Immune response against toxoplasmosis—some recent updates RH: Toxoplasma gondii immune response

Abstract

Aims

Cytokines, soluble mediators of immunity, are key factors of the innate and adaptive immune system. They are secreted from and interact with various types of immune cells to manipulate host body’s immune cell physiology for a counter-attack on the foreign body. A study was designed to explore the mechanism of Toxoplasma gondii (T. gondii) resistance from host immune response.

Methods and results

The published data on aspect of host (murine and human) immune response against T. gondii was taken from Google scholar and PubMed. Most relevant literature was included in this study. The basic mechanism of immune response starts from the interactions of antigens with host immune cells to trigger the production of cytokines (pro-inflammatory and anti-inflammatory) which then act by forming a cytokinome (network of cytokine). Their secretory equilibrium is essential for endowing resistance to the host against infectious diseases, particularly toxoplasmosis. A narrow balance lying between Th1, Th2, and Th17 cytokines (as demonstrated until now) is essential for the development of resistance against T. gondii as well as for the survival of host. Excessive production of pro-inflammatory cytokines leads to tissue damage resulting in the production of anti-inflammatory cytokines which enhances the proliferation of Toxoplasma. Stress and other infectious diseases (human immunodeficiency virus (HIV)) that weaken the host immunity particularly the cellular component, make the host susceptible to toxoplasmosis especially in pregnant women.

Conclusion

The current review findings state that in vitro harvesting of IL12 from DCs, Np and MΦ upon exposure with T. gondii might be a source for therapeutic use in toxoplasmosis. Current review also suggests that therapeutic interventions leading to up-regulation/supplementation of SOCS-3, IL12, and IFNγ to the infected host could be a solution to sterile immunity against T. gondii infection. This would be of interest particularly in patients passing through immunosuppression owing to any reason like the ones receiving anti-cancer therapy, the ones undergoing immunosuppressive therapy for graft/transplantation, the ones suffering from immunodeficiency virus (HIV) or having AIDS. Another imortant suggestion is to launch the efforts for a vaccine based on GRA6Nt or other similar antigens of T. gondii as a probable tool to destroy tissue cysts.

Introduction

Toxoplasmosis is a zoonotic infectious disease caused by an intracellular protozoon pathogen called Toxoplasma gondii (T. gondii). It causes the abortion or congenital abnormalities (hydrocephalus and retinochoroiditis) in pregnant women which are more susceptible to Toxoplasma infection. ¹ Generally, the immune system of immunocompetent individuals builds a protective immune response upon the interaction of antigen with antigen-presenting cells (APC) which induces the translocation of nuclear factor kappa (NF-κB) to initiate production of pro-inflammatory cytokines (IL1b, IL12, IL18, and IFNγ).^2,3 The host lymphocytes and myeloid cells not only secrete a network of cytokines for signaling pathways upon exposure to antigen but also up-regulate certain chemokines (CXC, C, and CX₃C) and toll-like receptors (TLR) on their surfaces for acting as signal-recipients against any antigen.^3,4 In response to specific intracellular signals, various pro-inflammatory (IL1β, IL12, IL18, TNFα, IFNγ) and anti-inflammatory (IL4, IL10, TGFβ) cytokines (Table 1) give rise to a cytokinome that acts for specific immunological stimulus to develop immune response for susceptibility or resistance (Figure 1) to toxoplasmosis.^4–7

Table 1.

This table illustrates the role of specific cytokines either in resistance or susceptibility against T. gondii. The cytokines’ source/s, main function/s, synergistic, and antagonistic relations with other cytokines.

Name of cytokine	Immunity response	Source	Main functions	Synergistic relationship	Antagonistic relationship	References
INFγ	Resistant	CD4+, CD8+, and NK cells	Renders protection against T. gondii by activation of MΦ, NO, and GTPase signaling	TNFα and IL1β	IL4 and IL10	[60]
TNFα	Resistant	MΦ, T cells, and basophils	Involves in acute inflammatory response	IFNγ and IL12	IL4 and IL10	[59-61]
IL1β	Resistant	Endothelial cells	Acute phase response mediator	TNFα	IL4	[57]
IL2	Resistant	CD4+ cells	Induces growth of T cells and the release of IFNγ, involved in the lytic activity of MΦ and NK cells	IFNγ	IL4	[45]
IL4	Susceptible	Basophils	Antagonizes the products of Th1 cells, long exposure leads to chronic toxoplasmosis	Th2 cytokines	Th1 cytokines	[53]
IL5	Resistant (chronic) and susceptible (acute)	Mast cells	Plays a counter protective role in acute toxoplasmosis and protective role in chronic toxoplasmosis	IL4 (acute infection)	IL12	[94]
IL6	Resistant	MΦ, endothelial cells, monocytes, fibroblasts, myelomatous	Plays a pleotropic role in immunity; builds barriers in early ocular and encephalitis toxoplasmosis, enhanced activities of NK cells, and maturation of T and B cells	IL1β and TNFα	IL12, IFNγ, and IL27	[97,111]
IL7	Resistant	DCs, hepatocytes, endothelial cells	Plays a crucial role in the development of memory CD8+ T cells	IL15	IL10 and IL4	[113]
IL10	Susceptible	CD4+ cells, MΦ, B cells, DCs, mastocytes	Controls hyper-inflammation, keeps check on protective functioning of CD4+ cells, and plays a suppressive microbicidal function for MΦ and Np	IL6	IL12 and IFNγ	[201,202]
IL12	Resistant	DCs, MΦ, Np	Central inducer of IFNγ	TNFα	IL4 and IL10	[24]
IL15	Resistant	Mononuclear phagocytes	Required for optimal role of NK cells, CD8+ cells, and IELs	IL12 and IL7	IL10 and IL4	[61]
IL17A	Resistant	CD8+, γδT cells, NK cells	Mainly involves in innate immunity by the recruitment of Np	IL12, IFNγ, and IL6	IL10 and IL4	[151,164]
IL18	Resistant	MΦ and some other cells	Involves in production of IFNγ by NK cells and T cells	IL12	IL10 and IL6	[182]
IL23	Resistant	MΦ and DCs	Stimulates NK cells and T cells more specifically in the absence of IL-12	IL12	IL10, IL4 and IL6	[158,159)
TGFβ	Susceptible	Intraepithelial lymphocyte	Anti-inflammatory role in brain, eyes and intestine	IL10	TNFα, TNFβ, IFNγ, IL6, and IL12	[15]

IFN (Interferon), IL (Interleukin), TNF (Tumor Necrosis Factor), TGF (Transforming Growth Factor).

