Atividade oxidativa e não oxidativa de células fagocitárias expostas a protozoários do género Leishmania

As leishmanioses, causadas por protozoários do género Leishmania, são um problema de saúde pública e veterinária. No homem, a doença classifica-se clinicamente em leishmaniose cutânea, visceral e mucocutânea. A leishmaniose cutânea pode ser produzida por L. amazonensis, L. shawi e L. guyanensis, entre muitas outras espécies. A leishmaniose visceral zoonótica causada por L. infantum apresenta manifestações clínicas graves que podem ser fatais. Neutrófilos ou células polimorfonucleares (PMN) têm função crucial na imunidade inata, sendo as primeiras a ser recrutadas para o local de infeção. Os monócitos/macrófagos (MФ) desempenham o duplo papel de serem células fagocíticas apresentadoras profissionais de antigénios e as hospedeiras por excelência de Leishmania. O presente trabalho teve como objetivo analisar a atividade leishmanicida de neutrófilos de murganhos BALB/c e de MФ (linha celular P388D1 derivada de murganhos) quando expostos in vitro a espécies de Leishmania do subgénero Leishmania (L. infantum e L. amazonensis) e Viannia (L. shawi e L. guyanensis) através da avaliação da (i) expressão dos sensores celulares NOD1, NOD2, TLR2, TLR4 e TLR9 por real time PCR, (ii) ativação dos mecanismos oxidativos (superóxido nos neutrófilos e óxido nítrico e ureia nos MФ), (iii) importância dos mecanismos enzimáticos e (iv) produção de NET por neutrófilos. Estudo idêntico foi realizado em neutrófilos e MФ de canídeos infetados in vitro com L. infantum. Neutrófilos de murganhos internalizam parasitas dos subgéneros Leishmania e Viannia. Os mecanismos oxidativos e enzimáticos são ativados e geradas NET, contribuindo para o controlo da infeção. No entanto, o contato com os parasitas não promove a expressão génica dos sensores celulares. MФ P388D1 fagocitam as diferentes espécies de Leishmania, porém não ocorre ativação da via clássica, mas sim da via alternativa, assegurando a sobrevivência intracelular do parasita. Nestas células, a exposição às diferentes espécies de Leishmania conduziu a aumentos pontuais da expressão génica de NOD1 e TLR2 e também de TLR9, com exceção de L. shawi. No entanto, as espécies do subgénero Viannia induziram aumento da expressão génica de NOD2. Em conjunto, estes resultados sugerem que cada espécie elabora estratégias próprias de ativação dos sensores celulares. Os neutrófilos caninos também internalizaram L. infantum, ativaram os mecanismos oxidativos e produziram NET capazes de aprisionar extracelularmente promastigotas. Porém, apenas ocorreu exocitose da elastase neutrofilica sugerindo que este parasita restringe a actividade enzimática de neutrófilos. MФ caninos infetados por L. infantum ativaram a via alternativa e apresentaram unicamente expressão aumentada de TLR2, o que desencadeia a ativação dos mecanismos oxidativos e produção de citocinas pró-inflamatórias. Este estudo contribuiu para clarificar o efeito da infeção por espécies cutâneas e viscerais de Leishmania na ativação dos mecanismos oxidativos e não oxidativos das células fagocitárias de canídeos e modelo roedor. Foi demonstrado pela primeira vez que a infeção por Leishmania spp. na célula hospedeira está intimamente associada ao aumento da expressão de TLR2 e, consequentemente, à provável ativação desta via metabólica. A compreensão dos fatores que inibem ou estimulam os sensores celulares de imunidade inata, cruciais no reconhecimento do parasita, pode ser importante no desenvolvimento de novas estratégias terapêuticas para a leishmaniose.Caused by protozoa of the genus Leishmania, leishmaniases, are considered an important public health issue and a veterinary disease. In humans, the disease can be clinically classified into cutaneous, visceral and mucocutaneous leishmaniasis. Cutaneous leishmaniasis can be produced by L. amazonensis, L. shawi and L. guyanensis among many others species. The zoonotic visceral leishmaniasis caused by L. infantum presents severe clinical manifestations that can be fatal if left untreated. Neutrophils or polymorphonuclear cells (PMN) play a crucial role in innate immunity, being the first cells to be recruited to the site of infection. Monocytes/macrophages (MФ) play the dual role of being phagocytic and antigenic presenting cells and the definitive host cells of Leishmania parasite. This study was designed to investigate the leishmanicidal activity of neutrophils isolated from BALB/c mice and of MФ (cell line P388D1 differentiated from mice) exposed in vitro to two species of subgenus Leishmania (L. amazonensis and L. infantum) and two species of subgenus Viannia (L. shawi and L. guyanensis) by evaluating (i) gene expression of cell sensors NOD1, NOD2, TLR2, TLR4 and TLR9 through real time PCR, (ii) the activation of oxidative mechanisms (superoxide by neutrophils and nitric oxide and urea by MФ), (iii) the importance of enzymatic mechanisms and (iv) the production of NET by neutrophils. Similar studies were carried out in dog’ neutrophils and MФ infected in vitro by L. infantum. Murine neutrophils were able to internalize parasites of Leishmania and Viannia subgenera. Oxidative and enzymatic mechanisms were activated and NET were generated, leading to control of infection. However, the parasite did not induced the gene expression of cell sensors. MФ P388D1 phagocytosed the different species of Leishmania although without activating the macrophage classical pathway. Instead, parasites activated the MФ alternative pathway, ensuring intracellular survival. In such cells, the exposition to the different species of Leishmania leaded to transient increase NOD1 and TLR2 gene expression and, also of TLR9 with the exception of L. shawi. However, only the species of the subgenus Viannia caused the increase of NOD2 gene expression. Taken together, these results suggest that each parasite species develop their own strategy to promote or, by he contrary to avoid the activation of cell sensors. Dog neutrophils also internalized L. infantum parasites, activatedoxidative mechanisms and generated NET able to ambush extracellular promastigotes. However, only occurs exocytosis of neutrophilic elastase, suggesting that this parasite restricts the enzymatic activity of neutrophils. Dog MФ L. infantum-infected activated the alternative pathway and enhanced the gene expression of TLR2, stimulating oxidative mechanisms and the production of pro-inflammatory cytokines. This study sheeds light on the effect of cutaneous and visceral Leishmania parasites in the activation of oxidative and non-oxidative mechanisms of mice and dog phagocytic cells. It is also demonstrated for the first time that the establishment of infection by Leishmania spp. in the definitive host cell is closely associated with the increase of TLR2 gene expression and likely, by the activation of this metabolic pathway. Understanding the factors that inhibit or, by the contrary, stimulate innate cell sensors, which are crucial for parasite recognition, might be important in the development of new therapeutic strategies for leishmaniasis

