The Biological Significance Of Inflammasome Activation In Dendritic Cells By Fas Signal And The Underlying Molecular Mechanism

Fas (CD95/Apo-1), a representative of tumor necrosis factor (TNF) receptor superfamily, triggers cell apoptosis following engagement by Fas ligand (FasL, CD95 ligand, TNFSF6) or by agonistic anti-Fas antibodies. It is generally accepted that Fas-mediated apoptosis is essential for the maintenance of immune homeostasis and immune privilege in brain, eye and testis. Fas-mediated apoptosis is considered to be a non-inflammatory process, leading to the resolution of immune and inflammatory responses. However, accumulating evidence indicates that Fas also initiates proliferative and activating signals, contributing to inflammatory responses. It is now well established that Fas signal can trigger the secretion of chemotactic factors and proinflammatory cytokines. Fas ligation has been reported to stimulate chemokine production by dendritic cells (DCs), early passage fetal human astrocytes and human vascular smooth muscle cells. Moreover, Fas also triggers the production of inflammatory cytokines such as TNFa, IL-6 and IL-8 in macrophages, fibroblasts, epithelial cells and synoviocytes. In addition, activation of CD95 also triggers the secretion of key inflammatory cytokine IL-1β. As yet the mechanisms how Fas ligation triggers the secretion of IL-1βremain elusive.In contrast to other inflammatory cytokines, IL-1βis produced as inactive cytoplasmic precursors (pro-IL-1β), which is cleaved by caspase-1 (IL-1β-converting enzyme [ICE]) to its mature active form. IL-18 and IL-33 also need to be processed by caspase-1 to become their active forms as IL-1β. It is well documented that caspase-1 activation requires a molecular complex termed the "inflammasome". Inflammasome recognizes specific ligands such as muramyl dipeptide, ATP or uric acid, leading to conformational change of the complex and activation of caspase-1. ATP, a well known danger signal, binds purinergic P2X7 receptor (P2X7R) to trigger NALP3 inflammasome activation. NOD-like receptor family members such as NALP3, NALP1 or IPAF is responsible for the detection of the danger signals or PAMPs (pathogen-associated molecular patterns).Dendritic cells (DCs) are professional antigen-presenting cells (APCs), which are crucial for initiating immune responses. Several studies have demonstrated that DCs, irrespective of their maturation state, are resistant to Fas-mediated apoptosis even though the existence of Fas on the surfaces of DCs. Increased expression of the anti-apoptotic protein FLICE-inhibitory protein (FLIP) may account for the resistance of DCs to Fas-mediated apoptosis. Our previous studies also showed that Fas ligation induces DCs maturation and survival instead of apoptosis. Thus, Fas signals an alternative pathway rather than the classical apoptotic signaling pathway in DCs, which may benefit DCs to fulfill antigen presentation in initiating immune response in vivo. However, Fas-triggered non-apoptotic signaling pathway in DCs remains unclear.Our previous studies showed that Fas ligation on DCs triggers the secretion of IL-1p, and this encourages us to explore whether Fas signal induces inflammasome assembly. In the present study, we demonstrate that Fas ligation by the agonistic anti-Fas monoclonal antibody (mAb) Jo-2 causes the release of endogenous ATP from DCs. Then the released ATP activates P2X7R in DCs via autocrine loop and subsequently triggers a series of signaling pathway that induces NALP3 inflammasome activation, leading to IL-1βsecretion and maturation of DCs.1. Fas signal induces IL-1βsecretion from DCs by activating NALP3 inflammasomeTo investigate the role of Fas signal in DCs functions, we first tested whether Fas signal induces apoptosis in DCs. In our experimental context, we used the agonistic anti-Fas monoclonal antibody (mAb) Jo-2 to stimulate CD11c positive DCs derived from bone marrow. In accordance with previous studies, we found that Jo-2, even in a high concentration with 24 hours incubation, failed to induce apoptosis in both immature DCs (imDCs) and mature DCs (mDCs). In contrast, the thymocytes, as positive control, were sensitive to Jo-2-induced cell apoptosis, but not thymocytes from Fas deficient mice. These results suggest that DCs, irrespective their maturation state, were not sensitive to Fas-mediated apoptosis.We next examined the effects of Jo-2 on the cytokine production from DCs. We showed that Jo-2 specifically elicited IL-1βproduction but not TNF-a or IL-6 from DCs. The control antibody showed no any effects on cytokine production from DCs. FasL showed similar effects on cytokine production from DCs as Jo-2. Interestingly, IL-1βproduction induced by Jo-2 stimulation could be inhibited by Caspase-1 specific inhibitor YVAD. We confirmed the Caspase-1 activation induced by Jo-2 stimulation using Western blot assay. Since the Caspase-1 activation is a hallmark of inflammasome activation, we presumed a link between Fas signal and inflammasome in DCs. More interestingly, we found that Jo-2 stimulation induced a striking increase in IL-1βproduction from LPS-primed DCs, and the increase in IL-1βproduction could be inhibited by Caspase-1 specific inhibitor YVAD or ATP channel antagonists. It is well known that LPS turns on the gene expression of components of NALP3 inflammasome. Jo-2 induced large amounts of IL-1βproduction from LPS-primed DCs may be associated with inflammasome activation and ATP channel P2X7R.To elucidate the association of Fas signal and inflammasome, we performed Real-time PCR analysis to measure the effects of Jo-2 on inflammasome expression. Real-time PCR analysis showed that Jo-2 increased the expression of NALP3 and IL-1β, but did not affect ASC and Caspase-1 mRNA expression. NF-κB specific inhibitor PDTC blocked the increase in NALP3 and IL-1βmRNA expression induced by Jo-2 stimulation. Therefore, Jo-2 