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Journal of Parasitic Diseases: Official Organ of the Indian Society for Parasitology logoLink to Journal of Parasitic Diseases: Official Organ of the Indian Society for Parasitology
. 2023 Dec 5;48(1):33–45. doi: 10.1007/s12639-023-01635-1

Modulation of CXCL10 activity as a therapeutic target of ocular toxoplasmosis in diabetic mice

Mennat-Elrahman Ahmed Fahmy 1,✉, Amany Ahmed Abdel-Aal 2,3, Maisa Ahmed Shalaby 1, Ragaa Issa 4, Manal Badawi 5, Marwa A Fouly 6
PMCID: PMC10908887  PMID: 38440758

Abstract

Ocular toxoplasmosis is likely the most common cause of infectious posterior uveitis worldwide. CXCL10 chemokine has an important role in the maintenance of the T-cell response and the control of Toxoplasma gondii in the eye during chronic infection. Drugs that can modulate the chemokine activity could be effective against the parasite. In this work, CXCL10 local retinal expression was investigated in a diabetic mouse model with ocular toxoplasmosis for the first time. In addition, the efficacy of naphthoquinones and quinolones was compared to spiramycin (SP) in treating the infection and modulating the chemokine expression. Our results revealed that chloroquine (CQ) achieved the best results regarding the reduction of cerebral cyst burden (84.36%), improving the retinal histopathological changes, cellular infiltrates, and vasculitis significantly (P < 0.005), and balancing the strong CXCL10 expression caused by the infection. Buparvaquone-treated mice showed a significant percentage of reduction of brain cysts (76.25%), moderate improvement of histopathology, and mild to moderate CXCL10 expression. While SP showed the least efficacy against the parasite in the eye in the form of mild improvement of histopathological changes and downregulation of retinal chemokine expression with the least reduction rate of cerebral parasitic burden (57%). In conclusion, Optimal control of pathogens probably needs a balanced immune response with an optimum expression of chemokines. So, targeting the modulation of retinal CXCL10 may eventually be beneficial in the management of ocular toxoplasmosis plus its potential to act as a marker for predictive local immunological response during the infection.

Keywords: Ocular toxoplasmosis, Retinal CXCL10, Diabetic mice, Chloroquine, Buparvaquone

Introduction

Chemokines are chemotactic cytokines produced in tissues during homeostatic conditions and infections, they play a pivotal role in orchestrating and balancing the immune response against parasitic infections. They are essential regulators of cell recruitment and trafficking for controlling parasite replication and limiting pathology (McGovern and Wilson 2013; Sokol and Luster 2015; Cecchinato et al. 2023).

Chemokines are grouped into four main classes; one of which is the CXCL group. Chemokine ligand 10 (CXCL10) is a member of the CXC chemokine family. It is an IFN-γ-induced protein with potent chemoattractant properties for activated T cells, NK cells, monocytes, and neutrophils via binding either with the CXCR3 receptor or Toll-like receptor 4 (TLR4) (Sokol and Luster 2015; Lee et al. 2017; Gudowska-Sawczuk and Mroczko 2022).

CXCL10 is essential for guiding antigen-specific CD4 and CD8 T cells into infected organs during infection with Toxoplasma gondii (T. gondii), an intracellular Apicomplexan parasite that is acquired mainly through the consumption of contaminated food or drink with oocysts or tissue cysts infecting about 25% to 30% of the human population. Ocular toxoplasmosis is the most common manifestation in chronically infected subjects. It is considered the primary cause of infectious uveitis, presenting with retinochoroiditis (Khan et al. 2000; Arruda et al. 2021; Marín-García et al. 2022).

As an opportunistic parasite, conditions associated with immune system dysfunction, especially T cell suppression can lead to severe toxoplasmic retinochoroiditis (Norose et al. 2011). Type 1 diabetes mellitus (T1D) is a chronic autoimmune condition caused by the destruction of pancreatic β-cells triggered by the binding of CXCL10 expressed in the pancreas to TLR4 and associated with pathogenic effector T cells, and defective regulatory T cells (Tregs) (Schulthess et al. 2009; Zhou et al. 2021).

