Reactive oxygen species-induced actin glutathionylation controls actin dynamics in neutrophils

Abstract

The regulation of actin dynamics is pivotal for cellular processes such as cell adhesion, migration, and phagocytosis and thus is crucial for neutrophils to fulfill their roles in innate immunity. Many factors have been implicated in signal-induced actin polymerization, but the essential nature of the potential negative modulators are still poorly understood. Here we report that NADPH oxidase-dependent physiologically generated reactive oxygen species (ROS) negatively regulate actin polymerization in stimulated neutrophils via driving reversible actin glutathionylation. Disruption of glutaredoxin 1 (Grx1), an enzyme that catalyzes actin deglutathionylation, increased actin glutathionylation, attenuated actin polymerization, and consequently impaired neutrophil polarization, chemotaxis, adhesion, and phagocytosis. Consistently, Grx1-deficient murine neutrophils showed impaired in vivo recruitment to sites of inflammation and reduced bactericidal capability. Together, these results present a physiological role for glutaredoxin and ROS- induced reversible actin glutathionylation in regulation of actin dynamics in neutrophils.

Figure 1. NADPH oxidase-mediated ROS production induced actin glutathionylation in chemoattractant-stimulated neutrophils

(A) Protein glutathionylation in chemoattractant-stimulated human neutrophils. (B) Treatment with DTT leads to reduction of glutathione mixed disulfides. (C) Biotinylated glutathione (BioGEE)-modified proteins were pulled down from neutrophil lysates using Streptavidin agarose beads and probed with a β-actin antibody. (D) Actin was immunoprecipitated from BioGEE labeled neutrophil lysates (5 min after fMLF stimulation) and probed for Biotinylated-glutathione modification using Streptavidin-HRP. ASK1, a cytosolic protein, was used as a negative control. (E) Actin glutathionylation in fMLF-stimulated human neutrophils is dependent on NADPH oxidase activation. Human neutrophils pretreated with 50 µM DPI for 30 min were stimulated with 100 nM fMLF. Ratio of glutathionylated-actin to total actin is reported as actin-glutathionylation in the bar-graph (right). ROS production was evaluated by monitoring chemiluminescence (left). (F) Actin glutathionylation in fMLF-stimulated murine neutrophils stimulated with 1 µM fMLF. Data represents mean±SD from n=3 wells from one experiment representative of three. *, p < 0.001, versus WT. #, p < 0.001 versus time 0.

Figure 2. Both NADPH oxidase-mediated ROS production and protein glutathionylation occurred at pseudopodia of migrating human neutrophils, colocalizing with actin

(A) Human neutrophils were labeled with ROS dye CM-H2DCFDA and exposed to a micropipette tip filled with 1 µM fMLF (denoted by solid white circle). Leading edges of migrating cells were marked by white arrows. Scale bars represent 10 µm. (B) Quantification of NADPH oxidase-dependent ROS localization at the front of migrating neutrophils. Four to five regions of interest (ROIs, red circles in Figure 2A) were randomly drawn at the front and back of the cell, as well as background regions of the image. Data are represented as mean±SD from n=12 cells. *, p < 0.001, versus untreated neutrophils. (C) Murine bone marrow neutrophils were labeled with 1 CM-H2DCFDA and exposed to a uniform bolus of chemoattractant (50 nM fMLF). Fluorescence images show randomly migrating neutrophils in three consecutive frames with 15 sec interval. Arrow heads point towards ROS localization at pseudopodia. Scale bars represent 5 µm. (D) Front to back ratio and ROS fluorescence intensity at the front of the untreated or DPI treated WT or CGD neutrophils were measured as described above. Data are represented as mean±SD from n=10 cells. *, p < 0.001, versus untreated WT neutrophils. (E) Mechanism of selective detection of intracellular H₂O₂ by PF6-AM. (F) Subcellular localization of H₂O₂ in chemotaxing neutrophils in a fMLF gradient. Human neutrophils were labeled with 1 µM of PF6-AM. Fluorescence intensity was measured by scanning a line (white line) through a cell with IPLab software. Shown are profiles of two representative cells. In this experiment, chemotactic gradient was generated with a micropipette filled with 10 µM fMLF. Scale bars represent 10 µm. (G) Front to back ratio and ROS fluorescence intensity at the front of the cell were calculated from images of untreated and 50 µM DPI-treated cells. Data are represented as mean±SD from n=15 cells. *, p<10⁻⁵ versus untreated neutrophils. (H) Distribution of glutathionylation and actin in fMLF-stimulated human neutrophils. Scale bars represent 5 µm. (I) Glutathionylation staining was conducted in the presence of 10 mM DTT. (J) Addition of excess amount of reduced GSH to primary antibody mix abrogated glutathionylation localization at the leading edge but did not affect detection of actin localization using the actin antibody. (K) Neutrophils were treated with 50 µM DPI before the fMLF stimulation. Glutathionylation staining was conducted as described in (H). The scale bars in I–K represent 20 µm.

