TRIF signaling is required for caspase-11-dependent immune responses and lethality in sepsis
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Caspase-11, a cytosolic receptor of bacterial endotoxin (lipopolysaccharide: LPS), mediates immune responses and lethality in endotoxemia and experimental sepsis. However, the upstream pathways that regulate caspase-11 activation in endotoxemia and sepsis are not fully understood. The aim of this study is to test whether TIR-domain-containing adapter-inducing interferon-β (TRIF) signaling is critical for caspase-11-dependent immune responses and lethality in endotoxemia.
Mice of indicated genotypes were subjected to endotoxemia or cecum ligation and puncture (CLP) and monitored daily by signs of a moribund state for lethality. Serum interleukin (IL)-1α, IL-1β, IL-6 and tumor necrosis factor (TNF) were measured by ELISA. Data were analyzed by using student’s t-test or one-way ANOVA followed by post-hoc Bonferroni test. Survival data were analyzed by using the log-rank test.
Blockade of type 1 interferon signaling or genetic deletion of TRIF or guanylate-binding proteins (GBPs) prevented caspase-11-dependent immune responses, organ injury and lethality in endotoxemia and experimental sepsis. In vitro, deletion of GBPs blocked cytosolic LPS-induced caspase-11 activation in mouse macrophages.
These findings demonstrate that TRIF signaling is required for caspase-11-dependent immune responses and lethality in endotoxemia and sepsis, and provide novel mechanistic insights into how LPS induces caspase-11 activation during bacterial infection.
KeywordsNon-canonical inflammasome Caspase-11 Innate immunity Sepsis
Cecum ligation and puncture
NLR pyrin domain-containing protein 3
Bacterial outer membrane vesicles
Receptor interacting protein 3
Transcription activator-like effector nucleases
Tumor necrosis factor-α
TIR-domain-containing adapter-inducing interferon-β
Increased levels of circulating LPS are encountered in sepsis and removal of LPS is beneficial to septic patients (Angus and van der Poll 2013; Ronco et al. 2010). Endotoxemia-induced lung injury and lethality depends on the activation of caspase-11, an intracellular LPS receptor that triggers a lytic form of cell death, termed pyroptosis (Kayagaki et al. 2011; Hagar et al. 2013; Kayagaki et al. 2013; Wang et al. 1998; Cheng et al. 2017). In this context, activated caspase-11 cleaves gasdermin D (GSDMD) into pore-forming peptides that disrupt the cell membranes (Kayagaki et al. 2015; Ding et al. 2016). This process leads to the release of alarmins, such as interleukin (IL)-1α, and non-canonical activation of the NLR pyrin domain-containing protein 3 (NLRP3) inflammasome, an intracellular protein complex that mediates the maturation of IL-1β through caspase-1 (Kayagaki et al. 2011; Kayagaki et al. 2015). Genetic deletion of Caspase-11 or GSDMD confers significant protection against lethal endotoxemia (Kayagaki et al. 2011; Hagar et al. 2013; Kayagaki et al. 2013; Wang et al. 1998; Cheng et al. 2017; Kayagaki et al. 2015). Pharmacological inhibition of caspase-11 by oxidized phospholipid (oxPAPC) or stearoyl lysophosphatidylcholine significantly promotes survival in endotoxemia (Chu et al. 2018; Li et al. 2018). However, the upstream pathways that regulate caspase-11 activation in endotoxemia are not fully understood.
Recent studies show that bacterial outer membrane vesicles, membrane-enclosed entities released by variety of bacteria, could efficiently delivers LPS into the cytosol and subsequently leads to activation of caspase-11 (Meunier et al. 2014; Finethy et al. 2017). We and others further demonstrate that the TIR-domain-containing adapter-inducing interferon-β (TRIF) signaling is critical for OMVs-induced caspase-11 activation (Santos et al. 2018; Gu et al. 2018). In this scenario, TRIF signaling mediates the production of type 1 interferon, which in turn induces the expression of guanylate-binding proteins (GBPs). The latter is required for OMVs- or Gram-negative bacteria-induced caspase-11 activation (Santos et al. 2018; Gu et al. 2018). These observations prompt us to test whether TRIF signaling is critical for caspase-11-dependent immune responses and lethality in endotoxemia.