Medications used against ocular toxoplasmosis include; pyrimethamine, sulfadiazine, intravitreal clindamycin, trimethoprim-sulphamethoxazole, spiramycin, azithromycin, atovaquone, and tetracycline. Unfortunately, they helped to limit the replication of the parasite but did not fully eliminate it (Bonfioli and Orefice 2005; Norose et al. 2011; Yogeswaran et al. 2022).

Chloroquine (CQ), a quinoline derivative drug that is commonly used in the treatment of malaria and other parasitic infections, displays direct and indirect anti-Toxoplasma effects (Gamea et al. 2022). It is also known for its immunomodulatory and anti-inflammatory effects via different mechanisms. Some of them are; the inhibition of pro-inflammatory cytokines, blockage of nucleic acid-sensing toll-like receptors (TLRs), and modulation of chemokines (Wittebole et al. 2010; Richard et al. 2020). CQ is generally well tolerated systemically with a good safety profile, however chronic use (> 5 years) of high doses is known to develop retinopathy. So, according to the ophthalmology guidelines, adjustment of the dose is important (Gaynes et al. 2008; Schrezenmeier and Dörner 2020).

As for buparvaquone (BPQ), it is a hydroxynaphthoquinone with a strong theilericidal activity in addition to in vivo and in vitro inhibition of Neospora caninum and Toxoplasma gondii. The drug acts through the inhibition of several enzymes involved in mitochondrial electron transport (Mhadhbi et al. 2015; Spalenka et al. 2018; Fahmy et al. 2023).

With the rising number of type 1 diabetic cases worldwide, being at great risk of both retinal problems resulting from diabetes and toxoplasmic retinochoroiditis resulting from altered immune status, an urgent need for proper diagnosis and development of novel ways of treatment has emerged as there are no enough studies on experimental toxoplasmosis in diabetic models. So, in this study, for the first time, an STZ-T1D nonobese diabetic (NOD) mouse model was established for further exploration of the contribution of retinal CXCL10 in the local immune response against ocular toxoplasmosis in such condition and its possible role as an indicator of the severity of infection, in addition to the efficacy of CQ and BPQ compared to spiramycin in modulating the chemokine and treating the infection.

Materials and methods

Laboratory animals and ethics statement

Male Swiss albino mice of CD1 strain weighing 25–30 g and aged six to eight weeks were bred and maintained in the animal house of Theodor Bilharz Research Institute (TBRI) with provided food and water ad libitum. Handling mice during the experiment was carried out according to the ethical considerations of the institutional ethical committee for animal research to minimize suffering (Approval number: PT 761).

Study design

A total of 60 mice were randomly divided into 6 groups each of 10 mice (Wu et al. 2022) as follows; G1: normoglycemic control, G2: hyperglycemic only, G3: hyperglycemic infected, G4: hyperglycemic infected and treated with SP, G5: hyperglycemic infected and treated with BPQ, and G6: hyperglycemic infected and treated with CQ.

Induction and monitoring of type 1 diabetes mellitus

Intraperitoneal (IP) injection of streptozotocin (STZ) (Sigma-Aldrich, Missouri, United States) at a dose of 40 mg/kg/day for 5 consecutive days was used to induce type 1 Diabetes Mellitus in fasted mice. While the normal control group mice were injected with an equal volume of citrate buffer (pH 4.5) according to Furman (2015). To ensure hyperglycemia in the STZ-treated mice, blood glucose levels were measured from tail-vein blood samples using a glucometer [ACCU-CHEK Active; Germany] on experimental day 14, and mice with fasting glucose concentrations ≥ 150 mg/dl were included in the study. For monitoring the chronic diabetic state in the STZ-treated mice, blood glucose level measurements were repeated at week 7 and by the end of the experiment. Blood sugar levels were also checked in the control normal group (Furman 2015).

