The Outcome of Therapeutic Trials Using Iron and Iron Chelator in Acute Murine Toxoplasmosis: Histopathological and Immunohistochemical study

• Toxoplasmosis cause severe diseases in both animals and humans.
• It is imperative to discover new treatment to overcome drug tolerance and resistance
• Iron plays a crucial role in the different cellular processes and the vital biological functions of the parasite
• The iron chelator, deferoxamine, could be a promising drug to control acute T. gondii infection due to the limitation of iron availability.

Iron plays a crucial role in different cellular processes, including oxygen transport, respiration, and DNA synthesis. Moreover, iron is essential for many vital biological functions such as heme biosynthesis [14], biogenesis of iron-sulphur clusters [15], and acting as a cofactor for a number of crucial proteins, including propyl hydroxylases [16] and catalase [17].

The hexadentate iron chelator deferoxamine (DFO) mesylate possesses antioxidant properties [18]. In cases of iron storage diseases, it is utilized as a chelator in clinical medicine to remove iron. Iron bound by DFO making it metabolically inactive, preventing or reversing free radical production's effects. Therefore, it reduces oxidative damage in vital organs and it may even lead to improved survival in vivo and in vitro studies [19].

Iron acquisition and utilization have been investigated in some pathogenic parasites, such as Trichomonas [20]. Additionally, they have been extensively studied in blood flagellates, like Leishmania [21] and Trypanosoma [22]. Interleukin 6 (IL-6) is the main mediator responsible for hepatocytic production of acute inflammatory phase proteins. Further, in murine toxoplasmosis, a gradual increase in serum IL-6 is correlated with clinical signs [23]. Transforming growth factor-β (TGF-β) is well-known for its immunosuppressant action. Also, the anti-inflammatory action of this cytokine makes it possible to control the development of immunopathological phenomena related to a type-1 immune response [24]. Yet research on iron use in toxoplasma is still lacking. Therefore, the present study aims to evaluate the effect of adding or removing iron on the outcome of T. gondii infection in the liver of mice using histopathological and Immunohistochemical analysis of IL-6 and TGF- β expression.

PATIENTS/MATERIALS AND METHODS

"The avirulent (Me49) strain of T. gondii:"

This experimental study was conducted from July 2023 to December 2023 at the Parasitology Department, Faculty of Medicine, Zagazig University in Egypt. Me49 type II cystogenic Toxoplasma strain was generously obtained from the Medical Parasitology Department, Faculty of Medicine, Alexandria University. Using a 22-gauge blunt feeding needle and a quantity of brain homogenate calibrated to produce 10 tissue cysts to retain the survival rate of mice. The strain was maintained by oral passage as it is the most naturalistic route of infection in outbred Swiss albino mice through administering cysts per mouse orally; the cysts were obtained from brain homogenate of mice that had been infected with the Me49 strain of T. gondii for at least eight weeks. Tissue cysts were counted in 10 μL of the brain suspension using light microscopy [25].

Experimental animals

Sample size was calculated to be not less than 40.  The sample size was calculated using the Open Epi program with a confidence level of 95% and power of 80%.

In our experiment, sixty Swiss albino male mice were free of parasites that were 3-4 weeks old and weighed 20-25 grams.  The mice were kept in well-ventilated cages and could accommodate 20 mice per cage. The bedding was changed daily to ensure a clean environment for the mice. The animals were provided with conventional pelleted food and had unrestricted access to water. They were kept in compliance with the research protocols following the recommendations of the National Institutes of Health Guidelines for the Use of Animals in Research.

Ethical consideration

Permission and official approval to carry out the study was obtained. The ethical committee at the Faculty of Medicine, Zagazig University approved the experimental protocol. Approval number: ZU-IACUC/3/F/449/2023

Experimental design

Sixty mice were classified into four groups; 15 mice each (GI): Control negative non-infected non-treated group. (GII): Control positive Infected non-treated group injected intraperitoneally (I.P) with vehicle (phosphate buffered saline) (PBS) the day before infection and for seven more days following infection. (GIII): Infected iron sucrose-treated group. (GIV): Infecteddeferoxamine (DFO)-treated group.