Figure 1.

Outcome of toxoplasmosis in case of resistance and susceptibility.

Immunological studies on human infections have clearly concluded that cell-mediated immunity and IFNγ are paramount in the control of any infection particularly caused by the intracellular pathogen (T. gondii). ⁸ The inability of the humoral response alone (antibodies) to prevent Toxoplasma reactivation is evident by the fact that most of the HIV-infected people lack effective immunity hence exhibit symptoms of T. gondii infection and reactivation, even in the presence of high titers of specific IgG. ⁹

Pro-inflammatory cytokines are among the key factors to initiate and maintain innate as well as acquired immunity to restrict proliferation of Toxoplasma. A variety of cytokines are produced upon activation of APCs and cells of the adaptive immune system (B and T cells). The differences in cytokinome can be speculated at the different stages of infection, due to intra- or extra-cellular nature of pathogens as well as due to diversity of the host genetic makeup. ¹⁰ The indirect role of cytokines against T. gondii in leading to either resistance or susceptibility also depends upon the stage of parasite in the host and the induction and modulation of pro-inflammatory cytokines driven by the particular parasite strain as well as the robustness of the host’s immune profile. Different typical and atypical strains of T. gondii exist globally, and have been specifically studied in America and Europe. This parasite is identified having three distinct genotypic lineages in humans: type I strain (RH-88), type II strain (ME49, and DEG), and type III strains (CEP and VEG). ¹¹ The genetic moieties in these strains result into a highly varied level of virulence^7,12 which inflicts diverse pathological effects in host by a variety of cytokine pathways as well as owing to the wide range of interactions through a vast diversity of host and parasite molecules interacting each other, with some known and some unknown footprints. The known footprints include but not limited to inherent-oxidative stress, ¹³ a diversity of IRGs (Immunity Related GTPases) ¹² of host with a locus on chromosome 11, ¹⁴ Z-DNA binding protein 1 (ZBP1), receptor-interacting serine/threonine-protein kinase 3 (RIPK3) of host. ¹⁵ Likewise, the wide range of parasite molecules includes a diversity of rhoptry proteins (e.g. ROP5, ROP18),^12,16 dense granule proteins (e.g., GRA5, GRA12, GRA16, GRA24) ¹⁷ ; small GTPase immunity-associated proteins (GIMAPs 4, 5 & 6) ¹⁸ of T. gondii. This review describes the cytokinome (Table 1) in toxoplasmosis and their interactive role for development of host’s susceptibility and resistance toward T. gondii infection.

Database search

A search was conducted on Science Direct (https://www.sciencedirect.com/science/search) and PubMed (https://www.ncbi.nlm.nih.gov/pubmed) using “host immune response and resistance of toxoplasmosis” to achieve the relevant literature for this study. The searched period ranged from 1980 until 15 September 2021, yield 649 publications. The literature having mechanism of immune response and pathogen resistance were included in this study. Whereas for the last 5 years, the data were searched from PubMed using the same key words as mentioned above and majority of the articles with novel insights into the immune response mechanism as well as host parasite interactions were included in this review. Although maximum efforts have been done to review the literature on “host immune response and resistance of toxoplasmosis”, certainly there are limitations of this review. Hence, this should not be considered a review encompassing all the new literature on ‘Toxoplasma gondii” and “Toxoplasmosis”. This is because of the fact that 24,912 and 13,928 results are retrieved from Science Direct and PubMed, respectively, while using a key word “Toxoplasmosis” with the same time period as mentioned above. Likewise, 23,158 and 15,750 results are displayed from Science Direct and PubMed, respectively, with a key word “Toxoplasma gondii” for the same time period. Similarly, slightly modified key words, that is, “host immune response of toxoplasmosis” displays almost 7000 (6969) results in Science Direct.

• 1) Virulence of T. gondii strains

In humans, the regulatory cytokines profile depends on the T. gondii strain. Among three notable strains (type I, II and III) of T. gondii, the type I is more virulent as compared to type II and type III strains. In type I (RH strain) infected cells, the translocation of NF-κB does not take place resulting in the production of anti-inflammatory (IL10, IL27, and TGFβ1) cytokines which are higher as compared to uninfected cells.^19–23 These anti-inflammatory cytokines enhance the proliferation of T. gondii. It was found that with a significantly high level of TGFβ1 in the blood as well as in the aqueous humor of the acutely intraocular Toxoplasma-infected host, it may adversely interfere with the effective cellular immune response leading to an increased mortality and extensive ocular tissue damage with tachyzoites observed in the pigment epithelium layers. ²⁴ Consequently, Th2 and Treg responses are enhanced in comparison with a primary ocular infection. ²⁵ ME49 is a type II strain that induces the translocation of NF-κB-light-chain-enhancer from the cytoplasm to the nucleus of activated B cells, splenocytes ²⁶ and bone marrow–derived MΦ ²⁷ which induce the production of pro-inflammatory cytokines (IL1β, IL12, IL18, TNFα, IFNγ, and IL12p40) by thioglycolated MΦ/cell lines^19,28. Whereas, the production of anti-inflammatory cytokines are lower in ME49-infected cells than that of uninfected cells ²¹ speculating CD36-mediated engagement of low virulence strains, with macrophages. ²⁹ Recent evidence also demonstrates the role of Toxoplasma’s parasitophorous vacuole-membrane–associate dense granule proteins in modulating parasitic virulence while interacting the host body’s resistance mechanisms like GRA12 of Toxoplasma was identified as a major virulence factor to counter the host’s IFNγ. ³⁰

