The Immunobiology of Leishmania Infections
Leishmaniasis is a disease that demonstrates the complex and highly specialised interactions that result from the co evolution of parasite and host. The Leishmania spp. have evolved not only to avoid destruction by the sophisticated mammalian immune system, but to exploit its mechanisms to ensure survival and reproductive success. A Protozoan of 12 pathogenic species, Leishmania is endemic to tropical and subtropical regions as well as parts of Southern Europe (Herwaldt 1999). Carried by a Sandfly vector, it enters the skin as a promastigote to differentiate into a non-flagellated amastigote in the host cells (Handman & Bullen 2002). It causes disease at varying degrees of severity from relatively mild (cutaneous) to seriously debilitating (visceral and mucosal) (Herwaldt 1999) and with 12 million people currently infected through out the world (WHO Leishmaniasis), represents a significant health problem. Many investigations have been carried out, particularly using lab strains of mice, to help elucidate the immunobiology of this disease.
Upon entry into the skin following a sandfly bite, the Promastigote Leishmania evades lysis by the complement system in the blood due to the presence of various surface molecules. An important one is the Lishmanial Protein Kinase 1 (LPK-1) that phosphorylates several subunits of the complement structure (Hermoso et al 1991). These were shown to be the alpha chains on C3 and C5 in humans and the 71 KDa part of the C9 protein (Hermoso et al 1991). One of the most important complement components is the C3 protein, as the parasite disables its antimicrobial function and uses it to gain entry into host macrophages (Mosser & Edelson 1987). LPK-1 appears to phosphorylate a serine residue at position 71 of the C3a protein, which is close to the active site, to disrupt the complement cascade (Hermoso et al 1991). Another surface structure, gp63, degrades the C3b component to iC3b so that the Membrane Attack Complex (MAC) can no longer be activated and to serve as a marker for phagocytosis (Bittingham & Mosser 1996). In addition, lipophosphoglycans (LPGs) also on the promastigote surface can become elongated to form a protective layer against MAC to avoid lysis (Bittingham & Mosser 1996).
Leishmania infect leukocytes, predominantly macrophages, to evade host defences and reproduce. In a study by Akarid et al (2004), it was shown that the presence of at least 10 intracellular promastigotes in bone marrow derived macrophages (BMDMs) was enough to prevent apoptosis of cells in C57BL/6 mice (Akarid et al 2004). These cells were able to survive at least four hours when exposed to the apoptosis inducing factor staurosporine, probably due to the inhibition of cytochrome c release into the cytosol from the cell's mitochondria (Akarid et al 2004). The amastigotes are able to reproduce in the phagolysosomes of the macrophage, until they reach such a number that the cell either bursts open, releasing the progeny, or possibly leak out gradually into the blood (Handman & Bullen 2002). The increase in the number of macrophages in response to infection provides a large number of macrophage precursors in BALB/c mice, which L. major is able to infect (Mirkovich 1986). The pathways necessary to kill foreign bodies are immature in these cells, allowing the parasites to exist without immune challenge in these so called ‘safe targets' (Mirkovich 1986). Neutrophils are the initial, transient host of Leishmania as they are the first leukocytes to migrate into the infection site (Ritter 2009 & van Zandbergen 2004). The high levels of MIP-1β secreted by parasite containing neutrophils, cause macrophages to phagocytose the neutrophil, resulting in the subsequent infection of the macrophage (van Zandbergen 2004). This is referred to as the ‘Trojan horse' model by Ritter (2009).
A number of surface structures on the Leishmania promastigote and host macrophage interact in a receptor-ligand fashion (Wilson et al 1988) to cause the phagocytosis of the parasite. The Mannose Fructose Receptor (MFR) on the macrophage membrane associates with mannose, fructose and N-acetyl-glucosamine-terminal glycoconjugates on the promastigote membrane to trigger endocytosis (Wilson et al 1988, Guy & Belosevic 1993). Inhibition of these interactions has been shown to prevent phagocytosis in murine macrophages (Wilson et al 1988). The Fc receptor on the macrophage is another example of the receptor-ligand relationship, as it binds to IgG1 on opsonised amastigotes in BALB/c mice (less common antibodies include IgG2b and IgM) to induce phagocytosis (Guy & Belosevic 1993). The complement breakdown product iC3b binds to LPG and gp63 on the surface of the promastigote and functions as an opsonising product (Peters et al 1995 & Wilson et al 1988). This then binds to the complement receptor 3 (CR3) on the macrophage to induce phagocytosis (Peters et al 1995 & Wilson et al 1988). This is assisted by MAC-1 on the macrophage which stabilises the iC3b-CR3 interaction (Brittingham& Mosser 1996). There is also evidence that CR3 and MFR may modulate each other during the phagocytic process (Wilson 1988). Other important receptors include that for fibronectin and advanced glycosylation end products (AGE) (Guy & Belosevic 1993).
