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Cited by 15Neutrophil, Myeloperoxidase and Oxidative MechanismsProtein Kinase Regulation and GTPase SignalingS100 Proteins and Annexins

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Nascent RHOH acts as a molecular brake on actomyosin-mediated effector functions of inflammatory neutrophils

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Peng, Shuang, Stojkov, Darko, Gao, Jian, Oberson, Kevin, Latzin, Philipp, Casaulta, Carmen, Yousefi, Shida, Simon, Hans-Uwe·2022·PLOS Biology
DOI 10.1371/journal.pbio.3001794PMC PMC9514642
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Abstract

In contrast to molecular changes associated with increased inflammatory responses, little is known about intracellular counter-regulatory mechanisms that control signaling cascades associated with functional responses of neutrophils. Active RHO GTPases are typically considered as effector proteins that elicit cellular responses. Strikingly, we show here that RHOH, although being constitutively GTP-bound, limits neutrophil degranulation and the formation of neutrophil extracellular traps (NETs). Mechanistically, RHOH is induced under inflammatory conditions and binds to non-muscle myosin heavy chain IIA (NMHC IIA) in activated neutrophils in order to inhibit the transport of mitochondria and granules along actin filaments, which is partially reverted upon disruption of the interaction with NMHC IIA by introducing a mutation in RhoH at lysine 34 (RhoH K34A). In parallel, RHOH inhibits actin polymerization presumably by modulating RAC1 activity. In vivo studies using Rhoh-/- mice, demonstrate an increased antibacterial defense capability against Escherichia coli (E. coli). Collectively, our data reveal a previously undefined role of RHOH as a molecular brake for actomyosin-mediated neutrophil effector functions, which represents an intracellular regulatory axis involved in controlling the strength of an antibacterial inflammatory response.

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TL;DR

RHOH is induced in neutrophils by inflammatory signals and acts as a molecular brake on degranulation and NET formation by binding NMHC IIA and inhibiting actomyosin-dependent organelle transport and Rac1-driven actin polymerization. Rhoh-/- mice show enhanced NET formation and E. coli killing in experimental peritonitis.

Key findings

  • RHOH expression is barely detectable in freshly isolated human and mouse neutrophils but is up-regulated by GM-CSF in less than 3 h; high RHOH is also detected in neutrophils from COVID-19 patients and mouse tumor-infiltrating neutrophils by scRNA-seq. CF neutrophils show high RHOH, impaired degranulation (reduced CD63/CD66b and elastase activity), and no detectable NETs/dsDNA release.
  • RhoH interacts with non-muscle myosin heavy chain IIA (NMHC IIA; Myh9/myosin-9) in activated mouse and human neutrophils; mass spectrometry identified NMHC IIA with 45.1% coverage. The RhoH K34A mutation decreases this interaction, restores granule/mitochondria binding to F-actin, and partially rescues degranulation and dsDNA release.
  • RhoH overexpression reduces F-actin formation and Rac1 activity, but not Rac2, Cdc42, or RhoA activation; K34A abolishes the Rac1 inhibition. RhoH does not affect myosin IIA phosphorylation at T18/S19 or S1943.
  • In E. coli-induced peritonitis, Rhoh-/- mice had significantly reduced bacterial loads at 8 h, higher dsDNA and neutrophil elastase in peritoneal lavage fluid, and enhanced NET formation, with no significant difference in absolute neutrophil numbers; n = 10 per group unless mentioned otherwise.

Methods

  • Human blood neutrophils from healthy donors and CF patients, mouse bone marrow neutrophils from WT and Rhoh-/- mice, and SCF-dependent HoxB8-immortalized myeloid progenitors reconstituted with HA-RhoH WT, K34A, Y33F, or double mutants, or Myh9 shRNA. Functional assays included flow cytometry for degranulation and ROS, PicoGreen dsDNA quantification, confocal microscopy for NETs/F-actin/microtubules, immunoblotting, co-immunoprecipitation, mass spectrometry, granule/mitochondria fractionation, actin-binding centrifugation, GTPase pulldown, and molecular docking.
  • In vivo model: 50 × 10^6 E. coli-GFP injected intraperitoneally into WT and Rhoh-/- mice; mice sacrificed at 8 h; bacterial burden measured as CFU, peritoneal lavage fluid assayed for dsDNA and NE activity, and peritoneal cells analyzed by flow cytometry and immunoblotting.
  • Statistics: data presented as means ± SD; unpaired two-tailed Student t test, one-way or two-way ANOVA as indicated; P < 0.05 considered significant.

