Neutrophils discriminate live from dead bacteria by integrating signals initiated by Fprs and TLRs.

The mechanisms whereby neutrophils respond differentially to live and dead organisms are unknown. We show here that neutrophils produce 5- to 30-fold higher levels of the Cxcl2 chemokine in response to live bacteria, compared with killed bacteria or isolated bacterial components, despite producing similar levels of Cxcl1 or pro-inflammatory cytokines. Secretion of high levels of Cxcl2, which potently activates neutrophils by an autocrine mechanism, requires three signals. The first two signals are provided by two different sets of signal peptides released by live bacteria, which selectively activate formylated peptide receptor 1 (Fpr1) and Fpr2, respectively. Signal 3 originates from Toll-like receptor activation by microbial components present in both live and killed bacteria. Mechanistically, these signaling pathways converge at the level of the p38 MAP kinase, leading to activation of the AP-1 transcription factor and to Cxcl2 induction. Collectively, our data demonstrate that the simultaneous presence of agonists for Fpr1, Fpr2, and Toll-like receptors represents a unique signature associated with viable bacteria, which is sensed by neutrophils and induces Cxcl2-dependent autocrine cell activation.

Streptococcus pneumoniae binds collagens and C1q via the SSURE repeats of the PfbB adhesin.

The binding of Streptococcus pneumoniae to collagen is likely an important step in the pathogenesis of pneumococcal infections, but little is known of the underlying molecular mechanisms. Streptococcal surface repeats (SSURE) are highly conserved protein domains present in cell wall adhesins from different Streptococcus species. We find here that SSURE repeats of the pneumococcal adhesin plasminogen and fibronectin binding protein B (PfbB) bind to various types of collagen. Moreover, deletion of the pfbB gene resulted in a significant impairment of the ability of encapsulated or unencapsulated pneumococci to bind collagen. Notably, a PfbB SSURE domain is also bound to the complement component C1q that bears a collagen-like domain and promotes adherence of pneumococci to host cells by acting as a bridge between bacteria and epithelial cells. Accordingly, deletion of PfbB or pre-treatment with anti-SSURE antibodies markedly decreased pneumococcal binding to C1q as well as C1q-dependent adherence to epithelial and endothelial cells. Further data indicated that C1q promotes pneumococcal adherence by binding to integrin α2 ß1 . In conclusion, our results indicate that the SSURE domains of the PfbB protein promote interactions of pneumococci with various types of collagen and with C1q. These repeats may be useful targets in strategies to control S. pneumoniae infections.

Role of Endosomal TLRs in Staphylococcus aureus Infection.

Identification of the receptors involved in innate immune recognition of Staphylococcus aureus, a major cause of morbidity and mortality in humans, is essential to develop alternative strategies to treat infections caused by antibiotic-resistant strains. In the current study, we examine the role of endosomal TLRs, which sense the presence of prokaryotic-type nucleic acids, in anti-staphylococcal host defenses using infection models involving genetically defective mice. Single deficiencies in TLR7, 9, or 13 resulted in mild or no decrease in host defenses. However, the simultaneous absence of TLR7, 9, and 13 resulted in markedly increased susceptibility to cutaneous and systemic S. aureus infection concomitantly with decreased production of proinflammatory chemokines and cytokines, neutrophil recruitment to infection sites, and reduced production of reactive oxygen species. This phenotype was significantly more severe than that of mice lacking TLR2, which senses the presence of staphylococcal lipoproteins. Notably, the combined absence of TLR7, 9, and 13 resulted in complete abrogation of IL-12 p70 and IFN-ß responses to staphylococcal stimulation in macrophages. Taken together, our data highlight the presence of a highly integrated endosomal detection system, whereby TLR7, 9, and 13 cooperate in sensing the presence of staphylococcal nucleic acids. We demonstrate that the combined absence of these receptors cannot be compensated for by cell surface-associated TLRs, such as TLR2, or cytosolic receptors. These data may be useful to devise strategies aimed at stimulating innate immune receptors to treat S. aureus infections.

Neutrophils Enhance Their Own Influx to Sites of Bacterial Infection via Endosomal TLR-Dependent Cxcl2 Production.

