Evaluation of the participation of Galectin-1 and Galectin-4 in the outcome of the experimental infection by Leishmania (Leishmania) amazonensis

A leishmaniose é uma doença negligenciada causada por protozoários do gênero Leishmania. Esta doença parasitária é um grave problema de saúde pública, cuja incidência vem crescendo em várias partes do mundo e o seu combate demanda tratamentos farmacológicos mais eficazes e a oferta de vacina. Estudos mostram que a resolução dessa infecção é dependente de uma resposta imunológica homeostática do hospedeiro. No entanto, ainda existem muitas lacunas na compreensão de sua patogênese. O papel das galectinas, proteínas que reconhecem glicanas com motivos ?-galactosídeos, tem sido descrito em várias doenças infecciosas. No entanto, existem poucos relatos sobre a participação de galectinas na leishmaniose. O objetivo deste trabalho foi avaliar o papel das galectinas-1 e -4 na infecção experimental por L. (L) amazonensis. Nós demonstramos que a deficiência do gene da Gal-1 (Gal-1) em camundongos da linhagem BALB/c (BALB/c Lgals1-/-), mas não em camundongos da linhagem C57BL/6, promoveu uma maior restrição da infecção por L. (L.) amazonensis em comparação com o respectivo camundongo selvagem (BALB/c Lgals+/+). Este fenótipo de menor susceptibilidade foi caracterizado pelo aumento da produção de IFN-?, diminuição de IL-4, IL-10, IL-12p70 e TNF-?, redução do tamanho da lesão e menor carga parasitária nos linfonodos drenantes. A Gal-1 não se ligou a Leishmania, sugerindo que a susceptibilidade à leishmaniose detectada nos camundongos BALB/c selvagens não foi devido a uma interação direta entre Gal-1 e L. (L.) amazonensis. Por outro lado, a Gal-4 completa, e não os seus domínios isolados, reconheceu L. (L.) amazonensis de modo sensível à lactose. Ainda, a Gal-4 reconheceu o lipofosfoglicano (LPG) purificado, um glicoconjugado expresso na superfície de celular de Leishmania, independentemente de seu polimorfismo estrutural intraespécie. O pré-tratamento de L. (L.) amazonensis com Gal-4 não alterou a sua internalização por macrófagos obtidos de camundongos BALB/c Lgals+/+ e favoreceu o controle de sua replicação por estes fagócitos. Curiosamente, a injeção intravenosa da forma radiomarcada da Gal-4 causou o acúmulo desta lectina no sítio de infecção por L. (L.) amazonensis em camundongos BALB/c Lgals+/+. Este trabalho foi pioneiro em descrever a participação da Gal-1 e da Gal-4 na resposta imunológica contra L. (L.) amazonensis de modo independente ou dependente de sua interação direta com este parasito, respectivamente. Além disso, estes achados poderão contribuir para o desenvolvimento de novas abordagens terapêuticas e/ou diagnósticas aplicáveis a esta importante doença negligenciada.Leishmaniasis is a neglected disease caused by protozoa of the genus Leishmania. This parasitic disease is a serious health problem with growing incidence in several parts of the world that demands more effective pharmacological treatments and the offer of vaccine in order to fight it. Studies have shown that the resolution of this infection is dependent of homeostatic immune response from the host. However, there are still many gaps in understanding its pathogenesis. The role of galectins, proteins that recognize glycans with beta-galactoside motifs, has been described in several infectious diseases. Nevertheless, there are few reports on the involvement of galectins in Leishmaniasis. The aim of this work is to evaluate the role of galectins 1 and 4 in experimental infection by Leishmania sp. We demonstrated that galectin-1 (Gal-1) deficiency in mice with the BALB/c background (Lgals1-/--BALB/c), whereas not in C57BL/6 mice, promotes better restriction of L. (L.) amazonensis infection compared to wild type BALB/c mice (Lgals1+/+-BALB/c). This outcome was characterized by increased IFN-? production, while decreased for other cytokines (IL-4, IL-10, IL-12p70, and TNF-?), decreased lesion size and parasite load at the draining lymph nodes. Gal-1 did not bind to the parasite, suggesting that the susceptibility to leishmaniasis detected in Lgals1+/+-BALB/c mice was not due to a direct interaction between galectin-1 and Leishmania. On the other side, full length Gal-4, and not their isolated domains, recognizes L. (L.) amazonensis in lactose-sensitive manner. Furthermore, Gal-4 recognizes purified lipophosphoglycan (LPG), a glyconjugate expressed on the cell surface of L. (L.) amazonensis, independently of its intra-species structural polymorphism. The Gal-4 treatment of parasite cells did not change its internalization by non-Gal-1 deficient macrophages and favored the control of intracellular replication of the parasite compared to non-Gal-1 deficient macrophages. Curiously, the radiolabeled Gal-4 intravenous injection in infected Lgals1+/+-BALB/c mice promotes great accumulation of this protein at the parasitic infection site. The data provides the first demonstration of galectin-4 and -1 in the immune response against L. (L.) amazonensis through direct parasite recognition or not, respectively. In addition, this work may open new approaches for therapeutic and/or diagnostic strategies applicable to this important neglected diseas

