Abstract
Purpose
Searching for a novel early diagnostic biomarker for toxoplasmosis, real-time-PCR was currently used to measure the serum mmu-miR-511-5p level in male Swiss-albino mice infected with either; ME49 or RH Toxoplasma gondii (T. gondii) strains.
Methods
Three mice groups were used; (GI) constituted the non-infected control group, while (GII) and (GIII) were experimentally infected with ME49 or RH strains, respectively. GII mice were orally infected using 10 or 20 ME49 cysts (ME-10 and ME-20), both were subdivided into; non-treated (ME-10-NT and ME-20-NT) and were further subdivided into; immunocompetent (ME-10-IC and ME-20-IC) [euthanized 3-days, 1, 2, 6 or 8-weeks post-infection (PI)], and immunosuppressed using two Endoxan® injections (ME-10-IS and ME-20-IS) [euthanized 6- or 8-weeks PI], and spiramycin-treated (ME-10-SP and ME-20-SP) that received daily spiramycin, for one-week before euthanasia. GIII mice individually received 2500 intraperitoneal RH strain tachyzoites, then, were subdivided into; non-treated (RH-NT) [euthanized 3 or 5-days PI], and spiramycin-treated (RH-SP) that were euthanized 5 or 10-days PI (refer to the graphical abstract).
Results
Revealed significant upregulation of mmu-miR-511-5p in GII, one-week PI, with gradually increased expression, reaching its maximum 8-weeks PI, especially in ME-20-NT group that received the higher infective dose. Immunosuppression increased the upregulation. Contrarily, treatment caused significant downregulation. GIII recorded significant upregulation 3-days PI, yet, treatment significantly decreased this expression.
Conclusion
Serum mmu-miR-511-5p is a sensitive biomarker for early diagnosis of ME49 and RH infection (as early as one-week and 3-days, respectively), and its expression varies according to T. gondii infective dose, duration of infection, spiramycin-treatment and host immune status.
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Introduction
Toxoplasma gondii is an obligate intracellular protozoan parasite that infects almost all the warm-blooded animals and about 30–50% of the world’s human population [1, 2].
Caused by T. gondii, toxoplasmosis is presented by wide spectrum of clinical presentations, based on; inoculum size, organism virulence, and host immunological status, ranging from asymptomatic or flu-like symptoms in immunocompetent patients, up to encephalitis in immunocompromised patients [1, 3, 4]. Moreover, reactivation of latent toxoplasmosis in patients with immunodeficiencies can result in high mortality rates [1, 5].
Classical serological techniques for detection of T. gondii antigens or antibodies are the commonly used for diagnosis nowadays. However, several limitations confront them, including; absence of parasite-specific antibodies during the early stages of infection, particularly in immunocompromised patients who fail to produce significant titers of specific antibodies. Moreover, they usually remain positive even after successful treatment, so, they are of low benefit for early detection, and cannot assess treatment [6,7,8].
Monataya et al. (2002) described the limitations in diagnostic techniques for T. gondii. They stated that IgG antibodies usually appear within 2 weeks of acquisition of the infection, peak within 1–2 months, decline at various rates, and usually persist for life. Thus, it can’t detect very early infection nor differentiate between new and old infections [9]. As for IgM antibodies, although the commercial test kits measuring them are widely distributed, these tests often have low specificity, and their results are frequently misinterpreted [10]. Regarding IgA antibodies, they may be detected in the sera of acutely infected adults and congenitally infected infants, using ELISA, and they may persist for more than a year [11]. For this reason, they are of little additional assistance for diagnosis of acute infections in adults.
The current diagnostic tools also involve histopathological examination that entails tissue biopsies [12], which can be cumbersome for the patients.
Moving on to molecular tests for toxoplasmosis diagnosis, although they are becoming more frequently applied, yet, they are still controversial, and there is no consensus about the best method or target to be used. In addition, T. gondii DNA concentration during chronic infection is usually undetectable, even when molecular sensitivity tests are performed [12]. As a result, diagnosis of T. gondii infection is still difficult and the development of alternative methods with higher specificity and sensitivity is crucial [13].
MicroRNAs (miRNAs) are endogenous 18–25 nucleotide non-coding RNA molecules that have fundamental roles in regulating immune response outcomes in eukaryotes, through the miRNA induced silencing complex. Mature miRNAs can directly affect the expression of thousands of genes, by inhibiting protein expression via translational repression and/or mRNA degradation [14, 15].
MiRNAs are stable enough to be detected in the serum, plasma and tissues. Circulating miRNAs are protected from the endogenous RNAse activity [16]. Thus, they have been recognized as appealing biochemical markers for many ailments, including infectious diseases [17,18,19,20,21].
In their review, Judice et al. (2016) reported that, many Apicomplexan parasites would alter their host miRNA expression [22]. One of them is, T. gondii parasite, which was reported in an earlier study to cause dysregulation in the plasma levels of multiple miRNAs in T. gondii infected mice [23]. Among these miRNAs, three miRNAs; (mmu-miR-712-3p, mmu-miR-511-5p, and mmu-miR-217-5p) kept the induced expression in the parasitized mice with both; RH or ME49 strains of T. gondii [23].
Because it was not thoroughly investigated in the literature, mmu-miR-511-5p was chosen by the current work’s authors to further assess its role as a tool for early diagnosis of toxoplasmosis and for follow up of its therapy. This was done by monitoring the marker level in the sera of immunocompetent and immunosuppressed Swiss-albino mice experimentally infected with the avirulent (ME49) or the virulent (RH) T. gondii strains.
Materials and Methods
Maintenance of Toxoplasma gondii
Both avirulent ME49 and virulent RH HXGPRT (-) T. gondii strains were maintained in Swiss-albino mice in the Medical Parasitology Department, Alexandria Faculty of Medicine.
