Loss of XIAP facilitates switch to TNFα-induced necroptosis in mouse neutrophils

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.

Fangjifuling Ameliorates Lipopolysaccharide-Induced Renal Injury via Inhibition of Inflammatory and Apoptotic Response in Mice

“…It was shown that inflammatory mediator channels, cell junctions, and apoptotic process were closely related to the anti-inflammatory mechanism of FF. LPS is known to induce the secretion of inflammatory cytokines including IL-1β and COX-2 [17], which are closely associated with AKI [18]. Except for iNOS, NF-κB and IL-1β were detected in bioinformatics analysis, and other inflammatory-related cytokines including MCP-1, TNF-α, and COX-2 were also affected in an in vivo test.…”

TNFR2 induced priming of NLRP3-inflammasome via RIPK1 leads to pyroptosis in XIAP deficient cells

“…There are various mutations found in all over the gene encoding XIAP, contributing to immune hyper-activation and tissue inflammation in XLP-2 patients (Prokop et al, 2017). To determine whether these mutations in XIAP cause sensitivity to TNFR2 induced cell death, we utilized the HoxB8 progenitor system (Wicki et al, 2016). Briefly, lineage negative cells from the bone marrow were transduced with 4-hydroxytamoxifen (4-OHT) inducible HoxB8 and immortalized with stem cell factors.…”

“…However, differentiated xiap -/granulocytes were sensitive to TNFR2 induced cell death but not to TR1-TNF ( Figure 1E). To assess the contribution of either the BIR or RING domain of XIAP to the observed cell death, different XIAP constructs containing mutations found in XLP-2 patients were re-introduced into the HoxB8 progenitor cells (Wicki et al, 2016) (Figure 1F). Xiap -/-HoxB8 granulocytes with XIAP wildtype reintroduced (xiap +/+ ) remained insensitive to either TR1-or TNC-TNF induced cell death ( Figure 1G).…”

“…TNFR1-Fas and TNFR2-Fas expressing mouse fibroblasts were obtained from Anja Krippner-Heidenreich (Krippner-Heidenreich et al, 2002) and were cultured in DMEM containing 1g/L glucose, 1% (v/v) penicillin/streptomycin/glutamine and 10% FBS. HOXB8 progenitor cells were cultured in RPMI 1640 media with 10% (v/v) heat-inactivated FBS, 1% (v/v) penicillin/streptavidin, 7% (v/v) SCF from CHO/SCF conditioned medium and 1µM 4hydroxytamoxifen (4-OHT, MedChemExpress) (Wicki et al, 2016). To differentiate granulocytes from the HOXB8 progenitors, cells were washed twice with PBS and resuspended at a concentration of 2.5x10 4 cells/mL in media without 4-OHT and differentiated for 5 days.…”

“…In response to LPS, XIAP deficient myeloid cells undergo necroptosis dependent on RIPK3, mediated by TNF (Lawlor et al, 2015;Wicki et al, 2016;Yabal et al, 2014). The loss of XIAP pre-disposes myeloid cells to LPS-induced sensitivity allowing for inflammasome activation (Lawlor et al, 2015;Wicki et al, 2016;Yabal et al, 2014). The activation of the inflammasome, which can be led by NLRP3, ASC and pro-caspase-1 or non-canonical pathway caspase-11, leading to pyroptosis.…”