Much of the infection related mortality, morbidity and overwhelming economic burden in the developing nations of the world is the result of infection by bacteria and protozoa that reside within cells. Classic examples include diseases caused by Mycobacteria, Salmonella, Lieshmania, Trypanosoma, Toxoplasma, Rickettsia, Anaplasma, Chlamydia, Histoplasma and Plasmodia. In many cases there are problems of increasing drug resistance and a lack of effective vaccines to treat and prevent these infections. In some cases veterinary species act as reservoirs and diseases are zoonotic. In this context it becomes important to realise that human and animal health are inextricably linked. The host has many levels of potentially effective immune defence but the huge success of intracellular organisms is testimony to their cunning means of immune evasion. This brief review will focus on important mechanisms of host immune response to intracellular infection as well as mechanisms evolved by infectious agents that inhabit this niche environment to evade immunity. Some useful review literature is listed at the end of this article (1-3).

1. Non-specific innate immune response

The host's first line of defence is the rapid response innate immune system which precedes the time consuming clonal-expansion of antigen specific lymphocytes. The innate immune system is comprised of non-phagocytic cells and phagocytic cells in tissues and circulation, and complement and various other plasma proteins in the extracellular space.

Epithelial cells not only provide an effective physical and chemical barrier but they also produce intracellular antimicrobial elements that kill Rickettsia which specifically target endothelium. Phagocytic cells recognise and engulf pathogens via germ-line encoded pattern recognition receptors (a classic example being the Toll receptors) for generic microbial molecules (a typical example being bacterial LPS). Phagocytes also express receptors for complement (CR) and antibodies (FcR), important in complement and antibody mediated elimination of intracellular pathogens. Natural killer cells (NK) play a crucial innate role in immunity to intracellular infections through their cytotoxic attack of infected target cells (through perforin and granzymes) and via their activation of macrophages through the synthesis of IFNγ. Granzymes released into target cells activate apoptotic pathways causing host cell apoptosis. IFNγ is a very potent activator of intracellular macrophage killing mechanisms (such as NO generation) and is produced by NK cells. Cytokines play a pivotal role in defence against intracellular pathogens. Experiments with neutralising antibodies and gene KO mice have demonstrated conclusively that without the activity of IFNγ, resistant hosts are rendered fully susceptible to numerous intracellular bacterial and protozoal organisms. More recently the role of IL-12 and IL-18 have been evaluated as these cytokines seem to be released very early in infection and play a fundamental role in the Th1/Th2 bias of subsequent T-cell responses. Cytokines released later in infections such at TNF have also been shown to be important in experiments with KO mice although the role of TNF appears to be less crucial in early direction of immunocyte bias. Early induction of chemokines following pathogen invasion is important for the recruitment of professional antigen presenting cells (APC) and the function of phagocytes. RANTES, MIP-1α and MIP1β are examples of such molecules and they increase the uptake and destruction of trypomastigotes by human and murine macrophages in an NO dependent way. RANTES has the same effect on human hepatocytes and endothelial cells by inducing an NO-dependant anti-rickettsial effect. Ligation of CCR5 provides an important signal for the induction of IL-12 synthesis by CD8α⁺ dendritic cells which establish an IFNγ dependant resistance to T. gondii. Once pathogens are internalised by host cells and localised within membrane bound vesicles, the phagosomes fuse with lysosomes to form phagolysosomes. Lysosomal hydrolases are activated at low pH and play an important microbicidal role.

2. Evasion of innate immunity by infectious organisms

T. cruzi infective metacyclic and blood stream trypomastigotes are resistant to complement degradation due to the expression of a glycoprotein (gp 160) which is a homologue of the host complement-regulatory protein decay-accelerating factor (DAF). Like DAF, gp 160 binds to C3b and C4b preventing convertase formation and lysis of the parasite. Leishmania employ another interesting strategy to evade complement-mediated lysis whilst using complement activation to target their host cell. Infective metacyclic forms alter their membranes to prevent insertion of the lytic C5a-C9 membrane attack complex (MAC). This is also associated with the generation of a surface proteinase gp63, which cleaves C3b to the inactive form (iC3b) which opsonises the parasite for phagocytosis through the complement receptors CR3 and CR1, thereby targeting the parasite to the macrophage, its host cell. Other serum proteins play an important role in innate defence as shown in primates that have specific trypanosome lysis factors (TFL's) in their serum making them resistant to the veterinary Trypanosoma species.

