Mouse Macrophages Completely Lacking Rho Subfamily GTPases (RhoA, RhoB, and RhoC) Have Severe Lamellipodial Retraction Defects, but Robust Chemotactic Navigation and Altered Motility

Königs, Volker; Jennings, Richard T.; Vogl, Thomas; Horsthemke, Markus; Bachg, Anne C.; Xu, Yan; Grobe, Kay; Brakebusch, Cord; Schwab, Albrecht; Bähler, Martin; Knaus, Ulla G.; Hanley, Peter J.

doi:10.1074/jbc.m114.563270

Cited by 42 publications

(40 citation statements)

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“…We recently demonstrated that the inhibition of the RhoA-ROCK1 pathway induces Nfix expression in fetal myoblasts and numerous studies have shown that the inhibition of RhoA-ROCK1 increases the phagocytosis, while its stimulation prevents phagocytosis [39][40][41][42][43]. Thus, we treated WT MPs with Y27632, an inhibitor of ROCK1, and after 1 h of treatment, the phosphorylation of ROCK1-target Mypt decreased, meaning that the inhibition of RhoA-ROCK1 pathway was effective ( Figure S5e).…”

Section: Phagocytosis Induces the Expression Of Nfixmentioning

confidence: 85%

The Transcription Factor Nfix Requires RhoA-ROCK1 Dependent Phagocytosis to Mediate Macrophage Skewing during Skeletal Muscle Regeneration

Saclier

Lapi

Bonfanti

et al. 2020

Cells

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Macrophages (MPs) are immune cells which are crucial for tissue repair. In skeletal muscle regeneration, pro-inflammatory cells first infiltrate to promote myogenic cell proliferation, then they switch into an anti-inflammatory phenotype to sustain myogenic cells differentiation and myofiber formation. This phenotypical switch is induced by dead cell phagocytosis. We previously demonstrated that the transcription factor Nfix, a member of the nuclear factor I (Nfi) family, plays a pivotal role during muscle development, regeneration and in the progression of muscular dystrophies. Here, we show that Nfix is mainly expressed by anti-inflammatory macrophages. Upon acute injury, mice deleted for Nfix in myeloid line displayed a significant defect in the process of muscle regeneration. Indeed, Nfix is involved in the macrophage phenotypical switch and macrophages lacking Nfix failed to adopt an anti-inflammatory phenotype and interact with myogenic cells. Moreover, we demonstrated that phagocytosis induced by the inhibition of the RhoA-ROCK1 pathway leads to Nfix expression and, consequently, to acquisition of the anti-inflammatory phenotype. Our study identified Nfix as a link between RhoA-ROCK1-dependent phagocytosis and the MP phenotypical switch, thus establishing a new role for Nfix in macrophage biology for the resolution of inflammation and tissue repair.

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Section: Phagocytosis Induces the Expression Of Nfixmentioning

confidence: 85%

The Transcription Factor Nfix Requires RhoA-ROCK1 Dependent Phagocytosis to Mediate Macrophage Skewing during Skeletal Muscle Regeneration

Saclier

Lapi

Bonfanti

et al. 2020

Cells

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“…28 RhoA-deficient mice are not viable but floxed mice exist that have been used to create different RhoAdeficient cell types such as fibroblasts, keratinocytes, and macrophages. 29,30 However, intestinal-specific RhoA KO mice have not been described. Analyzing colitis development using such mice will definitely be an attractive in vivo approach to analyze Rho/ROCK signaling in intestinal barrier regulation in the future.…”

Section: Discussionmentioning

confidence: 99%

Cortactin deficiency causes increased RhoA/ROCK1-dependent actomyosin contractility, intestinal epithelial barrier dysfunction, and disproportionately severe DSS-induced colitis