Male wild-type (WT) C57BL/6 mice, B6.129S4 (D2)-Casp4tm1Yuan/J (Caspase-11 KO) mice, C57/B6 trif-LPS2 (TRIF KO) mice and B6.129S2-Ifnar1tm1Agt/Mmjax (IFNaβR KO) mice were purchased from the Jackson Laboratory. GBPchr3 KO mice and GBP2 KO mice were generated as described previously (Yamamoto et al. 2012; Degrandi et al. 2013). Receptor interacting protein 3 (Rip3) KO mice were generated by the transcription activator-like effector nucleases (TALENs)-mediated gene-disruption method in a C57BL/6 background, as described previously (Wu et al. 2013).
Mice were bred in the animal facilities of Central South University. Experimental protocols were approved by the Institutional Animal Care and Use Committees of Central South University.
Ultrapure LPS (E. coli 0111:B4) for in vitro experiments were obtained from InvivoGen. LPS (E. coli 0111:B4) for endotoxemia experiments were obtained from Sigma.
Male or female mice that were 25 to 30 g in weight were injected intraperitoneally with 10 mg/kg LPS (E. coli 0111:B4, Sigma). Serum samples were collected at 16 h after LPS injection for the detection of IL-1α, IL-1β, TNF-α and IL-6. Mice injected intraperitoneally with 10 mg/kg LPS were sacrificed 8 h later to measure serum alanine aminotransferase (ALT), creatinine (Cre) levels. Lung specimens were stained with H&E.
For survival experiments, mice were injected intraperitoneally with 40 mg/kg LPS and monitored daily by signs of a moribund state for lethality.
Experimental sepsis was induced by cecal ligation and puncture (CLP). Male or female mice that were 25 to 30 g in weight were used. The skin was disinfected with a 2% iodine tincture. Laparotomy was performed under 2% isoflurance (Piramal Critical Care) with oxygen. To cause death in around 40–50% of CLP mice, 50% of the cecum was ligated and punctured twice with a 20-gauge needle. Saline (1 mL) was given subcutaneously for resuscitation immediately after operation. Mice were sacrificed at 18 h after CLP.Serum samples were collected for the detection of IL-1α, IL-1β, TNF-α, IL-6. alanine aminotransferase (ALT) and creatinine (Cre). Lung specimens were stained with H&E. To cause death in around 80% of CLP mice, 75% of the cecum was ligated and punctured twice with an 20-gauge needle. Mice were monitored daily by signs of a moribund state for lethality.
Macrophages preparation and stimulation
Mouse peritoneal macrophages were isolated and cultured as described previously (Lu et al. 2012). Briefly, mice (7–12 wk. old) were intraperitoneally injected with 3 mL of sterile 4% thioglycollate broth to elicit peritoneal macrophages. Cells were collected by lavage of the peritoneal cavity with 5 mL of RPMI medium 1640 (Gibco)72 h later. After washing, cells were resuspended in RPMI medium 1640 (Gibco) supplemented with 10% heat-inactivated FBS and antibiotics (Gibco). Peritoneal macrophages (106 cells per well) plated in 12-well plates were stimulated with LPS or CTB plus LPS. Supernatants were collected 16 h later for ELISA and LDH assay.
Measuring ALT and creatinine
Serum samples were collected form indicated genotypes mice,ALT and Creatinine were measured by Automatic Biochemical Analyzer (Chemray240).
ELISA and Cell death assay
Plasma and cell culture supernatant samples were analyzed using IL-1α (eBioscience), IL-1β (eBioscience), TNF-α (eBioscience), IL-6(eBioscience) ELISA kits. Cell death was assessed by LDH Cytotoxicity Assay kit (Beyotime Biotechnology).
