Actin polymerization as a key innate immune effector mechanism to control Salmonella infection

Salmonellosis is one of the leading causes of food poisoning worldwide. Controlling bacterial burden is essential to surviving infection. Nucleotide-binding oligomerization domain-like receptors (NLRs), such as NLRC4, induce inflammasome effector functions and play a crucial role in controlling Salmonella infection. Inflammasome-dependent production of IL-1β recruits additional immune cells to the site of infection, whereas inflammasome-mediated pyroptosis of macrophages releases bacteria for uptake by neutrophils. Neither of these functions is known to directly kill intracellular salmonellae within macrophages. The mechanism, therefore, governing how inflammasomes mediate intracellular bacterial-killing and clearance in host macrophages remains unknown. Here, we show that actin polymerization is required for NLRC4-dependent regulation of intracellular bacterial burden, inflammasome assembly, pyroptosis, and IL-1β production. NLRC4-induced changes in actin polymerization are physically manifested as increased cellular stiffness, and leads to reduced bacterial uptake, production of antimicrobial molecules, and arrested cellular migration. These processes act in concert to limit bacterial replication in the cell and dissemination in tissues. We show, therefore, a functional link between innate immunity and actin turnover in macrophages that underpins a key host defense mechanism for the control of salmonellosis.A critical step in disease pathogenesis for many clinically important bacteria is their ability to infect and survive within host cells such as macrophages. Salmonella enterica, a pathogen that resides and replicates within macrophages, causes a range of life-threatening diseases in humans and animals, and accounts for 28 million cases of enteric fever worldwide each year (1). S. enterica infects phagocytes by a process that requires cytoskeletal reorganization (2). This bacterium resides in a Salmonella-containing vacuole (SCV) within host macrophages, and this intracellular lifestyle enables them to avoid extracellular antimicrobial killing, evade adaptive immune responses, and potentially to spread to new sites to seed new infectious foci within host tissue, which eventually develop into granulomas (3). Survival and growth of S. enterica within phagocytes is critical for virulence (4) and host restriction of the intracellular bacterial load is, therefore, paramount in surviving salmonellosis. Salmonella delivers microbial effector proteins into the host cell via the type III secretion systems (T3SS), mediated by the Salmonella pathogenicity island-1 and -2 (SPI-1 and SPI-2), to subvert cellular functions and facilitate intracellular survival (5).Microbes are recognized by macrophages through pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain-like receptors (NLRs), which initiate innate immune responses, including cytokine production and pathogen killing (6). NLRs drive the formation of inflammasomes—macromolecular protein complexes—comprising one or more NLRs, usually an adaptor protein (ASC) and the effector protein caspase-1, which then cleaves prointerleukin-1β (IL-1β) and pro–IL-18 into biologically active cytokines, and initiates macrophage cell death by pyroptosis (7). NLRC4, in concert with NAIPs 1, 2, 5, and 6, is a key PRR that forms an inflammasome complex upon sensing flagellin and/or the inner rod or needle proteins (PrgJ and PrgI, respectively) of the SPI-1 T3SS of S. enterica serovar Typhimurium (S. Typhimurium) (8–11). Activation of the NLRC4 inflammasome by Salmonella infection results in IL-1β and IL-18 production driven by an ASC-dependent pathway and macrophage pyroptosis driven by an ASC-independent pathway (12, 13). A second, noncanonical, NLR signaling pathway has been described, which requires caspase-11 to initiate delayed cell death and NLRP3 inflammasome activation (14–16). Effective clearance of Salmonella infection in host cells may therefore require a coordinated effort between different inflammasome signaling pathways.We, and others, have shown that NLRC4 is important in regulating bacterial burden of S. Typhimurium in vivo (17–19). A recent study revealed that Salmonella-infected epithelial cells are extruded from the intestinal epithelium in a process that requires NLRC4 (20). The molecular mechanism behind how NLRC4 restricts bacterial burden in macrophages infected with Salmonella is still unknown. Here, we identify an actin-dependent mechanism that controls NLRC4-mediated regulation of bacterial replication in macrophages infected with S. Typhimurium. Activation of NLRC4 in infected macrophages mediates the production of reactive oxygen species (ROS) to inhibit bacterial replication and limits additional bacterial uptake by inducing mechanical stiffening the cell via actin polymerization. Overall, we describe a previously unidentified effector mechanism, governed by actin and the NLRC4 inflammasome, to control Salmonella infection in macrophages.     

Human NAIP and mouse NAIP1 recognize bacterial type III secretion needle protein for inflammasome activation

Inflammasome mediated by central nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) protein is critical for defense against bacterial infection. Here we show that type III secretion system (T3SS) needle proteins from several bacterial pathogens, including Salmonella typhimurium, enterohemorrhagic Escherichia coli, Shigella flexneri, and Burkholderia spp., can induce robust inflammasome activation in both human monocyte-derived and mouse bone marrow macrophages. Needle protein activation of human NRL family CARD domain containing 4 (NLRC4) inflammasome requires the sole human neuronal apoptosis inhibitory protein (hNAIP). Among the seven mouse NAIPs, NAIP1 functions as the mouse counterpart of hNAIP. We found that NAIP1 recognition of T3SS needle proteins was more robust in mouse dendritic cells than in bone marrow macrophages. Needle proteins, as well as flagellin and rod proteins from five different bacteria, exhibited differential and cell type-dependent inflammasome-stimulating activity. Comprehensive profiling of the three types of NAIP ligands revealed that NAIP1 sensing of the needle protein dominated S. flexneri-induced inflammasome activation, particularly in dendritic cells. hNAIP/NAIP1 and NAIP2/5 formed a large oligomeric complex with NLRC4 in the presence of corresponding bacterial ligands, and could support reconstitution of the NLRC4 inflammasome in a ligand-specific manner.Innate immunity in mammals relies on a group of germline-encoded pattern recognition receptors (PRRs) to sense conserved pathogen-associated molecular patterns (PAMPs) and defend against microbial infections (1). Cytosolic nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) proteins, characterized by an N-terminal caspase recruitment domain or a pyrin domain, a central NOD, and a C-terminal leucine-rich repeat domain, are a family of PRRs with increasingly appreciated function in innate immune defense (2, 3). The NLR family contains 23 members in humans and 34 members in mice, many of which are known or thought to form large oligomeric inflammasome complexes in response to particular stimulation. The inflammasome is present mostly in macrophage and dendritic cells, and functions as a signaling platform for caspase-1 autoprocessing and activation (4). Activated caspase-1 further cleaves IL-1β and IL-18 into mature forms, and also induces macrophage inflammatory death, or pyroptosis (5), both of which play important roles in restricting microbial infection (6).The physiological function of most NLRs is not established, and only very few NLRs have defined ligands and stimulation signals. The NLRC4 inflammasome senses a wide spectrum of bacterial infections, including Legionella pneumophila, Salmonella typhimurium, Pseudomonas aeruginosa, enteropathogenic Escherichia coli (EPEC), and Shigella flexneri. NLRC4-dependent IL-1β production by intestinal phagocytes can discriminate pathogenic from commensal bacteria, contributing to immune defense against enteric bacterial infections (7). The NLRC4 inflammasome senses cytosolic flagellin as well as the rod component of bacterial type III secretion system (T3SS) (8–10). T3SS translocates effector proteins into host cells and is a general virulence mechanism for many Gram-negative pathogens (11).How does NLRC4 sense the two different bacterial molecules? Recent identification of the NAIP family of inflammasome receptors provides significant insights into this question (12, 13). NAIPs are a family of NLRs with seven paralogs in mice (NAIP1–7) but only one family member in humans (hNAIP). NAIP5/6 and NAIP2 bind directly to bacterial flagellin and T3SS rod components, respectively (12, 13), mediating caspase-1 activation through direct interaction with NLRC4 (13). Intriguingly, the NLRC4 inflammasome in human U937 monocytes does not respond to flagellin and T3SS rod protein, but instead is activated by T3SS needle protein CprI in Chromobacterium violaceum infection (13).Here we report that T3SS needle proteins can activate NLRC4 inflammasome in both human and mouse macrophages, and identify hNAIP and its mouse ortholog NAIP1 as responsible for recognizing cytosolic T3SS needle proteins. Recognition of the needle protein by hNAIP/NAIP1 stimulates formation of the large hetero-oligomeric hNAIP/NAIP1-NLRC4 inflammasome complex in 293T cell reconstitution. Further profiling of the inflammasome-stimulation activities of flagellin and T3SS rod and needle proteins from five bacterial pathogens reveals that each NAIP-bacterial ligand pair contributes differently to NLRC4-mediated innate immune detection of a particular bacterial infection. This extensive profiling also reveals a dominant role of NAIP1 recognition of T3SS needle protein in inflammasome detection of S. flexneri infection.     

