The Orai-1 and STIM-1 complex controls human dendritic cell maturation

Abstract

Ca(2+) signaling plays an important role in the function of dendritic cells (DC), the professional antigen presenting cells. Here, we described the role of Calcium released activated (CRAC) channels in the maturation and cytokine secretion of human DC. Recent works identified STIM1 and Orai1 in human T lymphocytes as essential for CRAC channel activation. We investigated Ca(2+) signaling in human DC maturation by imaging intracellular calcium signaling and pharmalogical inhibitors. The DC response to inflammatory mediators or PAMPs (Pathogen-associated molecular patterns) is due to a depletion of intracellular Ca(2+) stores that results in a store-operated Ca(2+) entry (SOCE). This Ca(2+) influx was inhibited by 2-APB and exhibited a Ca(2+)permeability similar to the CRAC (Calcium-Released Activated Calcium), found in T lymphocytes. Depending on the PAMPs used, SOCE profiles and amplitudes appeared different, suggesting the involvement of different CRAC channels. Using siRNAi, we identified the STIM1 and Orai1 protein complex as one of the main pathways for Ca(2+) entry for LPS- and TNF-α-induced maturation in DC. Cytokine secretions also seemed to be SOCE-dependent with profile differences depending on the maturating agents since IL-12 and IL10 secretions appeared highly sensitive to 2-APB whereas IFN-γ was less affected. Altogether, these results clearly demonstrate that human DC maturation and cytokine secretions depend on SOCE signaling involving STIM1 and Orai1 proteins.

Figure 1. Expression of maturation markers in human dendritic cells according to extracellular Ca²⁺ concentration.

DC were cultured with increasing concentrations of Ca²⁺ (in mM). Cells were harvested and double or quadruple staining was assessed by FACS analysis for the 4 conditions. A – CD83 expression is increased by extracellular Ca2+ concentration. The population showed on this panel is gated on DC-Sign labeling. The percentage of double (CD86 and CD83) positive cells is shown in panel A. Results are representative of 7 independent experiments. B – Expression of maturation markers in relation to extracellular Ca²⁺ concentrations. The same population expressed CD80, CD86, HLA-DR and CD83 in increasing proportions in increasing extracellular Ca²⁺ concentration. The percentage of quadruple positive cells is shown in panel B. Graph bars represent the mean of 6 independent experiments (mean ± SD, *p<0,05). C – CD25 expression in relation to extracellular Ca²⁺. Grey histograms represent the expression of the cell-surface marker CD25. Results represent one out of 7 independent experiments.

Figure 2. Identification of SOCE during the earlier events of DC maturation.

DC were treated with thapsigargin (TG) in calcium free PSS, then PSS supplemented with 2 mM Ca²⁺ was added (A). 100 µM 2-APB PSS solution induced a rapid and reversible decrease of [Ca²⁺]_i (Grey trace). According to different maturation signals (LPS, B; TNF-α, C or zymosan, D), the variations of [Ca^2+] _i in the presence of SOCE inhibitor (Grey trace, at 100 µM 2-APB) or its absence (Black trace) were observed by microspectrofluorimetry on left panels. Results are representative of 7 independent experiments. Central panels represent the maximal amplitude mean of CCE according to the treatment. Right panels represent the CCE amplitude mean at 7.5 and 15 min. Means were obtained from 7 independent experiments.

Figure 3. 2-APB decreased the expression of maturation markers in human DC.

In panel A, DC were cultured in medium with maturating agents (TG (750 nM), LPS (50 ng/ml), Zymosan (25 µg/ml) or TNF-α (20 ng/ml)) in the absence or presence of 2-APB (mean of percentage ± SD, *p<0,05, n = 7). In panel B, DC were cultured in medium with the maturating agents in the absence or presence of SKF 96365 (100 µM) (n = 7 for DCi, TG and LPS conditions and n = 4 for Zymosan and TNF-α). The marker expressions were analyzed by FACS as described for figure 1B. In panel C, ORAI-1 and STIM-1 protein expressions were analyzed by western blotting to control Si-RNA efficacy.

Figure 4. 2-APB decreased the cytokine secretions in human DC.

IL-12 (panel A), IL-10 (panel B) and IFN-γ (panel C) were measured in supernatants of DC treated by maturating agents (TG (750 nM), LPS (50 ng/ml), zymosan (25 µg/ml) or TNF-α (20 ng/ml)) for 18 h in the presence of 100 µM 2-APB. In each graph, the black line represents the mean of 7 experiments, (*p<0,05).

Figure 5. Localization of STIM-1 and Orai-1 in DC.

The expression of Orai-1 and STIM-1 was analyzed at the transcriptional (A and B) or translational (C) level. Quantitative RT-PCR analysis of Orai-1 (A) and STIM-1 (B) mRNA showed their expression profile for different maturating stimuli (mean ± SD). The protein expressions of STIM-1 and Orai-1 were analyzed by confocal microscopy using specific antibodies coupled with a secondary antibody: Alexa Fluor®-488 for STIM-1 (green) and Alexa Fluor®-555 for Orai-1 (red). The cells were treated with maturating agents (TG (750 nM), LPS (50 ng/ml), zymosan (25 µg/ml) or TNF-α (20 ng/ml)) during 18 hours.

Figure 6. Inhibition of SOCE in DC by si-RNA (si-STIM-1 and si-Orai-1).

CCE was analyzed on DC treated by specific Orai-1 (grey line) and STIM-1 (light grey line) si-RNAs for 36 h; DC were exposed to TG (in Ca²⁺-free solution) next, Ca²⁺was re-introduced (PSS, Ca²⁺ at 2 mM), a PSS solution with 2-APB (100 µM) was perfused on si-Ctl cells (black line) by microspectrofluorimetry (A). In panel B, the mean amplitude of intracellular Ca²⁺ concentration is represented by bar graphs (5 experiments, mean ± SD, *p<0,05). The siRNA efficiencies were controlled by protein expression analysis using western blotting (C).

Figure 7. Orai-1 and STIM-1 inhibition decreased the maturation marker expressions on DC.

DC treated by si-Ctl (white), by si-STIM-1 (light grey) or by si-Orai-1 (dark grey) were maturated by following maturating agents (TG at 750 nM, LPS at 50 ng/ml, zymosan at 25 µg/ml or TNF-α at 20 ng/ml) for 18 h. Then, cells were harvested and quadruple staining (CD80, CD86, CD83, CD25) was assessed by FACS analysis for each condition. The results represent the mean of percentage of quadruple positive cells from 5 independent experiments (mean ± SD, *p<0,05).