Impact of toxoplasmosis on apoptotic activity and natural killer cell cytotoxicity among Iraqi pregnant women

This study was aimed at assessing NK cell cytotoxicity, soluble Fas ligand (sFasL) serum concentration among Toxoplasmosis patients, and healthy controls. To understanding the role of these parameters in the immunity and pathophysiology of toxoplasmosis, correlations between these parameters were evaluated. NK cell cytotoxicity was estimated by the cultivation of Candida cells on Sabouraud`s dextrose agar (SDA) plate which had been incubated for two hours with NK cells. Colony-forming inhibition assay (CFIA) by calculating anticandidal index (ACI) has been estimated in two ratios of target: effector (T: E) cells 1:3 (NK R1) and 1:30 (NK R2) for both patients and controls. Enzyme-linked immunosorbent assay (ELISA) kit sFasL were used to estimate serum level in both patients and controls. There is a decrease in sFasL in toxoplasmosis compared to controls.


Introduction
Toxoplasma gondii infects a wide range -blooded vertebrate including humans and is one of the world's most successful parasite, as a member of the phylum Apicomplexia (Sibly et al., 2007) The main infection route is ingestion of cyst from raw or badly-cooked meat, ingestion of oocyst from substrates contaminated with the feces of infected feline and congenital transmission by tachyzoites (Lopiset et al., 2007). Natural killer cells are a subset of mononuclear cells, they are nonphagocytic large granular lymphocyte which is cytotoxic for certain tumor cells lines and virally infected cells, NK cells play several important roles in the host defense against certain microorganisms (Denker and Gazzinalli, 1998). Natural killer cells represent an arm of the innate immune system. Cellmediated immune responses involving CD4+ and CD8+ Tcells and NK cells play a protective role in T. gondii primary infection (Laneir, 2005).
Macrophage and NK cells and cytokine are the main component involved in the innate immunity responses against T. gondii (Advenson, 2006). Apoptosis or program cell death plays essential roles in the regulation of the immune responses and as innate and adaptive effects or mechanisms against intracellular pathogens (Luder and Cruoss, 2005). The FAS/FASL system is implicated as an important pathway mediating apoptosis (Arefet et al., 2004). In the immune system FAS/FASL plays an important function in down regulatory immune response and deletion of the autoreactive T-lymphocyte peripheral (Jouet et al., 1998). In the case of infectious diseases, the fas ligand (FSL) triggers an apoptotic waterfall and has a critical role in the effect of the parasites on type I cells which transmitted FAS/CD95, without the use of the mitochondrial loop T. gondii death receiver pathways. (Hippeet et al., 2008).

Study subjects
The research was performed in the Center Public Health Laboratory/Babylon on patients referred from Babylon Maternity Teaching Hospital and private clinic through the history from January 2019 to July 2019. All these subjects

Original Research Article
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ISSN: 2582-6697
were with a past abortion and were suspected of having toxoplasmosis.

Patient groups
Screening tests for toxoplasmosis antibodies by using IgM, IgG specific toxoplasma ELISA kit, were done for 30 referred women with a history of abortion. Eighteen patients were found to be with positive IgM, IgG using the ELISA technique. Positive toxoplasma patients with only IgG Immunoglobulin 9 and only IgM was 3, their ages were ranged from 18 -33 years.

Control groups
Ten, apparently healthy age-matched women with negative toxoplasma antibodies and with a history of abortion were selected as a control group.

Blood sample collection
Four ml of the blood sample was obtained from each patient by vein puncture using a syringe with needle gauge 23, two ml transported to a non-heparinized blood collecting tube and then centrifuged at 1800 XG to separate serum then labeled and stored at -20ºC until used, no preservative was, added. The other 2 ml were transported to a heparinized (20 U of heparin/ml) vacutainer tube then used for separation of polymorph blood monocyte (PBMC).

Statistical analysis
Statistical analysis was done according to percentages to compare between samples using SPSS V.25 computer software.

