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BackgroundSYNB1891 is a live, modified strain of probiotic E. coli Nissle engineered to produce cyclic dinucleotides under hypoxia leading to stimulator of interferon genes (STING)-activation in phagocytic antigen-presenting cells in tumors and activating complementary innate immune pathways.MethodsThis first-in-human study (NCT04167137) enrolled patients with refractory advanced solid tumors to receive intratumoral (IT) injections of SYNB1891 monotherapy or in combination atezolizumab. Patients enrolled in the monotherapy arms received doses of 1x106 - 3x108 live cells on Days 1, 8 and 15 of the first 21-day cycle and then on Day 1 of each subsequent cycle. Patients enrolled in the 2 combination cohorts received doses of 1x107 - 3x107 live cells in combination with atezolizumab administered on a 21-day cycle. The primary objective of the study was to evaluate safety and tolerability of SYNB1891 alone and in combination with atezolizumab. Other objectives include SYNB1891 kinetics in blood and injected tumor, STING-target engagement as assessed by IT gene expression and serum cytokines, and tumor responses.ResultsThis interim analysis includes 23 patients across 6 monotherapy cohorts dosed at 1x106, 3x106, 1x107, or 3x107, 1x108 and 3x108 live cells, and 7 patients dosed in 2 combination therapy cohorts (1x107 and 3x107 live cells). The mean (range) age was 61 (25–82); 19 patients were female. There were 4 cytokine release syndrome events in monotherapy cohorts, including one grade 3 event which met the criterion for dose limiting toxicity at 3x108 live cells; there were no other SYNB1891-related serious adverse events. There were no SYNB1891-related infections. SYNB1891 was not detected in the blood at 6 or 24 hours after the first dose or intratumorally 7 days following the first dose. Treatment with SYNB1891 demonstrated activation of the STING pathway and target engagement as assessed by upregulation of interferon-stimulated genes (ISG15, IFIT1, IFIt2), chemokines/cytokines (CXCL9, CXCL10, TNFRS18, TNFSF10) and T-cell response genes (GZMA, CD4, PD-L2) in core biopsies obtained pre-dose and 7 days following the third weekly dose. In addition, there was a dose-response increase in serum cytokines. Durable, stable disease was observed in two patients treated with SYNB1891 monotherapy refractory to prior PD-1/L1 antibodies with vulvar melanoma (1x106 live cells; RECIST -28%) and small cell lung cancer (1x107 live cells; RECIST -12%).ConclusionsRepeat IT injection of SYNB1891 as monotherapy and in combination atezolizumab in this ongoing study is safe and well-tolerated up to at least 1x108 live cells, and shows evidence of STING pathway target engagement.AcknowledgementsWe thank Inessa Vulfova for her clinical support in conduct of this study.Trial Registration clinicaltrials.gov (NCT04167137)Ethics ApprovalThe study protocol, the informed consent form (ICF), and printed subject information materials were reviewed and approved by the institutional review board (IRB) at the investigational site before any study procedures were performed. Written informed consent to participate in the study was obtained from each subject before any study-specific procedures were performed.The Ohio State University Cancer Institutional Review Board; Approval ID: 2020C0194MD Anderson Cancer Center Institutional Review Board; Approval ID: 2019–0576Mary Crawley Medical Research Center Institutional Review Board; Approval ID: 19–31 SYNB1891-CP-001North Texas Institutional Review Board; Approval ID: 2019.040WIRB Approval ID: 20192779University of Pittsburgh Institutional Review Board Approval ID: STUDY20010116

Introduction: SYNB1891 is a live, modified strain of the probiotic E. coli Nissle engineered to produce cyclic dinucleotides under hypoxia leading to stimulator of interferon genes (STING)-activation in phagocytic antigen-presenting cells in tumors and activating complementary innate immune pathways. Methods: This first-in-human study (NCT04167137) is enrolling patients with refractory advanced solid tumors or lymphoma to receive an intratumoral (IT) injection of SYNB1891 on Days 1, 8 and 15 of the first 21-day cycle and then on Day 1 of each subsequent cycle. Dose escalation is planned across 7 cohorts (1x106 - 1x109 live cells) with Arm 1 consisting of escalating doses of SYNB1891 as monotherapy, and Arm 2 in combination with atezolizumab. The primary objective is to determine the single-agent maximum tolerated dose as monotherapy and the recommended Phase 2 dose in combination with atezolizumab. Other objectives include SYNB1891 kinetics in blood and the injected tumor, STING-target engagement as assessed by IT gene expression and serum cytokines, and tumor responses. Results: This interim analysis includes the first 11 patients across 4 cohorts dosed at 1x106, 3x106, 1x107, or 3x107 live cells, with a total of 59 doses administered. The mean (range) age was 56 (25-70); 9 patients were female. There were no dose-limiting toxicities, SYNB1891-related infections or discontinuations due to adverse events. There was one SYNB1891-related serious adverse event in a patient who experienced a grade 2 cytokine release syndrome that resolved within one day, and one patient experienced a grade 2 injection site reaction of erythema which resolved. SYNB1891 was not detected in the blood at 6 or 24 hours after the first dose or intratumorally 7 days following the first dose. Treatment with SYNB1891 demonstrated activation of the STING pathway and target engagement as assessed by upregulation of interferon-stimulated genes (ISG15, IFIT1, IFIt2), chemokines/cytokines (CXCL9, CXCL10, TNFRS18, TNFSF10) and T-cell response genes (GZMA, CD4, PD-L2) in core biopsies obtained pre-dose and 7 days following the third weekly dose. In addition, there was a dose-response increase in serum cytokines at dose levels of 1x107 and 3x107 live cells (IL-6, TNFα, IFNγ, IL-1Ra) at 6 hours post-dose. Durable, stable disease was observed in two patients refractory to prior PD-1/L1 antibodies with vulvar melanoma (1x106 live cells) and small cell lung cancer (1x107 live cells). Conclusions: Repeat IT injection of SYNB1891 as monotherapy is safe and well-tolerated up to at least 3x107 cells and shows evidence of STING pathway target engagement. These data support the continued dose escalation of SYNB1891 as monotherapy, and initiation of Arm 2 in combination with atezolizumab. Citation Format: Filip Janku, Jason J. Luke, Aoife Brennan, Richard Riese, Mary Varterasian, Michael B. Armstrong, Karen L. Kuhn, Anna Sokolovska, James F. Strauss. Intratumoral injection of SYNB1891, a synthetic biotic designed to activate the innate immune system, demonstrates target engagement in humans including intratumoral STING activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT110.

