RESUMEN
Envenomation by Loxosceles spiders can result in severe systemic and local reactions, which are mainly triggered by Sphingomyelinase D (SMase D), a toxic component of Loxosceles venom. SMase D induces a systemic inflammatory condition similar to the reaction observed during an endotoxic shock. Considering the potent pro-inflammatory potential of Loxosceles venom and the SMase D, in this study we have used the whole human blood model to study the endotoxic-like shock triggered by SMase D. Recombinant purified SMase D from L. intermedia venom, similarly to LPS, induced activation of blood leukocytes, as observed by the increase in the expression of CD11b and TLR4, production of reactive oxygen and nitrogen species (superoxide anion and peroxynitrite) and release of TNF-α. Complement consumption in the plasma was also detected, and complement inhibition by compstatin decreased the SMase D and LPS-induced leukocyte activation, as demonstrated by a reduction in the expression of CD11b and TLR4 and superoxide anion production. Similar results were found for the L. intermedia venom, except for the production of TNF-α. These findings indicate that SMase D present in Loxosceles venom is able to activate leukocytes in a partially complement-dependent manner, which can contribute to the systemic inflammation that follows envenomation by this spider. Thus, future therapeutic management of systemic Loxosceles envenomation could include the use of complement inhibitors as adjunct therapy.
Asunto(s)
Proteínas del Sistema Complemento/fisiología , Leucocitos/efectos de los fármacos , Hidrolasas Diéster Fosfóricas/farmacología , Venenos de Araña/enzimología , Animales , Granulocitos/efectos de los fármacos , Granulocitos/fisiología , Humanos , Leucocitos/fisiología , Activación de Macrófagos/efectos de los fármacos , Monocitos/efectos de los fármacos , Monocitos/fisiología , Estrés Oxidativo/efectos de los fármacos , Hidrolasas Diéster Fosfóricas/fisiología , Especies Reactivas de Oxígeno/metabolismo , Venenos de Araña/farmacología , ArañasRESUMEN
While C57BL/6 mice infected in the ear with L. major mount a vigorous Th1 response and resolve their lesions, the Th1 response in C57BL/6 mice infected with L. mexicana is more limited, resulting in chronic, non-healing lesions. The aim of this study was to determine if the limited immune response following infection with L. mexicana is related to a deficiency in the ability of monocyte-derived dendritic cells (mo-DCs) to prime a sufficient Th1 response. To address this issue we compared the early immune response following L. mexicana infection with that seen in L. major infected mice. Our data show that fewer monocytes are recruited to the lesions of L. mexicana infected mice as compared to mice infected with L. major. Moreover, monocytes that differentiate into mo-DCs in L. mexicana lesions produced less iNOS and migrated less efficiently to the draining lymph node as compared to those from L. major infected mice. Treatment of L. mexicana infected mice with α-IL-10R antibody resulted in increased recruitment of monocytes to the lesion along with greater production of IFN-γ and iNOS. Additionally, injection of DCs into the ear at the time of infection with L. mexicana also led to a more robust Th1 response. Taken together, these data suggest that during L. mexicana infection reduced recruitment, activation and subsequent migration of monocytes and mo-DCs to the draining lymph nodes may result in the insufficient priming of a Th1 response.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/parasitología , Leishmania mexicana/inmunología , Leishmaniasis Cutánea/inmunología , Monocitos/inmunología , Monocitos/parasitología , Animales , Oído/parasitología , Femenino , Leishmania major/inmunología , Leishmania major/patogenicidad , Leishmania mexicana/patogenicidad , Ratones , Ratones Endogámicos C57BL , Células TH1/inmunologíaRESUMEN
Leishmania major-infected TNF receptor 1 deficient (TNFR1 KO) mice resolve parasitism but fail to resolve lesions, while wild-type mice completely heal. We investigated the cell composition, cytokine production, and apoptosis in lesions from L. major-infected TNFR1 KO and wild-type (WT) mice. Chronic lesions from L. major-infected TNFR1 KO mice presented larger number of CD8+ T and Ly6G+ cells. In addition, higher concentrations of mRNA for IFN-γ CCL2 and CCL5, as well as protein, but lower numbers of apoptotic cells, were found in lesions from TNFR1 KO mice than in WT, at late time points of infection. Our studies showed that persistent lesions in L. major-infected TNFR1 KO mice may be mediated by continuous migration of cells to the site of inflammation due to the presence of chemokines and also by lower levels of apoptosis. We suggest that this model has some striking similarities to the mucocutaneous clinical form of leishmaniasis.
Asunto(s)
Leishmania major/inmunología , Leishmaniasis Cutánea/genética , Leishmaniasis Cutánea/inmunología , Receptores Tipo I de Factores de Necrosis Tumoral/deficiencia , Animales , Apoptosis/inmunología , Citocinas/inmunología , Citocinas/metabolismo , Leishmaniasis Cutánea/parasitología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Carga de Parásitos , Receptores Tipo I de Factores de Necrosis Tumoral/genéticaRESUMEN
Sphingomyelinase D (SMase D) present in the venoms of Loxosceles spiders is the principal component responsible for local and systemic effects observed in the loxoscelism. By using "expressed sequencing tag", it was possible to identify, in a L. laeta venom gland library, clones containing inserts coding for proteins with similarity to SMase D. One of these clones was expressed and the recombinant protein compared with the previously characterized SMase I from L. laeta, in terms of their biological, biochemical and structural properties. The new recombinant protein, SMase II, possesses all the biological properties ascribed to the whole venom and SMase I. SMase II shares 40% and 77% sequence similarity with SMase I and Lb3, respectively; the latter, a SMase D isoform from L. boneti, catalytically inactive. Molecular modeling and molecular dynamics simulations were employed to understand the structural basis, especially the presence of an additional disulfide bridge, in an attempt to account for the observed differences in SMases D activity.