Elsevier

Acta Tropica

Volume 210, October 2020, 105589
Acta Tropica

Comparative transcriptome analysis of normal and CD44-deleted mouse brain under chronic infection with Toxoplasma gondii

https://doi.org/10.1016/j.actatropica.2020.105589Get rights and content

Highlights

  • “After T. gondii infection, significant changes of gene expression profiles were observed between wild type mice and CD44 deleted mice.”.

  • “Genes related with immune response were significantly up-regulated in infected brains of normal mice but not in CD44-deleted mice. The lack of CD44 would be expected to significantly decrease the ability of mice to resist T. gondii infection.”.

  • “Provides a basis data for further understanding of the immune protection mechanism of T. gondii.”.

Abstract

Toxoplasma gondii is a globally-distributed intracellular parasitic protozoon with wide host range. Chronic infection is the most prevalent form of T. gondii infection, which can lead to significant damage. CD44 plays an important role in body's immune response, however, little is known about the function and mechanism of CD44 in T. gondii infection until now. In the present study, total RNA isolated from four groups including C57BL/6 mouse (C57), C57BL/6△CD44 mouse(C57△CD44), C57BL/6 mouse infected with T. gondii (C57-TG) and C57BL/6△CD44 infected with T. gondii (C57△CD44-TG)were subjected to comparative transcriptome analyses using RNA-seq techniques to explore the possible function of CD44 in mouse brain during chronic Toxoplasma infection. The results indicated a total of 35,908, 54,428, 51,473 and 22,387 unigenes were annotated in KOG, Swissprot, GO and KEGG databases by transcriptome analysis, respectively, and all the databases shared 9,833 unigenes. Subsequently, differentially expressed GO terms and enriched KEGG Pathways showed 20,303 unigenes were annotated belonging to three main GO categories (namely biological process, cellular component and molecular function) and six main KEGG categories (cellular processes, environmental information processing, genetic information processing, human diseases, metabolism and organismal systems) between normal C57 and C57△CD44 mice, as well as for C57-TG and C57△CD44-TG mice. For up-regulated genes, Mid1, Ttr and Cd4 were significantly up-regulated in the C57△CD44 mouse compared with the C57 mouse, and Pcp2, Ppp1r17 and Nrk were significantly up-regulated in the C57△CD44-TG mouse compared with the C57-TG mouse. As to down-regulated genes, AC114588.1, Cbln3 and Pmch were significantly down-regulated in the C57△CD44 the mouse compared with the C57 mouse, and down-regulated genes were enriched for immunoglobulins, major histocompatibility complex (MHC) class II antigens, chemokines ligands and interferon (IFN)-inducible GTPase families in the C57△CD44-TG mouse compared with the C57-TG mouse. The present study is the first trial for exploring the function of CD44 in the mouse brain during chronic infection with T. gondii at the transcriptional level, which can provide a basis for the study of the host immune defense mechanism against T. gondii infection.

Introduction

Toxoplasmosis is an important zoonosis caused by T. gondii, an obligate intracellular apicomplexan parasite with a wide host range in almost all the warm-blood animals including humans (Tenter et al., 2000; Montoya and Liesenfeld, 2004; Liu et al., 2015). Over two billion people in the world were infected with T. gondii (Robert-Gangneux et al., 2012; Galvan-Ramirez Mde et al., 2012) . Generally, T. gondii infection is asymptomatic for humans with normal immunity, but is severe or even fatal for immune-compromised individuals or for the fetus (Luft and Remington, 1992; Dubey, 2004; Almeida et al., 2015; Arantes et al., 2015; Machala et al., 2015). Clinical infection is common and asymptomatic during clinical toxoplasmosis, usually with the longtime existing of cysts in the brain of patients (Hakimi et al., 2017) . However, cysts in the brain can be re-activated in practice and cause serious damage to the host when the immunity decreases (Krishnamurthy and Konstantinou, 2017).

