The release of membrane-enclosed microvesicles is a property evolutionarily conserved for both prokaryotic and eukaryotic organisms (1–2). Mammalian cell-released microvesicles, termed exosomes, are able to deliver intracellular proteins, RNA, and lipids to neighboring cells or even cells in distal organs (2). Similar to that of exosomes, the major function of bacterial outer membrane vesicles (OMVs) is to facilitate intercellular communication and transport of molecules (1–2). Enriched in microbial components, toxins, and virulent factors, OMVs play critical roles in host–pathogen interaction and can facilitate pathogenic bacteria to invade the host at multiple levels (3). For examples, proteins located on the OMVs, such as SabA and BabA, could promote the adherence of bacteria to the epithelial lining of gastrointestinal or pulmonary tract. This not only helps pathogenic bacteria resist the physical elimination, but also facilitates their invasion to the host tissue (4). Furthermore, OMVs-containing toxins, such as leukotoxin and ClyA, are more potent than their soluble forms (5–6). These toxins and OMVs-associated virulent factors help bacteria to invade host through hijacking host machinery for nutrient acquisition or evade host immune system (1–2). OMVs also could increase the coaggregation of bacteria in the biofilms, and thus protects bacteria from adverse effects of antimicrobial agents (1, 7). Therefore, immune detection of OMVs is important for the host to combat invading bacteria.
Host immune system is armed with a series of pattern-recognition receptors (PRRs) that recognize molecules structurally conserved among microbes, termed pathogen-associated molecular patterns (PAMPs) (8). Some of the PRRs, such as toll like receptor (TLR) family, are expressed on cytoplasmic membranes that detect PAMPs in the extracellular space or within phagosomes (8). LPS or peptidoglycan on the surface of OMVs could trigger inflammatory responses through TLR4 or TLR2, respectively (8). As OMVs have the capacity to deliver their cargo into the cytosol of host cells, intracellular PRRs such as NOD1 and NOD2 in the host immune cells could be activated by OMVs (9). Intriguingly, OMVs could induce robust activation of caspase-11, a cytosolic LPS receptor, by delivering LPS into the cytosol of immune cells through unknown mechanisms (10). Activated caspase-11 triggers a cell-lytic and proinflammatory form of programmed cell death, termed pyroptosis, through enzymatic cleavage of its substrate gasdermin D (11–14). This event critically orchestrates antimicrobial immune responses and importantly contributes to pathogenesis of septic shock when the activation of caspase-11 is excessive (11–14). A recent study shows that guanylate binding protein 2 (GBP2) is essential for OMVs-induced caspase-11 activation in interferon-γ primed macrophages (15). However, how LPS is transported from engulfed OMVs to the cytosol for the caspase-11 activation remains largely unknown.
In this study, we show that OMVs-induced TLR4 signaling licenses the cytosolic delivery of LPS from OMVs into the cytosol and the subsequent caspase-11 activation. Of the TLR4 signaling molecules, TRIF mediates the cytosolic delivery of LPS and the activation of caspase-11 upon OMVs stimulation. Importantly, TRIF is required for caspase-11-dependent immune responses and lethality in mice challenged with Escherichia coli-derived OMVs. These effects depend on the production of type 1 interferon the expression of GBPs. Deletion of TRIF or GBPs prevents pyroptosis and lethality induced by OMVs or OMVs-releasing E. coli. Taken together, these findings not only provide novel insight into how host coordinates extracellular and intracellular LPS sensing to orchestrate immune responses during gram-negative infection, but also might confer a new avenue to treat septic shock.
MATERIALS AND METHODS
Experimental protocols were approved by the Institutional Animal Care and Use Committees of the Central South University. The TLR4 KO/TRIF KO/caspase-11 KO/IFN-α/βR1 KO mice were purchased from the Jackson Laboratory. Wild-type (WT) C57BL/6 mice were originally purchased from the Jackson Laboratory. The GBP2 KO/GBP chr3 KO mice were generous gifts from Petr Broz, PhD. The RIPK3 KO mice on a C57BL/6 background were from Jiahuai Han in Xiamen University. The PKR KO mice were previously described (16). Transgenic mice used for experiments were confirmed to be desired genotype via standard genotyping techniques.