For infection by avirulent ME49 strain, cysts were obtained from the brains of infected mice 8 to 15-weeks post-infection (PI). These brains were homogenized in sterile phosphate buffered saline (PBS) (pH = 7.4), Then cysts were counted in the homogenate, and mice were orally infected with 10 or 20 cysts according to their groups [24].
As regards virulent RH HXGPRT(-) strain, it was propagated by serial intraperitoneal passages of tachyzoites in the mice. Tachyzoites were harvested from mice peritoneal lavage on the 5th day PI (dpi) and were kept in sterile PBS at 4○C until used [25]. Each mouse was intraperitoneally infected with 2500 tachyzoites [21].
Grouping of Experimental Animals
The study was performed using 126 male Swiss-albino mice, 4–6 weeks old, weighing 20–25 g each. Animals were housed in dry, clean, well-ventilated cages, with free access to water and food. They were kept in the animal house, in the Medical Parasitology Department, Faculty of Medicine, Alexandria University, according to the national guidelines of animal experimentation. The followed experimental design is shown in Fig. 1. Experimental mice were divided into three groups as follows:
Group I (Non-Infected Control Group)
Six non-infected mice were used as control.
Group II (ME Group)
Ninety-six mice were infected with the avirulent ME49 strain, and were divided equally, according to the dose of infection, into two subgroups:
Subgroup ME-10
Forty-eight mice were orally infected with 10 cysts of ME49 strain and were further subdivided into spiramycin-treated (ME-10-SP) and non-treated (ME-10-NT).
ME-10-SP subgroup included 6 mice, that received oral spiramycin (Rovac®), 8-weeks PI in a dose of 400 mg/kg, daily for 7-days [26]. One-day after the end of treatment, mice were anesthetized with ether inhalation, then, they were euthanized using cervical dislocation.
While ME-10-NT subgroup included 42 mice, that were further subdivided into immunocompetent (ME-10-IC) and immunosuppressed (ME-10-IS). ME-10-IC included 30 mice which were subgrouped according to the time of euthanasia, into 5 subgroups; ME-10-IC-3d, ME-10-IC-1w, ME-10-IC-2w, ME-10-IC-6w, and ME-10-IC-8w. Each subgroup contained 6 mice that were euthanized; 3-days, 1, 2, 6, and 8-weeks PI, respectively.
Regarding, ME-10-IS subgroup, it included 12 mice, which were immunosuppressed using intra-peritoneal injection of cyclophosphamide (Endoxan®) in 2 doses of 70 mg/kg each, 1-week apart, starting 4-weeks PI [21]. They were divided into; ME-10-IS-6w and ME-10-IS-8w, that were euthanized 6 and 8-weeks PI, respectively.
Subgroup ME-20
This included 48 mice, that were orally infected with 20 ME49 cysts and were subdivided into spiramycin-treated (ME-20-SP) and non-treated (ME-20-NT).
ME-20-SP included 6 mice, that received 400 mg/kg of oral spiramycin, 8-weeks PI, daily for one-week [26], then, they were euthanized one-day after the end of treatment.
While, ME-20-NT subgroup included 42 mice that were further subdivided into immunocompetent (ME-20-IC) and immunosuppressed (ME-20-IS). ME-20-IC included 30 mice which were divided according to the time of euthanasia into 5 subgroups; ME-20-IC-3d, ME-20-IC-1w, ME-20-IC-2w, ME-20-IC-6w, and ME-20-IC-8w. Each subgroup contained 6 mice.
Regarding ME-20-IS, it included 12 immunosuppressed mice according to the method of Mogahed et al. (2018) [21]. They were equally subgrouped into ME-20-IS-6w and ME-20-IS-8w.
Group III (RH Group)
This included 24 mice, which were, individually, infected by 2500 tachyzoites of the RH virulent strain, then they were equally subdivided into non-treated (RH-NT) and spiramycin-treated (RH-SP) subgroups.
RH-NT subgroup included 12 mice which were equally subgrouped into; RH-NT-3d and RH-NT-5d, where mice were euthanized 3 or 5-dpi.
While, RH-SP included 12 mice, which received a daily dose of 400 mg/kg of oral spiramycin, starting on the 1st day of infection. Then, they were equally subdivided into: RH-SP-5d, RH-SP-10d. RH-SP-5d mice were treated for 5-days, and were euthanized at the end of the treatment, to be comparable to the corresponding non-treated subgroup (RH-NT-5d). RH-SP-10d mice were treated for 7-days, and were euthanized 10-dpi.
Collection of Mice’ Blood Samples
The collected blood samples from different mice via cardiac puncture were left at room temperature for 30 minutes to allow complete coagulation. Aspirated sera were used for RNA extraction [27].
Study of mmu-miR-511-5p Expression in the Sera of the Studied Groups by Reverse Transcription-Real Time Quantitative PCR (RT-qPCR) [23, 28]
RNA Extraction
Total RNA isolation was carried out using the miRNeasy Mini Kit (QIAGEN, Maryland, USA, Catalogue No. 217004) according to the manufacturer’s instructions. The final step involved eluting the extracted RNA using RNase free water.
RNA Quantitation and Quality Assessment
The concentration and purity of RNA were measured at wavelengths of 260, 280 and 230 nm using NanoDrop 2000 spectrophotometer. Samples with 260:230 ratio greater than 1.7 and a 260:280 ratio greater than 2.0 were included in the study.
Reverse Transcription (RT)
TaqMan® MicroRNA Reverse Transcription Kit was used (ThermoFisher Scientific, catalogue number: 4366596) to convert RNA to complementary DNA (cDNA). The volumes shown in the supplementary section (a) were added to prepare the RT reaction master mix.
After preparing the master mix, the RT reaction was prepared by adding 7 μl master mix, 3 μl primer, and volume containing a concentration of 100 ng of extracted RNA. Nuclease free water was added to reach a total volume of 15-μl. Samples were applied to 25 thermal cycler which was programmed at 30 min hold at a temperature of 16°C, 30 min hold at a temperature of 42°C, 5 min hold at a temperature of 85°C, then lowering the temperature to 4°C and stopping the run [29]. Samples were chilled on ice for at least one min. cDNA was stored at − 20°C till used in real time qPCR.