Results and Discussion
Ten patients (25%) out of these forty patients were with a history of abortion but with seronegative toxoplasmosis, their age range 20-37 years considered as a control, and 30 (75%) of them showed seropositive toxoplasmosis (IgM+ or IgG+ or both). Table 2, figure 4 showed that out of those 30 positive result, 9 (30%) gave IgG positive and IgM negative toxoplasmosis, 3 (10 %) gave positive IgM and negative IgG, and 18 (60 %) gave both IgG and IgM positive toxoplasmosis. Toxoplasma infection stimulates humoral immune response as antibody production which includes IgM and IgG (Darcy and Santora, 1994) Serological diagnosis of toxoplasmosis is based on a variety of techniques the Dye test (DT), the immunofluorescence (IF), and the indirect haemagglutination test (IHAT), each has its limitation, but all are still widely used in clinical medicine (Wielard et al., 1983) these testing for IgM/IgG anti-toxoplasma antibodies may fail to differentiate between a recent and pillar-infection (Iqbal and Nabila, 2007) but for detection of IgM antibody, the ELISA method is preferred because of its greater sensitivity (Jalan, 2009). The different immunoglobulins are considered as marking of the acute or chronic phase of the infection as it is explained next IgM: It is the first antibody in appearing. It is the best activator of the complement, makes an excellent agglutination thanks to its structure, increases the level of cytotoxicity and its main target is the antigens of the surface of the parasite, classically it was considered as a marker of the acute phase of the disease nevertheless at present is known that the titers of IgM can even remain detectable during many months and years after produced the primary infection.
The main value of the IgM is that its absence practically discards the recent infection although it is necessary to consider that in the case of a newborn the answer of IgM antibodies can delay several months after the infection. The presence of IgM on the contrary implies the necessity to continue the study because the simple detection of this antibody is difficult to evaluate if data of the titers of IgG and IgM in series samples or with the results of other tests are not had (IgA and IgE) that suggests recent infection. IgG: This immunoglobulin is second in appearing, facilitates the mechanism of dependent cytotoxicity of antibodies, opzoniza to the antigen, activates to the complement, can cross the placenta and their main target is the antigens of the surface of the parasite, the presence of IgG antibodies implies that at some time of the life the patient has been in contact with the parasite and it is only possible to assure that exists a recent infection if between two separated samples 3-4 weeks a seroconversión of the titer of IgG exists (Filiseti and Candolf, 2004).

Diagnosis of acute infection with the protozoan parasite
Toxoplasma gondii generally relies on serological methods (Remington and Desmonts, 1990). However, serodiagnosis might be indistinct in immune-compromised patients, during pregnancy, or in patients with congenital toxoplasmosis. Whereas immunoglobulin M (IgM) antibodies have been shown to be reliable markers for acute infection, this class of immunoglobulins might have only limited value for diagnosis in these patients; the observed persistence of IgM antibodies in some female patients might complicate diagnosis, especially during pregnancy.
In contrast, IgM antibodies might not be detectable in patients with congenital toxoplasmosis or during reactivation of a chronic infection in immunocompromised individuals (Fucillo et al., 1987).
The detection of IgM specific antibody can be of major importance in the diagnosis of congenital Toxoplasmosis in the neonate because IgM class antibodies do not cross the placental barrier (Remington, 2015). It is also helpful in differentiating recently acquired (acute) toxoplasmosis from chronic infection. The sensitivity, specificity, and reproducibility of enzyme-linked immunoassays are comparable to other serological tests for antibodies, such as immunofluorescence, complement fixation, hemagglutination, and radio immunoassays (Engwal et al., 2011).
The level of specific IgG antibody in sera was revealed high, after deletion positive cases from it, meant chronic toxoplasmosis between the mothers whose neonates were confirmed in hospital (Kompalic et al., 2004), the chronic toxoplasmosis was considered when only IgG was positive (Peterson et al., 2001), and it's the immunoglobulin was to appears in toxoplasmosis after IgM (Bancroft et al., 2013).  Table 3 showed that mean toxoplasma IgM index results have a high value with the group of IgM +ve IgG -ve compared to a decreasing value at the group of IgM +ve IgG +ve , while in the case of IgM -ve IgG +ve , IgM index showed a negative result. The mean level of IgG index showed an increasing value IgMve IgG +ve group compared to that in the group of IgM +ve , IgG +ve .
According to the general concept which states that the acute stage is associated with high IgM, and the transitional stage associated with mixed IgM and IgG followed with a high level of IgG at the chronic stage, it can be concluded that the group of IgM +ve IgG -ve represents the acute stage, and a group of IgM +ve IgG +ve represents the transitional stage, while IgMve IgG +ve group represents a chronic stage of the disease. NK cells are involved in innate immunity against a broad range of intracellular pathogens, especially T.gondii, in the earliest stage of infection (Bancroft et al 2013).
The levels of the NK activity have been found elevated in children with malaria and in mice infected with Trypanosoma cruzi and Toxoplasma gondii, suggesting a possible effector role of these cells in parasitic disease (Hatoher et al., 2000). T . gondii is capable of triggering the non-specific activation of macrophage and NK cells (Hauser et al., 1983). inhuman congenital toxoplasmosis, elevated circulating NK cell numbers have been linked to protection. Natural killer cytotoxicity is one of the important parameters used in this study to evaluate the natural killer cells' activity in the acute and chronic stages.

