SphK1 and LIGHT mRNA induction is regulated by Syk and IL-10.

<p>(A, B) Wild type BMDMs were stimulated with 200 µg/ml zymosan in the presence or absence of 4 µM Syk Inh II. The induction of SphK1 (A) and LIGHT (B) mRNA was determined by qPCR. (C, D) Wild type or IL-10 knockout BMDMs were stimulated for 8 h with 200 µg/ml zymosan and the induction of SphK1 (C) and LIGHT (D) mRNA was determined by qPCR. (E, F) Wild type or MSK1/2 knockout BMDMs were stimulated for 8 h with 200 µg/ml zymosan and the induction of SphK1 (E) and LIGHT (F) mRNA was determined by qPCR. Error bars represent the standard deviation of independent cultures from 4 mice per genotype. P values (Student's t-test) of less than 0.05 are indicated by * and less than 0.01 by **.</p

Inhibition of MAPK pathways modulates IL-10 induction in response to dectin-1 selective ligands.

<p>Wild type BMDMs were treated with 2 µM PD184352 or 5 µM SB203580 as indicated. Cells were the stimulated with 10 µg/ml curdlan. IL-10 secretion was measured after 8 h (A). Alternatively at 1 h IL-10 mRNA (B) or primary transcript (C) levels were determined by qPCR. (D–F) As above, except cells were stimulated with 200 µg/ml depleted zymosan. Error bars represent the standard deviation of independent cultures from 4 mice per genotype. P values (Student's t-test) of less than 0.05 are indicated by * and less than 0.01 by **.</p

MSKs regulate IL-10 mRNA transcription via CREB.

<p>BMDMs were cultured from wild type, MSK1/2 knockout, CREB S133A knockin or MSK1/2 knockout/CREB S133A knockin mice. Cells were then stimulated with 200 µg/ml zymosan for either 1 h or for the indicated times. The induction of nur77 (A), TNFα (B) and IL-10 (C) mRNA was determined by qPCR. (D) Wild type or MSK1/2 knockout BMDMs were pretreated with 10 µM SB-747651A for 1 h where indicated and then stimulated with zymosan for 1 h. IL-10 mRNA induction was determined by qPCR. (E) MSK1/2 knockout BMDMs were treated with 2 µM PD184352 or 5 µM SB203580 as indicated before stimulation with zymosan for 1 h. IL-10 mRNA induction was then determined by qPCR. Error bars represent the standard deviation of independent cultures from 4 mice per genotype. In A to C, a P value (Student's t-test) relative to the wild type cells of less than 0.05 is indicated by * and less than 0.01 by **. In D and E a P value of less than 0.01 relative to the no inhibitor control is indicated by **.</p

Zymosan induced MAPK activation and IL-10 production in part through dectin-1.

<p>(A) BMDMs were isolated from wild type or dectin-1 knockout animals on a C57/Bl6 background and stimulated with 200 µg/ml zymosan for 8 h. The levels of IL-10 protein secreted into the media were measured. (B) Wild type BMDMs were treated with 4 µM Syk Inhibitor II where indicated for 1 h before stimulation with 200 µg/ml zymosan for 8 h. Secreted IL-10 levels were then determined. In A and B error bars represent the standard deviation of independent cultures from 4 mice per genotype. P values (Student's t-test) of less that 0.001 are indicated by **. (C) BMDMs from wild type or dectin-1 knockout on a 129SvJ background were stimulated for the indicated times with 200 µg/ml zymosan. The levels of phospho and total ERK1/2, phospho and total p38 and phospho CREB/ATF1 were then determined by immunoblotting. (D) Wild type BMDMs were treated with 2 µM PD184352 or 5 µM SB203580 where indicated for 1 h before stimulation with 200 µg/ml zymosan for 30 min. The levels of total and phospho (T581) MSK1, phospho and total ERK1/2, phospho and total p38α and phospho CREB/ATF1 were then determined by immunoblotting. (E) Wild type or MSK1/2 double knockout BMDMs were stimulated with 200 µg/ml zymosan for the indicated times, and protein levels determined as in (D). In addition to MSK1, the total MSK1 antibody also picks up a non-specific band (ns) that runs at a molecular weight just below MSK1. The blots shown are representative of one (C) or three (D and E) separate experiments.</p

MSK and CREB regulate IL-10 secretion in BMDCs.