Limitations

  • Stated: COVID-19 restrictions limited the sample size for CF patient experiments; the molecular mechanism inducing RhoH expression remains unidentified; the mechanism by which RhoH suppresses Rac1 activation is unresolved; the authors cannot rule out NMHC IIA involvement in Rac1 activation; other myosin proteins may participate in organelle translocation.
  • Evident from the text: many mechanistic assays rely on HoxB8 overexpression/reconstitution rather than primary human neutrophils, and several human CF comparisons use small sample sizes (n = 3–4 in Fig 1). The in vivo validation uses a single E. coli peritonitis model and a single 8 h time point.

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Crossref · 10.1371/journal.pbio.3001794

Cites

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Figures

Fig 1 Neutrophils from CF patients exhibit enhanced expression of RHOH associated with reduced degranulation and NET formation. (A) Representative images of neutrophils from HD and CF patients (n = 4). (B) RHOH expression in neutrophils from HD and CF patients were analyzed by immunoblotting. (C–H) Freshly isolated neutrophils from HD and CF patients were treated with the indicated stimuli. (C) ROS activity was assessed by flow cytometry (n = 4). (D) Neutrophil degranulation was determined by flow cytometry (n = 3, sample number: 1, 2, 3). (E) Quantification of released NE in the culture supernatants (n = 3, sample number: 1, 2, 3). (F) Extracellular DNA fibers were stained with MitoSOX Red and the nucleus with Hoechst 33342 and analyzed by confocal microscopy (n = 3, sample number: 1, 2, 3). Scale bars, 10 μm. (G) Quantification of released dsDNA in the culture supernatants (n = 4). (H) NET formation indicated by the colocalization of NE (green) with released DNA (PI, red) was analyzed by confocal microscopy (n = 3, sample number: 1, 2, 4). Scale bars, 10 μm. Values are means ± SD. Two-way ANOVA with Tukey’s multiple comparison post-test was applied. The underlying data for Fig 1C–E and 1G can be found in S1 Data. The underlying data for Fig 1B can be found in S1 Raw images. AU, arbitrary units; CF, cystic fibrosis; dsDNA, double-stranded DNA; MFI, mean fluorescence intensity; NET, neutrophil extracellular trap; PI, propidium iodide; RFU, relative fluorescence units.
Fig 1 Neutrophils from CF patients exhibit enhanced expression of RHOH associated with reduced degranulation and NET formation. (A) Representative images of neutrophils from HD and CF patients (n = 4). (B) RHOH expression in neutrophils from HD and CF patients were analyzed by immunoblotting. (C–H) Freshly isolated neutrophils from HD and CF patients were treated with the indicated stimuli. (C) ROS activity was assessed by flow cytometry (n = 4). (D) Neutrophil degranulation was determined by flow cytometry (n = 3, sample number: 1, 2, 3). (E) Quantification of released NE in the culture supernatants (n = 3, sample number: 1, 2, 3). (F) Extracellular DNA fibers were stained with MitoSOX Red and the nucleus with Hoechst 33342 and analyzed by confocal microscopy (n = 3, sample number: 1, 2, 3). Scale bars, 10 μm. (G) Quantification of released dsDNA in the culture supernatants (n = 4). (H) NET formation indicated by the colocalization of NE (green) with released DNA (PI, red) was analyzed by confocal microscopy (n = 3, sample number: 1, 2, 4). Scale bars, 10 μm. Values are means ± SD. Two-way ANOVA with Tukey’s multiple comparison post-test was applied. The underlying data for Fig 1C–E and 1G can be found in S1 Data. The underlying data for Fig 1B can be found in S1 Raw images. AU, arbitrary units; CF, cystic fibrosis; dsDNA, double-stranded DNA; MFI, mean fluorescence intensity; NET, neutrophil extracellular trap; PI, propidium iodide; RFU, relative fluorescence units.CC BY · PubMed Central
