Inhibition of Lipopolysaccharide-Induced Macrophage IL-12 Production byLeishmania mexicanaAmastigotes: The Role of Cysteine Peptidases and the NF-κB Signaling Pathway

Abstract

Infection with lesion-derived Leishmania mexicana amastigotes inhibited LPS-induced IL-12 production by mouse bone marrow-derived macrophages. This effect was associated with expression of cysteine peptidase B (CPB) because amastigotes of CPB deletion mutants had limited ability to inhibit IL-12 production, whereas preincubation of cells with a CPB inhibitor, cathepsin inhibitor IV, was able to suppress the effect of wild-type amastigotes. Infection with wild-type amastigotes resulted in a time-dependent proteolytic degradation of IκBα and IκBβ and the related protein NF-κB. This effect did not occur with amastigotes of CPB deletion mutants or wild-type promastigotes, which do not express detectable CPB. NF-κB DNA binding was also inhibited by amastigote infection, although nuclear translocation of cleaved fragments of p65 NF-κB was still observed. Cysteine peptidase inhibitors prevented IκBα, IκBβ, and NF-κB degradation induced by amastigotes, and recombinant CPB2.8, an amastigote-specific isoenzyme of CPB, was shown to degrade GST-IκBα in vitro. LPS-mediated IκBα and IκBβ degradation was not affected by these inhibitors, confirming that the site of degradation of IκBα, IκBβ, and NF-κB by the amastigotes was not receptor-driven, proteosomal-mediated cleavage. Infection of bone marrow macrophages with amastigotes resulted in cleavage of JNK and ERK, but not p38 MAPK, whereas preincubation with a cysteine peptidase inhibitor prevented degradation of these proteins, but did not result in enhanced protein kinase activation. Collectively, our results suggest that the amastigote-specific cysteine peptidases of L. mexicana are central to the ability of the parasite to modulate signaling via NF-κB and consequently inhibit IL-12 production.

Down-regulation of dendritic cell signaling pathways by Leishmania amazonensis amastigotes

“…Whether La infection triggers the activation of a host cellular phosphatase(s) awaits further investigation. Together with previous findings of the cysteine peptidase-dependent degradation of MHC class II and IκB molecules in La-or L. mexicana-infected MΦs (Cameron et al, 2004;De Souza Leao et al, 1995), we conclude that parasite-derived protease/peptidase or proteosome may contribute to their virulence, as well as disease progression, by disrupting the early signaling events crucial for DC activation.…”

“…Cameron et al found that the inhibition of LPS-induced IL-12 production in L. mexicana amastigote-infected MΦs was due to the degradation of IκB and NF-κB by amastigotes-derived cysteine peptidase B (Cameron et al, 2004). Another study showed that L. major amastigotes selectively inhibited p50/p65 translocation, but induced p50/c-Rel complex formation, which was correlated to the production of TNF-α and IL-10 from human monocytes (Guizani-Tabbane et al, 2004).…”

“…5D). The lack of inhibitory effect for E64 on live amastigotes was probably due to its non-permeablility to live cells (Cameron et al, 2004). Pretreatment of live amastigotes with a phosphatase inhibitor (bpV) had no demonstrable effects on parasite-induced reduction of p-STATs, suggesting a negligible role for parasite phosphatase(s) in de-phosphorylation of tested STATs (data not shown).…”

Leishmania, the phagosome, and host responses: The journey of a parasite

“…Another pathogenicity factor, the cysteine proteinase B (CPB), contributes to L. mexicana virulence as knock-out parasite induced a protective Th1 response and poor lesion growth in BALB/c mice [48]. L. mexicana CPB was also shown to alter macrophages signaling and functions through the alteration of NF-B, STAT-1, and AP-1 which inhibits IFN- responses, production of nitric oxide, and lipopolysaccharide-induced macrophages IL-12 production [49,50]. Recently, our group reported that L. mexicana CPB regulates virulence through the control of GP63 expression.…”

“…More recently, GP63 was shown to target the nuclear envelop, where it degrades nucleoporins of the nuclear pore complexes [59]. These signaling and transcription modifications were proposed to inhibit host responses associated to the control of infection, such as the release of TNF, IL-12, and NO, which will contribute to the survival of the parasite and facilitate its replication [50,55,60]. In contrast, our group recently described a role for GP63 in inducing cytokine release, whereby GP63 cleaves synaptotagmin XI, a SNARE present on recycling endosomes and lysosomes and that negatively regulatesthe secretion of preformed stored TNF and IL-6 [61].…”