Increased abscess formation and defective chemokine regulation in CREB transgenic mice - PubMed (original) (raw)

Comparative Study

Increased abscess formation and defective chemokine regulation in CREB transgenic mice

Andy Y Wen et al. PLoS One. 2013.

Abstract

Cyclic AMP-response element-binding protein (CREB) is a transcription factor implicated in growth factor-dependent cell proliferation and survival, glucose homeostasis, spermatogenesis, circadian rhythms, and synaptic plasticity associated with memory. To study the phenotype of CREB overexpression in vivo, we generated CREB transgenic (TG) mice in which a myeloid specific hMRP8 promoter drives CREB expression. CREB TG mice developed spontaneous skin abscesses more frequently than wild type (WT) mice. To understand the role of CREB in myeloid function and innate immunity, chemokine expression in bone marrow derived macrophages (BMDMs) from CREB TG mice were compared with BMDMs from WT mice. Our results demonstrated decreased Keratinocyte-derived cytokine (KC) in CREB TG BMDMs but not TNFα protein production in response to lipid A (LPA). In addition, mRNA expression of KC and IL-1β (Interleukin)-1β was decreased in CREB TG BMDMs; however, there was no difference in the mRNA expression of TNFα, MCP-1, IL-6 and IL-12p40. The mRNA expression of IL-1RA and IL-10 was decreased in response to LPA. Nuclear factor kappa B (NFκB) expression and a subset of its target genes were upregulated in CREB TG mouse BMDMs. Although neutrophil migration was the same in both CREB TG and WT mice, Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity was significantly increased in neutrophils from CREB TG mice. Taken together, CREB overexpression in myeloid cells results in increased abscess formation in vivo and aberrant cytokine and chemokine response, and neutrophil function in vitro.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Comparison of abscess location and types of infection.

(A) Numbers of mice that developed skin and soft-tissue abscesses were evaluated and the location of abscesses was recorded. The majority (90%) of skin and soft-tissue abscesses were located in the genital region. The remaining abscesses were discovered on the tail (2%), involved the eyes, kidney, and abdomen (4%), or not recorded (4%). (B) Types of bacteria identified in 14 abscesses cultured from CREB TG mice, the numbers of positive cultures, and percent of each type of bacteria are listed. (C) Estimated Cumulative Incidence of skin infections in CREB TG and WT mice. Kaplan-Meier analysis was performed to take censoring into account in estimating the cumulative risk with age of developing a first skin abscess or non-abscess dermatitis in CREB TG and WT, if male and female mice could be followed indefinitely under breeding colony conditions. (A) CREB TG males developed skin abscesses alone earlier and with a higher overall risk than WT male mice (p<0.0001 by log-rank test). Similarly, CREB TG male mice were at greater risk of developing abscesses or non-abscess dermatitis than WT mice (p<0.0001). (B) CREB TG female mice developed skin abscesses earlier than WT female mice (p = 0.0008). However, the time to first appearance of abscess or non-abscess dermatitis did not appear significantly different between CREB TG female and WT female mice (p = 0.168).

Figure 2

Figure 2. Purification of BMDMs and expression of CREB.

(A) Whole bone marrow was obtained from CREB TG and WT mice. Flow cytometry was performed after staining cells for CD11b and F4/80 cell surface markers confirming 93% CD11b+/F4/80+ BMDM population. (B) Western blot analysis was performed with CREB antisera (UBI, Upstate, NY) and normalization for Actin expression (Santa Cruz Biotechnology, Santa Cruz, CA). To quantitate the band, ImageJ software was used (

http://rsb.info.nih.go/ij/

). CREB TG mice BMDMs had 4-fold greater CREB expression than WT mice.

Figure 3

Figure 3. CREB overexpression results in reduced LPA-induced KC production and differential chemokine and cytokine expression.

(A) ELISA were performed to quantify KC and TNF-α production and (B) qRT-PCR was performed to compare mRNA expression in BMDMs isolated from CREB TG or WT mice exposed for 2 hours to 10 ng/ml LPA. KC and TNF-α production was measured using ELISA and mRNA levels were measured using qRT-PCR and normalized to GAPDH mRNA levels. The data represent an average ± SEM of four mice analyzed in triplicate for ELISA and three independent experiments with three mice each for qRT-PCR experiments; *p<0.05, **p<0.01, ***p<0.001, ns means “not significant”.

Figure 4

Figure 4. Expression of known nuclear factor kappa B (NFκB) target genes.

(A-E) qRT-PCR was performed with RNA from BMDMs from CREB and WT mice. Chemokine ligand 5 (CCL5), Colony Stimulating Factor 1 or Macrophage Colony Stimulating Factor (CSF1 or M-CSF), Interferon Factor 1 (IRF1), and NFkB were upregulated in CREB BMDMs (***p<0.005). Chemokine ligand 2 (CCL2) expression was downregulated. The data represent an average ± SEM of four mice analyzed in triplicate for ELISA and two independent experiments. ***p<0.001.

Figure 5

Figure 5. NBT Oxidase Activity.

Neutrophils from CREB TG or WT bone marrow were incubated with 10 uM formyl-Leu-Met-Phe (fMLP) and 0.125% NBT. WT = wild type mice, CREB = CREB TG mice. P-values are represented above the data bars, * p<0.05, ** p<0.01. Error bars represent the standard error.

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