Abstract

Serum Response Factor (SRF) is a ubiquitously expressed transcription factor and master regulator of the actin cytoskeleton. We have previously shown, that SRF is essential for megakaryocyte maturation and platelet formation and function. Here we elucidate the role of SRF in neutrophils, the primary defense against infections. To study the effect of loss of SRF in neutrophils, we crossed Srffl/fl mice with select Cre-expressing mice and studied neutrophil function in vitro and in vivo. Despite normal neutrophil numbers, neutrophil function is severely impaired in mice in whom Srf is selectively deleted in the hematopoietic system (Srf KO). Srf KO neutrophils fail to migrate to sites of inflammation in vivo. In a model mimicking lung infection by nebulization of lipopolysaccharide (LPS), significantly fewer neutrophils are recruited to the inflamed lungs 4 and 24 hours after LPS administration as evident by cell numbers retrieved in the bronchoalveolar lavage fluid (BAL, total cells: WT 0.568 ± 0.093x106 vs. KO 0.128 ± 0.024x106 at 4 hours and WT 1.337 ± 0.369x106 vs. KO 0.347 ± 0.045x106 at 24 hours; p <0.005 at 4 hours, p < 0.05 at 24 hours). Similarly, in an in vivo peritonitis model, where all other immune cells normally express Srf, significantly fewer Srf KO than WT neutrophils are recruited to the inflamed peritoneal space resulting in significantly reduced Srf KO neutrophil numbers in the peritoneal lavage fluid. To directly assess neutrophil migration and chemotaxis we performed in vitro transwell assays and assessed neutrophil migration in the Dunn chamber assay. Srf KO neutrophils show a significant migration defect in response to fMLP and KC. We next assessed actin polymerization in Srf WT and KO neutrophils. Srf KO neutrophils fail to polymerize globular actin to filamentous actin in response to fMLP. Neutrophil polarization in response to cytokine stimuli is markedly decreased. In addition, Srf KO neutrophils show markedly reduced adhesion. Integrins play an essential role in neutrophil adhesion and migration. Srf KO neutrophils show normal expression of the integrin LFA1 (CD11a/CD18), however, Mac1 (CD11b/CD18) expression is markedly increased on the cell surface as assessed by flow cytometry and Immunofluorescent staining, despite reduced mRNA expression. We find that trafficking of Mac1, essential for directed cell migration, is disrupted in Srf KO neutrophils, likely contributing to the migratory defect. Migration and cellular adhesion are essential for normal cell function, but also for malignant processes such as metastasis, thus underscoring an essential function for SRF and its pathway in health and disease.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.