Hepatic coccidiosis in rabbits is caused by Eimeria stiedai. First discovered more than 100 years ago, it has a high host specificity, infectivity, and lethality. Post-weaning rabbits are particularly susceptible, with mortality as high as 80% (Gomez-Bautista et al., 1987; Pakandl, 2009). Unlike the other 10 species of rabbit intestinal coccidia, E. stiedai lives in the rabbit liver to complete its entire development (Duszynski and Couch, 2013). The life cycle of E. stiedai occurs both in the external environment and within the host rabbit, and generally experiences three stages, Sporogony, Schizogony or Merogony, and Gametogony. Firstly, sporulated oocysts are consumed by rabbits and rupture, then released sporozoites invade the liver and bile duct epithelial cells where merogony occurs. Merozoites multiply within and are released from epithelial cells and continue to increase until they complete a repeat of at least four generations. Lastly, the life cycle enters the gametogony stage, macrogametes and microgametes combine into zygotes and eventually develop into oocysts passed out in the feces. E. stiedai causes severe damage to the hepatobiliary epithelial structure, affects the normal hepatobiliary function, leads to slow growth and high mortality in diseased rabbits (Oliveira et al., 2011; Jing et al., 2016), and causes severe harm to the rabbit industry. It is listed as a second-class animal disease by the Ministry of Agriculture of the People’s Republic of China.

T helper cells with specific cytokines (IFN-γ, IL4, IL10, IL17A, IL21, IL9, TNF-α, and TGF-β1) play essential roles in parasitic diseases (Pesce et al., 2006; Seki et al., 2012; Pang et al., 2014; Li et al., 2017; Kalantari et al., 2019; Bai et al., 2021; Al-Masoudi et al., 2021; Parhizgar et al., 2021; Li et al., 2022). Usually, interferon-γ (IFN-γ) is induced by TBX2, but in New Zealand white rabbits infected with Schistosoma japonicum (S. japonicum), it was found that the transcription factor TBX2 was positively correlated to TNF-α, but not to IFN-γ, which was correlated to another Th1 transcriptional factor, IRF8 (Farwa et al., 2018); thus, our study aimed to evaluate the correlation between T helper cells, transcription factors (TBX2, GATA3, Foxp3, RORC, SPI1, and BCL6), and cytokines (IFN-γ, IL4, IL10, IL17A, IL21, IL9, TNF-α, and TGF-β1) in rabbits infected with E. stiedai.

The Th1 cytokine IFN-γ sometimes protects against damage, while the Th2 cytokine IL4 enhances fibrogenesis (McManus et al., 2010; Du et al., 2011). Treg cells that produce IL10 cytokines play a crucial role in the immune balance; it acts as an immunosuppressant or a fibrotic inducer (Layland et al., 2010; Adalid-Peralta et al., 2011; Zhou et al., 2015). For example, in Schistosoma mansoni-infected mice, higher expression levels of hepatic IL17A were found in CBA mice, suggesting that its lesions may be associated with IL17A produced by Th17 cells (Kalantari et al., 2019). However, lacking the IL21/IL21R axis in S. mansoni-infected mice attenuated granulomatous inflammation and liver fibrosis formation in mice (Pesce et al., 2006). There were significantly increased levels of Th9 (IL9) in the liver of C57BL/6 mice infected by S. japonica in the early stage, suggesting that IL9 may be involved in liver injury (Li et al., 2017). However, whether these Th subsets and their cytokines also play an immuno-protective or damaging role in E. stiedai infection is unknown.

TBX2 induces the production of interferon-γ (IFN-γ) and orchestrates the Th1 cell migration by regulating the expression of chemokines and chemokine receptors. TBX2 and GATA3 regulate Th1/Th2 cell fate using a synchronous mechanism, by directly binding to the genetic locus of cytokines: TBX2 binds IFN-γ whereas GATA3 binds IL4 and other Th2 cytokines such as IL5 and IL13, leading to the expression of these proteins (Kanhere et al., 2012).

