BACKGROUND AND PURPOSEThe two longest C‐termini of the purinergic P2X receptors occur in the P2X2 and P2X7 receptors and are thought to interact with multiple cytoplasmic proteins, among which are members of the cytoskeleton, including microtubules. In this work we asked whether disrupting the microtubule cytoskeleton might affect the functions of these receptors.EXPERIMENTAL APPROACHFunctions of heterologously expressed P2X2 and P2X7 receptors were evaluated with electrophysiology and dye uptake following ATP application. Permeabilization and secretion of pro‐inflammatory agents were quantified from fresh or cultured peritoneal mouse macrophages, treatedin vitroorin vivowith colchicine.KEY RESULTSDisrupting the microtubule network with colchicine did not affect currents generated by ATP in P2X2 and P2X7 receptor‐expressing cells but inhibited uptake of the dye Yo‐Pro‐1 inXenopusoocytes and HEK293 cells expressing these channels. Peritoneal mouse macrophages showed less ATP‐induced permeabilization to ethidium bromide in the presence of colchicine, and less reactive oxygen species (ROS) formation, nitric oxide (NO) and interleukin (IL)‐1β release. Colchicine treatment did not affect ATP‐evoked currents in macrophages. Finally,in vivoassays with mice inoculated with lipopolysaccharide and ATP showed diminished ROS, IL‐1β, interferon‐γ and NO production after colchicine treatment.CONCLUSIONS AND IMPLICATIONSColchicine has known anti‐inflammatory actions and is used to treat several conditions involving innate immunity, including gout and familial Mediterranean fever. Here we propose a new mechanism of action – inhibition of pore formation induced by activation of P2X receptors – which could explain some of the anti‐inflammatory effects of colchicine.LINKED ARTICLEThis article is commented on by Pelegrín, pp. 908–911 of this issue. To view this commentary visithttp://dx.doi.org/10.1111/j.1476‐5381.2011.01325.x

The origin and functional specialization of dermal macrophages in cutaneous infections have been little studied. In this paper, we show that a strain of Leishmania major (L. major Seidman [LmSd]) that produces nonhealing cutaneous lesions in conventionally resistant C57BL/6 mice was more efficiently taken up by M2-polarized bone marrow (BM)–derived macrophages (BMDMs) in vitro and by mannose receptor (MR)^hi dermal macrophages in vivo compared with a healing strain (L. major Friedlin V1). Both in steady and in T helper type 1 (Th1) cell–driven inflammatory states, the MR^hi dermal macrophages showed M2 characteristics. The dermal macrophages were radio resistant and not replaced by monocytes or adult BM-derived cells during infection, but were locally maintained by IL-4 and IL-10. Notably, the favored infection of M2 BMDMs by LmSd in vitro was MR dependent, and genetic deletion of MR or selective depletion of MR^hi dermal macrophages by anti–CSF-1 receptor antibody reversed the nonhealing phenotype. We conclude that embryonic-derived, MR^hi dermal macrophages are permissive for parasite growth even in a strong Th1-immune environment, and the preferential infection of these cells plays a crucial role in the severity of cutaneous disease.

IL-4/CCL24–mediated interaction with eosinophils maintains dermis-resident macrophages as replicative niches for Leishmania major .

There is substantial experimental evidence to indicate that Leishmania infections that are transmitted naturally by the bites of infected sand flies differ in fundamental ways from those initiated by needle inocula. We have used flow cytometry and intravital microscopy (IVM) to reveal the heterogeneity of sand fly transmission sites with respect to the subsets of phagocytes in the skin that harbor L. major within the first hours and days after infection. By flow cytometry analysis, dermis resident macrophages (TRMs) were on average the predominant infected cell type at 1 hr and 24 hr. By confocal IVM, the co-localization of L. major and neutrophils varied depending on the proximity of deposited parasites to the presumed site of vascular damage, defined by the highly localized swarming of neutrophils. Some of the dermal TRMs could be visualized acquiring their infections via transfer from or efferocytosis of parasitized neutrophils, providing direct evidence for the “Trojan Horse” model. The role of neutrophil engulfment by dermal TRMs and the involvement of the Tyro3/Axl/Mertk family of receptor tyrosine kinases in these interactions and in sustaining the anti-inflammatory program of dermal TRMs was supported by the effects observed in neutrophil depleted and in Axl^-/-Mertk^-/- mice. The Axl^-/-Mertk^-/- mice also displayed reduced parasite burdens but more severe pathology following L. major infection transmitted by sand fly bite.

