The role of Leishmania RNA vírus in the modulation of the innate imune response

Author(s)

Renan Villanova Homem de Carvalho

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2019-09-20

Examining board members

Dario Simões Zamboni; José Carlos Farias Alves Filho; Daniel Youssef Bargieri; Marcelo Torres Bozza

• Advisor: Dario Simões Zamboni

Abstract

Parasites from Leishmania genus are the causative agents of Leishmaniasis, an important human disease. It is estimated that over 12 million people are infected worldwide, and 350 million are under risk of acquiring the disease. The cutaneous form is the mildest type of the disease, which may evolve to a devastating outcome, named mucocutaneous leishmaniasis, in where parasites may methastize and reach nasopharingeal tissues. In order to do so, parasites must subvert several imune functions displayed by the host to efficiently establish their replication and dissemination. Leishmania braziliensis and Leishmania guyanensis are some species that may be found variably infected by the Leishmania RNA virus (LRV) in nature, and its presence has been associated with the development of mucocutaneous disease in patients. However, the mechanisms by which LRV enhances disease severity remain largely unknown. Macrophages are the main cell type in which the parasites replicate, where they negatively modulate several innate immune signaling pathways. Among them, we highlight the cytosolic sensor NLRP3, which has recently been shown to be play an important role in parasite control. This sensor is able to sense cellular damage and oligomerize to form a cytoplasmic platform known as the inflammasome, composed by NLRP3, ASC (the adaptor molecule) and Caspase-1. NLRP3 may be activated via the canonical pathway, through reactive oxygen species and potassium efflux, or via the non-canonical pathway, which happens via cytosolic detection of bacterial LPS via caspase- 11. In the latter case, the role of caspase-11 in the detection of parasitic infections, such as Leishmania, is still unknown. In the current study, we evaluated the possible role of LRV in the modulation of the innate immune response, with special focus given to NLRP3. By analyzing lesion aspirates obtained from patients displaying either cutaneous or mucocutaneous disease, we observed a positive correlation between the presence of LRV and the likelihood of developing the most severe form of the disease, while virus presence correlated with decreased activation of the inflammasome, suggesting that LRV could interfere with NLRP3 activation by the parasite. To better investigate this hypothesis, we generated a LRV negative clone by the M4147 L. guyanensis strain, which harbors high levels of the virus. Through a combination of experiments performed both in mouse and human macrophages, we found that LRV is found within extracelular vesicles secreted by the parasite, which binds to TLR3 via its double-stranded RNA, recruiting TRIF and driving type I IFN production. This cytokine induces autophagy in an autocrine or paracrine manner, which degrades NLRP3 and ASC in the cytoplasm, then favouring parasite replication in vitro and in vivo. Moreover, we show that LRV is capable of blocking caspase-11 and the noncanonical pathway induced by the parasites. Taken together, our results demonstrate an evasion mechanism triggered by LRV in the context of Leishmania infection, favouring parasite replication and the severity of the disease. This work unravels several molecular targets with potential for pharmacological intervention, aiming the treatment and prevention of the devastating disease displayed by people suffering from mucocutaneous Leishmaniasis. (AU)

• FAPESP's process: 15/25404-2 - Role of the Leishmania RNA virus (LRV1-4) present in Leishmania guyanensis M4147 strain in the modulation of the innate immune response
• Grantee: Renan Villanova Homem de Carvalho
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)