FAS mediates non-canonical IL-1β and IL-18 maturation via caspase-8 in a Rip3-independent manner

Abstract

Fas, a tumor necrosis factor family receptor, is activated by the membrane protein Fas ligand (FasL) expressed on various immune cells. Fas signaling triggers apoptosis and induces inflammatory cytokine production. Among the Fas induced cytokines, the IL-1β family cytokines require proteolysis to gain biological activity. Inflammasomes, which respond to pathogens and danger signals, cleave IL-1β cytokines via caspase-1. The mechanisms, by which Fas regulates IL-1β activation, however, remain unresolved. Here, we demonstrate that macrophages exposed to TLR ligands upregulate Fas, which renders them responsive to receptor engagement by Fas ligand. Fas signaling activates caspase-8 in macrophages and dendritic cells leading to the maturation of IL-1β and IL-18 independently of inflammasomes or Rip3. Hence, Fas controls a novel non-canonical IL-1β activation pathway in myeloid cells, which could play an essential role in inflammatory processes, tumor surveillance and control of infectious diseases.

Introduction

Fas is a membrane protein that belongs to the tumor necrosis factor receptor (TNFR) family. Binding of its physiological ligand, Fas ligand (FasL), to Fas causes apoptosis, a process that is thought to be critical for the control of tumor cells, infected or otherwise damaged cells (1). Fas-mediated killing of immune cells is important for immune homeostasis as mice with spontaneous mutations in Fas or FasL develop autoimmunity (2, 3) and a fraction of human ALPS (autoimmune lymphoproliferative syndrome) patients carry inherited mutations in Fas (4). Similar to other death-inducing signaling receptors, Fas signaling also has non-apoptotic functions in cellular proliferation, differentiation and cytokine activation (1). However, little is known about the consequences of Fas signaling on cytokine activation for the control of tumorous, damaged or infected cells or for the development of pathologies in autoimmune diseases. In cells of the adaptive immune system, Fas signaling is important for the regulation of apoptosis and therefore essential for the establishment of self-tolerance (5, 6).

Of importance for inflammatory conditions, Fas signaling in cells of the innate immune system such as neutrophils, dendritic cells or macrophages, can mediate the production and activation of pro-inflammatory cytokines of the IL-1β family as well as chemokines (7-9). The pro-inflammatory effect of Fas signaling on innate immune cells is particularly noticeable in microbially infected cells or in cells that have been primed by stimulants such as TLR ligands (10).

The pro-inflammatory IL-1β family of cytokines are controlled at the transcriptional and post-transcriptional levels and produced as biologically inactive precursors, which upon proteolytic processing become active cytokines. Several proteases have been implicated in the processing of IL-1β cytokines (11). Among these the best-studied protease known to activate IL-1β cytokines is caspase-1. Caspase-1 activity is controlled by inflammasomes, which are multi-protein signaling complexes that detect microbial derived molecular signatures or endogenous danger signals (12). Fas-mediated IL-1β activation, however, is caspase-1 independent and thus it remains to be determined how Fas induces IL-1β activation (7, 8).

Several lines of evidence suggest the existence of a non-canonical IL-1β activation pathway that involves caspase-8 and Rip kinases. In primed macrophages, activation of the TRIF-engaging TLRs 3 and 4 led to IL-1β maturation via caspase-8 in conditions where protein synthesis was pharmacologically inhibited (13). Furthermore, experiments employing antagonists of inhibitors of apoptosis proteins (IAPs) revealed IL-1β maturation via the NLRP3 inflammasome and caspase-1 as well as via a caspase-8 dependent pathway. Notably, the protein kinase receptor interacting protein 3 (Rip3), a key enzyme in the crossroads between apoptosis and necrosis, and reactive oxygen species was required for both these pathways (14). Finally, dectin-1 can activate IL-1β via caspase-8 in a pathway that requires the inflammasome adapter molecule ASC (15).

Here, we demonstrate that Fas-mediated IL-1β activation does not require the inflammasome components NLRP3, ASC or caspase-1 but instead proceeds in a process requiring the adapter molecule Fas-associated death domain (FADD) and caspase-8. In contrast to previously described caspase-8 activation pathways, Fas-induced IL-1β maturation via caspase-8 proceeds independently of Rip3 kinase.

