Kruppel-like factor 2 is a transcriptional regulator of chronic and acute inflammation

Abstract

Although myeloid cell activation is requisite for an optimal innate immune response, this process must be tightly controlled to prevent collateral host tissue damage. Kruppel-like factor 2 (KLF2) is a potent regulator of myeloid cell proinflammatory activation. As an approximately 30% to 50% reduction in KLF2 levels has been observed in human subjects with acute or chronic inflammatory disorders, we studied the biological response to inflammation in KLF2(+/-) mice. Herein, we show that partial deficiency of KLF2 modulates the in vivo response to acute (sepsis) and subacute (skin) inflammatory challenge. Mechanistically, we link the anti-inflammatory effects of KLF2 to the inhibition of NF-κB transcriptional activity. Collectively, the observations provide biologically relevant insights into KLF2-mediated modulation of these inflammatory processes that could potentially be manipulated for therapeutic gain.

Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Deficiency of KLF2-accelerated LPS-induced sepsis symptoms. A and B: WT and KLF2 hemizygous mice were subjected to LPS-induced sepsis by i.p. injection of LPS. Surface body temperature was recorded by thermal infrared camera (A), and core body temperature was recorded by rectal probe (B). C and D: Age- and sex-matched WT and KLF2 hemizygous mice were subjected to LPS-induced sepsis and were monitored for alteration in systolic blood pressure (tail cuff blood pressure monitor system; C) and shock index (D). E and F: WT and KLF2 hemizygous mice were subjected to LPS-induced sepsis, and plasma levels of TNF-α (E) and MCP-1 (F) were measured by ELISA after 4 hours of LPS injection. G: Age- and sex-matched WT and KLF2 hemizygous (HET) mice were subjected to LPS-induced sepsis. The mice were observed for 48 hours for survival. The combined data from three experiments are shown in each case. Data are presented as means ± SD. ∗P < 0.05 versus the indicated control group.

Figure 2

Deficiency of KLF2 enhanced LPS-induced proinflammatory gene expression. A–C: WT and KLF2 hemizygous peritoneal macrophages were induced with 100 ng/mL of LPS, and total RNA was analyzed for expression of critical proinflammatory molecules, such as TNF-α (A), cyclooxygenase-2 (COX-2) (B), and MCP-1 (C), and was normalized to 36B4. D–F: Peritoneal macrophages derived from WT and KLF2 hemizygous mice were subjected to 100 ng/mL of LPS stimulation. Total RNA from these cells were extracted and analyzed for expression of systemic inflammatory response syndrome mediators, such as IFN-γ (D), IL-6 (E), and iNOS (F). 36B4 was used as an internal control. The combined data from three experiments are shown in each case. Data are presented as means ± SD. ∗P < 0.05 versus the indicated control.

Figure 3

KLF2 modulated the progression of cutaneous inflammation. A: WT and KLF2 hemizygous mice were subjected to the TPA-induced cutaneous inflammation model. Ear weights from the control and experimental groups were recorded using an analytical balance, and percentage increase in ear weight compared with the control group is indicated. B: WT and KLF2 hemizygous mice were subjected to TPA-induced inflammation. Ear tissues from mice from these experiments were immunohistochemically analyzed using anti-MAC3 antibody for macrophage localization. C: Total protein extracts from ear tissue from mice derived through the TPA-induced inflammation model were subjected to myeloperoxidase (MPO) activity analysis, and data are presented as MPO activity units/1 μg of total protein. D–F: Total RNA was isolated from ears of WT and KLF2 hemizygous mice that were subjected to the TPA-induced inflammation model. Expression of IL-1β (D), IL-6 (E), and MCP-1 (F) was analyzed by quantitative PCR and normalized to 36B4. The combined data from three experiments are shown in each case. Data are presented as means ± SD. ∗P < 0.05 versus the indicated control.

Figure 4

Deficiency of KLF2 enhanced carrageenan-induced acute inflammation. A: WT and KLF2 hemizygous mice were subjected to carrageenan-induced acute inflammation. Paw weights from the control and experimental groups were recorded using an analytical balance, and percentage increase in paw weight compared with the control group is indicated. B and C: WT and KLF2 hemizygous mice received 0.1 mL of saline or 1% carrageenan. The mice were euthanized, and paw morphologic features of the control and experimental group were documented (B). Paw tissue from WT and KLF2 hemizygous mice from carrageenan-induced inflammation experiments were subjected to immunohistochemical analysis using anti-MAC3 antibody (C). D: WT and KLF2 hemizygous mice were subjected to thioglycolate-induced peritonitis. Total cells from the peritoneal cavity were harvested using sterile PBS after 72 hours of thioglycolate injection and then were counted. E and F: Peritoneal macrophages derived from WT and KLF2 hemizygous mice were induced with 5 ng/mL of IFN-γ for 12 hours. MCP-2 (E) and MCP-5 (F) mRNA expression was analyzed by quantitative PCR and normalized to 36B4. The combined data from three experiments are shown in each case. Data are presented as means ± SD. ∗P < 0.05 versus the indicated control.

Figure 5

KLF2 negatively regulated proinflammatory gene expression by modulating recruitment of critical NF-κB coactivators. A: RAW264.7 cells were cotransfected with NF-κB concatemer luciferase reporter plasmid in the presence of KLF2 or pcDNA3.1. These cells were stimulated with IFN-γ or LPS, and induction in luciferase was documented and is indicated as relative luciferase activity over the control group. B and C: RAW264.7 cells were cotransfected with iNOS promoter luciferase reporter plasmid in the presence of KLF2 or pcDNA3.1. These cells were stimulated with IFN-γ or LPS for 12 hours, and luciferase activity was measured and is indicated as relative luciferase activity over control (B). RAW264.7 cells were transfected with pcDNA3.1 or KLF2 plasmid and stimulated with IFN-γ or LPS for 12 hours. Total RNA from these cells was isolated, and iNOS mRNA expression was analyzed by quantitative PCR and normalized to 36B4 (C). D: RAW264.7 cells infected with Ad-GFP or Ad-KLF2 were stimulated with LPS for 45 minutes. Chromatin immunoprecipitation was performed using the indicated antibody on the iNOS promoter flanking the NF-κB binding site. The combined data from three experiments are shown in each case. Data are presented as means ± SD. ∗P < 0.05 versus the indicated control.