Flagellin-induced NLRC4 phosphorylation primes the inflammasome for activation by NAIP5

The Nlrc4 inflammasome contributes to immunity against intracellular pathogens that express flagellin and type III secretion systems, and activating mutations in NLRC4 cause autoinflammation in patients. Both Naip5 and phosphorylation of Nlrc4 at Ser533 are required for flagellin-induced inflammasome activation, but how these events converge upon inflammasome activation is not known. Here, we showed that Nlrc4 phosphorylation occurs independently of Naip5 detection of flagellin because Naip5 deletion in macrophages abolished caspase-1 activation, interleukin (IL)-1β secretion, and pyroptosis, but not Nlrc4 phosphorylation by cytosolic flagellin of Salmonella Typhimurium and Yersinia enterocolitica. ASC speck formation and caspase-1 expression also were dispensable for Nlrc4 phosphorylation. Interestingly, Helicobacter pylori flagellin triggered robust Nlrc4 phosphorylation, but failed to elicit caspase-1 maturation, IL-1β secretion, and pyroptosis, suggesting that it retained Nlrc4 Ser533 phosphorylating-activity despite escaping Naip5 detection. In agreement, the flagellin D0 domain was required and sufficient for Nlrc4 phosphorylation, whereas deletion of the S. Typhimurium flagellin carboxy-terminus prevented caspase-1 maturation only. Collectively, this work suggests a biphasic activation mechanism for the Nlrc4 inflammasome in which Ser533 phosphorylation prepares Nlrc4 for subsequent activation by the flagellin sensor Naip5.Inflammasomes contribute critically to immunity and antimicrobial host defense of mammalian hosts. Their activation is tightly controlled because aberrant inflammasome signaling is harmful to the host, and results in inflammatory diseases (1, 2). Inflammasomes are a set of cytosolic multiprotein complexes that recruit and activate caspase-1, a key protease that triggers secretion of the inflammatory cytokines interleukin (IL)-1β and IL-18. In addition, caspase-1 induces pyroptosis, a proinflammatory and lytic cell death mode that contributes to pathogen clearance (3, 4). Several inflammasomes respond to a distinctive set of microbial pathogens (5). Activating mutations in the nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) member Nlrc4 were recently shown to induce autoinflammation in patients (6–8). Moreover, the inflammasome assembled by Nlrc4 is critically important for clearing a variety of bacterial infections, including Salmonella enterica serovar Typhimurium (S. Typhimurium), Shigella flexneri, Pseudomonas aeruginosa, Burkholderia thailandensis, and Legionella pneumophila (3, 9–17). These intracellularly-replicating bacteria have in common that they propel themselves with flagella (18) and/or express bacterial type III secretion systems (T3SS) to translocate effector proteins into infected host cells (19). Members of the NLR apoptosis-inhibitory protein (Naip) subfamily recognize the cytosolic presence of the building blocks of these evolutionary conserved bacterial structures, and trigger Nlrc4 to assemble an inflammasome (20–25). C57BL/6J mice express four Naip proteins, Naip1, -2, -5, and -6, which are expressed from a multigene cluster located on chromosome 13qD1 (26). Mouse Naip1 and human NAIP bind T3SS needle proteins, Naip2 interacts with the T3SS basal rod component PrgJ, and Naip5 and Naip6 recognize flagellin (20, 22–25).In addition to these Naip sensors, recent work showed that phosphorylation of Nlrc4 at Ser533 is critical for activation of the Nlrc4 inflammasome following infection with S. Typhimurium and L. pneumophila, or transfection of purified S. Typhimurium flagellin (27). Reconstitution of immortalized Nlrc4^−/− macrophages with wild-type Nlrc4 restored S. Typhimurium- and L. pneumophila-induced inflammasome activation, whereas cells reconstituted with Nlrc4 S533A mutant were specifically defective in maturation of caspase-1, secretion of IL-1β, assembly of ASC (apoptosis-associated speck-like protein containing a CARD) specks and induction of pyroptosis by these pathogens (27). However, a central outstanding question is how these upstream events (i.e., bacterial recognition by Naip members and Nlrc4 phosphorylation) relate to each other. Naip binding of bacterial components may trigger Nlrc4 phosphorylation to induce inflammasome activation. Alternatively, Nlrc4 phosphorylation and Naip sensing of flagellin and T3SS may converge independently onto Nlrc4 inflammasome activation.Here, we approached this question by breeding Nlrc4^Flag/Flag mice that express Nlrc4 fused to a carboxy-terminal 3× Flag tag from both Nlrc4 alleles (27) with Naip5-deficient mice (22, 28). We found S. Typhimurium infection and cytosolic delivery of S. Typhimurium flagellin, S. Typhimurium PrgJ and Yersinia enterocolitica flagellin to induce Nlrc4 phosphorylation at Ser533 independently of Naip5. Interestingly, Helicobacter pylori (H. pylori) flagellin induced robust Nlrc4 Ser533 phosphorylation without caspase-1 activation, suggesting that Nlrc4 Ser533 phosphorylation and caspase-1 activation are molecularly decoupled. In agreement, the S. Typhimurium flagellin D0 domain was required and sufficient for Nlrc4 phosphorylation, whereas caspase-1 activation required the flagellin carboxy-terminus. Collectively, this work suggests a biphasic activation mechanism for the Nlrc4 inflammasome in which Ser533 phosphorylation primes Nlrc4 for subsequent activation by the flagellin sensor Naip5.     