The influx of neutrophils to infection sites is a fundamental step in host defenses against the frequent human pathogen group B Streptococcus (GBS) and other extracellular bacteria. Using a mouse model of GBS-induced peritonitis, we show in this study that the chemokines Cxcl1 and Cxcl2 play distinctive roles in enhancing the recruitment and the antibacterial activities of neutrophils in a manner that is linked to differences in the cellular sources of these mediators. Cell depletion experiments demonstrated that neutrophils make a significant contribution to the in vivo production of Cxcl2 but not Cxcl1. In vitro, neutrophils responded weakly to LPS but released high levels of Cxcl2 after stimulation with GBS or other bacteria. Neutrophil-derived Cxcl2 acted in an autocrinous manner to increase its own production and to enhance antibacterial activities, including the release of oxygen radicals. In both neutrophils and macrophages, the production of Cxcl1/2 largely required the presence of functional UNC93B1, a chaperone protein involved in signaling by endosomal TLRs. Moreover, the phenotype of UNC93B1-defective phagocytes could be recapitulated by the simultaneous absence of TLR7, 9, and 13 but not by the absence of individual TLRs. Collectively, our data show that neutrophils recognize Gram-positive and Gram-negative bacteria by means of multiple phagosomal TLRs, resulting in de novo synthesis of Cxcl2, amplification of neutrophil recruitment, and potentiation of their antibacterial activities. These data may be useful to devise alternative therapeutic strategies aimed at enhancing the recruitment and the functional activities of polymorphonuclear leukocytes during infections caused by antibiotic-resistant bacteria.

The TLR4 adaptor TRAM controls the phagocytosis of Gram-negative bacteria by interacting with the Rab11-family interacting protein 2.

Phagocytosis is a complex process that eliminates microbes and is performed by specialised cells such as macrophages. Toll-like receptor 4 (TLR4) is expressed on the surface of macrophages and recognizes Gram-negative bacteria. Moreover, TLR4 has been suggested to play a role in the phagocytosis of Gram-negative bacteria, but the mechanisms remain unclear. Here we have used primary human macrophages and engineered THP-1 monocytes to show that the TLR4 sorting adapter, TRAM, is instrumental for phagocytosis of Escherichia coli as well as Staphylococcus aureus. We find that TRAM forms a complex with Rab11 family interacting protein 2 (FIP2) that is recruited to the phagocytic cups of E. coli. This promotes activation of the actin-regulatory GTPases Rac1 and Cdc42. Our results show that FIP2 guided TRAM recruitment orchestrates actin remodelling and IRF3 activation, two events that are both required for phagocytosis of Gram-negative bacteria.

Bacterial recognition by TLR7 in the lysosomes of conventional dendritic cells.

Little is known of how and where bacterial recognition triggers the induction of type I interferon. Whether the type of recognition receptor used in these responses is determined by the subcellular location of bacteria is not understood. Here we show that phagosomal bacteria such as group B streptococcus, but not cytosolic bacteria, potently induced interferon in conventional dendritic cells by a mechanism that required Toll-like receptor 7, the adaptor MyD88 and the transcription factor IRF1, all of which localized together with bacterial products in degradative vacuoles bearing lysosomal markers. Thus, this cell type-specific recognition pathway links lysosomal recognition of bacterial RNA with a robust, host-protective interferon response.

Characterization of an immunogenic cellulase secreted by Cryptococcus pathogens.

Members of the C. neoformans/C. gattiii species complex are an important cause of serious humans infections, including meningoencephalitis. We describe here a 45 kDa extracellular cellulase purified from culture supernatants of C. neoformans var. neoformans. The N-terminal sequence obtained from the purified protein was used to isolate a clone containing the full-length coding sequence from a C. neoformans var. neoformans (strain B-3501A) cDNA library. Bioinformatics analysis indicated that this gene is present, with variable homology, in all sequenced genomes of the C. neoformans/C. gattii species complex. The cDNA clone was used to produce a recombinant 45 kDa protein in E. coli that displayed the ability to convert carboxymethyl cellulose and was therefore designated as NG-Case (standing for Neoformans Gattii Cellulase). To explore its potential use as a vaccine candidate, the recombinant protein was used to immunize mice and was found capable of inducing T helper type 1 responses and delayed-type hypersensitivity reactions, but not immune protection against a highly virulent C. neoformans var grubii strain. These data may be useful to better understand the mechanisms underlying the ability C. neoformans/C. gattii to colonize plant habitats and to interact with the human host during infection.

The Streptococcus agalactiae cell wall-anchored protein PbsP mediates adhesion to and invasion of epithelial cells by exploiting the host vitronectin/αv integrin axis.

