Lee et al. demonstrate that mannose receptor–mediated infection of M2-like dermal macrophages plays a critical role in nonhealing cutaneous infection by L. major. The dermal macrophages are radio resistant and self-renewed and efficiently maintain their M2 phenotype during Th1 immunity.

Functions 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, treated in vitro or in vivo with 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 in Xenopus oocytes 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)-1b release. Colchicine treatment did not affect ATP-evoked currents in macrophages. Finally, in vivo assays with mice inoculated with lipopolysaccharide and ATP showed diminished ROS, IL-1b, interferon-g 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 visit http://dx.doi.org/10.1111/ j. 1476-5381.2011.01325.x Abbreviations DAPI, 4′,6-diamidino-2-phenylindole; DCFH2DA, dichlorodihydrofluorescein diacetate; DMEM, Dulbecco's modified Eagle's medium; EB, ethidium bromide; IFN-g, interferon-g; IL-1b, interleukin-1b; LPS, lipopolysaccharide; PGP, P-glycoprotein; PMA, phorbol 12-myristate 13-acetate; ROS, reactive oxygen species BJP British Journal of Pharmacology

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.

Tissue-resident macrophages (TRMs) maintain tissue homeostasis, but they can also provide a replicative niche for intracellular pathogens such as Leishmania. How dermal TRMs proliferate and maintain their M2 properties even in the strong TH1 environment of the L. major infected dermis is not clear. Here, we show that, in infected mice lacking IL-4/13 from eosinophils, dermal TRMs shifted to a proinflammatory state, their numbers declined, and disease was attenuated. Intravital microscopy revealed a rapid infiltration of eosinophils followed by their tight interaction with dermal TRMs. IL-4–stimulated dermal TRMs, in concert with IL-10, produced a large amount of CCL24, which functioned to amplify eosinophil influx and their interaction with dermal TRMs. An intraperitoneal helminth infection model also demonstrated a requirement for eosinophil-derived IL-4 to maintain tissue macrophages through a CCL24-mediated amplification loop. CCL24 secretion was confined to resident macrophages in other tissues, implicating eosinophil-TRM cooperative interactions in diverse inflammatory settings.

SummaryNucleotides are released into the extracellular milieu from infected cells and cells at inflammatory sites. The extracellular nucleotides bind to specific purinergic (P2) receptors and thereby induce a variety of cellular responses including antiparasitic effects. Here we investigated whether extracellular nucleotides affect leishmanial infection in macrophages, and found that UTP reduces strongly the parasite load in peritoneal macrophages. Ultrastructural analysis of infected cells revealed that UTP induced morphological damage in the intracellular parasites. Uridine nucleotides also induced dose-dependent apoptosis of macrophages and production of ROI and RNI only in infected macrophages. The intracellular calcium measurements of infected cells showed that the response to UTP, but not UDP, increased the sensitivity and amplitude of cytosolic Ca 2+ changes. Infection of macrophages with Leishmania upregulated the expression of P2Y2 and P2Y4 receptor mRNA. The data suggest indirectly that Leishmania amazonensis infection induces modulation and heteromerization of P2Y receptors on macrophages. Thus UTP modulates the host response against L. amazonensis infection. UTP and UTP homologues should therefore be considered as novel components of therapeutic strategies against cutaneous leishmaniasis.

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 B 4 (LTB 4 ) production and release. Therefore, we investigated whether L . amazonensis elimination by P2X7 receptor and LTB 4 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 LTB 4 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 LTB 4 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 LTB 4 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 LTB 4 was dependent on non-canonical NLRP3 inflammasome activation, ROS production, and IL-1β signaling.

Leishmania and other parasites display several mechanisms to subvert host immune cell function in order to achieve successful infection. The ATP receptor P2X7, an agonist-gated cation channel widely expressed in macrophages and other cells of the immune system, is also coupled to inflammasome activation, IL-1 beta secretion, production of reactive oxygen species, cell death and the induction of the permeabilization of the plasma membrane to molecules of up to 900 Da. P2X7 receptors can function as an effective microbicidal triggering receptor in macrophages infected with several microorganisms including Mycobacteria tuberculosis, Chlamydia and Leishmania. We have previously shown that its expression is up-regulated in macrophages infected with L.amazonensis and that infected cells also display an increase in P2X7-induced apoptosis and membrane permeabilization to some anionic fluorescent dyes. In an independent study we recently showed that the phenomenon of macrophage membrane permeabilization can involve at least two distinct pathways for cations and anions respectively. Here, we re-addressed the effects of ATP-induced P2X7-associated phenomena in macrophages infected with L.amazonensis and demonstrated that the P2X7-associated dye uptake mechanisms are differentially modulated. While the membrane permeabilization for anionic dyes is up-modulated, as previously described, the uptake of cationic dyes is strongly down-modulated. These results unveil new characteristics of two distinct permeabilization mechanisms associated with P2X7 receptors in macrophages and provide the first evidence indicating that these pathways can be differentially modulated in an immunologically relevant situation. The possible importance of these results to the L.amazonensis escape mechanism is discussed.