Parasite and infection

After confirming the hyperglycemic state on the 14th experimental day, mice were infected by oral gavage with ME49 T. gondii cysts obtained from the brains of previously infected mice 2 months before the experiment according to Ashour (2018). After preparing brain homogenate and counting the cysts under the microscope (40×), the infection dose needed for each mouse was adjusted to 100 cysts in 0.2 ml of PBS while control mice received 0.2 ml of PBS only (Dias et al. 2014).

Drugs

Drugs were administrated 6 weeks post-inoculation. Both SP and CQ were provided in tablet form and were administered orally to mice via gavage after dissolution in PBS and adjustment of the dose. While BPQ was provided as a powder that was emulsified in corn oil and heated to 37 °C to enhance the solubility of the drug before oral gavage (Müller et al. 2017).

Each mouse in G4 received spiramycin® [Pharaonia Pharmaceuticals, Egypt] once daily in a dose of 200 mg/kg/day for 10 successive days (Etewa et al. 2018). Each mouse in G5 received buparvaquone [Sigma-Aldrich, Inc.] once daily in a dose of 50 mg/kg/day for five successive days (Müller et al. 2017). While each mouse in G6 received chloroquine (Alexoquine®) [Alexandria Co. for Pharmaceuticals & Chemical Industries, Egypt] once daily in a dose of 20 mg/kg/day for 4 successive days (Fahmy et al. 2023).

Euthanasia and tissue preparation

At the end of the experiment (70 days), the surviving mice were euthanized by intraperitoneal injection of an anesthetic-anticoagulant solution (500 mg/kg thiopental and 100 units/mL heparin) (Laferriere et al. 2020). Eyeballs were removed from each mouse in all groups. Additionally, the brains of infected groups were removed. Eyes were fixed in a mixture of ethyl alcohol 30 ml, formalin 20 ml, and glacial acetic acid 10 ml to be processed by dehydration, clearing, then paraffin embedding. Sections from the paraffin-embedded eyes were stained with hematoxylin and eosin to be evaluated for pathological changes while, other sections were exposed to de-waxing, blocking, then incubation with rabbit anti-CXCL10 antibodies [Dako, Carpinteria, CA, USA] Slides were counterstained with hematoxylin before the examination. (Lyons et al. 2001; Norose et al. 2011; Ramos-Vara and Miller 2014; Yang et al. 2021). Each removed brain was emulsified in 1 ml of PBS.

Histopathological evaluation of eye sections

Pathological changes of retinal architecture, inflammatory cellular infiltrates, and vasculitis. Each was scored as follows (Lyons et al. 2001; Norose et al. 2011):

Retinal pathological changes

(0) normal histology; (1) mild edema; (2) obvious inflammatory reaction with one focal lesion of the retina; (3) few focal lesions associated with folding of the retinal layers; (4) intensive necrotic retinitis and retinal destruction or endophthalmitis.

Retinal inflammatory cellular infiltration

(0) no; (1) mild (< 10 cells/field × 400); (2) moderate (11–20 cells/field × 400); (3) severe (21–30 cells/field × 400).

Retinal vasculitis

(0) normal histology; (1) vessel dilation; (2) < 10 inflammatory cells around the vessels; (3) 10–30 inflammatory cells; (4) > 30 inflammatory cells.

Immunohistochemical evaluation

To measure the expression of the local retinal CXCL10 chemokine, a real-time automated image analyzer [LEICA Imaging Systems Ltd., Cambridge, England] connected with a video monitor in The National Research Centre (Giza, Egypt) was used. Optical density (OD) was automatically calculated in 10 fields for the CXCL10 marker.

Staining intensities of CXCL10 positive cells were graded on a numerical scale of 0 to 3 compared to the secondary antibody control. So that, 0 represents no difference from the control; 1, slightly greater than the control; 2, moderate staining; and 3, intense staining (Yoon et al. 2010).

Estimation of brain cyst count

Three aliquots of 20 μl of the brain suspension were examined under a light microscope (40×) for the presence and count of the T. gondii cysts. In addition, the concentration of cysts per ml of brain suspension was calculated according to Mokua Mose et al. (2017).

