P-value was adjusted using Benjamini and Hochberg's approach. 21 Adjusted P-value <.05 was set as the threshold for significantly differential expression. GO enrichment analysis of the differential expressed genes (DEGs) was implemented by the GOseq R packages based Wallenius noncentral hypergeometric distribution. 22

| Western blot analysis
Liver tissues were homogenized and lysed in RIPA assay buffer.
Proteins (25-35 µg) were separated using 12.5% sodium dodecyl sulphate/polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes (Millipore). After blocking, membranes were incubated with appropriate dilutions of primary antibodies, horseradish peroxidase-conjugated secondary antibodies, respectively, and visualized using the ECL system.

| Statistical analysis
All experiments were performed for at least three times. All statistical analyses were carried out using SPSS 19.0. The data values were presented as the mean ± standard deviation. Differences in mean The KOG annotation of assembled unigenes. X-axis, percentage of unigene annotated in the group; Y-axis, the name of 26 groups in KOG values between two groups were analysed by two-tailed t test, and the mean values of more than two groups were compared with oneway analysis of variance. Significant differences were shown by an asterisk (*P < .05, ** P < .01, *** P < .001).

| Illumina sequencing and data assembly
In order to analyse the expression profiles of the DEGs in the liver of M. fortis, we performed the comparative transcriptome to analyse the resistance-related genes. It was reported that massive haemorrhage in the lungs, vacuolar degeneration in the hepatocytes and dilated liver sinusoids were the major pathophysiological changes observed in M. fortis in 6-10 days after infected with S. japonicum, and these changes would gradually be recovered from the 20 th day after infection. 24 After removing adaptors and low-quality reads,   and other species (21 602, 29.4%) (Figure 1).
We then utilized GO analysis to categorize 41 162 annotated genes into three groups according to their functions, including biological process, cellular component and molecular function ( Figure 2). In the first group, 19 332, 7 016 and 534 unigenes were enriched in metabolic process, response to stimulus and immune system, respectively, (Table S1) (Figure 3 and Table S2).

| DEGs expression patterns between control and infected groups at different time points
To

| Immune-function related DEGs
KEGG analysis showed that up-regulated DEGs were present in five 'immune system' pathways, most of which were significantly enriched  Table 6).

| qRT-PCR verification of the RNA-Seq data
Eight DEGs were randomly selected just to verify the results of the RNA-Seq analysis by qRT-PCR. The results from the tested genes displayed significant differential expression among the control and infected groups at different time points, which was similar to the DEGs pattern obtained from RNAseq (Figure 8). The OAS1A, OAS2,

| Infection-induced IL-10 and JAK/STAT signalling pathway
HSP90α was profoundly increased after infected with S. japonicum

| D ISCUSS I ON
In the current study, we utilized RNA-Seq to obtain the transcriptome profile of M. fortis infected with S. japonicum for 10 days and 20 days. By performing GO and KEGG analysis of the DEGs, we found that the JAK/STAT signalling pathway was one of the most prominent enriched pathways involved in the pathological process of M. fortis infected with S. japonicum at different time points, and JAK2 and STAT1 were among the up-regulated DEGs that showed same expression patterns between the two groups infected at different time points. Moreover, IL-10 is a prototypic anti-inflammatory cytokine, and enhanced whole-blood IL-10 secretion was detected in response to cercarial excretory/secretory products after schistosome infection. 25 The IL-10-JAK-STAT axis has been suggested to participate in the pathogenesis of microbe infection. 26 In our previous study, Mf-HSP90α, KPNA2 and albumin were found to be the resistance-associated proteins of M. fortis. [17][18][19] In order to explore the resistance mechanism of Mf-HSP90α, KPNA2 and ALB, RNA-Seq was performed to obtain the transcriptome profile of the liver of M. fortis infected with S. japonicum for 10 days TA B L E 6 Immune system-related signalling pathway of DEGs in KEGG enrichement

Immune system-related signalling pathway
Sample number

Background number
Corrected
IL-10 is able to activate the JAK2/STAT1 pathway ( Figure S2) and activate promoter of HSP90α directly. 29 Moreover, HSP90 is a direct target of STAT1. 31  There were some limitations of this study. The number of the animals used in this study has not been calculated.
In conclusion, we showed that the anti-schistosome effect of Mf-HSP90α is likely to be activated by the phosphorylated JAK2/ STAT1 pathway followed by increased IL-10 secretion. Our results suggested that the IL-10-JAK2/STAT1-Mf-HSP90α axis may be responsible for resistance effect of M. fortis to S. japonicum infection.

ACK N OWLED G EM ENT
We thank the Novogene Bioinformatics Technology Co., Ltd. for conducting part of bioinformatics analysis.

CO N FLI C T O F I NTE R E S T
The authors declare no potential conflicts of interest.