the complexity of host's effective immune response against a polymorphic parasitic disease

This review is aimed at providing a comprehensive outline of the immune response displayed against cutaneous leishmaniasis (CL), the more common zoonotic infection caused by protozoan parasites of the genus Leishmania. Although of polymorphic clinical presentation, classically CL is characterized by leishmaniotic lesions on the face and extremities of the patients, which can be ulcerative, and even after healing can lead to permanent injuries and disfigurement, affecting significantly their psychological, social, and economic well-being. According a report released by the World Health Organization, the disability-adjusted life years (DALYs) lost due to leishmaniasis are close to 2.4 million, annually there are 1.0-1.5 million new cases of CL, and a numerous population is at risk in the endemic areas. Despite its increasing worldwide incidence, it is one of the so-called neglected tropical diseases. Furthermore, this review provides an overview of the existing knowledge of the host innate and acquired immune response to cutaneous species of Leishmania. The use of animal models and of in vitro studies has improved the understanding of parasite-host interplay and the complexity of immune mechanisms involved. The importance of diagnosis accuracy associated with effective patient management in CL reduction is highlighted. However, the multiple factors involved in CL epizoology associated with the unavailability of vaccines or drugs to prevent infection make difficult to formulate an effective strategy for CL control.publishersversionpublishe

from in uterus to elderly

Immune system recognize and fight back foreign microorganisms and inner modifications that lead to deficient cell and tissue functions. During a dog's life, the immune system needs to adapt to different physiological conditions, assuring surveillance and protection in a careful and controlled way. Pregnancy alters normal homeostasis, requiring a balance between immunity and tolerance. The embryos and fetus should be protected from infections, while the female dog must tolerate the growing of semi-allografts in her uterus. After birth, newborn puppies are at great risk of developing infectious diseases, because their immune system is in development and immune memory is absent. Passive transfer of immunity through colostrum is fundamental for puppy survival in the first weeks of life, but hampers the development of an active immune response to vaccination. At the end of life, dogs experience a decline in the structure and functional competence of the immune system, compromising the immune responses to novel antigenic challenges, such as infections and vaccines. Therefore, the current article reviews the general processes related to the development of the dog´s immune system, providing an overview of immune activity throughout the dog's life and its implications in canine health, and highlighting priority research goals.publishersversionpublishe

New Insights on Innate Immune Response by Blood Macrophages and Liver Kupffer Cells to Leishmania infantum Parasites