Statistical analysis of data

Quantitative values of the measured parameters were expressed as mean ± standard deviation (SD). The data were analyzed by one-way ANOVA for comparing between groups followed by post hoc test least significant difference (LSD). The probability of significant differences between the qualitative variables was determined by a chi-square test followed by pairwise comparisons for the immunohistochemical results. The difference was considered statistically significant when P < 0.005, and not significant when P ˃ 0.05. Statistical Package for Social Sciences (SPSS) version 14.0 was used.

Results

Blood sugar levels

Fasting blood sugar levels in the normal control group were (80–90 mg/dl), while in the diabetic groups ranged from 238.2 ± 36.32 to 436 ± 84.5 mg/dl. The highest levels were found in the diabetic-infected group in the 10th week (G3). At the end of the experiment, the fasting blood sugar levels among diabetic mice were significantly lower in both the diabetic-only group (G2) (313.2 ± 103.83) and the diabetic-infected and CQ-treated group (G6) (342.5 ± 45.76) (Fig. 1).

Fig. 1.

Fig. 1

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Fasting blood sugar levels among different diabetic study groups at different time intervals (BG: blood glucose, W: week)

Brain cyst burden

Significant rates of reduction of cerebral Toxoplasma cysts burden were achieved in all treated groups (P < 0.005) compared to the infected non-treated group (G3) with the best result seen after CQ treatment (G6) (84.36%) (Table 1).

Table 1.

One-way ANOVA and LSD post hoc tests were used for pairwise comparisons a, b, and c

Groups Number of cysts/mL brain homogenate (mean ± SD) Percentage of reduction of brain cysts
G3 551.67 ± 76.5 a
G4 237 ± 30.57 b 57%
G5 131 ± 32.26 c 76.25%
G6 86.25 ± 24.97 c 84.36%
Open in a new tab

There is a statistically significant difference (P < 0.005) between study groups having different letters

There is no statistically significant difference (P > 0.05) between study groups having the same letter

Histopathology

Retinal histopathological changes induced by diabetes and infection

The preserved retinal architecture of the normal group (Fig. 2A) was disturbed by either diabetes alone or diabetes and infection. The retinae of the mice in the diabetic group (G2) showed dilatation of the superficial vascular plexus in the ganglion cell layer (GCL) and vessels in the inner plexiform layer, edema of both outer plexiform and inner nuclear layers with few focal retinal foldings (Fig. 3A). These histopathological changes were aggravated by Toxoplasma infection (G3) with disturbed retinal architecture, severe vasculitis, and multiple focal inflammatory cellular infiltrates and areas of hemorrhages in the inner plexiform layer (IPL) (Fig. 4A, B and C and Tables 2, 3 and 4). Parasites appeared as solitary cysts in the outer layer of the retina (Fig. 4C).

Fig. 2.

Fig. 2

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A; Section of control mouse eye showing normal retina with different layers (H&E, X200). B; Section of control normal mouse eye showing normal retina with moderate immunoreactions in endothelial cells (CXCL10, X200)

Fig. 3.

Fig. 3

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A; Section of control diabetic mouse eye showing areas of some irregularities in the retinal layer, dilated capillaries (black arrows), and edema of the outer plexiform layer (OPL) and the outer nuclear layer of the retina (red arrow) (H&E, X200). B; Section of control diabetic mouse eye showing highly moderate immunoreactions in endothelial cells (CXCL10, X200)

Fig. 4.

Fig. 4

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A; Section of diabetic infected mouse eye showing dilated capillaries with reactive endothelium and distended with RBCs in the retina (arrow) (H&E, X200). B; Section of diabetic infected mouse eye showing dilated capillaries surrounded by a focal collection of inflammatory cells in the retina (arrow) (H&E, X100). C; Section of diabetic infected mouse eye showing interstitial hemorrhage and Toxoplasma cysts in the retina (H&E, X200). D; Section of control diabetic infected mouse eye showing relatively strong immunoreactions in endothelial cells (CXCL10, X200)

Table 2.

