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Bridging the gap between innate and adaptive immunity in dog leishmaniasis
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A leishmaniose é um grupo de doenças causadas por diferentes espécies de Leishmania que afecta maioritariamente populações pobres de países subtropicais e tropicais e também animais selvagens e domésticos, como o cão. A leishmaniose canina (CanL) é uma doença endémica de preocupação mundial causada principalmente pelo protozoário L. infantum . O cão é um bom reservatório de L. infantum, uma vez que mantém a infeção durante muito tempo antes de desenvolver a doença facilitando a transmissão do parasita. Além disso, pensa-se que o cão também pode ser o reservatório de espécies americanas de Leishmania, como é o caso de L. amazonensis. A imunidade inata que é a primeira linha de defesa contra agentes patogénicos inclui células fagócitárias e células Natural Killer (NK). As células dendríticas (CD) têm a principal função de capturar agentes patogénicos e processar antigénios, desempenhando um papel crucial na proliferação e activação de células T, estabelecendo assim uma ponte entre a imunidade inata e a adquirida. As células NK influenciam o processo infeccioso através do reconhecimento e eventual destruição de células infectadas e da libertação de mediadores imunitários celulares que promovem microambientes inflamatórios. Considerando a reduzida informação existente sobre o papel das CD e das células NK durante a infeção por Leishmania, este estudo visou analisar in vitro a atividade das CD derivadas do sangue periférico (moCDs) canino e também abordar o papel das NK na resposta imunitária celular e a sua inter-relação com as moCDs. Após confirmação da diferenciação in vitro, as moCDs foram infectadas com L. infantum e L. amazonensis, e também expostas a vesículas extracelulares (EVs) libertadas por L. infantum (LiEVs) e L. amazonensis (LaEVs ). A infecção por L. infantum aumentou a expressão génica do receptor toll-like (TLR) 4 das moCDs em sinergia com a activação e translocação do factor nuclear (NF)-B para o núcleo e subsequente geração de citocinas pró-inflamatórias [interleucina (IL)-1β e IL-18]. Este parasita também induziu o predomínio da subpopulação de moCDs expressando moléculas de classe I do complexo maior de histocompatibilidade (MHCI) e aumentou a molécula de co-estimulação CD86 que, juntamente com a libertação da quimiocina CXCL16, poderá atrair e activar linfócitos T citotóxicos (CD8+) que podem causar a apoptose das células infetadas. Em contraste, L. amazonensis parece induzir moCDs anergicos, indicando que estes parasitas estabelecem diferentes relações imunitárias com as CDs, o que pode ser uma consequência da co-evolução parasita-hospedeiro. Os moCDs infectados com parasitas em co-cultura com células NK parecem promover a predominio da subpopulação de moDCs expressando moléculas de classe II do MHC, sugerindo a possibilidade de apresentação de antigénios às células T CD4+. Além disso, apesar da desgranulação das células NK e da libertação de perforina, não houve aumento da apoptose dos moCDs, e os parasitas internalizados pelos moCDs mostraram-se viáveis. Quando expostos a EVs, os moDCs também mostram diferenças na actividade imunitária. As LaEVs parecem sinalizar moCDs através de TLR2, causando a upregulação de CD80/CD86 e LiEVs promovem a expansão de moCDs MHCI+ aumentam a expressão génica de CD86. Ambas as EVs induziram a libertação de CXCL16, atraindo leucocitos. Os moCDs estimuladas por EVs induziram as células NK a gerar quimiocinas, atraindo outros leucócitos e promovendo a desgranulação que, no entanto, não induziam a apoptose dos moCDs. Quando em contacto com as células NK, os moDCs com LiEVs provocaram a expansão das subpopulações de MHCI+ e MHCII+ moCDs, enquanto as moCDs expostos a LaEVs mostraram aumento de moDCs MHCI+.Estes resultados indicam que as EVs podem modular a atividade imunitária das DC caninas. L.infantum e L.amazonensis, assim como as respetivas EVs, modulam a ativação das CD caninas, embora de formas diferentes. A compreensão detalhada das vias de ativação das CDs assim como a interação com as NK pode conduzir ao desenvolvimento de novas estratégias de controlo da leishmaniose.
