Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Wen, Xian‐Huan; Jin, Tian; Xu, Xuehua

doi:10.3791/54511

Cited by 7 publications

(7 citation statements)

References 20 publications

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“…HL-60 cells were maintained in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% FBS, and were incubated at 37°C in 5% CO 2 . HL-60 cells were differentiated into neutrophil-like cells for 5 days in culture media containing 1.2% DMSO (Collins et al, 1979; Wen et al, 2016). Cell differentiation was confirmed by nitro blue tetrazolium reduction assay (Collins et al, 1979).…”

Section: Methodsmentioning

confidence: 99%

Streptococcus pneumoniae Evades Host Cell Phagocytosis and Limits Host Mortality Through Its Cell Wall Anchoring Protein PfbA

Yamaguchi

Hirose

Takemura

et al. 2019

Front. Cell. Infect. Microbiol.

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Streptococcus pneumoniae is a Gram-positive bacterium belonging to the oral streptococcus species, mitis group. This pathogen is a leading cause of community-acquired pneumonia, which often evades host immunity and causes systemic diseases, such as sepsis and meningitis. Previously, we reported that PfbA is a β-helical cell surface protein contributing to pneumococcal adhesion to and invasion of human epithelial cells in addition to its survival in blood. In the present study, we investigated the role of PfbA in pneumococcal pathogenesis. Phylogenetic analysis indicated that the pfbA gene is highly conserved in S. pneumoniae and Streptococcus pseudopneumoniae within the mitis group. Our in vitro assays showed that PfbA inhibits neutrophil phagocytosis, leading to pneumococcal survival. We found that PfbA activates NF-κB through TLR2, but not TLR4. In addition, TLR2/4 inhibitor peptide treatment of neutrophils enhanced the survival of the S. pneumoniae Δ pfbA strain as compared to a control peptide treatment, whereas the treatment did not affect survival of a wild-type strain. In a mouse pneumonia model, the host mortality and level of TNF-α in bronchoalveolar lavage fluid were comparable between wild-type and Δ pfbA -infected mice, while deletion of pfbA decreased the bacterial burden in bronchoalveolar lavage fluid. In a mouse sepsis model, the Δ pfbA strain demonstrated significantly increased host mortality and TNF-α levels in plasma, but showed reduced bacterial burden in lung and liver. These results indicate that PfbA may contribute to the success of S. pneumoniae species by inhibiting host cell phagocytosis, excess inflammation, and mortality by interacting with TLR2.

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Section: Methodsmentioning

confidence: 99%

Streptococcus pneumoniae Evades Host Cell Phagocytosis and Limits Host Mortality Through Its Cell Wall Anchoring Protein PfbA

Yamaguchi

Hirose

Takemura

et al. 2019

Front. Cell. Infect. Microbiol.

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“…To precisely determine the function of C2GAP1 in chemotaxis in response to gradients at different concentrations, we monitored the chemotaxis behavior of WT and c2gapA − cells in the gradients with a wide concentration range (Figure 4A). For the experiments, we used EZ-TAXIScan to apply a well-controlled linear gradient (Wen et al, 2016). Without a gradient, c2gapA − cells displayed a bigger random walk than WT cells.…”

Section: Sensitivity Of C2gapa − Cells Is Increased In Response To Chemoattractant Stimulusmentioning

confidence: 99%

Membrane Targeting of C2GAP1 Enables Dictyostelium discoideum to Sense Chemoattractant Gradient at a Higher Concentration Range

Bhimani

Pots

et al. 2021

Front. Cell Dev. Biol.

