THE ROLE of PROTOZOA in IMMUNE SUPPRESSION and INFLAMMATORY DISORDERS Are P24, P40, P60

THE ROLE OF PROTOZOA IN IMMUNE SUPPRESSION AND INFLAMMATORY DISORDERS – Are p24, p40, p60, p120, p160 of protozoan or viral origin?

Beldeu Singh - Research Article

Keywords – protozoa, immune suppression, AIDS, inflammation, actins, glycoproteins, cancers

ABSTRACT

About 20% of people in developed societies are infected with micro-parasites. Toxoplasmosis is one of the most epidemic parasitic diseases in human beings and animals. About 35-40% of the world’s adult population is estimated to carry Toxoplasmainfection and demonstrates varying clinical manifestations1. In Africa, the infection rate may be as high as 60-80%. Many will remain asymptomatic. Homosexual men have a much higher incidence of parasitic infections. A long co-evolution of eukaryotic pathogens with the host vertebrate immune system yielded the critical ability to the micro-parasites to evade or suppress or deactivate the humoral effector mechanisms and hijack cells of the innate immune defenses and hide in vacuoles in cells or in free form with various degrees of molecular mimicry and ability to recruit host cell mitochondria enabling these microorganisms to typically produce long-lasting chronic infections after a variable window period. Infected cells undergo remodeling of the phagosomal compartments and lymphocytes undergo transformation upon blocking of apoptotic signalling. Microparasites are an integral part in inducing immune suppression and are a factor in inflammatory problems. Hence, the incidence of immune suppression varies in different parts of the world and in different groups according to incidence of protozoan infections. Glycoproteins p160, p120, p60, p40, p24, p10 are produced in an orchestrated manner by different infective stages of protozoa to protect them from host innate immune responses and to promote their propagation. The p24 antigen is said to be highly specific for infection but it also appears to be cleavage product of gp160 under oxidative stress that occurs within the cell. Further research is required to streamline data for better diagnostics and better understanding on the primary cause of immune suppression.

INTRODUCTION

Various protozoan species infect humans, producing a spectrum of clinical manifestations. It is estimated that 350 million people are at risk, with a global yearly incidence. Since, protozoan infections tend to suppress the immune system, there is a setting of co-development of AIDS2. Many protozoan species, including Toxoplasma, Cryptosporidium, Microsporidia, Trypanosoma cruzi and different Leishmania specie have been found in AIDS patients3.

Protozoa elicit an initial strong inflammatory response from the host immune system which is subsequently suppressed by the protozoa. This is orchestrated through a set of actins produced by the protozoa that aids its infective stages. The infective forms can invade cells of the immune system. Subsequently, a concerted effort is established through an elaborate biochemistry that involves blocking apoptosis, hijacking the cellular machinery, cell remodeling and transformation involving recruitment of host cell mitochondria by the bradicytes. About 6-7% of infected people will develop clinical manifestation of immune suppression. The internal cell environment is altered through influx of Ca2+ and triggering of reactive intermediates and TNF-α and NFkappaβ. This can initiate inflammatory disorders or aggravates problems in people with inflammation-related problems as in diabetics. The abnormally activated NFkappaβ migrates into nucleus and binds to DNA domains. This modifies gene expression, leading to production of abnormal proteins and abnormal genes that, over time, can lead to formation of cancer cells and tumors.

Elimination of the micro-parasite through non-toxic interventions appears to be the most favorable approach in people with such infections instead of treating the inflammation and managing the symptoms. This is important because “while part of the clinical manifestations of toxoplasmosis results from direct tissue destruction by the parasite, inflammatory cytokine-mediated immunopathologic changes may also contribute to disease progression4.

INITIAL PROTOZOAN INFECTION INDUCES HIGH INFLAMMATORY RESPONSE

Initial protozoan infection induces IFN-gamma (IFNγ) secretion as a response that activates macrophages for ROS generation and prolonged activation may be problematic with regard to excess production of proinflammatory factors. Tachyzoites of protozoa infect cells of the immune system and can precipitate immune suppression in which viral loads are found as opportunistic infections like other pathogens. “Toxoplasma gondiiis among the most successful parasites. It is capable of infecting all warm-blooded animals and causing opportunistic disease in humans.5”

It has been demonstrated that live, as well as heat-inactivated, tachyzoites ofNeospora caninum, aToxoplasma-like protozoan, directly trigger production of IFN-γ from purified, IL-2-activated bovine NK cells6. Live Leishmania promastigotes (the form that is injected by the vector) can directly induce human peripheral blood NK cells from healthy donors to secrete IFNγ in the absence of IL-12 and killing of promastigotes abolishes this property7.