Drugs

(Iron sucrose) iron saccharate (C₁₂H₂₉Fe₅Na₂O₂₃): Scrofer, manufactured and provided by Amoun Pharmaceutical Company, Egypt. The dosage chosen for the current work was therapeutic dose to treat the iron deficiency anaemia of 80mg/kg/ day [26]. Deferoxamine mesylate (C₂₆H₅₂N₆O₁₁S ): Desferal, provided by Novartis, Egypt. The chosen dosage for this study was 300mg/kg/day[27]. Both drugs were administered intraperitoneally one day prior to infection and for a further seven days following infection, according to the drug table ofPaget and Barnes[28].  Assessment during acute stage after completing the treatment. all animals were euthanized by cervical dislocation following the last dose of therapy. Hepatic tissue was taken for doing the following assessments.

I. Histopathological assessment:

Haematoxylin and eosin staining

After fixing in 10% formalin for 24 hours, samples were rinsed for twelve hours in water and dried in progressively increasing ethanol concentrations (120 minutes at 70% ethanol, 90 minutes at 90% ethanol, and one hour at 100% ethanol (two cycles). Following a one-hour immersion in a 50% ethanol and 50% xylene combination, the samples were cleaned for a further 1.5 hours in pure xylene. The samples were then covered with paraffin wax. The paraffin slices (4–5 μm) were stained with Haematoxylin and eosin [29]. The degree of inflammatory cell infiltration in the hepatic tissue was assessed. Five histological sections were examined in each mouse. Then, ten low-power fields (100×) from each of the examined sections were determined to calculate the average score (A mild reaction is denoted by +1, a moderate reaction by +2, and an extreme reaction by +3) [30].

 Iron Staining of the infected mice's liver:

The assessment of iron accumulation in the liver was conducted using Perl's staining. Sections that had been deparaffinized were first immersed in a solution containing 10% potassium ferrocyanide for five minutes at room temperature (Merck KGaA, Darmstadt, Germany). They were incubated for twenty minutes in a solution containing hydrochloric acid 10% (Merck S.A., Rio de Janeiro, RJ, Brazil). To counterstain the slides, nuclear fast red was used (Merck KGaA, Darmstadt, Germany) and then examined using a 40_˟ objective by light microscopy [31].

Congo red staining

Some paraffin-embedded tissue sections of GIII were stained with Congo red stain for detection of amyloidosis because iron overload accelerates the production of amyloid β. We prepared the stock solution as the following:Congo red 0.2% solution:  Add the Congo red dye (0.1 g) (cat. no. C6767, Sigma-Aldrich) to 500 ml of saturated NaCl solution, making sure to leave a layer of NaCl at the bottom. Before using, let the mixture stir for a whole night. A dark glass bottle will keep the solution fresh for a very long period. Filtration is required before using this solution [32].

II. Immunohistochemical assessment:

Immunohistochemical study was carried out to investigate the expression of IL-6 and TGF- β levels. Liver sections were embedded in paraffin after being fixed in formalin 10%, after that, the sections were mounted on glass slides with a positive charge (Biogenex, USA). Expression of IL-6 and TGF-β levels were evaluated using the biotin-peroxidase-ABC method. The slices were incubated in phosphate-buffered saline (PBS) for five minutes; the sections were submerged in citrate buffer solution (pH 6) in a microwave for twenty minutes to facilitate antigen retrieval. An endogenous peroxidase-blocking reagent containing sodium azide and hydrogen peroxide was incubated with tissue sections (DAKO, Cat. No. S2001) in compliance with the guidelines suggested by the manufacturer. After rinsing, tissue sections were incubated with primary monoclonal antibodies against both IL-6 (Cat. No. 406-ML-005) and TGF-β (Cat. No. NBP2-11921B) (Novus Biologicals, LLC, USA). Next, the avidin-biotin-peroxidase complex was incubated in each section. (ABC kit, PK-4000). 3, 5 diaminobenzidines (DAB) substrate was used for visualization (DAKO Corp, Fremont, CA, USA). y. These sections were inspected with a light microscope (Olympus, Hamburg, Germany) using a grading scale: 0=no cells, 1=few cells (low densities), 2=many cells (high densities) [29].

IL-6 and TGF-b1 immuno-activity quantitation was made by digital image analysis through the image J analysis software on five fields from each slide. This software could measure the total positive stained brown color/areas throughout the unstained cells. From this data an index (positively stained cells per a total of 1000 cells) can be computed (JID801D).

Statistical analysis

The collected data were computerized and statistically analyzed using the Statistical Package for Social Sciences SPSS program version 27.0.  Quantitative data were expressed as mean ± SD (Standard deviation). ANOVA test was used to calculate the difference between quantitative variables in more than two groups in normally distributed data. Image J software analytical estimation was used. Statistical comparisons were done with significance Level of P-value ≤ 0.05 indicates significant, p 0.05 indicates a non-significant difference.

