Mechanisms for suppression of interleukin-6 expression in peritoneal macrophages from docosahexaenoic acid-fed mice

Abstract

Consumption of the trichothecene mycotoxin deoxynivalenol (DON) induces interleukin-6 (IL-6)-dependent IgA nephropathy (IgAN) in mice. This effect can be prevented by feeding long-chain n-3 polyunsaturated fatty acids (PUFAs) found in fish oil. The purpose of this study was to identify the signal transduction pathways by which DON up-regulates IL-6 in the peritoneal macrophage and how consumption of fish oil enriched with the n-3 PUFA docosahexaenoic acid (DHA) suppresses these processes. Incubation with DON induced IL-6 expression in naïve macrophages maximally at 3 h. Knockdown of the transcription factor cAMP response element-binding protein (CREB) or pharmacologic inhibition of the CREB kinases Akt1/2, MSK1 and RSK1 down-regulated this expression. Inhibition of double-stranded RNA-activated protein kinase (PKR) suppressed not only IL-6 expression but also phosphorylation of CREB and its upstream kinases, Akt1, MSK1 and RSK1. Phosphorylations of PKR, CREB kinases and CREB were markedly impaired in peritoneal macrophages isolated from mice that consumed DHA-enriched fish oil for 6 to 8 weeks. DHA's effects were not explainable by increased activity of protein phosphatase 1 and 2A since both were suppressed in mice consuming the DHA diet. Although cells cultured directly with DHA expressed less IL-6 compared to cells cultured with arachidonic acid (AA), neither fatty acid treatment affected DON-induced protein phosphorylation. Furthermore, DHA and AA similarly inhibited cell-free protein kinase activity. These data suggest that DON-induced IL-6 expression is CREB mediated and PKR dependent, and that requisite kinase activities for these pathways were suppressed in macrophages from mice fed DHA for an extended period.

Fig. 1

Kinetics of DON-induced IL-6 mRNA expression in peritoneal macrophages. Naïve peritoneal macrophages were cultured with DON (250 ng/ml) for different time periods. Total RNA was extracted and IL-6 mRNA was analyzed by real-time PCR. Data are means ± SEM. Data points with different letters differ (p<0.05). All data were normalized against β2-microglobulin and expressed relative to the value at time 0. Results are representative of two independent experiments.

Fig. 2

Transcription factor CREB knockdown inhibits IL-6 mRNA expression induced by DON. siRNA specific to CREB or scrambled siRNA was transfected by electroporation into naïve peritoneal macrophages. (A) To evaluate CREB knockdown efficiency, total protein was collected after 48 h and CREB measured by Western blot. RI indicates relative intensity which is the percentage of the maximal fluorescence in the same lane. (B) To detect the role of CREB on IL-6 mRNA expression, cells were treated with DON 48 h after transfection. Total RNA was collected and IL-6 mRNA was analyzed by real-time PCR. Data are means ± SEM. Bars with different letters differ (p<0.05). Results are representative of two independent experiments.

Fig. 3

Inhibition of CREB kinases suppresses DON-induced IL-6 expression. Naïve peritoneal macrophages were cultured for 1 h with CREB kinase inhibitors dissolved in DMSO or the DMSO vehicle, incubated with DON (250 ng/ml) for 3 h and then IL-6 mRNA measured by real-time PCR‥ Compounds used were (A) MSK1/RSK1 inhibitor Ro 31–8220, (B ) Akt inhibitor IV, (C) Akt inhibitor V (25 µM) and (D) Akt inhibitor VIII (25 µM). Data are means ± SEM. Bar with asterisk differs from that of DON treatment without inhibitor (p<0.05). Results are representative of at least two independent experiments.

