Search

Background The availability of L-arginine in tumors is a key determinant of an efficient anti-tumor T cell response. Consequently, the elevation of typically low L-arginine levels within the tumor may greatly potentiate the anti-tumor responses of immune check point inhibitors, such as PD-L1 blocking antibodies. However, currently no means are available to locally increase intra-tumoral L-arginine levels. Methods We used a synthetic biology approach to develop an engineered probiotic Escherichia coli Nissle 1917 strain that colonizes tumors and continuously converts ammonia, a metabolic waste product that accumulates in tumors, into L-arginine. Results Colonization of tumors with these bacteria elevated intra-tumoral L-arginine concentrations, increased the amount of tumor-infiltrating T cells, and had striking synergistic effects with PD-L1 blocking antibodies in the clearance of tumors. The anti-tumor effect of the living therapeutic was mediated by L-arginine and was dependent on T cells. Conclusions These results show that engineered microbial therapies enable metabolic modulation of the tumor microenvironment leading to enhanced efficacy of immunotherapies.

Tetraspanins are a family of proteins possessing four transmembrane domains that help in lateral organization of plasma membrane proteins. These proteins interact with each other as well as other receptors and signaling proteins, resulting in functional complexes called “tetraspanin microdomains.” Tetraspanins, including CD82, play an essential role in the pathogenesis of fungal infections. Dectin-1, a receptor for the fungal cell wall carbohydrate β-1,3-glucan, is vital to host defense against fungal infections. The current study identifies a novel association between tetraspanin CD82 and Dectin-1 on the plasma membrane of Candida albicans–containing phagosomes independent of phagocytic ability. Deletion of CD82 in mice resulted in diminished fungicidal activity, increased C. albicans viability within macrophages, and decreased cytokine production (TNF-α, IL-1β) at both mRNA and protein level in macrophages. Additionally, CD82 organized Dectin-1 clustering in the phagocytic cup. Deletion of CD82 modulates Dectin-1 signaling, resulting in a reduction of Src and Syk phosphorylation and reactive oxygen species production. CD82 knockout mice were more susceptible to C. albicans as compared with wild-type mice. Furthermore, patient C. albicans–induced cytokine production was influenced by two human CD82 single nucleotide polymorphisms, whereas an additional CD82 single nucleotide polymorphism increased the risk for candidemia independent of cytokine production. Together, these data demonstrate that CD82 organizes the proper assembly of Dectin-1 signaling machinery in response to C. albicans.

Integrin signalling triggers cytoskeletal rearrangements, including endocytosis and exocytosis of integrins and other membrane proteins. In addition to recycling integrins, this trafficking can also regulate intracellular signalling pathways. Here we describe a role for αv integrins in regulating Toll-like receptor (TLR) signalling by modulating intracellular trafficking. We show that deletion of αv or β3 causes increased B-cell responses to TLR stimulation in vitro, and αv-conditional knockout mice have elevated antibody responses to TLR-ligand-associated antigens. αv regulates TLR signalling by promoting recruitment of the autophagy component LC3 (microtubule-associated proteins 1 light chain 3) to TLR-containing endosomes, which is essential for progression from NF-κB to IRF signalling, and ultimately for traffic to lysosomes where signalling is terminated. Disruption of LC3 recruitment leads to prolonged NF-κB signalling and increased B-cell proliferation and antibody production. This work identifies a previously unrecognized role for αv and the autophagy components LC3 and atg5 in regulating TLR signalling and B-cell immunity., Integrins can regulate antigen-specific and innate immune receptor signalling, thereby affecting immune cell function. Here the authors show that avß3 integrin controls Toll-like receptor (TLR) signalling by regulating its trafficking to limit TLR-mediated B-cell proliferation and antibody production.