One-way ANOVA and LSD post hoc tests were used for pairwise comparisons a, b, c, d, e

Retinal pathological changes Total X2 P
Normal histology Mild edema Obvious inflammatory reaction with one focal lesion Few focal lesions
Groups G2 N 0 6 0 3 9d 29.72 0.003
% 0.0% 66.7% 0.0% 33.3% 100.0%
G3 N 0 0 2 4 6a
% 0.0% 0.0% 33.3% 66.7% 100.0%
G4 N 0 0 4 3 7b
% 0.0% 0.0% 57.1% 42.9% 100.0%
G5 N 0 3 3 1 7c
% 0.0% 42.9% 42.9% 14.3% 100.0%
G6 N 3 3 2 0 8e
% 37.5% 37.5% 25.0% 0.0% 100.0%
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There is a statistically significant difference (P < 0.005) between study groups having different letters

There is no statistically significant difference (P > 0.05) between study groups having the same letter

Table 3.

One-way ANOVA and LSD post hoc tests were used for pairwise comparisons a, b, and c

Retinal inflammatory cellular infiltration Total X2 P
No Mild Moderate Severe
Groups G2 N 7 2 0 0 9c 30.55 0.002*
% 77.8% 22.2% 0.0% 0.0% 100.0%
G3 N 0 0 2 4 6a
% 0.0% 0.0% 33.3% 66.7% 100.0%
G4 N 0 2 3 2 7a
% 0.0% 28.6% 42.9% 28.6% 100.0%
G5 N 1 2 3 1 7b
% 14.3% 28.6% 42.9% 14.3% 100.0%
G6 N 3 4 1 0 8c
% 37.5% 50.0% 12.5% 0.0% 100.0%
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There is a statistically significant difference (P < 0.005) between study groups having different letters

There is no statistically significant difference (P > 0.05) between study groups having the same letter

Table 4.

One-way ANOVA and LSD post hoc tests were used for pairwise comparisons a, b, and c

Retinal vasculitis Total X2 P
Normal histology Vessel dilation  < 10 Inflamm. cells 10–30 Inflamm. cells  > 30 Inflamm. cells
Groups G2 N 0 7 2 0 0 9 c 39.69 0.001
% 0.0% 77.8% 22.2% 0.0% 0.0% 100.0%
G3 N 0 0 0 3 3 6 a
% 0.0% 0.0% 0.0% 50.0% 50.0% 100.0%
G4 N 0 1 3 3 0 7 b
% 0.0% 14.3% 42.9% 42.9% 0.0% 100.0%
G5 N 0 2 4 1 0 7 b
% 0.0% 28.6% 57.1% 14.3% 0.0% 100.0%
G6 N 1 5 2 0 0 8 c
% 12.5% 62.5% 25.0% 0.0% 0.0% 100.0%
Open in a new tab

There is a statistically significant difference (P < 0.005) between study groups having different letters

There is no statistically significant difference (P > 0.05) between study groups having the same letter

Improvement of histopathological changes caused by ocular toxoplasmosis after treatment

Mild improvement of the histopathology was observed after SP treatment (G4) with focal dilated vessels with extravasation of RBCs with moderate edema and moderate inflammatory infiltrates and vasculitis with no statistical difference compared to G3, while retinae of mice treated with BPQ showed moderate improvement of retinal structure with mild edema, mild to moderate inflammatory infiltrates, and the vasculitis with no significant difference compared to group 4. CQ-treated mice revealed preserved retinal architecture with focal mild edema and mild inflammatory infiltrations and vasculitis with no statistical difference compared to G2 with degenerated small-sized cysts in the outer retinal layer (Fig. 5 A, C and E and Tables 2, 3 and 4).

Fig. 5.

