Sialylation regulates neutrophil transepithelial migration, CD11b/CD18 activation, and intestinal mucosal inflammatory function
Veronica Azcutia, Matthias Kelm, Dylan Fink, Richard D. Cummings, Asma Nusrat, Charles A. Parkos, Jennifer C. Brazil
Veronica Azcutia, Matthias Kelm, Dylan Fink, Richard D. Cummings, Asma Nusrat, Charles A. Parkos, Jennifer C. Brazil
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Research Article Immunology Inflammation

Sialylation regulates neutrophil transepithelial migration, CD11b/CD18 activation, and intestinal mucosal inflammatory function

Abstract

Polymorphonuclear neutrophils (PMNs) play a critical role in clearing invading microbes and promoting tissue repair following infection/injury. However, dysregulated PMN trafficking and associated tissue damage is pathognomonic of numerous inflammatory mucosal diseases. The final step in PMN influx into mucosal lined organs (including the lungs, kidneys, skin, and gut) involves transepithelial migration (TEpM). The β2-integrin CD11b/CD18 plays an important role in mediating PMN intestinal trafficking, with recent studies highlighting that terminal fucose and GlcNAc glycans on CD11b/CD18 can be targeted to reduce TEpM. However, the role of the most abundant terminal glycan, sialic acid (Sia), in regulating PMN epithelial influx and mucosal inflammatory function is not well understood. Here we demonstrate that inhibiting sialidase-mediated removal of α2-3–linked Sia from CD11b/CD18 inhibits PMN migration across intestinal epithelium in vitro and in vivo. Sialylation was also found to regulate critical PMN inflammatory effector functions, including degranulation and superoxide release. Finally, we demonstrate that sialidase inhibition reduces bacterial peptide–mediated CD11b/CD18 activation in PMN and blocks downstream intracellular signaling mediated by spleen tyrosine kinase (Syk) and p38 MAPK. These findings suggest that sialylated glycans on CD11b/CD18 represent potentially novel targets for ameliorating PMN-mediated tissue destruction in inflammatory mucosal diseases.

Authors

Veronica Azcutia, Matthias Kelm, Dylan Fink, Richard D. Cummings, Asma Nusrat, Charles A. Parkos, Jennifer C. Brazil

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Figure 4

Sialidase inhibition prevents degranulation and ROS release in human and murine PMN.

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Sialidase inhibition prevents degranulation and ROS release in human and...
(A and B) Human PMN were exposed to 5 mM Sia, 5 mM Gal, 5 mM 2-DN, or 5 mM KDO for 30 minutes at 37°C, followed by stimulation with 1.25 μM LaB and 5 μM fMLF to induce degranulation before assessment of surface expression of CD66b and CD63 by flow cytometry. Data shown are fold-change in mean fluorescence intensity (MFI) comparing treatment with sialidase inhibitors against relevant control (Gal or KDO). Data are expressed as mean ± SEM and were analyzed by 1-way ANOVA followed by Bonferroni post hoc testing (n = 4–6 PMN donors, ***P < 0.001, ****P < 0.0001). (C and D) Murine PMN were exposed to 5 mM Sia, 5 mM Gal, 5 mM 2-DN, or 5 mM KDO for 30 minutes at 37°C followed by stimulation with 1.25 μM LaB and 10 μM fMLF to induce degranulation before assessment of surface expression of CD66b and CD63 by flow cytometry. Data shown are fold-change in mean fluorescence intensity (MFI) comparing treatment with sialidase inhibitors against relevant control (Gal or KDO). Data are expressed as mean ± SEM and were analyzed by 1-way ANOVA followed by Bonferroni post hoc testing for PMN isolated from 3–5 mice (**P < 0.01, ***P < 0.001). (E) Human PMN incubated with 5 mM Gal, 5 mM Sia, 5 mM 2-DN, or 5 mM KDO were exposed to 100 μM cytochrome C. Reduction of cytochrome C in response to 500 nM fMLF was measured by quantifying changes in absorbance at 550 nm at 2, 5, 10, 15, 20, and 60 minutes. Data are fold change in absorbance relative to time 0, are expressed as mean ± SEM, and were analyzed by 1-way ANOVA followed by Bonferroni post hoc testing (n = 3 independent human PMN donors, *P < 0.05, **P < 0.01, ***P < 0.001). (F) Murine PMN incubated with 5 mM Gal, 5 mM Sia, 5 mM 2-DN, or 5 mM KDO were exposed to 100 μM cytochrome C. Reduction of cytochrome C in response to 1 μM fMLF was measured by quantifying changes in absorbance at 550 nm at 2, 5, 10, 15, 20, and 60 minutes. Data are fold change in absorbance relative to time 0; data are expressed as mean ± SEM and were analyzed by 1-way ANOVA followed by Bonferroni post hoc testing (n = 3 mice, *P < 0.05, **P < 0.01, ***P < 0.001).