MicroRNA 155 Contributes to Host Immunity Against Toxoplasm Gondii

Background: Toxoplasma gondii is known to infect almost all the mammalian including human beings and avian species, with worldwide distribution, and cause serious toxoplasmosis, posing regards with public health problem. The role of microRNAs in the pathogenesis of T. gondii has not been well described. The objective of the present study was to investigate the role of microRNA-155 (miR-155) in mediating innate and adaptive immune responses during T. gondii infection. Methods: The survival and parasite burden in T. gondii-infected miR-155-/- and WT C57BL6 mice were compared. In these two mouse models, ELISA were used for analysis of Th1-associated, Th-2 associated, and Th-17 associated cytokines, and ow cytometry were used for analysis of the subpopulations of NK, NKT, CD8 + T, CD4 + T cells and Tregs, as well as Ly6Chi inammatory monocytes and DCs. Proinammatory mediators and CD8 + T cells responses were also analyzed by using qRT-PCR and ow cytometry, respectively. In the end, the expression of the direct target of miR-155, SHIP-1 and SOCS1 was analyzed by using qRT-PCR. Results: The lack of miR-155 led to increased parasite burden and decreased survival of infected mice in contrast to WT mice. Innate and adaptive immune responses were reduced in the absence of miR-155, associated with diminished Proinammatory mediators, Th1-associated and Th-2 associated cytokines and accumulation of lymphocyte subpopulations. Also, CD8 + T cells exhaustion was also worsened in the absence of miR-155 via targeting to SHIP-1 and SOCS1, showing as up-regulated recruit of Tregs and expression of PD-1 and, and down-regulated expression of IFN-γ and TNF-α in CD8 + T cells. Conclusion: miR-155 is a critical immune regulator for the control of T. gondii infection, suggesting that miR155 can be explored as a potential molecular target for boosting immunity against T. gondii.


Background
As the causative agent of toxoplasmosis, Toxoplasma gondii can infect all warm-blooded animals, and has infected approximately 30% of the world's population throughout the world [1,2]. Primary infection in immune-potent individuals is usually asymptomatic or presented as a mild, u-like illness, concomitant with parasite conversion to dormant bradyzoite within a tissue cyst [3]. While severe toxoplasmosis in immunocompromised individuals could develop following reactivation of bradyzoites into disseminating tachyzoites, leading to toxoplasmic encephalitis (TE), eye disease, neurological problems and even death [2,3]. Congenital infection can occur in the newborns resulting from the infection of pregnant women, associated with dysplasia, hydrocephaly and chorioretinitis [4]. Toxoplasma gondii infection can also cause large economic losses to the stock-raising industry [2]. Despite T. gondii acute infections could be controlled by medications, there are still no effective medications can be used for completely eliminate the chronic infection, and more effective chemotherapeutic agents are needed for this organism [5].
Additionally, there are no clinical licensed vaccines available for human infection [6]. Disclosing the immune response to T. gondii is vital for the design of effective vaccines and also drug targets.
It is well documented that the immune response in T. gondii infection is complex, including innate and adaptive immune response, which involves various immune cells, such as CD4 + and CD8 + T cells, natural killer (NK) cells, dendritic cells (DCs), macrophages and neutrophils [7]. CD8 + T cells and their responses are essential for the control of infection by acting synergistically to CD4 + T cells [8]. Primarily, T helper 1 (Th1) cell-mediated protective immunity to T. gondii drive the production of high levels of interleukin-12 (IL-12) and interferon-γ (IFN-γ) following by the migration of innate immune cells (e.g. DCs, macrophages and neutrophils) to the site of infection, which are indispensable for host resistance against T. gondii, and thus limit the parasite's proliferation and the progression of infection via multiple intracellular mechanisms with the production of various antiparasitic factors [9,10]. The expansion of Th2 cells and those anti-in ammatory cytokines production of IL-4, IL-10, IL-13, IL-27, and transforming growth factor-β (TGF-β) are responsible for controlling the effects of excessive immune activation and preventing the immunogic pathology [10]. Collectively, these innate and adaptive immune responses, mainly Th1-and Th2-associated cytokines are contributed to protective immunity to T. gondii infection.
MicroRNAs (miRNAs), as class of non-coding RNAs, are involved in gene regulation at both transcriptional and post-transcriptional levels [11]. Through several mechanisms including translational repression and messenger RNA (mRNA) degradation, they have been shown to mediate and regulate some physiological processes and also to be related to human disease, such as carcino-genesis, and even extended to the immune system [12,13]. It has been revealed that miRNAs can regulate the immune system biology, including both lymphocyte development and function and host immune responses, which involved in in ammation associated with CD4 + T cell differentiation and CD8 + T cell responses [13]. In particularly, miRNA-155 is a well-characterized miRNA, which is shown as a chief regulator of monocytes, macrophages, T-cells and B-cells by interference the expression of pro-in ammatory and antiin ammatory cytokines [14,15]. MiR-155 has a regulative role in visceral leishmaniasis by up-regulating of both Th1 and Th2 immune responses, which is contributed to the control of the infection [16].  has been demonstrated to be an important immune regulatory molecule critical for the control of Trypanosoma cruzi infection [17]. Likewise, miR-155 is ascertained to play a critical role in maintaining the survival of Mtb-infected macrophages and the function of Mtb-speci c T-cells during Mycobacterium tuberculosis infection [18].
Despite the fact that miR-155 showed an elevated level in chronic T. gondii infection [19], but its speci c role in involved in the innate and adaptive immune responses against this infection has not been uncovered. To determine if miRNA-155 in uences the progression of T. gondii infection and immune responses in mice models, we have investigated the role of miRNA-155 during immunity to T. gondii infection in miR155 gene-de cient mice and those of their age-and sex-matched wild-type (WT) counterparts. Our results show that miR155 is required for control of T. gondii infection via mediating a wide spectrum of immune compartments.

