Synthetic immunostimulatory glycans interference with host cell apoptosis upon of Toxoplasma gondii infection, in vitro

Toxoplasmosis is a protozoan infection of humans and animals caused by Toxoplasma gondii, and it’s continuous public health and food safety issue. The tachyzoites (Tg) of T. gondii are the most important stage, as they come in direct contact with immune cells such as a macrophage. Tg can modulate and prevent apoptosis of immune cells while promoting survival of the pathogen. Infections caused by Tg can be eradicated if immune cells could stimulate apoptosis and kill pathogens upon exposure. Apoptosis is characterized by the release of mediators, namely Caspases (Cas). New means are required for inducing apoptosis and enhance immunity in the infected host cell to control toxoplasmosis. The present study investigated whether Synthetic Immuno-stimulatory Glycans (SIGs) influence Cas and Nitric oxide (NO) release and led to Tg damage. Galβ13Gal-PAA-fluor (SIG1), Fucα1-4GlcNAcβ-PAA-fluor (SIG2) and GlcNAcβ1-3GalNAcα-PAA-fluor (SIG3) constituted samples studied principally. Murine macrophage had been exposed to the Tg then the SIGs effects on Cas and NO production were determined after 20 hours of pathogen phagocytosis. Here we report that the SIGs had potent in vitro activity against T. gondii; SIG2 was more effective than SIG1 and SIG3, representative by SIG2 treated infected macrophages can induced infected macrophages to release Cas1, 3, and 9. Maximum production of NO by infected macrophages was noticed following the expoxure to all SIGs. Therefore the present study provided the method for the selection of SIGs ligands bearing immunostimulatory factor and apoptotic stimuli properties.


Introduction
T. gondii is a unique obligatory intracellular protozoan.It infects a variety of cells in almost all warm blooded animals which in turn causes toxoplasmosis, a disease with acute and chronic infections (1).Toxoplasmosis is a zoonotic disease can be transmitted from animal to humans, the individuals at risk for life-threatening toxoplasmosis's forms, such as encephalitis, myocarditis, and retinitis, include in immunocompromised patients, pregnant women and animals, and fetuses (2,3).Chemotherapy of toxoplasmosis is mandatory; however, available treatments are limited, side effects are common, and are effective against the tachyzoite stage only (1,4).To date, there is no drug which is effective against chronic Toxoplasma infections and no licensed vaccine available for human (5,6).
A class of cysteine proteases (Caspases) are synthesized as inactive preforms (i.e., zymogens) (7,8).Triggering of (Cas) preludes the final commitment to death after cells have encountered intrinsic or extrinsic apoptotic promoter (9).Apoptosis has recently been recognized as an important defense system against viral, bacterial, and parasitic pathogens during innate and adaptive immune responses (10,11).Although, it has been demonstrated that macrophages are essential immune cells of resistance and restrict parasite replication during toxoplasmosis (12,13), but Tg have improved mechanisms to inhibit host cell apoptosis which helps Tg to survive and replicate (14), and macrophages under some conditions of Toxoplasma infection may act as Trojan horses to spread the Tg (15).Therefore, it is important to induce early apoptosis host cell upon Tg infection.
The production of nitric oxide (NO) by activated macrophages was recognized to be the primary antiparasitic effector mechanism (16,17).There is good evidence that the macrophages infected with T. gondii are resistant to multiple inducers of apoptosis (18).Also previously reported that NO levels increased during toxoplasmosis (17,19) and potentially induce apoptosis (20,21) which decreased T. gondii replication (17,19,22,23).
Carbohydrates bearing molecules (glycans or lectins) are pivotal in biological activities such as immunity, development, and phagocytosis (24), and they play a crucial role in biological communication between adjacent cells (25) and immune cells (26).Several of Tg receptors are natural carbohydrate-bearing molecules (27)(28)(29) which are important and allow the Tg to interact with responding to the external environment (30).Researchers investigated the ability of naturally occurring carbohydrates and synthetic carbohydrates to restrict or prevent T. gondii infection (31).
Synthetic Immuno-stimulatory Glycans (SIGs) are suitable ligands for the recognition and binding of pathogens (32).It was reported that SIGs selectively early recognize and bind to Tg (33).Also SIGs stimulate NO production and increase macrophage resistance during acute toxoplasmosis (34).In addition, Natural Carbohydrates or SIGs were reported to have a longer shelf live than proteins or antibodies, (35)(36)(37).Correspondingly using SIGs either in stimulation the individual immunity or treatment would provide a greater advantage over proteins both economically and regarding molecular stability (38).Thus, current study might produce promising candidates for SIGs which can influence and upregulate apoptosis of the infected macrophages and may be used for therapy against toxoplasmosis (39).
T. gondii tachyzoites type I (RH) strain, are highly virulent and they were used as a pathogen model in this research.The aim of this study was investigating whether SIGs influence Cas release by murine macrophage (M) after 20 hours of Tg in vitro phagocytosis and analyzing whether NO production is associated with Cas release.

