Ptpn6 is a cytoplasmic phosphatase that functions to prevent autoimmune and interleukin-1 (IL-1) receptor-dependent, caspase-1-independent inflammatory disease. Conditional deletion of Ptpn6 in neutrophils (Ptpn6∆PMN) is sufficient to initiate IL-1 receptor-dependent cutaneous inflammatory disease, but the source of IL-1 and the mechanisms behind IL-1 release remain unclear. Here, we investigate the mechanisms controlling IL-1α/β release from neutrophils by inhibiting caspase-8-dependent apoptosis and Ripk1–Ripk3–Mlkl-regulated necroptosis. Loss of Ripk1 accelerated disease onset, whereas combined deletion of caspase-8 and either Ripk3 or Mlkl strongly protected Ptpn6∆PMN mice. Ptpn6∆PMN neutrophils displayed increased p38 mitogen-activated protein kinase-dependent Ripk1-independent IL-1 and tumor necrosis factor production, and were prone to cell death. Together, these data emphasize dual functions for Ptpn6 in the negative regulation of p38 mitogen-activated protein kinase activation to control tumor necrosis factor and IL-1α/β expression, and in maintaining Ripk1 function to prevent caspase-8- and Ripk3–Mlkl-dependent cell death and concomitant IL-1α/β release.
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Mlkl−/− mice were provided by W. S. Alexander (Walter and Eliza Hall Institute of Medical Research). Ripk3−/− mice were provided by V. Dixit (Genentech). Ripk1−/−, conditional Ripk1fl/fl and Ripk1D138N mice were provided by M. Kelliher and M. Pasparakis, supported by NIH/AID grant RO1 AI075118. Casp8 antibody was provided by A. Strasser (Walter and Eliza Hall Institute of Medical Research). This work was supported by NIH grant 5RO1HL124209 (to B.A.C.), the American Asthma Foundation (to B.A.C.), ISF grants 1416/15 and 818/18 (to M.G.), Alpha-1 Foundation grant 615533 (to M.G.), the Recanati Foundation and Varda and Boaz Dotan Research Center (to M.G.), United States–Israel Binational Science Foundation grant 2017176 (to M.G. and B.A.C.), the Australian National Health and Medical Research Council (NHMRC) Dora Lush Scholarship (to J.A.O.) and NHMRC grants 637367, 1145788 and 1162765. This work was supported by a NHMRC Independent Research Institutes Infrastructure Support Scheme grant (9000220), and a Victorian State Government Operational Infrastructure Support grant, support from the Novo Nordisk Foundation provided to the Center for Biosustainability at the Technical University of Denmark (NNF10CC1016517 to N.E.L.) and NIGMS (R35 GM119850 to I.S.). Live-cell imaging performed at Boston Children’s Hospital Intellectual Developmental Disabilities Research Center is supported by grant 1U54HD0902565.
The authors declare no competing interests.
Peer review information Laurie A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
a) Gene Set Enrichment Analysis of RNA-Seq transcriptomic data derived from bone marrow neutrophils of female Ptpn6ΔPMNcaspase-8ΔPMNRipk3-/-Mlkl-/- mice and controls. b) Age-dependent effects on the neutrophil transcriptome in Ptpn6ΔPMNcaspase-8ΔPMNRipk3-/-Mlkl-/- mice and controls. Values are gene-wise z-scores of counts normalized using the variance stabilized transformation from DESeq2.
Extended Data Fig. 2 Flow cytometry gating strategy for hematopoietic stem and progenitor cells (HSPC).
For gating of HSPC in Fig. 3, bone marrow cells were sorted after enrichment for hematopoietic progenitor cells by magnetic bead-based depletion of lineage-positive hematopoietic cells. a) Definition and gating strategy for lineage restricted progenitors (LRP), multi-potent progenitors (MPP), and hematopoietic stem cells (HSC), (b) common myeloid progenitors (CMP), megakaryocyte erythroid progenitors (MEP), granulocyte macrophage progenitors (GMP), and (c) common lymphoid progenitors. Lineage-negative cells and gating were defined using isotype control antibodies.
a-b) Live-cell imaging of CTG-labeled wild-type, Ripk1ΔPMN, and Ripk1D138N/D138N neutrophils treated with 2 μM BPT and/or 10 μM z-VAD-fmk + /- 100 ng/mL TNFα. PI and Annexin V were used to monitor changes in viability. Mean and SEM, n = 3 biologically independent samples, and triplicate fields of view per independent biological sample. c) Live-cell imaging of CTG-labeled wild-type, Ptpn6ΔPMN, Ripk1ΔPMN, and Ptpn6ΔPMNRipk1ΔPMN neutrophils treated with saline or 100 ng/mL TNFα. PI and Annexin V were used to monitor changes in viability. Mean and SEM of technical replicates shown. Data are representative of two independent experiments.
Live-cell imaging of CellTracker Orange-labeled wild-type, Ripk3-/-, Ripk1ΔPMN, and Ptpn6∆PMNRipk1ΔPMN neutrophils treated with 2 μM BPT and/or 10 μM z-VAD-fmk + /- 100 ng/mL TNFα. CellEvent caspase-3/7 Green Detection Reagent and Draq7 were used to monitor changes in caspase activation and viability. Mean and SEM, n = 3 technical triplicate samples from triplicate fields of view.
Comparison of CTG-positive proportion of a) z-VAD-fmk and b) birinapant treatment of TNF-stimulated Ripk1ΔPMN and wild-type neutrophils. Red curves indicate predicted profiles for wild-type, blue for Ripk1ΔPMN, with line patterns indicating predicted profile for treatment.
Extended Data Fig. 6 A model illustrating the role of Ptpn6 in regulation of cell death signaling in neutrophils.
Ptpn6 function is controlled in part by Y208−dependent anchoring to the actin−myosin−9 cytoskeleton. In the absence of Ptpn6, the negative regulatory functions of Ripk1 are lost but the kinase domain remains active to influence apoptotic and necroptotic cell death. In Ptpn6ΔPMN neutrophils lacking RIPK1 kinase activity or RIPK1, necroptotic and apoptotic cell death proceed unabated.
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Speir, M., Nowell, C.J., Chen, A.A. et al. Ptpn6 inhibits caspase-8- and Ripk3/Mlkl-dependent inflammation. Nat Immunol 21, 54–64 (2020). https://doi.org/10.1038/s41590-019-0550-7
Targeting protein phosphatases for the treatment of inflammation-related diseases: From signaling to therapy
Signal Transduction and Targeted Therapy (2022)
Arthritis Research & Therapy (2020)