Materials And Methods
Mice and parasite Age-matched 6-8 week old female miR155KO C57BL/6 and WT C57BL/6 mice were purchased from purchased from Beijing Vital River Laboratory Animal Technology Co., China. All mice were maintained and bred in strict accordance according to the Animal Ethics Procedures and Guidelines of the People's Republic of China. Animal experiments were approved by the ethical committee of Ningbo University (permission: SYXK(ZHE)2019-0005).
The PRU strain (Type II) of T. gondii were used for the in vivo challenge of mice, which were propagated and harvested as described in our previous studies [20]. The tachyzoites-forming of PRU strain (Type II) of T. gondii were also used for preparation of soluble tachyzoite antigens (TLA) as previously described [20,21].
Cytokine ELISA According to our previously described method [20,21], splenocytes were prepared by push the spleens through a wire mesh, and then puri ed by removing the red blood cells using RBC erythrocyte lysis buffer, and then re-suspended in DMEM medium supplemented with 10% FCS, 1% penicillin-streptomycin, and 1% HEPES. Following by plated at a concentration of 5 ×10 6 cells/ml and stimulated for 72 h with 15 µg /ml TLA, cell-free supernatants were collected and assayed for IL-2 and IL-4 at 24 h, for IL-22 activity at 48 h, for IL-13, IL-17A, IL-17F activity at 72 h, and for IFN-γ activity at 96 h using commercial ELISA kits according to the manufacturer's instructions (Biolegend, USA).

Flow cytometry
Single-cell suspensions were prepared according to the method mentioned above. Followed by blocking of FC receptors with addition of normal mouse serum, cells were stained with the following antibodies:

qRT-PCR
The expression of IL-1α, IL-1 β, IL-6, SHIP-1 and SOCS1 were analyzed by qRT-PCR. Total RNA was isolated from three puri ed splenocytes of mice in each group by using Trizol reagent (Invitrogen, USA), as per the manufacturer's instructions. RNAs were dissolved in RNase-free ddH 2 O (TaKaRa, China) and the cDNA was synthesized using a GoScript™ Reverse Transcription System (Promega, Madison, WI, USA), which were used as templates for quantitative real-time polymerase chain reaction (qRT-PCR). qRT-PCR was performed using the Light Cycler 480 SYBR Green I Master (Roche, Switzerland). The primers used for ampli cation are listed in Table 1. qRT-PCR analysis was performed on the Light Cycler 480 (Roche, Switzerland) and data were calculated using the comparative cycle threshold (CT) method (2 −ΔCT ).