RESULTS

Mice death during the experimental study: There was no significant difference in the survival and mortality rates among the different studied groups as we started our experiment with 45 infected mice, and 15 mice as negative control. By the end of the experiment, 8 infected mice, and 2 control mice had died, while 37 and 13 mice, respectively survived till scarification. Survival rate in the infected and negative control group was 82.2% and 86.6% respectively. While the mortality among the studied groups rate was 17.7% and 13.3% respectively  

I. Histopathological Results 

 Haematoxylin and Eosin stain:

Group I (non-infected control group):

Normal hexagonal hepatocyte plates were visible in serial sections obtained from the liver of this group. The hepatocytes are large and polyhedral cells. They may contain about two and four nuclei. The perisinusoidal space (also known as the space of Disse) is generated when the hepatocytes expand the villi into the perisinusoidal vascular space. Satellite cells inhabit space of Disse. There were no discernible signs of necrotic, apoptotic, degenerative, or inflammatory alterations (Fig.1a). 

Group II (T. gondii Infected group):

Examined sections from liver of this group pointed out marked histopathological changes represented by portal biliary reaction, lymphocytic cholangitis, lymphoplasmacytic infiltration and interstitial aggregations with associated focal hepato-cellular necrosis and individual cells apoptosis. The most prominent feature was reticulo-endothelial cells proliferation. Some of the proliferating cells mostly arise from hepatic satellite cells (HSCs). These characteristic cells appeared as large, rounded, apple-shaped or atypically formed with intracellular nuclear proliferative activities and abundant eosinophilic cytoplasm (Fig.1b).  

Group III (Iron-treated group):

Characteristic hepatic lesions were represented by marked portal and interstitial round cells aggregations, focal hepatocellular degeneration, scattered necrosis and apoptosis. Von-Kupffer cells proliferation, hypertrophic changes and focal endotheliosis. Also, Hepatic satellite cells with metamorphic and nuclear reactive changes were observed. Moreover, Focal and diffuse, per-arteriolar and subendothelial deposition of homogenous eosinophilic amorphous structureless materials were detected (Fig.1c).

Group IV (Deferoxamine -treated group)

Examined sections from liver of this group declared a less significant micromorphological lesions compared with the infected group as the changes were represented by mild to moderate hepatocellular degenerative changes with a few scattered necrotic and apoptotic cells. Mild portal lymphocytic infiltration, Von-Kupffer cells hyper reactive changes and transformation of some hepatic satellite stem cells to large typical and atypical metamorphic cells were recorded (Fig.1d).

Iron staining in the liver of infected mice:

The characteristic iron deposition (hemosiderin granules) was demonstrated in the cytoplasm of some hepatocytes, vascular endothelial cells, Von-Kupffer cells and macrophages. Stained hemosiderin particles were amorphous basophilic structures of variable shapes and sizes. The staining reactivity was noticeable in iron supplemented-Infected group (GIII) (Fig.2c), meanwhile, most of the examined sections of groups I, II and IV were negatively stained (Fig.2a, b, d).

Congo red stain:

Amyloid deposition was markedly demonstrated in group III with presence of homogenous eosinophilic structureless materials, mostly sub endothelially between hepatocytes and portal- peri-arteriolar, in H&E stained sections. Such materials were specifically stained as magenta red materials in the same locations using Congo red stain (Fig.3).

II. Immunohistochemical Results:

A. Interleukin 6 (IL-6):

Expressions of IL-6 have been identified as a reactive cytoplasmic brownish protein in some of the hepatic endothelial and Von-Kupffer cells. The hepatic tissues of the control free group (GI) exhibited a negative reaction. Moreover, almost identical reactivity was observed in the hepatic tissues of the group treated with deferoxamine (GIV) and seemed to be clear within the endothelial cells associated with amyloid deposition. Moderate to strong brownish cytoplasmic reactivity was pronounced in some hepatocytes, the hepatic peri-portal and para-portal inflammatory cells, vascular endothelial and activated Von-Kupffer cells.  Such reactivity was detected in T.gondii infected control group (GII) and in the iron-supplemented infected group (GIII) which appeared with a comparable more potent immune reactivity (Fig. 4).