T. gondii propagation and purification
T. gondii RH strain (Tg) was maintained as tachyzoites by serial passage on monolayers of HFFs at 37 0 C with 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillinstreptomycin.The viability of the parasites was determined by trypan blue (40).

Isolation of murine peritoneal cavity macrophages
C57BL/6 mice were injected intraperitoneally with 1.5 ml of 3% Thioglycolate broth.Five days after injection, mice were euthanized, and peritoneal exudate cells were collected by lavage with 5.0 ml RPMI 1640.Peritoneal macrophages were plated at 1.2×10 6 cells/ well in 6-well culture plate then incubated at 37 0 C in 5% CO 2 for 2 hours.Non-adherent cells were removed by washing with fresh RPMI 1640 media without antibiotics.Macrophages were infected at a tachyzoites-to-cells ratio of 2:3 (8×10 5 tachyzoites/ 1.2×10 6 cell).Each well was immediately treated with 0.03 µg of SIGs and incubated for 20 hours at 37 0 C in 5% CO 2. Cas and NO productions were determined on (1) macrophages treated with SIG (M+SIG); (2) macrophages exposed to either tachyzoites only (M+Tg) or (3) macrophages treated with SIG after tachyzoites exposure (M+SIG+Tg) and comparing those conditions to untreated macrophages (M only).

In vitro nitric oxide (NO) analysis
SIGs effect on macrophages activation was investigated by determining nitric oxide production 20 hours after infection or post phagocytosis.Culture supernatants (100 μl) were assayed for nitric oxide determination using the Griess assay.The nitrite ion (NO2−) concentration, indicative of NO, was determined using NaNO2 as a standard (41).The cell culture supernatant (100μl) was mixed with an equal volume of Griess reagent [0.1% (w/v) N-(1 naphthyl) ethylenediamine dihydrochloride and 1% (w/v) sulfanilamide in 5% (v/v) phosphoric acid].The samples were incubated at room temperature for 20 minutes, and absorbance was measured at 490 nm using a Bio-Tek Ex800 plate reader.

In vitro Caspases activities
The caspase proteolytic activity in lysates macrophages was determined with Caspases 1, 3, and 9 Colorimetric Assay Kits.Cas peptides were obtained from the postphagocytosis studies of macrophages exposed to Tg. Cas peptides isolation was performed according to manufacturer protocol (BioVision Research Products).Briefly, after 20 hours of incubation, the macrophages were harvested and resuspended in 50μl of chilled cell lysis buffer.Isolated cells were incubated on ice for 10 min followed by centrifugation of the suspension for 1min in a microcentrifuge (10,000x g).Finally, the supernatant (or cytosolic extract) was transferred to a fresh tube and kept on ice for further assay.
Cas peptides were determined based on spectrophotometric detection of the cleavaged chromophore p-nitroanilide (pNA) from the labeled substrate Cas-pNA sequences including YVAD, DEVD, and LEHD according to the company protocol (reference from the biovesion company).Above amino acid sequences allow detecting Cas 1, 3, and 9 respectively.The cytosolic extract was diluted to concentrations 50-200μg protein using 50μl cell lysis buffer and plated into each well of a 96-wells plate.Soon after, 50μl of 2× reaction buffer (10μM DTT) was added to each sample.Then, 5μl of the 4mM Cas-pNA substrate (200μM final concentration) was introduced, and plates were incubated at 37 0 C for 2 hours protected from light.After incubation, samples were read at 405-nm using a Bio-Tek Ex800 plate reader.