Phagocytosis is a fundamental cellular process that is pivotal for immunity as it coordinates microbial killing, innate immune activation and antigen presentation. An essential step in this process is phagosome acidification, which regulates many functions of these organelles that allow phagosomes to participate in processes that are essential to both innate and adaptive immunity. Here we report that acidification of phagosomes containing Gram-positive bacteria is regulated by the NLRP3 inflammasome and caspase-1. Active caspase-1 accumulates on phagosomes and acts locally to control the pH by modulating buffering by the NADPH oxidase NOX2. These data provide insight into a mechanism by which innate immune signals can modify cellular defenses and establish a new function for the NLRP3 inflammasome and caspase-1 in host defense.

Autophagy plays an important role in maintaining cell homeostasis by providing nutrients during periods of starvation and removing damaged organelles from the cytoplasm. A marker in the autophagic process is the reversible conjugation of LC3, a membrane scaffolding protein, to double membrane autophagosomes. Recently, a role for LC3 in the elimination of pathogenic bacteria and fungi, including Candida albicans (C. albicans), was demonstrated, but these organisms reside in single membrane phagosomes. This process is distinct from autophagy and is termed LC3-associated phagocytosis (LAP). This review will detail the hallmarks of LAP that distinguish it from classical autophagy and review the role of autophagy proteins in host response to C. albicans and other pathogenic fungi.

Background: While both T-cell priming and blockade of immune suppression play critical roles in the generation of an efficacious antitumor immune response, most therapies fail to support both processes as a single agent. At Synlogic we are using synthetic biology and engineered bacteria programmed with immune-modulatory circuits to develop treatments or “Synthetic Biotic Medicines” capable of simultaneously manipulating multiple pathways relevant for the treatment of cancer and autoimmunity. Recent studies have demonstrated that activation of the stimulator of interferon genes (STING) pathway can play a critical role in the initiation of an antitumor immune response via activation of antigen presenting cells (APCs) and presentation of tumor antigens. Additionally, metabolites derived from biosynthetic pathways, such as conversion of tryptophan into kynurenine by indoleamine 2,3 dioxygenase (IDO), have also been recently appreciated as major components of the immune-suppressive tumor microenvironment which lead to T cell disfunction and exhaustion. Here we present results showing the development of two circuits, an immune “initiator” STING activating circuit (SYN-STING) and an immune “sustainer” Kynurenine consuming circuit (SYN-Kyn), in engineered strains of E. coli Nissle. Methods and Results: Synthetic biological techniques were employed to generate bacterial strains expressing various protein components capable of (1) generating the STING agonist cyclic-di-AMP (SYN-STING) or (2) actively transporting and metabolizing kynurenine (SYN-Kyn). In in vitro biochemical and functional assays, SYN-STING generated high levels of cyclic-di-AMP and triggered the expression of IFNβ1 and IL-6 upon co-culture with APCs, while SYN-Kyn actively depleted test media containing kynurenine at levels 20-fold higher than those found in tumors of cancer patients. In mice bearing subcutaneous syngeneic tumors the intratumoral administration of SYN-STING resulted in an early rise of innate cytokines which several days later shifted towards molecules indicative of an effector-T-cell response. Additionally the administration of a single dose of SYN-Kyn led to significant decreases in tumor kynurenine levels for up to 72 hours post dose. Finally, administration of a combination of either SYN-STING with anti-PD1 or SYN-Kyn with anti-CTLA4 antibodies in tumor-bearing mice led to significant anti-tumor effects over those observed for the antibodies alone. Conclusions: Taken together, these results demonstrate that the use of synthetic biology to engineer bacteria is a viable approach to deliver profound efficacy in experimental models of cancer. The data supports the further development of Synthetic Biotic Medicines capable of locally targeting multiple immune pathways as single immuno-oncology therapeutic agents. Citation Format: Daniel Leventhal, Kip West, Adam Fisher, Anna Sokolovska, Starsha Kolodziej, Ning Li, Chris Plescia, Carey Gallant, Mary Castillo, Paul Miller, Jose Lora. Activation of innate and adaptive immunity via combinatorial immunotherapy using Synthetic Biotic™ Medicines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-131.

