The human parasite Leishmania amazonensis downregulates iNOS expression via NF-κB p50/p50 homodimer: role of the PI3K/Akt pathway

Abstract

Leishmania amazonensis activates the NF-κB transcriptional repressor homodimer (p50/p50) and promotes nitric oxide synthase (iNOS) downregulation. We investigated the role of PI3K/Akt in p50/p50 NF-κB activation and the effect on iNOS expression in L. amazonensis infection. The increased occupancy of p50/p50 on the iNOS promoter of infected macrophages was observed and we demonstrated that both p50/p50 NF-κB induction and iNOS downregulation in infected macrophages depended on PI3K/Akt activation. Importantly, the intracellular growth of the parasite was also impaired during PI3K/Akt signalling inhibition and in macrophages knocked-down for Akt 1 expression. It was also observed that the increased nuclear levels of p50/p50 in L. amazonensis-infected macrophages were associated with reduced phosphorylation of 907 Ser p105, the precursor of p50. Corroborating these data, we demonstrated the increased levels of phospho-9 Ser GSK3β in infected macrophages, which is associated with GSK3β inhibition and, consequently, its inability to phosphorylate p105. Remarkably, we found that the levels of pPTEN 370 Ser, a negative regulator of PI3K, increased due to L. amazonensis infection. Our data support the notion that PI3K/Akt activity is sustained during the parasite infection, leading to NF-κB 105 phosphorylation and further processing to originate p50/p50 homodimers and the consequent downregulation of iNOS expression.

Leishmaniaparasitophorous vacuole membranes display phosphoinositides that create conditions for continuous Akt activation and a target for miltefosine inLeishmaniainfections

“…In contrast, induction of Akt1 shRNA with doxycycline resulted in a 40% reduction in the number of infected cells ( Figure 4c). This result was in agreement with the results of Calegari-Silva et al (2015) who showed that in THP-1 human cells transfected with Akt1 shRNA, the survival of Leishmania parasites was significantly reduced. We then proceeded to assess the efficacy of miltefosine in these cells with doxycycline co-incubation.…”

RIPK1 and PGAM5 Control Leishmania Replication through Distinct Mechanisms

“…In this regard, it is noteworthy that L. amazonensis infection in macrophages activates the p50/p50 homodimer, a NF-κB transcriptional repressor. Inhibition of NF-κB-dependent gene transcription by p50/p50 homodimer has been attributed to cause downregulation of iNOS expression in L. amazonensis infection (43, 44). It is therefore tempting to speculate that RIPK1 may promote canonical NF-κB activation to counteract this inhibitory signal from p50/p50.…”

PI3K signaling in Leishmania infections

“…In their study Calegari-Silva et al (2015) (14) observed a reduction in infection in cells in which PKB/AKT levels were suppressed by siRNA treatment. This suggests that Leishmania survival in macrophages, even in the absence of any additional activation stimulus is dependent on activation of PKB/AKT.…”

“…As stated earlier, Lambertz et al (2012) (6) presented a comprehensive review of studies that demonstrated the critical of PKB/Akt signaling in Leishmania infections. More recent studies include a report by Calegari-Silva et al (2015) (14) who evaluated the role of PKB/Akt in the inhibition of nitric oxide release in L. amazonensis infections. Specifically, they considered the role of PKB/Akt in the activation of NFκB, which has been shown to control the transcription of the iNOS gene.…”