Fig 2 Induction of RhoH expression by GM-CSF stimulation or overexpression of HA-RhoH impairs neutrophil degranulation and dsDNA release. (A) Neutrophils purified from bone marrow of WT and Rhoh-/- mice were incubated with GM-CSF for the indicated times followed by immunoblotting. (B–D) Neutrophils from WT and Rhoh-/- were pretreated with GM-CSF for the indicated times followed by activation with C5a for 15 min. (B) Neutrophil degranulation was determined by flow cytometry. (C) Extracellular DNA fibers were analyzed by confocal microscopy. Scale bars, 10 μm. (D) Quantification of released dsDNA in the culture supernatants. (E) Expression of RhoH in the indicated HoxB8 neutrophils was analyzed by immunoblotting. (F–H) Mature HoxB8 neutrophils were treated with the indicated stimuli. (F) Neutrophil degranulation was determined by flow cytometry. (G) Extracellular DNA fibers were analyzed by confocal microscopy. Scale bars, 10 μm. (H) Quantification of released dsDNA in culture supernatants. (A, C, E, G) Similar results were obtained from 3 independent experiments. (B, D, F, H) Each symbol represents a single experiment. Values are means ± SD. (B, D) Two-way ANOVA with Tukey’s multiple comparisons test. (F, H) Two-way ANOVA with Šídák’s multiple comparisons test. The underlying data for Fig 2B, 2D, 2F, and 2H can be found in S1 Data. The underlying data for Fig 2A and 2E can be found in S1 Raw images. dsDNA, double-stranded DNA; GM-CSF, granulocyte/macrophage colony-stimulating factor; WT, wild-type.
Fig 2 Induction of RhoH expression by GM-CSF stimulation or overexpression of HA-RhoH impairs neutrophil degranulation and dsDNA release. (A) Neutrophils purified from bone marrow of WT and Rhoh-/- mice were incubated with GM-CSF for the indicated times followed by immunoblotting. (B–D) Neutrophils from WT and Rhoh-/- were pretreated with GM-CSF for the indicated times followed by activation with C5a for 15 min. (B) Neutrophil degranulation was determined by flow cytometry. (C) Extracellular DNA fibers were analyzed by confocal microscopy. Scale bars, 10 μm. (D) Quantification of released dsDNA in the culture supernatants. (E) Expression of RhoH in the indicated HoxB8 neutrophils was analyzed by immunoblotting. (F–H) Mature HoxB8 neutrophils were treated with the indicated stimuli. (F) Neutrophil degranulation was determined by flow cytometry. (G) Extracellular DNA fibers were analyzed by confocal microscopy. Scale bars, 10 μm. (H) Quantification of released dsDNA in culture supernatants. (A, C, E, G) Similar results were obtained from 3 independent experiments. (B, D, F, H) Each symbol represents a single experiment. Values are means ± SD. (B, D) Two-way ANOVA with Tukey’s multiple comparisons test. (F, H) Two-way ANOVA with Šídák’s multiple comparisons test. The underlying data for Fig 2B, 2D, 2F, and 2H can be found in S1 Data. The underlying data for Fig 2A and 2E can be found in S1 Raw images. dsDNA, double-stranded DNA; GM-CSF, granulocyte/macrophage colony-stimulating factor; WT, wild-type.CC BY · PubMed Central
Fig 3 RhoH interacts with NMHC IIA. (A) Coomassie blue staining of HA pulldown proteins in HoxB8 neutrophils after stimulation with the indicated triggers. The proteins interacting with RhoH were identified by mass spectrometry analysis. (B, C) Immunoblot analysis after Co-IP with anti-HA antibody in Hoxb8 neutrophils (B) and anti-NMHC IIA antibody in human neutrophils (C). Quantification of the precipitated NMHC IIA (B) or RHOH (C) was shown below the representative images. (D–F) Pretreated human neutrophils with vehicle control or myosin IIA inhibitors for 30 min were subsequently activated with GM-CSF in combination with C5a. (D) Neutrophil degranulation was determined by flow cytometry. (E) Quantification of dsDNA released into the supernatants. (F) NET formation indicated by the colocalization of NE (green) with released DNA (PI, red) was analyzed by confocal microscopy. Scale bars, 10 μm. (G) Protein expression of NMHC IIA in mature HoxB8 neutrophils treated with control (Ctrl) or Myh9 shRNA was analyzed by immunoblot. (H, I) Mature HoxB8 neutrophils were treated as indicated. (H) Neutrophil