Funding Information: Funding: This study was supported by FCT-Foundation for Science and Technology, I.P., through research grant PTDC/CVT-CVT/28908/2017 and PTDC/CVT-CVT/0228/2020, and by national funds within the scope of Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA, UIDB/00276/2020) and Global Health and Tropical Medicine (GHTM, UID/04413/2020). Publisher Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland.L. infantum is the aetiological agent of zoonotic visceral leishmaniasis (ZVL), a disease that affects humans and dogs. Leishmania parasites are well adapted to aggressive conditions inside the phagolysosome and can control the immune activation of macrophages (MØs). Although MØs are highly active phagocytic cells with the capacity to destroy pathogens, they additionally comprise the host cells for Leishmania infection, replication, and stable establishment in the mammal host. The present study compares, for the first time, the innate immune response to L. infantum infection of two different macrophage lineages: the blood macrophages and the liver macrophages (Kupffer cells, KC). Our findings showed that L. infantum takes advantage of the natural predisposition of blood-MØs to phagocyte pathogens. However, parasites rapidly subvert the mechanisms of MØs immune activation. On the other hand, KCs, which are primed for immune tolerance, are not extensively activated and can overcome the dormancy induced by the parasite, exhibiting a selection of immune mechanisms, such as extracellular trap formation. Altogether, KCs reveal a different pattern of response in contrast with blood-MØs when confronting L. infantum parasites. In addition, KCs response appears to be more efficient in managing parasite infection, thus contributing to the ability of the liver to naturally restrain Leishmania dissemination.publishersversionpublishe

Insights on Host–Parasite Immunomodulation Mediated by Extracellular Vesicles of Cutaneous Leishmania shawi and Leishmania guyanensis

Funding Information: This study was supported by FCT-Foundation for Science and Technology, I.P., through research grant PTDC/CVT-CVT/28908/2017 and PTDC/CVT-CVT/0228/2020 and by national funds within the scope of Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA, UIDB/00276/2020) and Global Health and Tropical Medicine (GHTM, UID/04413/2020). Publisher Copyright: © 2023 by the authors.Leishmaniasis is a parasitic disease caused by different species of Leishmania and transmitted through the bite of sand flies vector. Macrophages (MΦ), the target cells of Leishmania parasites, are phagocytes that play a crucial role in the innate immune microbial defense and are antigen-presenting cells driving the activation of the acquired immune response. Exploring parasite–host communication may be key in restraining parasite dissemination in the host. Extracellular vesicles (EVs) constitute a group of heterogenous cell-derived membranous structures, naturally produced by all cells and with immunomodulatory potential over target cells. This study examined the immunogenic potential of EVs shed by L. shawi and L. guyanensis in MΦ activation by analyzing the dynamics of major histocompatibility complex (MHC), innate immune receptors, and cytokine generation. L. shawi and L. guyanensis EVs were incorporated by MΦ and modulated innate immune receptors, indicating that EVs cargo can be recognized by MΦ sensors. Moreover, EVs induced MΦ to generate a mix of pro- and anti-inflammatory cytokines and favored the expression of MHCI molecules, suggesting that EVs antigens can be present to T cells, activating the acquired immune response of the host. Since nano-sized vesicles can be used as vehicles of immune mediators or immunomodulatory drugs, parasitic EVs can be exploited by bioengineering approaches for the development of efficient prophylactic or therapeutic tools for leishmaniasis.publishersversionpublishe

Extracellular vesicles shed by trypanosoma brucei brucei manipulate host mononuclear cells

Funding Information: Funding: This study was supported by FCT—Foundation for Science and Technology, I.P., through research grant PTDC/CVT-CVT/28908/2017 and by national funds within the scope of Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA, UIDB/00276/2020) and Global Health and Tropical Medicine (GHTM, UID/04413/2020). Funding Information: This study was supported by FCT?Foundation for Science and Technology, I.P., through research grant PTDC/CVT-CVT/28908/2017 and by national funds within the scope of Centro de Investiga??o Interdisciplinar em Sanidade Animal (CIISA, UIDB/00276/2020) and Global Health and Tropical Medicine (GHTM, UID/04413/2020). Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.African trypanosomiasis or sleeping sickness is a zoonotic disease caused by Trypanosoma brucei, a protozoan parasite transmitted by Glossina spp. (tsetse fly). Parasite introduction into mammal hosts triggers a succession of events, involving both innate and adaptive immunity. Macrophages (MΦ) have a key role in innate defence since they are antigen-presenting cells and have a micro-bicidal function essential for trypanosome clearance. Adaptive immune defence is carried out by lymphocytes, especially by T cells that promote an integrated immune response. Like mammal cells, T. b. brucei parasites release extracellular vesicles (TbEVs), which carry macromolecules that can be transferred to host cells, transmitting biological information able to manipulate cell immune response. However, the exact role of TbEVs in host immune response remains poorly understood. Thus, the current study examined the effect elicited by TbEVs on MΦ and T lymphocytes. A combined approach of microscopy, nanoparticle tracking analysis, multiparametric flow cytometry, colourimetric assays and detailed statistical analyses were used to evaluate the influence of TbEVs in mouse mononuclear cells. It was shown that TbEVs can establish direct communication with cells of innate and adaptative immunity. TbEVs induce the differentiation of both M1-and M2-MΦ and elicit the expansion of MHCI+, MHCII+ and MHCI+ MHCII+ MΦ subpopulations. In T lymphocytes, TbEVs drive the overexpression of cell-surface CD3 and the nuclear factor FoxP3, which lead to the differentiation of regulatory CD4+ and CD8+ T cells. Moreover, this study indicates that T. b. brucei and TbEVs seem to display opposite but complementary effects in the host, establishing a balance between parasite growth and controlled immune response, at least during the early phase of infection.publishersversionpublishe