Material and Methods

Reagents

Antibodies: anti-IL-1β (R&D Systems), anti-ASC (Santa Cruz), anti-Caspase-1 (eBiosciences). Nigericin, and poly-dAdT was from Sigma, zVAD was from Promega, Z-IEDT-FMK, Z-DQMD-FMK and Z-VEID-FMK was from EMD Biosciences. The IL-1β Elisa kit was from BD-Biosciences. The IL-18 Elisa was performed using rat anti-mouse IL-18 capture antibody (clone 74) and biotinylated rat anti-mouse IL-18 detection antibody (clone 93-10C) with recombinant murine IL-18 cytokine standard (MBL).

Mice

The following mice were used: NLRP3-KO, ASC-KO (Millennium Pharmaceuticals), Caspase 1/11 DKO (R. Flavell, Yale University, New Haven, CT), Caspase 8/RIP3 DKO (E. Mocarski), RIP3 KO (E. Mocarski or F. Chan, UMASS, Worcester, MA) and FLIP^+/-Rip3^+/-FADD^+/-, FLIP^+/-Rip3^-/-FADD^+/-, FLIP^+/-Rip3^-/-FADD^-/- (D. R. Green). The Fas^lpr/lpr mice were backcrossed 8 generations from MRL to BALB/c. C57BL/6 were from Jackson Laboratories.

Cell stimulation and analysis

Bone marrow-derived macrophages (BMDM) or bone marrow-derived dendritic cells (BMDC) were primed with 20 ng/ml ultrapure LPS or with 1 μg/ml CLO97 (Invivogen) as indicated. Cell-free membrane-bound FasL-expressing microvesicles (mFasL) or control vesicles (Neo) from empty vector expressing cells were prepared from transfected Neuro2a cells as described (9). For WB analysis, serum-free supernatants (SN) were precipitated by chloroform/methanol. The caspase-8 activity assay (Promega) was performed per the manufacturer's instructions and read after 90 min. Cell death was determined by flow cytometry (LSRII, BD Biosciences) using TOPRO-3 (Invitrogen) or 7AAD (BD Pharmingen) dyes.

Results and Discussion

Priming of macrophages leads to Fas expression

Fas signaling can efficiently be studied using cell-free membrane-bound FasL-containing microvesicles (mFasL) prepared from FasL expressing cells (16, 17). Using these vesicles, we have previously shown that peritoneal mFasL administration induced resident peritoneal macrophages to transcribe a number of pro-inflammatory cytokines and chemokines. Of note, upon mFasL exposure, peritoneal macrophages produce large amounts of IL-1β and other inflammatory cytokines, prior undergoing apoptosis. Activated IL-1β results in the subsequent recruitment of high numbers of neutrophils into the peritoneum (9). Hence, Fas signaling in peritoneal macrophages induced an inflammatory response in vivo that is very similar to that seen in response to inflammasome activators (18, 19). Inflammasomes can be activated in response to necrosis-inducing agents (20) and since inflammasomes control IL-1β maturation, we hypothesized that inflammasomes may be engaged downstream of Fas.

BMDMs require a priming signal for the upregulation of NLRP3 and pro-IL-1β that allow inflammasome activation by danger signals (21). While a two-hour priming step is sufficient to render BMDMs responsive to NLRP3 activators, we found that IL-1β release in response to mFasL required significantly longer priming periods (Fig. 1A and supplemental Fig 1A). Microvesicles prepared from control vector transfected cells (Neo) did not stimulate IL-1β release at either time point after LPS priming. In contrast to resident peritoneal macrophages, BMDMs constitutively express only low amounts of Fas (9). We therefore assessed whether priming of cells could induce Fas expression on BMDMs. Indeed, TLR4 or TLR7 priming for 24 hours led to increased staining of membrane Fas. Notably, the IL-1β response (Fig. 1 A, C) correlated with the surface expression of Fas (Fig. 1B) and priming was required for Fas-dependent cell killing (Fig. 1D). In agreement with the notion that mFasL stimulation of cells is dependent on Fas expression, mFasL stimulation of primed cells isolated from Fas^lpr/lpr mice failed to respond while being responsive to NLRP3 activators (supplemental Fig. 1B). Furthermore, BMDC also released large amounts of IL-1β in response to mFasL (supplemental Fig. 1C). Together, these studies suggest that Fas signaling is sufficient to induce IL-1β release from myeloid cells and TLR-induced priming licenses IL-1β cleavage via regulating Fas expression.