Fig. 5

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A; ocular section of a treated mouse with SP showing focal dilated capillaries distended by blood with extravasation (arrow) in the retina with area of moderate interstitial edema (H&E, X200). B; Section of diabetic infected treated mouse eye with SP showing relatively low immunoreactions in endothelial cells (CXCL10, X200). C; Section of treated mouse eye with BPQ showing mild edema in the inner layer of the retina (H&E, X100). D; Immunostained section showing relatively low to moderate immunoreactions in endothelial cells (CXCL10, X200) E; ocular section of a treated mouse with CQ showing mild edema in the inner layer and degenerated Toxoplasma cyst in the outer layer of the retina (H&E, X100). F; Immunohistochemically stained section showing relatively moderate immunoreactions in endothelial cells (CXCL10, X200

Immunohistochemistry

Diabetes type 1 and infection increased retinal CXCL10 expression

The retinae of diabetic mice showed a relative increase in the local CXCL10 expression (Fig. 3B) compared to the normal moderate expression (Fig. 2B). While a strong significant expression was detected in the diabetic-infected non-treated group (Fig. 4D and Table 5).

Table 5.

One-way ANOVA and LSD post hoc tests were used for pairwise comparisons a, b, c, d, e, and f

CXCL10 immunostaining intensity Total X2 P
No Slight Moderate Intense
Groups G1 N 0 2 8 0 10 d 44.05  < 0.001
% 0.0% 20.0% 80.0% 0.0% 100.0%
G2 N 0 1 6 2 9 b
% 0.0% 11.1% 66.7% 22.2% 100.0%
G3 N 0 0 1 5 6 a
% 0.0% 0.0% 16.7% 83.3% 100.0%
G4 N 2 4 1 0 7 f
% 28.6% 57.1% 14.3% 0.0% 100.0%
G5 N 0 4 3 0 7 e
% 0.0% 57.1% 42.9% 0.0% 100.0%
G6 N 0 2 5 1 8 c
% 0.0% 25.0% 62.5% 12.5% 100.0%
Open in a new tab

There is a statistically significant difference (P < 0.005) between study groups having different letters

There is no statistically significant difference (P > 0.05) between study groups having the same letter

Treatment decreased retinal CXCL10 expression significantly

Immunohistochemical staining of the retinae of the infected SP-treated mice showed mild CXCL10 expression. While the infected BPQ-treated showed mild to moderate expression. After CQ treatment, the retinae of the mice showed moderate significant expression (Fig. 5B, D and F, and Table 5).

Discussion

Clearance of intracellular pathogens possibly relies on an immune response with well-regulated cytokine signaling (McGovern and Wilson 2013). CXCL10 chemokine expression in the eye was known to be implicated in the inflammatory responses to ocular infections (Carr et al. 2003). In the present study for the first time, we measured the local expression of retinal CXCL10 and evaluated the retinal histopathological changes in diabetic mice infected with chronic toxoplasmosis before and after treatment. In addition, the efficacy of antimalarial drugs in alleviating toxoplasmic retinochoroiditis and modulating local retinal CXCL10 expression plus reducing the cerebral parasitic burden was evaluated. Wallace and Stanford (2008) stated that the eye is not a favored site for parasite encystment, therefore the effect of the administered drugs on the cerebral parasitic burden was evaluated.

A positive association between type-1 diabetes and Toxoplasma gondii was reported by Catchpole et al. (2023) who explained it by either the decreased immune function with increasing susceptibility to infections or the ability of toxoplasmosis to induce autoimmunity with autoantibodies production.

Our results revealed a moderate to high immunostaining intensity of retinal CXCL10 in the diabetic non-infected group, statistically higher than the normal expression in G1. Similarly, Sun et al. (2022) found an up-regulation of the chemokine in the retinae of STZ-induced Diabetic retinopathy (DR) mice. In addition, Vujosevic et al. (2016) and Chen et al. (2017) documented that CXCL10 was significantly higher in the aqueous humor of Diabetic Retinopathy (DR) patients compared to non-diabetic patients. This finding may be explained by the implication of the CXCL10/CXCR3 axis in the autoimmune process in T1D as reported by Corrado et al. (2014) who also found an increase in the serum CXCL10 level in T1D patients.