Isolation of cytosol fraction from mouse peritoneal macrophages and LPS activity assay
Subcellular fractions of mouse peritoneal macrophages were isolated by a digitonin-based fractionation method as described previously with modifications (Vanaja et al. 2016). Briefly, 5 × 106 cells were stimulated with LPS (1 μg/ml) or CTB plus LPS (1 μg/ml) or CTB alone. After 2 h of treatment, the cells were washed with sterile cold PBS 4 times. Cells were subsequently treated with 300 μl of 0.005% digitonin extraction buffer for 20 min on ice and the supernatant containing cytosol was collected. The residual cell fractions containing cell membrane, organelles and nucleus were collected in 300 μl of 0.1% CHAPS buffer. BCA assay was used for protein quantification and LPS activity assay was used for LPS quantification. In addition, the fractions were subjected to immunoblot for Na+/K+ ATPase, Rab7, LAMP1, and beta-Actin to confirm the purity of cytosolic fraction.
All data were analyzed using GraphPad Prism software (version 5.01). Data were analyzed by using two-tailed student’s t-test for comparison between two groups. Data were analyzed by using one-way ANOVA followed by post-hoc Bonferroni test for comparison between multiple groups. Survival data were analyzed using the log-rank test. A p-value < 0.05 was considered statistically significant for all experiments. All values are presented as the mean ± SD.
TRIF is required for caspase-11-dependent immune responses in endotoxemia
TRIF is required for caspase-11-dependent organ injury and lethality in endotoxemia
TRIF is critical for caspase-11-dependent immune responses, organ injury and lethality in sepsis
Type 1 interferon signaling is critical for caspase-11-dependent immune responses, organ injury and lethality in endotoxemia
GBPs are essential for caspase-11-dependent immune responses and lethality in endotoxemia
To test this end, we utilized the GBP chr3 KO mice, in which GBP1, GBP2, GBP3, GBP5 and GBP7 have been deleted (Meunier et al. 2014). Notably, deletion of GBPs markedly reduced the release of IL-1α and IL-1β in endotoxemia (Fig. 5a-b). In accordance, GBPs deficiency blocked pulmonary leukocyte infiltration (Fig. 5c), elevation of the serum levels of ALT and Cre (Fig. 5d-e), and lethality in endotoxemia (Fig. 5f). Together, GBP family proteins play redundant roles in mediating caspase-11 activation in endotoxemia.
Type 1 IFNs-GBPs pathway is critical for caspase-11-dependent immune responses, organ injury and lethality in sepsis
TRIF-interferon-GBPs pathway is required for caspase-11 activation in vitro
We next investigated how TRIF-interferon-GBPs pathway regulates caspase-11 activation. Extracellular LPS is able to induce caspase-11-dependent pyroptosis of cultured mouse macrophages in the presence of cholera toxin B3. Using this in vitro caspase-11 activation model, we found that TRIF, type 1 IFNs and GBPs are required for caspase-11-dependent release of IL-1α, IL-1β and LDH (Fig. 7b-d). To exclude the possibility that TRIF-interferon-GBPs pathway is essential for the translocation of extracellular LPS to the cytosol, we isolated cytosol devoid of cytoplasmic membranes, endosomes and lysosomes using low concentrations of digitonin on mouse macrophages treated with LPS and cholera toxin B (Fig. 7e). LPS levels in the cytosolic fraction were comparable among WT, TRIF KO, IFN-α/βR KO, or GBPchr 3 KO macrophages (Fig. 7f). As TRIF-type 1 IFN signaling is essential for the expression of GBPs, these data indicate that GBPs are critical for cytosolic LPS-induced activation.