Fig. 1 the IgM and IgG index results in toxoplasmosis patients at different stages
To determine NK cytotoxic activity, CFI (candidal formula index) assay was applied. In this procedure, target cells (T=Candida) were incubated with effecter cells (E=NK cell) at different T: E ratios; 1:3 and 1:30 (1:3 called R1 and 1:30 called R2). Figure 3 showed the cells of normal murderers' cytotoxicity in control, acute, chronic, and transitional stages of patients with toxoplasmosis were increased in both ratios R1 and R2 due to an increase in the stimulation of body defense response. Gazzanilli et al. (1998) reported that Toxoplasmagondii has the ability to activate the NK cells from the innate compartment of the immune system. Table 4 and figure 4 showed that the cytotoxicity index (CI%) of NK cells in acute toxoplasmosis (IgM +ve , IgG -ve ) at ratio 1 (R1) was 64.6 % while in the patient control CI% was 59.5% and it was 99%in the R2 compared with the control patient 84.5 %, from these results it can be concluded that Toxoplasmagondii upregulates the NK cell cytotoxicity in the acute stage of disease more than in control, and also the increase in NK ratio from R1 to R2 lead to change P-value from nonsignificant 0.34 to a significant difference of P = 0.03 which indicates that upregulation will have a prominent effect when it goes in parallel with the increase in NK cell population. In the chronic stage (IgM -ve , IgG +ve ), as in table 5 and figure 3, the NK cells cytotoxicity in R1 increased to 65% in comparison with the control patient 59.5 % and with R2 NK cell cytotoxicity increased to 88.7 % in comparison with control patients 84.5%. Table 6 and figure 6 showed the final stage of the acute infection and the first stage of the chronic infection, the IgM and IgG were both seropositive (IgM +ve , IgG +ve ) which represent transitional stage. In this stage both acute (IgM) and chronic (IgG) parameter showed a seropositive result, but when NK cytotoxicity in R2 of the patient (89.3%) compared to NK cytotoxicity at R2 in acute (99%) and chronic stage (88.8%) it is found that NK cytotoxicity is near to the chronic result than to the acute result. These might be explained on the basis that this stage will proceed to the chronic more than acute and enable this proposal that this is the transitional stage of the disease. The difference in CI % between R1 and R2 in the acute stage was 27 while in CI% between R1 and R2 in the chronic stage was 18, which indicates that Toxoplasma infection had a higher impact on C I % of NK cells at the acute stage compare to the chronic stage, as in figure 7, also it reveals that the increasing number rise of NK cells (effecter cells) is not a determinative factor in NK cells cytotoxicity but the toxoplasma infection stage.