degranulation was determined by flow cytometry. (I) Quantification of released dsDNA in culture supernatants. (J) Extracellular DNA fibers were analyzed by confocal microscopy. Scale bars, 10 μm. (A–C, F, G, J) Three independent experiments were performed. (B–E, H, I) Values represent means ± SD. (B–E) One-way ANOVA with Dunnett’s multiple comparisons test. (H, I) Two-way ANOVA with Šídák’s multiple comparisons test. The underlying numerical data for Fig 3B–E, 3H, and 3I can be found in S1 Data. The uncropped immunoblots for Fig 3A–C and 3G can be found in S1 Raw images. Co-IP, co-immunoprecipitation; dsDNA, double-stranded DNA; GM-CSF, granulocyte/macrophage colony-stimulating factor; NE, neutrophil elastase; NET, neutrophil extracellular trap; NMHC IIA, non-muscle myosin heavy chain IIA; PI, propidium iodide.
Fig 3 RhoH interacts with NMHC IIA. (A) Coomassie blue staining of HA pulldown proteins in HoxB8 neutrophils after stimulation with the indicated triggers. The proteins interacting with RhoH were identified by mass spectrometry analysis. (B, C) Immunoblot analysis after Co-IP with anti-HA antibody in Hoxb8 neutrophils (B) and anti-NMHC IIA antibody in human neutrophils (C). Quantification of the precipitated NMHC IIA (B) or RHOH (C) was shown below the representative images. (D–F) Pretreated human neutrophils with vehicle control or myosin IIA inhibitors for 30 min were subsequently activated with GM-CSF in combination with C5a. (D) Neutrophil degranulation was determined by flow cytometry. (E) Quantification of dsDNA released into the supernatants. (F) NET formation indicated by the colocalization of NE (green) with released DNA (PI, red) was analyzed by confocal microscopy. Scale bars, 10 μm. (G) Protein expression of NMHC IIA in mature HoxB8 neutrophils treated with control (Ctrl) or Myh9 shRNA was analyzed by immunoblot. (H, I) Mature HoxB8 neutrophils were treated as indicated. (H) Neutrophil degranulation was determined by flow cytometry. (I) Quantification of released dsDNA in culture supernatants. (J) Extracellular DNA fibers were analyzed by confocal microscopy. Scale bars, 10 μm. (A–C, F, G, J) Three independent experiments were performed. (B–E, H, I) Values represent means ± SD. (B–E) One-way ANOVA with Dunnett’s multiple comparisons test. (H, I) Two-way ANOVA with Šídák’s multiple comparisons test. The underlying numerical data for Fig 3B–E, 3H, and 3I can be found in S1 Data. The uncropped immunoblots for Fig 3A–C and 3G can be found in S1 Raw images. Co-IP, co-immunoprecipitation; dsDNA, double-stranded DNA; GM-CSF, granulocyte/macrophage colony-stimulating factor; NE, neutrophil elastase; NET, neutrophil extracellular trap; NMHC IIA, non-muscle myosin heavy chain IIA; PI, propidium iodide.CC BY · PubMed Central
Fig 4 NMHC IIA associates with neutrophil organelles and mediates their binding to actin filaments. (A) Phosphorylation of myosin IIA in human neutrophils treated as indicated was analyzed by immunoblotting. Threonine 18 (T18); Serine 19 (S19); Serine 1943 (S1943). (B–D) The PNL from unstimulated or GM-CSF primed and C5a-activated human neutrophils was isolated and collected equally into 1–10 fractions, from top to bottom. (B) Immunoblot analysis of density gradient fractions. (C) NE activity of density gradient fractions. (D) Bacteria killing mediated by density gradient fractions. (E) Immunoblot analysis of NMHC IIA in cytosolic and mitochondrial fractions from control and activated human neutrophils. C, cytosolic fraction. M, mitochondrial fraction. (F) Granular or mitochondrial localization of NMHC IIA in unstimulated and activated human neutrophils was analyzed by confocal microscopy. Scale bars, 10 μm. Numerical analysis was performed on 10 cells in each group and the Pearson’s coefficient in colocalized volume between NMHC IIA and CD63 or MTO was calculated using Imaris software. (G) Enriched granules and mitochondria from mature HoxB8 neutrophils treated with control (Ctrl) or Myh9 shRNA were