Figure 3. Suppression of NADPH oxidase-mediated ROS production led to elevated actin polymerization in neutrophils

(A) Actin polymerization in human neutrophils treated with (or without) DPI. (B) Actin polymerization in WT and CGD murine neutrophils. Results are the means (±SD) of three independent experiments. *p < 0.01 versus WT neutrophils (Student’s t test).

Figure 4. Reducing actin glutathionylation led to increased F-actin, formation of multiple pseudopods, and defective chemotaxis

(A) Actin glutathionylation is a dynamic reversible process that can be regulated by glutaredoxin. (B) Overexpression of Grx1 in neutrophil like differentiated HL60 cells. (C) Grx1 overexpression reduced actin glutathionylation. Data represents means ± SD of three experiments. * p<0.01 versus the control. (D) Grx1 overexpression augmented the amount of F-actin. fMLF-elicited actin polymerization was calculated as the increase of F-actin amount compared to time 0. Data shown are means ± SD of five experiments. * p<0.01 versus the control (vector only). (E) Cells overexpressing Grx1 displayed defective chemotaxis. Data are represented as mean±SD for n=20 cells, * p<0.05 versus control neutrophils. (F) Cells overexpressing Grx1 displayed multiple pseudopodia. The multiple pseudopodia was identified during cell chemotaxis in the EZ-taxiscan device. Scale bars represent 5 µm. (G) Sodium arsenite treatment inhibits actin glutathionylation in human neutrophils. Human neutrophils pretreated with (or without) 50 µM sodium arsenite for 30 min were stimulated with 100 nM fMLF for 5 min. * p<0.05 versus untreated neutrophils. (H) Effect of sodium arsenite treatment on human neutrophil chemotaxis. Neutrophils were evaluated for migration speed, directionality, upward directionality (n=16 cells, *, p<0.0005 by Student’s t-test), and percentage of multiple pseudopodia (n>30 cells, **, p<0.05, Fisher’s exact test, 2-tail).

Figure 5. Neutrophil chemotaxis defects induced by ROS depletion could be rescued by inhibiting glutaredoxin

(A) Actin glutathionylation was augmented in cells treated with CdCl₂. Data represents means ± SD of three experiments. *, p<0.01 versus untreated dHL60 cells. (B) Augmenting protein glutathionylation by CdCl₂ rescued DPI-induced chemotaxis defect. Neutrophils chemotaxis was analyzed as described in Figure 4E. *, p<0.05 versus untreated neutrophils; *, p<0.05 versus DPI treated neutrophils. (C) siRNA silencing of Grx1 in differentiated HL60 cells. (D) Actin glutathionylation was enhanced in cells transfected with glutaredoxin siRNA. Data represents means ± SD of three experiments. *, p<0.01 versus dHL60 cells transfected with control siRNA. (E) siRNA silencing of glutaredoxin rescued DPI-induced chemotaxis defect. **, p<0.005 versus dHL60 cells transfected with control siRNA. (F) ROS depletion-induced formation of multiple pseudopodia and impaired directional migration were partially rescued by treatment with latrauculin B. Mouse neutrophils were pretreated with 50 µM DPI, 5 nM latrauculin B (LAT), 5 nM LAT + 2 µM CdCl₂, or left untreated, and then exposed to a chemoattractant gradient generated by addition of 100 nM fMLF (1 µl) in the EZ-taxiscan device. *, p<0.005. (G) Schematic representation of wild-type and mutant forms of human β-actin fused with GFP. (H) Cys³⁷⁴ is critical for ROS-elicited actin glutathionylation. dHL60 cells overexpressing indicated protein were stimulated with 1µM fMLF. Overexpression of wild-type and mutant forms of GFP-actin was confirmed by western blotting with a GFP antibody. Ratio of glutathionylated GFP-actin to total GFP-actin was calculated as actin-glutathionylation. Shown in the bar-graph are the fold increases of actin-glutathionylation compared to unstimulated cells (time 0). Data represents means ± SD of three experiments. *, p<0.01 versus unstimulated cells. (I) Glutathionylation at actin-Cys³⁷⁴ is a key regulatory mechanism for efficient neutrophil chemotactic migration. Cells overexpressing EGFP-actin were identified by their higher fluorescent intensity compared with untransfected cells. The chemotaxis of transfected dHL60 cells was analyzed using an EZ-taxiscan device as described above (n>20 cells; *, p<0.005 versus dHL60 cells expressing wild-type actin).

