Degranulating Neutrophils Promote Leukotriene B4 Production by Infected Macrophages To Kill Leishmania amazonensis Parasites.

Neutrophils mediate early responses against pathogens, and they become activated during endothelial transmigration toward the inflammatory site. In the current study, human neutrophils were activated in vitro with immobilized extracellular matrix proteins, such as fibronectin (FN), collagen, and laminin. Neutrophil activation by FN, but not other extracellular matrix proteins, induces the release of the granules' contents, measured as matrix metalloproteinase 9 and neutrophil elastase activity in culture supernatant, as well as reactive oxygen species production. Upon contact with Leishmania amazonensis-infected macrophages, these FN-activated neutrophils reduce the parasite burden through a mechanism independent of cell contact. The release of granule proteases, such as myeloperoxidase, neutrophil elastase, and matrix metalloproteinase 9, activates macrophages through TLRs, leading to the production of inflammatory mediators, TNF-α and leukotriene B4 (LTB4), which are involved in parasite killing by infected macrophages. The pharmacological inhibition of degranulation reverted this effect, abolishing LTB4 and TNF production. Together, these results suggest that FN-driven degranulation of neutrophils induces the production of LTB4 and TNF by infected macrophages, leading to the control of Leishmania infection.

Understanding the mechanisms controlling Leishmania amazonensis infection in vitro: the role of LTB4 derived from human neutrophils.

Neutrophils are rapidly recruited to the site of Leishmania infection and play an active role in capturing and killing parasites. They are the main source of leukotriene B4 (LTB4), a potent proinflammatory lipid mediator. However, the role of LTB4 in neutrophil infection by Leishmania amazonensis is not clear. In this study, we show that L. amazonensis or its lipophosphoglycan can induce neutrophil activation, degranulation, and LTB4 production. Using pharmacological inhibitors of leukotriene synthesis, our findings reveal an LTB4-driven autocrine/paracrine regulatory effect. In particular, neutrophil-derived LTB4 controls L. amazonensis killing, degranulation, and reactive oxygen species production. In addition, L. amazonensis infection induces an early increase in Toll-like receptor 2 expression, which facilitates parasite internalization. Nuclear factor kappa B (NFkB) pathway activation represents a required upstream event for L. amazonensis-induced LTB4 synthesis. These leishmanicidal mechanisms mediated by neutrophil-derived LTB4 act through activation of its receptor, B leukotriene receptor 1 (BLT1).

Lutzomyia longipalpis saliva drives apoptosis and enhances parasite burden in neutrophils.

Neutrophils are considered the host's first line of defense against infections and have been implicated in the immunopathogenesis of Leishmaniasis. Leishmania parasites are inoculated alongside vectors' saliva, which is a rich source of pharmacologically active substances that interfere with host immune response. In the present study, we tested the hypothesis that salivary components from Lutzomyia longipalpis, an important vector of visceral Leishmaniasis, enhance neutrophil apoptosis. Murine inflammatory peritoneal neutrophils cultured in the presence of SGS presented increased surface expression of FasL and underwent caspase-dependent and FasL-mediated apoptosis. This proapoptosis effect of SGS on neutrophils was abrogated by pretreatment with protease as well as preincubation with antisaliva antibodies. Furthermore, in the presence of Leishmania chagasi, SGS also increased apoptosis on neutrophils and increased PGE(2) release and decreased ROS production by neutrophils, while enhancing parasite viability inside these cells. The increased parasite burden was abrogated by treatment with z-VAD, a pan caspase inhibitor, and NS-398, a COX-2 inhibitor. In the presence of SGS, Leishmania-infected neutrophils produced higher levels of MCP-1 and attracted a high number of macrophages by chemotaxis in vitro assays. Both of these events were abrogated by pretreatment of neutrophils with bindarit, an inhibitor of CCL2/MCP-1 expression. Taken together, our data support the hypothesis that vector salivary proteins trigger caspase-dependent and FasL-mediated apoptosis, thereby favoring Leishmania survival inside neutrophils, which may represent an important mechanism for the establishment of Leishmania infection.

Interação entre neutrófilos humanos e macrófagos contribui no desfecho da infecção por Leishmania amazonensis / Interaction between human neutrophils and macrophages contributes to the outcome of infection by Leishmania amazonensis

Os neutrófilos são células que atuam na primeira linha de defesa do organismo contra patógenos e estão constitutivamente programadas para a morte celular por apoptose, sendo então, fagocitados por macrófagos. No presente estudo, avaliamos inicialmente o efeito da fagocitose de neutrófilos humanos em repouso: viáveis, apoptóticos ou necróticos, por macrófagos humanos infectados por Leishmania amazonensis. A interação com neutrófilos em repouso apoptóticos, mas não com células viáveis, leva a um aumento na taxa de infecção e carga parasitária em macrófagos. O favorecimento do crescimento intracelular do parasita em presença de neutrófilos apoptóticos foi dependente da produção de TGF-f3 e PG~. Por outro lado, houve uma diminuição da infecção dos macrófagos por L. amazonensis, assim como da carga parasitária, na presença de neutrófilos necróticos. A atividade leishmanicida nessas co-culturas foi dependente da produção de TNF-a, elastase neutrofilica (NE) e superóxido. A presença de neutrófilos ativados viáveis e apoptóticos pelo contato com fibronectina, mas não com outras proteínas de matriz extracelular, também reduz a taxa de infecção e a carga parasitária dos macrófagos infectados com L. amazonensis. O efeito microbicida nas co-culturas com neutrófilos ativados foi independente de contato e envolve a participação de enzimas neutrofilicas NE e MPO, bem como outros mediadores pró-inflamatórios como TNF-a, LTB4 e ROI. Os resultados apresentados neste estudo demonstram que a interação com neutrófilos humanos em repouso ou ativados é capaz de modular a resposta de macrófagos contribuindo para o desfecho da infecção por L. amazonensis.