incubated for 30 min in the presence or absence of F-actin. The pellet (P) and supernatant (S) were evaluated by immunoblotting (left). The ratios of F-actin in the pellet (P) to the supernatant (S) were quantified (right). (A, B, E–G) Data are representative of 3 independent experiments. (C, D, F, G) Values are means ± SD. (F, G) Unpaired 2-tailed Student t test was applied. The underlying numerical data for Fig 4C, 4D, 4F, and 4G can be found in S1 Data. The uncropped immunoblots for Fig 4A, 4B, 4E, and 4G can be found in S1 Raw images. GM-CSF, granulocyte/macrophage colony-stimulating factor; NE, neutrophil elastase; NMHC IIA, non-muscle myosin heavy chain IIA; PNL, postnuclear lysate.
Fig 4 NMHC IIA associates with neutrophil organelles and mediates their binding to actin filaments. (A) Phosphorylation of myosin IIA in human neutrophils treated as indicated was analyzed by immunoblotting. Threonine 18 (T18); Serine 19 (S19); Serine 1943 (S1943). (B–D) The PNL from unstimulated or GM-CSF primed and C5a-activated human neutrophils was isolated and collected equally into 1–10 fractions, from top to bottom. (B) Immunoblot analysis of density gradient fractions. (C) NE activity of density gradient fractions. (D) Bacteria killing mediated by density gradient fractions. (E) Immunoblot analysis of NMHC IIA in cytosolic and mitochondrial fractions from control and activated human neutrophils. C, cytosolic fraction. M, mitochondrial fraction. (F) Granular or mitochondrial localization of NMHC IIA in unstimulated and activated human neutrophils was analyzed by confocal microscopy. Scale bars, 10 μm. Numerical analysis was performed on 10 cells in each group and the Pearson’s coefficient in colocalized volume between NMHC IIA and CD63 or MTO was calculated using Imaris software. (G) Enriched granules and mitochondria from mature HoxB8 neutrophils treated with control (Ctrl) or Myh9 shRNA were incubated for 30 min in the presence or absence of F-actin. The pellet (P) and supernatant (S) were evaluated by immunoblotting (left). The ratios of F-actin in the pellet (P) to the supernatant (S) were quantified (right). (A, B, E–G) Data are representative of 3 independent experiments. (C, D, F, G) Values are means ± SD. (F, G) Unpaired 2-tailed Student t test was applied. The underlying numerical data for Fig 4C, 4D, 4F, and 4G can be found in S1 Data. The uncropped immunoblots for Fig 4A, 4B, 4E, and 4G can be found in S1 Raw images. GM-CSF, granulocyte/macrophage colony-stimulating factor; NE, neutrophil elastase; NMHC IIA, non-muscle myosin heavy chain IIA; PNL, postnuclear lysate.CC BY · PubMed Central
Fig 5 RhoH inhibits NMHC IIA-mediated granules and mitochondria binding to F-actin. (A) Enriched granules and mitochondria from mature HoxB8 neutrophils expressing HA-RhoH or the corresponding EV were incubated for 30 min in the presence or absence of F-actin. After centrifugation, the pellet (P) and supernatant (S) were evaluated by immunoblotting (left). The ratios of F-actin in the pellet (P) to the supernatant (S) were quantified (right). (B) Docking analysis for predicting the binding sites between RHOH and NMHC IIA (upper). Sequence alignment of RAC1, CDC42, RHOA, and RHOH (lower). (C) Immunoblot analysis of Co-IP with anti-HA antibody in cell lysates from activated HoxB8 neutrophils expressing WT or mutated HA-RhoH. Quantification of the precipitated NMHC IIA was shown below the representative images. (D) Enriched granules and mitochondria were incubated for 30 min in the presence or absence of F-actin. The pellet (P) and supernatant (S) were evaluated by immunoblotting (left). The ratios of F-actin in the pellet (P) to the supernatant (S) were quantified (right). (E–G) Mature HoxB8 neutrophils expressing WT or mutated HA-RhoH or EV were treated as indicated. (E) Neutrophil degranulation was determined by flow cytometry. (F) Extracellular DNA fibers were analyzed by confocal microscopy. Scale bars, 10 μm. (G) Quantification of dsDNA released into the supernatants. (A, C, D, F) Data are representative of 3 independent experiments. Values are means ± SD. Two-tailed Student t test (A, D); 1-way ANOVA with Dunnett’s multiple comparisons test (C); 2-way ANOVA with Tukey’s multiple comparisons test (E, G). The underlying numerical data for Fig 5A, 5C–E, and 5G can be found in S1 Data. The uncropped immunoblots for Fig 5A, 5C, and 5D can be found in S1 Raw images. Co-IP, co-immunoprecipitation; dsDNA, double-stranded DNA; EV, empty vector; NMHC IIA, non-muscle myosin heavy chain IIA; WT, wild-type.