Cytosolic flagellin-induced lysosomal pathway regulates inflammasome-dependent and -independent macrophage responses

NAIP5/NLRC4 (neuronal apoptosis inhibitory protein 5/nucleotide oligomerization domain-like receptor family, caspase activation recruitment domain domain-containing 4) inflammasome activation by cytosolic flagellin results in caspase-1–mediated processing and secretion of IL-1β/IL-18 and pyroptosis, an inflammatory cell death pathway. Here, we found that although NLRC4, ASC, and caspase-1 are required for IL-1β secretion in response to cytosolic flagellin, cell death, nevertheless, occurs in the absence of these molecules. Cytosolic flagellin-induced inflammasome-independent cell death is accompanied by IL-1α secretion and is temporally correlated with the restriction of Salmonella Typhimurium infection. Despite displaying some apoptotic features, this peculiar form of cell death do not require caspase activation but is regulated by a lysosomal pathway, in which cathepsin B and cathepsin D play redundant roles. Moreover, cathepsin B contributes to NAIP5/NLRC4 inflammasome-induced pyroptosis and IL-1α and IL-1β production in response to cytosolic flagellin. Together, our data describe a pathway induced by cytosolic flagellin that induces a peculiar form of cell death and regulates inflammasome-mediated effector mechanisms of macrophages.Flagellin, the monomeric subunit of flagella present in Gram-negative and Gram-positive bacteria, is one of the few protein structures that can activate both transmembrane and cytosolic pattern recognition receptors of the innate immune system. Extracellular flagellin is recognized by the transmembrane Toll-like receptor (TLR)5 (1). On the other hand, flagellin can be directly delivered into the cytosol by transport systems, such as the type III secretion system (T3SS) of Salmonella (2) and the type IV secretion system (T4SS) of Legionella (3). Once in the cytosol, flagellin is sensed by the inflammasome complex comprised of the NOD-like receptor (NLR) proteins neuronal apoptosis inhibitory protein (NAIP)5 and NLRC4 [NLR family, caspase activation recruitment domain (CARD) domain-containing 4] (2–5).Both TLR5 and NAIP5/NLRC4 receptors recognize conserved regions of flagellin. TLR5 is thought to detect a region of flagellin located in the D1 domain (6), whereas a sequence of three leucine residues that is present in the C-terminal D0 domain of flagellin is required to activate the NAIP5/NLRC4 inflammasome (7). Despite some redundant roles that are attributed to NLRC4 and NAIP5 in flagellin-mediated macrophage activation (7), a new model for NAIP5/NLRC4 inflammasome activation in response to flagellin was recently proposed (8, 9). In this model, NAIP5 acts as an immune sensor protein that specifically binds to flagellin (9). The interaction between NAIP5 and flagellin promotes the recruitment of NLRC4 through the NOD domain. The formation of this protein complex leads to the association of NLRC4 with procaspase-1 via CARD-CARD interactions. Additionally, NLRC4 can recruit the adaptor protein ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain), which also contains a CARD domain and is able to recruit and process procaspase-1.Caspase-1 activation results in the cleavage and secretion of biologically active forms of the inflammatory cytokines interleukin (IL)-1β and IL-18 (10) and the induction of a form of cell death named pyroptosis (11). The activation of caspase-1 in response to cytosolic flagellin by the NAIP5/NLRC4 inflammasome complex can also induce other effector mechanisms to restrict infections, such as caspase-7–dependent phagosome maturation (4, 12) and the activation of inducible nitric oxide synthase (iNOS) by macrophages (13). Both of these effector mechanisms lead to the inhibition of Legionella pneumophila replication. Importantly, caspase-1–induced IL-1β and IL-18 are not involved in phagosome maturation (4, 12), induction of pyroptosis (14), or iNOS activation (13), suggesting that caspase-1 mediates independent effects that cooperate to clear infections.Although the NAIP5/NLRC4 inflammasome complex is involved in the control of many bacterial infections, such as infection with Salmonella Typhimurium (2, 5), Shigella flexneri (15), Pseudomonas aeruginosa (16, 17), L. pneumophila (3, 4), and Listeria monocytogenes (18), the precise effector mechanism mediated by these receptors is not completely understood. Among the NAIP5/NLRC4 inflammasome-mediated effector mechanisms that have been implicated with intracellular bacterial replication control, pyroptosis has received great attention.Pyroptosis has been described as a programmed cell death pathway that uniquely depends on caspase-1 (19). Recently, it was demonstrated that the enteric pathogenic bacteria Escherichia coli, Citrobacter rodentium, and Vibrio cholerae and the cholera toxin B subunit can trigger the activation of a noncanonical inflammasome that targets caspase-11 (also known as caspase-4 in humans and related to caspase-1) (20). These stimuli induce cell death in a caspase-11–dependent fashion, but the process is not dependent on ASC, NLRC4, or caspase-1. Interestingly, this process of cell death (also named pyroptosis) is accompanied by the secretion of IL-1α but not by the secretion of IL-1β (which requires caspase-1). Importantly, the 129 mouse strain that was used to generate the first caspase-1^−/− mutants (21, 22) harbors a mutation in the caspase-11 locus that impairs caspase-11 function. Because of the close proximity in the genome between the caspase-1 and caspase-11 genes, the two proteins cannot be segregated by recombination. Therefore, these caspase-1^−/− mice are also defective for caspase-11 (20).Importantly, although pyroptosis is regulated by caspase activation, similarly to apoptosis, inhibition of or genetic deficiency in apoptotic caspase does not rescue cells from pyroptosis (11, 23). In addition, pyroptosis and apoptosis provide distinct outcomes for the immune response, which may be explained by the different morphological and biochemical changes that are observed in cells undergoing these forms of cell death (24, 25). Activation of caspase-1/11 results in the rapid formation of pores in the plasma membrane that dissipate cellular ionic gradients. This process allows the influx of water into the cells, resulting in cell swelling, osmotic lysis, and the release of intracellular contents (25, 26). The loss of plasma membrane integrity and the secretion of inflammatory mediators during pyroptosis, including IL-1β and IL-18, results in the induction of a strong inflammatory response (27). The inflammatory milieu produced by pyroptosis could result in the recruitment of effector cells to the site of infection as a mechanism of pathogen clearance. Recently, it was demonstrated that the ectopic expression of the Salmonella flagellin protein FliC during the intracellular phase of infection triggers pyroptosis of infected cells in vivo (14). The bacteria released by the pyroptotic macrophages were controlled by infiltrating neutrophils through a reactive oxygen species-dependent mechanism.Despite the evidence implicating pyroptosis as an important host defense mechanism to clear intracellular pathogens, the molecular regulation of pyroptosis is poorly understood. Here, we analyzed the regulation of macrophage death using purified flagellin as a single, death-inducing stimulus. Our data demonstrate that cytosolic flagellin is able to induce cell death in the absence of caspase-1/11. Although displaying some apoptotic features, such as cell shrinkage and the formation of membrane blebs, cytosolic flagellin-induced caspase-1/11–independent cell death does not require apoptotic caspases but depends on lysosomal events. Similar to pyroptosis, cytosolic flagellin-induced caspase-1/11–independent cell death results in the release of intracellular inflammatory contents. Caspase-1/11–independent cell death also contributes to the control of Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) infection by macrophages, supporting the existence of an effector mechanism important to restrict bacterial infection. Finally, our data provide evidences that lysosomal cathepsins also regulate IL-1β secretion and pyroptosis in response to cytosolic flagellin. Taken together, our results suggests lysosome events as a central regulator of both inflammasome-dependent and inflammasome-independent macrophage responses induced by cytosolic flagellin.     

