Rho Kinase regulates neutrophil NET formation that is involved in UVB-induced skin inflammation

Abstract

Objective. Ultraviolet B (UVB) is an important trigger of skin inflammation and lupus with recruitment of leukocytes to inflamed skin. Neutrophil NETosis has been implicated in pathogenesis of lupus erythematosus. We recently reported that nuclear envelope rupture and NET formation is driven by PKCα-mediated nuclear lamin B disassembly, with involvement of NETosis in UVB-induced skin inflammation. Studies have shown that cytoskeletal networks are involved in NETosis. However, it is still unknown how cytoskeletal networks and their upstream ROCK are involved in NET formation as well as UVB-induced skin inflammation. Methods. The involvements of actomyosin cytoskeletal networks and ROCK1 in NETosis were studied in vitro. Mice with hematopoietic specific ROCK1 deficiency were irradiated by UVB to investigate NET formation in vivo and its involvement in skin inflammation. Results. In time course studies, PKCα nuclear translocation was very well matched with actin polymerization and ROCK1 activation. Inhibition of actin polymerization or ROCK/MLCK/myosin pathway with chemical inhibitors decreased nuclear translocation of PKCα and NETosis in vitro. Furthermore, genetic deficiency of ROCK1 inhibited NET formation ex vivo. Hematopoietic specific ROCK1 deficiency ameliorated UVB-induced skin inflammation in mice with reduced skin thickness by attenuating NET formation and the extracellular display of NET-associated IL-17A, TNFα, IFNγ, and IFNα in the inflamed skin. Conclusion. ROCK1 regulated neutrophils for NET formation by modulation of PKCα nuclear translocation through actomyosin cytoskeletal networks. ROCK1 deficiency ameliorates UVB-induced skin inflammation by inhibition of NETosis and display of NET-associated cytokines. This study provides insights into novel therapeutics on NETosis-related diseases.

KRIT1‐mediated regulation of neutrophil adhesion and motility

“…On the other hand, based on data from endothelial cells, it has been proposed that Rap1 binding to KRIT1 facilitates the inactivation of RhoA/ROCK [31,51,52]. As RhoA/ROCK activity is also important for neutrophil function [53][54][55][56][57], that KRIT1 could also regulate RhoA in neutrophils is an attractive hypothesis. Our data suggest that KRIT1 deficient neutrophils exhibit increased baseline RhoA/ ROCK activity, as shown by increased pMLC staining in vehicle-treated cells.…”

“…Studies in endothelial cells have shown that KRIT1 is a negative regulator of RhoA and its effector Rho kinase (ROCK); loss of KRIT1 expression leads to increased RhoA/ROCK activity, phosphorylation of myosin light chain (MLC) and increased actin-myosin contractility [51,52]. In neutrophils, RhoA/ROCK has been shown to regulate adhesion and migration on multiple substrates [53][54][55][56][57]. Therefore, we examined the effect of KRIT1 depletion on RhoA/ROCK activity in neutrophils by measuring phospo-MLC levels using immunofluorescent microscopy.…”

Moonlighting chromatin: when DNA escapes nuclear control

“…However, NET formation is a caspase-independent process [43,47,48], during which caspase-3 remains inactive [43]. Recent studies indicate that protein kinase C-α (PKC-α)-mediated lamin B phosphorylation and disassembly is responsible for nuclear envelope rupture [43,49]. In addition, cyclin dependent kinase 4/6 (CDK4/6) controls NET formation through modulation of lamin A/C phosphorylation, resulting in nuclear envelope rupture [38,50].…”

“…Thus, kinase-mediated nuclear lamina phosphorylation-disassembly [43,50], but not proteolytic cleavage [38], is responsible for nuclear envelope rupture during NET formation. PKCα and CDK4/6 are located in the cytoplasm of resting neutrophils, and their nuclear translocation requires a functional actin cytoskeleton in the early stage of neutrophil activation [38,49,52]. Genetic [49,53] or pharmacologic [54] inhibition of actin assembly or its upstream regulatory molecules, Rho kinase [49] or Wiskott-Aldrich syndrome protein [53], impair NET formation.…”

“…PKCα and CDK4/6 are located in the cytoplasm of resting neutrophils, and their nuclear translocation requires a functional actin cytoskeleton in the early stage of neutrophil activation [38,49,52]. Genetic [49,53] or pharmacologic [54] inhibition of actin assembly or its upstream regulatory molecules, Rho kinase [49] or Wiskott-Aldrich syndrome protein [53], impair NET formation. This argues for a crucial role of the actin cytoskeleton in NET formation [38,52].…”