The Decreased Treg Cells Number Associated with Retinal Lesion Size in Ocular Toxoplasmosis

Introduction The imbalance of the immune response is an important factor contributing to the incidence of ocular toxoplasmosis (OT). Regulatory T cells (Treg) play a key role in maintaining the balance between Th1 and Th17 immune responses, while interleukin-27 (IL-27) levels are related to the differentiation of Th17 cells. This study analyzes the differences in the number of Treg cells and the level of IL-27 between OT patients and seropositive individuals without ocular lesions and its correlation with retinal lesion size. Methods This analytic observational study, conducted for 8 months, involved 11 OT patients and 10 seropositive individuals without ocular lesions. All subjects underwent a comprehensive ophthalmological examination. Retinal lesions were documented by fundus photographs and the size was measured using Digimizer 4.2.2.0 software. Isolation of peripheral blood mononuclear cells (PBMC) was performed to measure the number of Treg cells using flow cytometry and interleukin-27 levels were assessed using the Sandwich enzyme-linked immunosorbent assay (ELISA) technique. Data were analyzed with SPSS. Result The number of Treg cells in the OT group (47.16 ± 15.66%) was lower than in the seropositive group without the ocular lesions (62.86 ± 17.08%) (p = 0.029). The serum IL-27 levels in the OT group were not significantly different from the seropositive group without the ocular lesions (p = 0.360). The number of Treg cells was significantly related to retinal lesion size (p = 0.043), with a correlation coefficient of −0.648, indicating a strong and inverse correlation. There was no significant correlation between serum IL-27 levels and retinal lesion size (p = 0.556). Conclusion Ocular toxoplasmosis patients have a low number of Treg cells that are inversely related to the retinal lesion size. The size of the retinal lesion increases as the number of Treg cells decreases.


Introduction
Ocular toxoplasmosis (OT) is caused by Toxoplasma gondii (T.gondii) infection in the eye and is the most common cause of posterior uveitis worldwide.It is estimated that one-third of the world's population has been infected with T. gondii but with diferent manifestations.A study in the United States stated that 2% of people with seropositive could manifest ocular toxoplasmosis [1][2][3][4].In Indonesia, Aditama et al. mentioned that the prevalence of toxoplasmosis is about 2%-80%, which varies in each region.Te study by Sofa and Hariyono at Dr. Saiful Anwar General Hospital Malang for three years found 48 eyes (38 patients) with retinal lesions due to T. gondii infection [5,6].
Te high prevalence of seropositivity, but only the small percentage that leads to ocular manifestations, raises the question of what factors cause the appearance of clinical manifestations in the eyes of individuals who are both infected with T. gondii.Tree factors play a major role in clinical manifestations due to T. gondii infection: host immunity, parasite inoculum, and strains or genotypes of T. gondii [7,8].
Te exact mechanism of how T. gondii infection can reach the eye and cause clinical manifestations in patients is still being studied.Te immune response as an essential factor in T. gondii infection is thought to be closely related to the role of cytokines.Various studies have found that T17 cells play a role in eye tissue damage due to T. gondii infection.It is also mentioned that IL-27 is a potent inhibitor in T17 cell diferentiation.Regulatory T cells (Treg) are associated with the T17 immune response, which plays a key role in maintaining the balance of immune response to maintain immune homeostasis.Treg cells are a subpopulation of T cells that function to suppress the excessive response of B cells and T cells to other antigens so that the immune response does not continue after it is unnecessary [9][10][11][12][13].
Several studies have been conducted related to OTand its correlation with immune responses, but the research in humans is still limited [14][15][16].Most studies have been conducted in rats, and current human studies are limited to aqueous humor samples, which collection process is invasive.Tis study evaluates the role of Treg cells and IL-27 in ocular toxoplasmosis and their correlation with the retinal lesion size, using blood serum samples from OT patients.
Te retinal lesion size is selected as the main parameter as it is one of the clinical manifestations of OT.Te lesions on the retina can be active, i.e., yellow-colored focal lesions or hyperpigmented retinochoroidal scars in inactive cases.

