Neutrophil extracellular trap (NET) formation can generate short-term, functional anucleate cytoplasts and trigger loss of cell viability. We demonstrated that the necroptotic cell death effector mixed lineage kinase domain-like (MLKL) translocated from the cytoplasm to the plasma membrane and stimulated downstream NADPH oxidase-independent ROS production, loss of cytoplasmic granules, breakdown of the nuclear membrane, chromatin decondensation, histone hypercitrullination, and extrusion of bacteriostatic NETs. This process was coordinated by receptor-interacting protein kinase-1 (RIPK1), which activated the caspase-8-dependent apoptotic or RIPK3/MLKL-dependent necroptotic death of mouse and human neutrophils. Genetic deficiency of RIPK3 and MLKL prevented NET formation but did not prevent cell death, which was because of residual caspase-8-dependent activity. Peptidylarginine deiminase 4 (PAD4) was activated downstream of RIPK1/RIPK3/MLKL and was required for maximal histone hypercitrullination and NET extrusion. This work defines a distinct signaling network that activates PAD4-dependent NET release for the control of methicillin-resistant (MRSA) infection.

ObjectivesThe endoplasmic reticulum aminopeptidase (ERAP1) haplotype Hap10 encodes for a variant allotype of the endoplasmic reticulum (ER)-resident peptide-trimming aminopeptidase ERAP1 with low enzymatic activity. This haplotype recessively confers the highest risk for Behçet’s diseases (BD) currently known, but only in carriers of HLA-B*51, the classical risk factor for the disease. The mechanistic implications and biological consequences of this epistatic relationship are unknown. Here, we aimed to determine its biological relevance and functional impact.MethodsWe genotyped and immune phenotyped a cohort of 26 untreated Turkish BD subjects and 22 healthy donors, generated CRISPR-Cas9 ERAP1 KOs from HLA-B*51+ LCL, analysed the HLA class I-bound peptidome for peptide length differences and assessed immunogenicity of genome-edited cells in CD8 T cell co-culture systems.ResultsAllele frequencies of ERAP1-Hap10 were similar to previous studies. There were frequency shifts between antigen-experienced and naïve CD8 T cell populations of carriers and non-carriers of ERAP1-Hap10 in an HLA-B*51 background. ERAP1 KO cells showed peptidomes with longer peptides above 9mer and significant differences in their ability to stimulate alloreactive CD8 T cells compared with wild-type control cells.ConclusionsWe demonstrate that hypoactive ERAP1 changes immunogenicity to CD8 T cells, mediated by an HLA class I peptidome with undertrimmed peptides. Naïve/effector CD8 T cell shifts in affected carriers provide evidence of the biological relevance of ERAP1-Hap10/HLA-B*51 at the cellular level and point to an HLA-B51-restricted process. Our findings suggest that variant ERAP1-Hap10 partakes in BD pathogenesis by generating HLA-B51-restricted peptides, causing a change in immunodominance of the ensuing CD8 T cell response.

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