Figure 3.
RvD1 reduces ex vivo human neutrophil adhesion and in vivo neutrophil counts in humanized SCD mice, which show decreases in plasma RvD1 values after DHA administration. (A) Kinetics of DHA conversion to proresolving mediator RvD1 following oral administration in AA and SS mice. Levels of RvD1 were determined, using a competitive enzyme immunoassay, in plasma collected from AA and SS mice at the indicated times following DHA gavage. Data are mean ± SD (n = 3). *P < .05 vs baseline for AA mice. (B) Adhesion of neutrophils (green) to TNF-α–activated human microvascular endothelial cell line (HMEC). Blood samples from a healthy donor (AA) and an SCD patient (SS) were incubated for 10 minutes with vehicle or 17R-RvD1 (1 μM) (left panels). A second dose of vehicle or 17R-RvD1 (1 μM) was added before perfusing blood for 40 minutes (at 1 dyn/cm2) through the biochip channels containing activated HMEC-1 monolayers. Percentage of neutrophil adhesion to TNF-α–activated HMECs from AA (n = 6) and SS (n = 5) blood samples incubated with vehicle or 17R-RvD1 (right panel). ***P < .001 vs the corresponding vehicle group. (C) Representative images showing reduced neutrophil recruitment after 17R-RvD1 administration in sickle cell mice (left panels). All of these experiments suggest that 17R-RvD1 influences neutrophil recruitment in SS mice, especially in the context of sustained and intense neutrophil recruitment specific to experimental SCD. Neutrophil adhesion density, defined as the number of adherent neutrophils per square millimeter of endothelial surface, in TNF-α–inflamed venules after 17R-RvD1 treatment in SS mice (SS TNF-α 17R-RvD1) or after dual endothelin receptor antagonism (SS TNF-α bosentan) (upper right panel). Data are mean ± standard error of the mean. P < .001 for SS mice treated with TNF-α and 17-RvD1 vs with TNF-α and vehicle alone (SS TNF-α 17R-RvD1), 2-way analysis of variance (ANOVA). *P < .05, **P < .01 vs vehicle-treated SS with TNF-α, 2-way ANOVA followed by the Tukey multiple-comparison test. Extravascular volume in inflamed venules after 17R-RvD1 or bosentan vs vehicle administration in SS mice (lower right panel). Emigrated neutrophils were visualized and quantified by optical sectioning and 2-dimensional maximum intensity projection. Twenty venules (5 mice) were analyzed in each group. Data are mean ± standard error of the mean. P < .001 for SS mice treated with TNF-α and 17R-RvD1 vs with TNF-α and vehicle alone, 2-way ANOVA. Data for heterozygous AS littermates are provided as control reference for neutrophil adhesion and emigration to tissue after TNF-α. *P < .05, **P < .01 vs vehicle-treated SS with TNF-α, 2-way ANOVA, followed by the Tukey multiple-comparison test.

RvD1 reduces ex vivo human neutrophil adhesion and in vivo neutrophil counts in humanized SCD mice, which show decreases in plasma RvD1 values after DHA administration. (A) Kinetics of DHA conversion to proresolving mediator RvD1 following oral administration in AA and SS mice. Levels of RvD1 were determined, using a competitive enzyme immunoassay, in plasma collected from AA and SS mice at the indicated times following DHA gavage. Data are mean ± SD (n = 3). *P < .05 vs baseline for AA mice. (B) Adhesion of neutrophils (green) to TNF-α–activated human microvascular endothelial cell line (HMEC). Blood samples from a healthy donor (AA) and an SCD patient (SS) were incubated for 10 minutes with vehicle or 17R-RvD1 (1 μM) (left panels). A second dose of vehicle or 17R-RvD1 (1 μM) was added before perfusing blood for 40 minutes (at 1 dyn/cm2) through the biochip channels containing activated HMEC-1 monolayers. Percentage of neutrophil adhesion to TNF-α–activated HMECs from AA (n = 6) and SS (n = 5) blood samples incubated with vehicle or 17R-RvD1 (right panel). ***P < .001 vs the corresponding vehicle group. (C) Representative images showing reduced neutrophil recruitment after 17R-RvD1 administration in sickle cell mice (left panels). All of these experiments suggest that 17R-RvD1 influences neutrophil recruitment in SS mice, especially in the context of sustained and intense neutrophil recruitment specific to experimental SCD. Neutrophil adhesion density, defined as the number of adherent neutrophils per square millimeter of endothelial surface, in TNF-α–inflamed venules after 17R-RvD1 treatment in SS mice (SS TNF-α 17R-RvD1) or after dual endothelin receptor antagonism (SS TNF-α bosentan) (upper right panel). Data are mean ± standard error of the mean. P < .001 for SS mice treated with TNF-α and 17-RvD1 vs with TNF-α and vehicle alone (SS TNF-α 17R-RvD1), 2-way analysis of variance (ANOVA). *P < .05, **P < .01 vs vehicle-treated SS with TNF-α, 2-way ANOVA followed by the Tukey multiple-comparison test. Extravascular volume in inflamed venules after 17R-RvD1 or bosentan vs vehicle administration in SS mice (lower right panel). Emigrated neutrophils were visualized and quantified by optical sectioning and 2-dimensional maximum intensity projection. Twenty venules (5 mice) were analyzed in each group. Data are mean ± standard error of the mean. P < .001 for SS mice treated with TNF-α and 17R-RvD1 vs with TNF-α and vehicle alone, 2-way ANOVA. Data for heterozygous AS littermates are provided as control reference for neutrophil adhesion and emigration to tissue after TNF-α. *P < .05, **P < .01 vs vehicle-treated SS with TNF-α, 2-way ANOVA, followed by the Tukey multiple-comparison test.

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