Caspase-8 mediates caspase-1 processing and innate immune defense in response to bacterial blockade of NF-κB and MAPK signaling

Significance

Pathogenic organisms express virulence factors that can inhibit immune signaling pathways. Thus, the immune system is faced with the challenge of eliciting an effective inflammatory response to pathogens that actively suppress inflammation. The mechanisms that regulate this response are largely undefined. The Yersinia virulence factor YopJ blocks NF-κB and MAPK signaling, resulting in reduced cytokine production and target cell death. Here, we find that caspase-8, RIPK1, and FADD are required for YopJ-induced cell death and show that mice lacking caspase-8 are severely susceptible to Yersinia infection and have defective proinflammatory cytokine production. These findings highlight a possible mechanism of immune defense that can overcome pathogen inhibition of cell-intrinsic proinflammatory immune responses.

Abstract

Toll-like receptor signaling and subsequent activation of NF-κB– and MAPK-dependent genes during infection play an important role in antimicrobial host defense. The YopJ protein of pathogenic Yersinia species inhibits NF-κB and MAPK signaling, resulting in blockade of NF-κB–dependent cytokine production and target cell death. Nevertheless, Yersinia infection induces inflammatory responses in vivo. Moreover, increasing the extent of YopJ-dependent cytotoxicity induced by Yersinia pestis and Yersinia pseudotuberculosis paradoxically leads to decreased virulence in vivo, suggesting that cell death promotes anti-Yersinia host defense. However, the specific pathways responsible for YopJ-induced cell death and how this cell death mediates immune defense against Yersinia remain poorly defined. YopJ activity induces processing of multiple caspases, including caspase-1, independently of inflammasome components or the adaptor protein ASC. Unexpectedly, caspase-1 activation in response to the activity of YopJ required caspase-8, receptor-interacting serine/threonine kinase 1 (RIPK1), and Fas-associated death domain (FADD), but not RIPK3. Furthermore, whereas RIPK3 deficiency did not affect YopJ-induced cell death or caspase-1 activation, deficiency of both RIPK3 and caspase-8 or FADD completely abrogated Yersinia-induced cell death and caspase-1 activation. Mice lacking RIPK3 and caspase-8 in their hematopoietic compartment showed extreme susceptibility to Yersinia and were deficient in monocyte and neutrophil-derived production of proinflammatory cytokines. Our data demonstrate for the first time to our knowledge that RIPK1, FADD, and caspase-8 are required for YopJ-induced cell death and caspase-1 activation and suggest that caspase-8–mediated cell death overrides blockade of immune signaling by YopJ to promote anti-Yersinia immune defense.

The innate immune response forms the first line of defense against pathogens. Microbial infection triggers the activation of pattern recognition receptors, such as Toll-like receptors (TLRs) on the cell surface or cytosolic nucleotide binding domain leucine-rich repeat family proteins (NLRs) (1). TLRs induce NF-κB and MAPK signaling to direct immune gene expression, whereas certain NLRs direct the assembly of multiprotein complexes known as inflammasomes that provide platforms for caspase-1 or -11 activation (2). Active caspase-1 and -11 mediate cleavage and secretion of the IL-1 family of proteins and a proinflammatory cell death termed pyroptosis. However, microbial pathogens can interfere with various aspects of innate immune signaling, and the mechanisms that mediate effective immune responses against such pathogens remain poorly understood. Pathogenic Yersiniae cause diseases from gastroenteritis to plague and inject a virulence factor known as YopJ, which inhibits NF-κB and MAPK signaling pathways in target cells (2–4). YopJ activity inhibits proinflammatory cytokine production (4) and induces target cell death (5). YopJ activity induces processing of multiple caspases, including caspases-8, -3, -7, and -1 (6–8). Nevertheless, Yersinia-infected cells exhibit properties of both apoptosis and necrosis (9, 10), and no specific cellular factors have been identified as being absolutely required for YopJ-induced caspase activation and cell death. We previously found that the inflammasome proteins NLR CARD 4 (NLRC4), NLR Pyrin 3 (NLRP3), and apoptosis-associated speck-like protein containing a CARD (ASC), are dispensable for YopJ-induced caspase-1 processing and cell death (11). Thus, additional pathways likely mediate YopJ-induced caspase-1 activation and cell death.