Reagents and antibodies
Pam3CSK4, Ultra pure LPS, and Poly (I:C) (high molecular weight) were obtained from InvivoGen. Western blot antibody against mouse caspase 11 (clone 17D9) was from Sigma, antibodies against IL-1α were from Abcam, and antibody against β-actin (8H10D10) was from Cell Signaling Technology.
Macrophages preparation and stimulation
Mouse peritoneal macrophages were isolated and cultured as previously described (17). Briefly, mice were injected with 2 mL of sterile 4% thioglycollate broth intraperitoneally to elicit peritoneal macrophages. Cells were collected by lavage of the peritoneal cavity with 5 mL of sterile 11.6% sucrose 72 h later. After washing, cells were resuspended in RPMI medium 1640 (Gibco) supplemented with 10% heat-inactivated FBS and antibiotics (Gibco). Peritoneal macrophages plated in plates were stimulated with Ultra pure LPS or OMV. In some experiments, cell lysates and supernatants were collected 16 h later for immunoblotting, ELISA, and LDH release.
Bacterial culture and OMV isolation
E. coli BL21was grown in Luria-Bertani broth (LB) overnight at 37°C. OMVs were purified from E. coli as previously described with modification (18). Briefly, for isolation of OMVs, the bacterial strains were grown in 200 mL of LB till OD600 of ∼0.5 and the bacteria-free supernatant was collected by centrifugation at 10,000 × g for 10 min at 4°C. This supernatant was further filtered through a 0.45 μm filter, the supernatant was collected by centrifugation at 10,000 × g for 10 min at 4°C, again, then this supernatant was further filtered through a 0.22 μm filter and OMVs were pelleted by ultracentrifugation at ∼100,000 × g for 4 h at 4°C in a Beckman (optima L-100 XP) rotor. After removing the supernatant, OMVs were resuspended in 300 μL sterile PBS. The OMVs were filtered through a 0.22 μm filter. Purified OMVs were subjected to agar plating to ensure lack of bacterial contamination and Limulus Amebocyte Lysate (LAL) assay according to the manufacturer's instructions to quantify LPS. The protein content of OMV preparations was assessed by Pierce BCA protein assay kit according to the manufacturer's instructions.
ELISA and cell death assay
Plasma and cell culture supernatant samples were analyzed using IL-1α (eBioscience), IL-1β (eBioscience) ELISA kits. Cell death was assessed by LDH Cytotoxicity Assay kit (Beyotime Biotechnology).
Proteins from cell-free supernatants were extracted by methanol/chloroform precipitation and cell extracts. Samples were separated by 12% SDS-PAGE and transferred onto PVDF membranes (Millipore). Antibodies to IL-1α, cas11 were used at 1:1,000 dilution. Blots were normalized to β-actin expression (1:5,000 dilution).
Isolation of cytosol fraction from mouse peritoneal macrophages and LPS activity assay
Subcellular fractionation of mouse peritoneal macrophages was conducted by a digitonin-based fractionation method as previously described with modifications. Briefly, 2.5 × 106 cells were stimulated with OMV (10 μg/mL) or LPS (2.5 μg/mL). After 4 h of treatment, the cells were washed with sterile cold PBS 4 times. Cells were subsequently treated with 150 μL of 0.005% digitonin extraction buffer for 20 min on ice and the supernatant containing cytosol was collected. The residual cell fraction containing cell membrane, organelles, and nucleus was collected in 150 μL of 0.1% CHAPS buffer. BCA assay was used for protein quantification and LPS activity assay for LPS quantification.
Proximity ligation assay
A Proximity Ligation Assay kit (Sigma) was used to study the interaction between LPS or caspase11 protein in mouse peritoneal macrophages, which is a unique method developed to visualize subcellular localization and protein–protein interactions in situ. Macrophages were cultured and stimulated on a six-well object glass in RPMI medium 1640. To assess LPS and caspase11 interaction, cells were primed with 100 ng/mL LPS for 4 h before this treatment. After fixation with 4% formaldehyde and permeabilization with Triton, cells were incubated over night with primary antibody pair of different species directed to LPS (mouse monoclonal 2D7/1), caspase11 (rat monoclonal 17D9). In situ proximity ligation assay (PLA) was performed according to manufacturer's instructions. Briefly, after incubation with primary antibodies, the cells were incubated with a combination of corresponding PLA probes, secondary antibodies conjugated to oligonucleotides (mouse PLUS and rat MINUS for LPS and caspase11 interaction). Subsequently, ligase was added forming circular DNA strands when PLA probes were bound in close proximity, along with polymerase and oligonucleotides to allow rolling circle amplification. Fluorescently labeled probes complementary in sequence to the rolling circle amplification product were hybridized to the rolling circle amplification product. Thus, each individual pair of proteins generated a spot that could be visualized using fluorescent microscopy. Images were taken using a Leica confocal laser scanning microscope and quantified using Image-J software.
