Regulation of CCL2 Gene Expression Figure 8. Overexpression of MKP-1 decreases the IL-1b-mediated activation of the CCL2 gene. A–C. 832/13 cells were transfected with siRNA duplexes targeting either a negative control sequence (siScramble) or siMKP-1. 24 h post-transfection cells were harvested and MKP-1 mRNA abundance was quantified (A), MKP-1 protein abundance was analyzed via immunoblot (B inset) or further treated concurrently with 10 nM Dex and 1 ng/mL IL-1b, then analyzed for CCL2 mRNA abundance (B) or CCL2 release into the media was measured via ELISA (C). #p,0.01 vs. siScramble. D. 832/13 cells were transduced with recombinant adenoviruses encoding either bGAL or MKP-1 (at multiple doses) overnight. Immunoblot analysis was done using whole cell lysates. The image shown represents two independent experiments. E. 832/13 cells were treated overnight with either bGAL or the lowest concentration of MKP-1 shown in D. The next day these cells were pre-treated with either DMSO (-) or 10 nM Dex for 6 h, followed by a 15 min exposure to 1 ng/mL IL-1b. Whole cell lysates were separated via SDS-PAGE. F. 832/13 cells were treated with either bGAL or three increasing concentrations of MKP-1; 24 h post-transduction cells were stimulated for 6 h with IL-1b. CCL2 mRNA abundance was measured and normalized to RS9. *p,0.05 vs. bGAL. G. 832/13 cells were treated with adenoviruses as in (F) for 4 h, transfected with 3.6-Luc and stimulated with IL-1b for 4 h. Promoter activity was normalized to total cellular protein. **p,0.01 vs. bGAL. H. 832/13 cells were transduced with either bGAL or MKP-1 adenovirus for 12 h and then incubated with IL-1b for a further 12 h. CCL2 release into the media was quantified via ELISA and data were normalized to total protein. *p,0.05. Data showing promoter activity, mRNA abundance and ELISA data are represented as means 6 SEM from 3–4 individual experiments; promoter activity experiments were performed in duplicate or triplicate. doi:10.1371/journal.pone.0046986.g008 of leukocyte recruited and the immune response pattern initiated (e.g., Th1 vs. Th2 responses). In this study, we observed both synthesis and secretion of CCL2 in response to the pro-inflammatory cytokine IL-1b (Figure 1) a cytokine strongly implicated in the development of T1DM [66]. We further discovered that inhibition of the p38 MAPK strongly impaired both the synthesis and secretion of CCL2 (see Figures 5, 7 & 8). Our results may thus explain data from a previous report demonstrating that administration of a p38 inhibitor to NOD mice decreases islet immune cell infiltration, which delayed the development of overt diabetes [28]. Taken together, the data reported herein reveals a heretofore undescribed potential explanation for the decrease in insulitis during p38 inhibitor delivery to NOD mice in vivo (i.e., reduction in secreted CCL2 decreases insulitis). Additionally, we also established that discrete synthetic glucocorticoids prevented the IL-1b-stimulated increase in expression of the CCL2 gene (see Figure 6). These novel findings may also have relevance to autoimmunity in the NOD mouse since these animals exhibit decreases in glucocorticoid receptor abundance prior to onset of diabetes [67]. The decline in GR protein may render the animals less sensitive to circulating glucocorticoids and therefore predispose the mice towards autoimmune and autoinflammatory conditions, including the development of T1DM. Treatment of T1DM by islet transplantation is still a work in progress [68,69]. Strategies to improve islet function and prevent graft rejection are critical barriers to successful transplantation therapy. Acute treatment of human islets ex vivo with glucocorti- coids prior to transplantation improved their function after transplantation [70]. This improvement in function could be due in large part to suppression of soluble secreted factors (e.g., CCL2, IL-8, etc.) that stimulate the recruitment of immune cells into grafted tissue. In addition, GC-mediated increases in anti- inflammatory proteins, such as MKP-1 (discussed below), are likely to also play key roles. While chronic glucocorticoid usage is associated with multiple negative side effects and was removed from some islet transplantation protocols, we propose that shorter term exposures may be useful for opposing the powerful pro- inflammatory signals that impair b-cell function and viability. Furthermore, modest doses of dexamethasone injected into rats enhanced glucose-stimulated insulin secretion in the subsequently isolated islets [71]. Thus, it is plausible that an enhancement in islet function may initially be possible in acute glucocorticoid treated islets, but long-term, chronic activation of the glucocor- ticoid receptor systemically could be detrimental to both pancreatic islets as well as peripheral tissues. Consequently, alternative strategies to suppress inflammation with the goal of retaining long-term islet function would circumvent the harmful side effects of chronic glucocorticoid exposure. It is therefore Figure 9. Overexpression of MKP-1 does not impair glucose- stimulated insulin secretion. A. 832/13 cells were transduced with the indicated recombinant adenoviruses overnight, followed by static incubation in either 3 mM or 15 mM glucose, or 15 mM glucose plus 5 mM forskolin. B. Isolated rat islets were transduced with the indicated adenoviruses for 48 h, followed by incubation in either 2.5 or 16.7 mM glucose. A, B. Insulin secreted into the culture media was measured by radioimmunoassay. A. *p,0.05 vs. respective glucose concentrations, n.s. = not significant; B. *p,0.05. All bar graphs shown represent means 6 SEM from 3–4 individual experiments, some of which were performed in duplicate. doi:10.1371/journal.pone.0046986.g009 PLOS ONE | www.plosone.org 15 October 2012 | Volume 7 | Issue 10 | e46986