Caspase-1 autoproteolysis is differentially required for NLRP1b and NLRP3 inflammasome function

Inflammasomes are caspase-1–activating multiprotein complexes. The mouse nucleotide-binding domain and leucine rich repeat pyrin containing 1b (NLRP1b) inflammasome was identified as the sensor of Bacillus anthracis lethal toxin (LT) in mouse macrophages from sensitive strains such as BALB/c. Upon exposure to LT, the NLRP1b inflammasome activates caspase-1 to produce mature IL-1β and induce pyroptosis. Both processes are believed to depend on autoproteolysed caspase-1. In contrast to human NLRP1, mouse NLRP1b lacks an N-terminal pyrin domain (PYD), indicating that the assembly of the NLRP1b inflammasome does not require the adaptor apoptosis-associated speck-like protein containing a CARD (ASC). LT-induced NLRP1b inflammasome activation was shown to be impaired upon inhibition of potassium efflux, which is known to play a major role in NLRP3 inflammasome formation and ASC dimerization. We investigated whether NLRP3 and/or ASC were required for caspase-1 activation upon LT stimulation in the BALB/c background. The NLRP1b inflammasome activation was assessed in both macrophages and dendritic cells lacking either ASC or NLRP3. Upon LT treatment, the absence of NLRP3 did not alter the NLRP1b inflammasome activity. Surprisingly, the absence of ASC resulted in IL-1β cleavage and pyroptosis, despite the absence of caspase-1 autoprocessing activity. By reconstituting caspase-1/caspase-11^−/− cells with a noncleavable or catalytically inactive mutant version of caspase-1, we directly demonstrated that noncleavable caspase-1 is fully active in response to the NLRP1b activator LT, whereas it is nonfunctional in response to the NLRP3 activator nigericin. Taken together, these results establish variable requirements for caspase-1 cleavage depending on the pathogen and the responding NLR.Anthrax is a zoonotic disease caused by the Gram-positive bacterium Bacillus anthracis. B. anthracis provokes a shock-like syndrome that can prove fatal to the host (1) and has recently gained notoriety as a potential bioterrorism agent. Anthrax pathogenicity relies on its ability to secrete three virulence proteins, which combine with each other to form two toxins. The protective antigen (PA) combines with the edema factor (EF) to form the edema toxin (2, 3). EF is an adenylate cyclase that causes edema of the infected tissue. The binary combination of PA with lethal factor (LF) gives rise to the most virulent factor, called lethal toxin (LT), responsible for the systemic symptoms and death of the infected animal. To escape the host immune response, LT impairs the host innate immunity by killing macrophages (4–6). The PA protein interacts with LF and binds to cell surface receptors, enabling endocytosis of the LT complex. In the acidic compartment, PA forms pores allowing the delivery of LF to the cytosol. LF is a zinc metalloprotease that was shown to cleave the N-terminal region of many MAP kinase kinases and to induce apoptosis of macrophages. LT also triggers pyroptosis through the formation of a caspase-1–activating platform, named “inflammasome” (6–8).Inflammasomes are multiprotein complexes of the innate immune response that control caspase-1 activity and pro–IL-1β and pro–IL-18 maturation. Most inflammasomes are composed of specific cytosolic pathogen recognition receptors (PRRs), as well as the apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (CARD) (ASC) adaptor protein that enables the recruitment and activation of the caspase-1 protease. Once caspase-1 is oligomerized within an inflammasome platform, the enzyme undergoes autoproteolysis to form heterodimers of active caspase-1 (9–12). In the mouse, at least five distinct inflammasomes have been described, distinguished by the PRR that induces the complex formation. The PRRs capable of participating in inflammasome platform formation are either members of the nod-like receptor (NLR) family (e.g., NLRP1, NLRP3, or NLRC4) or of the PYrin and HIN (PYHIN) family (e.g., AIM2) (13, 14). ASC is composed of a pyrin domain (PYD) and a caspase activation and recruitment domain (CARD). ASC interacts with a PYD-containing PRR via its PYD domain and recruits the CARD domain of caspase-1 via its CARD domain. Thus, ASC is essential to the formation of the inflammasome by receptors such as NLRP3 or AIM2 (15–18). However, its presence is dispensable for NLRC4, which contains a CARD in place of a PYD, allowing direct interaction with the CARD domain of caspase-1 (19, 20).Past studies have determined that certain mouse strains are more sensitive than others to LT cytotoxicity, and genetic studies identified NLRP1b as the factor conferring mouse strain susceptibility to anthrax LT (21). The mouse genome contains three different NLRP1 isoforms (a, b, and c) and a functional NLRP1b was found to be expressed by the mouse strains sensitive to LT (e.g., BALB/c or 129 background). Expression of NLRP1b was shown to mediate IL-1β release and caspase-1–mediated cell death in response to LT (7, 21, 22). Mouse NLRP1b differs structurally from human NLRP1 in that it lacks the N-terminal PYD (23). The absence of the PYD suggests that NLRP1b can directly engage caspase-1 without a requirement for ASC. However, studies dissecting the mechanism of NLRC4 inflammasome activation demonstrated that ASC is required for the amplification of caspase-1 autoprocessing and IL-1β secretion but not for pyroptosis (19, 20). Cell lysis mediated by LT was shown to be dependent on sodium and potassium fluxes (24), and high extracellular potassium inhibited IL-1β secretion upon LT treatment, suggesting a role for the NLRP3 inflammasome in LT sensing (22, 25). Therefore, we investigated whether NLRP3 and/or ASC were required for caspase-1 activation in response to LT. The NLRP3, ASC, and caspase-1 mouse knockout strains were backcrossed into the BALB/c background and the response of macrophages and dendritic cells (DCs) to LT intoxication was studied. Our data reveal that (i) in response to LT, ASC is dispensable for caspase-1 activation, but uncleavable caspase-1 is fully active; and (ii) upon activation of the NLRP3 inflammasome, uncleavable caspase-1 is inactive.     