Interleukin 32γ (IL-32γ) is highly expressed in cutaneous and mucosal lesions of American Tegumentary Leishmaniasis patients: association with tumor necrosis factor (TNF) and IL-10

Submitted by Luciana Ferreira ([email protected]) on 2018-10-26T13:57:10Z No. of bitstreams: 2 Artigo - Hélio Galdino Júnior - 2014.pdf: 1388972 bytes, checksum: a574207e186a1f30c5f815ae5aaebfaf (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)Approved for entry into archive by Luciana Ferreira ([email protected]) on 2018-10-29T11:10:43Z (GMT) No. of bitstreams: 2 Artigo - Hélio Galdino Júnior - 2014.pdf: 1388972 bytes, checksum: a574207e186a1f30c5f815ae5aaebfaf (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)Made available in DSpace on 2018-10-29T11:10:43Z (GMT). No. of bitstreams: 2 Artigo - Hélio Galdino Júnior - 2014.pdf: 1388972 bytes, checksum: a574207e186a1f30c5f815ae5aaebfaf (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014Background: The interleukin 32 (IL-32) is a proinflammatory cytokine produced by immune and non-immune cells. It can be induced during bacterial and viral infections, but its production was never investigated in protozoan infections. American Tegumentary Leishmaniasis (ATL) is caused by Leishmania protozoan leading to cutaneous, nasal or oral lesions. The aim of this study was to evaluate the expression of IL-32 in cutaneous and mucosal lesions as well as in peripheral blood mononuclear cells (PBMC) exposed to Leishmania (Viannia) braziliensis. Methods: IL-32, tumour necrosis factor (TNF) and IL-10 protein expression was evaluated by immunohistochemistry in cutaneous, mucosal lesions and compared to healthy specimens. The isoforms of IL-32α, β, δ, γ mRNA, TNF mRNA and IL-10 mRNA were assessed by qPCR in tissue biopsies of lesions and healthy skin and mucosa. In addition, PBMC from healthy donors were cultured with amastigotes of L. (V.) braziliensis. In lesions, the parasite subgenus was identified by PCR-RFLP. Results: We showed that the mRNA expression of IL-32, in particular IL-32γ was similarly up-regulated in lesions of cutaneous (CL) or mucosal (ML) leishmaniasis patients. IL-32 protein was produced by epithelial, endothelial, mononuclear cells and giant cells. The IL-32 protein expression was associated with TNF in ML but not in CL. IL-32 was not associated with IL-10 in both CL and ML. Expression of TNF mRNA was higher in ML than in CL lesions, however levels of IL-10 mRNA were similar in both clinical forms. In all lesions in which the parasite was detected, L. (Viannia) subgenus was identified. Interestingly, L. (V.) braziliensis induced only IL-32γ mRNA expression in PBMC from healthy individuals. Conclusions: These data suggest that IL-32 plays a major role in the inflammatory process caused by L. (Viannia) sp or that IL-32 is crucial for controlling the L. (Viannia) sp infection