Adaptation to the intracellular lifestyle necessitates overcoming the hostile environment of the phagolysosome. T. gondii resides in a parasitophorous vacuole and restricts its fusion with host endosomes and lysosomes because its membrane lacks integral membrane proteins of host cell origin having been extensively modified by secreted parasite proteins. T. cruzi trypomastigotes enter cells and reside briefly in lysosomes but escape rapidly into the cell cytosol through the secretion of a parasite protein, Tc-TOX, which has a membrane pore-forming activity at acidic pH. Leishmania metacyclic promastigotes are taken up by receptor-mediated phagocytosis and then phagosome maturation is possibly transiently inhibited by a parasite surface lipophosphoglycan (LPG) that becomes incorporated in the phagosome membrane. The replicating amastigote stage ultimately resides within a phagolysosome where they survive via production of cell-surface and secreted glycoconjugates.

One of the primary defence mechanisms in macrophages is the generation of reactive oxygen intermediates through oxidative metabolism through the multi-component enzyme NADPH oxidase and the synthesis and release of arachidonic acid metabolites. The malarial pigment hemozoin formed as a result of haemoglobin use by the parasite is capable of inhibiting protein kinase C (PKC), an enzyme critical in the initiation of the NADPH cascade. Leishmania also seems capable of inhibiting PCK and thus inhibiting macrophage oxidative burst. One of the more striking dysfunctions observed in protozoa infected macrophages is their inability to produce IL-12, which is the main physiological inducer of IFNγ and hence Th1 T-cell differentiation. Indeed, IL-12 is an essential cytokine in the development of acquired immunity to most intracellular pathogens. Excess IL-12 would be detrimental to both host (inducing a 'cytokine storm' and death) and parasite (causing complete elimination). Inhibition of IL-12 is through the selective inhibition of the Janus-kinase-signal transducers and activators of transcription (Jak-STAT) signalling pathway. Toxoplasma has yet another way of regulating immune gene expression through modulation of the NF-κB family of transcription factors. This family of transcription factors is essential in the regulation of numerous immune genes, including those encoding IL-12, IFNγ, TNFα, iNOS and adhesion molecules as well as several genes involved in cell proliferation and survival. Toxoplasma inhibits NF-κB translocation to the nucleus. In addition to being directly inhibited by parasites, IL-12 can be suppressed through the synthesis of down regulatory molecules like IL-10 and TGF-β which can themselves be upregulated by infection. One final means of down modulating host cell immune responses evolved by some parasites is through the inhibition of infected cell apoptosis. Apoptotic pathways in Leishmania and T. gondii infected macrophages were strongly inhibited, perhaps by parasite induced upregulation of Bcl-2 homologs.

A fascinating and very topical method employed by some organisms to evade innate immune responses is through the modulation of dendritic cell (DC) function. DC's are professional APC's and the most potent APC known. These are the only cells capable of activating naïve T-cells and as such as a vital link between the adaptive and acquired arms of the immune system. Whereas many parasites actively inhibit IL-12 production by the target host cell the macrophage, IL-12 secretion by DC's is spared in many cases (although the role of costimulatory molecules and IFNγ is usually required). A particularly intriguing example of the inhibition of DC function has been demonstrated in Plasmodium falciparum infection. Parasite clones that result in red cell adhesion to DC's cause the down regulation of MHC II, adhesion molecules and co-stimulatory molecules, thus rendering the DC unresponsive to stimulation by LPS which is normally a potent stimulator of DC activation. Leishmania is capable of inhibiting the migration of DC's in lymphoid tissue, thus inhibiting the presentation of antigen to T-cells. IL-12 synthesis by DC's, although not completely inhibited by infection, does appear to be very carefully regulated. This was shown using a soluble antigen extract of T. gondii (STAg). Injection of the antigen causes a short-lived IL-12 secretion that cannot be recalled by a second injection for up to a week. Taken together these findings indicate that once IL-12 has been induced and a Th1 response activated, the synthesis of this cytokine is actively inhibited, probably to the joint benefit of both host and parasite.