Previous studies show that TRIF signaling and GBPs are critical for vacuolar Gram-negative bacteria-induced caspase-11 activation (Meunier et al. 2014; Rathinam et al. 2012). In this context, vacuolar Gram-negative bacteria induce the expression of GBPs, which target the bacteria-containing vacuoles and subsequently induce lysosomal rupture (Meunier et al. 2014). This event results in the leakage of LPS into the cytoplasm, leading to caspase-11 activation (Meunier et al. 2014). However, recent advance reveals that extracellular Gram-negative bacteria are also capable of activating caspase-11 in adjacent macrophages (Vanaja et al. 2016). In this regards, phagocytosis of the whole bacteria and lysosomal rupture are not necessary for the activation of caspase-11 (Vanaja et al. 2016). One of the underlying mechanisms is that Gram-negative bacteria-released OMVs deliver LPS into the cytoplasm (Vanaja et al. 2016). Another mechanism through which Gram-negative bacterial infection triggers caspase-11-dependent immune responses is endotoxemia. Endotoxemia is common in sepsis; and removal of circulating LPS is beneficial to septic patients (Angus and van der Poll 2013; Ronco et al. 2010). Importantly, endotoxemia is able to trigger robust caspase-11-dependent immune responses in a manner similar to Gram-negative bacteremia (Kayagaki et al. 2011; Hagar et al. 2013; Kayagaki et al. 2013; Wang et al. 1998; Cheng et al. 2017). However, the upstream pathways that regulate endotoxemia-induced caspase-11 activation remain largely unknown. In current study, we showed for the first time that TRIF-type 1 IFN-GBPs signaling is critical for caspase-11-dependent immune responses, organ injury and lethality in both endotoxemia and polymicrobial sepsis.
Among GBP protein family, GBP2 plays the dominant role in vacuolar Gram-negative bacteria-induced caspase-11 activation (Meunier et al. 2014). Interestingly, we found that GBP2 deficiency fails to significantly inhibit caspase-11-dependent immune responses in endotoxemia; whereas genetic deletion of GBP1, GBP2, GBP3, GBP5 and GBP7 simultaneously blocked endotoxemia-induced caspase-11 activation. These observations suggest that GBP proteins play redundant and distinct roles in mediating caspase-11 activation in responses to different stimuli. The mechanisms by which GBPs mediate caspase-11 activation are not fully understood. Early work shows that GBPs enable LPS leaking into the cytosol by inducing lysosomal rupture (Meunier et al. 2014). However, accumulated evidence reveals that GBPs are also essential for intracellular LPS-induced caspase-11 activation (Santos et al. 2018; Pilla et al. 2014). In line with these findings, we found that GBPs are required for the caspase-11 activation but not the cytoplasmic translocation of LPS in cholera toxin B + LPS-stimulated macrophages. As GBPs could physically bind LPS, one intriguing possibility is that GBPs might function as a co-receptor of intracellular LPS for caspase-11. Taken together, our study identifies the TRIF-type 1 IFN-GBPs signaling as an upstream pathway that mediates caspase-11 activation, and suggests that targeting this pathway might be potential therapeutic strategy to treat sepsis.
Together, our findings demonstrate that TRIF signaling is required for caspase-11-dependent immune responses and lethality in endotoxemia and sepsis, and provide novel mechanistic insights into how LPS induces caspase-11 activation during bacterial infection.
The authors thank Dr. Petr Broz for sharing key mouse strains (GBPchr3 KO mice and GBP2 KO mice).
This work was supported by National key scientific project 2015CB910700 (B.L.), National Natural Science Foundation of China (No. 81422027 (B.L.), No. 81400149 (Y.T.), and No. 81470345 (B.L.).
Availability of data and materials
BL conceived the project and wrote the paper; YT designed experiments, performed the experiments, analyzed the data and wrote the paper; RZ, XQ, RM, XW, YY, XL performed the experiments; HW analyzed the data; XX and TRB supervised the study. All authors read and approved the final manuscript.
Experimental protocols were approved by the Institutional Animal Care and Use Committees of Central South University.
Consent for publication
All the authors contributed to, read and approved the final manuscript for submission.
The authors declare that they have no competing interests.
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