Figure 6. Disruption of glutaredoxin resulted in reduced actin polymerization and impaired neutrophil migration, polarization, adhesion, and phagocytosis

(A) Grx1 protein expression was completely abolished in neutrophils isolated from Grx1^−/− mice. (B) Glutaredoxin disruption resulted in increased amount of glutathionylated actin in neutrophils. (C) Disruption of Grx1 reduced actin polymerization. (D) Chemotaxis of mouse neutrophils in response to chemoattractant fMLF. (E) Cell tracks of migrating neutrophils (cells that move at least 65 µm from the bottom of the channel) (n=20). (F) Disruption of Grx1 reduced the efficiency of neutrophil chemotaxis. * p<0.05. (G) The percentage of cells that stopped or slowed down during chemotaxis. * p<0.0001 versus WT neutrophils. (H) Chemoattractant-induced ruffling in WT and Grx1^−/− neutrophils. Scale bar represents 10 µm. (I) The percentage of polarized neutrophils (cells that ruffled or extended pseudopods) was calculated at indicated time after fMLF stimulation. Data shown are mean ± SD collected from (n=3) separate preparations of neutrophils. *, P<0.01 versus WT neutrophils. ** p<0.0001 versus WT neutrophils. (J) Adhesion of WT and Grx1^−/− neutrophils on fibronectin-coated flow chambers in response to fMLF stimulation. Scale bar represents 50 µm. (K) Grx1 disruption reduced fMLF induced cell adhesion on fibronectin-coated flow chambers. * p<0.001 versus WT neutrophils. (L) In vitro phagocytosis assay. FITC-labeled Zymosan A bioparticles were opsonized with mouse serum and incubated with neutrophils at 37°C for indicated time. Extracellular fluorescence was quenched by trypan blue. The results shown are representative of three experiments. (M) Phagocytosis index (PI) was expressed as the number of bioparticles engulfed by 100 neutrophils. (N) Binding index was expressed as the number of bioparticles bound to 100 neutrophils (4°C for 30 min). (O–P) In vitro phagocytosis assay using pHrodo labeled E.coli bioparticles. Results are the means (±SD) of three independent experiments. *p < 0.01 versus WT neutrophils (Student’s t test). Scale bars in L represent 10 µm. Scale bars in O represent 5 µm.

Figure 7. Disruption of Grx1 led to reduced neutrophil recruitment and impaired bacterial killing

(A) Disruption of Grx1 led to reduced neutrophil accumulation in the inflamed peritoneal cavity. Data shown are mean ± SD of n=3 mice. *p < 0.01 by Student's t test. (B) Disruption of Grx1 led to impaired bacterial killing. Images of representative culture plates are shown. (C) Total numbers of survived E.coli in the inflamed peritoneal cavity. Data are means ± SD of 4 independent experiments. *p < 0.01 versus WT mice by Student's t test. (D) Schematic diagram of neutrophil adoptive transfer assay. Bone marrow neutrophils from WT and Grx1^−/− mice were labeled with different colors (SNARF1 or CFSE labeled), mixed 1:1 and then intravenously injected into WT recipient mice that have been challenged with E.coli (intraperitoneally injected). The relative recruitment of Grx1^−/− and WT neutrophils in the WT recipient was evaluated 60 min after the challenge. (E) The amount of adoptively transferred neutrophils recruited to the peritoneal cavity. Shown are representative FACS plots of an input control (left) and a mouse transplanted with labeled neutrophils (right). The double negative cells are the unlabeled endogenous cells. (F) Relative recruitment of neutrophil was calculated as the ratio of indicated populations in the peritoneal cavity. Results are the means (±SD) of three independent experiments. *, p < 0.01 versus WT neutrophils (Student’s t test).