Natural killer (NK) cells are a key component of innate immunity, particularly crucial during the early phase of immune responses against certain viruses, parasites and microbial pathogens. However, as they are endowed with the entire cytolytic arsenal, they promptly become capable of attacking fetal and maternal tissues during infection and inflammation8 through excess IFN-γ production and the resultant cellular damage may be diagnosed as an auto-immune disorder.

Protozoan infection induce IL-12 that in high levels triggers NK cells to secrete IFNγ which in turn activates macrophages to kill tumor cells and activates the reactive oxygen species (ROS) that destroys intracellular pathogens9. The parasite induces high levels of gamma interferon (IFN-γ) during initial infection as a result of early T-cell as well as natural killer (NK) cell activation. During chronic infection, IL-12 stimulates differentiation of Th1 into parasite-specific T lymphocytes release high levels of IFN-γ, which is required to prevent cyst reactivation.

Many studies show that T. gondii and T. cruzi have the capacity to nonspecifically trigger cytokine synthesis by macrophages and it has been shown that infection with either live tachyzoites or live trypomastigotes induces cytokine synthesis by different types of macrophages. Live parasite forms trigger the synthesis of a wide range of cytokines (i.e., IL-1ß, IL-10, IL-12 and TNF-a) by inflammatory macrophages in vitro10.

IFNγ response may not succeed in killing all the intracellular microparasites as many infective forms invade cells and hide in parasitophorous vacuoles inside cells. Later on there is T-cell-mediated cytolytic activity against infected cells11. These microparasites have developed an adaptive mechanism in which their tachyzoites can infect cells on the immune system. Protozoa have a system of glycoproteins based on molecular mimicry that bind to receptors on host cells, including receptors of cells of the immune system to downregulate the initial high IFNγ and NFkappaβ immune response against them which helps their infective stages. Non-soluble components secreted by protozoa hiding in vacuoles continue to trigger NK cells to trigger IFNγ, thereby keeping macrophages in a continuous activated state during which they secrete ROS that may add to the oxidative stress in blood and maintain a state of inflammation. On the other hand, some infected cells will be eliminated by cytolytic activity against them, thereby depleting cells of the immune system that may precipitate various degrees of immune suppression.

INFECTIVE FORMS OF PROTOZOA INVADE CELLS OF THE IMMUNE SYSTEM

The tachyzoites of some protozoan species can cross the placenta and many protozoan species (eg. N. caninum)can infect different immune cell types including dendritic cells, macrophages and NK cellsin a similar fashion asT. gondii12and the tachyzoite-infected dendritic cells (DC) in mice resulted in increased parasitic loads in various organs. The tachyzoites are able to infect cultured NK cells, in which tachyzoites proliferate rapidly inside parasitophorous vacuoles13. Suppression of infected NK cells compromises its function and shuts down IFNγ activity against invading protozoan forms as well as against newly formed cancer cells.

It has been demonstrated that tachyzoites differentially infect and replicate in human monocytes, neutrophils, dendritic cells, and lymphocytes. Although tachyzoites are able to infect each of these cell types, they do not infect them equally14. Infection of peripheral blood cells and dendritic cells by tachyzoites is parasite dose dependent. Multiple infections of monocytes, dendritic cells and neutrophils were observedto increase degeneration of neutrophils15. When tachyzoites were incubated with human peripheral blood leukocytes in vitro, more monocytes and dendritic cells than neutrophils or lymphocytes were infected16. By hijacking the machinery of infected monocytes, the tachyzoites transform to bradyzoites and induce the production and release of IL-10 which will quickly downregulate the IFNγ immune response from NK cells against the infective forms and aid the progress of the infection as NK cells are the first to respond against the protozoan infections. The accumulating research information confirms that infection of monocytes, neutrophils, dendritic cells, lymphocytes and NK cells occurs during the tachyzoite stage in the life cycle of protozoa. “Tachyzoite infection suppresses immunity and reduces the number of CD4+T-cells17” while T-cell mediated cytolytic activity against the infected T-cells depletes T-cells. Many protozoan species are reported in AIDS patients. Enterocytozoon bieneusihas also been reported in HIV-infected patients18. In other animals too, protozoa can cause similar problems. Enterocytozoon salmonis, an intranuclear microsporidian of salmonid mononuclear leukocytes suppresses the humoral response following infection and the degree of suppression increased as the severity of the infection progressed. Subsequently, the response to mitogen-induced lymphoproliferation is impaired19.