Binding of microbial pathogens to host vitronectin (Vtn) is a common theme in the pathogenesis of invasive infections. In this study, we characterized the role of Vtn in the invasion of mucosal epithelial cells by Streptococcus agalactiae (i.e. group B streptococcus or GBS), a frequent human pathogen. Moreover, we identified PbsP, a previously described plasminogen-binding protein of GBS, as a dual adhesin that can also interact with human Vtn through its streptococcal surface repeat (SSURE) domains. Deletion of the pbsP gene decreases both bacterial adhesion to Vtn-coated inert surfaces and the ability of GBS to interact with epithelial cells. Bacterial adherence to and invasion of epithelial cells were either inhibited or enhanced by cell pretreatment with, respectively, anti-Vtn antibodies or Vtn, confirming the role of Vtn as a GBS ligand on host cells. Finally, antibodies directed against the integrin αv subunit inhibited Vtn-dependent cell invasion by GBS. Collectively, these results indicate that Vtn acts as a bridge between the SSURE domains of PbsP on the GBS surface and host integrins to promote bacterial invasion of epithelial cells. Therefore, inhibition of interactions between PbsP and extracellular matrix components could represent a viable strategy to prevent colonization and invasive disease by GBS.

PbsP, a cell wall-anchored protein that binds plasminogen to promote hematogenous dissemination of group B Streptococcus.

Streptococcus agalactiae (Group B Streptococcus or GBS) is a leading cause of invasive infections in neonates whose virulence is dependent on its ability to interact with cells and host components. We here characterized a surface protein with a critical function in GBS pathophysiology. This adhesin, designated PbsP, possesses two Streptococcal Surface Repeat domains, a methionine and lysine-rich region, and a LPXTG cell wall-anchoring motif. PbsP mediates plasminogen (Plg) binding both in vitro and in vivo and we showed that cell surface-bound Plg can be activated into plasmin by tissue plasminogen activator to increase the bacterial extracellular proteolytic activity. Absence of PbsP results in a decreased bacterial transmigration across brain endothelial cells and impaired virulence in a murine model of infection. PbsP is conserved among the main GBS lineages and is a major plasminogen adhesin in non-CC17 GBS strains. Importantly, immunization of mice with recombinant PbsP confers protective immunity. Our results indicate that GBS have evolved different strategies to recruit Plg which indicates that the ability to acquire cell surface proteolytic activity is essential for the invasiveness of this bacterium.

The Human Pathogen Streptococcus pyogenes Releases Lipoproteins as Lipoprotein-rich Membrane Vesicles.

Bacterial lipoproteins are attractive vaccine candidates because they represent a major class of cell surface-exposed proteins in many bacteria and are considered as potential pathogen-associated molecular patterns sensed by Toll-like receptors with built-in adjuvanticity. Although Gram-negative lipoproteins have been extensively characterized, little is known about Gram-positive lipoproteins. We isolated from Streptococcus pyogenes a large amount of lipoproteins organized in vesicles. These vesicles were obtained by weakening the bacterial cell wall with a sublethal concentration of penicillin. Lipid and proteomic analysis of the vesicles revealed that they were enriched in phosphatidylglycerol and almost exclusively composed of lipoproteins. In association with lipoproteins, a few hypothetical proteins, penicillin-binding proteins, and several members of the ExPortal, a membrane microdomain responsible for the maturation of secreted proteins, were identified. The typical lipidic moiety was apparently not necessary for lipoprotein insertion in the vesicle bilayer because they were also recovered from the isogenic diacylglyceryl transferase deletion mutant. The vesicles were not able to activate specific Toll-like receptor 2, indicating that lipoproteins organized in these vesicular structures do not act as pathogen-associated molecular patterns. In light of these findings, we propose to name these new structures Lipoprotein-rich Membrane Vesicles.

FbsC, a novel fibrinogen-binding protein, promotes Streptococcus agalactiae-host cell interactions.