Transmission electron microscopy
For negative staining of OMVs, 5 μg of OMVs sample was added to carbon coated mesh grid, stained with 1% uranylacetate for 1 min and imaged with a Hitachi H-7700 Transmission Electron Microscope.
In vivo OMV stimulation and infection
Male or female mice that were 25 to 30 g in weight were injected intraperitoneally with 200 μg purified OMVs or PBS control and sacrificed at 6 h after injection. In addition, mice were infected intraperitoneally with 109 CFU of E. coli and sacrificed at 12 h after injection. For survival experiments, mice were monitored for 48 h after injection.
All data were analyzed using GraphPad Prism software (version 5.01). Data were analyzed using by Student t test used for comparison between two groups or one-way ANOVA followed by Dunnett test for multiple comparisons. A P < 0.05 was considered statistically significant for all experiments. All values are presented as the mean ± SD, except for bacterial counts, for which median values are designate.
TLR4 is critical for OMVs-induced cytosolic delivery of LPS and caspase-11 activation
A recent study establishes that gram-negative bacteria-derived OMVs are able to deliver LPS into the cytosol of immune cells and thus trigger caspase-11-dependent pyroptosis (10). Consistent with these findings, we observed that E. coli-derived OMVs induced robust release of LDH, IL-1α, and IL-1β in WT but not caspase-11 KO mouse peritoneal macrophages (Fig. 1A). Next, we investigated whether immune cells passively allow OMVs to deliver LPS into the cytosol or actively uptake LPS from OMVs through a highly regulated mechanism. OMVs from E. coli BL21 were purified using established methods (18) and verified to be devoid of bacterial contamination by electron microscopy (Fig. 1F) and agar plating (data not shown). LPS on the surface of OMVs can be recognized by host immune receptor TLR4. This prompted us to determine whether engagement of TLR4 with LPS is required for OMVs-induced caspase-11 activation. To test this end, WT or TLR4 KO mouse peritoneal macrophages were primed with Pam3Csk4, a TLR2 agonist, to induce the expression of caspase-11, and then stimulated with E. coli-derived OMVs (Fig. 1, B and C). WT or TLR4 KO mouse peritoneal macrophages expressed comparable levels of caspase-11 after Pam3Csk4 priming (Fig. 1C). However, genetic deletion of TLR4 blocked E. coli-derived OMVs-induced caspase-11-dependent pyroptosis and IL-1α, IL-1β release in Pam3Csk4-primed mouse macrophages (Fig. 1, B and C), indicating that TLR4 is required for OMVs-induced caspase-11 activation.
Next, we determined whether TLR4 is essential for OMVs-mediated cytosolic delivery of LPS using two independent approaches. First, we isolated cytosol devoid of cytoplasmic membranes, endosomes, and lysosomes using low concentrations of digitonin (10) on WT or TLR4 KO macrophages stimulated with OMVs (Fig. 1D). We observed that LPS was present in the cytosol of WT but not TLR4 KO macrophages after stimulation of OMVs (Fig. 1D). As caspase-11 is an intracellular LPS receptor that only recognizes LPS in the cytosol, we next used proximity ligation assay (PLA) to quantitatively measure caspase-11 and LPS interaction. Notably, OMVs delivered LPS into caspase-11 in WT mouse macrophages but not Pam3Csk4-primed TLR4 KO mouse macrophages (Fig. 1E). Together, these observations demonstrated that OMVs are insufficient to deliver LPS into the cytosol of immune cells by themselves, and that TLR4 is required OMVs-induced cytosolic delivery of LPS and subsequent caspase-11 activation.