Death receptors, such as TNF receptor and Fas, mediate caspase-8–dependent apoptosis via a death-inducing signaling complex containing receptor-interacting serine/threonine kinase 1 (RIPK1), caspase-8, and Fas-associated death domain (FADD) (12, 13). Whether these proteins are required for Yersinia-induced cell death, and whether this death contributes to antibacterial immune responses, is not known. The Ripoptosome complex, which contains RIPK1, FADD, caspase-8, as well as RIPK3 and cFLIP, regulates apoptosis, programmed necrosis, and survival in response to various stimuli including signaling by the TLR adaptor TRIF (14, 15). Because YopJ-induced cell death is inhibited in the absence of either TLR4 or TRIF (17) we sought to determine whether YopJ-dependent cell death and caspase-1 activation is regulated by caspase-8 or RIPK3 and to define the role of YopJ-dependent cell death in host defense. Here, we describe a previously unappreciated requirement for RIPK1, FADD, and caspase-8, but not RIPK3, in YopJ-induced caspase-1 activation and cell death. Critically, loss of caspase-8 in the hematopoietic compartment resulted in a failure of innate immune cells to produce proinflammatory cytokines in response to Yersinia infection and severely compromised resistance against Yersinia infection. Our data suggest that caspase-8–mediated cell death in response to blockade of NF-κB/MAPKs by YopJ allows for activation of host defense against Yersinia infection. This cell death may thus enable the immune system to override inhibition of immune signaling by microbial pathogens.

Results

RIPK1 Is Required for Yersinia-Induced Cell Death and Caspase-1 Activation.

Activation of multiple caspases, including caspase-1, -3, -7, and -8, is triggered owing to YopJ activity in Yersinia-infected cells (6, 7, 11, 16). Casp1^−/−Casp11^−/− bone marrow-derived macrophages (BMDMs) exhibit a significant delay in cell death in response to Yersinia pseudotuberculosis (Yp), whereas both Yp-induced cell death and caspase-1 activation are indistinguishable between B6 and Casp11^−/− or 129SvImJ BMDMs (Fig. S1 A–C and Table S1). These data suggest that caspase-1 plays a functional role in YopJ-induced cell death. Surprisingly, NLRC4, NLRP3, ASC, and type I IFN receptor alpha are dispensable for Yersinia-induced caspase-1 processing and cell death, suggesting that these events occur via a distinct pathway (Fig. S1 D–H and ref. 11).

