RIPK1 and PGAM5 Control Leishmania Replication through Distinct Mechanisms

Abstract

Leishmaniasis is an important parasitic disease found in the tropics and sub-tropics. Cutaneous and visceral leishmaniasis affect an estimated 1.5 million people worldwide. Despite its human health relevance, relatively little is known about the cell death pathways that control Leishmania replication in the host. Necroptosis is a recently identified form of cell death with potent anti-viral effects. RIPK1 is a critical kinase that mediates necroptosis downstream of death receptors and toll-like receptors. Heme, a product of hemoglobin catabolism during certain intracellular pathogen infections, is also a potent inducer of macrophage necroptosis. We found that human visceral leishmaniasis patients exhibit elevated serum levels of heme. Therefore, we examined the impact of heme and necroptosis on Leishmania replication. Indeed, heme potently inhibited Leishmania replication in bone marrow derived macrophages (BMDMs). Moreover, we found that inhibition of RIPK1 kinase activity also enhanced parasite replication in the absence of heme. We further found that the mitochondrial phosphatase phosphoglycerate mutase family member 5 (PGAM5), a putative downstream effector of RIPK1, was also required for inhibition of Leishmania replication. In mouse infection, both PGAM5 and RIPK1 kinase activity are required for IL-1β expression in response to Leishmania. However, PGAM5, but not RIPK1 kinase activity, was directly responsible for Leishmania-induced IL-1β secretion and nitric oxide production in BMDMs. Collectively, these results revealed that RIPK1 and PGAM5 function independently to exert optimal control of Leishmania replication in the host.

Heme Drives Oxidative Stress-Associated Cell Death in Human Neutrophils Infected with Leishmania infantum

“…We have previously demonstrated that induction of HO-1 favors Leishmania infantum replication and infection in human and murine macrophages by reduction of its leishmanicidal mechanisms, as nitric oxide and ROS ( 12 ). We have also described increased serum levels of HO-1 and total heme in VL patients compared to healthy endemic controls ( 12 , 13 ).…”

“…Human neutrophils were experimentally infected with L . infantum and incubated in the absence or presence of heme for 3 h. We have used 30 μM of heme in the experiments because this was the average level detected in serum from patients with VL ( 13 ), as similar to what has been described in malaria ( 25 , 26 ) and sickle cell disease ( 27 ). In addition, doses close to 30 µM have been commonly used in in vitro experiments published from other groups ( 8 , 9 , 28 ).…”

“…Both HO-1 and heme have been previously described as important biomarkers associated to VL ( 12 , 13 ). Serum LDH is usually elevated in hemolytic diseases, reflecting the degree of cell/tissue damage ( 24 ).…”

“…Serum LDH is usually elevated in hemolytic diseases, reflecting the degree of cell/tissue damage ( 24 ). In the present study, we re-examined the data from a patient cohort previously published by our group ( 12 , 13 ) to assess correlations between circulating levels of total heme with LDH, HO-1, or peripheral blood neutrophils counts. Serum samples were obtained from patients with VL and from sex-matched endemic controls, all from a highly endemic area in the northeast of Brazil.…”

“…In this setting, hemoglobin and other heme proteins are degraded, releasing free heme ( 36 ). We have previously described that serum concentrations of both total heme ( 13 ) and of HO-1 ( 12 ) are substantially elevated in patients with VL than in uninfected healthy endemic controls. Besides that, it has been shown that HO-1 induction favors L. infantum replication inside dog macrophages ( 39 ).…”