Long-Time Treatment by Low-Dose <i>N</i>-Acetyl-L-Cysteine Enhances Proinflammatory Cytokine Expressions in LPS-Stimulated Macrophages

<div><p><i>N</i>-acetyl-L-cysteine is known to act as a reactive oxygen species scavenger and used in clinical applications. Previous reports have shown that high-dose <i>N</i>-acetyl-L-cysteine treatment inhibits the expression of proinflammatory cytokines in activated macrophages. Here, we have found that long-time <i>N</i>-acetyl-L-cysteine treatment at low-concentration increases phosphorylation of extracellular signal-regulated kinase 1/2 and AKT, which are essential for the induction of proinflammatory cytokines including interleukin 1β and interleukin 6 in lipopolysaccharide-stimulated RAW264.7 cells. Furthermore, long-time <i>N</i>-acetyl-L-cysteine treatment decreases expressions of protein phosphatases, catalytic subunit of protein phosphatase-2A and dual specificity phosphatase 1. On the other hand, we have found that short-time <i>N</i>-acetyl-L-cysteine treatment at low dose increases p53 expression, which inhibits expressions of proinflammatory cytokines. These observations suggest that long-time low-dose <i>N</i>-acetyl-L-cysteine treatment increases expressions of proinflammatory cytokines through enhancement of kinase phosphorylation.</p></div

The effect of NAC treatment on TLR signal pathway in LPS-stimulated RAW264.7 cells.

<p>RAW264.7 cells were plated at 1.0×10⁵ cells/well, and cultured in DMEM containing 10% FCS with 2 mM NAC for 30 hours. 100 ng/ml LPS was added into the conditioned media, followed by further 3 hour incubation. A. The cell lysates were subjected to western blot analysis using anti-MyD88, cot/Tpl2, or IκBα antibody. B. The pNFκB-Luc plasmid was stably transfected into RAW264 cells. Cells were treated with 2 mM NAC for 1 hour, 9 hours or 30 hours, and stimulated with 100 ng/ml LPS for 3 hours. Luciferase activities in the cell lysates were normalized by the protein contents.</p

LPS-stimulated phosphorylation of MAPKs to induce proinflammatory cytokines.

<p>RAW264.7 cells were plated at 1.0×10⁵ cells/well and cultured in DMEM containing 10% FCS for 2 days. Cells were pretreated with various concentrations of U0126, SB203580 (SB), SP600125 (SP) or LY2904002 (LY) for 30 min, and stimulated with 100 ng/ml LPS for 20 min (A) or 3 hr (B). A. The cell lysates were subjected to western blot analysis using anti-pERK1/2, ERK1/2, pp38, p38, pJNK1/2, and JNK1/2. B. The gene expression of IL-1β and IL-6 was detected by real time RT-PCR. The mRNA levels were normalized by Ubc mRNA levels. *<i>p</i><0.05 versus control.</p

Reduction of interleukin expressions in LPS-stimulated RAW264.7 cells by over-expression of p53.

<p>RAW264.7 Tet-On stable cells were stably transfected with pTRE2Hyg-tp53, and treated with or without doxycycline. 48 hours later, the cells were treated with or without 100 ng/ml LPS for 3 hours, and the cell lysates and total RNA were extracted. A. Protein levels of p53 in the cell lysates were analyzed by western blot analysis. B. IL-1β and IL-6 mRNAs were detected by real time RT-PCR in total RNA from RAW264.7 Tet-On cells stably transfected with pTRE2Hyg-tp53. The mRNA levels were normalized by Ubc mRNA levels. (C) RAW264.7 Tet-On cells stably transfected with pTRE2Hyg-p53 were further transfected with pGV-IL1p. The resultant cells were treated with doxycycline for 48 hours. Luciferase activity in the cell lysates was measured and normalized by the protein content.</p

Effects of NAC treatment on p53 protein levels in the nuclear fraction of LPS-stimulated macrophages.

<p>A, B. RAW264.7 cells were pretreated with 2(A) or 20 mM (B) NAC for the indicated time. Cell lysates and nuclear extracts were prepared before (upper panels) and after (lower panels) stimulation with 100 ng/ml LPS for 3 hours, and then subjected to western blot analyses with anti-p53, anti-TBP, or anti-GAPDH antibody. C. RAW264.7 cells, pretreated with 2 mM NAC for the indicated time, were stimulated with 100 ng/ml LPS for further 3 hours, and then total RNA was extracted from the cells. p53 mRNA expression was analyzed by real time RT-PCR. The mRNA levels were normalized by Ubc mRNA levels. *<i>p</i><0.05 versus time 0.</p

Reduction of phosphatase expressions by long-time NAC treatment.

<p>RAW264.7 cells were treated with 2(A) or 20 mM (B) NAC following the time schedule shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087229#pone-0087229-g001" target="_blank">Fig. 1A</a>. Total RNA and cell lysates were extracted from the cells. The gene expression of PP2Ac and DUSP1 was detected by real time RT-PCR. The mRNA levels were normalized by Ubc mRNA levels. *<i>p</i><0.05 versus time 0.</p

The effect of NAC treatment on ROS production in RAW264.7 cells.

<p>RAW264.7 cells were treated with 2 µM H₂DCFDA-containing HBSS for 2 hours. The fluorescence emission of the cell suspensions was measured. *<i>p</i><0.05 versus time 0, relatively. RFU, relative fluorescence units.</p

MAP3K8 in humans is associated with higher BMI and cytokine expression.

<p>MAP3K8 mRNA expression in human subcutaneous adipose tissue, associated with (a) BMI, (b) plasma insulin values, (c) plasma glucose levels, (d) HOMA-IR, (e) adipocyte sell size cell size and (f) crown-like structures. *p<0.05. n = 51, 50, 71, 70 respectively. HOMA-IR  =  Homeostatic Model Assessment for insulin resistance.</p

Inflammatory profile of the adipose tissue of HFD-fed WT and MAP3K8-ko animals.

<p>MAP3K8-ko and WT mice were fed a LFD or HFD during 16 weeks. (a–f) qPCR analysis for cytokines (a) TNF-α, (b) IFNγ, (c) IL-1β, (d) CXCL-1, (e) IL-6 and (f) IL-1Ra. n = 9 mice per group. Relative phosphorylation of NFκB p65 (g) and ERK 1/2 (h) in eWAT of MAP3K8-ko and WT animals after HFD-feeding (i). * p<0.05, ** p<0.01, *** p<0.001.</p

Obesity and macrophage influx in adipose tissue of HFD-fed WT and MAP3K8-ko animals.

<p>MAP3K8-ko and WT mice were fed a LFD or HFD during 16 weeks. (a) Bodyweight development upon LFD or HFD feeding. (b) Epididymal white adipose tissue (eWAT) weight after 16 weeks of LFD or HFD. (c) Liver weight after 16 weeks of LFD or HFD. (d) Plasma CXCL1 levels after 16 weeks of LFD or HFD (e) Macrophage influx into the adipose tissue as determined by immunohistochemistry, F4/80 (serotec) staining: 20× magnification or 40× as indicated: (f) Number of crown-like structures per field. (g–i) qPCR analysis for macrophage infiltration markers, (g) CD68, (h) F4/80, (i) MCP-1 in adipose tissue of MAP3K8-ko and WT animals. * p<0.05, ** p<0.01, *** p<0.001.</p