Challenge and parasite burdens
A total of ve mice in each group was challenged with 10 PRU tissue cysts of T. gondii PRU strain orally and the survival periods were recorded daily until all mice were dead. In the meanwhile, mean brain cyst loadings were counted at 4 weeks after the challenge, as described in our previous studies [20,21]. All samples were counted in triplicate. The parasite reduction rate of brain cysts is relative to that of the control.
Statistical analysis.
Statistical analysis was conducted using GraphPad Prism 8. Student's unpaired t test was used to determine statistical signi cance of differences among the groups. P values < 0.05 were considered to statistically signi cant.
Results miR155 de ciency enhanced the susceptibility to T. gondii MiR-155 has recently been uncovered to be contributed to the resistance to the experimental parasitic diseases [16,17]. To test if the lack of miR-155 would potentiate the chronic T. gondii in mice models, miR-155 −/− mice were orally inoculated with 10 PRU tissue cysts. All of the miR-155 −/− infected mice died after 17 days of infection, but WT mice infected with T. gondii showed a prolonged survival time (Fig. 1A).
To further con rm the T. gondii infection in WT and miR-155 −/− mice, the mean brain cyst loadings were counted. T. gondii-infected miR-155 −/− mice showed more brain cyst (Fig. 1B). These data demonstrate that the absence of miR-155 enhance susceptibility to T. gondii in mice models. miR155 de ciency impairs both Th1 and Th2 immune responses in the spleens of T. gondii-infected mice.
As miR-155 has been shown to be critical for regulating T cell responses as well as in ammatory responses and cytokine signals, which have the well-documented roles in the control of intracellular pathogens, including T. cruzi and Leishmania donovani [16,17], we further analyzed the production of cytokines Th1-associated IFN-γ, IL-2, Th2-associated IL-4 and IL-13, Th17-associated IL-17A and IL-17F by splenic cells harvested from WT and miR155KO (miR-155 −/− ) mice after infection with T. gonii PRU cysts.
After ELISA analysis of TLA-stimulated spleen cells supernatants, it has been shown that the production of IFN-γ and IL-2 was signi cantly reduced in T. gondii-infected miR-155 −/− mice compared to WT controls (Fig. 2). Likewise, the IL-13 production by splenic cells from T. gondii-infected miR-155 −/− mice were dramatically decreased in contrast to WT controls (Fig. 2). However, there were no signi cant changes of the levels of IL-4, and IL-17A and IL-17F in these T. gondii-infected miR-155 −/− mice and WT controls (Fig. 2). Taken together, these data suggest that this T. gondii infection in miR-155 −/− is due to the lower protective cytokines production.
Lack of miR155 contributes to the impaired recruitments of CD4 + T, CD8 + T, NK and NK-T cells in the spleens of T. gondii-infected mice.
NK cells, NK-T, and T cells are usually recruited to kill T. gondii by hosts through the production of IFN-γ and the perforin-independent mechanism [10,22]. To investigate whether the miR155 de ciency has an effect in these immune cells recruitment, the splenic cell population of CD4 + T cells, CD8 + T cells, NK cells and NK-T cells were analyzed from T. gondii-infected miR155 −/− and WT mice by ow cytometry. We have found that these indicated splenic cell populations of T. gondii-infected miR-155 −/− mice have been signi cantly decreased in contrast to WT counterparts (Fig. 3). These results suggest that miR155 plays a role in mediating recruitment of NK, NK-T cells, CD4 + T cells and CD8 + T cells, that control the T. gondii infection, while these decreased recruited cells in miR-155 −/− mice could not activate the protective immunity effectively, leading to the subsequent aggravated parasitic infection and even death.