In our study, the highest significant retinal chemokine expression was reported among the diabetic-infected mice, which was in agreement with Norose et al. (2011) who documented upregulation of CXCL10 in the retina during chronic experimental toxoplasmosis. This finding was attributed to the crucial role of this chemokine in controlling ocular infection and maintenance of the T-cell response against Toxoplasma gondii. The intense expression of CXCL10 found in this work was accompanied by a relatively disturbed retinal architecture, severe vasculitis, multiple focal inflammatory cellular infiltrates, and areas of hemorrhages in the inner retinal layers. Gudowska-Sawczuk et al. (2022) observed overexpression of CXCL10 chemokine associated with intense inflammation and tissue damage. These findings highlight the serious condition of diabetic patients with ocular toxoplasmosis and the urgent need for an effective treatment.

On the other hand, many authors tested the efficacy of spiramycin (SP) as an anti-toxoplasmic treatment for ocular toxoplasmosis either in vivo or in vitro. In this study, a significant reduction of cerebral cysts (57%) and mild improvement of retinal histopathological changes were found with the downregulation of CXCL10 after SP treatment at a dose of 200 mg/kg/day for 10 successive days. However, Ayachit et al. (2016) reported a case of ocular toxoplasmosis in a 12-year-old female child treated with oral SP 1500 mg/day twice daily with prednisolone for 6 weeks which resulted in regression of chorioretinitis lesions without recurrence. Tavares et al. (2021) also documented the in vitro efficacy of SP against the Toxoplasma gondii RH strain without inducing human retinal pigment epithelial cell death using spiramycin-loaded poly(lactic-co-glycolic) acid (PLGA) ocular implant. Using different doses, duration, and formulas of SP could be the cause. As CXCL10 is an IFN-γ induced chemokine (Rashighi et al. 2015), decreased IFN-γ levels in Toxoplasma gondii-infected cells after SP treatment as stated by Franco et al. (2011) could be the explanation for the weak immunostaining intensity of the chemokine and therefore the mild improvement of the retinal pathology found in our study. CXCL10 deficiency affects the infiltration of several leukocyte populations into infected tissue hindering the immune response against infections (Wuest and Carr 2008). Norose et al. (2011) found that neutralization of CXCL10 during chronic ocular toxoplasmosis resulted in disorganization of the retinal architecture with a significant decrease in CD4 T-cells. This may explain the limited ability of SP to treat ocular toxoplasmosis in our study.

Immunohistochemical results of the BPQ-treated mice in our study showed mild to moderate CXCL10 retinal expression with moderate histopathological improvement and significant reduction of cerebral parasitic burden (76.25%). The exact mechanism by which the drug can exert an effect on modulating the chemokine locally is not fully understood. However, da Costa-Silva et al. (2017) described in vitro immunomodulatory actions of BPQ via upregulation of cytokines such as tumor necrosis factor (TNFα), monocyte chemoattractant protein 1, interleukin-10 (IL-10), and IL-6 in Leishmania-infected macrophages compared to untreated L. infantum-infected macrophages. TNFα is known to be a weak inducer of CXCL10 (Tokunaga et al. 2018) which would elucidate the mild to moderate expression of the chemokine after BPQ treatment and the moderate improvement of the pathological changes caused by the infection.

Interestingly, we observed a balanced retinal CXCL10 expression after CQ treatment with the best reduction rate of cerebral Toxoplasma cysts (84.36%) and the most significant resolution of the retinal histopathological changes caused by the parasite. Studies have demonstrated the anti-inflammatory and immunomodulatory actions of CQ. Some of them are related to inhibition of the production of IFN-α and IFN-γ and/or TNF-α cytokines, and downregulation of TLR-9 and TLR-4 mRNA secretion (Richard et al. 2020). CXCL10 possibly exerts its actions by binding to its receptor CXCR3. In addition, CXCL10 has been shown to activate toll-like receptor 4 (TLR4) (Lee et al. 2017). A potential relationship between TLR4 and Toxoplasma pathogenesis was described by Zare-Bidaki et al. (2014) who reported that deficiency in TLR4 expression may suppress immune responses against T. gondii. However, Molteni et al. (2016) documented that dysregulation of TLR4 pathways could contribute to disease progression. So, in this study, CQ was shown to be effective in regulating CXCL10 retinal expression which may be explained by its ability to modulate the loop between IFN-γ producing Th1 cells and resident cells producing CXCL10 and modifying the TLR4 pathway. Wallace and Stanford (2008) stated that inflammatory cell products, such as lysosomal enzymes play an important role in developing retinal damage caused by Toxoplasma retinochoroiditis, and since Mahon et al. (2004) reported that CQ could disrupt lysosomal function in retinal neurons and RPE in vitro, this could explain the excellent results of histopathology after CQ treatment obtained in this work.

