Dynamic modulation of innate immune response by varying dosages of lipopolysaccharide (LPS) in human monocytic cells - PubMed (original) (raw)
Dynamic modulation of innate immune response by varying dosages of lipopolysaccharide (LPS) in human monocytic cells
Matthew C Morris et al. J Biol Chem. 2014.
Abstract
Innate monocytes and macrophages can be dynamically programmed into distinct states depending upon the strength of external stimuli. Innate programming may bear significant relevance to the pathogenesis and resolution of human inflammatory diseases. However, systems analyses with regard to the dynamic programming of innate leukocytes are lacking. In this study, we focused on the dynamic responses of human promonocytic THP-1 cells to lipopolysaccharide (LPS). We observed that varying dosages of LPS differentially modulate the expression of selected pro- and anti- inflammatory mediators such as IL-6 and IL-33. Super-low dosages of LPS preferentially induced the pro-inflammatory mediator IL-6, while higher dosages of LPS induced both IL-6 and IL-33. Mechanistically, we demonstrated that super-low and high doses of LPS cause differential activation of GSK3 and Akt, as well as the transcription factors FoxO1 and CREB. Inhibition of GSK3 enabled THP-1 cells to express IL-33 when challenged with super-low dose LPS. On the other hand, activation of CREB with adenosine suppressed IL-6 expression. Taken together, our study reveals a dynamic modulation of monocytic cells in response to varying dosages of endotoxin, and may shed light on our understanding of the dynamic balance that controls pathogenesis and resolution of inflammatory diseases.
Keywords: Innate Immunity; Lipopolysaccharide (LPS); Monocyte; Systems Biology; inflammation.
© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
Figures
FIGURE 1.
Relative induction of pro- and anti-inflammatory genes in THP-1 cells by 4 h stimulation with different concentrations of LPS. Total RNAs were harvested from THP-1 cells challenged as indicated. Real-time RT-PCR was performed to determine the expression levels of IL-6 (A) TNFα (B), and IL-33 (C). Different letters denote statistically significant differences between groups (p < 0.05, Holm-Sidak).
FIGURE 2.
Relative induction of pro-and anti-inflammatory genes in human THP-1 cells by low- and high-dose LPS. Total RNA was isolated from THP-1 cells treated with either 100 ng/ml LPS (A) or 50 pg/ml LPS (B) for 4 h. Real-time RT-PCR was performed to determine the expression levels of both IL-6 and IL-33. Data are representative of three separate experiments (*, p < 0.05; Student's t test).
FIGURE 3.
Differential regulation of GSK3 and Akt in THP-1 cells by LPS. THP-1 cells were treated with either 50 pg/ml or 100 ng/ml LPS for various time periods as indicated. Whole cell lysates were separated on SDS-PAGE. The levels of pAkt-S473 and total Akt (A) pGSK3β-Y216 and total GSK3β (B), and pPyk2-Y402 and total Pyk2 (C) were determined by Western blot with specific antibodies. Data are representative of three separate experiments.
FIGURE 4.
Differential regulation of FoxO1 and CREB by LPS. THP-1 cells were treated with either 50 pg/ml or 100 ng/ml LPS for various time periods as indicated. Whole cell lysates were separated on SDS-PAGE. The levels of pFoxO1 and total FoxO1 (A) and pCREB-S133 and total CREB (B) were determined by Western blot with specific antibodies. Data are representative of three separate experiments.
FIGURE 5.
Effect of GSK3 inhibition on cellular responses to LPS stimulation. A, THP-1 cells were treated with the combination of 50 pg/ml LPS alone or together with 10 μ
m
indirubin for 4 h. Total RNA was harvested and used for real-time RT-PCR analysis for IL-6. B, total protein lysates were harvested from THP-1 cells treated as indicated and used for Western blot analyses for FoxO1 and GAPDH. C, THP-1 cells were treated with the combination of 50 pg/ml LPS alone or together with 10 μ
m
SB216763 for 4 h. Total RNA was harvested and used for real-time RT-PCR analysis for IL-33. D, total protein lysates were harvested from THP-1 cells treated as indicated, and used for Western blot analyses for CREB and GAPDH. Data are representative of three separate experiments (*, p < 0.05, Student's t test).
FIGURE 6.
Activation of CREB suppresses pro-inflammatory gene induction and promotes anti-inflammatory gene induction by LPS in THP-1 cells. THP-1 cells were treated with various dosages of LPS with 500 n
m
adenosine for 4 h. The levels of IL-6 (A), TNFα (B), IL-10 (C), and IL-33 mRNA (D) were measured by real-time RT-PCR (*, p < 0.05; Student's t test). E, THP-1 cells were treated with various dosages of LPS with 500 n
m
adenosine for 2 h. Nuclear protein lysates were separated on SDS-PAGE and probed with specific antibodies against pCREB-S133, CREB, or Lamin-B1. Data are representative of three separate experiments.
FIGURE 7.
The proposed signaling network contributing to the pro-inflammatory skewing of innate immunity by super-low dose LPS. GSK3 and Akt are engaged in competitive inhibition while simultaneously driving the activity of downstream transcription factors, ultimately promoting the expression of pro- or anti-inflammatory genes. High-dose LPS robustly activates both arms of this response, while super-low dose LPS preferentially causes mild activation of GSK3.
References
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