Transcriptional factor Foxp3 serves as a lineage specification factor of Treg cells, and is induced either during native Treg (nTreg) development in the thymus or in the presence of TGF-β1 and retinoic acid. High levels of IL17 is expressed by Th17 under the transcriptional regulation of RORC. In some studies, it was suggested that the signature transcription factor RORC of Th17 cells is also induced by TGF-β1, thus leading to differentiation of Treg and Th17 lineages. In the absence of a second signal derived from proinflammatory cytokines, Foxp3 can inhibit RORC function and promote Treg differentiation. However, when cells also receive signals from proinflammatory cytokines (e.g., IL6), the function of Foxp3 is inhibited and Th17 differentiation is induced. Thus, it is the balance between Foxp3 and RORC function that determines the fate of CD4+T cells and the type of immune response that would be generated (Ziegler and Buckner, 2009; Ren and Li, 2017).

PU.1 (SPI1) is a transcription factor that belongs to the E-twenty six (ETS) family of proteins and is a key factor for the differentiation of the IL9-producing Th9 subset, in humans and mice (Chang et al., 2010; Ramming et al., 2012). BCL6 served as a master regulator of Th21 differentiation, and Th21 differentiation does not occur in vivo in the absence of BCL6 (BCL6 −/−) (Johnston et al., 2009; Nurieva et al., 2009; Yu et al., 2009). While BCL6 is vital in both GC B cells and Th21 cells, it can control Th21 differentiation mainly by regulating genes of GC B cells (Crotty et al., 2010).

In E. stiedai infection, it is unclear whether different mechanisms are involved. For these reasons, we measured dynamic changes in Th cells, including Th1, Th2, Th17, Treg, Th9, and Th21, during different stages of E. stiedai infection. Our findings provide a theoretical basis to control for protective and therapeutic vaccinations.

We studied dynamic changes in Th mRNA expression levels of their transcript—TBX2 (Th1), GATA3 (Th2), RORC (Th17), Foxp3 (Treg), SPI1 (Th9), and BCL6 (Th21)—and analyzed the potential effects of their imbalance on disease outcomes in the rabbit. Moreover, E. stiedai infections provide an excellent model to study the roles of Th cell transcriptional factors. In the early stage of the infection with E. stiedai (on day 5 PI), Th1 dominated at both transcriptional and protein levels. We also found a Treg response in the late stage of infection. Furthermore, our study also showed that Th17, Th9, and Th21 were involved in E. stiedai infection. However, helminth infection is characterized by Th2-dominated immune responses, which may contribute to chronic infection and the host’s inability to clear the parasites (Kim et al., 2012; Uddin et al., 2012). For Th1, the transcription factor TBX2 was positively correlated to TNF-α, but not IFN-γ, which Farwa found was correlated to another Th1 transcriptional factor IRF8 (Farwa et al., 2018). Mechanistically, TBX2-dependent innate IFN-γ is essential in T. gondii infection T-bet to produce IFN-γ, which is essential for host resistance to intracellular infection by maintaining IRF8 inflammatory dendritic cells at the infection site (Lopez-Yglesias et al., 2021). A report showed that loss of TBX2 had some effect on IFN-γ production in T cells but not parasite-specific T cells (Harms Pritchard et al., 2015).

For Th2, the expression levels of GATA3 and IL4 appeared to be elevated on days 5 and 15 PI, and GATA3 mRNA expression was uncorrelated to IL4, similar to GATA3 mRNA expression, which was essential for IL13 production but not IL4 in the experiment group, whereas IL4 appeared to be a STAT6-dependent stimulation (Zhu, 2010). Regarding Treg, the expression levels of Foxp3 also appeared to be elevated. TGF-β1 was also induced simultaneously, similar to an observation by Tang et al. (2015) on day 5 PI. Foxp3 appeared to have elevated expression levels, and the same result appeared in S. japonicum infection, which was a potent inducer for Foxp3 (Finlay et al., 2014). For Th17, RORC expression was correlated to IL17A. On days 5 and 15 PI, Th17 mRNA and IL17A expression levels were elevated, similarly in S. mansoni-infected mice, in which the liver developed significant hepatic granulomatous inflammation, with higher expression levels of IL17 and RORC, suggesting that its lesions may be associated with IL17 produced by Th 17 cells (Kalantari et al., 2019). For Th9, the expression levels of SPI1 and IL9 appeared to be elevated on days 5 and 15 PI, and SPI1 expression was correlated to IL9. The expression of IL9 and its transcription factor SPI1 in hydatid patients was significantly higher in focal tissue, suggesting the formation of chronic granuloma in hydatid by upregulating the expression of Th9 cells, IL9, and transcription factor SPI1 during infection (Tuxun et al., 2015). The expression levels of Th21, BCL6, and IL21 appeared to be elevated on days 5 and 15 PI, and BCL6 expression was correlated to IL21. A correlation study of IL21 and transcription factor BCL6 showed that IL21 was positively correlated to BCL6, which was upregulated on Th21 cells, and IL21 is the primary effector molecule of Tfh cells. (Geng et al., 2018).