Streptococcus agalactiae (group B Streptococcus or GBS) is a common cause of invasive infections in newborn infants and adults. The ability of GBS to bind human fibrinogen is of crucial importance in promoting colonization and invasion of host barriers. We characterized here a novel fibrinogen-binding protein of GBS, designated FbsC (Gbs0791), which is encoded by the prototype GBS strain NEM316. FbsC, which bears two bacterial immunoglobulin-like tandem repeat domains and a C-terminal cell wall-anchoring motif (LPXTG), was found to be covalently linked to the cell wall by the housekeeping sortase A. Studies using recombinant FbsC indicated that it binds fibrinogen in a dose-dependent and saturable manner, and with moderate affinity. Expression of FbsC was detected in all clinical GBS isolates, except those belonging to the hypervirulent lineage ST17. Deletion of fbsC decreases NEM316 abilities to adhere to and invade human epithelial and endothelial cells, and to form biofilm in vitro. Notably, bacterial adhesion to fibrinogen and fibrinogen binding to bacterial cells were abolished following fbsC deletion in NEM316. Moreover, the virulence of the fbsC deletion mutant and its ability to colonize the brain were impaired in murine models of infection. Finally, immunization with recombinant FbsC significantly protected mice from lethal GBS challenge. In conclusion, FbsC is a novel fibrinogen-binding protein expressed by most GBS isolates that functions as a virulence factor by promoting invasion of epithelial and endothelial barriers. In addition, the protein has significant immunoprotective activity and may be a useful component of an anti-GBS vaccine.

Role of Toll-like receptor 13 in innate immune recognition of group B streptococci.

Murine Toll-like receptor 13 (TLR13), an endosomal receptor that is not present in humans, is activated by an unmethylated motif present in the large ribosomal subunit of bacterial RNA (23S rRNA). Little is known, however, of the impact of TLR13 on antibacterial host defenses. Here we examined the role of this receptor in the context of infection induced by the model pathogen group B streptococcus (GBS). To this end, we used bacterial strains masked from TLR13 recognition by virtue of constitutive expression of the ErmC methyltransferase, which results in dimethylation of the 23S rRNA motif at a critical adenine residue. We found that TLR13-mediated rRNA recognition was required for optimal induction of tumor necrosis factor alpha and nitrous oxide in dendritic cell and macrophage cultures stimulated with heat-killed bacteria or purified bacterial RNA. However, TLR13-dependent recognition was redundant when live bacteria were used as a stimulus. Moreover, masking bacterial rRNA from TLR13 recognition did not increase the ability of GBS to avoid host defenses and replicate in vivo. In contrast, increased susceptibility to infection was observed under conditions in which signaling by all endosomal TLRs was abolished, i.e., in mice with a loss-of-function mutation in the chaperone protein UNC93B1. Our data lend support to the conclusion that TLR13 participates in GBS recognition, although blockade of the function of this receptor can be compensated for by other endosomal TLRs. Lack of selective pressure by bacterial infections might explain the evolutionary loss of TLR13 in humans. However, further studies using different bacterial species are needed to prove this hypothesis.

Activation of the NLRP3 inflammasome by group B streptococci.

Group B Streptococcus (GBS) is a frequent agent of life-threatening sepsis and meningitis in neonates and adults with predisposing conditions. We tested the hypothesis that activation of the inflammasome, an inflammatory signaling complex, is involved in host defenses against this pathogen. We show in this study that murine bone marrow-derived conventional dendritic cells responded to GBS by secreting IL-1ß and IL-18. IL-1ß release required both pro-IL-1ß transcription and caspase-1-dependent proteolytic cleavage of intracellular pro-IL-1ß. Dendritic cells lacking the TLR adaptor MyD88, but not those lacking TLR2, were unable to produce pro-IL-1ß mRNA in response to GBS. Pro-IL-1ß cleavage and secretion of the mature IL-1ß form depended on the NOD-like receptor family, pyrin domain containing 3 (NLRP3) sensor and the apoptosis-associated speck-like protein containing a caspase activation and recruitment domain adaptor. Moreover, activation of the NLRP3 inflammasome required GBS expression of ß-hemolysin, an important virulence factor. We further found that mice lacking NLRP3, apoptosis-associated speck-like protein, or caspase-1 were considerably more susceptible to infection than wild-type mice. Our data link the production of a major virulence factor by GBS with the activation of a highly effective anti-GBS response triggered by the NLRP3 inflammasome.

The Mannoprotein Cig1 supports iron acquisition from heme and virulence in the pathogenic fungus Cryptococcus neoformans.