Statistical analysis
SAS® statistics software was used for all statistical analysis.The data represents the mean of three wells per experimental group and results were accepted as statistically significant at p-values <0.05 using Student's ttest and ANOVA.The Tukey test was performed for post ANOVA to indicate which group was not statistically significant compared to the others.

Results and discussion
The current research aims to gain deeper insight into whether SIGs could make macrophages capable of undergoing early apoptosis on T. gondii tachyzoites exposure, by controlling Cas cascade and associated with NO releasing.
NO is a radical with a small molecular weight released by immune cells like macrophages.We studied the effect of SIG1, SIG2, and SIG3 on NO production by macrophages infected with Tg (Figure 1).SIGs alone activate macrophages and increase NO release.The NO release by untreated macrophages (M only) was considered as the baseline of NO release and compared with both, T. gondii treated sample (M+Tg) and SIGs treated sample (M+SIG1-3), while M only, M+Tg, and M+SIG1-3 were used as controls.
Exposure of macrophages to SIGs alone (M+SIG1, M+SIG2, and M+SIG3) activate macrophages and increase NO release.When we were statistically comparing M+SIG1, M+SIG2, and M+SIG3 to M and M+Tg, the results showed significant differences (P< 0•05) in the expression of NO production.Conversely, there were no significant differences among M+SIG1, M+SIG2, and M+SIG3; they all showed a similar pattern of NO release.This result is in agreement with those of Lahiani (42).
Recent results indicated that murine macrophages slightly increase NO production after 20 hours of Tg infection, as it was shown in other previous work (43).In addition, our results showed that after 20 hours of Tg infection the SIG-treated macrophages (M+SIG1+Tg), (M+SIG2+Tg), and (M+ SIG3+Tg) displayed significantly higher NO production (*P< 0•05) compared with M+Tg and M+SIG1-3.Macrophages treated with SIG2 (M+SIG2+Tg) released a high level of NO (1830.16μM/ml)upon exposure to the tachyzoites M+SIG2+Tg compared to M+SIG1+Tg and M+SIG3+Tg (*P< 0•05).Released NO is an indication of the activation of macrophages in killing protozoa (44), and triggers the apoptotic pathway to induce macrophages death (45).Our present study has shown that NO production increased affectedly in early phagocytosis when the infected macrophages were treated with SIGs after 20 hours of Tg in vitro infection.We believe these findings represent the ability of NO production in stimulating Cas cascade which are very important in inducing early apoptosis of infected macrophages in order to combat Tg or to limit the Tg multiplication.
The cells apoptotic process commenced by a cascade that involves the activation of initiators and executioner caspases (7,8).Additional steps drive to cleavage or stimulation of molecules bring about most of the morphological and biochemical characteristics of cell death, activate Cas 1 inflammatory mediators (48,49) and eradicate the unwanted cells (old, cancer, and infected cells) from healthy individual bodies (50,51).
It has been reported that T. gondii activates the host survival response, thereby increasing the overall resistance of infected cells to apoptotic stimuli (52).The effect of SIGs on Cas production by macrophage (M) upon Tg exposure after 20 hours of phagocytosis were studied.Present result has shown that macrophages only (M) demonstrated low level of Cas release as shown in Figure 2.While macrophages infected with tachyzoites (M+Tg) increased the levels of Cas1, 3, and 9 (3 and 4-fold above the M only at 20 hours of infection).
Consequently, further studies will be needed for assessing the effect of SIG1 and SIG3 on Cas production within timeframe of T. gondii infection.Also, the level of Cas that released by the infected cells M+Tg should compared to the level of Cas that released by the uninfected M and infected cells M+Tg during exposure to apoptotic stimuli such as TNFa.Since Cas release by macrophages is used as an indicator of cell apoptosis, the inducing effects of SIG2 on macrophages and increase the level of Cas production that may contribute to the parasites ability to inhibit host cell apoptosis (53).
Previous studies have shown that T. gondii downregulates in vitro induced apoptosis by downregulates activation of caspases 3 and 9 (54,14).In agreement with present results which showed that the level of 3 and 9 reduced, therefore we proposed that SIGs can activated Cas cascade during early Tg infection and induced macrophages apoptosis lead to block the Tg reproduction.
Figure 1 confirmed that NO production was increased after treatment of T. gondii infected with SIG2 (M+SIG2+Tg), the data adds evidence for a role of NO in Cas 1, 3, and 9 production and the induction of apoptosis.This results agree with research done by Nishikawa team (55).
Depend on achieved results, in vitro mechanism action of SIG influence on NO and caspase Cas production after 20 hours phagocytosis was suggested.SIG present will bind either to cell (M) or pathogen (Tg).This make the Tg more prone to engulfed by macrophages lead to stimulate early immune defense and cell apoptosis by induce NO and Cas release (Figure 3).