immune activation of canine hepatic spheroids exposed to leishmania infantum

The application of innovative three-dimensional (3D) spheroids cell culture strategy to Parasitology offers the opportunity to closely explore host–parasite interactions. Here we present a first report on the application of 3D hepatic spheroids to unravel the immune response of canine hepatocytes exposed to Leishmania infantum. The liver, usually considered a major metabolic organ, also performs several important immunological functions and constitutes a target organ for L. infantum infection, the etiological agent of canine leishmaniasis (CanL), and a parasitic disease of major veterinary and public health concern. 3D hepatic spheroids were able to sense and immunologically react to L. infantum parasites, generating an innate immune response by increasing nitric oxide (NO) production and enhancing toll-like receptor (TLR) 2 and interleukin-10 gene expression. The immune response orchestrated by canine hepatocytes also lead to the impairment of several cytochrome P450 (CYP450) with possible implications for liver natural xenobiotic metabolization capacity. The application of meglumine antimoniate (MgA) increased the inflammatory response of 3D hepatic spheroids by inducing the expression of Nucleotide oligomerization domain (NOD)-like receptors 1 and NOD2 and TLR2, TLR4, and TLR9 and enhancing gene expression of tumour necrosis factor α. It is therefore suggested that hepatocytes are key effector cells and can activate and orchestrate the immune response to L. infantum parasites.publishersversionpublishe

Bridging the gap between innate and adaptive immunity in dog leishmaniasis

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

Development of Dog Immune System: From in Uterus to Elderly

Immune system recognize and fight back foreign microorganisms and inner modifications that lead to deficient cell and tissue functions. During a dog’s life, the immune system needs to adapt to different physiological conditions, assuring surveillance and protection in a careful and controlled way. Pregnancy alters normal homeostasis, requiring a balance between immunity and tolerance. The embryos and fetus should be protected from infections, while the female dog must tolerate the growing of semi-allografts in her uterus. After birth, newborn puppies are at great risk of developing infectious diseases, because their immune system is in development and immune memory is absent. Passive transfer of immunity through colostrum is fundamental for puppy survival in the first weeks of life, but hampers the development of an active immune response to vaccination. At the end of life, dogs experience a decline in the structure and functional competence of the immune system, compromising the immune responses to novel antigenic challenges, such as infections and vaccines. Therefore, the current article reviews the general processes related to the development of the dog´s immune system, providing an overview of immune activity throughout the dog’s life and its implications in canine health, and highlighting priority research goals

Exploiting <i>Leishmania</i>—Primed Dendritic Cells as Potential Immunomodulators of Canine Immune Response

Dendritic cells (DCs) capture pathogens and process antigens, playing a crucial role in activating naïve T cells, bridging the gap between innate and acquired immunity. However, little is known about DC activation when facing Leishmania parasites. Thus, this study investigates in vitro activity of canine peripheral blood-derived DCs (moDCs) exposed to L. infantum and L. amazonensis parasites and their extracellular vesicles (EVs). L. infantum increased toll-like receptor 4 gene expression in synergy with nuclear factor κB activation and the generation of pro-inflammatory cytokines. This parasite also induced the expression of class II molecules of major histocompatibility complex (MHC) and upregulated co-stimulatory molecule CD86, which, together with the release of chemokine CXCL16, can attract and help in T lymphocyte activation. In contrast, L. amazonensis induced moDCs to generate a mix of pro- and anti-inflammatory cytokines, indicating that this parasite can establish a different immune relationship with DCs. EVs promoted moDCs to express class I MHC associated with the upregulation of co-stimulatory molecules and the release of CXCL16, suggesting that EVs can modulate moDCs to attract cytotoxic CD8+ T cells. Thus, these parasites and their EVs can shape DC activation. A detailed understanding of DC activation may open new avenues for the development of advanced leishmaniasis control strategies