et al. 2017

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The intestinal epithelium constitutes a first line of defense of the innate immune system. Epithelial dysfunction is a hallmark of intestinal disorders such as inflammatory bowel diseases (IBDs). The actin cytoskeleton controls epithelial barrier integrity but the function of actin regulators such as cortactin is poorly understood. Given that cortactin controls endothelial permeability, we hypothesized that cortactin is also important for epithelial barrier regulation. We found increased permeability in the colon of cortactin-KO mice that was accompanied by reduced levels of ZO-1, claudin-1, and E-cadherin. By contrast, claudin-2 was upregulated. Cortactin deficiency increased RhoA/ROCK1-dependent actomyosin contractility, and inhibition of ROCK1 rescued the barrier defect. Interestingly, cortactin deficiency caused increased epithelial proliferation without affecting apoptosis. KO mice did not develop spontaneous colitis, but were more susceptible to dextran sulfate sodium colitis and showed severe colon tissue damage and edema formation. KO mice with colitis displayed strong mucus deposition and goblet cell depletion. In healthy human colon tissues, cortactin co-localized with ZO-1 at epithelial cell contacts. In IBDs patients, we observed decreased cortactin levels and loss of co-localization with ZO-1. Thus, cortactin is a master regulator of intestinal epithelial barrier integrity in vivo and could serve as a suitable target for pharmacological intervention in IBDs.

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“…Using this system and time-lapse, phase-contrast microscopy, we developed an assay to image mouse resident peritoneal macrophages migrating in a chemotactic complement C5a gradient 31,34,35,36 . This assay, combined with knockout mouse models, proved instrumental in the investigation of the roles of various Rho GTPases and motor proteins in macrophage morphology, motility, and chemotaxis 31,34,35,36,37 . We have also used this approach to image human peripheral blood monocytes migrating on a 2D surface or in a 3D collagen type I matrix…”

mentioning

confidence: 99%

Time-lapse Imaging of Mouse Macrophage Chemotaxis

Bos

Walbaum

Horsthemke

et al. 2020

JoVE

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Chemotaxis is receptor-mediated guidance of cells along a chemical gradient, whereas chemokinesis is the stimulation of random cell motility by a chemical. Chemokinesis and chemotaxis are fundamental for the mobilization and deployment of immune cells. For example, chemokines (chemotactic cytokines) can rapidly recruit circulating neutrophils and monocytes to extravascular sites of inflammation. Chemoattractant receptors belong to the large family of G protein-coupled receptors. How chemoattractant (i.e., ligand) gradients direct cell migration via G protein-coupled receptor signaling is not yet fully understood. In the field of immunology, neutrophils are popular model cells for studying chemotaxis in vitro. Here we describe a real-time two-dimensional (2D) chemotaxis assay tailored for mouse resident macrophages, which have traditionally been more difficult to study. Macrophages move at a slow pace of ~1 µm/min on a 2D surface and are less well suited for point-source migration assays (e.g., migration towards the tip of a micropipette filled with chemoattractant) than neutrophils or Dictyostelium discoideum, which move an order of magnitude faster. Widely used Transwell assays are useful for studying the chemotactic activity of different substances, but do not provide information on cell morphology, velocity, or chemotactic navigation. Here we describe a time-lapse microscopybased macrophage chemotaxis assay that allows quantification of cell velocity and chemotactic efficiency and provides a platform to delineate the transducers, signal pathways, and effectors of chemotaxis. Video Link The video component of this article can be found at https://www.jove.com/video/60750/ 17. At that time, the molecular identities of chemotactic factors were unknown. In the 1960s, Stephen Boyden 18 recognized that techniques to study the chemotactic activity of soluble substances were lacking. He devised a chamber, subsequently known as the Boyden chamber, with two compartments separated by a filter paper membrane. A cell

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Mouse Macrophages Completely Lacking Rho Subfamily GTPases (RhoA, RhoB, and RhoC) Have Severe Lamellipodial Retraction Defects, but Robust Chemotactic Navigation and Altered Motility

Cited by 42 publications

References 50 publications

The Transcription Factor Nfix Requires RhoA-ROCK1 Dependent Phagocytosis to Mediate Macrophage Skewing during Skeletal Muscle Regeneration

The Transcription Factor Nfix Requires RhoA-ROCK1 Dependent Phagocytosis to Mediate Macrophage Skewing during Skeletal Muscle Regeneration

Cortactin deficiency causes increased RhoA/ROCK1-dependent actomyosin contractility, intestinal epithelial barrier dysfunction, and disproportionately severe DSS-induced colitis

Time-lapse Imaging of Mouse Macrophage Chemotaxis

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