3. Specific immune responses

The specificity of cell-mediated immunity against microorganisms is a function of lymphocytes. CD4⁺ T-cells recognise pathogen antigens in the context of MHC II molecules and accessory costimulatory molecules on APC's, become activated, and, depending on the antigen type, dose, organ in which presentation occurs, and probably most importantly the cytokine milieu in which presentation occurs, differentiate into Th1 or Th2 type cells. Elimination of intracellular pathogens depends classically on a Th1 response. This is characterised by the secretion of IFNγ by T-cells in response to IL-12 and/or IL-18 by the APC, which is then responsible for the activation of macrophages that supposedly kill or at least severely limit pathogen fitness. In addition to macrophage activation, CD4⁺ T-cells also activate CD8⁺ cytotoxic T-cells (CTL's) and B-cells for antibody production.

CD8⁺ T-cells recognise antigen in the context of MHC I molecules and then mediate their function through the production of IFNγ and/or direct cytolytic action mediated by perforins and granzyme. In addition CTL's can target infected cells through Fas mediated apoptosis. CTL's are known to play a role in immunity to R. conorii as resistant mice depleted of this phenotype succumb to infection and adoptive transfer to sensitive mice is protective. Protection against the liver stage sporozoite of Plasmodium also depends on CTL activity. CTL mediated apoptosis or lysis of infected host cells could be critical when Rickettsia infected host macrophages are incapable of eliminating replicating organisms because of their defective activation or overwhelming infection. A major feature of apoptosis of infected cells is the surface expression of specific receptors that allow phagocytes to recognise organisms containing apoptotic bodies thus avoiding spillage of intracellular contents that could cause inflammation or spread of infection.

It is commonly believed that antibodies play no role against intracellular infections and that cell mediated mechanisms are the sole defensive mechanism. This is based on the belief that antibodies cannot enter infected cells. It has however been shown that there is an intracellular-mediated inhibition of listerial growth inside infected macrophages preventing bacterial escape from the phagosome. The anti-listerial monoclonal antibody blocking growth is independent of FcγR expression, IFNγ signalling and the production of NO or ROI but is dependant on intracellular neutralisation by a bacteria specific monoclonal antibody. Antibodies have been shown to be protective in immunodeficient and immunocompetent mice against intracellular infection with E. chaffeensis. The cytophilic complement fixing antibody isotype IgG2a is more effective than IgG3. This isotype is also the consequence of a Th1 type immune bias whereas IgG3 is associated with a Th2 bias.

4. Evasion of specific immune responses by infecting organisms

A strategy commonly adopted by organisms for avoiding detection by specific antibodies is antigenic variation. Good examples of this are seen in African trypanosomes, Giardia and the intraerythrocytic phase of the malaria parasite in which a family of genes (the var genes) have been shown to code for the highly polymorphic Pfemp-1 antigen on the red cell surface.

There is abundant evidence that protozoan parasites can actively regulate adaptive T cell responses resulting in suppressed effector functions. A striking example of this is the recent demonstration that Leishmania major actively induces IL-10 producing CD4⁺/CD25⁺ T regulatory cells to prevent complete clearance of the parasite. In a rodent model of malaria it has been shown that infection results in very specific apoptotic deletion of clone specific anti-malarial CD4⁺ T-cells that leads to significantly impaired immunity to the parasite.

References

1.  Alexander, J., A. R. Satoskar, and D. G. Russell. 1999. Leishmania species: models of intracellular parasitism. J Cell Sci 112 Pt 18:2993-3002.

2.  Ismail, N., J. P. Olano, H. M. Feng, and D. H. Walker. 2002. Current status of immune mechanisms of killing of intracellular microorganisms. FEMS Microbiol Lett 207:111-20.

3.  Sacks, D., and A. Sher. 2002. Evasion of innate immunity by parasitic protozoa. Nat Immunol 3:1041-7.