A novel role for c-Src and STAT3 in apoptotic cell-mediated MerTK-dependent immunoregulation of dendritic cells

Abstract

Dendritic cells (DCs) play an instrumental role in regulating tolerance to self-antigens and preventing autoimmunity. One mechanism by which "tolerogenic" DCs are established is through the inhibitory effects of apoptotic cells (ACs). Immature DCs encountering ACs are resistant to stimuli that activate and mature DCs. We have shown that the Mer receptor tyrosine kinase (MerTK) plays a key role in transducing inhibitory signals upon binding of ACs, which in turn involve the phosphatidylinositol 3-kinase (PI3K) pathway. Nevertheless, the molecular basis for AC-induced inhibition of DCs is ill defined. In the current study, the proximal signaling events induced by MerTK after AC binding were studied. AC treatment of bone marrow-derived or splenic DCs established a complex consisting of MerTK, the nonreceptor tyrosine kinase c-Src, the transcription factor STAT3, and PI3K. In contrast, AC treatment of DCs lacking MerTK expression failed to increase c-Src and STAT3 activation. In addition, the inhibitory effects of ACs were blocked by treating DCs with pharmacologic inhibitors or siRNA specific for c-Src and STAT3. These findings demonstrate that AC-induced inhibition of DCs requires MerTK-dependent activation of c-Src and STAT3, and provide evidence for novel roles for c-Src and STAT3 in the immunoregulation of DCs.

Figure 1

AC-induced STAT3 activation in DCs is MerTK dependent. MerTK^+/+ BMDCs (A-C) or MerTK^+/+ and MerTK^−/− sDCs (D) were treated with ACs for the indicated times. pSTAT3, STAT3, and β actin were measured in whole-cell lysates via Western blot using the same membranes (A,D), or DNA-binding activity of nuclear STAT3 and SP1 determined by EMSA (B) and a supershift assay (C). (E) MerTK^+/+ and MerTK^−/− sDCs were treated with Gas6 (150 nM) for the indicated times, and pSTAT3 and STAT3 were measured in whole-cell lysates via Western blot using the same membrane. (D-E) Fold induction of pSTAT3 was determined by normalizing densitometric readings against control cultures. (F) MerTK^+/+ BMDCs were incubated with ACs for the indicated times, MerTK was immunoprecipitated from whole-cell lysates, and Western blot membranes were probed for STAT3 and MerTK protein using the same membrane. Data are representative of a minimum of 3 experiments.

Figure 2

STAT3 activation is necessary for AC-induced inhibition of NF-κB activation in DCs. MerTK^+/+ BMDCs (A) or sDCs (B) were incubated with the STAT3 inhibitors Jsi124 (10 nM) or sttatic (20 nM) for 1 hour, cocultured with ACs or left untreated for 3 hours, and stimulated with LPS (50 ng/mL) for 0.5 hours. Nuclear NF-κB and SP-1 DNA-binding activity were determined via EMSA, and IκBα and β actin protein expression was detected by Western blot. (C-D) MerTK^+/+ BMDCs were transfected with STAT3-specific or scrambled control siRNA, cocultured with ACs for 3 hours, stimulated with LPS, and either (C) nuclear NF-κB and SP-1 DNA-binding activity and IκBα or β actin protein was measured as described for panels A and B or (D) IL-12 secretion was measured by enzyme-linked immunosorbent assay; *P < 10⁻³, DCs treated with STAT3-specific siRNA versus mock-transfected DCs or DCs transfected with control siRNA (Student t test). Error bars indicate ± SEM. (E) MerTK^+/+ BMDCs were pretreated with the PI3K inhibitors Wort and LY for 1 hour and incubated with ACs for the indicated times, and pSTAT3 and STAT3 protein was measured. (F) MerTK^+/+ or MerTK^−/− BMDCs were incubated with ACs for the indicated times, and p-STAT1 and STAT1 proteins were measured by Western blot. (G) MerTK^+/+ BMDCs were treated with STAT1 inhibitor fludarabine (20 μM) for 1 hour, and cocultured with ACs for 3 hours or left untreated, and stimulated with LPS (50 ng/mL). Nuclear NF-κB or SP-1 DNA-binding activity, and IκBα and β actin protein were measured as described for panels A-B. Data are representative of a minimum of 3 experiments.

Figure 3

c-Src is activated in a MerTK-dependent manner upon AC treatment of DCs. (A-B) MerTK^+/+ or MerTK^−/− sDCs were incubated with ACs for the indicated times, and pc-Src and c-Src protein was measured by Western blot. Fold induction of pc-Src was determined by normalizing densitometric readings against control cultures. (C) MerTK^+/+ BMDCs were treated with ACs, c-Src was immunoprecipitated from whole-cell lysates, and in vitro c-Src kinase activity and c-Src protein were measured by Western blot. (D) MerTK^+/+ BMDCs were incubated with ACs for the indicated times, MerTK was immunoprecipitated from whole-cell lysates, and c-Src and MerTK protein were determined by Western blot. (E) MerTK^+/+ BMDCs were pretreated with αMerTK or isotype Ab for 1 hour, and then incubated with ACs. Whole-cell lysates were examined via Western blot for pSTAT3, pc-Src, and β actin. Data are representative of a minimum of 3 experiments.

Figure 4

c-Src activation is required for AC-induced inhibition of DCs. MerTK^+/+ BMDCs (A) or sDCs (B) were incubated with the c-Src inhibitors E804 (10 nM) or PP1 (20 nM) for 1 hour and cocultured with ACs for 3 hours or left untreated, and stimulated with LPS (50 ng/mL). Nuclear NF-κB or SP-1 DNA-binding activity was determined via EMSA, and IκBα and β actin protein measured by Western blot. (C-D) MerTK^+/+ BMDCs were transfected with c-Src–specific or scrambled control siRNA, cocultured with ACs for 3 hours or left untreated, and stimulated with LPS (50 ng/mL for 0.5 hours or 1 μg/mL for 24 hours for IL-12 detection), and nuclear NF-κB and SP-1 DNA-binding activity and IκBα or β actin protein were examined (C), and IL-12p70 secretion was measured (D); *P < 10⁻³, DCs treated with c-Src–specific siRNA versus mock-transfected DCs or DCs transfected with control siRNA (Student t test). Data are representative of a minimum of 3 experiments. Error bars in panel D indicate ± SEM.

Figure 5

c-Src activation is an upstream event of PI3K and STAT3 activation. MerTK^+/+ BMDCs were pretreated with E804 (10 nM) for 1 hour and incubated with ACs for indicated times. Whole-cell lysates were prepared to detect p-AKT, AKT, p-STAT3, and STAT3 via Western blot (A-B) or MerTK was immunoprecipitated with MerTK Ab and Western blots were probed for STAT3, PI3Kp85, p-Tyr, and MerTK (C-D).