TGF-β is a potent regulatory cytokine that suppresses expression of inducible NO synthase and IFN-γ, and suppresses Th1 and Th2 cell development. We examined whether functionally active TGF-β is present in the local environment surrounding the invading protozoan Leishmania chagasi. Our prior data showed that TGF-β levels are significantly increased in L. chagasi-infected mice. In the current study, we found TGF-β was also abundant in bone marrows of humans with acute visceral leishmaniasis but not in those of uninfected controls. Furthermore, L. chagasi infection caused an increase in biologically active TGF-β in human macrophage cultures without changing the total TGF-β. Therefore, we investigated the means through which leishmania could augment activated but not total TGF-β. Incubation of latent TGF-β with Leishmania sp. promastigotes caused active TGF-β to be released from the latent complex. In contrast, the nonpathogenic protozoan Crithidia fasciculata could not activate TGF-β. TGF-β activation by leishmania was prevented by inhibitors of cysteine proteases and by the specific cathepsin B inhibitor CA074. Physiologic concentrations of TGF-β inhibited killing of intracellular L. chagasi in macrophages, although the phagocytosis-induced respiratory burst remained intact. In contrast, supraphysiologic concentrations of TGF-β had no effect on parasite survival. We hypothesize that the combined effect of abundant TGF-β stores at extracellular sites during infection, and the ability of the parasite to activate TGF-β in its local environment, leads to high levels of active TGF-β in the vicinity of the infected macrophage. Locally activated TGF-β could, in turn, enhance parasite survival through its effects on innate and adaptive immune responses.

SummaryCaveolae are membrane microdomains enriched in cholesterol, ganglioside M1 (GM1) and caveolin-1. We explored whether caveolae facilitate the entry of Leishmania chagasi into murine macrophages. Transient depletion of macrophage membrane cholesterol by 1 h exposure to methyl-b -cyclodextrin (M b CD) impaired the phagocytosis of non-opsonized and serum-opsonized virulent L. chagasi . In contrast, M b CD did not affect the phagocytosis of opsonized attenuated L. chagasi . As early as 5 min after phagocytosis, virulent L. chagasi colocalized with the caveolae markers GM1 and caveolin-1, and colocalization continued for over 48 h. We explored the kinetics of lysosome fusion. Whereas fluorescent-labelled dextran entered macrophage lysosomes by 30 min after addition, localization of L. chagasi in lysosomes was delayed for 24-48 h after phagocytosis. However, after transient depletion of cholesterol from macrophage membrane with M b CD, the proportion of L. chagasi -containing phagosomes that fused with lysosomes increased significantly. Furthermore, intracellular replication was impaired in parasites entering after transient cholesterol depletion, even though lipid microdomains were restored by 4 h after treatment. These observations suggest that virulent L. chagasi localize in caveolae during phagocytosis by host macrophages, and that cholesterol-containing macrophage membrane domains, such as caveolae, target parasites to a pathway that promotes delay of lysosome fusion and intracellular survival.

Lipid bodies (LB; lipid droplets) are cytoplasmic organelles involved in lipid metabolism. Mammalian LBs display an important role in host-pathogen interactions, but the role of parasite LBs in biosynthesis of prostaglandin F2α (PGF2α) has not been investigated. We report herein that LBs increased in abundance during development of Leishmania infantum chagasi to a virulent metacyclic stage, as did the expression of PGF2α synthase (PGFS). The amount of parasite LBs and PGF2α were modulated by exogenous arachidonic acid. During macrophage infection, LBs were restricted to parasites inside the parasitophorous vacuoles (PV). We detected PGF2α receptor (FP) on the Leishmania PV surface. The blockage of FP with AL8810, a selective antagonist, hampered Leishmania infection, whereas the irreversible inhibition of cyclooxygenase with aspirin increased the parasite burden. These data demonstrate novel functions for parasite-derived LBs and PGF2α in the cellular metabolism of Leishmania and its evasion of the host immune response.

Leishmania spp. are protozoans that survive and replicate intracellularly in mammalian macrophages. Antileishmanial immunity requires gamma interferon (IFN-␥)-mediated macrophage activation and generation of microbicidal effector molecules. The presence of intracellular Leishmania sp. impairs macrophage responses to IFN-␥, which has led to the description of macrophages as deactivated. It has recently become apparent that in addition to classical activation, macrophages can be activated by distinct triggers to express noninflammatory or anti-inflammatory genes. These nonclassical activation programs have been called alternative or type II pathways. We hypothesized that during initial contact with a phagocyte, leishmaniae activate one of these nonclassical pathways, resulting in expression of genes whose products suppress microbicidal responses. Using DNA microarrays, we studied gene expression in RNAs from BALB/c bone marrow macrophages with and without Leishmania chagasi infection. Some changes were verified by an RNase protection assay, reverse transcription-PCR, immunoblotting, or a bioassay. The pattern of genes activated by leishmania phagocytosis differed from the pattern of genes activated by bacteria or lipopolysaccharide and IFN-␥. Genes encoding some proinflammatory cytokines, receptors, and Th1-type immune response genes were downmodulated, and some genes associated with anti-inflammatory or Th2-like immune responses were up-regulated. Nonetheless, some markers of alternative (arginase) or type II activation (interleukin-10, tumor necrosis factor alpha) were unchanged. These data suggest that macrophages infected with L. chagasi exhibit a hybrid activation profile that is more characteristic of alternative or type II activation than of classical activation but does not strictly fall into either of these categories. We speculate that the pattern of genes upregulated by leishmania phagocytosis optimizes the chance of parasite survival in this hostile environment.Leishmania spp. are parasitic protozoans with a two life stages that correspond to the transit of the organisms between two hosts. The extracellular promastigote is found in the gut of the sand fly vector, and the obligate intracellular amastigote resides in the mammalian host macrophage. During a blood meal the sand fly inoculates the promastigote into a pool of blood in the skin, where it is phagocytosed by a resident mononuclear phagocyte. Inside the macrophage the parasite changes to an amastigote, which is the obligate intracellular form and the only form of the parasite found in a mammalian host. Intracellular parasites spread to other mononuclear