miR155 de ciency exacerbated CD8 + T cell exhaustion
Since miR-155 have recently demonstrated to restrain CD8 + T cell functional exhaustion in chronic virus infection and tumor, through targeting to several inhibitors of cytokine signaling, including SOCS1, SHIP1 [23,24]. Thus, to better understand the role of miR-155 on CD8 + T cell exhaustion during chronic infection with T. gondii, we analyzed the expression of IFN-γ, TFN-α and PD-1 in CD8 + T cells and the number of regulatory cells (Tregs) by ow cytometry (Fig. 4). As we expected, miR155KO mice signi cantly decreased the numbers of IFN-γ CD8 + and TNF-α CD8 + T cells in contrast to WT mice, suggesting a decreased effector T cells in miR155KO mice (Fig. 4). In the meanwhile, the number of Tregs were also up-regulated by T. gondii-infected miR155KO mice, suggesting the enhanced immune-inhibitory effects followed by the de cient expression of miR155.
It is known that PD-1 expression by the myeloid cell population plays an important role in immune regulation in various infectious diseases as well as in several cancer models [25][26][27]. Since PD-1 has been implicated in suppressing T cell response in T. gondii [28], ow cytometric analysis of the expression PD-1 in CD8 + T cell revealed that T. gondii-infected miR155KO mice up-regulated the expression PD-1 in CD8 + T cell in contrast to T. gondii-infected WT mice. Furthermore, miR155KO mice showed increased expression of both SHIP-1 and SOCS1 by qRT-PCR (Fig. 4), which are known to be direct targets of miR155 [23,24]. Together, these ndings indicate that miR155 de ciency contribute to the increased T cell exhaustion by suppressing T cell responses via targeting to SHIP-1 and SOCS1. miR155 de ciency decreased accumulation of splenic in ammatory monocytes and DCs and expression of pro-in ammatory mediators.
Recent studies have established the critical role of CD11b + Ly6C + cells and DCs in T. gondii infection [29,30]. Flow cytometric analysis of phagocyte populations has revealed that T. gondii-infected miR-155 −/− mice contained signi cantly lower numbers of CD11b + Ly6C + cells and DCs than their WT counterparts (Fig. 5). In the meanwhile, we further determined whether decreased numbers of CD11b + Ly6C + cells and DCs in miR155KO mice consequently impaired the expression of pro-in ammatory mediators by Flow cytometric analysis. In consistent with decreased numbers of CD11b + Ly6C + cells and DCs, the expression of IL-1α, IL-1β and IL-6 was signi cantly reduced in contrast to their T. gondii-infected WT counterparts (Fig. 5). These data suggest that miR155 de ciency leads to the decreased accumulation of DCs and Ly6C + in ammatory monocytes, in combination with reduced production of pro-in ammatory mediators could contribute to high parasitic burdens in miR155KO mice.