Background: The immunosuppressive milieu found within the tumor microenvironment (TME) has long been understood to be a key driver of tumor initiation and progression. More recently it has been appreciated that metabolites derived from biosynthetic pathways are major components in forming this immune-privileged niche. For example, the conversion of tryptophan into kynurenine by indoleamine 2,3 dioxygenase (IDO) or the reduction of adenosine triphosphate to adenosine by the ectoenzymes CD39 and CD73 leads to T cell dysfunction and exhaustion, and a significantly blunted antitumor immune response. At Synlogic we are using synthetic biology in combination with natural probiotics to develop engineered bacteria or “Synthetic Biotic Medicines,” which are programmed with precision to correct disease-causing and -promoting metabolic defects. Here we present results showing the development of two engineered bacterial strains that have been designed to consume either kynurenine or adenosine, two molecules known to play central roles in promoting tumor immune tolerance, with the goal of relieving TME-associated immunosuppression and promoting antitumor immunity. Methods and Results: Synthetic biologic techniques were employed to generate the adenosine-consuming strain (SYN-Ade) or the kynurenine-consuming strain (SYN-Kyn) by introduction of genetic elements that were highly efficient in the metabolism of adenosine or kynurenine, respectively. In in vitro biochemical assays, SYN-Ade and SYN-Kyn were able to deplete test media containing levels of adenosine and kynurenine that are ~100-fold and 20-fold higher than the adenosine or kynurenine levels found in the tumors of cancer patients, (180uM of adenosine or 80uM of kynurenine, respectively) to undetectable levels within 2 hours. For the kynurenine-consuming strain, this in vitro kynurenine consumption translated to robust in vivo pharmacodynamic activity. In mice bearing subcutaneous CT26 tumors, the administration of SYN-Kyn by intratumoral (IT) injection led to significant decreases in tumor kynurenine levels, which was equivalent to small-molecule inhibition of the IDO enzyme. Importantly, the combination of SYN-Kyn with an anti-CTLA4 antibody in the CT26 tumor model or the combination of SYN-Kyn or SYN-Ade with a cocktail of anti-PD1/CTLA4 antibodies in MC38 tumor-bearing mice led to significant antitumor effects over those observed with the antibodies alone. Conclusions: Taken together, these results demonstrate that the application of synthetic biology to engineer nonpathogenic bacteria is a viable approach to deliver profound efficacy in experimental models of cancer, and support the further development of these Synthetic Biotic medicines as potential immuno-oncology therapies. Citation Format: Kip A. West, Adam Fisher, Dan Leventhal, Anna Sokolovska, Ning Li, Chris Plescia, Mary Castillo, Vincent Isabella, Starsha Kolodziej, Paul Miller, Jose M. Lora. Metabolic modulation of the tumor microenvironment using Synthetic Biotic™ Medicines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2920.

NOD2 (nucleotide-binding oligomerization domain containing 2) is an important cytosolic pattern recognition receptor that activates NF-kappaB and other immune effector pathways such as autophagy and antigen presentation. Despite its intracellular localization, NOD2 participates in sensing of extracellular microbes such as Staphylococcus aureus. NOD2 ligands similar to the minimal synthetic ligand muramyl dipeptide (MDP) are generated by internalization and processing of bacteria in hydrolytic phagolysosomes. However, how these derived ligands exit this organelle and access the cytosol to activate NOD2 is poorly understood. Here, we address how phagosome-derived NOD2 ligands access the cytosol in human phagocytes. Drawing on data from Drosophila phagosomes, we identify an evolutionarily conserved role of SLC15A transporters, Drosophila Yin and PEPT2, as MDP transporters in fly and human phagocytes, respectively. We show that PEPT2 is highly expressed by human myeloid cells. Ectopic expression of both Yin and PEPT2 increases the sensitivity of NOD2-dependent NF-kappaB activation. Additionally, we show that PEPT2 associates with phagosome membranes. Together, these data identify Drosophila Yin and PEPT2 as evolutionarily conserved phagosome-associated transporters that are likely to be of particular importance in delivery of bacteria-derived ligands generated in phagosomes to cytosolic sensors recruited to the vicinity of these organelles.

