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 9

Inhibition or depletion of CD45 reduces CD11b/CD18 surface expression and activation on PMNs.

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Inhibition or depletion of CD45 reduces CD11b/CD18 surface expression an...
(A and B) Human PMNs exposed to 500 nM CD45 inhibitor VI or vehicle control were incubated with a FITC-conjugated anti-CD11b mAb or isotype control mAb and assessed by flow cytometry. Data represent fold change in CD11b mean fluorescence intensity (MFI) normalized to Ctrl (n = 3 independent biological experiments). (C and D) Human PMNs exposed to 500 nM CD45 inhibitor VI or vehicle control were assessed for CD11b activation by flow cytometry. Data represent fold change in CBRM1/5 MFI normalized to Ctrl (n = 3 independent experiments). (E and F) CD11b surface expression detected by flow cytometry of CD45KO HL60, SCR HL60, or non-transduced HL60 cells. Data represent fold change in CD11b MFI normalized to non-transduced HL60 cells (n = 3 independent experiments). (G and H) Surface expression of active CD11b in CD45KO HL60, SCR HL60, or non-transduced HL60 cells detected by flow cytometry. Data represent fold change in CBRM1/5 MFI normalized to non-transduced HL60 cells (n = 3 independent experiments). (I and J) WT murine PMNs were exposed to 500 nM CD45 inhibitor VI or vehicle control and incubated with an APC-conjugated anti-CD11b mAb or an isotype control mAb before flow cytometric analysis of CD11b surface expression. Data represent fold change in CD11b MFI normalized to vehicle control (n = 4 independent experiments). (K and L) PMNs from MRP8-Cre;Cd45fl/fl mice or Cd45fl/fl mice were incubated with an APC-conjugated anti-CD11b mAb or isotype control mAb before analysis of CD11b surface expression by flow cytometry. Data represent fold change in CD11b MFI normalized to Cd45fl/fl PMNs (n = 4 independent experiments). Data represent mean ± SEM and were analyzed by 1-way ANOVA with Tukey’s post hoc testing (B, D, F, H) or unpaired, 2-tailed t test (J, L). *P < 0.05; **P < 0.01.