<p>(A) BMDCs were isolated from wild type and MSK1/2 knockout mice. Following stimulation with 200 µg/ml zymosan or 10 µg/ml curdlan for 8 h the levels of IL-10 secreted into the media was determined. (B) as (A) except BMDCs were from wild type or CREB Ser133Ala knockin macrophages. In both panels measurements were made for independent preparations of BMDCs from 4 mice per genotype. Individual measurements (open symbols) as well as average and standard deviation are shown. A p value (Student's t-test) of less that 0.01 is shown by **.</p

Dectin-1 specific ligands activate MSKs.

<p>(A) BMDMs from wild type or dectin-1 knockout on a 129SvJ background were treated with 10 µg/ml of curdlan for the indicated times and the levels of phospho and total ERK1/2, phospho and total p38α and phospho CREB/ATF1 determined by immunoblotting. (B) As (A) except cells were stimulated with 200 µg/ml of depleted zymosan. (C) Where indicated, wild type BMDMs were treated with 2 µM PD184352 or 5 µM SB203580 for 1 h. Cells were then stimulated with 10 µg/ml of curdlan for 30 min and the levels of total and phospho (T581) MSK1, phospho ERK1/2, phospho and total p38α and phospho CREB/ATF1 determined by immunoblotting. (D) Wild type or MSK1/2 knockout BMDMs were stimulated with 10 µg/ml of curdlan for the indicated times and the levels of the indicated proteins determined by immunoblotting. The blots shown are representative of one (B) or two (A, C, D) separate experiments.</p

MSKs promote IL-10 transcription.

<p>Wild type and MSK1/2 knockout BMDMs were isolated and stimulated with either 200 µg/ml of zymosan (A, B, C), 10 µg/ml curdlan (D, E, F) or 200 µg/ml of depleted zymosan (Dep Zym, G, H, I) for the indicated times. IL-10 protein levels secreted into the media were measured (A, D, G) and IL-10 mRNA (B, E, H) or IL-10 1° transcript (C, F, I) induction determined by qPCR. Error bars represent the standard deviation of independent cultures from 4 mice per genotype. P values (Student's t-test) of less than 0.05 are indicated by * and less than 0.01 by **. ns indicates a p value of more than 0.05.</p

SphK1 and LIGHT mRNA induction is partially regulated via dectin-1.

<p>BMDMs were isolated from either wild type or dectin-1 mice on a C57/Bl6 background. Cells were stimulated with either 200 µg/ml zymosan or 10 µg/ml curdlan for 8 h. Total RNA was then isolated and SphK1 (A) and LIGHT (B) mRNA levels determined. Error bars represent the standard deviation of independent cultures from 4 mice per genotype. P values (Student's t-test) of less than 0.01 are indicated by **.</p

ERK1/2 regulate IL-10 transcription via MSK1/2 dependent and independent mechanisms.

<p>Dectin-1 is activated by complex β-glucan containing particles that induce clustering of dectin-1 at the membrane and formation of a phagocytic synapse. This leads to Syk recruitment to the ITAM like sequence in the cytoplasmic domain of dectin-1. Syk then mediates the activation of downstream signaling including the ERK1/2 and p38α MAPK cascades. Both ERK1/2 and p38α phosphorylate and activate the protein kinases MSK1 and 2. These in turn phosphorylate CREB on the IL-10 gene promoter, which stimulates IL-10 mRNA transcription. In addition, ERK1/2 also activate a MSK and p38 independent pathway that inhibits IL-10 mRNA transcription. The identity of this pathway is not clear. In addition to the ERK1/2 and p38α pathways shown dectin-1 also activates NFκB that likely also plays a role in inducing IL-10 transcription.</p

Zymosan induces STAT3 phosphorylation via IL-10.

<p>(A) BMDMs were isolated from wild type or MSK1/2 knockout mice and stimulated for the indicated times with 200 µg/ml zymosan. Cells were lysed and the levels of total and phospho (Y705) STAT3 as well as GAPDH were determined by immunoblotting. (B, C) BMDMs from either wild type or MSK1/2 knockout mice were stimulated for the indicated times with 200 µg/ml zymosan. Secreted IL-12p40 protein (B) and IL-12p40 mRNA (C) levels were determined. (D) Wild type, IL-10 knockout, MSK1/2 double knockout or MSK1/2/IL-10 triple knockout BMDMs were treated with 200 µg/ml zymosan for 8 h. Secreted IL-12p40 levels were then determined. The average for each genotype is shown by a bar and the BMDMs from individual mice by X. Error bars (B–D) represent the standard deviation of independent cultures from 4 mice per genotype. P values (Student's t-test) of less than 0.05 are indicated by * and less than 0.01 by **.</p