ELMO1 Deficiency Reduces Neutrophil Chemotaxis in Murine Peritonitis
<p>ELMO1 plays a role in acute peritonitis by regulating infiltration of neutrophils. (<b>A</b>) H&E-stained peritoneum and liver sections. The inflammatory cell infiltration is indicated by black arrows. Scale bar, 50 μm. (<b>B</b>) Total number of cells in peritoneal lavage fluid. The number of TG-induced cells in peritoneal lavage fluid were detected by flow cytometry. (<b>C</b>,<b>D</b>) Relative content levels of TNF-α and IL-6 in peritoneal lavage fluid detected by ELISA. The data are presented as means ± SEM (<span class="html-italic">n</span> = 6). (<b>E</b>,<b>F</b>) Immunofluorescence-stained histological peritoneum and liver sections. Yellow arrows indicate inflammatory cell infiltration. Scale bar: 50 μm. Statistical significance was assessed by <span class="html-italic">t</span>-test, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001, and <sup>ns</sup> <span class="html-italic">p</span> > 0.05. <span class="html-italic">n</span> = 6.</p> "> Figure 2
<p>Elmo1 deficiency reduces the chemotaxis and adhesion of neutrophils. (<b>A</b>) The percentage of neutrophils number in the peritoneal lavage fluid. TG-induced neutrophil aggregation was stained with CD11b and Gr-1 to detect the ratio by flow cytometry (different colors means different cell population). (<b>B</b>) The percentage of CD11b- and Gr-1-positive cells were quantified with FlowJo software and the data are presented as means ± SEM (<span class="html-italic">n</span> = 5). (<b>C</b>,<b>D</b>) Primary neutrophils (<span class="html-italic">n</span> = 4) and HL-60 cells (<span class="html-italic">n</span> = 5) migration assay. Cells migrate under stimulation of fMLP for 2 h. Migrated cell numbers in the bottom chamber were counted by flow cytometry. The data are presented as means ± SEM. (<b>E</b>,<b>F</b>) Primary neutrophils and HL-60 cells adhesion assay. The fMLP-stimulated cells were incubated at 37 °C for 2 h and stained with crystal violet followed by 2% SDS dissolving for measuring absorbance at 570 nm. The data are presented as means ± SEM (<span class="html-italic">n</span> = 5). Each group had 3 replicates. Statistical significance was assessed by <span class="html-italic">t</span>-test, KD groups versus CON group by one-way ANOVA. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001, and <sup>ns</sup> <span class="html-italic">p</span> > 0.05.</p> "> Figure 3
<p>Deficiency of Elmo1 down-regulates fMLP-induced actin polymerization. (<b>A</b>,<b>B</b>) Neutrophil F-actin polymerization assay. Confocal micrographs of fMLP-stimulation cells stained with Alexa Fluor 633 phalloidin and DAPI. The percentage of phalloidin-positive cells in vision were quantified with ImageJ software and the data are presented as means ± SEM (<span class="html-italic">n</span> = 7). (<b>C</b>) The fMLP-stimulated primary neutrophils were stained with phalloidin to detect the F-actin by flow cytometry (<span class="html-italic">n</span> = 5). (<b>D</b>,<b>E</b>) F-actin polymerization assay in HL-60 cells (<span class="html-italic">n</span> = 5). (<b>F</b>) The fMLP-stimulated HL-60 cells F-actin polymerization assay (<span class="html-italic">n</span> = 5). MFI: mean fluorescence intensity. Yellow arrows indicate F-actin polymerization cells. Each group had 3 replicates. Statistical significance was assessed by <span class="html-italic">t</span>-test, * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01 and *** <span class="html-italic">p</span> < 0.001.</p> "> Figure 4
<p>ELMO1 regulates fMLP-triggered Rac activation independent of PI3K and mTORC2 signaling pathways. (<b>A</b>) fMLP-induced Rac1 activation assay. Total proteins of neutrophils were collected after 5 and 15 s of fMLP stimulation, incubated with PAK-PBD beads and then subjected to SDS-PAGE and analyzed by immunoblotting for detecting for Rac1. (<b>B</b>) The ratio of Rac1-GTP to total Rac1 was quantified and graphed. Data are presented as mean ± SEM representation (<span class="html-italic">n</span> = 3). (<b>C</b>,<b>F</b>) fMLP-stimulated neutrophils and HL-60 cells. Phosphorylated AKT and total AKT were detected by SDS-PAGE Western blotting. (<b>D</b>–<b>H</b>) The levels of phosphorylated AKT-T308 (<span class="html-italic">n</span> = 3) and AKT-S473 (<span class="html-italic">n</span> = 3) were density-determined using ImageJ software and expressed as the ratio of phosphorylated form to total protein. Data are presented as means ± SEM. Each group had 3 replicates. Statistical significance was assessed by <span class="html-italic">t</span>-test, * <span class="html-italic">p</span> < 0.05, and <sup>ns</sup> <span class="html-italic">p</span> > 0.05.</p> ">
Abstract
:1. Introduction
2. Results
2.1. ELMO1 Plays a Role in Acute Peritonitis by Regulating the Infiltration of Neutrophils
2.2. Elmo1 Deficiency Reduces the Chemotaxis and Adhesion of Neutrophils
2.3. Deficiency of Elmo1 Down-Regulates fMLP-Induced Actin Polymerization
2.4. ELMO1 Regulates fMLP-Triggered Rac Activation Independent of PI3K and mTORC2 Signaling Pathways
3. Discussion
4. Materials and Methods
4.1. Mice
4.2. Primary Neutrophil Isolation and Identification
4.3. Cell Culture, Infection, and Differentiation
4.4. H&E, Immunofluorescence, and Phalloidin Staining Assay
4.5. Generation of the Peritonitis Mouse Model
4.6. Enzyme-Linked Immunosorbent Assay (ELISA)
4.7. Adhesion Assay
4.8. Transwell Migration Assay
4.9. Rac Activity Assay
4.10. Western Blot Analysis
4.11. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Acknowledgments
Conflicts of Interest
References
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Yu, S.; Geng, X.; Liu, H.; Zhang, Y.; Cao, X.; Li, B.; Yan, J. ELMO1 Deficiency Reduces Neutrophil Chemotaxis in Murine Peritonitis. Int. J. Mol. Sci. 2023, 24, 8103. https://doi.org/10.3390/ijms24098103
Yu S, Geng X, Liu H, Zhang Y, Cao X, Li B, Yan J. ELMO1 Deficiency Reduces Neutrophil Chemotaxis in Murine Peritonitis. International Journal of Molecular Sciences. 2023; 24(9):8103. https://doi.org/10.3390/ijms24098103
Chicago/Turabian StyleYu, Shuxiang, Xiaoke Geng, Huibing Liu, Yunyun Zhang, Xiumei Cao, Baojie Li, and Jianshe Yan. 2023. "ELMO1 Deficiency Reduces Neutrophil Chemotaxis in Murine Peritonitis" International Journal of Molecular Sciences 24, no. 9: 8103. https://doi.org/10.3390/ijms24098103