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 4

Knockout of CD45 in PMN-like HL60 cells reduces degranulation and phagocytosis.

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Knockout of CD45 in PMN-like HL60 cells reduces degranulation and phagoc...
CD45 was knocked out by CRISPR/Cas9 in the human promyelocytic cell line HL60 (CD45KO). (A) Lysates from HL60, SCR HL60, or CD45KO HL60 cells differentiated into a PMN- like state were immunoblotted for CD45 or GAPDH. (B) Densitometry revealed a less than 98% reduction in CD45 expression (normalized to GAPDH) in CD45KO HL60 cells relative to non-transduced HL60 cells or SCR HL60 cells. Data were normalized to GAPDH and relative to non-transduced HL60 cells (n = 3 independent experiments). (C and D) Flow cytometric analysis using a PerCP-labeled anti-CD45 mAb and a PerCP-labeled IgG matched control mAb. Data represent mean fluorescence intensity (MFI) normalized to HL60 SCR IgG control (n = 3 independent experiments). (E and F) Differentiated SCR HL60 cells and CD45KO HL60 cells were stimulated with 1.25 mM LaB and 5 mM fMLF to induce degranulation. Data are the fold change in MFI for CD63 normalized to SCR HL60 IgG (n = 3 independent experiments). (G and H) Differentiated SCR HL60 and CD45KO HL60 cells were stimulated with 100 nM fMLF for 60 minutes at 37°C before fluorescent microsphere phagocytosis/uptake was quantified by measuring changes in total levels of APC fluorescence by flow cytometry. Data represent APC MFI (n = 4 independent experiments). (I and J) Differentiated CD45KO HL60 or SCR HL60 cells were incubated with 20 mg pHrodo-conjugated E. coli bioparticles in the presence of 10 nM fMLF. Phagocytosis was quantified by measuring changes in APC fluorescence by flow cytometry. Data are MFI values (n = 3 independent experiments). Data are shown as 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). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.