Werth, V. | bioRxiv Science

bioRxiv2021

Deficiency of Rho Kinase 1 (ROCK1) attenuates neutrophil NETosis and ameliorates UVB-induced skin inflammation

ObjectiveUltraviolet B (UVB) is an important trigger of skin inflammation and lupus with leukocyte recruitment to inflamed skin. We recently reported the involvement of neutrophil NETosis in UVB-induced skin inflammation, and that NETotic nuclear envelope rupture is driven by PKC-mediated nuclear lamin B disassembly. To address the role of Actin cytoskeleton in NETosis, we investigated the effects of Rho kinase (ROCK) and its downstream actomyosin cytoskeletal networks on PKC nuclear translocation and NET formation, as well as their involvement in UVB-induced skin inflammation. MethodsWe studied the dynamic changes of ROCK and actomyosin cytoskeletal networks during NETosis induction and their involvement in PKC nuclear translocation. Using mice with hematopoietic-specific ROCK1 deficiency, we investigated the effects of ROCK1 deficiency on NETosis, and its involvement in UVB-induced skin inflammation. ResultsOur time course studies demonstrated the dynamic changes of actin polymerization and ROCK activation, support the role of actin cytoskeleton in nuclear translocation of cytosolic PKC in early stage of NETosis induction. Inhibition of actin polymerization or key molecules of the ROCK/MLCK/myosin pathway decreased PKC nuclear translocation and NET formation. Genetic deficiency of ROCK1, inhibited NETosis ex vivo and in vivo, decreased extracellular display of NET-associated IL-17A, TNF, IFN{gamma}, and IFN in inflamed skin, which were correlated with the ameliorated skin inflammation in UVB-irradiated mice with hematopoietic-specific ROCK1 deficiency. ConclusionsROCK regulated NETosis through modulation of PKC nuclear translocation via actomyosin cytoskeletal networks in neutrophils. ROCK1 deficiency ameliorated UVB- induced skin inflammation by attenuation of NETosis and NET-associated cytokines.

pathology↗

bioRxiv2019

Nuclear lamin B is crucial to the nuclear envelope integrity and extracellular trap release in neutrophils

Its not clear how nuclear envelope (NE) is ruptured for chromatin externalization during NETosis. The membrane rupture during neutrophil NET release was described as a membrane lysis process, this notion, however, has been questioned. Here, we found that lamin B, the structural NE component, was involved in NETosis. Unexpectedly, lamin B was not fragmented by destructive proteolysis, but rather disassembled into its intact full-length molecule, in NETotic cells with ruptured NE. In the mechanistic study, our experiments demonstrated that cytosolic PKC translocated to the nucleus, where it serves as a NETotic lamin kinase to induce lamin B phosphorylation, following by lamina disassembly and NE rupture. To determine causality, we found that decreasing lamin B phosphorylation, by PKC inhibition or genetic deletion, or mutation at the PKC consensus phosphorylation sites of lamin B, attenuated extracellular trap formation. Importantly, strengthening NE by lamin B overexpression attenuated neutrophil NETosis in vivo and alleviated exhibition of NET-associated inflammatory cytokines in UVB irradiated skin of lamin B transgenic mice. These findings advance our understanding of NETosis process and elucidate a cellular mechanism that PKC-mediated lamin B phosphorylation drives nuclear envelope rupture for NET release in neutrophils.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC=\"FIGDIR/small/647529v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (49K):\norg.highwire.dtl.DTLVardef@7bd231org.highwire.dtl.DTLVardef@124bf6dorg.highwire.dtl.DTLVardef@19396ecorg.highwire.dtl.DTLVardef@9989cb_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