Fig. 6 NK cells cytotoxicity in transitional stage of disease and control
The FAS / FAS L dysregulation contributes to infectious disease pathogenesis (Nigro et al. 1994). FASL activates and plays a critical role in the apoptotic cascading during infectious disease and the effect of the parasite on apoptosis in type I cells translating FAS/CD95 via death receptor pathways without mitochondrial looping need. Toxoplasma gondii significantly reduces FAS/CD95 triggered apoptosis by impairing activation of the pro-caspase 8 (Polya et al. 2007). And also Toxoplasma gondii inhibition FAS/CD95-mediated apoptosis in type II cells primarily by decreasing the apoptogenic function to mitochondria (Hippe et al. 2008). In this study, FAS L was determined at three stages; The first stage was acute, the mean value of the result of FAS L in this stage was 0.21 pg/ml, and the second stage (transitional stage ) was 0.22 pg /ml, and the third stage (chronic stage) value was 0.25 pg/ml. While the mean value of FASL concentration in the control group was 0.53 pg /ml, as showed in table.7. From these results, it can be concluded that FAS L concentration at both acute and transitional stages showed a decrease in their values compared to the chronic stage. The results also showed that FAS L concentration at the transitional stage (0.22 pg/ml) was mostly deflected towards the acute stage (0.21 pg/ml) more than chronic stage (0.25 pg/ ml).    The correlation between sFasL and the age groups Table 7 showed those age groups have a positive correlation with sFasL in the control (r=0.094) and patients with toxoplasmosis (Table 8) have a positive correlation (r=0.030) but not significant. These results could be compared with the result of other studies. Mysliwiec et al. 2006, found a positive correlation between sFas and age in Graves' disease patients. No big variations were made in FasL concentrations between those studied groups of patients. However, a negative correlation was formed between FasL and age in all Hashimoto thyroiditis and Graves' disease patients. Goto 2008, showed The sFasL in Werner syndrome (WS), a level comparable to that in healthy elderly ages 83 to 95 years, had significantly increased (p < 0.05) compared to that in young healthy individuals ages 15 to 70 years, and Jiang et al 2008, showed the plasma sFasL levels were examined as a function of age, There has been a considerable connection between the sFasL and age in the non-age-related macular degeneration (AMD) subjects, and also showed the plasma FasL increased with age and AMD. Table 9. correlation between age, NK CI% and sFasL among controls **Correlation is significant at the 0.01 level (2-tailed).

Table 10 correlation between age, NK CI% and sFasL among toxoplasmosis patients
The correlation between sFasL and the NK CI%. Table 7 showed those NK CI% in control have a positive correlation with FASL (r =0.14) but not significant (P > 0.01). While in patient with toxoplasmosis (table 8) NK CI% have a negative correlation with FASL concentration, these correlation indicate that any increased in NK activity showed a greater decline in FASL concentration. These results could be compared with the result of other studies such as: Tanaka et al 1996, showed that High levels of sFasL in serum have been generally detected in several hematological diseases including NK-cell leukemia, NK cell lymphoma, and the Peripheral nasal T/NK cell lymphomas coexpressFas, FasL, and perforin/granzyme. This is in good correlation with the observation that in NK cell lymphoma, mFasL was constitutively expressed, and sFasL was shed, whereas NK cells of healthy volunteers expressed FasL only upon activation. Kern et al. 2000, reported that a significant negative correlation was found between initial sFasL levels on the first day and both total lymphocyte and T-cell counts during the Plasmodium falciparum disease course, sFasL levels significantly declined. Massaro et al. 2005, showed that FasL interacting with Fas at NK cell surface causes NK cell suicide, as apoptosis of NK cells was inhibited by blocking FasL/Fas interaction with specific mAbs, and doughty et al. 2002, reported that Increased sFasL was associated with a viral infection and lymphoproliferative disease.

Conclusion
When comparing NK R1 to NK R2 the results revealed that NK cell function increase with the increase in number in both patients and controls, but in patients the level of the increase is more than the controls. A negative correlation was detected between sFasL and NK cell cytotoxicity in both toxoplasmosis patients and controls. The positive correlation between sFasL and NK cell cytotoxicity in controls was changed to a negative correlation in Toxoplasmosis. There is a significant positive correlation between NK cell cytotoxicity and age group in controls. While in toxoplasmosis a positive correlation was detected between age groupand sFasL and a negative correlation between age group and NK cell cytotoxicity.