stimulates the expression of NALP3 inflammasome components via NF-κB signaling pathway. We confirmed Jo-2 induced NF-κB activation as evidenced by the phosphorylation and degradation of NF-κB inhibitor protein IκB. To further investigate the relationship of Fas signal and NALP3 inflammasome activation, we measured Jo-2 stimulation induced IL-1βproduction after knockdown of components of NALP3 inflammasome. We found that IL-1βproduction induced by both LPS and Jo-2 was significantly inhibited after knockdown of NALP3 inflammasome components. These results suggest that Fas-signal induced IL-1βproduction was dependent on NALP3 inflammasome.2. Fas signal triggers the release of endogenous ATP to activate NALP3 inflammasomeSince our results indicate that Jo-2 induced IL-1 p secretion was correlated with ATP, we next examined the effects of Fas signal on ATP release using reporter gene assay. We found that LPS elicited ATP release from DCs, whereas FasL or Jo-2 induced large amounts of ATP release that could not be inhibited by pan Caspase inhibitor zVAD. Ecto-ATPase inhibitor ARL increased ATP concentration about two folds. These results suggest that Jo-2 elicited endogenous ATP release which was not correlated with cell death. Accordingly, LPS and Jo-2 induced IL-1βproduction that could be inhibited by pan Caspase inhibitor zVAD, this may associate with Caspase-1 inhibition. ARL increased IL-1βproduction from DCs induced by LPS and Jo-2 stimulation. LDH release assay showed that both LPS and Jo-2 failed to trigger LDH release from DCs. These results demonstrate that Jo-2 specifically elicits endogenous ATP release from DCs without inducing cell death.Extracellular ATP engages on P2X7R on cell surface to initiate intracellular signal transduction, leading to NALP3 inflammasome activation. We next focused on the role of P2X7R in Fas signal-induced IL-1βsecretion. Pretreatment DCs with P2X7R specific inhibitors compromised Jo-2-induced IL-1βsecretion and DCs maturation as indicated by lab and CD86 staining. We confirmed the prerequisite role of IL-1βduring Jo-2-induced DCs maturation. Actually, the antagonists of P2X7R inhibited Jo-2-induced DCs maturation via inhibition of IL-1βsecretion.We next explored signal transduction pathways in DCs elicited by ATP. Western blot assay showed that Jo-2 stimulation induced potent ERK activation after engagement on DCs. Whereas P2X7R antagonists could reduce Jo-2-induced both ERK and Caspase-1 activation dose dependently, indicating that ATP acted upstream of ERK and Caspase-1. In addition, ERK specific inhibitor PD98059 also inhibited Jo-2-induced both ERK and Caspase-1 activation dose dependently. Therefore, ERK signaled upstream of Caspase-1. Accordingly, both PD98059 and Caspase-1 inhibitor YVAD inhibited IL-1βproduction and DCs maturation. In sum, we showed that the signaling axis ATP-P2X7R-ERK-Caspase-1 was required for Fas signal-induced IL-1βsecretion and DCs maturation.3. Fas signal promotes DCs to prime T cells by inducing endogenous ATP release and IL-1βsecretionThe source of ATP in vivo remains elusive since ATP is quickly degraded by ATPase once secreted into extracelluar compartment. The concentration of ATP is tightly controlled in vivo. Since our results suggested that Fas signal triggered the release of endogenous ATP, we presumed that FasL-Fas cross-linking may play important role in DC-T interaction by inducing ATP release.We examined the role of ATP in DC-T interaction by detecting peptide-specific T cell proliferation triggered by peptide-pulsed DCs. We introduced P2X7R antagonist KN-62 and ATPase (apyrase) into the DC-T coculture system. ELISA assay showed that both KN-62 and apyrase significantly inhibited IL-1βproduction during DC-T cognate interaction. In addition, KN-62 or apyrase also impaired the production of IL-2 and IFN-γ, and the inhibitory effects on cytokine production by KN-62 could be partially restored by recombinant IL-1β. More importantly, both KN-62 and apyrase also inhibited peptide specific T cell proliferation, and the inhibition of T cell proliferation by KN-62 could be restored by recombinant IL-1β. These results suggest that ATP involves in the initiation of antigen-specific T cell proliferation via regulation of IL-1βor other cytokine production during DC-T cognate interaction. To directly document the presence of ATP during the priming of T cells by DCs, we measured the ATP level in the supernatants of DC-T co-culture system. We found that ATP was released quickly into the DC-T co-culture supernatant and peaked (approximately 100nM) at four hours in the presence of antigen specific peptide, then decreased and maintained at almost 50nM level for more than 40 hours. Fas signal in DCs was not required for the peak of ATP release but was crucial in the maintenance of ATP concentration in the later period. In addition, we did not found evident LDH release into the DC-T cu-culture supernatant. Therefore, our data showed the presence of ATP during the priming T cells by DCs, and Fas signal in DCs is crucial for ATP release, and consequently Fas signal-induced ATP release may contribute to the priming of T cells by DCs.In conclusion, Fas signal triggers the release of endogenous ATP from DCs, which functions in an autocrine manner to initiate the axis ATP-P2X7R-ERK-Caspase-1-IL-1βwith resultant IL-1βsecretion. IL-1βalso acts through an autocrine manner to promote the maturation of DCs, leading to the enhanced proliferation of antigen specific T cells. Our study provides the direct evidence of Fas signal in inflammation and raises new view of DCs in immune regulation. This will contribute to a better understanding of the relationship between FasL-Fas and inflammation, and provide insight into the pathogenesis of inflammation-associated diseases. Moreover, our study might also identify potential targets for therapeutic intervention of inflammation-associated diseases.