TRIF is required for OMVs-induced cytosolic delivery of LPS and caspase-11 activation
Of the TLR4 signaling molecules, TRIF is reported to regulate bacteria-induced macrophage cell death or LPS-induced necroptosis (19–20). Thus, we next examined whether TRIF is required for OMVs-induced cytosolic delivery of LPS and caspase-11 activation. Indeed, TRIF deficiency blocked OMVs-induced release of LDH, IL-1α, and IL-1β from mouse peritoneal macrophages (Fig. 2, A and B). However, genetic deletion of TRIF did not affect the OMVs-induced caspase-11 expression (Fig. 2B), indicating that TRIF is required for the caspase-11 activation rather than the priming. Furthermore, deletion of RIPK3, the key regulator of necroptosis (20), failed to inhibit OMVs-induced release of LDH, IL-1α, and IL-1β (Fig. 2A).
To determine whether TRIF is required for OMVs-mediated cytosolic delivery of LPS, we isolated the cytosolic fraction of WT or TRIF KO macrophages stimulated with OMVs. Notably, OMVs delivered LPS into the cytosol of WT but not TRIF KO macrophages (Fig. 2C). Accordingly, deletion of TRIF blocked LPS–caspase-11 interaction in OMVs-stimulated macrophages (Fig. 2D). Taken together, these findings establish that TRIF is required for OMVs-induced cytosolic delivery of LPS and caspase-11 activation.
Type 1 interferon signaling is essential for OMVs-induced cytosolic delivery of LPS and caspase-11 activation
Upon TLR4 activation, TRIF triggers the production of type 1 interferon through recruitment of its downstream factor interferon regulatory factor 3 (IRF3). TLR4–TRIF signaling also induces the activation of RNA-dependent protein kinase (PKR), which is reported to mediate bacteria- or LPS-induced macrophage cell death (19). Thus, we next examined whether type 1 IFN signaling or PKR is required for OMVs-induced caspase-11 activation and macrophage pyroptosis. Deletion of IFN-α/βR1, the receptor for type 1 IFN, markedly inhibited OMVs-induced release of LDH, IL-1α, and IL-1β from mouse peritoneal macrophages (Fig. 3, A and B). In contrast, PKR deficiency failed to inhibit OMVs-induced pyroptosis (Fig. 3, A and B).
We next determined whether type 1 IFN signaling is critical for OMVs-mediated cytosolic delivery of LPS. Cytosolic fractions of WT or IFN-α/βR1 KO macrophages were isolated after stimulation with OMVs. Notably, OMVs delivered LPS into the cytosol of WT but not IFN-α/βR1 KO macrophages (Fig. 3C). Accordingly, deletion of IFN-α/βR1 or TRIF blocked LPS–caspase-11 interaction in OMVs-stimulated macrophages (Fig. 3D). In contrast, PKR deficiency did not affect OMVs-induced LPS–caspase-11 interaction. Collectively, these findings establish that type 1 IFN signaling is required for OMVs-induced cytosolic delivery of LPS and caspase-11 activation.
OMVs-induced caspase-11 activation and cytosolic delivery of LPS depends on GBPs
Type 1 IFNs stimulation leads to expression of hundreds of genes, termed IFN-stimulated genes (ISGs), in both immune and nonimmune cells. Among the most strongly upregulated ISGs were interferon-induced GTPases, such as GBPs (21). Previous works established that GBPs are critical for gram-negative bacteria-induced caspase-11 activation, with GBP2 playing the dominant role in this scenario (22). A recent study reported that GBP2 is required for OMVs-induced caspase-11 activation in IFN-γ-primed mouse macrophages (15). Notably, we found that OMVs were still able to trigger robust release of LDH, IL-1α, and IL-1β in unprimed mouse macrophages (Fig. 4, A and B). However, deletion of GBP2 did reduce the magnitude of OMVs-induced pyroptosis by approximately 30%, as compared with that of WT macrophages (Fig. 4, A and B). Accordingly, deletion of GBP2 partially reduced OMVs-induced caspase-11–LPS interaction (Fig. 4C). This partial dependence on GBP2 implicates the redundant roles of GBP family proteins in mediating the OMVs-induced caspase-11 activation.
To test whether GBP family proteins are required for OMVs-induced cytosolic delivery of LPS and caspase-11 activation, we used the GBP chr3 KO mice, in which GBP1, GBP2, GBP3, GBP5, and GBP7 have been deleted (22). Notably, OMV failed to induce the release of LDH, IL-1α, and IL-1β in GBP chr3 KO macrophages (Fig. 5, A and B). In accordance, OMVs stimulation led to caspase-11–LPS interaction in WT but not GBP chr3 KO mice (Fig. 5C). Together, GBP family proteins play redundant roles in mediating OMVs-induced cytosolic delivery of LPS and caspase-11 activation.