Methods
Tis study is an analytic observational with a cross-sectional approach which was conducted at the Ophthalmology Outpatient Clinic and Clinical Pathology Laboratory of Dr.We used a consecutive sampling method, which recruited all patients with OT and seropositive individuals without ocular lesions who met the inclusion criteria.Te inclusion criteria were aged 10-60 years, immunocompetent, agreed to participate, and signed the informed consent.Meanwhile, individuals with reactive VDRL/TPHA, those unable to complete all examinations, or those with damaged blood samples were excluded.Te seropositive subjects without ocular lesions were recruited from healthy volunteers.Te pregnancy test was performed in female subjects of childbearing age.All of the subjects agreed to participate in this study.

Ophthalmological Examination.
Primary data collection was carried out by history taking from all subjects.In female subjects of childbearing age, a pregnancy test is carried out; if it is negative, the subject is recruited.A complete ophthalmological examination was then performed.Te anterior segment was examined using a slit-lamp biomicroscope and the posterior segment was evaluated using a binocular indirect ophthalmoscope and fundus photograph.

Serological Examination.
Te blood samples were taken and divided into two parts.A blood sample in a serum separator tube (SST) was used to examine IgG and IgM anti-Toxoplasma.A serological test was performed to confrm the exposure of T. gondii.

Te Isolation of Peripheral Blood Mononuclear Cells (PBMC).
Blood samples with EDTA were immediately processed to isolate unstimulated PBMC.A 15 ml centrifuge tube was prepared and flled with Ficoll-Hypaque d � 1.077 g/ ml (ratio 1 : 1) with the number of blood samples and then reversed slowly to make a homogeneous solution, mixed 1 : 1 with Phosphate Bufer Saline (PBS), and centrifuged at room temperature at a speed of 1600 rpm for 30 minutes.After centrifugation, the PBMC ring was slowly taken using a micropipette and placed in a new 15 ml centrifuge tube.
Te PBMC solution was washed with 10 ml PBS and centrifuged at room temperature at a speed of 1200 rpm for 10 minutes.Te supernatant was discarded and the cell pellets formed were washed with the PBS, at a room temperature of 1200 rpm for ten minutes, repeated twice.After centrifugation, the pellets (PBMC cells) are formed at the base of the tube.Te pellets are dissolved with PBS 1 ml in an Eppendorf 1.5 ml tube and stored in a refrigerator at a temperature of −40 °C.
Te PBMC cell pellets were stained with cell surface antibodies (CD4+ and CD25+) frst and then with intracellular (nuclear) markers for FCM (Treg).
Cell pellets are washed with the fow cytometry staining bufer and then centrifuged at 2500 rpm for 3 minutes at 4 °C.Te supernatant is removed and the cell pellets formed are ready to be stained with the cell surface antibody.First, mix the antihuman CD4 FITC-conjugated and antihuman CD25 PE/Cy.5-conjugated with the fow cytometry staining bufer.A 50 μl solution is mixed with cell pellets and then incubated at room temperature for 20 minutes in the dark.After that, rehomogenized for continued intracellular (nuclear) staining.
Te suspension was centrifuged at 2500 rpm for 3 minutes, at 4 °C.Te cell pellets formed are washed by adding 500 μl of 1x FoxP3 fxation/permeabilization bufer solution and then centrifuged like before.Te cell pellets formed are washed by adding 500 μl of 1x FoxP3 fxation/permeabilization bufer solution, homogenized, and then incubated at room temperature for 20 minutes in the dark.After incubation, they are centrifuged like before.Ten, they repeated the procedure, without incubating in the dark room.Te cell pellets were mixed with 500 μl of 1x FoxP3 permeabilization bufer solution and then incubated at a room temperature for 20 minutes in the dark.Te cell pellets are ready to be stained with the intracellular nuclear antibody.Ten, antihuman FoxP3 PEconjugated antibody with 1x FoxP3 permeabilization bufer was mixed.A 50 μl solution was mixed with cell pellets and then incubated at room temperature, for 20 minutes, in the dark.500 μl of 1x FoxP3 permeabilization bufer solution was added and then centrifuged at 2500 rpm for 3 minutes, at 4 °C.Te cell pellets were mixed with 300 μl fow cytometry staining bufer and then transferred to the FCM cuvette and ready to read in fow cytometry device.

Te Retinal Lesion Size Measurement.
Te retinal lesion size in the OT group was examined using fundus photographs and measured with the Digimizer 4.2.2.0 software, performed in disk-diameter (dd), as shown in Figure 1.In patients with bilateral retinal lesions, measurements are taken in the active lesion or the larger lesion if both lesions are active or inactive.Te results of the retinal lesion measurements were then confrmed by an infection and immunology ophthalmologist consultant.