Aluminum-containing adjuvants are widely used in licensed human and veterinary vaccines. However, the mechanism by which these adjuvants enhance the immune response and predominantly stimulate a T(H)2 humoral immune response is not well understood. In this study, the effects of aluminum hydroxide and aluminum phosphate adjuvants on antigen presentation, expression of costimulatory molecules and cytokines by mouse dendritic cells (DCs) and the ability of DCs to induce T helper cell differentiation were investigated. Dendritic cells pulsed with ovalbumin (OVA) adsorbed to aluminum-containing adjuvants activated antigen-specific T cells more effectively than DCs pulsed with OVA alone. Aluminum hydroxide adjuvant had a significantly stronger effect than aluminum phosphate adjuvant. Both aluminum-containing adjuvants significantly increased the expression of CD86 on DCs but only aluminum hydroxide adjuvant also induced moderate expression of CD80. Aluminum-containing adjuvants stimulated the release of IL-1beta and IL-18 from DCs via caspase-1 activation. DCs incubated with LPS and OVA induced T(H)1 differentiation of naïve CD4(+) T cells. In contrast, DCs incubated with aluminum/OVA activated CD4(+) T cells to secrete IL-4 and IL-5 as well as IFN-gamma. Addition of neutralizing anti-IL-1beta antibodies decreased IL-5 production and addition of anti-IL-18 antibodies decreased both IL-4 and IL-5 production. Inhibition of IL-1beta and IL-18 secretion by DCs via inhibition of caspase-1 also led to a marked decrease of IL-4 and IL-5 by CD4(+) T cells. These results indicate that aluminum-containing adjuvants activate DCs and influence their ability to direct T(H)1 and T(H)2 responses through the secretion of IL-1beta and IL-18.

Brucella abortusstrain RB51 is an attenuated rough mutant used as the live vaccine against bovine brucellosis in the United States and other countries. We previously reported the development of strain RB51 as a bacterial vaccine vector for inducing Th1-type immune responses against heterologous proteins. Because safety concerns may preclude the use of strain RB51-based recombinant live vaccines, we explored the ability of a gamma-irradiated recombinant RB51 strain to induce heterologous antigen-specific immune responses in BALB/c mice. Exposure of strain RB51G/LacZ expressingEscherichia coliβ-galactosidase to a minimum of 300 kilorads of gamma radiation resulted in complete loss of replicative ability. These bacteria, however, remained metabolically active and continued to synthesize β-galactosidase. A single intraperitoneal inoculation of mice with 109CFU equivalents of gamma-irradiated, but not heat-killed, RB51G/LacZ induced a β-galactosidase-specific Th1-type immune response. Though no obvious differences were detected in immune responses toB. abortus-specific antigens, mice vaccinated with gamma-irradiated, but not heat-killed, RB51G/LacZ developed significant protection against challenge with virulentB. abortus. In vitro experiments indicated that gamma-irradiated and heat-killed RB51G/LacZ induced maturation of dendritic cells; however, stimulation with gamma-irradiated bacteria resulted in more interleukin-12 secretion. These results suggest that recombinant RB51 strains exposed to an appropriate minimum dose of gamma radiation are unable to replicate but retain their ability to stimulate Th1 immune responses against the heterologous antigens and confer protection againstB. abortuschallenge in mice.