RIPK1, RIPK3, caspase-8, and FADD regulate cell survival and death fate decisions downstream of TRIF as a result of RIP homotypic interaction motif-driven interactions between TRIF and RIPK1 (15, 19). This interaction can promote apoptosis through a RIPK1/FADD/caspase-8 complex (20), or programmed necrosis via RIPK1 and RIPK3 (21). We therefore investigated the potential involvement of RIPK1 in Yersinia-induced cell death. Necrostatin-1 (Nec-1) inhibits RIPK1 kinase activity and prevents TLR-induced necrosis (21, 22) but was previously reported not to inhibit Yersinia pestis-induced cell death (10). Surprisingly, we observed that both RIPK1-deficient BMDMs, and B6 BMDMs treated with Nec-1, exhibited reduced levels of Yp-induced caspase-1 and -8 processing (Fig. 1 A–D) and cell death (Fig. 1 E and F and Fig. S2 A and B). Tlr4^−/− BMDMs are partially protected from Yersinia-induced death (17). TLR4 signaling induces programmed necrosis in cells treated with the pan-caspase inhibitor zVAD-fmk via a TRIF- and RIPK3-dependent pathway (21). Interestingly, zVAD-fmk still sensitized Trif^−/− BMDMs to cell death in response to WT Yp, but not YopJ-deficient Yp (ΔYopJ) infection, whereas B6 BMDMs were sensitized to both (Fig. 1 E and F and Fig. S2C). In contrast, Ripk1^−/− cells were not sensitized to death in the presence of zVAD-fmk in response to either ΔYopJ or Yp (Fig. 1E and Fig. S2D). These data indicate that YopJ induces cell death through RIPK1 via both TRIF-dependent and -independent pathways. Notably, although Nec-1 treatment protects Yp-infected cells from death, expression of NF-κB–dependent cytokines was not restored (Fig. S2 E–G). These data demonstrate that YopJ-mediated blockade of cytokine production is independent of YopJ-induced cell death and imply that this cell death may serve an alternative function. Nec-1 did inhibit YopJ-induced secretion of IL-18, consistent with its effect on caspase-1 activation (Fig. 1G). Altogether, these data demonstrate a key role for RIPK1 in Yersinia-induced death and caspase-1 activation and suggest that this cell death functions as a host defense against pathogen blockade of inflammatory signaling pathways.

Fig. 1.

RIPK1 is required for Yersinia-induced caspase-1 processing and cell death. (A and B) B6 BMDMs were either left uninfected (UI), LPS-primed (50 ng/mL) for 3 h followed by ATP (2.5 mM) for 1 h (LPS+ATP), or infected with WT Yersinia (Yp) or YopJ-deficient Yp (ΔYopJ) for 2 h or Salmonella (STm) for 1 h. Cell lysates were probed for caspase-1 or -8 processing by Western analysis. (C and D) Ripk1^+/+ and Ripk1^−/− fetal liver-derived macrophages (FLDMs) were infected as in A and B. (E) Percent cytotoxicity was measured by LDH release from B6 BMDMs (Left) and Ripk1^+/+ or Ripk1^−/− FLDMs (Right) that were uninfected or infected with ΔYopJ, Yp, or LPS+ATP for 4 h, or STm for 1 h. (F) Flow cytometry for PI uptake (percent PI⁺ cells) by cells as treated in E. (G) IL-18 assayed by ELISA and LDH release on cells treated as in E; 30 μM Nec-1 and 100 μM zVAD-fmk were used 3 h before infection where indicated. Grey peaks indicate uninfected cells. Error bars indicate mean ± SEM of triplicates and are representative of three or more independent experiments. ***P < 0.0001, **P < 0.001, *P < 0.01.

Caspase-8 Activity Is Required for YopJ-Induced Cell Death and Caspase-1 Processing.