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Chemotaxis, which is G protein-coupled receptor (GPCR)-mediated directional cell migration, plays pivotal roles in diverse human diseases, including recruitment of leukocytes to inflammation sites and metastasis of cancer. It is still not fully understood how eukaryotes sense and chemotax in response to chemoattractants with an enormous concentration range. A genetically traceable model organism, Dictyostelium discoideum, is the best-studied organism for GPCR-mediated chemotaxis. Recently, we have shown that C2GAP1 controls G protein coupled receptor-mediated Ras adaptation and chemotaxis. Here, we investigated the molecular mechanism and the biological function of C2GAP1 membrane targeting for chemotaxis. We show that calcium and phospholipids on the plasma membrane play critical roles in membrane targeting of C2GAP1. Cells lacking C2GAP1 (c2gapA–) displayed an improved chemotaxis in response to chemoattractant gradients at subsensitive or low concentrations (<100 nM), while exhibiting impaired chemotaxis in response to gradients at high concentrations (>1 μM). Taken together, our results demonstrate that the membrane targeting of C2GAP1 enables Dictyostelium to sense chemoattractant gradients at a higher concentration range. This mechanism is likely an evolutionarily conserved molecular mechanism of Ras regulation in the adaptation and chemotaxis of eukaryotes.

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“…To further determine the function of CAPRI in chemotaxis of neutrophils, we monitored chemotaxis behavior of CTL and capri kd cells in gradients of fMLP at different concentrations using an EZ-TAXIScan analysis (56). In a 1 μM fMLP gradient, capri kd cells, in a clear contrast to CTL cells, displayed a significant decrease in migrating speed (CTL: 20.86 ± 3.12 μm/min; caprikd: 12.16 ± 3.73 μm/min), directionality (CTL: 0.86 ± 0.09; caprikd: 0.73 ± 0.14 μm/min), total path length (CTL: 140.56 ± 27.…”

Section: Resultsmentioning

confidence: 99%

Ras Inhibitor CAPRI Enables Neutrophils to Chemotax Through a Higher-Concentration Range of Gradients

Wen

moosa

et al. 2020

Preprint

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Neutrophils sense and migrate through an enormous range of chemoattractant gradients through adaptation. Here, we reveal that, in human neutrophils, Calcium-promoted Ras inactivator (CAPRI) locally controls the GPCR-stimulated Ras adaptation. Human neutrophils lacking CAPRI (caprikd) exhibit chemoattractant-induced non-adaptive Ras activation; significantly increased phosphorylation of AKT, GSK3α/3β, and cofilin; and excessive actin polymerization. caprikd cells display defective chemotaxis in response to high-concentration gradients but exhibit improved chemotaxis in low- or subsensitive-concentration gradients of various chemoattractants as a result of their enhanced sensitivity. Taken together, our data reveal that CAPRI controls GPCR activation-mediated Ras adaptation and lowers the sensitivity of human neutrophils so that they are able to chemotax through a higher concentration range of chemoattractant gradients.Significance StatementNeutrophils provide first-line host defense by migrating through chemoattractant gradients to the sites of inflammation. Inappropriate recruitment and mis-regulated activation of neutrophils contribute to tissue damage and cause autoimmune and inflammatory disease. One fascinating feature of chemotactic neutrophils is their ability to migrate through an enormous concentration range of chemoattractant gradients (10−9 ∼ 10−5 M) through “adaptation,” in which cells no longer respond to the present stimuli, but remain sensitive to stronger stimuli. The inhibitory mechanism largely remains elusive, although many molecules of the excitatory signaling pathway have been identified. Our study reveals, for the first time, that the inhibitory component, CAPRI, is essential for both the sensitivity and the GPCR-mediated adaptation of human neutrophils.

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Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Cited by 7 publications

References 20 publications

Streptococcus pneumoniae Evades Host Cell Phagocytosis and Limits Host Mortality Through Its Cell Wall Anchoring Protein PfbA

Streptococcus pneumoniae Evades Host Cell Phagocytosis and Limits Host Mortality Through Its Cell Wall Anchoring Protein PfbA

Membrane Targeting of C2GAP1 Enables Dictyostelium discoideum to Sense Chemoattractant Gradient at a Higher Concentration Range

Ras Inhibitor CAPRI Enables Neutrophils to Chemotax Through a Higher-Concentration Range of Gradients

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