An important step in the induction of an immune response to vaccines is the internalization of antigens by antigen presenting cells, such as dendritic cells (DCs). Many current vaccines are formulated with antigens adsorbed to an aluminum-containing adjuvant. Following injection of the vaccine the antigens may either elute or stay adsorbed to the adjuvant surface. Antigens, which elute from the adjuvant surface, are internalized by dendritic cells through macropinocytosis while those that remain adsorbed are internalized with the adjuvant particle by phagocytosis. The relative efficiency of these two routes of internalization was studied. Alpha casein (AC) labeled with a green fluorescent dye was selected as the model antigen. In order to model vaccine antigens that elute from aluminum-containing adjuvants following administration, dendritic cells were incubated with a solution of fluorochrome-labeled alpha casein. To model vaccine antigens that do not elute from aluminum-containing adjuvants following administration, dendritic cells were exposed to fluorochrome-labeled alpha casein adsorbed to aluminum hydroxide adjuvant (AH). Alpha casein has eight phosphate groups and adsorbs to aluminum hydroxide adjuvant through ligand exchange. Alpha casein does not elute from aluminum hydroxide adjuvant upon exposure to cell culture media. The uptake of antigen by dendritic cells was determined at 0.5, 1, 2 and 3h by confocal microscopy and flow cytometry. Dendritic cells internalized both alpha casein in solution and alpha casein adsorbed to aluminum hydroxide adjuvant. However, the mean fluorescence intensity of dendritic cells incubated with adsorbed alpha casein was four times greater than dendritic cells incubated with alpha casein in solution. In addition, the internalization of alpha casein was enhanced when the mean aggregate diameter of the adjuvant in the cell culture media was reduced from 17 microm to 3 microm. It was concluded that antigen internalization by dendritic cells was enhanced when the antigen remained adsorbed to the aluminum-containing adjuvant following administration and the aggregate size of the adjuvant was smaller than dendritic cells which are approximately 10 microm in diameter.

Elimination of fungal pathogens by phagocytes requires phagosome maturation, a process that involves the recruitment and fusion of intracellular proteins. The role of Dectin-1, a β-1,3-glucan receptor, critical for fungal recognition and triggering of Th17 responses, to phagosomal maturation has not been defined. We show that GFP-Dectin-1 translocates to the fungal phagosome, but its signal decays after 2 h. Inhibition of acidification results in retention of GFP-Dectin-1 to phagosome membranes highlighting the requirement for an acidic pH. Following β-1,3-glucan recognition, GFP-Dectin-1 undergoes tyrosine phosphorylation by Src kinases with subsequent Syk activation. Our results demonstrate that Syk is activated independently of intraphagosomal pH. Inhibition of Src or Syk results in prolonged retention of GFP-Dectin-1 to the phagosome signifying a link between Syk and intraphagosomal pH. β-1,3-glucan phagosomes expressing a signaling incompetent Dectin-1 failed to mature as demonstrated by prolonged Dectin-1 retention, presence of Rab5B, failure to acquire LAMP-1 and inability to acidify. Phagosomes containing Candida albicans also require Dectin-1-dependent Syk activation for phagosomal maturation. Taken together, these results support a model where Dectin-1 not only controls internalization of β-1,3-glucan containing cargo and triggers proinflammatory cytokines, but also acts as a master regulator for subsequent phagolysosomal maturation through Syk activation.

Mannose-binding lectin (MBL) is a key soluble effector of the innate immune system that recognizes pathogen-specific surface glycans. Surprisingly, low-producing MBL genetic variants that may predispose children and immunocompromised individuals to infectious diseases are more common than would be expected in human populations. Since certain immune defense molecules, such as immunoglobulins, can be exploited by invasive pathogens, we hypothesized that MBL might also enhance infections in some circumstances. Consequently, the low and intermediate MBL levels commonly found in human populations might be the result of balancing selection. Using model infection systems with pseudotyped and authentic glycosylated viruses, we demonstrated that MBL indeed enhances infection of Ebola, Hendra, Nipah and West Nile viruses in low complement conditions. Mechanistic studies with Ebola virus (EBOV) glycoprotein pseudotyped lentiviruses confirmed that MBL binds to N-linked glycan epitopes on viral surfaces in a specific manner via the MBL carbohydrate recognition domain, which is necessary for enhanced infection. MBL mediates lipid-raft-dependent macropinocytosis of EBOV via a pathway that appears to require less actin or early endosomal processing compared with the filovirus canonical endocytic pathway. Using a validated RNA interference screen, we identified C1QBP (gC1qR) as a candidate surface receptor that mediates MBL-dependent enhancement of EBOV infection. We also identified dectin-2 (CLEC6A) as a potentially novel candidate attachment factor for EBOV. Our findings support the concept of an innate immune haplotype that represents critical interactions between MBL and complement component C4 genes and that may modify susceptibility or resistance to certain glycosylated pathogens. Therefore, higher levels of native or exogenous MBL could be deleterious in the setting of relative hypocomplementemia which can occur genetically or because of immunodepletion during active infections. Our findings confirm our hypothesis that the pressure of infectious diseases may have contributed in part to evolutionary selection of MBL mutant haplotypes.