Host immune response

• A) Innate immune response

In early stages of infection, dendritic cells (DC), macrophages (MΦ), natural killer (NK), and neutrophils (Np) interact in a coordinated way to provide the first line of defense in the form of innate immune response leading to develop adaptive immunity.^31-33 The innate immune response is elicited against toxoplasmosis in the form of IL12 production upon interaction with antigen (Ag). The release of IL12 from MΦ, DCs, and Np is essential for the release of IFNγ from NK cells (innate immune response) and T lymphocytes (adaptive immune response) via antigen presentation. ³⁴ IFNγ has been shown to induce guanylate binding proteins (GBPs) in a murine model of toxoplasmosis, thereby, these GBPs accumulate on the surface of intracellular parasites potentially causing parasitic destruction, thus displaying an active role of intracellular autonomous immunity. ³⁵ The increased susceptibility toward T. gondii infection is due to the depletion of NK cells, MΦ, or DCs which have a significant involvement for innate immune response against the infection. ³⁶ The mechanism of innate immune response initiate upon interaction of toll-like receptors (TLRs) with ligands expressed on T. gondii surface, thereby starting intracellular signaling pathways through engagement of the myeloid differentiation domain-88 (MyD88). These are the universal adaptor proteins involved in signaling of all TLRs except TLR-3. The study on MyD88 deficient mice model was impaired to induce primary protection in acute infection of T. gondii (RH strain). ³⁷ Studies on mouse with targeted inactivation of MyD88 showed that DCs work as antigen-presenting cells (APC) and are responsible for the increased susceptibility to T. gondii infection. However, there was no effect on MΦ and Np. The MyD88 deficient mice masked the production of IL12 from DCs and IFNγ by NKs to initiate innate immune response. It explains a central role of DCs in the coordination of innate immune response against T. gondii infection and predicts the increased susceptibility towards infection if DCs are recognized defective in early encounter of T. gondii infection. ³⁸

• B) Cellular immune response

In T. gondii infection, the strong resistance to re-infection as well as the hindrance to reactivation of chronic infection is based on the host’s cell-mediated immunity.^39,40 The synergistic role of CD4⁺ and CD8⁺ T cells for the development of acquired immunity was understood from targeted experiments on C57BL/6 mice vaccinated with temperature sensitive mutant strain of T. gondii (ME49).^41,42 The development of complete immunity against a virulent strain (type I) is dependent on IFNγ synthesis from NK and T cells. The immunocompetent host activates either T cells or NK cells for encountering parasitic invasion. In a previous study, the MHC I (lack of CD8⁺ cell stimulation) impaired mice (beta 2m-deficient mice) surprisingly showed high resistance against T. gondii following vaccination. This enhanced immunological response in the absence of CD8⁺ cells showed the involvement of NK cells activated by IL12 upon parasitic invasion. ⁴³

Generally, the CD8⁺ T cells are the major source of IFNγ production against most of the T. gondii strain. ⁴⁴ The c-Rel expression regulated by NF-κB is widely dominant in hematopoietic cells, ⁴⁵ which play a critical role in the development of resistance against T. gondii (ME49) infection. ⁴⁶ The role of c-Rel in influencing the CD8⁺ cell response was investigated in mice model (c-Rel–/– mice) with a special infection of T. gondii strain (replication-deficient strain) in C57BL/6, CD45.1, and Thy1.1 mice. The CD8 ⁺ cells impair to replicate in the absence/deficiency of c-Rel. Likewise, the c-Rel deficient mice remained unable to survive during infection with the replicating pathogen.^47,48

CD8⁺ T immune cells having a TCR Vβ8.1, 8.2+ phenotype produce protection against tissue cyst development in mouse model. Transfer of CD8⁺ cells induced by N-terminal of dense granule protein-6 (GRA6Nt) of parasite has been shown to clear T. gondii cysts from the brains of infected mice that were deficient in T cells (Sa et al. ⁴⁹ 2017), further highlighting the role of cytotoxic T cells in the induction of protective immunity against T. gondii. A genetically resistant strain of mice having a H-2d haplotype helped discover these specific type of cytotoxic T cells, whereas H-2Ld was found as a major antigen-presenting molecule to CD8⁺ T cells to achieve this objective of tissue cyst elimination.

The immune response of CD8⁺ cells is more dominant alike effector cells than CD4⁺ cells. Nevertheless, CD4⁺ helper T cells are direly required for an effective functioning of CD8⁺ cells.^50,51 The correlation of CD4⁺ and CD8⁺ `cells proved to be the main scaffold of cytokine trafficking in mice host.

Regulatory T (T reg) cells are required for the maintenance of immunological self-tolerance and immune homeostasis by actively suppressing the pathological and physiological immune responses.^52,53 An IL2 knocked-out mouse model orally infected with lethal dose of T. gondii, showed highly Th1 cell type-polarized mucosal immune response. Such effect contributed to the incapacity of T reg cells to perform effector responses and consequently led to immuno-pathogenesis.^54,55 Besides this, T reg cells of infected mice expressed lower levels of Bcl-2 and increased levels of apoptotic markers than that of naive mice. It is suggested that de-regulation in the T reg cells is a consequence of these impaired cells turnover. ⁵⁴ Besides this, there was found a gradual weight loss and significant delayed mortality in T reg-transferred toxoplasmosis-infected mice associated with lower level of IFNγ and TNFα. Additionally, higher cyst number and parasite load in brain of those mice were observed. ⁵⁶ Furthermore, activity of T reg cells results in the death of proliferating T cells which favors the multiplication of pathogen in the murine model. ⁵⁷ In a pregnant T. gondii-infected mouse model, numbers of splenic CD4⁺CD25⁺-T reg cells and placental Foxp3⁺ cells decreased synchronously. During infection, the reduction of splenic CD4⁺CD25⁺-T reg cells was associated with apoptosis (Bcl-2) induced by proliferating T cells. ⁵⁸ Additionally, injection of pregnant mice with excretory–secretory antigens (ESA) of T. gondii also causes fetal death associated with apoptosis of CD4⁺CD25⁺ T reg cells by down-regulating their Bcl-2 expressions and Bcl-2/Bax ratio. It could be partly prevented by adoptive transfer of CD4⁺CD25⁺ T reg cells from normal to infected pregnant mice. ⁵⁹