Assay of miRNA (mmu-miR-511-5p) Expression Using Real Time qPCR
This was done using TaqMan MicroRNA Assay for both target miRNA (mmu-miR-511-5p, catalogue number A25576) and housekeeping gene (U6, catalogue number: 4427975).On the day of the assay, the reagents were thawed and mixed then the tubes were gently vortexed and briefly centrifuged to suspend the assays. TaqMan® Universal Master was mixed by gently swirling the bottle. Reactions were performed in duplicates according to the manufacture’s recommendation. For each sample, the PCR reaction mix was prepared using the volumes shown in the supplementary section (b), where 20μL of PCR reaction mix were transferred to wells of the PCR strips. The plate was sealed and vortexed and the qPCR was programmed as follows: 1 cycle of enzyme activation for 10 min at temperature of 95°C, followed by 1 cycle of denaturation for 15 s at temperature of 95°C, then 40 cycles of annealing and extension for 60 s each at temperature of 60°C. Fluorescent signals from each sample were recorded at the endpoint of every cycle. Amplification plots of the studied miRNA and housekeeping gene were plotted and shown in Fig. 2 a, b
Analysis of miRNA Expression [30]
The fold change between a sample and a normal control for (mmu-miR-511-5p) was calculated with the relative quantification method (RQ = 2−ΔΔCt).
Statistical Analysis
Data were fed to the computer and analyzed using IBM SPSS software package version 20.0. Qualitative data were described using number and percent. The Shapiro–Wilk test was used to verify the normality of distribution. Quantitative data were described using mean and standard deviation. Normally distributed quantitative variables were analysed using F-test (ANOVA) to compare between more than two groups, and Post Hoc test (Tukey) for pairwise comparisons. Student t-test was used for normally distributed quantitative variables to compare between two studied groups. Significance of the obtained results was judged at the 5% level.
Results
The mean relative quantification (RQ) of the serum level of microRNA mmu-miR-511-5p was demonstrated in Fig. 3, and was as follows:
Group I (Non-Infected Group)
The RQ level was 0.0086 ± 0.0013, and this was referred to as a control.
Group II (ME Group)
The Non-Treated Immunocompetent ME Subgroups
There was a non-significant decrease in the miRNA level in subgroup ME-10-IC-3d when compared to (group I). Yet, a significant upregulation was observed in subgroups; ME-10-IC-1w, ME-10-IC-2w, and ME-10-IC-6w. This increased expression reached the maximum 8-weeks PI (Table 1).
On the other hand, compared to the control, a significant downregulation was recorded in subgroup ME-20-IC-3d. Mice sacrificed later showed a significant upregulation, starting one-week PI, then, increased gradually during the 2nd and 6th weeks PI, reaching its highest level during the 8th weeks PI (Table 1).
The comparison between ME-10-IC and the corresponding ME-20-IC subgroups, revealed that, miRNA of ME-20-IC-3d was significantly lower than that of ME-10-IC-3d. Contrarily, the mean RQ level in mice infected with 20 cysts and euthanized during the 1st, 2nd, 6th and 8th weeks was significantly higher than that of the corresponding subgroups infected with 10 cysts and euthanized at the corresponding time points (Table 1).
The Non-Treated Immunosuppressed ME Subgroups
Compared to the control, a significant upregulation was observed in the immunosuppressed subgroups infected with 10 ME49 cysts; ME-10-IS-6w and ME-10-IS-8w. This was significantly higher than that of the corresponding immunocompetent subgroups (Table 2). A significant upregulation was also recorded in the immunosuppressed subgroups infected with 20 cysts; ME-20-IS-6w and ME-20-IS-8w. Moreover, these were significantly higher than the corresponding immunocompetent subgroups ME-20-IC-6w and ME-20-IC-8w (Table 2).
Noteworthy that, the RQ level was significantly higher in the immunosuppressed subgroups infected with 20 cysts, than those infected with 10 cysts, and during the 8th week PI, than during the 6th week PI (Table 2).
Spiramycin-Treated ME Subgroups
The mean RQ level of ME-10-SP and ME-20-SP subgroups was significantly higher than that of the control. Yet, both showed a significant downregulation when compared to the corresponding non-treated subgroups; ME-10-IC-8w and ME-20-IC-8w (Table 3). There was non-significant difference between the treated subgroups infected with 10 and 20 cysts (Table 3).
Group III (RH Group)
Compared to the control, a significant upregulation was recorded in the non-treated subgroups as early as 3-dpi (RH-NT-3d). A further significant upregulation was evident 5-dpi (RH-NT-5d), compared to RH-NT-3d (Table 4).
Regarding the spiramycin-treated subgroups, the RQ level in RH-SP-5d was significantly lower than that of subgroup RH-NT-5d, although it was still significantly higher than that of the control. After the end of treatment, the level continued to decrease in RH-SP-10d, which was significantly lower than RH-SP-5d, yet, it was non-significant, compared to the control (Table 4).
Discussion
The worldwide T. gondii infects a plenty of human populations, resulting in a wide range of symptoms and complications, that differ according to the host immune status [31,32,33].
Though serological techniques are counted on in diagnosis of toxoplasmosis, yet, they are deficient in early diagnosis and in follow-up of treatment. Therefore, novel sensitive method is urgently needed [6,7,8].
The detection of miRNAs in body fluids has raised the interest of their use as potential diagnostic biomarkers [34]. miR-511 is a short non-coding RNA, that was reported by Jia et al. (2014), to have a role as a potential biomarker for T.gondii diagnosis. However, being rarely investigated in the literature has motivated the current work authors to investigate its role during T.gondii infection [23].