Phagocytes are an important part of host defense, playing a critical role in innate immune responses against pathogens and in the initiation of adaptive immunity. One of the main characteristics of these cells is their ability to recognize and internalize invading microorganisms into a phagosome. The internalized microbe is rapidly delivered into a mature phagolysosome where it is killed and degraded. However, numerous pathogens have evolved complex mechanisms to manipulate these intracellular organelles to establish a survival niche. Here, we describe several methods to assess important properties of phagosomes in macrophages, such as phagocytosis, acidification of the phagosome contents during the maturation process, and the ability of phagosomes to inactivate and kill pathogens. Phagocytosis and phagosome acidification assays are FACS-based assays where labeled bacteria are used as probes to monitor internalization into a phagosome and to detect the pH of the phagosome environment. The killing assay is based on the counting of bacterial colonies after recovery of internalized bacteria from macrophages.

Spontaneous mutations of the Sharpin (SHANK-associated RH domain-interacting protein, other aliases: Rbckl1, Sipl1) gene in mice result in systemic inflammation that is characterized by chronic proliferative dermatitis and dysregulated secretion of T helper1 (Th1) and Th2 cytokines. The cellular and molecular mechanisms underlying this inflammatory phenotype remain elusive. Dendritic cells may contribute to the initiation and progression of the phenotype of SHARPIN-deficient mice because of their pivotal role in innate and adaptive immunity. Here we show by flow cytometry that SHARPIN- deficiency did not alter the distribution of different DC subtypes in the spleen. In response to TOLL-like receptor (TLR) agonists LPS and poly I:C, cultured bone marrow-derived dendritic cells (BMDC) from WT and mutant mice exhibited similar increases in expression of co-stimulatory molecules CD40, CD80, and CD86. However, stimulated SHARPIN-deficient BMDC had reduced transcription and secretion of pro-inflammatory mediators IL6, IL12P70, GMCSF, and nitric oxide. Mutant BMDC had defective activation of NF-κB signaling, whereas the MAPK1/3 (ERK1/2) and MAPK11/12/13/14 (p38 MAP kinase isoforms) and TBK1 signaling pathways were intact. A mixed lymphocyte reaction showed that mutant BMDC only induced a weak Th1 immune response but stimulated increased Th2 cytokine production from allogeneic naïve CD4(+) T cells. In conclusion, loss of Sharpin in mice significantly affects the immune function of DC and this may partially account for the systemic inflammation and Th2-biased immune response.

Innate immunity is vital for protection from microbes and is mediated by humoral effectors, such as cytokines, and cellular immune defenses, including phagocytic cells (e.g., macrophages). After internalization by phagocytes, microbes are delivered into a phagosome, a complex intracellular organelle with a well-established and important role in microbial killing. However, the role of this organelle in cytokine responses and microbial sensing is less well defined. In this study, we assess the role of the phagosome in innate immune sensing and demonstrate the critical interdependence of phagocytosis and pattern recognition receptor signaling during response to the Gram-positive bacteria Staphylococcus aureus. We show that phagocytosis is essential to initiate an optimal MyD88-dependent response to Staphylococcus aureus. Prior to TLR-dependent cytokine production, bacteria must be engulfed and delivered into acidic phagosomes where acid-activated host enzymes digest the internalized bacteria to liberate otherwise cryptic bacterial-derived ligands that initiate responses from the vacuole. Importantly, in macrophages in which phagosome acidification is perturbed, the impaired response to S. aureus can be rescued by the addition of lysostaphin, a bacterial endopeptidase active at neutral pH that can substitute for the acid-activated host enzymes. Together, these observations delineate the interdependence of phagocytosis with pattern recognition receptor signaling and suggest that therapeutics to augment functions and signaling from the vacuole may be useful strategies to increase host responses to S. aureus.