The high level of Th1 cytokines (IL2 and IFNγ) were reported to be produced by CD4⁺ cells upon interaction with tachyzoite. ⁵⁰ It is found that MyD88 is effectively involved in the development of Th1 response. ⁶⁰ Generally, the Th1 cell-mediated immunity builds the resistance against the T. gondii infection by IFNγ production via Th1 effector cells. ⁶¹ The role of Th1 cytokines (IFNγ, IL12, and TNFα) for susceptibility to toxoplasmosis has been witnessed with the absence of any of these pro-inflammatory mediators as previously studied.^42,62 Moreover, other cytokines like IL2, IL6, IL7, IL15, IL18, and IL23 are also associated with the development of strong immunogenic response. ⁶⁰ Recent evidence demonstrates TLR-11–independent activation of inflammasome for driving CD4⁺ T-cell–derived IFNγ-mediated host resistance to T. gondii. ⁷ GRA24-driven protective immunity mediated through p38 MAPK activation, IL12 production, and independent of MyD88 pathway has been evidenced, recently, through use of bicistronic IL12YFP reporter mice on MyD88+/+ and MyD88-/- genetic backgrounds, MyD88+/+ and MyD88-/- bone marrow–derived macrophages as well as exploiting parasites species named as uracil auxotrophic Type-I stain of T. gondii cps1-1 and cps1-1:Δgra24. ⁶³

In contrast, various experiments have demonstrated that the cytokines involved in Th2 response also play a detrimental role for enhancing the susceptibility to T. gondii infection. ⁶⁴ The modulation of Th2 response is mainly carried out by IL4 and IL10. Both cytokines increase the host susceptibility to T. gondii in early infection. ¹⁰³ Nevertheless, the regulatory function of Th2 cytokines has been unveiled. The evidence from T. gondii infection (Type II stain) to the IL4-knockout mice resulted in less susceptibility to toxoplasmosis. ⁶⁵ Shoot-up levels of inflammatory cytokines were detected in IL10 knockout mice causing early resistance to Toxoplasma.^64,66

• C) Cytokines and other inflammatory mediators playing a role against T. gondii

Interferon-γ: Interferon-γ (IFNγ) is reckoned as the main pillar of cytokines induced by T cells (CD4⁺ and CD8⁺), γδT cells, and NK cells as protective immunity against either the acute or chronic phase of T. gondii infection.^42,67,68 The neutralization of INFγ (anti-INFγ antibodies) in in vivo makes the mice (Swiss-Webster) susceptible to the primary infection (acute infection) and reactivate parasite (ME49 strain) in chronic infection. ⁶⁹ The IFNγ is produced from activated MΦs to exhibit its specific immunological functions against T. gondii (C56 strain). ⁷⁰ The latest evidence demonstrating the CD4⁺, CD8⁺, γδT cells, and NK cells as the main producers of IFNγ during toxoplasmosis was based on the use of a newly developed mouse line named as “GREVEN” an IFNγ reporter mouse having a fusion protein of Venus and NanoLuc to analyze IFNγ producing cells. ⁶⁷ Likewise, the Lck-Cre/Ifngfl/fl mice were found highly susceptible to toxoplasmosis, further strengthening the role of T-cell-IFNγ in protection against toxoplasmosis. ⁶⁷ At the site of infection, the maintenance of IFR8+ inflammatory DCs is essential provision of host resistance against intracellular T. gondii, whereas this requires the production of IFNγ by ILC1 and NK cells through T-bet involvement. ⁶⁸ The extensive role of IFNγ was partly dependent on the release of TNFα by activated MΦs. ⁷¹ In the host, most documented immunological pathway against T. gondii (virulent RH strain) is nitric oxide–dependent. ²² It involves the collective role of IFNγ and TNFα for the production of nitric oxide that curtails the development of microbial pathogens. ⁷² IFNγ is involved in an alternative mechanism via inducing Ag-specific CD8⁺ (CTL) cells-dependent immunity. ⁷³ INFγ directs the antimicrobial response such as STAT-1, TNFα, IL1β, and CD40L by activating the transcription factor (NF-κβ) signaling pathway.^74,75 This signaling pathway has been believed to rely mainly through involvement of inducible nitric oxide synthase (iNOS) and immunity related GTPase (IRGs) which make grounds to resist T. gondii (ME49) infection, ⁷⁶ the nitric oxide-independent intracellular resistance mechanism has been evidenced recently through use of IFNγ-stimulated bone marrow–derived macrophages (BMDM). ⁷⁷ Likewise, recent evidence has shown IFNγ as an inducing agent of guanylate binding proteins (GBP) that accumulate on surface of intracellular stage of T. gondii; thus, mediating the role of an intracellular check on the excessive growth of this parasite that may become lethal to mouse host in the absence of GBP as has been demonstrated by the early death of the mice deficient in murine guanylate binding protein-7 (mGBP7), by T. gondii infection. ³⁵ The intracellular pathogen (T. gondii) has been highly adapted for combating host immune response by interfering with NF-κβ signaling pathway.^78,79 It also has the ability to inhibit IFNγ signaling by curtailing the function of STAT-1 and by augmenting the levels of IFNγ signaling suppressor molecules, named as suppressor of cytokine signaling molecule-1 (SOCs1).^80–82 Most of the Th2 cytokines function antagonistically to IFNγ.

Tumor necrosis factor-α (TNFα): TNFα is a pyrogenic factor, produced by MΦs, T lymphocytes and basophils, found to be responsible for the production of acute inflammatory response. It has the ability for microbicidal activity in MΦs via the production of IFNγ from NK cells. ⁸³ TNFα functions synergistically with IFNγ for the development of resistance against T. gondii (C56 strain) infection.^71,84 Hence, it is suggested to have a crucial role in the protective immunity against toxoplasmosis. However, certain researchers explained the role of TNFα to be doubtful. TNFα has also been reported to elicit cerebral and hepatic autoimmunity.^85,86 It has also been found to assist in intra-cerebral dissemination of T. gondii (ts-4 strain and virulent RH) in mice ((TNF(-/-), LTalpha(-/-), and TNF/LTalpha(-/-)). ⁸⁷

Interleukin-1: It is an acute phase response mediator cytokine that plays a synergistic role with TNFα enhancing inflammation during infection with T. gondii.^90,91 TNFα has been found to be associated with IL1β in regulating endothelial cells for the immunological and inflammatory role. In vitro studies elucidated an effect of these cytokines in hindering the intracellular multiplication of T. gondii in murine peritoneal MΦs or human fibroblast.^71,90 However, in vivo studies on mice showed the protective role of recombinant TNFα and/or recombinant IL1β during infection with tachyzoites of T. gondii (C56 strain and RH strain). ⁷¹ In female mouse models (BALB/c, and Swiss-Webster), IFNγ induced the anti-toxoplasmic activity by augmenting the production of TNFα when treated with recombinant cytokines (TNFα and IL1β).