Human caspase-4 mediates noncanonical inflammasome activation against gram-negative bacterial pathogens

Inflammasomes are critical for host defense against bacterial pathogens. In murine macrophages infected by gram-negative bacteria, the canonical inflammasome activates caspase-1 to mediate pyroptotic cell death and release of IL-1 family cytokines. Additionally, a noncanonical inflammasome controlled by caspase-11 induces cell death and IL-1 release. However, humans do not encode caspase-11. Instead, humans encode two putative orthologs: caspase-4 and caspase-5. Whether either ortholog functions similar to caspase-11 is poorly defined. Therefore, we sought to define the inflammatory caspases in primary human macrophages that regulate inflammasome responses to gram-negative bacteria. We find that human macrophages activate inflammasomes specifically in response to diverse gram-negative bacterial pathogens that introduce bacterial products into the host cytosol using specialized secretion systems. In primary human macrophages, IL-1β secretion requires the caspase-1 inflammasome, whereas IL-1α release and cell death are caspase-1–independent. Instead, caspase-4 mediates IL-1α release and cell death. Our findings implicate human caspase-4 as a critical regulator of noncanonical inflammasome activation that initiates defense against bacterial pathogens in primary human macrophages.Pattern recognition receptors (PRRs) of the innate immune system are critical for promoting defense against bacterial pathogens (1). Cytosolic PRRs are key for discriminating between pathogenic and nonpathogenic bacteria, because many pathogens access the host cytosol, a compartment where microbial products are typically not found (2). Cytosolic PRRs respond to patterns of pathogenesis that are often associated with virulent bacteria, such as the use of pore-forming toxins or injection of effector molecules through specialized secretion systems (3). A subset of cytosolic PRRs induces the formation of multiprotein complexes known as inflammasomes (4). In mice, the canonical inflammasome activates caspase-1, an inflammatory caspase that mediates cell death and IL-1 family cytokine secretion (5, 6). Additionally, the noncanonical inflammasome activates caspase-11 in response to many gram-negative bacteria (7–14). The canonical and noncanonical inflammasomes differentially regulate release of IL-1α and IL-1β (7). Caspase-11 mediates LPS-induced septic shock in mice (7, 15), and caspase-11 responds to cytoplasmic LPS independent of Toll-like receptor 4 (16, 17).In addition to its pathologic role in septic shock, the noncanonical inflammasome is critical for host defense in mice (11, 18). However, in humans, it is unclear whether an analogous noncanonical inflammasome exists. Whereas mice encode caspase-11, humans encode two putative functional orthologs: caspase-4 and caspase-5 (19–21). All three inflammatory caspases bind directly to LPS in vitro (22). In murine macrophages, caspase-1 and caspase-11 have both distinct and overlapping roles in the release of IL-1α and IL-1β and the induction of cell death (7). However, the precise role of the human inflammatory caspases in the context of infection by bacterial pathogens remains unclear.To elucidate how human inflammasome activation is regulated, we investigated the contribution of inflammatory caspases to the response against gram-negative bacterial pathogens in human macrophages. Here, we show that both canonical caspase-1–dependent and noncanonical caspase-1–independent inflammasomes are activated in primary human macrophages and that caspase-4 mediates caspase-1–independent inflammasome responses against several bacterial pathogens, including Legionella pneumophila, Yersinia pseudotuberculosis, and Salmonella enterica serovar Typhimurium (S. Typhimurium). Importantly, noncanonical inflammasome activation in human macrophages is specific for virulent strains of these bacteria that translocate bacterial products into the host cytosol via the virulence-associated type III secretion system (T3SS) or type IV secretion system (T4SS). Thus, caspase-4 is critical for noncanonical inflammasome responses against virulent gram-negative bacteria in human macrophages.     

Shigella IpaH7.8 E3 ubiquitin ligase targets glomulin and activates inflammasomes to demolish macrophages