In this study, a short-duration regimen of CQ was used to avoid exposure to retinal toxicity. It is proved that the development and character of experimentally induced chloroquine-related retinopathy in the mammalian eye depend on many factors such as; CQ concentration, duration of exposure, and mode of drug delivery (Gaynes et al. 2008). In our study, we used a short-duration regimen of CQ to avoid exposure to retinal toxicity.

Another interesting finding regarding mice treated with CQ is the significantly lower blood sugar levels compared to other treated groups. Growing evidence supports the in vitro beneficial anti-inflammatory activity of CQ as an alternative and adjuvant anti-inflammatory therapy to prevent type 1 diabetes complications and its protective action against T1D tubulopathy (de Almeida Júnior et al. 2020; Kang et al. 2020). In addition, Gaafar et al. (2002) reported an improvement in glucose tolerance after an oral glucose tolerance test in rats after short-term administration of chloroquine (10 mg/kg) for 6 days. However, Yuan et al. (2016) documented that the fasting blood glucose levels of STZ-induced mice did not differ significantly from those of the CQ-treated group (P > 0.05). Using different STZ and CQ doses for different durations on different mouse strains could be the reason for the variable results.

Optimal control of pathogens probably needs a balanced immune response with an optimum expression of chemokines. So, strategies targeting the modulation of CXCL10 may eventually be beneficial in the management of ocular toxoplasmosis. Therefore, retinal CXCL10 can be a candidate for a predictive marker of the immune response during the infection.

One of the limitations of our study is that there are some structural and metabolic differences between the mouse central retina and the human macula as stated by Volland et al. (2015) and Li et al. (2020). So, further studies on human retinal pigment epithelium culture would be needed for studying ocular toxoplasmosis.

In conclusion, CQ is shown to treat ocular toxoplasmosis, improve the retinal histopathological changes caused by the infection, balance local CXCL10 expression, and reduce the cerebral parasitic burden. In addition, targeting the modulation of retinal CXCL10 may eventually be beneficial in the management of ocular toxoplasmosis plus its potential to act as a marker for predictive local immunological response during the infection. Follow-up, early diagnosis, and proper treatment of toxoplasmosis in diabetic patients would be very important for the prevention of complications and decreasing morbidity.

Abbreviations

BPQ

Buparvaquone

CXCL10

Chemokine ligand 10

CQ

Chloroquine

IP

Intraperitoneal

NOD

Nonobese diabetic mouse

OD

Optical density

SP

Spiramycin

SD

Standard deviation

TZ

Streptozotocin

TLRs

Toll-like receptors

T. gondii

Toxoplasma gondii

TNFα

Tumor necrosis factor

T1D

Type 1 diabetes mellitus

Author contributions

Material preparation was performed by M-EAF and MAS; Data collection and analysis were performed by MB, MAF, RI, and AAA-A; Writing the paper was done by M-EAF, MAS, and AAA-A. All authors reviewed and approved the manuscript.

Funding

The authors declare that no funds were received throughout the preparation of this manuscript.

Data availability

The data that support the findings of this study are available from the corresponding author (Mennat-Elrahman Ahmed Fahmy), upon reasonable request.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

The study was approved by the ethical committee of Theodor Bilharz Research Institute (TBRI).

Consent to participate

No available consent to participate or consent for publication.

Footnotes

Publisher's Note

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References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author (Mennat-Elrahman Ahmed Fahmy), upon reasonable request.

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