Interleukin-2: Interleukin-2 is exclusively produced by CD4⁺ T cells. It is initially regarded as the primary T cell growth factor having significant role in the proliferation as well as in the development of antigen stimulated CD8⁺ T cells. ⁹¹ Various studies carried out on viral infections revealed an indispensable role of IL2 during primary response of T cells via effector cytotoxic T cell development and restoration of CD8⁺ memory T cells.^92,95 IL2 has also been found to induce T cells proliferation for IFN-γ production during the infection with T. gondii (virgin).^96–98 Whereas, the role of IL2 in secondary immune response to T. gondii (ME49) was disclosed by Sa et al., (2013) on CD8⁺ T cell hybridoma clones from the spleens of chronically infected female mice (BALB/c, BALB/c-background Rag1^−/−, and Swiss-Webster). They found an increased production of INFγ with exogenous input of IL2 to CD8⁺ T cell hybridomas. ⁹⁹ Various studies on murine models proved the protective role of IL2 against the infection with T. gondii. Increased survival rates and reduced number of cysts in the brains of mice were observed when treated with recombinant IL2. ⁹⁶ It also triggered the lytic activity of MΦs and NK cells. ¹⁰⁰

Interleukin-4: Interleukin-4 is a Th type 2 cytokine that down-regulates the effect of Th type 1 cytokines. The progressive toxoplasmic encephalitis found to be linked with the presence of mRNA transcripts in the brains of infected mice (C57BL/10 ScSn). ¹⁰¹ In acute infection of T. gondii, IL4 exerted a protective role by antagonizing the products of Th1 cells that reduced the number of mortalities. However, the prolonged exposure of IL4 made the mice (IL-42/2) susceptible to chronic toxoplasmosis with increased multiplication of parasite cysts in brain. ¹⁰²

Interleukin-6: Mainly, it is involved in early development of acute phase response, maintenance of hematopoiesis ¹⁰³ and immune barriers in ocular ¹⁰⁴ as well as cerebral Toxoplasmosis. ¹⁰⁵ It makes the NK cells to increment their cytotoxic activity and is also involved in the maturation of antibody secreting B lymphocytes and the differentiation of T lymphocytes. ¹⁰⁶ Different immune cells such as MΦs, endothelial cells, monocytes, myelomatous, and fibroblasts are involved in the production of IL6. This cytokine functions synergistically with IL1β and TNFα. Hence, it is regarded as a remarkable pyrogenic factor mediating dominantly the production of hepatocyte based acute inflammatory proteins. The signaling pathway of IL6 is associated with gp130 signal transducing component that leads toward the activation of STAT1 and STAT3 via the signaling of JAK1, JAK2, and TYk2. This pathway is referred as gp130-mediated JAK/STAT signaling pathway ¹⁰⁷ which is most effectively regulated by socs-3 signaling.^108,109 The host transcription factor STAT3, antagonizes host response and strengthens parasite survival which is activated by the cytokines (IL6 and IL10) during infection with certain intracellular pathogens.^110–112 Interestingly, T. gondii (ROP16-deficient type I) has been found to activate this factor by phosphorylation caused by rhoptry kinase.^20,113 This event is accompanied with impaired production of IL12p40 and TNFα response. To investigate the mechanism involved in the invasion of immune strategies, it was found that socs-3 up-regulates IL6 and IL10 which stimulate activation of STAT-3. The role of IL6 is more critical as in normal defense mechanism, socs-3 can curtail function of this cytokine by formation of gp130/IL6 receptor complex. ¹¹⁴ In the work conducted by Whitmarsh et al., (2011), the mice deletion with socs-3 in MΦs and neutrophils unexpectedly resulted in increased susceptibility to toxoplasmosis by reducing the levels of IL12 and IFN-γ. This study suggested more pronounced anti-inflammatory role of IL6 in MΦs particularly in the absence of socs-3. ¹¹⁵

Moreover, the gp130 transducing component has common structural features to IL6 and IL27. ¹¹⁶ This IL27 is critically involved in curtailing the infection induced inflammatory pathology and functions antagonistically to IL6. The mice lacking gp130 signal transducer (gp130 Y757F mice) upon infection with T. gondii (ME49), showed high parasite burdens and increased mortality having low IL12 and IFNγ titer. ¹¹⁷ IL6 is a cytokine that more pronouncedly functions to resist the Toxoplasma-induced encephalitis in murine model. ¹¹⁸ Previously, the protective role of IL6 was questioned in various studies that illustrated the role of IL6 in increased intracellular multiplication of T. gondii. ¹¹⁰ Later studies defined the critical role of IL6 in progression of T. gondii infection as the IL6-deficient mice rapidly switched to severe states of toxoplasmosis such as Toxoplasma encephalitis ¹¹⁹ (Figure 2).

Figure 2.

Pathways for host susceptibility and resistance in human Toxoplasmosis, particularly as a result of IL6.

Interleukin-7: It plays a crucial role for the development of memory CD8⁺ T cells ¹²⁰ which are the key producers of INF-γ in acquired immunity. ⁴¹ The IL2, IL7, and IL15 fall in the family of γ-chain cytokines that are involved in building CD8⁺ memory T cell.^121,122 The development of memory cells in the form of CD8⁺ T cells has been disclosed to be dependent on the significant role of IL7 as well as IL15. The in vivo neutralization of IL15 affected CD8⁺ memory T cells productions which are more susceptible to T. gondii (76K) re-infection. ¹²³ In memory cells development process, IL7 is recruited to provide the survival signals to naïve and memory CD8⁺ T cells. ¹²⁴ In a study conducted by Bhadra et al., (2010), the synergistic role of IL7 and IL15 was explored. Their findings indicated a severe impairment of memory cells in case of the absence of both of these cytokines. However, the absence of any of these cytokines resulted in minimal impact on the maturation of splenic CD8⁺ T cells. ¹²⁵