TLR signaling can trigger RIPK1-mediated cell death either via caspase-8 or RIPK3 (14, 21). The observation that zVAD-fmk-treatment did not protect B6 BMDMs from Yp-induced death suggests either caspases are not required for Yp-induced cell death or blocking caspase activity in Yp-infected cells triggers cell death via RIPK3-mediated necrosis. Caspase-8 catalytic activity prevents a lethal RIPK3-dependent programmed necrosis that occurs in early embryonic development (23). Notably, we found that Ripk3^−/− BMDMs had no defect in Yp-induced caspase-1 processing or cell death, whereas Ripk3^−/−Casp8^−/− BMDMs failed to process caspase-1 or undergo cell death in response to Yp infection, in contrast to Salmonella (STm) or LPS+ATP treatment (Fig. 2 A–C). This failure was not the result of insufficient back-crossing of these mice, because there were no single nucleotide variants between B6 and three different lines of 129 mice in caspase-8, cFLIP, FasL, or RIPK3, and the polymorphic genes immediately flanking caspase-8 are not linked to these cell death pathways (Fig. S3). Importantly, in Ripk3^−/− BMDMs, zVAD-fmk abrogated Yp-induced death (Fig. 2C). These data demonstrate that caspase inhibition induces RIPK3-dependent programmed necrosis in Yp-infected cells and that RIPK3 is not required for YopJ-induced caspase-1 processing or cell death in the presence of caspase-8. RIPK3 can activate caspase-1 in the absence of caspase-8 in response to LPS (24) or cIAP inhibitors (25). Therefore, caspase-8 and RIPK3 might play redundant roles in caspase-1 activation or cell death during Yp infection. Intriguingly, conditional deletion of caspase-8 by inducible (ERT2-Cre), or developmental (LysM-Cre) approaches significantly reduced YopJ-mediated processing of caspase-1 in RIPK3-sufficient cells, indicating that caspase-8 and RIPK3 play a nonredundant role in YopJ-induced caspase-1 processing (Fig. 2D and Fig. S4A). This caspase-8–dependent processing of caspase-1 was functionally important, because IL-18 secretion was significantly reduced in Ripk3^−/−Casp8^−/− but not in Ripk3^−/− BMDMs (Fig. 2E). However, conditional deletion of caspase-8 was not sufficient to protect BMDMs from Yp-induced cell death (Fig. S4 B and C), in keeping with the finding that zVAD-fmk leads to programmed necrosis in RIPK3-sufficient cells. Importantly, caspase-1 activation in response to STm was completely unaffected in caspase-8 conditional BMDMs, indicating that these cells retained the ability to undergo inflammasome activation (Fig. S4D). We also observed an apparent synergy between zVAD-fmk and Nec-1 in protection from LPS+ATP-treated cell death in B6 cells (Fig. 2C). However, this is most likely because zVAD-fmk–pretreated cells that are then exposed to LPS+ATP undergo programmed necrosis, as Ripk3^−/− cells treated with zVAD-fmk followed by LPS+ATP are protected from cell death (Fig. 2C).

Fig. 2.

Caspase-1 activation and IL-18 secretion in response to Yersinia infection require caspase-8. (A and B) Lysates from BMDMs left uninfected (UI), infected with Yp for 2 h, STm for 1 h, or LPS-primed for 3 h and ATP for 1 h (LPS+ATP) were probed for caspase-1 processing by Western analysis. (C) Percent cytotoxicity (LDH release) of BMDMs infected with Yp for 4 h, STm for 1 h, or treated with LPS+ATP as in A. (D) BMDMs treated with 4-hydroxytamoxifen (4-OHT) (50 nM) and infected as in A. (E) IL-18 assayed by ELISA from BMDMs left uninfected (UI), infected with isogenic Yp 32777, YopJ C172A, or STm, or LPS-primed for 4 h. (F and G) iR3^−/−C8^−/− BMDMs were reconstituted with empty vector, WT caspase-8 (WT C8), or noncleavable mutant caspase-8 (D3A C8). (F) LDH release 4 h postinfection. (G) Caspase-1 processing by Western analysis, MOI of 50:1; 30 μM Nec-1 and 100 μM zVAD-fmk were used 3 h before infection where indicated. N.D., not detected. Error bars indicate mean ± SEM of triplicates and are representative of three or more independent experiments. ***P < 0.0001.