The current results revealed that, the immunocompetent mice subgroups infected with 10 or 20 cysts of ME49 strain showed an upregulation in their mmu-miR-511-5p serum level, starting one-week PI, with a gradual significant increase, reaching its maximum 8-weeks PI. This could be explained by the progression of the infection and was in accordance to Jia et al. (2014) who found that, the circulating mmu-miR-511-5p has specifically increased in response to T. gondii infection [23].
This upregulation in miRNA level was also affected by the dose of infection, as evident by the significantly different increased expression among the subgroups infected with 10 cysts and corresponding subgroups infected with 20 cysts.
Toxoplasma gondii successful infection depends on the parasite ability to manipulate the host immune response, creating an environment that favors its survival and replication, through; balancing the pro- and anti-inflammatory cytokines, that control apoptosis and signaling pathways [35]. This is mediated via many mechanisms; one of them is manipulating the host miRNA, to establish favorable growth conditions, as reported by Zeiner et al. (2010) [36]. The authors reached a conclusion that, Toxoplasma infection has upregulated the levels of mature miR-17∼92-derived miRNAs in primary human cells. MiR-17∼92 cluster is crucial for apoptosis regulation, and accordingly, for T. gondii evasion [36].
Additionally, it was reported that, T. gondii causes abundant expression of mmu-miR-155-5p in murine immune cells involved in inflammatory responses, and an upregulation of mmu-miR-146a-5p, that is also involved in inflammation [37, 38]. The release of exosome-like vesicles containing several distinct miRNA species from cells infected with ME49 and RH strains was also reported, these miRNAs have immune regulatory functions [39].
Regarding mmu-miR-511-5p, the increased expression is multifactorial, as it has multiple predicted target genes, with many regulatory functions; involving immunological or pathological functions that can be affected by T. gondii infection with subsequent upregulation in its circulatory levels [40]. The miR-511 coding unit is located within the 5th intron of mannose receptor gene MRC1, that is expressed mainly in macrophages and dendritic cells and its expression regulates the expression of miR-511 [41].
The macrophages are known to be one of the main effector cells during the innate immune response against T. gondii infection [42]. Mosser and Edwards (2008) explored the full spectrum of macrophage activation and observed the macrophages’ remarkable plasticity that allows them to efficiently respond to the environmental signals and change their phenotype and physiology, that can be markedly altered by innate and adaptive immune responses. According to the stimulation type, macrophages can be either classically activated (M1) or alternatively activated (M2) [43]. The classically activated macrophage is activated through toll-like receptors (TLR) and interferon-γ (IFN-γ). It enhances killing of intracellular microorganisms, with increased cytokines’ secretion, and higher expression of co-stimulatory molecules. While the alternatively activated macrophage is activated by interleukin-4 (IL-4) or IL-13 and expresses IL-4 receptor-α, mannose receptor (CD206) and arginase-1 [43].
Later, Jensen et al. (2011) observed that different Toxoplasma strains could equally infect macrophages during the first three-days of intraperitoneal infection, with no differences in their recruitment [44]. These observations suggest that differences in parasite virulence might be partially due to how these strains interact with macrophages through effector molecules secreted by their rhoptries and dense granules that, accordingly, affect macrophage gene expression and control its polarization and activation to either M1 or M2.
In 2012, Hunter and Sibley, have identified a dense granule protein GRA15II, that is encoded by ME49 strain. This protein is secreted into the host cytosol, causing the activation of the classically activated macrophages (M1), while, ROP16 is secreted by RH strain and is responsible for the alternatively activated macrophages (M2) [42].
In addition to the aforementioned, Mazurek et al. (2022), outlined in their elegant paper [45], that, MiR-511-3p plays an important role in inflammation via the regulation of peroxisome proliferator-activated receptor γ (PPARγ) expression and Toll-like receptor 4 (TLR4) regulation in human dendritic cells (DCs). PPARγ is known to regulate many DC functions including pro-inflammatory cytokine production, migration, antigen presentation and activation. Downregulation of miR-511-3p increases PPARγ expression and leads to suppression of lipopolysaccharide-mediated inflammation (inhibition of NF-κB pathway). On the other hand, overexpression of miR-511-3p causes decreased activation of PPARγ and increased production of pro-inflammatory cytokines [46, 47]. This is consistent with the current findings where, miR-511 expression increased with progress of Toxoplasma burden and hence inflammation. It was also reported that Rho-associated coiled-coil containing protein kinase 2 (ROCK2), a serine/threonine kinase, is a direct target of miR-511-3p and promotes M2 polarization of macrophages by phosphorylating the interferon regulatory factor 4 transcription factor [48].
In 2015, Karo-Atar et al. showed that microRNA profiling of M1 and M2 macrophages revealed an opposing expression pattern for miR-511 [49]. Its level was downregulated in M1 which are activated in response to ME49 strain. On the other hand, miR-511 level was upregulated in M2 which are activated in response to RH strain. These can explain the current early downregulation of mmu-miR-511-5p recorded 3-dpi by the avirulent strain.
Contrarily, Jia et al. (2014) recorded upregulation in the plasma mmu-miR-511-5p level, 3-dpi [23], which can be attributed to the variation in the used mice strains, dose and route of infection between the current and the aforementioned study, as Jia et al. (2014) used female BALB/c mice, and individually, injected them intraperitoneally using 106 tachyzoites. These variations may affect the pathological, the immunological outcomes, and accordingly, miRNA levels.
