Cutting Edge: FAS (CD95) Mediates Noncanonical IL-1β and IL-18 Maturation via Caspase-8 in an RIP3-Independent Manner

Priming of macrophages leads to Fas expression

Fas signaling can be studied efficiently using cell-free membrane-bound mFasL prepared from FasL-expressing cells (16, 17). Using these vesicles, we showed previously that peritoneal mFasL administration induced resident peritoneal macrophages to transcribe a number of proinflammatory cytokines and chemokines. Of note, upon mFasL exposure, peritoneal macrophages produce large amounts of IL-1β and other inflammatory cytokines, prior to 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, because inflammasomes control IL-1β maturation, we hypothesized that they 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). Although a 2-h 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, Supplemental Fig 1A). Control microvesicles (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). Therefore, we assessed whether priming of cells could induce Fas expression on BMDMs. Indeed, TLR4 or TLR7 priming for 24 h led to increased staining of membrane Fas. Notably, the IL-1β response (Fig. 1A, 1C) 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, BMDCs 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 the regulation of Fas expression.

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FIGURE 1.

IL-1β activation by Fas is dependent on Fas receptor upregulation secondary to TLR priming in BMDMs. (A) IL-1β ELISA of SNs from BMDMs primed with LPS and stimulated for the indicated times with Neo or mFasL vesicles. (B) Graphs depicting the mean fluorescence intensity of Fas on live-gated BMDMs after 24 h of stimulation with 300 ng/ml CLO97 (open graph) or 20 ng/ml LPS (open graph) and untreated controls (shaded graph). (C) IL-1β ELISA of SNs from Neo- or mFasL-stimulated BMDMs, which were left untreated or primed for 24 h with LPS or CLO97. (D) Graphs of TOPRO-3–stained BMDMs in medium only (left panel), after the addition of mFasL without priming (middle panel), or after 24 h of priming with LPS and stimulation with mFasL for 6 h (right panel).

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

Fas activation normally leads to cellular apoptosis, but it also was 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 BMDMs and compared their IL-1β response to Asc-deficient or caspase-1/11–deficient BMDMs. The AIM2 activator dsDNA (dAdT) robustly activated wild-type, but not Asc- or caspase-1/11–deficient, BMDMs (23), whereas mFasL activated BMDMs 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.

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FIGURE 2.

Inflammasome and caspase-1/11–independent IL-1β processing in BMDMs stimulated with mFasL. (A) ELISA for IL-1β of SNs from LPS-primed (24 h) BMDMs from wild-type, ASC^−/−, or caspase-1/11^−/− mice, stimulated as indicated for an additional 6 h with decreasing concentrations of mFasL, Neo, or dAdT. (B) Immunoblot of SNs and cell lysates (CL) of wild-type and ASC^−/− BMDMs using 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 demonstrated that IL-1β cleavage downstream of the C-type lectin receptor dectin-1 in dendritic cells can proceed in a noncanonical 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 cellular inhibitor of apoptosis 1 and 2, resulted in both NLRP3-dependent and caspase-8–dependent activation of IL-1β. Both of these pathways were dependent on the RIP3-mediated production of reactive oxygen species (14). Because 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 BMDMs. Thus, we 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. However, caspase-8/Rip3 double-deficient mice are viable and, hence, allowed the testing of 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 BMDMs, whereas AIM2 or NLRP3 inflammasome activators failed to do so (Fig 3A, 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 BMDMs. We found that Fas signaling led to IL-1β processing in primed WT BMDMs, whereas primed caspase-8/Rip3 double-deficient BMDMs 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, 3D). 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 mFasL, we found that the secreted IL-18 was also released independently of caspase-1 or -11 in caspase-1/11 double-deficient BMDMs (Supplemental Fig. 2B). Because the adaptor protein FADD is also critical for signaling from Fas by recruiting caspase-8, we wondered whether its genetic deletion would also inhibit IL-1β maturation. Indeed, deletion of FADD also abolished IL-1β cleavage and secretion in BMDCs, 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 (caspase-3, caspase-6, and caspase-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).

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FIGURE 3.

FAS-induced IL-1β and IL-18 processing is caspase-8 dependent and RIP3 independent. (A) Caspase-8 activity in cell lysates from wild-type, caspase-8^−/−/Rip3^−/−, or Rip3^−/− BMDMs stimulated for 6 h with decreasing concentrations of mFasL. Immunoblot (B, F) or ELISA for IL-1β (C, E) and IL-18 (D) in SNs from wild-type, caspase-8^−/−/Rip3^−/−, or Rip3^−/− BMDMs that were stimulated as indicated.

We found that Fas activation mediates a nonapoptotic 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 antimicrobial defenses and that they play key roles during the development of adaptive immune responses (12). More recently, viruses were shown to block the activity of key innate immune pathways, such as the DNA- or RNA-sensing or inflammasome pathways (26). Thus, it is conceivable that infected cells that 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, because it would lead to the activation of IL-1β cytokines, thus alerting other immune cells of the infection and initiating an antimicrobial response.

However, the described Fas-dependent noncanonical IL-1β activation pathway 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 proinflammatory 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; therefore Fas could be of significance for the development of inflammatory conditions (29, 30). Because the activation of IL-1β cytokines can have dramatic consequences for the establishment of inflammation and immunity, it is not surprising that it is highly regulated. In the case of the NLRP3 inflammasome, transcriptionally active pattern recognition receptors or cytokine signaling receptors 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, such as Fas itself; most likely, other factors important for Fas signaling can be induced by TLR activation. However, a notable difference from inflammasome activation 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.