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 1

CD45 inhibition blocks PMN TEpM in vitro and in vivo.

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CD45 inhibition blocks PMN TEpM in vitro and in vivo. (A and B) Human PM...
(A and B) Human PMNs (1 × 106) were incubated with 500 nM CD45 inhibitor VI or vehicle control or 10 μg/mL anti-CD45 mAb (MEM-28) or isotype-matched control IgG mAb before addition to the basolateral surface of confluent inverted T84 monolayers. PMNs migrated in the physiologically relevant basolateral to apical direction for 1 hour in response to a 100 nM gradient of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLF). Numbers of migrated PMNs were quantified by myeloperoxidase assay (n = 5 independent experiments). (C and D) Human PMNs (1 × 106) were incubated with 500 nM CD45 inhibitor VI, vehicle control, 10 μg/mL anti-CD45 mAb MEM-28, or isotype-matched control IgG mAb before addition to the basolateral surface of confluent inverted human colonoid–derived monolayers of primary IECs. PMNs migrated for 1 hour in response to a 100 nM gradient of fMLF (n = 3 independent experiments). (E and F) Quantification of absolute number of PMNs recruited into the lumen of proximal colon loops following intraluminal injection of 1 nM LTB4 ± 500 nM CD45 inhibitor VI or intraperitoneal injection of 3 mg/kg body weight CD45 inhibitor VI (n = 3–4 independent experiments). Data are shown as mean ± SEM and were analyzed by 1-way ANOVA with Tukey’s post hoc testing. **P < 0.01; ***P < 0.001; ****P < 0.0001.