IL-1β is produced by monocytes when stimulated by antigens20. IL-1 β is required for IL-12 to stimulate IFNγ by NK cells21 and for stimulating the differentiation of naïve t-calls into parasitic-specific T-cells but protozoan-infected monocytes are inhibited, probably by p40, and do not produce IL-1β which retards the protective immunity of the infected host22. Hence, tachyzoites quickly infect a large number of monocytes early during infection together followed by infection of a large number of NK cells,23 that begins a process which can lead to various grades of immune suppression following a variable window period. Immune suppression is an integral result of protozoan infection that corresponds to the severity of infection.

THE GLYCOPROTEIN MECHANISM OF PROTOZOA – Molecular Mimicry and High Viral Loads

Toxoplasma gondii can actively infect any nucleated cell type, including cells from the immune system. “Infection is normally asymptomatic in healthy individuals because control of this pathogen results from the host's ability to mount a robust cell-mediated immune response which is dominated by production of Interferon-γ (IFN-γ) by NK cells during the earliest stages of infection and by parasite specific CD4+and CD8+T cells thereafter.24” All tachyzoite and antigen preparations at high doses stimulated high levels of interleukin-12 (IL-12), interferon-gamma (IFNγ) and tumor necrosis factor TNF-α25. IL-12 precedes and initiates synthesis of IFN-gamma, while the latter lymphokine directly controls parasite growth26 by killing it directly with NFkappaβ or indirectly through stimulating differentiation of cells that will target the micro-parasites. IL-12 is produced in response to the actins secreted by protozoa during the infective process. IL-12 triggers the differentiation of naïve T-cells into parasitic-specific T-cells for production of IFNγ against the infective stages of the invading protozoa.

However, apart from an elaborate and “smart” model of biochemistry to aid infection and propagation, protozoan have a strategy to infect key cells of the immune system to suppress their role and function against their infective stages. Since, a large number of monocytes and “natural killer (NK) cells become infected shortly after entering the host27” the IFN-γ production drops thus evading elimination by host responses and as infection is followed by dysregulation and hijacking of NK cell machinery leading to failure to control parasite replication by down regulating the IFNγ, IL-1β and IL-12 while cellular infection maintains a low grade inflammation through another biochemical framework resulting in a wide range of inflammatory disease conditions. Thus, “Toxoplasma gondii infection may cause a variety of symptoms involving virtually all organs and there may be immune suppression in 6-7% of infected people28.”

Many protozoa synthesize unusual glycol-carbohydrate structures, which are often antigenic are required for invasion of their hosts and these are binding proteins29. T. gondii tachyzoite invasion is an active process involving parasite actin-based motility30. T. gondii have a single gene to form the soluble actin within the cytoplasm31 and anothergene that encodes for p24.32 This actin glycoprotein fraction is produced by T. gondii inside the infected cell and is secreted. The bradicyte has one fully functional mitochondrion to produce p24. This glycoprotein can be obtained from supernatants.

Products released by protozoan-infected cancer cells will naturally include abnormal glyco-proteins as found in one study33 and its clinical value may lie in that it proves protozoan infection(s) with various degrees of immune suppression depending on its concentration. Glycoproteins are found throughout matrices. They act as binding-proteins that bind to receptors on cell surfaces. The glycoproteins produced from actins in the actin cytoskeleton of protozoan cells may be part of micro-parasite molecular mimicry as these actins, including p24, are released together with tachyzoites. Tachyzoite infection of cells of the immune system is an active process wherein the p24 actin binds to receptors on these cells to shut down their protective immune against tachyzoites. It is patently clear that only high concentrations of p24 found are associated with protozoa-induced immune suppression and this is expected in chronic infections. Hence, such a sero-test must be based on concentration levels to indicate immune suppression in contrast to one of viral origin that can be based on its absence or presence, especially when it is claimed that it is a viral-specific protein belonging to a unique retrovirus.

The mechanism of targeting infected cells mediated through specific T-cells is quite clear from research on the protozoan, Theileria parva. This parasitic protozoan infects and transforms bovine lymphocytes through its specific protein with a 3-4 weeks window period. The transformedlymphocytes are recognized by CD4+ parasite-specific T-cell clones derived from immune cattle. The antigen obtained from supernatants that correlates with antigenic activity is a 24 kilodalton protein (p24).34 A 24 kD protein is also found in T. gondii lysate35 which interestingly coincides with the reported p24 obtained from supernatants of AIDS patients but this p24 associated with immune suppression is obviously distinct from retroviral protein. “The 24 kilodalton protein (p24), associated with AIDS and immunologically distinct from proteins in most other retroviruses, is said to be a major structural core component of HIV-1.36” The fact that it is immunologically distinct from proteins in most other retroviruses seems to indicate that this p24 may not have originated from a retrovirus but from protozoa that can transform lymphocytes into leukemic cells while producing immune suppression.