Interleukin-10: It induces pleiotropic effect on cells and is found to be a suppressive cytokine that is purely an anti-inflammatory in its action. ¹²⁶ Immune cells including MΦs, CD4⁺, B cells, DCs, and mastocytes are the source of this cytokine.^127–129 The rapid production of IL10 by localized MΦs was reported upon infection with high doses of virulent (RH) strain in mice. ¹³⁰ However, it keeps a check on the protective functioning of CD4⁺ T cells in acute phase infection.^131,132 IL10 is also reported to be responsible for suppressive microbicidal activity of MΦs and neutrophils by minimizing the function of nitric oxide (NO) synthase enzyme, IFNγ and IL12. The NO inhibits the production of O₂ free radicals and prostaglandins.^133–135 The simultaneous production of these antagonizing cytokines may lead to demolish the defensive mechanism. Hence, it was reported that the production of IL10 is supported by an “activation signal” after the release of IL12. The IL12 lets the production of IFNγ by Th1 cells not only triggering the effector cells to play protective role but also passing a signal for the reactivation of IL10 gene expression. The study conducted by Gaddi et al., ¹³⁶ (2007) explained the negative feedback mechanism for the poised state of these pathogenic and regulatory cytokines via CD4⁺ T cell lineage. In the mice (BALB/c) susceptible to T. gondii, the increased levels of IL10 were found in their lymph nodes and central nervous system causing chronic toxoplasmic encephalitis. ¹³⁴ In a study, the detrimental role of IL10 was disclosed as to promote the development of intracellular tachyzoites by inhibiting MΦ L–arginine-based killing. ¹³³ However, making a comparison, severe combined immune-deficient (SCID) IL10 knockout T. gondii (RH strain) infected mice lived longer than the infected SCID mice. ⁶⁶

Interleukin-12: In innate immune response, certain cell populations (DCs, MΦ, and Np) are reported to produce IL12 in vitro against T. gondii (a virulent strain).^130,137,138 It is the central inducer of IFNγ in developing the protective immunity against T. gondii. It is evident from various experiments that the complete absence of immunity in INFγ as well as IL12p40, IL12p35, and STAT4 deficient mice leads to deaths in early acute infection.^139–141 The increased mortalities were reported when diphtheria toxin were used to deplete DCs in T. gondii (ME49 strain) infected organism which abolish the production of IL12. ¹⁴² The absence of p40 chain in IL12 heterodimer is responsible for the poor production of IFNγ in mice (IL12-deficient). ¹⁴³ During chronic Toxoplasmosis, the 12/15 lipoxygenase (12/15-LOX) is involved in the oxidation of unsaturated fatty acids in MΦs which deprives the production of IL12. In addition, the 12/15-LOX deficient mice were enabled to produce comparable levels of IL12 when stimulated with lipopolysaccharide (LPS). This finding was explained by the involvement of neutrophils and particularly DCs in inducing IL12 production in acute phase infection. ¹⁴⁴

Interleukin-15: It belongs to Th1 immune response and plays an effective role in enhancing the function and development of CD8⁺ T cells. ¹⁴⁵ IL15 serves to play a key role in the development of various lymphocyte population more importantly NK, CD8⁺ T cells and intraepithelial lymphocytes (IELs). ¹⁴⁶ The memory (CD8⁺) T cell response was found inadequate identified in IL15 knockout mice.^147,148 However, a study conducted by Lieberman et al., (2004) reported no role of IL15 for development of memory immune response. They found the mice deficient with this cytokine withstood the severe T. gondii (ME49) infection. ¹⁴⁹

Interleukin-17: The IL17A, IL17F, and IL22 are secreted from Th17 cells reported by Wu et al., (2018). But a variety of immune cells including CD8⁺,^150,151 γδ, ¹⁵² and NK cells^153,154 also secrete IL17. It is an inflammatory cytokine that provides innate immunity from the recruitment of neutrophils ¹⁵⁵ which make the host resistant against T. gondii infection. ¹⁵⁶ A subset of CD4⁺ T cells has been identified which produces IL6, IL17A, IL17F, and TNF in response to IL23.^157,158 In a study conducted by Kelly et al., (2005), IL17 knockout mice remained successful in developing a normal acquired immunity against T. gondii.^152,161,162 A study carried out on T and B lymphocytes deficient mice revealed that NK cells are the major IL17 producers. These cells are influenced to produce IL17 in the same manner as T cells are triggered. In addition, the researchers revealed a key role of IL6 to target NK cells for secretion of IL17. ¹⁶¹ The IL17A neutralization by antibodies had a partial protective effect against fatal T. gondii–associated inflammation. ¹⁶² Severe South American ocular toxoplasmosis is associated with decreased level of intraocular IFNγ and IL17A. ¹⁶³ But in ocular toxoplasmosis (with less severe clinical presentation and infected by non-virulent strains) in France, the IL17A level was augmented in toxoplasmic uveitis. ¹⁶⁴ Neutralizing IL17A decreased intraocular inflammation and parasite load in mice (Swiss-Webster). It is suggested that the local IL17A production by resident cells plays a central role in the pathology of ocular toxoplasmosis. ²⁵ Finally, there was observed a lower level of IL17 expressing CD4⁺ and CD8⁺ T lymphocytes in cells cultures from sero-negative and seropositive pregnant and non-pregnant women, respectively upon stimulation with tachyzoites. ¹⁶⁵ A recent study demonstrates an essential role of T cells expressing class I-restricted T cell-associated molecule (CRTAM), for IL17 production during toxoplasmosis. ¹⁶⁶ The study also highlights the importance of IL17 in regulating immunopathology whereby deficiency of IL17 can cause dysbiosis through the production of antimicrobial peptides as well as through translocating gut-bacterial flora to spleen and mesenteric lymph nodes. ¹⁶⁶ Overall, these results suggest that IL17-mediated responses may be useful for both protective and pathogenic effects.