Iron acquisition is critical for virulence of the human pathogenic fungus Cryptococcus neoformans. The cryptococcal transcript for the extracellular mannoprotein Cig1 is highly regulated by iron and abundant in iron-starved cells, suggesting a role in iron acquisition. Indeed, loss of Cig1 resulted in delayed growth on heme at physiological pH. Expression of CIG1 is regulated by the pH-responsive transcription factor Rim101, and loss of Rim101 also impaired growth on heme. A cig1Δ mutant was less susceptible than the wild-type strain to noniron metalloporphyrins, further indicating a role for Cig1 in heme uptake. Recombinant Cig1 exhibited the absorbance spectrum of a heme-binding protein upon heme titration, and Cig1 may therefore function as a hemophore at the cell surface. Cig1 contributed to virulence in a mouse model of cryptococcosis but only in a mutant that also lacked the high-affinity iron uptake system. Overall, Cig1-mediated heme uptake is a potential therapeutic target in C. neoformans.

Role of the SaeRS Two-Component Regulatory System in Group B Streptococcus Biofilm Formation on Human Fibrinogen.

Streptococcus agalactiae, also known as Group B Streptococcus or GBS, is a commensal colonizer of human vaginal and gastrointestinal tracts that can also be a deadly pathogen for newborns, pregnant women, and the elderly. The SaeRS two-component regulatory system (TCS) positively regulates the expression of two GBS adhesins genes, but its role in the formation of biofilm, an important step in pathogenesis, has not been investigated. In the present study, we set up a novel model of GBS biofilm formation using surfaces coated with human fibrinogen (hFg). Biofilm mass and structure were analyzed by crystal violet staining and three-dimensional fluorescence microscopy, respectively. GBS growth on hFg resulted in the formation of a mature and abundant biofilm composed of bacterial cells and an extracellular matrix containing polysaccharides, proteins, and extracellular DNA (eDNA). Enzymatic and genetic analysis showed that GBS biofilm formation on hFg is dependent on proteins and eDNA in the extracellular matrix and on the presence of covalently linked cell wall proteins on the bacterial surface but not on the type-specific capsular polysaccharide. In the absence of the SaeR regulator of the SaeRS TCS, there was a significant reduction in biomass formation, with reduced numbers of bacterial cells, reduced eDNA content, and disruption of the biofilm architecture. Overall, our data suggest that GBS binding to hFg contributes to biofilm formation and that the SaeRS TCS plays an important role in this process.

The SaeRS two-component system regulates virulence gene expression in group B Streptococcus during invasive infection.

Group B Streptococcus (GBS) is a pathobiont responsible for invasive infections in neonates and the elderly. The transition from a commensal to an invasive pathogen relies on the timely regulation of virulence factors. In this study, we characterized the role of the SaeRS two-component system in GBS pathogenesis. Loss-of-function mutations in the SaeR response regulator decrease virulence in mouse models of invasive infection by hindering the ability of bacteria to persist at the inoculation site and to spread to distant organs. Transcriptome and in vivo analysis reveal a specialized regulatory system specifically activated during infection to control the expression of only two virulence factors: the PbsP adhesin and the BvaP secreted protein. The in vivo surge in SaeRS-regulated genes is complemented by fine-tuning mediated by the repressor of virulence CovRS system to establish a coordinated response. Constitutive activation of the SaeRS regulatory pathway increases PbsP-dependent adhesion and invasion of epithelial and endothelial barriers, though at the cost of reduced virulence. In conclusion, SaeRS is a dynamic, highly specialized regulatory system enabling GBS to express a restricted set of virulence factors that promote invasion of host barriers and allow these bacteria to persist inside the host during lethal infection. IMPORTANCE: Group B Streptococcus (or GBS) is a normal inhabitant of the human gastrointestinal and genital tracts that can also cause deadly infections in newborns and elderly people. The transition from a harmless commensal to a dangerous pathogen relies on the timely expression of bacterial molecules necessary for causing disease. In this study, we characterize the two-component system SaeRS as a key regulator of such virulence factors. Our analysis reveals a specialized regulatory system that is activated only during infection to dynamically adjust the production of two virulence factors involved in interactions with host cells. Overall, our findings highlight the critical role of SaeRS in GBS infections and suggest that targeting this system may be useful for developing new antibacterial drugs.

Constitutive activation of two-component systems reveals regulatory network interactions in Streptococcus agalactiae.