To dissect how caspase-8 mediates activation of caspase-1, we reconstituted immortalized Ripk3^−/−Casp8^−/− (iR3^−/−C8^−/−) macrophages with either WT caspase-8 (WT C8) or mutant caspase-8 lacking the three critical aspartate residues required for autoprocessing (D3A C8). Critically, expression of WT C8 but not of D3A C8 restored both Yp-induced cell death and processing of caspase-1 in iR3^−/−C8^−/− cells (Fig. 2 F and G). Both forms of caspase-8 were expressed at similar levels in the reconstituted cells, but the D3A mutant lacked catalytic activity, consistent with observations that noncleavable forms of caspase-8 do not exhibit catalytic activity in the absence of forced dimerization (26) (Fig. S5 A and B). Noncleavable caspase-8 nonetheless retains some function, because it interacts with cFLIP to inhibit programmed necrosis and mediate protection from embryonic lethality (23, 27). Consistently, zVAD-fmk treatment abolished both caspase-8 and caspase-1 processing in response to Yersinia infection, but not in STm-infected cells (Fig. S5 C–E). These data indicate that zVAD-fmk does not block the autoprocessing of caspase-1 that occurs in canonical inflammasomes but inhibits caspase-8–mediated caspase-1 processing. Altogether, these data indicate that the cleaved active caspase-8 homodimer mediates YopJ-induced processing of caspase-1.

Inhibition of NF-κB and MAPK signaling by YopJ is necessary for Yp-induced cell death (28), but whether this is responsible for RIPK1- and caspase-8–mediated caspase-1 activation is not known. Intriguingly, inhibition of both iκB kinase beta (IKKβ) and p38 MAPK induced RIPK1-dependent processing of caspase-8 and -1 and RIPK1-dependent death of ΔYopJ-infected cells (Fig. S6). Similarly to WT Yp infection, this cell death was largely unaffected in Ripk3^−/− cells but was abrogated in the absence of both RIPK3 and caspase-8. Together, these data demonstrate that YopJ-dependent inhibition of NF-κB and MAPK signaling triggers RIPK1/caspase-8–mediated caspase-1 activation.

FADD Is Required for Yp-Induced Caspase-1 Processing and Cell Death.

These findings, together with previous observations that YopP induces formation of a caspase-8, RIPK1, FADD complex (16), and that FADD plays a role in apoptosis downstream of TRIF (14), suggested that FADD might play a role in YopJ-induced caspase-1 activation and cell death. Fadd^−/− mice exhibit embryonic lethality that is reverted with loss of RIPK3 (29). As anticipated, YopJ-induced caspase-1 processing was indeed abrogated in Ripk3^−/−Fadd^−/− cells but not in Ripk3^−/− cells (Fig. 3A), whereas STm-infected cells exhibited robust caspase-1 processing in the absence of RIPK3 and FADD (Fig. 3 A and B). Consistent with the recent study by Gurung et al. (30), Ripk3^−/−Fadd^−/− BMDMs also showed a noticeable reduction in LPS+ATP-induced caspase-1 processing and cell death (Fig. 3 B and C). Moreover, proinflammatory cytokine production in response to LPS alone was reduced in BMDMs lacking RIPK3 and caspase-8 or FADD (Fig. S7), consistent with recent findings that pro-IL-1β expression is reduced in these cells (30–32). Whereas Gurung et al. (30) observed that caspase-8 contributes to LPS+ATP-induced canonical inflammasome activation, unlike with FADD deficiency, we did not observe a consistent reduction in caspase-1 processing in Ripk3^−/−Casp8^−/− macrophages treated with LPS+ATP, and we were unable to detect caspase-8 processing in LPS+ATP-treated cells. The contribution of FADD and caspase-8 to LPS+ATP-induced caspase-1 activation may relate to differential effects on NLRP3 inflammasome priming.

Fig. 3.

FADD is required for Yersinia-induced caspase-1 processing and cell death in the absence of RIPK3. (A and B) BMDM cell lysates were probed for caspase-1 by Western analysis after infection with ΔYopJ or Yp for 2 h, STm for 1 h, treated with LPS for 3 h and ATP for 1 h (LPS+ATP), or left uninfected (UI). (C) Percent cytotoxicity (LDH release) of BMDMs left uninfected, infected with ΔYopJ or Yp for 4 h, STm for 1 h, or LPS+ATP as in A. N.D., not detected. Error bars indicate mean ± SEM of triplicates and are representative of three or more independent experiments. ***P < 0.0001.

YopJ-Induced Caspase-8 Activation Promotes Anti-Yersinia Immune Defense in Vivo.