B. Transforming growth factor beta (TGF-β):

 Immuno-reactivates of TGF-β signal were represented by mild brownish immunostaining of the Von-Kupffer cells of the liver in the non-infected control group (GI). On the contrary, the immunoreactive staining reaction was notable in T.gondii infected group (GII) where some of hepatic Von-Kupffer and vascular endothelial cells beside some of the centro-lobular and paracentral hepatocytes showed moderate brownish cytoplasmic stainability to the used marker. Immune reactivity of TGF-β in the infected-iron-supplemented group (GIII) denoted mild reactions in most of the examined hepatic cells, a part of some mildly stained hepatic Von-Kupffer cells. DFO treated group (GIV) demonstrated a strong number of reactive hepatocytes, reticulo-endothelial cells. Positive cells showed marked brownish cytoplasmic stainability to the used marker (Fig.5).   

Morphometric Findings:

Image J soft wear analytic estimation of immune- reactive cells of IL-6 and TGF-β  in the different studied groups were recorded. Hepatic control free group (GI) denoted the estimated percentages of positivities of IL-6 and TGFβ as 6.61 ± 1.59 and 1.22 ± 0.27 respectively in the liver tissues. Meanwhile in control-infected group (GII), the percentages of positivities of IL-6 and TGFβ were 39.54 ± 8.7 and 32.94 ± 9.88 in the liver tissues. Also in infected-iron supplemented group (GIII), the percentages of IL-6 and TGFβ were 46.29 ± 13.89 and 12.45 ± 3.61 in the liver tissues. Infected-DFO supplemented group (GIV) showed the percentages of IL-6 and TGFβ as 10.49 ± 3.04 and 49.18 ± 12.3 respectively in the liver tissues [Table 1].

Table 1: Comparison between the percentage of positivity of IL-6 and TGF-β in the liver tissues in the different studied groups 

         SD: Standard deviation          F: ANOVA test        *: highly significant (P<0.001)

DISCUSSION

T. gondii is important because it may avoid the immune system after an acute infection, causing a developmental transformation from rapid replicating tachyzoite to slow replicating latent bradyzoite in the brain. This shift from one parasite stage to the next produces chronic infection. [33]. In this study, we concentrated on the acute stage of infection before transformation of bradyzoite in brain

 The liver is an organ that stores excess bodily iron, thus it may play a crucial role in the parasite's development when iron supplements are taken [34]. Iron has a role in numerous biological reactions for instance, proteins essential to nucleic acid synthesis. From unicellular organisms to humans, all forms of life have evolved to retain these functions [35]. During infection whenever microorganisms and humans interact, the shared requirement for iron forms these interactions [36]. Further, iron is essential for metabolism, but because of its potential toxicity, storage and transportation must be carefully controlled [37]

In our study, several pathological changes were demonstrated in experimentally infected mice with T. gondii infection.  In GII, the pathological changes in the liver tissues of mice were in the form of lymphocytic cholangitis, individual cells apoptosis, reticulo-endothelial cells proliferation with the multi-focal nodular formation compared to negative control group (Fig.1a). This observation is harmonious with He et al. and Alvarado-Esquivel et al.[38, 39]who found that acute T.gondii infection in the liver is associated with lymphocytic infiltration and apoptosis. The mechanisms underlying T. gondii infection-induced liver damage and histological alterations may be related to the parasite's direct proliferative effect on the tissues, which results in tissue damage and cell death, or they may be attributed to the indirect effect of the infection, which is caused by an overreaction by the immune system to the parasite. Moreover, in GIII, pathological changes in liver sections showed higher degree of inflammatory infiltration compared to GII (Fig.1b,c). It was reported that infected-iron supplemented group is associated with more inflammatory reactions when compared with other groups [27]. According to Dziadek et al. [40] T. gondii requires iron in order to survive and proliferate within its host cells. In addition, different studies suggested the availability of particular human lactoferrin-binding parasite receptors that contribute to the supply of host iron to the tachyzoites [41].

As regard, DFO-treated mice (GIV), inflammatory and degenerative changes were significantly lower in the liver samples when compared to infected control group (GII) (Fig.1d). The results of Olivera et al. [27]who mentioned that DFO is associated with decreasing inflammatory score in experimentally infected mice with T. gondii corroborate these conclusions.

Interestingly,Dimier and Bout[42] stated that using the iron chelator, deferoxamine, inhibited the growth of the parasite intracellularly by limiting the availability of iron, a condition that was reserved by adding holotransferrin, an iron-saturated transferrin. Also, Almeida et al. [43] found that the DFO treatment had no impact on the cell viability suggesting that the reduction in iron availability to the parasite was what controlled the growth of T. gondii.      