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.

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.

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.

Adsorption of the antigen to an aluminum-containing adjuvant is considered an important aspect of vaccine formulation. Adsorption is described by two parameters: the maximum amount that can be adsorbed as a monolayer, which is characterized by the adsorptive capacity and the strength of the adsorption force, which is described by the adsorptive coefficient. Research to date has focused on the adsorptive capacity with the goal of complete adsorption of the antigen. In this study, the relationship between the adsorptive coefficient and immunopotentiation was investigated. Four vaccines were prepared in which the adsorptive coefficient was varied by altering the number of phosphate groups on the antigen (alpha casein and dephosphorylated alpha casein) or the number of surface hydroxyls on the adjuvant (aluminum hydroxide adjuvant and phosphate-treated aluminum hydroxide adjuvant). In vitro elution upon exposure to interstitial fluid or normal human plasma was inversely related to the adsorptive coefficient. The geometric mean antibody titer in mice was also inversely related to the adsorptive coefficient. T-cell activation was not observed in mice that received the vaccine with the greatest adsorptive coefficient (alpha casein/aluminum hydroxide adjuvant). This suggests that antigen processing and presentation to T-cells is impaired when the antigen is adsorbed too strongly.

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.

Signaling through Toll-like receptors (TLR) has been implicated in activation of immune cell populations such as B lymphocytes. TLR ligands associated with self-antigens can drive increased self-reactive B cell responses however TLR ligands present in pathogenic products or vaccines can also enhance protective antibody responses. Our recent work has identified a novel mechanism by which αv integrins, a family of adhesion molecules and the autophagy proteins limit excessive B cell TLR signaling. Specifically, we have found that αvβ3 integrin regulates TLR signaling (NFKB and IRF7 activation) by directing maturation of TLR containing endosomes, through activation of components of the autophagy pathway (LC3 and Atg5). B cells lacking either αv integrins or autophagy components show enhanced TLR signaling and increased proliferative responses to TLR ligands. Mice with disruption in this pathway develop increased autoantibodies with age and develop accelerated autoimmunity in murine lupus models. Therefore, we propose that αv-mediated regulation of TLR signaling exists to limit excessive B cell responses to self-antigens. In addition, we find that mice in which αv is specifically deleted from B cells mount stronger antibody responses when immunized with exogenous antigens containing TLR-ligand adjuvants. Immune responses to these antigens are characterized by increased expansion of germinal center cells, increased somatic hyper-mutation and higher levels of high-affinity class-switched antibodies. Therefore this αv mediated regulation of TLR signaling not only regulates B cell responses to self-antigens but it is also important for regulating germinal center B cell responses and production of high-affinity antibodies.

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.

A classic hallmark in the autophagic process is the reversible conjugation of LC3 to the autophagosomal membrane. Autophagy has been shown to target pathogens that reside in the cytosol or within autophagosomes for lysosomal degradation. Therefore, components of the autophagy pathway can participate in the effective elimination of viruses, bacteria, and parasites, though their role in fungal immunity is poorly understood. We show that activation of Dectin-1 by its ligand β-1,3-glucan leads to recruitment of LC3 to the phagosome and describe multiple intermediate signaling steps. The cytoplasmic tail of Dectin-1 contains an immunoreceptor tyrosine-based activation (ITAM)-like motif which is phosphorylated in response to binding of β-1,3-glucan, leading to relocation from the cell surface to the phagosome. We show that Dectin-1 activation by intact fungal pathogens or by chemically defined ligands leads to LC3 recruitment to the phagosomal membrane. Mutation of the tyrosine phosphorylation site in Dectin-1 abolished LC3 recruitment, suggesting the involvement of spleen tyrosine kinase (Syk) in this subcellular redistribution of LC3. Likewise, impairment of ROS generation by knockout of NADPH oxidase (NCF4) downstream of Syk inhibited LC3 recruitment to the phagosome, whereas knockout of Card9 did not affect LC3 recruitment. These observations suggest a previously unobserved signaling pathway connects Dectin-1 to the autophagic machinery via Syk signaling and ROS generation.

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.