Discussion
It is well known that IFN-γ and IL-4-associated signaling pathways are desired immunological responses for defending against T. gondii infection [7]. In particually, releasing of Th1 type cytokines, IL-12, IL-2 and IFN-γ are essential for the development of T cell immunity against T. gondii infection [22]. Besides, T helper type 2 (Th2) cells-associated cytokines, such as IL-4, IL-10 and IL-13, which also play an important role in coordinating the immune response by dampening the systemic Th1 type cytokine production and thus prevents lethal immunopathology [8]. Th17-associated immune responses are also shown as signi cant protective immunity against T. gondii infection [31]. Despite miR155 has been shown to be able to regulate the development and activity of Th1, Th12, and CD8 + T cells, its speci c role in immunity against T. gondii infection has yet to be well studied. In our study, we have established that miR155 contributes to host immunity against T. gondii infection through the regulation of Th1 and Th2 immune responses, but it showed no any effect in Th17-associated immune responses.
Some recent studies have shown the important role of microRNAs during the infection [23,24,32]. In our study, it has been revealed that lack of miR155 led to a signi cant increase in parasitic brain cysts and decrease survival time. Since miR155 has been shown as the key regulator of IFN-γ production via the targeting of SOCS1 [33], it is not surprise that this regulative effect should be ascribed to the inability of miR-155 −/− mice to mount an e cient Th1 immune response, with the reduced production of IFN-γ and IL-2. Besides, we have observed a decrease in Th2 immune responses in miR-155 −/− mice, which is similar to a previous study in vitro and in L. donovani-infected miR-155 −/− mice [16], indicating a potential role of miR155 that it is bene cial for the generation of optimal Th2 immune responses against T. gondii infection. These data demonstrate that miR155 plays a signi cant role in regulating both Th1 and Th2 immune responses during T. gondii infection.
MiR-155 regulates the activation of several immune subpopulations including CD8 + T cells, as well as NK and NKT cells [34,35], which is critical for immunity against T. gondii infection [31]. Previous studies have found that miR-155 is important in control of the Leishmania donovani and T. cruzi infection by mediating the regulation of T-cell proliferation and thus the activation of CD8 + T cells, NK and NKT cells [16,17]. In support with these studies, our data indicate that those decreased recruitment of CD8 + T cells, NK and NKT cells resulting from the absence of miR-155 expression, can lead to the lack of control of parasite infection in the miR-155 −/− mice.
As the rst line cell types in the initial stages of infection, in ammatory monocytes, especially Ly6C + monocytes are necessary to govern the control of chronic infection with T. gondii in mice [29], through the inductive production of proin ammatory mediators, such as IL-1α, IL-1β, IL-6, inducible NO synthase, TNF, and reactive oxygen intermediate. Additionally, DCs are essential for the expansion and subsequent T cell priming and activation of Ag-speci c CD8 + T cells during infection with T. gnodii [30]. Our studies have shown decreased accumulation of Ly6C + in ammatory monocytes and DCs in miR155KO mice, associated with a signi cantly increased parasite load and with the reduced expression of those proin ammatory mediators. However, it is contradictory that aberrant miR-155 expression has recently been shown to adversely affect Ly6C + in ammatory monocytes migration in L. donovani and T. cruzi, which is due to a fact that in ammatory monocytes facilitate the growth of the parasites in the spleen in visceral leishmaniasis and trypanosomosis [16,17]. These results demonstrate that miR155 de ciency exacerbated T. gondii infection through the down-regulation of DCs and in ammatory monocyte in ltration.
Recent studies showed that chronic infection with T. gondii led to CD8 + T cell exhaustion, concomitant with decreased CD8 + T cell effector response, which is characterized as up-regulated expression of inhibitory receptor PD-1 on these CD8 + T cells [28]. MiRNA-155 expression is essential for optimal CD8 + T cell responses toward chronic infection with LCMV, and cancer, involved in regulation of CD8 + T cell exhaustion [23,24]. In this study, our analysis of the effects of miR155 on cellular immune responses showed that the de ciency of miR-155 ablated CD8 T cell responses during chronic T. gondii infection, with up-regulation of PD-1, down-regulation of IFN-γ and TNF-α in CD8 + T cells. In the meanwhile, Tregs have also been augmented in miR-155 −/− mice, which are shown as the roles of immune-counter in cancer [36], indicating a possible immune-inhibitor effect in T. gondii, but its speci c role need further con rmation. As the direct target of miR-155, SHIP-1 and SOCS1 are indicated as negative regulators of IFN-γ production and T cell exhaustion [23,24]. The higher SHIP-1 and SOCS1 levels in infected miR155KO mice, together with these perturbed responses of CD8 + T cells suggest that T cell exhaustion during chronic T. gondii infection is mediated by miR155-dependent mechanisms.

Conclusions
We demonstrate the absence of miR-155 could enhance the susceptibility of mice to chronic T. gondii infection. It is clear that this effect of miR155 is ascribed to its contribution to host immunity via the regulation of both Th1 and Th2 protective immune responses, as well as in ammatory monocyte and DCs in ltration. It should be noted that miR155 was essential to mediate the T cell exhaustion in this model. Therefore, miR155 can be exploited as a potential target for reinforcing hosts' immunity against T. gondii.

Declarations
Authors' contributions YZ, CZ and JC designed the study. YZ, CQ and YX performed experiments. YZ, CQ and YX statistically analysed the data. LV, YZ, CQ and JC made major contributions to the writing of the manuscript. All authors read and approved the nal manuscript.

Availability of data and materials
Data supporting the conclusions of this article are included within the article and its additional les. The raw datasets used and analyzed during the present study are available from the corresponding author upon reasonable request.
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