Moreover,Amyloid deposition was markedly demonstrated in GIII with presence of homogenous eosinophilic structureless materials, mostly sub-endothelial between hepatocytes and portal-peri-arteriolar, in H&E and congo red stained section (Fig.1&3). This was in line with Atmaca and Atmaca[10]who demonstrated thatT.gondii infected mice exhibited a statistically significant progressively rise in serum amyloid A levels on the 2nd, 4th, and 6th days post infection. Also, Becerril-Ortega et al. [44] explained that iron overload accelerates the production of amyloid β and results in cognitive impairment in transgenic mice and associated with many degenerative diseases such as alzheimer’s disease. Researchers found that Amyloid peptide protein accelerates iron export by fixing the iron export protein, ferroportin, on the cell surface in a functional region [45]. Actually, multiple investigations have demonstrated that iron might increase the oxidative damage associated with Amyloid peptide aggregation [46].

In the current study, there was a notable expression of IL-6 in the liver tissues of mice (39.54 ± 8.7) in GII (Fig.4).  Additionally, it was shown that BeWo trophoblasts and human monocyte cell lineage THP-1 cells, raised IL-6 levels in reaction to infection with T. gondii [43].

Moreover, clinical symptoms of murine toxoplasmosis are associated with a progressive increase in serum IL-6 [36]. It seems that IL-6 enhances T. gondii's intracellular growth in mice [47].

After iron saccharate supplementation, expression of IL-6 significantly increased in liver tissues of mice (46.29 ± 13.89) in GIII (Fig.4). This observation is in an agreement with Olivera et al. [27] who stated that the infection raised IL-6 levels in the serum samples of both the infected group and the infected treated with iron.

IL-6 can regulate iron systemically via the induction of hepcidin hormone, which controls the iron entry to the bloodstream and imprisons it inside the enterocytes [48]. So, IL-6 needs to be adjusted since high IL-6 levels brought on by an oral T. gondii infection may promote the growth of the parasite, since the hepcidin-IL-6 axis makes iron more available inside the enterocytes, allowing the parasite to thrive [49].

As regards GIV (deferoxamine treated group), decrease of IL-6 expression (10.49 ± 3.04) in the liver sections of mice was detected (Fig.4). This finding was in line withAghabi et al.[37]who exhibited decrease inflammation and subsequently increase parasite survival after chelation of iron with deferoxamine.Furthermore, compared to infected control mice, deferoxamine therapy reduced IL-6 serum levels 1.65-fold, as demonstrated by Olivera et al. [27].

 In this study, we found that TGF-β expression was elevated in liver sections (49.18 ± 12.3) in GIV compared to GII (Fig.5). These results coincide with the research conducted by Misumi et al. [50] who noticed that in rats infected experimentally, DFO is linked to increased TGF-β expression.Furthermore, Qayoom et al.[51] noted that deferoxamine was linked to increased TGF-β expression and was crucial in limiting infection, particularly in rats that were infected experimentally. In the same side, we demonstrated that increased expression of TGF-β in intestinal tissues was noticed when iron deprivation occurred [52].

 Keep in mind that this cytokine stimulates regulatory T cells to modulate the immunological response. Overproduction and/or activation of TGF-β can influence the growth, migration, invasion, and survival of cells. Thus, a wide range of pathological conditions as tissue renewal result in increased TGF-β expression levels [53].

 In conclusion: The present work showed that hepatic lesions in GIII were represented by focal hepatocellular degeneration, scattered necrosis and apoptosis. Examined sections from liver of GIV declared a mild hepatocellular degenerative changes with a few scattered necrotic and apoptotic cells. Chelation of iron using deferoxamine treatment showed histopathological improvement in liver tissues of experimentally infected mice with T. gondii parasite. These findings were confirmed by immunohistochemical assessment. Therefore, deferoxamine might be a hopeful drug to control acute T. gondii infection due to the limitation of iron availability.   Iron supplementation in pregnant women can be beneficial for embryo/fetus development, but it can also promote parasite growth, increasing the chance of congenital infection. This paper lays the groundwork for future research on iron in T. gondii, which still has a lot of unanswered concerns.

Funding: None. Author funded

Conflict of Interest: None.

Ethical approval:

Permission and official approval to carry out the study was obtained. The ethical committee at the Faculty of Medicine, Zagazig University approved the experimental protocol. Approval number: ZU-IACUC/3/F/449/2023

Author contribution

We declare that the idea and design of the research were contributed to by all writers. All listed authors have made substantial contributions to the material preparation, the data collecting and analysis and writing the manuscript's initial draft. The final manuscript was read and approved by all writers.

Availability of data and materials:

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.