YopJ promotes dissemination of Yersinia to systemic tissues (33). However, increasing YopJ-mediated cytotoxicity paradoxically enhances immune clearance and reduces virulence (34). How this cytotoxicity contributes to host defense in vivo is not known. We therefore infected bone marrow chimeric mice lacking both RIPK3 and caspase-8 in the hematopoietic compartment, because these cells cannot undergo YopJ-inducd cell death. Critically, in contrast to B6, Ripk3^–/–, or littermate control bone marrow (BM) chimeras, mice with a caspase-8–deficient hematopoietic compartment rapidly succumbed to infection with a Yp strain that is nonlethal to WT mice (35) (Fig. 4A and Fig. S8A). Surprisingly, Ripk3^−/−Casp8^−/− chimeric mice were deficient in production of serum IFN-γ, IL-6, and IL-1β despite a several-log increase in bacterial cfu in infected tissues on day 6 postinfection, (Fig. 4 B and C and Fig. S8B). Both innate and adaptive Ripk3^−/−Casp8^−/− cells failed to produce cytokines; inflammatory monocytes in the mesenteric lymph nodes (mLNs) and splenic natural killer (NK) cells were all defective in production of TNF-α, and IFN-γ at day 6 postinfection (Fig. 4 D–G and Fig. S8C). In contrast, the chemokine MCP-1 was not affected in Ripk3^−/−Casp8^−/− chimeric mice (Fig. 4B). On day 3 postinfection, Ripk3^−/−Casp8^−/− chimeras had a higher proportion of TNF-producing inflammatory monocytes in the spleen (Fig. S9B), suggesting that caspase-8 deficiency in vivo does not lead to a failure of cell-intrinsic cytokine production per se. Cytokine production in the mLNs, where bacterial burdens were similar across genotypes, was defective on both days 3 and 5 (Fig. S9 A–C).

Fig. 4.

RIPK3/caspase-8–deficient mice are highly susceptible to Yersinia infection and have dysregulated cytokine production. Lethally irradiated B6.SJL mice were reconstituted with B6, Ripk3^+/−Casp8^+/−, Ripk3^−/−Casp8^+/−, Ripk3^−/−, or Ripk3^−/−Casp8^−/− BM and orally infected with 8–10 × 10⁷ cfu Yersinia (Yp) per mouse. Mice were assayed for (A) survival, (B) serum IFN-γ, IL-6, IL-1β, and MCP-1 by Luminex, (C) bacterial loads per gram of tissue, and (D) percent TNF⁺ inflammatory monocytes in mLNs by flow cytometry. (E–H) IFN-γ production from splenic NK cells (NK1.1⁺) was assessed by flow cytometry. (E) Representative plots, (F) total numbers, and (G) mean fluorescence intensity (MFI). (B–G) Day 6 postinfection. Dagger denotes 13 dead or moribund mice not harvested for cfus. Dotted lines represent limit of detection. Solid lines represent means (ELISA) or geometric means (cfu). Flow cytometry plots in D were gated on live CD45.2⁺, CD11c⁻, CD11b^hi, Ly6G^–, and Ly6C^hi cells and in E–G on live CD45.2⁺ CD3^– cells. R3C8, Ripk3Casp8. Statistical analysis in F and G was performed using the Mann–Whitney U test. Data representative of two or more independent experiments.