Result
3.1.Characteristics of the Subjects.Tere were 21 subjects consisting of 11 subjects in the OT group and 10 subjects in the seropositive group without ocular lesions.Te characteristics of the subjects are presented in Table 1, most of the subjects were 19-45 years of age in both groups (71.43%), in the OT group (45.45%) and in the seropositive group without ocular lesions (90%).Based on the sex predominance, most of the subjects are women (71.43%), either in the OT group (63.64%) or the seropositive group without ocular lesions (80%).Based on the result of serological tests of anti-Toxoplasma antibodies, 100% had positive IgG titers, while 23.81% had positive IgM titers.
Te ophthalmological conditions in the OT group are presented in Table 2.It was found that 63.64% of OT patients had unilateral lesions, and most of the lesions were active lesions (63.64%).Most of the active OTs were recurrent cases (85.71%).Ten patients (90.9%) had macular lesions.

Comparison between the Number of Treg Cells in OT and
Seropositive Groups without Ocular Lesions.Tere was no signifcant diference between the number of Treg cells in the active OT and inactive OT (p � 0.098) (Table 3).So, we further analyzed the diference in the number of Treg cells between the OT group and seropositive individuals without ocular lesions.
Te number of Treg cells in the OT group was signifcantly lower than the number of Treg cells in the seropositive group without ocular lesions (p � 0.029) (Figure 2).Measurement of the number of Treg cells in the OT group showed a signifcant correlation with the retinal lesion size (p � 0.043) (Figure 3).

Comparison between the IL-27
Levels in OT and Seropositive Groups without Ocular Lesions.Tis study found no signifcant diference between serum IL-27 levels in the OT and seropositive groups without ocular lesions (p � 0.360).Serum IL-27 levels also did not signifcantly correlate with retinal lesion size in OT (p � 0.556) (Figure 4).

Characteristics of Subjects.
Most of the study subjects (71.43%) were adults (19-45 years).Tis fnding was also found in the OT group, which was 45.45% and the seropositive group was 90%.Tis fnding is in accordance with the previous study by Cifuentes-González et al. in an epidemiological study of OT in California (2015-2019), where 65.2% of subjects were in the 15-49 year age group [17].Bustillo et al. also found that the highest incidence and prevalence of active OT each year is at 25-44 years old [18].Gilbert et al. 1999; Bosch-Drissen et al. 2002, also reported that patients aged 25-45 years were the largest group suffering from OT, 78-82% [19,20].
We also found that most of the subjects were women, with 63.64% in the OT group and 80% in the seropositive group without ocular lesions.Tabatabaei et al. reported the highest prevalence of OT in women, 60% (n = 24); Sofa and Hariyono reported that 66% (n = 25) of OT cases occurred in women; and Mendes et al. said that the highest proportion of seropositive toxoplasmosis were in women, 64.6% (n = 75) [6,17,21,22].Several studies reported no signifcant difference between males and females in the prevalence of OT [20,23,24].Ferreira et al. reported a total of 349 patients, 53.6% (n = 187) were male, and 46.4% (n = 162) were female [23].Te high prevalence of toxoplasmosis in women especially in childbearing age in this study may be because women have higher consideration to do anti-Toxoplasma antibody testing for screening purposes before planning a pregnancy.

Comparison between the Number of Treg Cells in OT and
Seropositive Groups without Ocular Lesions.Tis study found that the number of Treg cells in the OT group was lower than that in the seropositive group without ocular lesions.Tis is in line with the results of a study by Oldenhove et al. [14,25].A lower number of Treg cells may play an important role in the infection process.In the OT group, the number of Treg cells was less, so it may contribute to clinical manifestations.A study on Treg cells in another eye infection entity, ocular tuberculosis, by Basu et al. also found that the number and function of Treg cells were decreased [26].A study that analyzed the correlation between the number of Treg cells and the retinal lesion size has not been reported.Tis study found a signifcant correlation between the number of Treg cells and the retinal lesions size with a strong and negative correlation coefcient, indicating an inverse relationship between the number of Treg cells and the retinal lesions size.In previous research by Cordeiro et al. that analyzed the correlation between IL-10 cytokine and retinal lesion size, it was found that there was a negative correlation between retinal lesion size and IL-10 [27].