Fig. 4

PKR inhibition blocks DON-induced IL-6 expression and protein phosphorylation. (A) Naïve peritoneal macrophages were cultured for 45 min with PKR inhibitor (PKRI) or PKR inhibitor negative control (PKRI NC) and then with DON 250 ng/ml for 3 h. IL-6 mRNA was measured by real-time PCR. Data are means ± SEM. Bar with asterisk differs from that of DON treatment without inhibitor (p<0.05). (B) To detect the role of PKR activation in CREB phosphorylation, naïve macrophages were cultured for 45 min with PKR inhibitor (PKRI) or PKR inhibitor negative control (PKRI NC) and then with DON (250 ng/ml) for different periods. Phosphorylation of CREB and its upstream kinases was measured by Western blot. RI indicates relative intensity which is the percentage of the maximal fluorescence in the same lane. Results are representative of two independent experiments.

Fig. 5

DON-induced phosphorylation of CREB, Akt1, MSK1 and RSK1 is suppressed in macrophages from DHA-fed mice. Peritoneal macrophages from mice fed control or DHA diet were incubated with DON (250 ng/ml) for indicated time periods. (A) Cell lysates were subjected to Western blot. RI indicates relative intensity which is the percentage of the maximal fluorescence in the same lane. (B) RI and DON exposure time were plotted. Results are representative of three independent experiments.

Fig. 6

DON-induced Akt1activation is suppressed in macrophages from DHA-fed mice. Peritoneal macrophages from mice fed control or DHA diet for were incubated with DON (250 ng/ml) for indicated time points. Following cell lysis, Akt1 was immunoprecipitated and incubated with CREB and ATP. Phosphorylation of CREB was analyzed by Western blot. RI indicates relative intensity which is the percentage of the maximal fluorescence in the same lane.

Fig. 7

Phosphorylation of PKR in the peritoneal macrophage is inhibited by DHA consumption. Peritoneal macrophages from mice fed control or DHA diet were treated with DON (250 ng/ml) for indicated time periods. Cell lysates were subjected to Western blot. RI indicates relative intensity which is the percentage of the maximal fluorescence in the same lane.

Fig. 8

PP1 and PP2A phosphatase activities are not increased in macrophages from DHA-fed mice. (A) Peritoneal macrophage from control- or DHA-fed mice were treated with DON (250 ng/ml) for 0, 10, 30, or 60 min. PP1 and PP2A in the cell lysates were immunoprecipitated and analyzed respectively for phosphatase activities. Values were expressed relative to control at time 0. Data are means ± SEM. Points with asterisk differ from those of corresponding DHA group (p<0.05). (B) To evaluate effects of PP1 and PP2A on protein phosphorylation, peritoneal macrophages from mice fed control or DHA diet were pretreated with vehicle or calyculin A (20 nM) for 1 or 2 h and then incubated with DON for 30 min. Cell lysates were subjected to Western blot. RI indicates relative intensity which is the percentage of the maximal fluorescence in the same lane.

Fig. 9

Fatty acids differentially affect DON-induced IL-6 mRNA expression but not protein phosphorylation in peritoneal macrophages. Serum-deprived peritoneal macrophages were treated with 50 µM fatty acid complexed with BSA for 24 h. (A) For IL-6 measurement, RNA was extracted after 3-h DON (250 ng/ml) treatment and analyzed by real-time PCR. Data are means ± SEM. Bars with different letters differ (p<0.05). (B) To detect protein phosphorylation affected by different fatty acids, cells were incubated with DON for 20 min, and total protein was analyzed by Western blot. RI indicates relative intensity which is the percentage of the maximal fluorescence in the same lane.

Fig. 10

AA and DHA similarly decrease CREB kinase activities in the cell-free system. Protein kinases (Akt1, RSK1 and MSK1) were incubated with 12.5, 25, 50 and 100 µM fatty acids or with PBS vehicle before CREB and ATP were added. CREB phosphorylation was analyzed by Western blot. RI indicates relative intensity which is the percentage of the maximal fluorescence (for PBS vehicle control, far right) in the same row.

Fig. 11

Effects of DHA consumption on signal transduction pathways mediating DON-induced IL-6 expression in peritoneal macrophages ex vivo. Possible CREB kinases inhibited by DHA feeding include AKT, RSK1 and MSK1. The symbol ⊣ on the left indicates inhibition of the pathway step on the right.