FIGURE 1. IL-1β activation by FAS is dependent on FAS receptor upregulation secondary to TLR priming in BMDMs.

A, IL-1β ELISA of SN from BMDMs primed with LPS and stimulated for indicated times with Neo control or mFASL vesicles B, Histogram depicting the MFI of FAS on live-gated BMDM after 24h stimulation with 300 ng/ml CLO97 (line) or 20 ng/ml LPS (line) and untreated controls (tinted). C, IL-1β ELISA of SN from Neo or mFASL stimulated BMDMs, which were left untreated or primed for 24h with LPS or CLO97. D, Histograms of TOPRO-3 stained BMDMs in medium only (left), after addition of mFASL without priming (middle) or after 24h priming with LPS and stimulated with mFASL for 6h (right).

FasL activates IL-1β in an ASC- and caspase-1-independent manner in primed macrophages

Fas activation normally leads to cellular apoptosis, but has also been reported to induce necrosis (22), which was recently placed upstream of NLRP3 inflammasome activation (20). To test whether Fas engagement by mFasL activates an inflammasome, we isolated wild-type BMDM and compared their IL-1β response to ASC-deficient or caspase-1-deficient BMDM. The AIM2 activator dsDNA (dAdT) robustly activated wild-type but not ASC- or caspase-1-deficient BMDM (23), while mFasL activated BMDM independently of NLRP3, ASC or caspase-1 (Fig. 2A, supplemental Fig. 1D). Additionally and in contrast to most conditions that induce inflammasome activation, mFasL incubation of BMDMs led to a marked increase in pro-IL-1β production (Fig. 2B). This effect is consistent with the ability of Fas to activate NF-κB (24), which leads to further priming of cells. Together, these data suggested that Fas-induced an inflammasome-independent IL-1β activation pathway.

FIGURE 2. Inflammasome and caspase-1/11 independent IL-1β processing in BMDMs stimulated with mFASL.

A, ELISA for IL-1β of SN from LPS-primed (24h) BMDMs from wt (white bars), ASC^-/- (black bars) or Caspase-1/11^-/- (grey bars) mice stimulated as indicated for an additional 6h with decreasing concentrations of mFAS-L, Neo or dAdT. B, Immunoblot of SN and cell lysates (CL) of wt and ASC^-/- BMDMs of the same assay conditions as in A.

Fas activates IL-1β and IL-18 in a caspase-8- and FADD-dependent and Rip3-independent pathway

Recent work has demonstrated that IL-1β cleavage downstream of the C-type lectin receptor dectin-1 in dendritic cells can proceed in a non-canonical pathway involving the activation of caspase-8 (15). Interestingly, caspase-8 activation in this setting required the inflammasome adapter molecule ASC. Furthermore, an IAP antagonist, which leads to the inhibition of XIAP and degradation of cIAP-1 as well as cIAP-2 resulted in both NLRP3-dependent and caspase-8 dependent activation of IL-1β. Both of these pathways were dependent on Rip3-mediated reactive oxygen species production (14). Since Fas activation is also known to activate caspase-8 we hypothesized that Fas signaling could lead to cleavage of IL-1β via caspase-8 in primed BMDM. We thus sought to establish genetic evidence that caspase-8 is involved in IL-1β maturation. Caspase-8-deficient mice show embryonic lethality, which is thought to be a consequence of unperturbed Rip3 activity leading to increased necrosis. Caspase-8/Rip3 double-deficient mice, however, are viable and hence allowed testing BMDMs for mFasL-mediated IL-1β activation (25). We first tested whether caspase-8 was efficiently activated by mFasL. Indeed, Fas activation induced caspase-8 activity in BMDM while AIM2 or NLRP3 inflammasome activators failed to do so (Fig 3A and supplemental Fig. 2A). In addition, Rip3 single-deficient cells showed normal caspase-8 activity in response to Fas signaling (Fig. 3A) suggesting that Rip3 is dispensable for caspase-8 activity downstream of Fas. We next assessed whether caspase-8 was required for Fas signaling-mediated IL-1β maturation in BMDM. We found that Fas signaling led to IL-1β processing in primed WT BMDM, while primed caspase-8/Rip3 double-deficient BMDM failed to cleave IL-1β (Fig. 3B). Consistently, BMDMs from caspase-8/Rip3 double-deficient mice failed to secrete IL-1β and IL-18 in response to mFasL (Fig. 3C and D). To assess whether Rip3 plays a role in IL-1β or IL-18 activation, we next challenged primed Rip3 single-deficient BMDMs with mFasL. In contrast to previous reports implicating Rip3 in the activation of IL-1β downstream of IAPs (14), Fas signaling mediated IL-1β and IL-18 activation independently of Rip3 (Fig. 3D-F). In line with the inflammasome independent release of IL-1β by mFAS-L, we found that the secreted IL-18 was also released independently of caspase 1 or 11 in caspase 1/11-double deficient BMDM (supplemental Fig. 2B). Since the adaptor protein FADD is also critical for signaling from Fas by recruiting caspase-8, we asked if its genetic deletion would also inhibit IL-1β maturation. Indeed, deletion of FADD also abolished IL-1β cleavage and secretion in BMDC and this effect was paralleled by a resistance to cell death induction via mFASL (supplemental Fig. 2C-E). Finally, to confirm that IL-1β activation is mediated directly by caspase-8 and not by the downstream executioner caspases-3, -6 and -7, we tested a panel of inhibitors of executioner caspases and confirmed that IL-1β activation was only reduced after inhibition of caspase-8 (supplemental Fig. 2F).