With the disease progression and activation of the adaptive immune response, other effectors may affect miRNA level. Researchers showed that within 8-days of oral ingestion of Toxoplasma tissue cysts, susceptible mice develop severe ileitis resulting in mucosal villus necrosis. This ileitis is due to the strong T-helper 1 biased immune response, characterized by the overproduction of pro-inflammatory mediators including; IFN-γ, TNFα, IL1β, IL18, and NO, commonly known as “cytokine storm” [50]. These cytokines in turn help in M1 polarization, leading to downregulation of miR-511-5p as reported by Curtale et al. (2017), who demonstrated a parallel inhibition of miR-511-5p expression in response to IFN-γ challenge [46]. The cytokine storm is controlled by the release of transforming growth factor β (TGF- β) by intraepithelial lymphocytes, and IL10 by CD4+ T cells. TGF- β and IL10 downregulate the IFN-γ response and inhibit chemokine production from enterocytes, limiting the recruitment of more immune cells, and thus, reducing inflammation [50]. In response to TGF-β release, miR-511-5p level is upregulated in monocytes, resulting in their desensitization due to downregulation of TLR4 present on their surface, which controls severe inflammation and dramatically reduce TNFα and other cytokines that are involved in ileitis [46]. The TH1 biased response, the cytokine storm, and its control by TGF- β and IL-10, all explain the present early downregulation, followed by upregulation in miR-511-5p levels in the macrophages and monocytes which is in turn reflected on its level in the host serum.
Tserel et al. (2011) found that miR-511 is important for the proper differentiation of monocyte-derived dendritic cells which are considered an important effector against toxoplasmosis [41]. Moreover, Puimège et al. (2015) reported that, MiR-511 upregulation is a mechanism of T. gondii immune evasion, through downregulation of the tumor necrosis factor receptor 1 protein (TNFR1) expression and subsequent TNF resistance which is an important effector during Toxoplasma infection [40].
Regarding the current immunosuppressed ME49 subgroups, immunosuppression was found to cause a significant upregulation of mmu-miR-511-5p level in mice euthanized 6- and 8-weeks PI, compared to the corresponding immunocompetent mice. This upregulation was also affected by the infective dose.
Qi et al. (2018) reported that cyclophosphamide increases T-helper 2 (TH2) cytokines (IL-4 and IL-10) [51] and meanwhile, it decreases TH1 cytokines, mainly; IFN-γ. These TH2 cytokines stimulate the expression of mmu-miR-511 in the macrophages [49], and thus, cause their polarization to M2 phenotype [44]. This provides an explanation of the current increase in miR-511 in the immunosuppressed subgroups. The reactivation of Toxoplasma infection which increases the parasite burden and the disease pathology after latency in response to immunosuppression augments miR-511 increase. These results suggest that, monitoring serum miR-511-5p levels can help in diagnosis of toxoplasmosis in immunosuppressed patients. In 2018, Mogahed et al. reported an upregulation of miR-712_3p level in the plasma of immunosuppressed Swiss-albino mice, infected with RH strain [21].
As regards spiramycin-treated ME subgroups, the level of mmu-miR-511-5p was downregulated after treatment. Although the downregulation was statistically significant when compared to the non-treated subgroup, yet the level did not reach that of the control. This decrease may be directly related to the decreased parasite burden, or indirectly affected by the immune cells’ response to treatment. Matsui et al. (2016) found that, spiramycin-treatment caused a decrease in TH2 cells [52]. The subsequent decrease in TH2 cytokines may explain the decrease in the miR-511 [49].
Concerning the RH group, the level of mmu-miR-511-5p in the sera of infected non-treated mice showed a significant increase, that was recorded as early as 3-days after infection, compared to the control. A further upregulation was recorded 5-dpi (the end of follow-up period). These results indicate that infection with the virulent strain has an early and up-regulatory effect on the circulating mmu-miR-511-5p. Similarly, in 2014, Jia et al. recorded an upregulation in mmu-miR-511-5p level in mice plasma 72-h after intraperitoneal infection with RH strain [23]. Mogahed et al. (2018) also recorded a significant up-regulation of plasma miR-712_3p, 3-and 5-dpi, in mice infected with RH strain [21].
The upregulation of mmu-miR-511-5p following infection with RH strain may be attributed to that: T. gondii virulent strain was found to produce rhoptry protein kinase (ROP16) [44], that can activate signal transducer and activator of transcription 6 (STAT6), leading to the induction of arginase, an enzyme that associates alternatively activated macrophages M2, leading to increased miR-511 expression inside the macrophages, and subsequently an upregulation of its serum level [42, 49, 53, 54].
The current spiramycin-treated RH subgroup showed a decrease in the serum miR-511 level, 5-dpi. This downregulation was significant, when compared to corresponding RH infected non-treated subgroup. The level continued to decrease until 10-dpi, when it reached a level that was non-significant when compared to the control. This post-treatment downregulation of miR-511 level may be related to the decrease in the parasite burden and amelioration of pathological changes in response to spiramycin treatment as proved by Omar et al. (2021) [55]. Spiramycin itself may affect the immune response, by decreasing the T-helper cells, leading to decrease in TH2 cytokines, with subsequent decrease in the miR-511 [49, 52].
The current results shed a light on the importance of resorting to alternative biomarkers for diagnosis of toxoplasmosis. Some of the advantages and challenges of using microRNAs in such a domain can be summarized as follows.
The easiness of obtaining a blood sample to assess the miRNA, the stability of miRNA in the blood even after repeated freeze–thaw cycles and meanwhile, its ability to reflect the tissue pathology, all make it an attractive marker. Looking at previous research on miRNA in the context of other infectious diseases [28], highlights some advantages and gives hope that this could be applied to toxoplasmosis. Advantages of miRNA biomarkers over the currently established techniques include; early detection, which is critical to prognosis and limitation of disease spread, hence giving a better chance of effective treatment. Expression of microRNAs may precede IgM class antibodies in parasitic diseases, since that IgM antibodies may eventually be undetected within the first weeks following infection [56].This is, especially, crucial during pregnancy, where early diagnosis and treatment of infected mothers decreases the risk of toxoplasmosis transmission to the foetus, thus, improving clinical outcomes [57]. MiRNAs as diagnostic biomarkers can also help in improved pathogen identification, especially when the symptoms of infectious diseases that first appear, are non-specific ones such as malaise, fever and headache [13]. Furthermore, microRNAs are biomarkers of high sensitivity and specificity for diagnosis of different stages of infection. According to Judice et al. (2016), other Apicomplexans such as; Plasmodia and Cryptosporidium parvum do not induce miR-511-5p expression [22], reinforcing its use as potential T. gondii biomarker. Besides, miRNA has an advantage over the current diagnostic tools, in differentiating new infections from old ones [13].
