Abstract
Neutrophils are essential players in the first-line defense against invading bacteria and fungi. Besides its antiapoptotic role, the inhibitor of apoptosis protein (IAP) family member X-linked IAP (XIAP) has been shown to regulate innate immune signaling. Whereas the role of XIAP in innate signaling pathways is derived mostly from work in macrophages and dendritic cells, it is not known if and how XIAP contributes to these pathways in neutrophils. Here we show that in response to bacterial lipopolysaccharides (LPS), mouse neutrophils secreted considerable amounts of tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β) and, in accordance with earlier reports, XIAP prevented LPS-induced hypersecretion of IL-1β also in neutrophils. Interestingly, and in contrast to macrophages or dendritic cells, Xiap-deficient neutrophils were insensitive to LPS-induced cell death. However, combined loss of function of XIAP and cIAP1/-2 resulted in rapid neutrophil cell death in response to LPS. This cell death occurred by classical apoptosis initiated by a TNFα- and RIPK1-dependent, but RIPK3- and MLKL-independent, pathway. Inhibition of caspases under the same experimental conditions caused a shift to RIPK3-dependent cell death. Accordingly, we demonstrate that treatment of neutrophils with high concentrations of TNFα induced apoptotic cell death, which was fully blockable by pancaspase inhibition in wild-type neutrophils. However, in the absence of XIAP, caspase inhibition resulted in a shift from apoptosis to RIPK3- and MLKL-dependent necroptosis. Loss of XIAP further sensitized granulocyte–macrophage colony-stimulating factor (GM-CSF)-primed neutrophils to TNFα-induced killing. These data suggest that XIAP antagonizes the switch from TNFα-induced apoptosis to necroptosis in mouse neutrophils. Moreover, our data may implicate an important role of neutrophils in the development of hyperinflammation and disease progression of patients diagnosed with X-linked lymphoproliferative syndrome type 2, which are deficient in XIAP.
Highlights
Neutrophil granulocytes mature in the bone marrow and are released as terminally differentiated, short-lived cells into the blood where they constitute the most abundant leukocyte population in humans
LPS induced comparable TNFα and IL-6 levels in Xiap− / − neutrophils, but, interestingly, loss of X-linked IAP (XIAP) did not increase cell death, in contrast to previous reports on macrophages and dendritic cells (DCs).[27,28]. It has been shown for macrophages and DCs that in the absence of XIAP, or alternatively cIAP1/-2, LPS stimulation alone causes exacerbated IL-1β secretion.[28,35]. Consistent with these findings, GM-CSF priming followed by LPS stimulation promoted excessive IL-1β secretion in Xiap− / −, but not in WT, neutrophils that was preceded by proteolytic processing of procaspase-8 (Figures 1b, d, e and Supplementary Figures S1b–d and f)
Besides its first identified role as direct caspase inhibitor, XIAP has recently been implicated in a RING domain-dependent positive regulation of NOD1/2 signaling in response to intracellular bacteria and in restriction of RIPK3-dependent cell death and inflammasome activation downstream of TNFR1 and Toll-like receptor-4 (TLR4).28,31,33,35 Most of these data are derived from macrophages or DCs
Summary
Neutrophil granulocytes mature in the bone marrow and are released as terminally differentiated, short-lived cells into the blood where they constitute the most abundant leukocyte population in humans. Neutrophils express a large variety of surface receptors including innate immune receptors to recognize invading pathogens Activation of those receptors leads to elimination of pathogens by phagocytosis, secretion of granule contents, reactive oxygen species and release of neutrophil extracellular traps.[1] Toll-like receptor-4 (TLR4), a receptor for bacterial lipopolysaccharides (LPS), is one of the most important receptors constitutively expressed by neutrophils,[1,2] which upon activation enhances activity, cell survival and proinflammatory cytokine secretion.[2,3]. Similar to cIAP1/-2, XIAP contains a RING domain that functions as E3 ubiquitin ligase regulating NF-κB and MAPK signaling pathways.[14,29,30] The best example currently is the positive regulation of NOD1/2-dependent signaling by XIAP upon detection of intracellular bacteria.[31,32,33] A crucial physiological role of XIAP in immunity is supported by loss-offunction mutations in patients suffering from the primary immunodeficiency disorder X-linked lymphoproliferative syndrome 2 (XLP2).[34]
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