Impaired cytokine production by cpdm BMDC is correlated with selective defects in NF-κB signaling
There are a number of possible explanations for the defective cytokine secretion in stimulated cpdm BMDC, including 1) reduced surface expression of the LPS receptor complex, 2) increased production of anti-inflammatory mediators, 3) increased expression of negative regulators of TLR pathways, and 4) impaired TLR-induced signaling activation.
We determined the surface expression of the LPS receptor complex that comprises TLR4, the accessory proteins CD14 and myeloid differentiation factor 2 (MD2/LY96) [19]. Flow cytometric analysis shows that the expression levels of CD14 and TLR4/MD2 between WT and cpdm BMDC were similar (Fig. 5A). We then quantified the secretion of the suppressive cytokines IL10 that can inhibit IL12 secretion in an autocrine manner [20], [21]. The supernatants from LPS-stimulated cpdm BMDC contained significantly lower levels of IL10 than stimulated WT BMDC (Fig. 5B), suggesting that IL10 was not responsible for decreased secretion of IL12P70 by cpdm BMDC. Increased expression of a negative regulator of TLR signaling such as A20 [22] may also suppress cytokine secretion. However, the transcript level of A20 was lower in LPS-activated cpdm BMDC than WT controls (Fig. 5C), thereby ruling out overexpression of A20 as a factor in the reduced cytokine production.
10.1371/journal.pone.0031809.g005 Figure 5  Normal TLR4 and MD2 expression and decreased IL10 secretion and A20 expression by cpdm BMDC.
(A) Unstimulated WT and cpdm BMDC (5×105) were labeled with PE-labeled anti-TLR4/MD2 or anti-CD14 and then subject to flow cytometry analysis. Unstained cells serve as negative controls. (B) Cultured WT and cpdm BMDC (1×105 cells in 0.1 ml complete medium) were washed and stimulated with medium, 100 ng/ml LPS or 25 µg/ml poly I:C for 24 hours. Supernatants were collected for ELISA of IL10. (C) Cultured WT and cpdm BMDC (5×105 cells in 0.2 ml complete medium) were washed and stimulated with 100 ng/ml LPS or 25 µg/ml poly I:C. At 0, 1, and 2 hours, total RNA was extracted and subject to qRT-PCR to measure the production of A20.    The transcription of TLR3/4-induced proinflammatory intermediates is tightly regulated by cellular signaling pathways, in particular NF-κB, TBK1/IRF3, and MAPK [23]–[27]. We next determined if disrupted NF-κB, TBK1/IRF3, and/or MAPK signaling may underlie the impaired cytokine production from stimulated Sharpin-deficient BMDC. Stimulus-induced phosphorylation of the IκB kinase (IKK1/2) is an essential step in NF-κB signaling, allowing phosphorylation and proteasome-mediated degradation of the NF-κB inhibitor IκBα to release the NF-κB transcription factors into the nucleus. The amount of phosphorylated IKK1/2 (p-IKK1/2) and IκBα (p-IκBα) following incubation with LPS or poly I:C was severely decreased in cpdm BMDC as compared with WT controls (Fig. 6). The cpdm BMDC exhibited similar levels of TBK1, ERK1/2, and p38 phosphorylation to those of WT cells (Fig. 6). These results indicate that the absence of functional SHARPIN decreased NF-κB activation but did not affect TBK1/IRF3, ERK1/2, and p38 signaling in BMDC.
10.1371/journal.pone.0031809.g006 Figure 6  Inhibition of NF-κB signaling in cpdm BMDC.
WT and cpdm BMDC (2×106 cells in 0.5 mL complete medium) were stimulated with 100 ng/mL LPS (A) or 25 µg/mL poly I:C (B). At 0, 15, 30, and 60 minutes, whole-cell lysates were obtained and subject to immunoblots with antibodies against proteins involved in NF-κB, TBK1/IRF3, ERK1/2, and p38 signaling pathways. Beta-actin was used as loading control. (C) Cellular levels of p-IKK1/2 and p-IκBα in LPS- or poly I:C-stimulated BMDC were quantitated with ImageJ (NIH) and presented as trend lines. Results are representative of at least two independent experiments.

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