TLR4–TRIF–GBPs signaling pathway is critical for OMVs-induced caspase-11 activation in vivo
Next, we sought to determine whether the TLR4–TRIF–GBPs signaling pathway is essential for OMVs-induced caspase-11 activation in vivo. To test this end, WT, TLR4 KO, TRIF KO, GBP2 KO, GBP chr3 KO, and caspase-11 KO mice were injected with E. coli-derived OMVs. In line with in vitro findings, genetic deletion of TLR4, TRIF, or caspase-11 blocked OMVs-induced release of IL-1α and IL-1β, whereas deletion of GBP2 partially inhibited IL-1α and IL-1β release (Fig. 6, A and B). Furthermore, OMVs failed to induce IL-1α and IL-1β release in GBP chr3 KO mice (Fig. 6, A and B). Previous works implicate an important role of OMVs in septic shock. Notably, deletion of TRIF conferred considerable protection against lethal dose of OMVs as compared with that of WT mice (Fig. 6C). Loss of GBP2 partially reduced the OMVs-induced lethality (Fig. 6C). Together, these findings established that critical role of the TLR4–TRIF–GBPs signaling pathway in OMVs-induced caspase-11 activation in vivo. Besides, we injected mice with E. coli. As shown in Figure 7, E. coli could induce the similar effect as OMVs (Fig. 6).
Taken together, our study reveals that the TLR4–TRIF–GBPs signaling pathway is critical for OMVs-induced caspase-11 activation in vitro and in vivo. Accumulated evidence shows that OMVs could efficiently deliver their components, such as LPS or muramyl dipeptide (MDP), into the cytosol of host cells, and subsequently activate the intracellular immune receptors (9–10, 23). Unlike bacteria, OMVs are unable to induce lysosomal rupture, which could result in lysosomal contents leaking into the cytoplasm (10). However, how OMVs deliver their components into the cytosol remains largely unknown. Our study reveals that host immune cells actively transport LPS from OMVs into the cytosol through the TLR4–TRIF–GBPs signaling. As both TLR4 and caspase-11 are the key LPS receptors, our findings provide novel insight into how host immune system coordinates extracellular and intracellular LPS sensing to orchestrate immune responses during gram-negative bacterial infection.
This study not only identified the key signal pathway that mediates the cytosolic delivery of LPS from OMVs, but also provided the evidence for the first time that OMVs importantly contribute to lethality during bacterial infection. Genetic deletion of host TRIF or GBPs, which is essential for OMVs-mediated cytosolic delivery of LPS, confers considerable protection against lethal OMVs infusion or during bacterial infection. Thus, targeting host TRIF–GBPs signaling or microbial OMVs-producing machinery might open a new avenue to treat sepsis.
Although we and others found that GBPs are critical for the cytosolic delivery of LPS from OMVs, the underlying mechanisms remain unknown. Etienne et al. show that GBPs are recruited to intracellular bacterial pathogens and are necessary to induce lysosomal rupture, which subsequently exposes LPS to caspase-11 in the cytosol (20). Phagocytosis of OMVs is required for cytosolic delivery of LPS and caspase-11 activaion, raising the possibility that GBPs-induced lysis of OMVs-containing phagosomes might lead to LPS leaking into the cytosol. However, OMVs are unable to induce detectable lysosomal rupture. Instead, OMVs in the early endosomes are capable of delivering LPS into the cytosol without destabilizing the endosomal membranes (10). The exact mechanisms by which GBPs transport LPS from OMVs into the cytosol remain unclear.
In conclusion, this study identifies a critical role of the TLR4–TRIF–GBPs signaling in OMVs-induced caspase-11 activation, which contributes to the lethality in bacterial sepsis. As both TLR4 and caspase-11 are pattern-recognition receptors for LPS, our findings also provide novel insights into how host coordinates extracellular and intracellular LPS sensing to orchestrate immune responses during gram-negative bacterial infection. However, future studies are needed to address how GBPs transport LPS from OMVs into the cytosol.
The authors thank Petr Broz and Jiahuai Han for sharing key mouse strains (GBP2 KO/GBP chr3 KO and RIPK3 KO mice).
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Keywords:© 2019 by the Shock Society
Caspase-11; noncanonical inflammation; OMVs; sepsis