When nucleotide-binding oligomerization domain–like receptors (NLRs) sense cytosolic-invading bacteria, they induce the formation of inflammasomes and initiate an innate immune response. In quiescent cells, inflammasome activity is tightly regulated to prevent excess inflammation and cell death. Many bacterial pathogens provoke inflammasome activity and induce inflammatory responses, including cell death, by delivering type III secreted effectors, the rod component flagellin, and toxins. Recent studies indicated that Shigella deploy multiple mechanisms to stimulate NLR inflammasomes through type III secretion during infection. Here, we show that Shigella induces rapid macrophage cell death by delivering the invasion plasmid antigen H7.8 (IpaH7.8) enzyme 3 (E3) ubiquitin ligase effector via the type III secretion system, thereby activating the NLR family pyrin domain-containing 3 (NLRP3) and NLR family CARD domain-containing 4 (NLRC4) inflammasomes and caspase-1 and leading to macrophage cell death in an IpaH7.8 E3 ligase-dependent manner. Mice infected with Shigella possessing IpaH7.8, but not with Shigella possessing an IpaH7.8 E3 ligase-null mutant, exhibited enhanced bacterial multiplication. We defined glomulin/flagellar-associated protein 68 (GLMN) as an IpaH7.8 target involved in IpaH7.8 E3 ligase-dependent inflammasome activation. This protein originally was identified through its association with glomuvenous malformations and more recently was described as a member of a Cullin ring ligase inhibitor. Modifying GLMN levels through overexpression or knockdown led to reduced or augmented inflammasome activation, respectively. Macrophages stimulated with lipopolysaccharide/ATP induced GLMN puncta that localized with the active form of caspase-1. Macrophages from GLMN^+/− mice were more responsive to inflammasome activation than those from GLMN^+/+ mice. Together, these results highlight a unique bacterial adaptation that hijacks inflammasome activation via interactions between IpaH7.8 and GLMN.Inflammasome activation is a key defense mechanism against bacterial infection that induces innate immune responses such as caspase-1 activation and inflammatory cell death (1–3). Although the mechanisms through which various bacterial activities promote infection remain incompletely understood, some bacterial pathogens stimulate inflammasome activity by delivering cytotoxins, type III secretion (T3SS)-mediated effectors, T3SS components, flagellin, or cytotoxins to the host cell membrane and cytoplasm. These foreign components modify the host cell-surface architecture, induce membrane damage, subvert cell signaling, reorganize the actin cytoskeleton, and alter cell physiology (4) through interactions with various cytoplasmic receptors, e.g., nucleotide-binding oligomerization domain–like receptors (NLRs)—including NLRP1, NLR family CARD domain-containing 4 (NLRC4), NLR family pyrin domain-containing 3 (NLRP3), AIM2, IFI16, and RIG-1—as pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs) (2, 3, 5). Upon recognition of these PAMPs and DAMPs, NLRs induce the assembly of inflammasomes, which are composed of NLR, apoptosis-associated speck-like protein (ASC), and inflammatory caspases such as caspase-1. Inflammasome assembly ultimately results in the extracellular release of IL-1β and IL-18 and induces inflammatory cell death (called “pyroptosis”) (6). For example, NLRP3 senses membrane rupture that occurs during infection with Listeria monocytogenes, Shigella, Salmonella typhimurium, Staphylococcus aureus, Neisseria gonorrhoeae, and Chlamydia spp. and upon exposure to bacterial pore-forming toxins, leading to caspase-1 activation (7–10). NLRC4 detects L. monocytogenes and S. typhimurium infection and stimulates caspase-1 activation (11–14). NLRC4 also senses flagellin and the T3SS rod components of Legionella pneumophila, Pseudomonas aeruginosa, Shigella, and S. typhimurium (11, 15–20) and the T3SS needle components of Chromobacterium violaceum, S. typhimurium, enterohemorrhagic Escherichia coli, Burkholderia thailandensis, and Shigella (21). Therefore, NLR inflammasomes act as major cytoplasmic pattern-recognition receptors and as central platforms that transmit alarm signals to a variety of downstream innate immune systems.Some bacterial pathogens, such as S. typhimurium (22) and Yersinia pseudotuberculosis (23–25), can induce macrophage death after they have fully replicated, promoting the egress of bacteria from their replicative compartments and the subsequent dissemination of bacteria into new host cells. This causal relationship suggests that these pathogens may benefit from and exert control over host cell death and the inflammatory response. In the case of Shigella, the bacteria rapidly induce macrophage cell death at early stages of infection, which is accompanied by NLR inflammasome activation and inflammatory cell death through a T3SS-dependent mechanism (19, 22). Previous studies indicated that during replication in macrophages, LPS, peptidoglycan, and T3SS rod or needle components of Shigella are recognized by the NLRC4 and NLRP3 inflammasomes (8, 19–21). Interestingly, the mode through which NLRs recognize Shigella infections seems to vary across different infection conditions. At a low infectious dose [e.g., a multiplicity of infection (MOI) of 10–25], bacteria induce rapid NLRC4–caspase-1–dependent pyroptosis at 2–3 h postinfection through the recognition of the T3SS components or some uncharacterized T3SS-delivered substance(s) (19, 22). However, at a high infectious dose (e.g., an MOI over 50) and at later time points (6 h postinfection), the bacteria induce NLRP3-dependent but caspase-1–independent necrosis-like cell death with inflammation (called “pyronecrosis”) (8). Because pyroptosis results in the release of intracellular contents, including proinflammatory cytokines and chemokines, and because, in the case of Shigella, macrophage death is a prerequisite for the subsequent infection of surrounding epithelial cells (19, 26), it remains unclear whether Shigella-mediated rapid cell death is beneficial to the pathogen or to the host. Nevertheless, these studies strongly suggest that the bacteria deploy multiple mechanisms to manipulate macrophage cell-death pathways in a T3SS-dependent manner.Shigella flexneri, e.g., the YSH6000 strain, possesses three invasion plasmid antigen H (ipaH) genes, ipaH9.8, ipaH7.8, and ipaH4.5, on a large virulence plasmid (27, 28). These IpaH proteins, which originally were identified in the S. flexneri M90T strain (29, 30), recently were found to act as enzyme 3 (E3) ubiquitin ligases (31) and were thus named “novel E3 ligases” (32). The ipaH cognate genes are distributed among various Gram-negative bacterial pathogens, including Shigella, Salmonella, Yersinia, Edwardsiella ictaluri, Bradyrhizobium japonica, Rhizobium sp. strain NGR234, Pseudomonas putida, Pseudomonas entomophila, Pseudomonas fluorescens, and Pseudomonas syringae (31). IpaH protein family members share structural and functional similarity; they are composed of an N-terminal leucine-rich repeat (LRR) and a highly conserved C-terminal region (CTR) (33, 34). The conserved CTR contains a Cys residue, which is critical for E3 ubiquitin ligase activity (31, 35, 36). Each of the IpaH family effectors characterized to date (e.g., Shigella IpaH9.8 and IpaH2077, Salmonella SlrP, SspH1, and SspH2, Yersinia YopM, and Rhizobium Y4fR and BIpM) has distinct host protein targets in different host cell types. Some act as effectors to attenuate host inflammatory responses, whereas others modulate host defense responses in plants (37, 38). The existence of multiple effectors with E3 ligase activity suggests that an array of E3 ligases is required to promote bacterial infection and antagonize host innate defense responses.Fernandez-Prada et al. (39) previously reported that Shigella lacking the ipaH7.8 gene are less capable than the WT strain of escaping the phagocytic vacuole of macrophages and that Shigella infection of macrophages induces apoptotic-like cell death. Paetzold et al. (40) subsequently showed that Shigella lacking the ipaH7.8 gene had no effect on phagosome escape compared with the WT strain, but bacterial-induced cytotoxicity was low compared with that of the WT strain. Although the biological significance of IpaH7.8 as an E3 ubiquitin ligase during Shigella infection remains to be elucidated, these studies suggested that IpaH7.8 is involved in inducing macrophage cell death.In this context we wished to clarify the pathological role of IpaH7.8 as an E3 ubiquitin ligase in Shigella infection of macrophages and the modality of cell death. Here we provide evidence that IpaH7.8 potentiates macrophage killing in an IpaH7.8 E3 ligase-dependent manner, in which E3 ligase activity triggers NLR inflammasome-mediated macrophage cell death by targeting glomulin/FAP68 (GLMN); this activity ultimately appears to benefit the pathogen.     

