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

Abstract

It's not clear how nuclear envelope (NE) is ruptured for chromatin externalization duringNETosis. 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 PKCα 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 PKCα inhibition or genetic deletion, or mutation at the PKCα 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 PKCα-mediated lamin B phosphorylation drives nuclear envelope rupture for NET release in neutrophils. Papayannopoulos et al., 2010), while neutrophil elastase is dispensable for NET formation as neutrophils from neutrophil elastase deficient mice could still undergo NETosis in a recent study (Martinod et al., 2016).Release of the decondensed nuclear chromatin, through the broken nuclear and plasma membranes, is the key step for formation of the neutrophil NET backbone. Neutrophil NET release was described as a lytic cell death mechanism (Fuchs et al., 2007;Papayannopoulos et al., 2010;Yipp and Kubes, 2013). However, this notion has been questioned by several recent observations (Pilsczek et al., 2010;Amulic et al., 2017;Neubert et al., 2018). Rupture of the nuclear envelope/nuclear membrane appears to be a distinct process from the previously described lysis or dissolution of the nuclear envelope (Amulic et al., 2017;Neubert et al., 2018).Since nuclear chromatin is enclosed by the nuclear envelope, its breakdown is, therefore, a prerequisite, and a key step, for externalization of nuclear chromatin and extracellular trap formation. (Papayannopoulos et al., 2010;Yipp and Kubes, 2013) However, the relevant mechanisms that regulate nuclear envelope rupture in NETosis are still unclear.The nuclear envelope consists of outer and inner lipid nuclear membranes (ONM and INM) and nuclear lamina, a protein filament meshwork that provides structural scaffold to reinforce nuclear envelope integrity (Goldberg et al., 2008;Schreiber and Kennedy, 2013;Stewart et al., 2007).Nuclear lamins are categorized as either A-type (A, C) or B-type (B1, B2) lamins (Schreiber and Kennedy, 2013). A-type lamins form thick filament bundles that provide mechanical rigidity to the nucleus (Goldberg et al., 2008;Lammerding...

Cellular Mechanisms of NETosis

“…The formation of meshwork discontinuities could be the result of lamin posttranslational modifications. As such, lamin A/C (Amulic et al 2017) and PKCα-mediated lamin B1 (Li et al 2019) phosphorylation was reported in PMA-induced NETosis. Indeed, mutation of the PKC phosphorylation sites of lamin B1 impairs extracellular trap release in RAW264.7 macrophages (Li et al 2019).…”

“…As such, lamin A/C (Amulic et al 2017) and PKCα-mediated lamin B1 (Li et al 2019) phosphorylation was reported in PMA-induced NETosis. Indeed, mutation of the PKC phosphorylation sites of lamin B1 impairs extracellular trap release in RAW264.7 macrophages (Li et al 2019). Additionally, lamin B1 degradation has been observed during ionomycin-induced NETosis (Gößwein et al 2019), and PAD4 deficiency has been shown to delay the formation of lamin discontinuities and to diminish lamin B1 degradation in ionomycin-stimulated neutrophils (Gößwein et al 2019, Thiam et al 2020.…”

“…However, mechanical forces alone were reported to be sufficient for nuclear envelope rupture during immune and cancer cell migration under confinement (Denais et al 2016, Raab et al 2016. Similar to the rearrangement described during cell division and migration, DNA release into the cytosol during NETosis was shown to involve lamin remodeling as well as formation of holes in the nuclear envelope prior to its rupture (Chen et al 2018, Fuchs et al 2007, Gößwein et al 2019, Li et al 2019, Neubert et al 2018, Thiam et al 2020. Interestingly, vital NETosis is proposed to occur without nuclear membrane disruption with decondensed DNA leaving the nucleus in vesicles (Pilsczek et al 2010).…”

“…Dramatic changes in the lamin meshwork have been observed during NETosis (Figures 1 and 2). The appearance of local discontinuities in the lamin B1 and B2 networks has been reported in human and mouse neutrophils stimulated with PMA or ionomycin (Li et al 2019, Neubert et al 2018, Thiam et al 2020. Using high-resolution live-cell imaging of HL60-derived neutrophils stimulated with ionomycin, Thiam and colleagues (2020) found that decondensed DNA extruded from these lamin discontinuities.…”