Interleukin-18: It is a pleiotropic cytokine produced in a non-specific manner. IL18 is a potent cytokine involved in the production of IFNγ by NK cells and T lymphocytes. Hence, it is involved in building innate as well as acquired immune response against T. gondii infection. Attributable to its identical role in developing resistance, IL18 is referred as a potential enhancer of IL12 activity.^167,168 Structurally, it is closely related to IL1β cytokine family. ¹⁶⁹ Moreover, similar to IL1β signaling pathway, IL18 precede the activation of NF-κβ^170,171 that requires STAT4 factor for its activation.^172,173 The impaired role of IFNγ was observed in IL18 deficient mice with intracellular infection.^174–176 Interestingly, NK cell has certain receptors for IL18 which has synergistic role with IL12 in developing innate immunity against T. gondii infection.^{167,177–179} However, endogenous role of IL18 on SCID mice was demonstrated to be trivial having less influence on IFNγ production when the infected mice were treated with anti-IL18. In contrary, the exogenous role of IL18 was reported to increment the production of IFNγ ultimately boosting the resistance against T. gondii. ¹⁷⁹ IL18 reported to be involved in the immunopathology of intestine in mice accompanied by IL12. ¹⁸⁰

Interleukin -33: The host damage protein IL-33 has recently been shown to play an important role in the immune response by affecting the local environment of brain in the favour of both host and parasite survival through engagement of astrocytes via IL-33 Receptor. IL-33 is a host damage protein that is produced locally in the brain tissue from the oligodendrocytes and astrocytes. The evidence to this effect is very strong as it is based on the use of mice having IL:-33 receptor-deficient astrocytes. ¹⁸¹

Transforming Growth Factor-β: Transforming growth factor-β (TGFβ) is another immunosuppressant cytokine that plays a critical role in antagonizing the action of TNFα, TNFβ, IFNγ, and IL12.^182,183 Moreover, it is considered to be involved in limiting the immune-pathologies incited by Th1 cytokines specifically in central nervous system (CNS) as well as in intestine.^184,185 TGFβ has been reported to induce immunopathological effects on retinal cell line in an in vitro study by increasing the replication of T. gondii. ¹⁸⁶ The intraepithelial lymphocytes are the main producers of TGFβ that is involved in the down-regulation of pro-inflammatory cytokines (IFNγ, TNFα, IL1β, IL12, IL15, and IL18) in case of pathogenic lamina propria lymphocytes (LPL) response (hyper-Th1 response). When wild type mice were treated with active transfer of IELs, they showed no sign of ileitis. This study revealed the modulating role of IELs for LPL produced Th1 response in the intestine. ¹⁸⁷

In spite of gastrointestinal sites, TGFβ also has a dominant role as an anti-inflammatory agent in brain and eyes. ¹⁸⁸ TGFβ signals the spleen cells to secrete anti-inflammatory cytokine such as IL10 that is synergistically involved in checking the pro-inflammatory secretions from NK cells and CD4⁺ T cells in these immune-privileged sites (eyes, brain, and placenta). ¹⁸⁹ The production of IL6 by innate immune response functions antagonistically to TGFβ and suspends its protective role for immune privileged sites (eyes and brain) susceptible to hyper-inflammation. ¹⁹⁰

CCL22: T. gondii induces expression of CCL22, a chemokine that has been linked to Toxoplasma-induced activation of Wnt/beta-catenin signaling pathway, for resisting cellular check on parasite replication and favoring the parasite survival within Toxoplasma-exposed naïve BMDMs. This cellular-check (induced by IFNγ and independent of nitric oxide) significantly reduced intracellular parasite load when Wnt/beta-catenin signaling pathway was chemically antagonized using IWR-1-endo in naïve BMDMs, thus strengthening the hypothesis that Toxoplasma induces this signaling pathway to survive intracellular anti-parasitic immunity. This got further support when a significant reduction in the intracellular parasite load of RHASP5, a mutant strain of T. gondii lacking ability to secrete dense proteins into the host cell, was seen in naïve BMDMs. Additionally, T. gondii invading the BMDMs pre-stimulated with IFNγ, switched on its bradyzoite gene profile. ⁷⁷

• 2) Toxoplasmosis and pregnancy

Toxoplasmosis is more important in pregnant women and immune compromised patients with respect to abortion, hydrocephalus, and retinochoroiditis. In pregnancy, Th2 immune response becomes activated which favors the proliferation of Toxoplasma. Briefly, in acute phase of T. gondii infection, certain cytokines (TGFβ1, TNFα, IL4, IL5 IL7, IL10, and IL17A) and chemokines (CXC, C, and CX₃C families) play an important role as protective immune response.^191,192 These pro-inflammatory cytokines down-regulate anti-inflammatory cytokines which travel to immune-privileged sites (brain, eyes, and placenta) to favor the existence of corpus luteum in the presence of low progesterone and 17β estradiol in pregnant women. ²³ Apoptosis of placental cells may end up in fetal resorption, congenital anomalies (hydrocephalus and retinochoroiditis), or abortion ¹⁹¹ (Figure 1). Briefly, toxoplasmosis with lymphadenitis has been reported with higher levels of chemokines (CXCL8/IL8, CXCL9, and CXCL10) in pregnant women. Additionally, levels of VCAM1, CCL2, and CCL5 are lower in pregnant than in non-pregnant women. ¹⁹³ The levels of ICAM1, CXCL9, CXCL10, MCSF, and TNFβ were up-regulated in acutely toxoplasmosis-infected Colombian pregnant women. Whereas, the levels of Eotaxin (Et), TGFβ, TNFα, IFNγ, IL2, IL4, IL15, CXCL1, and stem cell factor (SCF) were down-regulated in pregnant American acute cohorts. ¹⁹⁴ In congenital toxoplasmosis, it was found that serum levels of IFNγ and IL5 were greatly increased during active stage of retinochoroiditis. In contrast, IL10 production was low during inflammatory stage and significantly higher in patients with inactive lesions. ¹⁹⁵ The cytokine profile of acute toxoplasmosis-infected patients varies with geographical localities.

• 4) Perspectives for immunomodulation, therapy, vaccine, and other anti-parasitic challenges

The exploration of deep knowledge on the role of cytokines in toxoplasmosis should open new avenues for therapeutic measures based on immunomodulation. For instance, the use of IL17A antagonist inhibited the ocular toxoplasmosis in European patients. ¹⁹⁶ Similarly, inhibition of parasite kinases in South American toxoplasmosis patients enhances the expression of IFNγ.^163,197,198 This difference in inhibition sites might be strain dependent. Recent analysis of the cytokines profile in congenital toxoplasmosis^199,200 indicates that modulation of cytokines through immuno-modulatory peptides could be assayed as immune adjuvants. ²⁰¹ Such approaches need to be explored for the control of toxoplasmosis in humans. Etanercept (a soluble TNF-receptor fusion protein), widely used to treat autoimmune disease, activates the conversion of bradyzoites (chronic toxoplasmosis) to tachyzoites (acute toxoplasmosis) through down-regulation of pro-inflammatory cytokines (TNF, IL-1beta, and IL6). ²⁰² It would be interesting to try to achieve a sterile immunity in an experimental model of chronic toxoplasmosis, at first transforming bradyzoites to tachyzoites through use of Etanercept but not too long after this, treating the tachyzoites to eliminate the parasite from the host body.