Fig 5 RhoH inhibits NMHC IIA-mediated granules and mitochondria binding to F-actin. (A) Enriched granules and mitochondria from mature HoxB8 neutrophils expressing HA-RhoH or the corresponding EV were incubated for 30 min in the presence or absence of F-actin. After centrifugation, the pellet (P) and supernatant (S) were evaluated by immunoblotting (left). The ratios of F-actin in the pellet (P) to the supernatant (S) were quantified (right). (B) Docking analysis for predicting the binding sites between RHOH and NMHC IIA (upper). Sequence alignment of RAC1, CDC42, RHOA, and RHOH (lower). (C) Immunoblot analysis of Co-IP with anti-HA antibody in cell lysates from activated HoxB8 neutrophils expressing WT or mutated HA-RhoH. Quantification of the precipitated NMHC IIA was shown below the representative images. (D) Enriched granules and mitochondria were incubated for 30 min in the presence or absence of F-actin. The pellet (P) and supernatant (S) were evaluated by immunoblotting (left). The ratios of F-actin in the pellet (P) to the supernatant (S) were quantified (right). (E–G) Mature HoxB8 neutrophils expressing WT or mutated HA-RhoH or EV were treated as indicated. (E) Neutrophil degranulation was determined by flow cytometry. (F) Extracellular DNA fibers were analyzed by confocal microscopy. Scale bars, 10 μm. (G) Quantification of dsDNA released into the supernatants. (A, C, D, F) Data are representative of 3 independent experiments. Values are means ± SD. Two-tailed Student t test (A, D); 1-way ANOVA with Dunnett’s multiple comparisons test (C); 2-way ANOVA with Tukey’s multiple comparisons test (E, G). The underlying numerical data for Fig 5A, 5C–E, and 5G can be found in S1 Data. The uncropped immunoblots for Fig 5A, 5C, and 5D can be found in S1 Raw images. Co-IP, co-immunoprecipitation; dsDNA, double-stranded DNA; EV, empty vector; NMHC IIA, non-muscle myosin heavy chain IIA; WT, wild-type.CC BY · PubMed Central
Fig 6 RhoH impairs actin rearrangement presumably by modulating Rac1 activity. (A, B) Mature HoxB8 neutrophils expressing WT or mutated HA-RhoH or EV were treated as indicated. (A) F-actin distribution was analyzed by confocal microscopy (left). Scale bars, 10 μm. Quantification of F-actin fluorescence intensity was performed by automated analysis of microscopic images using Imaris software. The 25 images (each containing 8–12 cells) from 3 independent experiments were included for each condition (right). (B) Phosphorylation of myosin IIA was analyzed by immunoblotting. (C, D) The levels of GTP-bound and total Rac1 and Rac2 proteins in the indicated mouse bone marrow (BM) neutrophils (C) and HoxB8 neutrophils (D) were examined by immunoblotting following pulldown assay. All data are representative of 3 independent experiments. Values are means ± SD. One-way ANOVA with Tukey’s multiple comparisons test was applied. The underlying numerical data for Fig 6A–D can be found in S1 Data. The uncropped immunoblots for Fig 6B–D can be found in S1 Raw images. EV, empty vector; WT, wild-type.