Leishmania parasites are able to survive intracellularly by inhibiting the production of oxygen metabolites produce by the antimicrobial respiratory burst (RB) in macrophages. Two inducers of the RB are PLS and macrophage activating factor (MAF). Buchmuller-Rouiller and Mauel showed that mouse peritoneal macrophages infected with L. enriettii and L. major produced a significantly reduced RB when exposed to PLS and MAF, due to the presence of the parasites (Buchmuller-Rouiller & Mauel 1987). The amastigotes produce surface molecules and enzymes that make them much better adapted to intracellular survival than their promastigotes precursors (Buchmuller-Rouiller & Mauel 1987). They possess a coat of GPI-anchored proteophosphoglycans and LPGs, the latter being the most abundant surface macromolecule (Ilgoutz & McConville 2001). The lipids are proposed by Buchmuller-Rouiller to detoxify hydrogen peroxide (H2O2) as they are used in peroxidation reactions that would otherwise damage the amastigote (Buchmuller-Rouiller & Mauel 1987). Scavenging enzymes such as catalase and glutathionine perioxidase are also utilised by amastigotes to neutralise H2O2 and superoxide intracellularly (Buchmuller-Rouiller & Mauel 1987). An investigation by Scott (1985) showed that IC-21 macrophages deficient in the RB pathway could use a non-oxidative mechanism to kill intracellular amastigotes but required the RB to kill log growth phase promastigotes, demonstrating the importance of RB in effective removal Leishmanial infections (Scott 1985).
The intracellular parasites are able to control whole immunobiological pathways by manipulating signalling cytokines. Arguably the most important of these is TGF-β, which is activated (inactive form secreted by B and T cells (Barral-Netto 1992)) by macrophages infected with L. donovani and L. brasiliensis to inhibit activation of antimicrobial pathways (Barral et al 1995). It has been shown that parasite numbers increased by ~50% in cells where TGF-β was added to the culture compared to those that had no additional TGF-β (Barral et al 1995). BALB/c mice are susceptible to Leishmania infection as they produce a predominantly TH2 response to L. major infection (Barral-Netto 1992). This is due to the production of IL-4 by CD4+ T cells in response to the infected macrophages (Launois 1997) producing the active TGF-β. Further regulation by TGF-β involves the inhibition of the antimicrobial effecter iNOS (Stenger 1994). Few parasites have been found in areas high in iNOS, so its inhibition allows Leishmania to survive in its host (Stenger 1994). TGF-β is able to neutralise IFN-γ, a key cytokine causing Leishmania resistance, to allow infection to become established (Barral-Netto 1992). In C57BL/6 mice, IL-12 is produced by macrophages to cause the TH1 response (Reiner 1994). This increases IFN-γ production, resulting in no TH2 response and the resistance of macrophages to Leishmania (Reiner 1994). However, Barral et al showed that even at high concentrations of IFN-γ (100 IU/ml) the number of intracellular parasites increased due to the presence of TGF-β (Barral et al 1995), indicating a possible dominance of TGF-β.
The intracellular parasites disrupt antigen presentation by macrophages and other antigen presenting cells (APCs), to reduce the activation of T cells (Kima 1996). When infected with L. major, it was shown that macrophages couldn't present processing independent antigen sequences (Chakroborty 2005), indicating the disruption of much of the antigen processing mechanism. This is believed to be due in part to the disruption of lipid rafts, microdomains rich in cholesterol that support MHC structures on the plasma membrane (Chakroborty 2005). It is believed that the parasites mediate this by increasing the membrane fluidity (Chakroborty 2005), so destabilising the base for the rafts. Kima (1996) believes antigen is sequestered from the MHC class II pathway by using parasitophorous vacuoles (Kima 1996). The two processes probably work together to prevent antigen reaching the macrophage membrane, the result being a lack of antigen exposure to CD4+ T cells and the subsequent lack of adaptive immune response to Leishmania.
The pathology of Leishmaniasis is the result of parasites overcoming a number of immunological mechanisms. The evolution of a complex immune system, such as that of the mammalian host, to protect against a vast number and variety of pathogenic organisms, opens up niches for exploitation by the coevolution of parasites. Although humans are an incidental host of Leishmania, infection can be persistent, disabling and bring attached social stigma. The growing reservoir of knowledge about Leishmania infections will hopefully bring us closer to the first vaccine against a human parasite as we disrupt its manipulation of host defences. This may be more attainable with the use of improved lab strains that are more representative of human or even wild type organisms that exist in situ; the BALB/c and C57BL/6 models are highly inbred so lack wild type genetic diversity. The problem of Leishmaniasis is exacerbated by the immunodeficiency caused by AIDS in parts of the world where the two coexist, making the problem likely to worsen if this disease continues to be neglected.
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