FIGURE 3. FAS-induced IL-1β and IL-18 processing is caspase-8 dependent and RIP3 independent.

A, Caspase-8 activity in CL from wt, Caspase-8^-/-/Rip3^-/- or RIP3^-/- BMDMs stimulated for 6h with decreasing concentrations of mFAS-L. B-F, Immunoblot (B,F) or ELISA for IL-1β (C,E) and IL-18 (D) of SN from wt, Caspase-8^-/-/Rip3^-/- or RIP3^-/- BMDMs stimulated as indicated.

We have revealed that Fas activation mediates a non-apoptotic pathway leading to an inflammasome-independent activation of IL-1β family cytokines. This pathway may be of great relevance for a number of processes downstream of Fas signaling. It is well established that IL-1β family members are important for the anti-microbial defenses and that they play key roles during the development of adaptive immune responses (12). More recently, viruses have been shown to block the activity of key innate immune pathways such as the DNA- or RNA-sensing or inflammasome pathways (26). It is thus conceivable that infected cells which upregulate Fas in response to infections could induce an inflammatory response to Fas signaling by autocrine or paracrine mechanisms or via the interaction with FasL on adaptive immune cells. A similar scenario could be relevant for infectious defense against microbes that target the Fas-mediated apoptosis pathways downstream of caspase-8, i.e., that block components of the extrinsic apoptosis pathway (26, 27). In such a scenario activation of Fas and caspase-8 would still benefit the host, since it would lead to the activation of IL-1β cytokines thus alerting other immune cells of the infection and initiating an antimicrobial response.

The described Fas-dependent non-canonical IL-1β activation pathway, however, could also cause harm under conditions where endogenous danger signals excessively trigger innate immune pathways, as seen for example in systemic lupus. Under these situations, upregulation of Fas could predispose innate immune cells to a pro-inflammatory response via FasL ligation that could further potentiate autoimmune pathology (28). In dendritic cells or microglia cells, apoptosis-inducing and inflammation-inducing Fas signaling is uncoupled and Fas could therefore be of significance for the development of inflammatory conditions (29, 30). Since the activation of IL-1β cytokines can have dramatic consequences for the establishment of inflammation and immunity, it is not surprising it is highly regulated. In the case of the NLRP3 inflammasome, transcriptionally active PRRs or cytokine signaling receptors act to prime cells leading to the induction of pro-IL-1β and NLRP3 itself. Activation of NLRP3 by a danger signal - the second signal – then leads to the proteolytic processing of IL-1β cytokines via caspase-1 (21). Similar to the NLRP3 inflammasome, Fas signaling in myeloid cells requires a licensing signal provided by pattern recognition receptors as Fas itself and most likely other factors important for Fas signaling can be induced by TLR activation. A notable difference to inflammasome activation however is that, once licensed, Fas signaling further induces pro-IL-1β and hence can provide the two signals required for IL-1β activation, i.e., the transcriptional induction of pro-IL-1β and the maturation of the cytokine via caspase-8. This suggests that once primed myeloid cells have upregulated Fas they can produce large amounts of key inflammatory cytokines of the IL-1β cytokine family in response to cues received from other immune cells via Fas, which could have important implications for the control of inflammation.