Kruppel-like factor 4 regulates neutrophil activation

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Presentation transcript:

Kruppel-like factor 4 regulates neutrophil activation by Yuyan Shen, Hong Hong, Panjamaporn Sangwung, Stephanie Lapping, Lalitha Nayak, Lilei Zhang, Mukesh K. Jain, and Xudong Liao BloodAdv Volume 1(11):662-668 April 25, 2017 © 2017 by The American Society of Hematology

Yuyan Shen et al. Blood Adv 2017;1:662-668 © 2017 by The American Society of Hematology

KLF4-deficient neutrophils exhibited impaired function ex vivo. KLF4-deficient neutrophils exhibited impaired function ex vivo. (A) Wild-type bone marrow neutrophils (BMNs) showed increased KLF4 expression after 30-minute LPS stimulation. (B) LPS-induced MMP-9 mRNA expression was blunted in KLF4-deficient BMNs. (C-D) The secreted MMP-9 and MPO in the condition medium from BMNs stimulated with LPS or E coli were measured by ELISA. (E) Caspase-3 activation in neutrophils assessed by western blot and fluorescent enzymatic activity assay. BMNs were incubated in culture medium without (Control) or with E coli (E. coli) for 2 hours at 37°C. (F) Extracellular ROS generation from Cre and K4-cKO BMNs in response to fMLF was determined by kinetic absorbance OD550. (G) KLF4-deficient neutrophils were defective in bacterial killing assay. The images on the left are 1× scans of 10-cm dishes. (H) LPS-induced iKBα phosphorylation and degradation. (I) LPS-induced gene expression of IκBα and TNF-α was attenuated in KLF4-deficient neutrophils. (J) CD14 mRNA expression in neutrophils. (K) CD14 protein expression on neutrophil cell surface as measured by flow cytometry. All gene expression levels were determined by qPCR. n = 3 to 5 in each group. *P < .05. Yuyan Shen et al. Blood Adv 2017;1:662-668 © 2017 by The American Society of Hematology

K4-cKO mice are susceptible to bacterial infection but resistant to EAE. (A) K4-cKO mice exhibited higher mortality rate than the Cre group after E coli intraperitoneal injection. n = 12 in each group. K4-cKO mice are susceptible to bacterial infection but resistant to EAE. (A) K4-cKO mice exhibited higher mortality rate than the Cre group after E coli intraperitoneal injection. n = 12 in each group. (B) Bacteria burden in the blood and peritoneal fluid harvested 14 hours postinjection. (C) TNF-α levels in serum at 4 hours postinjection. K4-cKO mice showed delayed occurrence and less disease severity after EAE induction, demonstrated by clinical score (D) and weight loss (E). n = 14 in each group. Difference was determined by 2-way ANOVA. For panel D, interaction P < .0001. For panel E, interaction P < .0021. For both panels D and E: time, P < .0001; genotype, P < .0001. (F) Spinal cord from lumbar 1 to 3 were taken from Cre and K4-cKO mice at disease peak (day 28) of EAE. Fluorescent myelin staining showed less demyelination in K4-cKO group. Hematoxylin and eosin (H&E) staining confirmed the cell infiltration in the foci. Representative data from 3 independent experiments. Original magnification ×200 (taken with a Nikon microscope; 20× objective, 10× eyepiece lens). (G) Immune cell infiltration in spinal cord as measured by flow cytometry at initiation of EAE (day 14). Neutrophils (CD45+CD11b+CD11c−Ly6G+), Th cells (CD3+CD4+), B cells (CD19+CD3−), and DCs (CD45+CD11b+CD11c+Ly6G−) were shown. n = 13 in each group. P < .05 was considered significant. Yuyan Shen et al. Blood Adv 2017;1:662-668 © 2017 by The American Society of Hematology