CD44 is a highly expressed cell adhesion molecule with hyaluronan (HA) as a principal ligand, a glycosaminoglycan that is a major component of the extracellular matrix (ECM) (Toole, 1997; Ponta et al., 2003; Su et al., 2017; Rios de la Rosa et al., 2017). Previous studies indicated that infection with T. gondii contributes to the increasing expression of CD44 of T, B, NK cells, and macrophages in mice (Blass et al., 2001). The role of CD44 in the inflammatory process is conducive to the transfer of leukocytes into local inflammatory site (Lesley et al., 1993; Pure and Cuff, 2001; Ehlers et al., 2018; Dong et al., 2018). Besides, CD44 can also regulate cytotoxicity, strengthen production of pro-inflammatory cytokines and chemokines, and provide costimulation for T cells (Matsumoto et al., 1998; Lee-Sayer et al., 2018). Furthermore, CD44 has an important role in the regulation of IFN-γ production which contributes to immunopathology during T. gondii infection (Blass et al., 2001; Saraav et al., 2019, Goldszmid et al., 2007). However, knowledge about the molecular mechanism of CD44 during the chronic infection of T. gondii is still unclear, clarifying this mechanism should provide a foundation for understanding the host's immune defense against T. gondii infection.

In the present study, the post infection total gene expression patterns between normal mouse brain and CD44-deleted mouse brain were investigated using RNA-seq strategies to understand on a transcriptional level the possible function of CD44 in the brain during chronic infection with T. gondii. Our findings indicated genes related with immune responses, such as immunoglobulins, MHC and chemokines ligands, were significantly up-regulated in mouse after T. gondii infection. The transcriptome data based on RNA-seq demonstrated that the immune responses were significantly stimulated in normal C57 mouse brain after chronic infection with T. gondii, but not for CD44-deleted mouse brain. The present study should provide basis for understanding the possible function of CD44 in the mouse brain after chronic infection with T. gondii, as well as contribute to the development of novel targets for the prevention and control of toxoplasmosis.

Section snippets

Mice

Ten-week-old male wild-type C57BL/6 female mice and CD44-deleted C57Bl/6 female mice in the present were obtained from Hubei Provincial Center for Diseases Control and Prevention (Wuhan, China). All the mice were kept under SPF condition of the experimental animal center of Hubei Province. The study was performed under the instructions and approval of Laboratory Animals Research Centre of Hubei province in P. R. China and the ethics committee of Huazhong Agricultural University (Permit number:

Illumina sequencing and read mapping

For each group, more than 3.3 × 107 raw sequences were generated (Table S2). By elimination of low-quality reads, poly-N reads, 3′ adaptor sequences, and the short reads, 32–48 million good sequences with average length of 150 bp were obtained from each group. For each group, more than 96% sequences were high-quality, indicating that the cDNA library quality was suitable for subsequent analysis.

Then, the clean reads were compared with the Mouse (M. musculus) and T. gondii genome, respectively.

Discussion

T. gondii infection results in the appearance of a population of CD44. Furthermore, administration of anti-CD44 to infected mice prevents the development of a CD4+ T cell-dependent, infection-induced inflammatory response in small intestine characterized by the over-expression of IFN-γ(Blass et al., 2001; Saraav et al., 2019). Thus, there is an urgent need to explore the function of CD44 in mouse during Toxoplasma infection. In this research, the gene expression profile between normal and

Conclusion

In summary, the present study is helpful to understand the response of the host to chronic T. gondii infection, and the results of the study serves as a model to investigate the important role of CD44 in assisting the host against chronic infection of T. gondii. CD44 was not a necessary cytokine for the host against T. gondii infection, but compared normal and CD44-deleted mice after T. gondii infection, four types of genes containing immunoglobulins, MHC II antigens, chemokines ligands and

Author contributions

RF conceived and designed the study. SYL and BH drafted the manuscript. SYL, BH, CHY, JY, LXW, XD, XKD performed the experiments and JLZ analyzed the data. All the authors read and approved the final manuscript.

Ethics approval and consent to participate

The study was performed under the instructions and approval of Laboratory Animals Research Centre of Hubei province in P. R. China and the ethics committee of Huazhong Agricultural University (Permit number: 4200695757).

Consent for publication

Not applicable.

Availability of data and materials

The datasets supporting the conclusions detailed in this article are available in the SRP repository (SRP155079, https://www.ncbi.nlm.nih.gov/sra/SRP155079).

