Cyclosporin A-mediated killing of Leishmania major by macrophages is independent of reactive nitrogen and endogenous TNF-α and is not inhibited by IL-10 and 13

Abstract

Murine macrophages were treated with various doses of cyclosporin A (CsA) to enhance the killing of Leishmania major parasites. CsA reduced the rate of infected cells from 75% in non-treated controls to less than 15% with 1lg CsA/ml in a dose-dependent manner. The leishmanicidal effect was also observed when CsA was added 48 h after the infection of macrophages. In contrast, FK506, another structural non-related immunosuppressive drug with antiparasitic activities, showed no effect on the ability of macrophages to kill intracellular Leishmania parasites. Since nitric oxide has been identified as a key molecule for the leishmanicidal function of macrophages, we analyzed the role of this molecule. There was no influence on the leishmanicidal effect of CsA when L-N-(1-iminoethyl)lysine, a potent and selective inhibitor of mouse inducible nitric oxide synthase, was added. Furthermore, the presence of the macrophage-inhibiting cytokines interleukin (IL)-10 and IL-13 simultaneously or prior to CsA did not inhibit leishmania killing, while both cytokines completely prevented parasite killing by macrophages activated with gamma interferon and tumor necrosis factor (TNF). CsA was fully active on macrophages from TNF-receptor p55 knockout mice arguing against autocrine activation by TNF. We therefore conclude that the antileishmanial effect of CsA is independent of effector mechanisms employed by macrophage-activating cytokines.

Cyclosporin A Treatment of Leishmania donovani Reveals Stage-Specific Functions of Cyclophilins in Parasite Proliferation and Viability

“…To our surprise, unlike CsA, FK506 did not exert a toxic effect on axenic amastigotes at concentrations between 5 and 15 µM (Fig. 6B), a fact previously observed in intracellular L. major amastigotes [57]. These data indicate that the toxic effect of CsA on amastigotes occurs likely through calcineurin-independent mechanisms, which may be directly linked to inhibition of stage-specific enzymatic functions of Leishmania CyPs.…”

“…This anti-parasitic activity has been subsequently confirmed for L. major in mouse and macrophage infection assays, and various modes of CsA action have been proposed [13], [14], [57]. The observation that CsA has no overt anti-microbial activity against L. major promastigotes in culture, but efficiently kills amastigotes in infected macrophages, provided support to the idea that the toxic effect of CsA on intracellular parasites depends on inhibition of host rather than Leishmania CyPs.…”

“…4A). This result demonstrates for the first time that the observed anti-leishmanial effect on intracellular amastigotes in mouse and macrophage infection [13], [14], [57] may rely mainly on direct inhibition of parasite CyPs by CsA, although a participation of host CyPs can not be excluded. We further investigated the mechanisms underlying the stage-specific effects of CsA using the unrelated antifungal macrolide inhibitor FK506.…”

“…In the following, we used the unrelated calcineurin inhibitor FK506 to analyze if the CsA effects on the parasite are mediated through inhibition of this phosphatase, a test that has been previously applied on Leishmania [57]. Log-phase promastigotes and axenic amastigotes (2×10 6 /ml) were cultured for 48 hours in medium containing either ethanol (vehicle) or FK506 at concentrations ranging from 5 to 25 µM, and incubated at 26°C, pH 7.4 (promastigote) or 37°C, pH 5.5 (amastigote).…”

Functional analysis of Leishmania major cyclophilin

“…Cyclosporin A (CsA) has been reported to inhibit infection by the human pathogenic protozoan parasites in vitro and in vivo (Chappell and Wastling, 1992;Meissner et al, 2003;Bua et al, 2004). Since main targets of CsA are cyclophilins, sensitivity of protozoan infection to CsA suggested an important role that cyclophilins may play in protozoan life cycle.…”