The protein tyrosine phosphatase CD45 promotes PMN transepithelial migration, antimicrobial function, and colonic mucosal repair
Jael Miranda, Dylan J. Fink, Zachary S. Wilson, Roland Hilgarth, Asma Nusrat, Charles A. Parkos, Jennifer C. Brazil
Jael Miranda, Dylan J. Fink, Zachary S. Wilson, Roland Hilgarth, Asma Nusrat, Charles A. Parkos, Jennifer C. Brazil
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Research Article Immunology Inflammation

The protein tyrosine phosphatase CD45 promotes PMN transepithelial migration, antimicrobial function, and colonic mucosal repair

Abstract

Polymorphonuclear neutrophils (PMNs) serve as frontline defenders against injury and infection, eliminating pathogens and initiating mucosal tissue repair. However, excessive PMN transepithelial migration (TEpM) contributes to chronic mucosal inflammatory disorders, including inflammatory bowel disease. PMN proinflammatory and pro-repair functions are regulated by incompletely defined signaling cascades involving kinases and phosphatases. Here, we determined how the protein tyrosine phosphatase CD45/PTPRC regulates PMN trafficking and effector functions in the gut. Pharmacologic inhibition of CD45 significantly reduced PMN colonic TEpM in vitro and in vivo and decreased intestinal PMN trafficking was observed in transgenic mice with PMN-specific deletion of Cd45 (MRP8-Cre;Cd45fl/fl). Beyond limiting TEpM, CD45 depletion impaired key antimicrobial functions, including degranulation and phagocytosis, indicating broader effects on PMN effector activity. Importantly, recovery from dextran sodium sulfate–induced colitis and biopsy-induced colonic wounding was delayed in MRP8-Cre;Cd45fl/fl mice, linking altered PMN function to defective mucosal healing. Mechanistically, CD45 depletion reduced surface expression of the β2 integrin CD11b/CD18 and inactivated the Src family kinase member Lyn. Together, these data highlight an important CD45/CD11b/Lyn signaling axis that regulates PMN trafficking and effector functions in the intestine and identify CD45 as a promising target for modulating PMN function to promote mucosal tissue repair.

Authors

Jael Miranda, Dylan J. Fink, Zachary S. Wilson, Roland Hilgarth, Asma Nusrat, Charles A. Parkos, Jennifer C. Brazil

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Figure 11

Inhibition of Lyn kinase with Bafetinib or Dasatinib mimics functional effects of CD45 inhibition or deletion.

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Inhibition of Lyn kinase with Bafetinib or Dasatinib mimics functional e...
Differentiated HL60 cells in suspension (A and B) or adhering to fibronectin (C and D) were incubated with 100 nM Bafetinib, 250 nM Dasatinib, or vehicle control before addition of pHrodo-conjugated E. coli particles in the presence of 10 nM fMLF. Phagocytosis was quantified by measuring changes in APC fluorescence by flow cytometry. Data represent mean fluorescence intensity (MFI) values normalized to vehicle control (n = 3 independent experiments). PMN-like HL60 cells in suspension (E and F) or adhering to fibronectin (G and H) were incubated with 100 nM Bafetinib, 250 nM Dasatinib, or vehicle control before stimulation with 1.25 mM LaB and 5 mM fMLF to induce degranulation followed by assessment of surface expression of CD63 by flow cytometry. Data shown are FITC/CD63 MFI values (n = 5 independent experiments). (I and J) Human PMNs adhering to fibronectin were incubated with 100 nM Bafetinib, 250 nM Dasatinib, or vehicle control before stimulation with 100 nM PMA to induce degranulation followed by assessment of surface expression of CD66b by flow cytometry (n = 3 independent experiments). Data represent mean ± SEM and were analyzed by 1-way ANOVA with Tukey’s post hoc testing. *P < 0.05; **P < 0.01.