Funding

This work was supported by the National Natural Science Foundation of China (R.F., No. 31572510), the Natural Science Foundation of Hubei Province (Grant No. 2017CFA020) and Da Bei Nong Group Promoted Project for Young Scholar of HZAU (Grant No. 2017DBN001).

Declaration of Competing Interest

The authors declare that they have no competing interests. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

Not applicable.

References (65)

  • E. Pure et al.

    A crucial role for CD44 in inflammation

    Trends Mol Med

    (2001)
  • C.V. Ramana et al.

    Stat1-dependent and -independent pathways in IFN-gamma-dependent signaling

    Trends Immunol

    (2002)
  • K. Sumpf et al.

    Histone deacetylase inhibitor MS-275 augments expression of a subset of IFN-γ-regulated genes in Toxoplasma gondii-infected macrophages but does not improve parasite control

    Exp. Parasitol.

    (2017)
  • W. Su et al.

    CD44 Transmembrane Receptor and Hyaluronan Regulate Adult Hippocampal Neural Stem Cell Quiescence and Differentiation

    J. Biol. Chem.

    (2017)
  • A.M. Tenter et al.

    Toxoplasma gondii: from animals to humans

    Int. J. Parasitol.

    (2000)
  • M. Yamamoto et al.

    A cluster of interferon-gamma-inducible p65 GTPases plays a critical role in host defense against Toxoplasma gondii

    Immunity

    (2012)
  • A. Agrawal et al.

    Bioinformatics: an undergraduate research/teaching tool

    South Carolina Acad. Sci.

    (2004)
  • F. Almeida et al.

    Toxoplasma gondii Chitinase Induces Macrophage Activation

    PLoS ONE

    (2015)
  • S. Anders et al.

    Differential expression analysis for sequence count data

    Genome Biol.

    (2010)
  • T.E. Arantes et al.

    Ocular Involvement Following Postnatally Acquired Toxoplasma gondii Infection in Southern Brazil: a 28-Year Experience

    Am. J. Ophthalmol.

    (2015)
  • M. Ashburner et al.

    Gene ontology: tool for the unification of biology

    Gene Ontol. Consortium. Nature Genet.

    (2000)
  • S. Audic et al.

    The significance of digital gene expression profiles

    Genome Res.

    (1997)
  • H. Bando et al.

    Toxoplasma Effector TgIST Targets Host IDO1 to Antagonize the IFN-γ-Induced Anti-parasitic Response in Human Cells

    Front. Immunol.

    (2018)
  • Y. Benjamini et al.

    Controlling the False Discovery Rate: a Practical and Powerful Approach to Multiple Testing

    J. R. Stat.Soc. Series B (Methodological)

    (1995)
  • S.L. Blass et al.

    A role for CD44 in the production of IFN-gamma and immunopathology during infection with Toxoplasma gondii

    J. Immunol.

    (2001)
  • L. Boding et al.

    Midline 1 directs lytic granule exocytosis and cytotoxicity of mouse killer T cells

    Eur. J. Immunol.

    (2014)
  • R.L. Brady et al.

    Crystal structure of domains 3 and 4 of rat CD4: relation to the NH2-terminal domains

    Science

    (1993)
  • L. Ehlers et al.

    Adoptive transfer of CD3+ T cells and CD4+ CD44high memory T cells induces autoimmune pancreatitis in MRL/MpJ mice

    J Cell Mol Med.

    (2018)
  • Galvan-Ramirez Mde L., Troyo R., Roman S., Calvillo-Sanchez C., Bernal-Redondo R. (2012) Parasit Vectors. A systematic...
  • R.S. Goldszmid et al.

    TAP-1 indirectly regulates CD4+ T cell priming in Toxoplasma gondii infection by controlling NK cell IFN-gamma production

    J. Exp. Med.

    (2007)
  • J. Guan et al.

    Purkinje cell protein-2 (Pcp2) stimulates differentiation in PC12 cells by Gbetagamma-mediated activation of Ras and p38 MAPK

    Biochem. J.

    (2005)
  • M.A. Hakimi et al.

    Toxoplasma Effectors Targeting Host Signaling and Transcription

    Clin. Microbiol. Rev.

    (2017)
  • 1

    The first two authors contributed equally to this paper

    View full text