These findings demonstrate that combined deficiency of RIPK3 and caspase-8 results in severe susceptibility to WT Yp infection, but whether this is due to a failure to respond to the activity of YopJ or to a more global failure to induce immune responses was not clear. Importantly, Ripk3^−/−Casp8^−/− bone marrow chimeric animals were capable of surviving infection with ΔYopJ Yp, had significantly lower bacterial burdens of ΔYopJ in their spleen and liver, and partially recovered production of intracellular TNF (Fig. 5 A–C). Despite elevated bacterial burdens, in the Ripk3^−/−Casp8^−/− chimeras these mice had smaller splenic lesions that lacked close association of neutrophils with bacteria, compared with control caspase-8–sufficient chimeras, which had extensive lesions containing bacterial colonies surrounded by visible cell debris and neutrophilic infiltrates (Fig. 5D, Fig. S9 D and E, and Table S2). These data are consistent with reduced levels of in vivo death of Ripk3^−/−Casp8^−/− cells. Intriguingly, ΔYopJ-infected Ripk3^−/−Casp8^−/− chimeras showed significantly elevated numbers and extent of liver lesions compared with Yp-infected animals, suggesting that in the absence of NF-κB blockade the Ripk3^−/−Casp8^−/− cells can generate protective inflammatory responses that correlate with increased cell death in vivo (Fig. 5 E–G). Together these findings demonstrate that YopJ-dependent triggering of caspase-8 plays a critical role in mediating anti-Yersinia immune defense in vivo and that loss of RIPK3 and caspase-8 results in dysregulated inflammatory responses and failure to control Yersinia.

Fig. 5.

RIPK3/caspase-8–deficient mice survive ΔYopJ infection and partially recover intracellular cytokine production. Lethally irradiated B6.SJL mice were reconstituted with B6, Ripk3^−/−, or Ripk3^−/−Casp8^−/− BM and orally infected with 8–10 × 10⁷ cfu Yersinia (Yp) or ΔYopJ Yersinia (ΔYopJ) per mouse. Mice were assayed for (A) survival, (B) bacterial loads per gram of tissue, and (C) percent TNF⁺ inflammatory monocytes in mLNs by flow cytometry. (D and E) Representative images of H&E-stained (D) spleen and (E) liver sections in naïve and infected chimeric mice, showing lesions of necrosuppurative inflammation (dashed white lines) and extracellular bacterial colonies (arrows) in infected mice. (Scale bars, 50 μm.) (F and G) Quantification of number and extent of lesions. (B) Day 5. (C) Representative plots from pooled data on days 5 and 6 (% ± SEM). (D–G) Day 6. Dagger denotes five dead or moribund mice not harvested for cfus. Dotted lines represent limit of detection. Solid lines represent means (histology) or geometric means (cfu). Flow cytometry plots were gated on live CD45.2⁺, CD11c⁻, CD11b^hi, Ly6G^–, Ly6C^hi cells. **P < 0.01, WT vs. ΔYopJ infected Ripk3^−/−Casp8^−/− monocytes. R3C8, Ripk3Casp8. Data representative of two or more independent experiments.

Discussion

Caspase-1 and caspase-8 activation are generally induced by distinct stimuli. In contrast to caspase-1, which causes pyroptosis independently of other caspases, caspase-8 induces apoptosis through caspases-3 and -7. Caspase-8 can also mediate cleavage and secretion of IL-1β and IL-18 downstream of Fas/FasL signaling or endoplasmic reticulum stress (36, 37) and was recently reported to regulate expression of pro-IL-1β (31, 32). We now demonstrate that caspase-8, RIPK1, and FADD mediate caspase-1 activation in response to the Yersinia virulence factor YopJ, thus revealing an unanticipated integration of caspase-1 and caspase-8 death pathways in response to Yersinia infection.

We cannot currently exclude the possibility that RIPK3 and caspase-8 function redundantly to mediate cell death, or that RIPK3-mediated necrosis contributes to host defense against Yersinia. Nevertheless, in caspase-8–sufficient cells, RIPK3 is dispensable for YopJ-induced cell death or caspase-1 activation, and in caspase-8 conditional knockout cells, RIPK3 is not sufficient for YopJ-induced caspase-1 processing. Under certain conditions, RIPK3 can mediate caspase-1 activation and cell death in response to LPS (24) or cIAP inhibitors (25). Thus, whereas TLR stimulation alone triggers RIPK3-dependent necrosis in caspase-8–deficient cells, our data demonstrate that YopJ triggers cell death and caspase-1 processing through caspase-8. Engagement of caspase-1 by both of these pathways may enable the release of caspase-1–dependent inflammatory signals by dying cells, even when this caspase-1 activation does not occur in a canonical inflammasome platform. Our data suggest a model (Fig. S10) whereby RIPK1, caspase-8, and FADD engage a cell death and caspase-1-activation pathway that promotes anti-microbial immune defense in response to pathogen-mediated interference with innate immune signaling pathways.