ATP is an important signaling molecule in the immune system, and it is able to bind the P2X7 purinergic receptor. Recently, our group showed that ATP-treated macrophages eliminate Leishmania amazonensis. It has been reported that leukotriene B4 (LTB4) reduces the parasitic load of infected macrophages. Additionally, it has been demonstrated that the P2X7 receptor can induce PLA2 activation and arachidonic acid mobilization. Based on these findings, we investigated whether LTB4 is produced upon P2X7 receptor activation and examined whether LTB4 modulates parasite elimination. Using macrophages lacking the P2X7 receptor, we observed that ATP was not able to reduce L. amazonensis load. This result suggests a role of the P2X7 purinergic receptor in parasite elimination. In addition, ATP was sufficient to induce LTB4 release from infected control macrophages but not from macrophages lacking the P2X7 receptor. Moreover, we found that ATP failed to decrease the parasitic load in 5-lipoxygenase (LO)–deficient macrophages. Treatment with the 5-LO inhibitor AA861 also impairs the ATP effect on parasitic loads. Furthermore, macrophages from 5-LO knockout mice eliminated L. amazonensis in the presence of exogenous LTB4, and macrophages obtained from P2X7 receptor knockout mice eliminated L. amazonensis when incubated with ionomycin. Finally, we demonstrated that in the presence of CP105696, an antagonist for LTB4 high-affinity receptor, ATP was not able to reduce parasitic load. These results indicate that P2X7 receptor activation leads to LTB4 formation, which is required for L. amazonensis elimination.

Leishmaniasis is a neglected tropical disease affecting millions of individuals worldwide. P2X7 receptor has been linked to the elimination of Leishmania amazonensis. Biological responses evoked by P2X7 receptor activation have been well-documented, including apoptosis, phagocytosis, cytokine release, such as IL-1β. It was demonstrated that NLRP3 inflammasome activation and IL-1β signaling participated in resistance against L. amazonensis. Furthermore, our group has shown that L. amazonensis elimination through P2X7 receptor activation depended on leukotriene B₄ (LTB₄) production and release. Therefore, we investigated whether L. amazonensis elimination by P2X7 receptor and LTB₄ involved NLRP3 inflammasome activation and IL-1β signaling. We showed that macrophages from NLRP3^-/-, ASC^-/-, Casp-1/11^-/-, gp91^{phox-/- ,} and IL-1R^-/- mice treated with ATP or LTB₄ did not decrease parasitic load as was observed in WT mice. When ASC^-/- macrophages were treated with exogenous IL-1β, parasite killing was noted, however, we did not see parasitic load reduction in IL-1R^-/- macrophages. Similarly, macrophages from P2X7 receptor-deficient mice treated with IL-1β also showed decreased parasitic load. In addition, when we infected Casp-11^-/- macrophages, neither ATP nor LTB₄ were able to reduce parasitic load, and Casp-11^-/- mice were more susceptible to L. amazonensis infection than were WT mice. Furthermore, P2X7^-/- L. amazonensis-infected mice locally treated with exogenous LTB₄ showed resistance to infection, characterized by lower parasite load and smaller lesions compared to untreated P2X7^-/- mice. A similar observation was noted when infected P2X7^-/- mice were treated with IL-1β, i.e., lower parasite load and smaller lesions compared to P2X7^-/- mice. These data suggested that L. amazonensis elimination mediated by P2X7 receptor and LTB₄ was dependent on non-canonical NLRP3 inflammasome activation, ROS production, and IL-1β signaling. Leishmania spp. is a protozoan parasite that infects human and causes several diseases. Leishmania amazonensis causes cutaneous leishmaniasis (CL) and mucocutaneous leishmaniasis (MCL). Leishmania parasites preferentially infect macrophages. In macrophages, several mechanisms have been described as controlling L. amazonensis infection. Here, we showed that P2X7 receptor and LTB₄ eliminated L. amazonensis in macrophages by a pathway dependent on non-canonical NLRP3 inflammasome activation and IL-1β signaling.