Bacterial two-component systems (TCSs) are signaling modules that control physiology, adaptation, and host interactions. A typical TCS consists of a histidine kinase (HK) that activates a response regulator via phosphorylation in response to environmental signals. Here, we systematically test the effect of inactivating the conserved phosphatase activity of HKs to activate TCS signaling pathways. Transcriptome analyses of 14 HK mutants in Streptococcus agalactiae, the leading cause of neonatal meningitis, validate the conserved HK phosphatase mechanism and its role in the inhibition of TCS activity in vivo. Constitutive TCS activation, independent of environmental signals, enables high-resolution mapping of the regulons for several TCSs (e.g., SaeRS, BceRS, VncRS, DltRS, HK11030, HK02290) and reveals the functional diversity of TCS signaling pathways, ranging from highly specialized to interconnected global regulatory networks. Targeted analysis shows that the SaeRS-regulated PbsP adhesin acts as a signaling molecule to activate CovRS signaling, thereby linking the major regulators of host-pathogen interactions. Furthermore, constitutive BceRS activation reveals drug-independent activity, suggesting a role in cell envelope homeostasis beyond antimicrobial resistance. This study highlights the versatility of constitutive TCS activation, via phosphatase-deficient HKs, to uncover regulatory networks and biological processes.

Recognition of fungal RNA by TLR7 has a nonredundant role in host defense against experimental candidiasis.

Despite convincing evidence for involvement of members of the Toll-like receptor (TLR) family in fungal recognition, little is known of the functional role of individual TLRs in antifungal defenses. We found here that TLR7 was partially required for the induction of IL-12 (IL-12p70) by Candida albicans or Saccharomyces cerevisiae. Moreover, the IL-12p70 response was completely abrogated in cells from 3d mice, which are unable to mobilize TLRs to endosomal compartments, as well as in cells from mice lacking either the TLR adaptor MyD88 or the IRF1 transcription factor. Notably, purified fungal RNA recapitulated IL-12p70 induction by whole yeast. Although RNA could also induce moderate TLR7-dependent IL-23 and tumor necrosis factor-alpha (TNF-α) secretion, TLR7 and other endosomal TLRs were redundant for IL-23 or TNF-α induction by whole fungi. Importantly, mice lacking TLR7 or IRF1 were hypersusceptible to systemic C. albicans infection. Our data suggest that IRF1 is downstream of a novel, nonredundant fungal recognition pathway that has RNA as a major target and requires phagosomal recruitment of intracellular TLRs. This pathway differs from those involved in IL-23 or TNF-α responses, which we show here to be independent from translocation of intracellular TLRs, phagocytosis, or phagosomal acidification.

The Spr1875 protein confers resistance to the microglia-mediated killing of Streptococcus pneumoniae.

By screening a whole-genome λ-display library of Streptococcus pneumoniae, we have previously identified a novel surface protein, named Spr1875, that exhibited immunogenic properties and was closely related to pneumococcal virulence. In the present study, we investigated the role of the Spr1875 antigen in the interaction of S. pneumoniae with microglia, the resident brain macrophages. By using an in vitro infection model, the BV2 microglial cell line was challenged with the S. pneumoniae strain DP1004 and its isogenic spr1875-deleted mutant (Δspr1875). Both strains were phagocytosed by microglia efficiently and to a similar extent; however, the DP1004 strain was more resistant than the Δspr1875 mutant to the intracellular killing, as assessed by antibiotic protection and phagosome maturation assays. Moreover, significant differences between the two strains were also observed in terms of susceptibility to microglia-mediated killing. Taken together, these results indicate that S. pneumoniae-microglial cell interplay is influenced by the presence of Spr1875, suggesting that this protein may play a role in the pathogenesis of pneumococcal meningitis.

Caspase-8 inhibition improves the outcome of bacterial infections in mice by promoting neutrophil activation.

During differentiation, neutrophils undergo a spontaneous pro-inflammatory program that is hypothesized here to be under caspase-8 control. In mice, intraperitoneal administration of the caspase-8 inhibitor z-IETD-fmk is sufficient to unleash the production of pro-inflammatory cytokines and neutrophil influx in the absence of cell death. These effects are due to selective inhibition of caspase-8 and require tonic interferon-ß (IFN-ß) production and RIPK3 but not MLKL, the essential downstream executioner of necroptotic cell death. In vitro, stimulation with z-IETD-fmk is sufficient to induce significant cytokine production in murine neutrophils but not in macrophages. Therapeutic administration of z-IETD-fmk improves clinical outcome in models of lethal bacterial peritonitis and pneumonia by augmenting cytokine release, neutrophil influx, and bacterial clearance. Moreover, the inhibitor protects mice against high-dose endotoxin shock. Collectively, our data unveil a RIPK3- and IFN-ß-dependent pathway that is constitutively activated in neutrophils and can be harnessed therapeutically using caspase-8 inhibition.