Leishmaniasis is a group of diseases caused by different species of Leishmania that af-flicts poor populations of subtropical and tropical low-income countries and causes dis-ease in wild and domestic animals, such as the domestic dog. Canine leishmaniasis (CanL) is an endemic disease of worldwide concern caused mainly by the protozoan L. infantum. The dog is a good reservoir of L. infantum since keeps the infection for a long time before developing the disease, facilitating parasite transmission. Furthermore, there is a growing belief that dogs also can be the reservoir of the American species of Leish-mania, as is the case of L. amazonensis. Innate immunity, the first line of defence against pathogens, includes phagocytes and natural killer (NK) cells. Dendritic cells (DC) have the principal function of capturing pathogens and processing antigens, playing a crucial role in the proliferation and activation of undifferentiated T cells, thereby establishing a bridge between innate and acquired immunity. NK cells influence the infectious process through the recognition and eventual destruction of infected cells and the release of cel-lular immune mediators that promote inflammatory microenvironments. Since there is limited information on the role of DCs and NK cells during Leishmania infection, this study aimed to analyze in vitro the activity of peripheral blood-derived DCs (moDCs) and tackle the NK role in the cellular immune response and its interrelation with moDCs. After confirmation of in vitro differentiation of moDCs cells were infected with L. infan-tum and L. amazonensis, and also exposed to extracellular vesicles (EVs) released by L. infantum (LiEVs) and L. amazonensis (EVs). The activity of moDCs and the interaction between moDCs and NK cells were analyzed. L. infantum infection increased the expres-sion of toll-like receptor (TLR) 4 genes of moDCs in synergy with the activation and translocation of nuclear factor (NF)-B to the nucleus and subsequent generation of pro-inflammatory cytokines [interleukin (IL)-1β and IL-18]. This parasite also induced the predominance of moDCs expressing class I molecules major histocompatibility complex (MHCI) and upregulated the co-stimulatory molecule CD86 that, together with the release of the chemokine CXCL16, could attract and activate cytotoxic T lymphocytes (CD8+) that can cause the apoptosis of infected cells. In contrast, L. amazonensis seems to induce anergic moDCs, indicating that these parasites establish different immune relationships with DCs, which may be a consequence of parasite-dog co-evolution. Parasite infected moDCs in co-culture with NK cells seem to promote the predominance of MHCII+ moDCs, suggesting the possibility of antigen presentation to CD4+ T cells. Furthermore, despite the degranulation of NK cells and perforin release, there was no increase in moDCs apoptosis, and the parasites internalized by the moDCs remained viable. When exposed to parasite EVs, moDCs also show different immune activity. LaEVs appear to signalize moDCs through TLR2, causing the upregulation of CD80/CD86 and LiEVs pro-moted the expansion of MHCI+ moDCs and upregulation of CD86. Both EVs induced the release of CXCL16. EVs-primed moDCs induced NK cells to generate chemokines, at-tracting other leukocytes and promoting degranulation that did not increase moDCs apop-tosis. When in contact with NK cells, LiEVs-primed moDCs caused the expansion of MHCI+ and MHCII+ moDC subsets, while moDCs exposed to LaEVs only showed in the expansion of MHCI+ moDCs. These findings indicate that EVs can modulate the immune activity of canine DCs. Therefore, L.infantum and L.amazonensis, as well as the respec-tive EVs, modulate the activation of canine DCs, albeit in different ways. A detailed un-derstanding of DC activation pathways and the interaction with NK cells can open new windows on leishmaniasis control strategies.
Leishmaniasis is a group of diseases caused by different species of Leishmania that af-flicts poor populations of subtropical and tropical low-income countries and causes dis-ease in wild and domestic animals, such as the domestic dog. Canine leishmaniasis (CanL) is an endemic disease of worldwide concern caused mainly by the protozoan L. infantum. The dog is a good reservoir of L. infantum since keeps the infection for a long time before developing the disease, facilitating parasite transmission. Furthermore, there is a growing belief that dogs also can be the reservoir of the American species of Leish-mania, as is the case of L. amazonensis. Innate immunity, the first line of defence against pathogens, includes phagocytes and natural killer (NK) cells. Dendritic cells (DC) have the principal function of capturing pathogens and processing antigens, playing a crucial role in the proliferation and activation of undifferentiated T cells, thereby establishing a bridge between innate and acquired immunity. NK cells influence the infectious process through the recognition and eventual destruction of infected cells and the release of cel-lular immune mediators that promote inflammatory microenvironments. Since there is limited information on the role of DCs and NK cells during Leishmania infection, this study aimed to analyze in vitro the activity of peripheral blood-derived DCs (moDCs) and tackle the NK role in the cellular immune response and its interrelation with moDCs. After confirmation of in vitro differentiation of moDCs cells were infected with L. infan-tum and L. amazonensis, and also exposed to extracellular vesicles (EVs) released by L. infantum (LiEVs) and L. amazonensis (EVs). The activity of moDCs and the interaction between moDCs and NK cells were analyzed. L. infantum infection increased the expres-sion of toll-like receptor (TLR) 4 genes of moDCs in synergy with the activation and translocation of nuclear factor (NF)-B to the nucleus and subsequent generation of pro-inflammatory cytokines [interleukin (IL)-1β and IL-18]. This parasite also induced the predominance of moDCs expressing class I molecules major histocompatibility complex (MHCI) and upregulated the co-stimulatory molecule CD86 that, together with the release of the chemokine CXCL16, could attract and activate cytotoxic T lymphocytes (CD8+) that can cause the apoptosis of infected cells. In contrast, L. amazonensis seems to induce anergic moDCs, indicating that these parasites establish different immune relationships with DCs, which may be a consequence of parasite-dog co-evolution. Parasite infected moDCs in co-culture with NK cells seem to promote the predominance of MHCII+ moDCs, suggesting the possibility of antigen presentation to CD4+ T cells. Furthermore, despite the degranulation of NK cells and perforin release, there was no increase in moDCs apoptosis, and the parasites internalized by the moDCs remained viable. When exposed to parasite EVs, moDCs also show different immune activity. LaEVs appear to signalize moDCs through TLR2, causing the upregulation of CD80/CD86 and LiEVs pro-moted the expansion of MHCI+ moDCs and upregulation of CD86. Both EVs induced the release of CXCL16. EVs-primed moDCs induced NK cells to generate chemokines, at-tracting other leukocytes and promoting degranulation that did not increase moDCs apop-tosis. When in contact with NK cells, LiEVs-primed moDCs caused the expansion of MHCI+ and MHCII+ moDC subsets, while moDCs exposed to LaEVs only showed in the expansion of MHCI+ moDCs. These findings indicate that EVs can modulate the immune activity of canine DCs. Therefore, L.infantum and L.amazonensis, as well as the respec-tive EVs, modulate the activation of canine DCs, albeit in different ways. A detailed un-derstanding of DC activation pathways and the interaction with NK cells can open new windows on leishmaniasis control strategies.
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Palavras-chave
Parasitologia Leishmaniose canina Células dendríficas Células NK