In addition, many cross-modulations were observed; TBX2 suppressed GATA3 expression on day 5 PI (Zhu, 2010). Cross-modulations between GATA3 and Foxp3 were observed in Treg cells, where GATA3 expression enhanced the accumulation of Treg in the inflammatory site (Turner et al., 2011). On days 5 and 23 PI, we also observed the cross-modulations between SPI1 and BCL6. The expression of B-cell lymphoma protein 6 (BCL6) depends on IRF8 and SPI1 in vivo, which is essential for developing germinal center B cells, and SPI1 acts as a regulator of BCL6 expression during GC B-cell development (Wang et al., 2019). GATA3 promotes Th2 differentiation while inhibiting Th1, and TBX2 has been shown to promote the development of Th1 and IFN-γ production (Szabo et al., 2000), but a study contradicted this finding (Cobb et al., 2010). A study showed that the IL21/IL21R signaling axis regulated the effector functions of T-cell subsets (e.g., production of cytokines) by enhancing TBX2 and STAT4 expression in human T cells, thereby enhancing IFN-γ production (Solaymani-Mohammadi et al., 2019). Treg only regulated TGF-β1 and was independent of type 2 cytokines (Mishra et al., 2014). In general, IL6 combined with TGF-β induced differentiation of Th17 cells from initial CD4+T cells and was also polarized into Treg cells after co-stimulation with IL2 and TGF-β (Kimura and Kishimoto, 2010). Our study found interesting findings: GATA3, Foxp3, RORC, and SPI1 mRNA and its relative cytokines (such as IL4, IL10, IL17A, and IL9 in the serum) were significantly decreased in the late stage, which suggests that they were suppressed.

In the experiment group, liver weight increased, and the significant proliferation of eggs of E. stiedai made liver lesions severe ( Table 3 ). In the liver sections, massive inflammatory cells were infiltrated around the portal areas accompanied by cholangiocyte proliferation on day 5 PI ( Figure 1D ). TBX2, GATA3, Foxp3, RORC, BCL6, SPI1, and relative cytokine expression levels were elevated, which resisted E. stiedai infection. Several merozoites were observed on day 15 PI ( Figure 1E ) (black arrow). TBX2, RORC, BCL6, SPI1, and relative cytokine expression levels were elevated, and Foxp3 expression levels were decreased, likely due to dynamic Treg/Th17 changes. On day 23 PI, the appearance of numerous eggs of E. stiedai was consistent with previous reports ( Figure 1F ), except TBX2 expression levels were elevated; GATA3, RORC, BCL6, and SPI1 expression levels were decreased; and Th1 dominated at both transcriptional and protein levels, but this contradicted a study carried out by Uddin et al. (2012). In addition, RORC, Foxp3, and SPI1 were negatively correlated to oocyst numbers on day 23 PI, and expression levels of cytokines including IL17A, IL10, IL9, and TGF-β1 decreased. No statistically significant difference of relationships between TBX2, GATA3, BCL6, and oocyst output was observed in the experiment groups at three time points ( Table 4 ). In the experiment group, the severe liver damage was observed during the proliferation of E. stiedai, likely through suppressing the expression of RORC, Foxp3, SPI1, cytokines (IL17A, IL10, and IL9), and TGF-β1.

Our findings provide the first evidence of dynamic changes in Th1/Th2/Th17/Treg/Th9/Th21 and confirm an imbalance of these cell populations in E. stiedai-infected rabbits. Additional studies are needed to elucidate the detailed roles of these Th cells in the pathogenesis of E. stiedai.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by the Institutional Animal Care Committee of Sichuan Animal Science Academy.

Conceptualization: X-DC, R-MK, and Y-GY. Methodology: X-DC. Software: LX, C-JM, and X-JW. Validation: J-FY, YW, and JX. Formal analysis: X-DC and YC. Resources: Y-GY. Data curation: R-MK and Y-GY. Writing—original draft preparation: X-DC. Writing—review and editing: Y-GY. Visualization: X-DC. Supervision: Y-GY. Project administration: R-MK and Y-GY. All authors contributed to the article and approved the submitted version.