Unfortunately, a limitation would be that the same microRNA might be over or under expressed in various infections or health conditions. According to He et al. (2013), serum levels of several miRNAs that were suggested as biomarkers for some diseases might be dysregulated in other diseases [58]. Wang et al. (2019) b revealed that miR-511-5p was abnormally expressed in colorectal cancer [59].
Moving on to challenges, like any potentially new diagnostic biomarker, that is being translated into clinical practice, extensive research on humans has to be done, including large set of patients under different conditions of infection (as that was attempted to be done in the current research, using different strains, doses and immune status models). miR-511 levels in Toxoplasma-infected patients have to be compared to patients bearing other infections and inflammatory conditions. A cutoff value for miR-511 for Toxoplasma infection should be determined if possible, including different cutoffs for different disease stages. In addition, miR-511 expression levels should be thoroughly examined within the context of other microRNAs, so that a certain microRNA signature specific for Toxoplasma infection can reach consensus rather than a single microRNA, an era of what is called ‘Personalized Medicine’ [60].
Conclusion
mmu-miR-511-5p could be a reliable biomarker for early diagnosis of infection with T. gondii ME49 strain, as early as one-week PI, and infection with the RH strain as early as 3-dpi. Its level is affected by the dose and progression of the infection. It can be also a promising diagnostic biomarker in the sera of immunosuppressed hosts, with the privilege of being less invasive than brain biopsy. Additionally, mmu-miR-511-5p serum level may be a useful indicator of successful treatment. So, it is a promising biomarker in immunocompetent, immunosuppressed, untreated and treated mice infected with ME49 or RH parasites.
To our knowledge, this is the first work assessing the expression of mmu-miR-511-5p in Swiss-albino mice sera using different infective doses of ME49 or RH T. gondii strains, in immunocompetent or immunosuppressed hosts, at different durations, before and after treatment with spiramycin.
Data availability
Not applicable.
References
Weiss LM, Dubey JP (2009) Toxoplasmosis: A history of clinical observations. Int J Parasitol 39(8):895–901. https://doi.org/10.1016/j.ijpara.2009.02.004
Flegr J, Prandota J, Sovičková M et al (2014) Toxoplasmosis-a global threat. Correlation of latent toxoplasmosis with specific disease burden in a set of 88 countries. PLoS ONE 9(3):e90203. https://doi.org/10.1371/journal.pone.0090203
Ahmadpour E, Daryani A, Sharif M et al (2014) Toxoplasmosis in immunocompromised patients in Iran: a systematic review and meta-analysis. J Infect Dev Ctries 8(12):1503–1510. https://doi.org/10.3855/jidc.4796
Elsheikha HM, Marra CM, Zhu XQ (2020) Epidemiology, pathophysiology, diagnosis, and management of cerebral toxoplasmosis. Clin Microbiol Rev 34(1):e00115-e119. https://doi.org/10.1128/CMR.00115-19
Derouin F, Sarfati C, Beauvais B et al (1990) Prevalence of pulmonary toxoplasmosis in HIV-infected patients. AIDS 4(10):1036 (PMID: 2261121)
Sensini A (2006) Toxoplasma gondii infection in pregnancy: opportunities and pitfalls of serological diagnosis. Clin Microbiol Infect 12(6):504–512. https://doi.org/10.1111/j.1469-0691.2006.01444.x
Contini C (2008) Clinical and diagnostic management of toxoplasmosis in the immunocompromised patient. Parassitologia 50(1–2):45–50
Liu Q, Wang ZD, Huang SY et al (2015) Diagnosis of toxoplasmosis and typing of Toxoplasma gondii. Parasit Vectors 28(8):292. https://doi.org/10.1186/s13071-015-0902-6
Montoya JG (2002) Laboratory diagnosis of Toxoplasma gondii infection and toxoplasmosis. J Infect Dis 185(s1):S73–S82. https://doi.org/10.1086/338827
Liesenfeld O, Press C, Montoya JG et al (1997) False-positive results in immunoglobulin M (IgM) Toxoplasma antibody tests and importance of confirmatory testing: the Platelia toxo IgM test. J Clin Microbiol 35:174–178. https://doi.org/10.1128/jcm.35.1.174-178.1997
Stepick-Biek P, Thulliez P, Araujo FG et al (1990) IgA antibodies for diagnosis of acute congenital and acquired toxoplasmosis. J Infect Dis 162(1):270–273. https://doi.org/10.1093/infdis/162.1.270
Conley FK, Jenkins KA, Remington JS (1981) Toxoplasma gondii infection of the central nervous system. Use of the peroxidase-antiperoxidase method to demonstrate toxoplasma in formalin fixed, paraffin embedded tissue sections. Hum Pathol 12(8):690–698. https://doi.org/10.1016/s0046-8177(81)80170-0
de Faria Junior GM, Murata FHA, Lorenzi HA et al (2021) The Role of microRNAs in the Infection by T. gondii in Humans. Front Cell Infect Microbiol 11:670548. https://doi.org/10.3389/fcimb.2021.670548
Xie X, Lu J, Kulbokas EJ et al (2005) Systematic discovery of regulatory motifs in human promoters and 3′ UTRs by comparison of several mammals. Nature 434(7031):338–345. https://doi.org/10.1038/nature03441
Gracias DT, Katsikis PD (2011) MicroRNAs: key components of immune regulation. Adv Exp Med Biol 780:15–26. https://doi.org/10.1007/978-1-4419-5632-3_2