Fig 6 RhoH impairs actin rearrangement presumably by modulating Rac1 activity. (A, B) Mature HoxB8 neutrophils expressing WT or mutated HA-RhoH or EV were treated as indicated. (A) F-actin distribution was analyzed by confocal microscopy (left). Scale bars, 10 μm. Quantification of F-actin fluorescence intensity was performed by automated analysis of microscopic images using Imaris software. The 25 images (each containing 8–12 cells) from 3 independent experiments were included for each condition (right). (B) Phosphorylation of myosin IIA was analyzed by immunoblotting. (C, D) The levels of GTP-bound and total Rac1 and Rac2 proteins in the indicated mouse bone marrow (BM) neutrophils (C) and HoxB8 neutrophils (D) were examined by immunoblotting following pulldown assay. All data are representative of 3 independent experiments. Values are means ± SD. One-way ANOVA with Tukey’s multiple comparisons test was applied. The underlying numerical data for Fig 6A–D can be found in S1 Data. The uncropped immunoblots for Fig 6B–D can be found in S1 Raw images. EV, empty vector; WT, wild-type.CC BY · PubMed Central
Fig 7 Rhoh-/- neutrophils display enhanced NET formation and bacteria killing in experimental peritonitis. (A) Peritoneal cells from WT mice challenged intraperitoneally with E. coli was analyzed by immunoblot. Protein lysates of bone marrow (BM) neutrophils from WT and Rhoh-/- mice were loaded as negative control. Data were representative of 3 independent experiments. (B–I) WT and Rhoh-/- mice were challenged intraperitoneally with E. coli and sacrificed after 8 h; n = 10 for each group unless mentioned otherwise. (B) Bacteria burden was quantified as CFU. (C) Quantification of released dsDNA in PLF supernatants. (D) Quantification of NE activity in PLF supernatants. (E) Total peritoneal cell count. (F) Infiltrated peritoneal neutrophils were defined as CD45+ and Ly6G+. Left, representative original flow cytometry data. Right, proportion of peritoneal neutrophils. (G) Number of peritoneal neutrophils. (H) Protein level of RhoH in peritoneal cells was analyzed by immunoblot (n = 5). (I) Representative confocal images of peritoneal cells stained with MitoSOX Red and Hoechst 33342, left. Scale bars, 10 μm. Quantification of cells releasing dsDNA fibers was performed in 2 randomly selected images from each mouse, right. Values are means ± SD. Statistical analyses were performed using unpaired 2-tailed Student t test. The underlying data for Fig 7B–E, 7G, and 7I can be found in S1 Data. The underlying data for Fig 7A and 7H can be found in S1 Raw images. CFU, colony-forming unit; dsDNA, double-stranded DNA; NE, neutrophil elastase; NET, neutrophil extracellular trap; PLF, peritoneal lavage fluid; WT, wild-type.
Fig 7 Rhoh-/- neutrophils display enhanced NET formation and bacteria killing in experimental peritonitis. (A) Peritoneal cells from WT mice challenged intraperitoneally with E. coli was analyzed by immunoblot. Protein lysates of bone marrow (BM) neutrophils from WT and Rhoh-/- mice were loaded as negative control. Data were representative of 3 independent experiments. (B–I) WT and Rhoh-/- mice were challenged intraperitoneally with E. coli and sacrificed after 8 h; n = 10 for each group unless mentioned otherwise. (B) Bacteria burden was quantified as CFU. (C) Quantification of released dsDNA in PLF supernatants. (D) Quantification of NE activity in PLF supernatants. (E) Total peritoneal cell count. (F) Infiltrated peritoneal neutrophils were defined as CD45+ and Ly6G+. Left, representative original flow cytometry data. Right, proportion of peritoneal neutrophils. (G) Number of peritoneal neutrophils. (H) Protein level of RhoH in peritoneal cells was analyzed by immunoblot (n = 5). (I) Representative confocal images of peritoneal cells stained with MitoSOX Red and Hoechst 33342, left. Scale bars, 10 μm. Quantification of cells releasing dsDNA fibers was performed in 2 randomly selected images from each mouse, right. Values are means ± SD. Statistical analyses were performed using unpaired 2-tailed Student t test. The underlying data for Fig 7B–E, 7G, and 7I can be found in S1 Data. The underlying data for Fig 7A and 7H can be found in S1 Raw images. CFU, colony-forming unit; dsDNA, double-stranded DNA; NE, neutrophil elastase; NET, neutrophil extracellular trap; PLF, peritoneal lavage fluid; WT, wild-type.CC BY · PubMed Central