Conclusion
Inhibition of host cell apoptosis by T. gondii is accompanied by reduced activation of the caspase cascade.Present results demonstrated that the SIG2 have a pivotal role in the increasing NO and Cas production which ultimately lead to induce the apoptosis of infected macrophages.Taken together, our data show that SIGs can used as immunostimulatory influence which enhance the infected macrophage NO response against Tg infection.In addition, SIG2 was more effective than SIG1, and SIG3 after 20 hours of phagocytosis.SIG2 might use as stimulatory factor for inducing the infected cell during Tg infection and controls Tg replication.Therefore the present study provided the method for the selection of SIGs ligands bearing immunostimulatory factor and apoptotic stimuli properties.

Figure 1 :
Figure 1: Nitric oxide (NO) production as a function of 20 hours of post-phagocytosis.After 20 hours, NO release by murine macrophages during tachyzoite infection was measured.Positive control results represent macrophages that were exposed to tachyzoites (M+Tg) and NO release by untreated macrophages (M) was considered as the baseline of NO release and compared to macrophages exposed to the tachyzoites (M+Tg).Macrophages during tachyzoites infection treated with SIG1, SIG2, or SIG3 (M+ SIG1+Tg, M+SIG2+Tg, and M+ SIG3+Tg) represented significant statistical difference compared to (M+Tg), *p<0.05.These results are the mean values of the NO production ± the SD of three independent experiments and analyzed by ANOVA.

Figure 2 :
Figure 2: Caspases (Cas) production as a function of 20 hours of post-phagocytosis.After 20 hours, Cas (1, 3, and 9) release by murine macrophages during tachyzoite infection was measured.Positive control results represent macrophages that were exposed to tachyzoites (M+Tg) and Cas release by untreated macrophages (M) was considered as the baseline of Cas release and compared to macrophages exposed to the tachyzoites (M+Tg).In compare to (M+Tg), SIG2 increase Cas 1, 3, and 9 release by cell (M) after 20 hours of treated with SIG2 (M+SIG2) *p<0.05, and increase Cas 1, 3, and 9 release by cell (M) after 20 hours of exposure to Tg simultaneously treated with SIG2 (M+SIG2+Tg) *p<0.005.Error bars represent the standard deviation for the means from three independent experiments conducted in triplicate and analyzed by ANOVA.Since Cas release by macrophages is used as an indicator of cell apoptosis, the inducing effects of SIG2 on macrophages and increase the level of Cas production that may contribute to the parasites ability to inhibit host cell apoptosis(53).Previous studies have shown that T. gondii downregulates in vitro induced apoptosis by downregulates activation of caspases 3 and 9(54,14).In agreement with present results which showed that the level of 3 and 9 reduced, therefore we proposed that SIGs can activated Cas cascade during early Tg infection and induced macrophages apoptosis lead to block the Tg reproduction.Figure1confirmed that NO production was increased after treatment of T. gondii infected with SIG2 (M+SIG2+Tg), the data adds evidence for a role of NO in Cas 1, 3, and 9 production and the induction of apoptosis.This results agree with research done by Nishikawa team(55).Depend on achieved results, in vitro mechanism action of SIG influence on NO and caspase Cas production after

Figure 3 :
Figure 3: The suggested in vitro mechanism action of Synthetic immunostimulatory Glycans (SIG) influence on Nitric oxide (NO) and caspase (Cas) production.A-Tachyzoites (Tg) actively attack non-treated macrophage (M) leading to partial inhibition of NO release and Cas production, thus prevent apoptosis and increase the chance of Tg replication.B-Supposedly, Tg are engulfed by macrophages that are treated with SIG will cause rise of NO and Cas production which in turn result in early apoptosis of macrophages (Apoptotic M).