Mitchell PS, Parkin RK, Kroh EM et al (2008) Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci USA 105(30):10513–10518. https://doi.org/10.1073/pnas.0804549105
Farr RJ, Joglekar MV, Taylor CJ et al (2013) Circulating non-coding RNAs as biomarkers of beta cell death in diabetes. Pediatr Endocrinol Rev 11(1):14–20 (PMID:24079075)
Morimura R, Komatsu S, Ichikawa D et al (2011) Novel diagnostic value of circulating miR-18a in plasma of patients with pancreatic cancer. Br J Cancer 105(11):1733–1740. https://doi.org/10.1038/bjc.2011.453
Zhang X, Guo J, Fan S et al (2013) Screening and identification of six serum microRNAs as novel potential combination biomarkers for pulmonary tuberculosis diagnosis. PLoS ONE 8(12):e81076. https://doi.org/10.1371/journal.pone.0081076
Chamnanchanunt S, Fucharoen S, Umemura T (2017) Circulating microRNAs in malaria infection: bench to bedside. Malar J 16(1):334. https://doi.org/10.1186/s12936-017-1990-x
Mogahed NM, Khedr SI, Ghazala R et al (2018) Can miRNA712_3p be a promising biomarker for early diagnosis of toxoplasmosis? Asian Pac J Trop Med 11(12):688–692. https://doi.org/10.4103/1995-7645.248341
Judice CC, Bourgard C, Kayano AC et al (2016) MicroRNAs in the host-Apicomplexan parasites interactions: A review of immunopathological aspects. Front Cell Infect Microbiol 2(6):5. https://doi.org/10.3389/fcimb.2016.00005
Jia B, Chang Z, Wei X et al (2014) Plasma microRNAs are promising novel biomarkers for the early detection of Toxoplasma gondii infection. Parasit Vectors 7:433. https://doi.org/10.1186/1756-3305-7-433
Martins-Duarte ES, Lemgruber L, de Souza W et al (2010) Toxoplasma gondii: fluconazole and itraconazole activity against toxoplasmosis in a murine model. Exp Parasitol 124(4):466–469. https://doi.org/10.1016/j.exppara.2009.12.011
Kalani H, Daryani A, Sharif M et al (2016) Comparison of eight cell-free media for maintenance of Toxoplasma gondii tachyzoites. Iran J Parasitol 11(1):104–109 (PMID:27095976)
Chew WK, Segarra I, Ambu S et al (2012) Significant reduction of brain cysts caused by Toxoplasma gondii after treatment with spiramycin coadministered with metronidazole in a mouse model of chronic toxoplasmosis. Antimicrob Agents Chemother 56(4):1762–1768. https://doi.org/10.1128/AAC.05183-11
Li Y, Kowdley KV (2012) Method for microRNA isolation from clinical serum samples. Anal Biochem 431(1):69–75. https://doi.org/10.1016/j.ab.2012.09.007
Meningher T, Lerman G, Regev-Rudzki N et al (2017) Schistosomal microRNAs isolated from extracellular vesicles in sera of infected patients: A new tool for diagnosis and follow-up of human schistosomiasis. J Infect Dis 215(3):378–386. https://doi.org/10.1093/infdis/jiw539
Basic principles of RT-qPCR https://www.thermofisher.com/eg/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/spotlight-articles/basic-principles-rt-qpcr.html. (accessed 16 February 2023)
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using Real-Time quantitative PCR and the 2−ΔΔCT method. Methods 25(4):402–408. https://doi.org/10.1006/meth.2001.1262
Carruthers VB (2002) Host cell invasion by the opportunistic pathogen Toxoplasma gondii. Acta Trop 81(2):111–122. https://doi.org/10.1016/s0001706x(01)00201-7
Montoya JG, Liesenfeld O (2004) Toxoplasmosis. Lancet 363(9425):1965–1976. https://doi.org/10.1016/S0140-6736(04)16412-X
Krings A, Jacob J, Seeber F et al (2021) Estimates of toxoplasmosis incidence based on healthcare claims data, Germany, 2011–2016. Emerg Infect Dis 27(8):2097–2106. https://doi.org/10.3201/eid2708.203740
Wang J, Chen J, Sen S (2016) MicroRNA as biomarkers and diagnostics. J Cell Physiol 231(1):25–30. https://doi.org/10.1002/jcp.25056
Cai Y, Shen J (2017) Modulation of host immune responses to Toxoplasma gondii by microRNAs. Parasite Immunol. https://doi.org/10.1111/pim.12417
Zeiner GM, Norman KL, Thomson JM et al (2010) Toxoplasma gondii infection specifically increases the levels of key host microRNAs. PLoS ONE 5(1):e8742. https://doi.org/10.1371/journal.pone.0008742
Hu RS, He JJ, Elsheikha HM et al (2018) Differential brain MicroRNA expression profiles after acute and chronic infection of mice with Toxoplasma gondii oocysts. Front Microbiol 2(9):2316. https://doi.org/10.3389/fmicb.2018.02316
Su YL, Wang X, Mann M et al (2020) Myeloid cell-targeted miR-146a mimic inhibits NF-κB-driven inflammation and leukemia progression in vivo. Blood 135(3):167–180. https://doi.org/10.1182/blood.2019002045
Kim MJ, Jung BK, Cho J et al (2016) Exosomes secreted by Toxoplasma gondii-Infected L6 cells: Their effects on host cell proliferation and cell cycle changes. Korean J Parasitol 54(2):147–154. https://doi.org/10.3347/kjp.2016.54.2.147
Puimège L, Van Hauwermeiren F, Steeland S et al (2015) Glucocorticoid-induced microRNA-511 protects against TNF by down-regulating TNFR1. EMBO Mol Med 7(8):1004–1017. https://doi.org/10.15252/emmm.201405010
Tserel L, Runnel T, Kisand K et al (2011) MicroRNA expression profiles of human blood monocyte-derived dendritic cells and macrophages reveal miR-511 as putative positive regulator of toll-like receptor. J Biol Chem 286(30):26487–26495. https://doi.org/10.1074/jbc.M110.213561
Hunter CA, Sibley LD (2012) Modulation of innate immunity by Toxoplasma gondii virulence effectors. Nat Rev Microbiol 10(11):766–778. https://doi.org/10.1038/nrmicro2858
