The parasitic infection mucocutaneous leishmaniasis can vary in severity. It emerges that the levels of an RNA virus within the parasite affect both the host's immune response and the parasite's persistence.
Protozoan parasites of the genus Leishmania are responsible for the infectious disease leishmaniasis. The disease mainly affects those living in tropical and sub-tropical parts of the world, and a unique form of it called mucocutaneous leishmaniasis causes a disfiguring disease in the Americas. Despite the century-old knowledge that Leishmania parasites of the Viannia subgenus cause leishmaniasis, how the disease progresses remains enigmatic. How does a primary cutaneous lesion lead to a secondary, metastasizing form reappearing months later in the nasopharyngeal tissues? Writing in Science, Ives et al.1 shed light on this process, showing that the parasite recruits help from within to counteract the host's immune responses, thus enabling it to spread farther afield.
Mucocutaneous leishmaniasis is associated with a persistent inflammatory response characterized by increased expression of pro-inflammatory mediators (TNF-α and some chemokines)2,3 that play a pivotal part in recruiting immune-system cells such as macrophages to the site of infection. Using previous approaches4,5, Ives et al. isolated clones of Leishmania guyanensis — a parasite of the Viannia subgenus — from infected hamsters.
The clones were either non-metastatic (L.g.M−) or metastatic (L.g.M+), and Ives et al. found that the L.g.M+ clones harboured a virus called Leishmania RNA virus 1 (LRV1). When the authors infected mouse macrophages with these clones, the cells rapidly expressed several cytokines and chemokines (including TNF-α and CXCL10) that are relevant to the pathology of mucocutaneous leishmaniasis. The strongest response was to L.g.M+ clones and to another clone isolated from patients with mucocutaneous leishmaniasis. By contrast, L.g.M− clones and their corresponding human isolates from cutaneous lesions led to only a modest, albeit significant, response compared with uninfected cells; this is consistent with earlier findings comparing different Leishmania species6.
Ives et al.1 show that the ability of the L.g.M+ clones to induce increased expression of pro-inflammatory molecules depends on their internalization into macrophages and their sequestration within phagolysosomes. A phagolysosome is an organelle in which engulfed pathogens can be destroyed; Leishmania, however, has evolved to survive its harsh environment.
An inflammatory immune response is often triggered by the recognition of a pathogen by specific receptors of the innate immune system. One such receptor family comprises the Toll-like receptors (TLRs), which are found on the cell surface or on vesicle membranes of macrophages and other cells of innate immunity. To determine how the inflammatory response to L. guyanensis comes about, Ives and colleagues studied macrophages that were functionally deficient in the various phagosomal TLRs (TLR3, 7 and 9) or in intracellular adaptors related to TLR signalling (MyD88 and TRIF).
They found that TLR3 and TRIF are essential for maximal production not just of pro-inflammatory mediators induced by L.g.M+, but also of IFN-β — a cytokine produced in response to TLR3 activation and which potentially can cause organ damage. Intriguingly, previous work on virus-free Leishmania species suggested that IFN-β7 and TLR38 could be involved, respectively, in the progression of leishmaniasis and in the parasite's recognition by IFN-γ-primed macrophages. Those studies did not, however, address the question of how the parasite modulates the host's inflammatory response.
Ives et al. find that, in metastasizing L.g.M+, LRV1 is responsible for the TLR3-dependent macrophage activation. TLR3, TLR7 and TLR9 recognize pathogenic nucleic-acid motifs, including double-stranded RNA (dsRNA). Indeed, purified dsRNA from LRV1 was as powerful as metastasizing parasites in inducing secretion of pro-inflammatory mediators, and this effect was completely abrogated in TLR3-deficient macrophages (Fig. 1).
This result is the cornerstone of the authors' study, revealing for the first time how a virus within a pathogen can modify its pathological development within the host. Nonetheless, although the authors report that TLR3-deficient mice have a lower parasite burden and make a reduced inflammatory response (as evidenced by a decreased response in the footpad-swelling test compared with wild-type animals), they found no significant difference in the development of mucocutaneous leishmaniasis.
A disadvantage of studying mucocutaneous leishmaniasis in mice is that these animals do not develop the nasopharyngeal pathology. It would be better to investigate the effects of TLR3 deficiency in hamsters to determine how L.g.M+ migrates from a primary lesion to the nasopharyngeal tissues to cause inflammation-mediated tissue destruction.
Ives et al.1 hypothesize that, early in the course of an infection, LRV1 dsRNA is released from dead parasites that could not survive within macrophages. Could this be so? The same team reported previously9 that L.g.M+ parasites are more resistant to reactive oxygen species and nitric oxide, which would make them better at resisting the harsh environment inside macrophage phagosomes compared with parasites that are not infected with viruses. This could be why, despite L.g.M+ parasites inducing a stronger immune response, L.g.M+-infected animals develop greater footpad swelling and a higher parasite burden.
Another question is whether the aflagellate intracellular form of the Leishmania parasite, which can migrate to the host's nasopharyngeal tissues, is as effective at inducing an inflammatory response as its predecessor flagellate form, which is inoculated into the host by the sand-fly vector. Without knowledge of the cellular and molecular mechanisms underlying the tropism of L.g.M+ parasites, how mucocutaneous leishmaniasis progresses remains a mystery. Nevertheless, Ives and co-workers' paper1 is important for providing the first evidence of how a 'pathogen' within a pathogen can modify the course of an infection.
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