In addition to controlling cell-extrinsic death, caspase-8 also plays a role in NF-κB signaling and gene expression (31, 32, 38, 39), potentially via cleavage of cFLIP (40, 41). However, YopJ-induced caspase-1 activation and cell death via caspase-8 and FADD are independent of LPS-induced priming, because they are triggered by NF-κB inhibition and are independent of ASC, NLRP3, and IFNAR. Indeed, LPS priming prevents YopJ-driven cell death (6, 11), either owing to up-regulation of NF-κB–dependent survival genes or inhibition of caspase-1 by YopM in LPS-primed cells (42).

A previous study by Zheng et al. (43) observed a role for ASC in caspase-1 activation in Y. pestis-infected cells, in contrast to our studies here. YopJ isoforms from different Yersinia isolates exhibit different degrees of NF-κB inhibitory activity, and this also correlates with their degree of ASC/NLRP3 inflammasome activation. An alternative possibility is the slightly different infection conditions in the Zheng et al. study, which infected cells for 20 min prior to gentamicin treatment. Future studies will dissect the underlying basis for this apparent difference.

Finally, whether cell death or dysregulated cytokine production, or both, are directly responsible for the inability of caspase-8-deficient animals to clear Yersinia infection remains to be determined. The drop in cytokine production over the course of Yp infection in Ripk3^−/−Casp8^−/− chimeric mice may be due to increased blockade of signaling pathways as a consequence of progressively greater bacterial burden over time. Caspase-8–mediated cell death and caspase-1 activation in response to blockade of innate signaling may thus mobilize bystander cells for rapid cytokine production and/or phagocytosis of pathogen-associated cell debris.

Materials and Methods

Cell Culture and Infections.

BMDMs were grown and infected as previously described (11). Briefly, Yersinia were grown overnight with aeration in 2×YT broth at 26 °C. On the next day, Yersinia were diluted into inducing media and grown with aeration for 1 h at 26 °C followed by 2 h at 37 °C. Salmonella was grown overnight in LB at 37 °C with aeration, then diluted into high-salt LB and grown standing at 37 °C. Cells were infected at a multiplicity of infection (MOI) of 20:1, centrifuged at 200 × g for 5 min, gentamicin (100 μg/mL) was added 1 h after infection, and cells/supernatants were harvested at indicated time points. Caspase processing in cell lysates was analyzed by Western blotting, percent cytotoxicity was assayed by lactate dehydrogenase (LDH) release or propidium iodide (PI) uptake, and release of cytokines was analyzed by ELISA as described in SI Text. All statistical analyses were performed using the two-tailed unpaired Student t test.

Animal Infections.

Mice (fasted 12–16 h) were orally inoculated with 8–10 × 10⁷ Yersinia (32777). Tissues and sera were collected on days 3, 5, and 6. Tissues were processed and plated on LB plates containing irgasan (2 μg/mL) to determine bacterial loads (colony-forming units per gram). Levels of serum cytokines were assayed by Luminex. Cells from spleens and mesenteric lymph nodes were cultured in the presence of brefeldin A and monensin for 5 h, stained for surface and intracellular cytokines, and analyzed by flow cytometry as described in SI Text. Spleen sections were fixed and stained for H&E. All animal studies were performed in accordance with University of Pennsylvania Institutional Animal Care and Use Committee approved protocols.