Mosser D, Edwards J (2008) Exploring the full spectrum of macrophage activation. Nat Rev Immunol 8:958–969. https://doi.org/10.1038/nri2448
Jensen KD, Wang Y, Wojno EDT et al (2011) Toxoplasma polymorphic effectors determine macrophage polarization and intestinal inflammation. Cell Host Microbe 9(6):472–483. https://doi.org/10.1016/j.chom.2011.04.015
Mazurek M, Mlak R, Homa-Mlak I et al (2022) High miR-511-3p Expression as a Potential Predictor of a Poor Nutritional Status in Head and Neck Cancer Patients Subjected to Intensity-Modulated Radiation Therapy. J Clin Med 11(3):805. https://doi.org/10.3390/jcm11030805
Curtale G, Renzi TA, Drufuca L et al (2017) Glucocorticoids downregulate TLR4 signaling activity via its direct targeting by miR-511-5p. Eur J Immunol 47(12):2080–2089. https://doi.org/10.1002/eji.201747044
Awuah D, Ruisinger A, Alobaid M et al (2020) MicroRNA-511-3p mediated modulation of the peroxisome proliferator-activated receptor gamma (PPARγ) controls LPS-induced inflammatory responses in human monocyte derived DCs. BioRxiv. https://doi.org/10.1101/2020.11.05.369967
Gunassekaran GR, Poongkavithai Vadevoo SM, Baek MC et al (2021) M1 macrophage exosomes engineered to foster M1 polarization and target the IL-4 receptor inhibit tumor growth by reprogramming tumor-associated macrophages into M1-like macrophages. Biomaterials 278:121137. https://doi.org/10.1016/j.biomaterials.2021.121137
Karo-Atar D, Itan M, Pasmanik-Chor M et al (2015) MicroRNA profiling reveals opposing expression patterns for miR-511 in alternatively and classically activated macrophages. J Asthma 52(6):545–553. https://doi.org/10.3109/02770903.2014.988222
Mukhopadhyay D, Arranz-Solís D, Saeij JPJ (2020) Influence of the host and parasite strain on the immune response during Toxoplasma infection. Front Cell Infect Microbiol 15(10):580425. https://doi.org/10.3389/fcimb.2020.580425
Qi Q, Dong Z, Sun Y et al (2018) Protective effect of Bergenin against cyclophosphamide-induced immunosuppression by immunomodulatory effect and antioxidation in Balb/c mice. Molecules 23(10):2668. https://doi.org/10.3390/molecules23102668
Matsui K, Tamai S, Ikeda R (2016) Effects of macrolide antibiotics on Th1 cell and Th2 cell development mediated by Langerhans cells. J Pharm Pharm Sci 19(3):357–366. https://doi.org/10.18433/J3Z32F
Butcher BA, Fox BA, Rommereim LM et al (2011) Toxoplasma gondii rhoptry kinase ROP16 activates STAT3 and STAT6 resulting in cytokine inhibition and arginase-1-dependent growth control. PLoS Pathog 7(9):e1002236. https://doi.org/10.1371/journal.ppat.1002236
Wang Y, Chang W, Zhang Y et al (2019) Circulating miR-22-5p and miR-122-5p are promising novel biomarkers for diagnosis of acute myocardial infarction. J Cell Physiol 234(4):4778–4786. https://doi.org/10.1002/jcp.27274
Omar M, Abaza BE, Mousa E et al (2021) Effect of spiramycin versus aminoguanidine and their combined use in experimental toxoplasmosis. J Parasit Dis 45(4):1014–1025. https://doi.org/10.1007/s12639-021-01396-9
Murata FH, Ferreira MN, Camargo NS et al (2016) Frequency of anti-Toxoplasma gondii IgA, IgM, and IgG antibodies in high-risk pregnancies. Brazil Rev Soc Bras Med Trop 49(4):512–514. https://doi.org/10.1590/0037-8682-0046-2016
Wallon M, Peyron F, Cornu C et al (2013) Congenital toxoplasma infection: monthly prenatal screening decreases transmission rate and improves clinical outcome at age 3 years. Clin Infect Dis 56(9):1223–1231. https://doi.org/10.1093/cid/cit032
He X, Sai X, Chen C et al (2013) Host serum miR-223 is a potential new biomarker for Schistosoma japonicum infection and the response to chemotherapy. Parasit Vector 6:272. https://doi.org/10.1186/1756-3305-6-272
Wang C, Fan HQ, Zhang YW (2019) MiR-511-5p functions as a tumor suppressor and a predictive of prognosis in colorectal cancer by directly targeting GPR116. Eur Rev Med Pharmacol Sci 23(14):6119–6130. https://doi.org/10.26355/eurrev_201907_18425
Tribolet L, Kerr E, Cowled C et al (2020) MicroRNA Biomarkers for Infectious Diseases: From Basic Research to Biosensing. Front Microbiol 11:1197. https://doi.org/10.3389/fmicb.2020.01197
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Rasha Fadly Mady, Mona Mohamed El-Temsahy and Safaa Ibrahim Khedr conceptualized the study; All authors contributed to the methodology; Yasmine Amr Issa and Aya Saied Zaghloul performed formal analysis and investigation; All authors contributed to original draft preparation and reviewed the manuscript; Safaa Ibrahim Khedr edited the whole text and made the final review of the manuscript, prepared the figures, formatted the tables and is the corresponding author. Safaa Ibrahim Khedr and Yasmine Amr Issa wrote the response to the reviewers.
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Mady, R.F., El-Temsahy, M.M., Issa, Y.A. et al. MicroRNA mmu-miR-511-5p: A promising Diagnostic Biomarker in Experimental Toxoplasmosis Using Different Strains and Infective Doses in Mice with Different Immune States Before and After Treatment. Acta Parasit. (2024). https://doi.org/10.1007/s11686-024-00851-w
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DOI: https://doi.org/10.1007/s11686-024-00851-w