Hemolysis-induced lethality involves inflammasome activation by heme

The increase of extracellular heme is a hallmark of hemolysis or extensive cell damage. Heme has prooxidant, cytotoxic, and inflammatory effects, playing a central role in the pathogenesis of malaria, sepsis, and sickle cell disease. However, the mechanisms by which heme is sensed by innate immune cells contributing to these diseases are not fully characterized. We found that heme, but not porphyrins without iron, activated LPS-primed macrophages promoting the processing of IL-1β dependent on nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3). The activation of NLRP3 by heme required spleen tyrosine kinase, NADPH oxidase-2, mitochondrial reactive oxygen species, and K⁺ efflux, whereas it was independent of heme internalization, lysosomal damage, ATP release, the purinergic receptor P2X7, and cell death. Importantly, our results indicated the participation of macrophages, NLRP3 inflammasome components, and IL-1R in the lethality caused by sterile hemolysis. Thus, understanding the molecular pathways affected by heme in innate immune cells might prove useful to identify new therapeutic targets for diseases that have heme release.Hemolysis, hemorrhage, and rhabdomyolysis cause the release of large amounts of hemoproteins to the extracellular space, which, once oxidized, release the heme moiety, a potentially harmful molecule due to its prooxidant, cytotoxic, and inflammatory effects (1, 2). Scavenging proteins such as haptoglobin and hemopexin bind hemoglobin and heme, respectively, promoting their clearance from the circulation and delivery to cells involved with heme catabolism. Heme oxygenase cleaves heme and generates equimolar amounts of biliverdin, carbon monoxide (CO) and iron (2). Studies using mice deficient for haptoglobin (Hp), hemopexin (Hx), and heme oxygenase 1 (HO-1) demonstrate the importance of these proteins in controlling the deleterious effects of heme. Both Hp^−/− and Hx^−/− mice have increased renal damage after acute hemolysis induced by phenyhydrazine (Phz) compared with wild-type mice (3, 4). Mice lacking both proteins present splenomegaly and liver inflammation composed of several foci with leukocyte infiltration after intravascular hemolysis (5). Hx protect mice against heme-induced endothelial damage improving liver and cardiovascular function (6–8). Lack of heme oxygenase 1 (Hmox1^−/−) causes iron overload, increased cell death, and tissue inflammation under basal conditions and upon inflammatory stimuli (9–15). This salutary effect of HO-1 has been attributed to its effect of reducing heme amounts as well as generating the cytoprotective molecules, biliverdin and CO.Heme induces neutrophil migration in vivo and in vitro (16, 17), inhibits neutrophil apoptosis (18), triggers cytokine and lipid mediator production by macrophages (19, 20), and increases the expression of adhesion molecules and tissue factor on endothelial cells (21–23). Heme cooperates with TNF, causing hepatocyte apoptosis in a mechanism dependent on reactive oxygen species (ROS) generation (12). Whereas heme-induced TNF production depends on functional toll-like receptor 4 (TLR4), ROS generation in response to heme is TLR4 independent (19). We recently observed that heme triggers receptor-interacting protein (RIP)1/3-dependent macrophage-programmed necrosis through the induction of TNF and ROS (15). The highly unstable nature of iron is considered critical for the ability of heme to generate ROS and to cause inflammation. ROS generated by heme has been mainly attributed to the Fenton reaction. However, recent studies suggest that heme can generate ROS through multiple sources, including NADPH oxidase and mitochondria (22, 24–27).Heme causes inflammation in sterile and infectious conditions, contributing to the pathogenesis of hemolytic diseases, subarachnoid hemorrhage, malaria, and sepsis (11, 13, 24, 28), but the mechanisms by which heme operates in different conditions are not completely understood. Blocking the prooxidant effects of heme protects cells from death and prevents tissue damage and lethality in models of malaria and sepsis (12, 13, 15). Importantly, two recent studies demonstrated the pathogenic role of heme-induced TLR4 activation in a mouse model of sickle cell disease (29, 30). These results highlight the great potential of understanding the molecular mechanisms of heme-induced inflammation and cell death as a way to identify new therapeutic targets.Hemolysis and heme synergize with microbial molecules for the induction of inflammatory cytokine production and inflammation in a mechanism dependent on ROS and Syk (24). Processing of pro–IL-1β is dependent on caspase-1 activity, requiring assembly of the inflammasome, a cytosolic multiprotein complex composed of a NOD-like receptor, the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), and caspase-1 (31–33). The most extensively studied inflammasome is the nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3). NLRP3 and pro–IL-1β expression are increased in innate immune cells primed with NF-κB inducers such as TLR agonists and TNF (34, 35). NLRP3 inflammasome is activated by several structurally nonrelated stimuli, such as endogenous and microbial molecules, pore-forming toxins, and particulate matter (34, 35). The activation of NLRP3 involves K⁺ efflux, increase of ROS and Syk phosphorylation. Importantly, critical roles of NLRP3 have been demonstrated in a vast number of diseases (34, 36). We hypothesize that heme causes the activation of the inflammasome and secretion of IL-1β. Here we found that heme triggered the processing and secretion of IL-1β dependently on NLRP3 inflammasome in vitro and in vivo. The activation of NLRP3 by heme was dependent on Syk, ROS, and K⁺ efflux, but independent of lysosomal leakage, ATP release, or cell death. Finally, our results indicated the critical role of macrophages, the NLRP3 inflammasome, and IL-1R to the lethality caused by sterile hemolysis.     

GsdmD p30 elicited by caspase-11 during pyroptosis forms pores in membranes

Gasdermin-D (GsdmD) is a critical mediator of innate immune defense because its cleavage by the inflammatory caspases 1, 4, 5, and 11 yields an N-terminal p30 fragment that induces pyroptosis, a death program important for the elimination of intracellular bacteria. Precisely how GsdmD p30 triggers pyroptosis has not been established. Here we show that human GsdmD p30 forms functional pores within membranes. When liberated from the corresponding C-terminal GsdmD p20 fragment in the presence of liposomes, GsdmD p30 localized to the lipid bilayer, whereas p20 remained in the aqueous environment. Within liposomes, p30 existed as higher-order oligomers and formed ring-like structures that were visualized by negative stain electron microscopy. These structures appeared within minutes of GsdmD cleavage and released Ca²⁺ from preloaded liposomes. Consistent with GsdmD p30 favoring association with membranes, p30 was only detected in the membrane-containing fraction of immortalized macrophages after caspase-11 activation by lipopolysaccharide. We found that the mouse I105N/human I104N mutation, which has been shown to prevent macrophage pyroptosis, attenuated both cell killing by p30 in a 293T transient overexpression system and membrane permeabilization in vitro, suggesting that the mutants are actually hypomorphs, but must be above certain concentration to exhibit activity. Collectively, our data suggest that GsdmD p30 kills cells by forming pores that compromise the integrity of the cell membrane.Pyroptosis is an inflammatory form of programmed cell death that occurs in response to microbial products in the cytoplasm or to cellular perturbations caused by diverse stimuli, including crystalline substances, toxins, and extracellular ATP (1, 2). Pyroptosis plays a critical role in the clearance of intracellular bacteria (3), but may also contribute to autoinflammatory and autoimmune disease pathology. Mechanistically, pyroptosis occurs when cytosolic nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), including NLRP1, NLRP3, and NLRC4, or the pyrin domain-containing protein AIM2, nucleate a canonical inflammasome complex that activates the protease caspase-1 (2). Alternatively, intracellular lipopolysaccharide (LPS) from Gram-negative bacteria can trigger noncanonical activation of mouse caspase-11 and human caspases 4 and 5 (4–7). Caspases 1, 4, 5, and 11 can each cleave Gasdermin-D (GsdmD) to mediate pyroptotic cell death (8, 9). It is the N-terminal p30 fragment of GsdmD that is cytotoxic to cells, but precisely how it kills cells is unknown.Here we show that the human GsdmD p30 fragment liberated by active caspase-11 forms ring-like structures within membranes that function as pores. Therefore, we propose p30 kills cells by directly compromising the integrity of cellular membranes. We also show that the GsdmD I105N mutant that was unable to mediate macrophage pyroptosis (9) is hypomorphic at cell killing in a transient overexpression system and at liposome permeabilization in vitro—conditions where p30 concentration favors oligomerization.     