Introduction Type I interferon response represents the hall mark of innate immune effector mechanisms. Although it has been well-documented that type I interferons are key players in coordinating anti-viral immunity, their role in bacterial infections remains poorly defined. Systemic infections with bacteria are characterized by high mortality rates due to the “sepsis syndrome”, a widespread and uncontrolled inflammatory response. The objective of this study was to determine the role of type I interferons in eliciting innate immune defense against bacterial pathogens. Here, we have identified a novel type I interferon pathway that licenses NLRP3 inflammasome activation by all Gram-negative bacteria. Methods Various knock-out mice and macrophages from them were used. Immunoblotting, ELISAs and qPCR were used to assess immune responses. Results A systematic investigation of inflammasome activation by enterohemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium , Gram-negative enteropathogens, revealed a role for TLR4 and TRIF in NLRP3 inflammasome activation as macrophages lacking TLR4 or TRIF failed to process caspase-1 and secrete IL-1β and IL-18. TRIF signaling downstream of TLR4 triggers interferon regulatory factor (IRF)-3 dependent IFN-β production. Indeed, TLR4-TRIF signaling was found to be essential for IFN-β production elicited by EHEC and C. rodentium . Importantly, we found that EHEC and C. rodentium -induced IFN-β subsequently targets the activation of caspase-11, which is a member of the caspase-1 subfamily of inflammatory proteases. A recent study identified caspase-11 as a key regulator of caspase-1 activation and IL-1β/IL-18 production in response to a set of enteric pathogens [1] . Unlike other caspases, caspase-11 is unique in that it is highly inducible. We determined that type I interferon regulates caspase-11 activation by up-regulating caspase-11 expression in an autocrine/paracrine manner in EHEC and C. rodentium infection. Remarkably the transcriptional induction of caspase-11 by IFN-β was both necessary and sufficient to promote caspase-11 auto-activation suggesting a model wherein transcriptional induction of pro-caspase-11 is coupled to its auto-activation. Caspase-11 activation via the TLR4-TRIF-IFN-β pathway ultimately synergizes with the NLRP3 pathway to coordinate caspase-1 dependent IL-1β and IL-18 processing and secretion. The requirement for IFN-β-caspase-11 axis for NLRP3 inflammasome activation was found to be highly specific for Gram-negative but not Gram-positive bacteria. Finally, in a well-established acute bacterial peritonitis and shock model using E. coli , we determined that IFN-β-caspase-11 pathway is essential for E. coli induced IL-1β and IL-18 production not only in vitro but also in vivo. Conclusion Thus, the identification of type I interferon as a regulator of caspase-11-dependent NLRP3 inflammasome activation provides new insights into the integration of TLR and NLR pathways during Gram-negative bacterial infections and unveils new targets that might be manipulated to prevent uncontrolled inflammation during septic shock.

Summary Systemic infections with Gram-negative bacteria are characterized by high mortality rates due to the "sepsis syndrome," a widespread and uncontrolled inflammatory response. Though it is well recognized that the immune response during Gram-negative bacterial infection is initiated after the recognition of endotoxin by Toll-like receptor 4, the molecular mechanisms underlying the detrimental inflammatory response during Gram-negative bacteremia remain poorly defined. Here, we identify a TRIF pathway that licenses NLRP3 inflammasome activation by all Gram-negative bacteria. By engaging TRIF, Gram-negative bacteria activate caspase-11. TRIF activates caspase-11 via type I IFN signaling, an event that is both necessary and sufficient for caspase-11 induction and autoactivation. Caspase-11 subsequently synergizes with the assembled NLRP3 inflammasome to regulate caspase-1 activation and leads to caspase-1-independent cell death. These events occur specifically during infection with Gram-negative, but not Gram-positive, bacteria. The identification of TRIF as a regulator of caspase-11 underscores the importance of TLRs as master regulators of inflammasomes during Gram-negative bacterial infection.