Neutrophils and macrophages cooperate in host resistance against Leishmania braziliensis infection.

Neutrophils play an active role in the control of infections caused by intracellular pathogens such as Leishmania. In the present study, we investigated the effect of neutrophil depletion at the time of Leishmania braziliensis infection of BALB/c mice and how neutrophils interact with the infected macrophage to promote parasite elimination. The in vivo depletion of neutrophils led to a significant increase in parasite load and enhanced the Th1-Th2 immune response in this experimental model of infection. BALB/c mice coinoculated with both parasites and live neutrophils displayed lower parasite burdens at the site of infection and in the draining lymph nodes. In vitro, we observed that live neutrophils significantly reduced the parasite load in L. braziliensis-infected murine macrophages, an effect not observed with Leishmania major. L. braziliensis elimination was dependent on the interaction between neutrophils and macrophages and was associated with TNF-alpha as well as superoxide production. Furthermore, cooperation between neutrophils and macrophages toward parasite elimination was also observed in experiments performed with L. braziliensis-infected human cells and, importantly, with two other New World Leishmania species. These results indicate that neutrophils play an important and previously unappreciated role in L. braziliensis infection, favoring the induction of a protective immune response.

Interactions with apoptotic but not with necrotic neutrophils increase parasite burden in human macrophages infected with Leishmania amazonensis.

Neutrophils are involved in the initial steps of most responses to pathogens. In the present study, we evaluated the effects of the interaction of apoptotic vs. necrotic human neutrophils on macrophage infection by Leishmania amazonensis. Phagocytosis of apoptotic, but not viable, neutrophils by Leishmania-infected macrophages led to an increase in parasite burden via a mechanism dependent on TGF-beta1 and PGE2. Conversely, infected macrophages' uptake of necrotic neutrophils induced killing of L. amazonensis. Leishmanicidal activity was dependent on TNF-alpha and neutrophilic elastase. Nitric oxide was not involved in the killing of parasites, but the interaction of necrotic neutrophils with infected macrophages resulted in high superoxide production, a process reversed by catalase, an inhibitor of reactive oxygen intermediate production. Initial events after Leishmania infection involve interactions with neutrophils; we demonstrate that phagocytosis of these cells in an apoptotic or necrotic stage can influence the outcome of infection, driving either parasite survival or destruction.

Role of costimulatory molecules in immune response of patients with cutaneous leishmaniasis.

T cell-mediated immunity is critical in resistance against Leishmania parasites, and T cell activation requires signals provided by costimulatory molecules. Herein we evaluated the role of costimulatory molecules on cytokine production and T cell surface molecule expression by peripheral blood mononuclear cells (PBMC) from cutaneous leishmaniasis (CL) patients. PBMC from CL patients were stimulated with soluble Leishmania antigen (SLA, 10 microg/ml), in the presence or absence of soluble CTLA4-Ig to block CD28-B7 interaction or in the presence or absence of anti-human CD40L to block CD40-CD40L interaction. Supernatants were harvested to evaluate tumor necrosis factor alpha (TNF-alpha), interleukin 10 (IL-10), transforming growth factor beta (TGF-beta) and interferon gamma (IFN-gamma) production by ELISA. Cells were harvested after 48 h of culture, stained for specific activation markers and analyzed by flow cytometry. Results show that the blockade of CD28-B7 interaction by CTLA4-Ig downmodulated IFN-gamma, IL-10, and TNF-alpha secretion by PBMC from CL patients. No alteration was detected on either TGF-beta production or the expression of CTLA44 or CD25 on CD4+ and CD8+ T cells. When the CD40-CD40L interaction was blockade using anti-CD40L, we did not observe changes in cytokine production or in surface molecule expression. The blockade of the CD28-B7 interactions by CTLA4-Ig also did not alter cytokine production in volunteers immunized against tetanus toxoid (TT). Taken together, these data suggest that the interaction of CTLA4 and CD28-B7 is a TGF-beta-independent mechanism that specifically downmodulates the immune response in cutaneous leishmaniasis patients.

Lutzomyia longipalpis salivary gland homogenate impairs cytokine production and costimulatory molecule expression on human monocytes and dendritic cells.

In this report, we describe an investigation of the effects of Lutzomyia longipalpis sand fly salivary gland homogenates (SGH) on cytokine production and expression of costimulatory molecules on human monocytes, macrophages (Mphis), and dendritic cells (DCs). SGH of L. longipalpis induced an increase in interleukin-6 (IL-6), IL-8 and IL-12p40 production but a decrease in tumor necrosis factor alpha and IL-10 production by lipopolysaccharida (LPS)-stimulated monocytes. We also examined the expression of costimulatory molecules on the surface of monocytes, Mphis, and DCs. Whereas SGH affected the expression of these molecules on monocytes and Mphis, it had little effect on these molecules on DCs. However, when DCs were generated from human monocytes in the presence of SGH, SGH inhibited the expression of costimulatory molecules. In addition, a decrease in the maturation of DCs induced by CD40L was observed in the presence of SGH. Finally, preincubating SGH with human sera containing anti-SGH-specific antibodies abolished the effects of SGH on cytokine production by LPS-stimulated monocytes.