Interdisciplinary Perspectives on Infectious Diseases
Increased number of Treg cells will increase the production of IL-10, decreasing T1 and IFN-c levels.An increased number of Treg cells will activate TGF-β, reducing the level of proinfammatory IL-17, so that retinal damage can be minimized.Meanwhile, if the number of Treg cells decreases, TGF-β and IL-10 will also decrease, while IL-17 and T1 will increase.Increased IL-17, which is a proinfammatory cytokine, will facilitate the occurrence of retinal damage.

Comparison between IL-27 Levels in OT and Seropositive
Groups without Ocular Lesions.Tis study showed that there was no diference between serum IL-27 levels in the OT group and seropositive group without ocular lesions.In a study by Maia et al., an analysis of the gene encoding IL-27 via PBMC mRNA was carried out between OT patients and seropositive without ocular lesions; it was found that there was no diference in IL-27 mRNA levels in the OT group and seropositive group without ocular lesions [28].Research by Tong et al. reported that there was a signifcant increase in the mRNA level of IL-27p28 starting on day 2 in ocular tissue and starting on day 6 in the cervical lymph tissue of the mice  Various studies have reported that IL-27 is pleiotropic in some T helper subsets.Te frst time IL-27 was discovered, its role was reported as a cytokine that induces T1 cells that are protective in infection processes.Several subsequent studies reported the efect of IL-27 as a potent inhibitor of T17 diferentiation in autoimmune encephalitis and cerebral toxoplasmosis [30].Te nature of IL-27 may explain the result of this study, which found no diference between serum IL-27 levels in the OT group and the seropositive group without ocular lesions.
Te mechanism underlying the occurrence of retinal damage in T. gondii infection is not fully understood, the immune response is thought to play a direct role in the pathogenesis of retinochoroiditis, and cytokines play a fundamental part.T17 cells, essential mediators in infammatory and autoimmune reactions in uveitis, are thought to play a signifcant role in the host's immune response to retinal tissue damage due to T. gondii infection [30,31].
A study by Carneior et al., which evaluated the efect of cytokines on OT, showed that in infants with OT, there was an increase in IL-1β and TNF-α levels, babies with active lesions of retinochoroiditis had increased levels of IFN-c and IL-17, infants with retinochoroidal scars have increased IL-10, IL-1β, and TNF-α, infants with active lesions and retinochoroidal scars have increased IL-10, and in general, there are increased levels of IFN-c and IL-12 in all clinical conditions [16].
Another study by Sauer et al. showed that in rats with OT, there was an increase in IL-17 levels in the primary infection, but these IL-17 levels then decreased, followed by an increase in IL-27 levels in recurrence cases.It was concluded that IL-27 is the main cytokine that plays a role in inhibiting the T17 immune response.IL-27 also plays a role in activating the T1 immune response, which plays an important role in the eradication process of parasites [15].
In this study, there was no correlation between serum IL-27 levels and the retinal lesion size in OT.According to the researchers, these fndings indicated that the process of retinal tissue damage is not directly infuenced by IL-27 activity but involves various other infammatory mediators that may be initiated by T17 cell activation.Other factors, such as T. gondii strains and genetic polymorphisms, may infuence the host's immune response to T. gondii infection [31][32][33].

Conclusion
Tis study showed that the number of Treg cells in the OT group was lower than in the seropositive group without ocular lesions and the number of Treg cells was inversely related to the retinal lesion size in OT.Meanwhile, there was no diference in serum IL-27 levels between the OT and seropositive groups without ocular lesions.

Figure 3 :Figure 4 :
Figure 3: Te correlation between the number of Treg cells and the retinal lesion size.Te greater the number of Treg cells, the smaller the retinal lesion size.
Saiful Anwar General Hospital Malang, as a tertiary referral hospital, and Biomedical Laboratory of the Faculty of Medicine, Universitas Brawijaya, for 8 months during 2022-2023.Tis study has been approved by the Health Research Ethics Commission of Dr. Saiful Anwar General Hospital Malang with number 400/155/K.3/102.7/2022.

Table 1 :
Characteristics of the subjects.

Table 2 :
Ophthalmological conditions of the OT group.

Table 3 :
Te number of Treg cells.Te number of Treg cells in the OT and seropositive groups without ocular lesions.Te average number of Treg cells in the OT group was lower than the seropositive group without ocular lesions.Interdisciplinary Perspectives on Infectious Diseases model infected with T. gondii compared to control mice.Tese results indicate that IL-27/IL-27R induced by T. gondii infection plays a role in the immunopathology of OT [29].