A recently identified drug target for T. gondii is an endonuclease named as cleavage and polyadenylation specificity factor subunit-3 (CPSF3) that has a role in mRNA processing in eukaryotes. This has been demonstrated by strong in vitro anti-parasitic activity by use of benzoxaborole (AN3661), a drug molecule that targets wild-type CPSF3. The parasites that were found resistant to this drug molecule displayed mutations in the TgCPSF3. Recapitulation of the similar resistant phenotype of the parasite through generation of mutations in the wild-type CPSF3 while exploiting CRISPR/Cas9, further strengthened the importance of this new therapeutic target against T. gondii. ²⁰³

One of the most exciting areas of research is to explore the means and effects of intervention strategies on how various strains of T. gondii can modulate host’s transcriptome ²⁰⁴ and non-coding RNAs including mircoRNA and long non-coding RNA. ²⁰⁵ Similarly, exploring how T. gondii exploits exosomes in modulating host immune response ²⁰⁶ as well as how therapeutic interventions designed for heme-deficient conditions affect infection outcome, ²⁰⁷ remains interesting areas of research.

Given the assumed fact that around one third population of world is harboring Toxoplasma in chronic form, that is, tissue cysts, why not to plan a vaccine to eliminate the tissue cysts from human population and other hosts seropositive to this infection, with a vaccine (based on GRA6Nt or other similar antigens) ⁴⁹ that should be capable of eliminating tissue cysts.

Conclusions

Different factors are responsible for the pathogenesis of Toxoplasmosis and the survival of host. These factors include versatile genetic makeup of different strains of T. gondii, complicated immunological background of hosts, biochemical interaction among certain cytokines, invasion strategies of parasite as well as the immunogenicity of antigens encountered with host’s immune cells. The type of cytokines production depends on the strain of Toxoplasma. The IL10 and TGFβ1 production were higher in type I strain and lower in type II and III strain of toxoplasmosis. The production of IL12 was higher upon exposure of pathogen to DCs, MΦ, Np, NK cells, and T cells which is essential for the release of IFNγ. The production of IL12 switches the NK cells for release of IFNγ which develops resistance against T. gondii infection in host. Impairment in the production of IL12 may lead to demolish IFNγ resulting to develop host sensitivity for T. gondii infection. Moreover, IL6 also has critical role for gp130 signaling pathway for the up- and down-regulation of SOCS-gene which is responsible for the susceptibility and resistance of toxoplasmosis (Figure 2). The basic switching of pro- and anti-inflammatory cytokines in acute and chronic phases of toxoplasmosis is direly required for understanding the development of disease. Such cytokines are involved in the development of resistance and susceptibility of Toxoplasma in host. Agonist and antagonist effect of host cytokines network leads to the chronic condition of disease. In vivo up- and down-regulation of desired cytokines (IFNγ, IL6, IL12, and SOCS-3) could be helpful to boost up the immune response of host for the control of toxoplasmosis. Moreover, the synergistic and antagonistic relations among cytokines need to be comprehended on molecular and biochemical basis. The most compelling results are related with a Th2-deviated response associated to virulent strains in South American patients. Type II strain has the ability to translocate NF-κB in the nucleus of mouse splenocytes and bone marrow–derived MΦ. It is the reason that Toxoplasma type I strain survives from host immune response rather than type II and III but the complete defeat of host’s immune response is not in the favor of parasite’s survival in the ecosystem. The survival of the host after entry of T. gondii, is essential for ensuring existence of both the host and the parasite as if parasite defeats the host’s immune response, it not only marks the death of the host but also of the parasite as parasite needs a viable host to ensure its own survival as well as for its transmission to next generations of the same host as well as to other host species. The current review findings state that in vitro harvesting of IL12 from DCs, Np and MΦ upon exposure with T. gondii might be a source for therapeutic use in toxoplasmosis. Current review suggests that therapeutic interventions leading to up-regulation/supplementation of SOCS-3, IL12, and IFNγ to the infected host could be a solution to sterile immunity against T. gondii infection. This would be of interest particularly in patients passing through immunosuppression owing to any reason like the ones receiving anti-cancer therapy, the ones undergoing immunosuppressive therapy for graft/transplantation, the ones suffering from immunodeficiency virus (HIV) or having AIDS.

Appendix.

Abbreviation

Ag

Antigen

APC

Antigen-presenting cells

BMDM

bone marrow–derived macrophages

CNS

Central nervous system

CPSF3

Cleavage and polyadenylation specificity factor subunit-3

DCs

Dendritic cells

ESA

Excretory–secretory antigens

GBPs

Guanylate Binding Proteins

GIMAPs

small GTPase immunity-associated proteins

HIV

Human immune deficiency virus

IELs

Intraepithelial lymphocyte

iNOS

Inducible nitric oxide synthase

LPL

Lamina propria lymphocytes

IRGs

Immunity-related GTPases

LPS

Lipopolysaccharide

MyD88

Myeloid differentiation domain-88

MΦ

Macrophages

NK

Natural killer

NP

Neutrophils

RIPK3

receptor-interacting serine/threonine-protein kinase 3

SCF

Stem cell factor

SCID

Severe combined immune-deficient

SOCs1

Suppressor of cytokine signaling molecule-1

T reg

Regulatory T

T. gondii

Toxoplasma gondii

TGFβ

Transforming growth factor-β

Th2

T helper cell 2

Th17

T helper cell 17

TLR

Toll-like receptors

ZBP1

Z-DNA binding protein-1

ORCID iDs

Imran Rashid https://orcid.org/0000-0002-0280-9335

Haroon Akbar https://orcid.org/0000-0002-8294-5862