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Metadata

Authors
Peng, S., Stojkov, D., Gao, J., Oberson, K., Latzin, P., Casaulta, C., Yousefi, S., & Simon, H.-U.
Affiliations
  • University of Bern (CH)
  • State Key Laboratory of Pharmaceutical Biotechnology (CN)
Corresponding author
Simon, Hans-Uwe
ORCID
Venue
PLOS Biology
Publisher
Public Library of Science (PLoS)
Published
2022-09-15
Volume
20
Issue
9
Pages
e3001794
ISSN
  • 1544-9173 (ISSN-L)
  • 1545-7885
DOI
10.1371/journal.pbio.3001794
PMC
PMC9514642
OpenAlex
W4295932318
Semantic Scholar
d5f73f24cfeba91bccb4c47210f12b9cb46d6bd4
Cited by
15
References
70
Citations by year
2026: 4 · 2025: 6 · 2024: 3 · 2023: 1 · 2022: 1
FWCI
1.03
Type
journal article
Language
en
Update notices
new_version (2022-09-27)
Open access
Licence
Locations
  • PLoS Biology (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Bern Open Repository and Information System (University of Bern) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Open Access CRIS of the University of Bern
Funders
  • Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (310030_184816, 31003A_173215)
  • Russian Government Program “Recruitment of the Leading Scientists into the Russian Institutions of Higher Education” (075-15-2021-600)
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › Neutrophil, Myeloperoxidase and Oxidative Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › S100 Proteins and Annexins
Keywords
RhoH, Rho GTPases, neutrophils, actomyosin, non-muscle myosin heavy chain IIA, neutrophil degranulation, neutrophil extracellular traps, mitochondrial transport, granule transport, actin polymerization, Rac1, antibacterial defense, Escherichia coli, inflammation
MeSH terms
Actins, Actomyosin, Animals, Anti-Bacterial Agents, Cytoskeletal Proteins, Escherichia coli, Guanosine Triphosphate, Lysine, Actin Cytoskeleton, Neutrophils, Transcription Factors, Myosin Heavy Chains, rho GTP-Binding Proteins, Mice

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

A language model drew the claims from the paper on 2026-11-12, under version 3 of the claim definition (model deepseek-v4.1-flash, prompt extract-v3).

A second model labelled each claim on 2026-11-12 (model jev-1.13-20260917, definition revision gold-guideline-v0.2).

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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