Salmonella exploits NLRP12-dependent innate immune signaling to suppress host defenses during infection

The nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain containing 12 (NLRP12) plays a protective role in intestinal inflammation and carcinogenesis, but the physiological function of this NLR during microbial infection is largely unexplored. Salmonella enterica serovar Typhimurium (S. typhimurium) is a leading cause of food poisoning worldwide. Here, we show that NLRP12-deficient mice were highly resistant to S. typhimurium infection. Salmonella-infected macrophages induced NLRP12-dependent inhibition of NF-κB and ERK activation by suppressing phosphorylation of IκBα and ERK. NLRP12-mediated down-regulation of proinflammatory and antimicrobial molecules prevented efficient clearance of bacterial burden, highlighting a role for NLRP12 as a negative regulator of innate immune signaling during salmonellosis. These results underscore a signaling pathway defined by NLRP12-mediated dampening of host immune defenses that could be exploited by S. typhimurium to persist and survive in the host.The nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family consists of a large number of intracellular pathogen recognition receptors that function as sensors of microbial-derived and danger-associated molecules in the cytoplasm of host cells. A subset of NLR proteins, including NLRP1, NLRP3, and NLRC4, activate caspase-1 via the formation of a cytosolic multiprotein complex termed the inflammasome (1). These inflammasome-forming NLRs mediate processing of the proinflammatory cytokines pro–IL-1β and pro–IL-18, which are then secreted by the cell. The non–inflammasome-forming members of the NLR family contribute to regulation of other key inflammatory pathways. For example, NOD1 and NOD2 activate NF-κB and MAPK pathways (2–5), whereas NLRP6, NLRC3, NLRC5, and NLRX1 have been demonstrated to regulate inflammation negatively (6–9).NLRP12 (NALP12, MONARCH-1, or PYPAF7) is a poorly characterized member of the NLR family. It has a tripartite domain structure, which consists of an N-terminal PYRIN domain, a central nucleotide binding site domain, and a C-terminal domain composed of at least 12 leucine-rich repeat motifs (10). In humans, NLRP12 is expressed in peripheral blood leukocytes, including granulocytes, eosinophils, monocytes, and dendritic cells (DCs) (10, 11). Similarly, mouse NLRP12 is highly expressed in bone marrow neutrophils and granulocytes, macrophages, and DCs (12, 13). Genetic studies in humans have shown that mutations in the NLRP12 gene are associated with periodic fever syndromes and atopic dermatitis (14–16). More recent studies have demonstrated that NLRP12 has both inflammasome-dependent and inflammasome-independent roles in health and disease. Our laboratory and others have previously reported that NLRP12 mediates protection against colon inflammation and tumorigenesis in vivo by negatively regulating inflammatory responses (12, 17).Recent studies have revealed a potential role for NLRP12 during infectious diseases. Vladimer et al. (18) reported that Nlrp12^−/− mice are hypersusceptible to Yersinia pestis infection, whereby NLRP12 is required to drive caspase-1 activation and IL-1β and IL-18 release. Another study found that WT and Nlrp12^−/− mice exhibit similar host innate responses in lung infections induced by Mycobacterium tuberculosis or Klebsiella pneumoniae (13). However, in vitro studies reported that a synthetic analog cord factor, trehalose-6,6-dimycolate (TDP), from M. tuberculosis and LPS from K. pneumoniae induced substantially elevated levels of TNF-α and IL-6 in Nlrp12^−/− bone marrow-derived DCs compared with their WT counterpart, although levels of secreted IL-1β were not changed (13). These results suggest that unlike the case in Yersinia infection, NLRP12 does not contribute to inflammasome-mediated protection against M. tuberculosis and K. pneumoniae infections. Overall, the physiological and functional relevance of NLRP12 in the host defense against infectious diseases is not fully understood.Salmonella enterica serovar Typhimurium (S. typhimurium) is a Gram-negative intracellular pathogen, and one of the most prevalent etiological agents of gastroenteritis worldwide. Salmonella infection accounts for 93.8 million cases of gastroenteritis annually in the world and is a leading cause of death among bacterial foodborne pathogens in the United States (19, 20). Previous studies have found that members of the Toll-like receptor (TLR) family, especially TLR4, are critical for the recognition and clearance of S. typhimurium (21, 22). One consequence of Salmonella-induced TLR activation is the production of inflammatory cytokines and antimicrobial compounds, including pro–IL-1β, pro–IL-18, IFN-γ, TNF-α, and reactive oxygen species, which are critical mediators for the control of bacterial growth in host tissues (23). In addition to TLR-mediated host responses, certain members of the NLR family, including NLRC4 and NLRP3, initiate inflammasome formation to drive processing and release of IL-1β and IL-18 following Salmonella infection (24, 25). Although the precise signals that trigger NLRP3 activation during Salmonella infection are unknown, NLRC4 is activated by NAIPs, a subset of receptors within the NLR family that detect Salmonella flagellin (mouse NAIP5 and NAIP6) or certain rod (mouse NAIP2) or needle (human NAIP and mouse NAIP1) proteins associated with the Salmonella type III secretion system (26–30). Nevertheless, the functional relevance of NLRP12 in response to Salmonella infection is unknown.Here, we show that NLRP12 negatively regulates antibacterial host defense during Salmonella infection independent of inflammasomes. NLRP12 inhibited TLR-induced NF-κB activation by dampening phosphorylation of IκBα and ERK, consequently enhancing intracellular bacterial survival. Together, our work unveiled an NLRP12-dependent innate immune pathway that may be strategically exploited by S. typhimurium to persist and survive in the host.     

Human NLRP3 inflammasome senses multiple types of bacterial RNAs

Inflammasomes are multiprotein platforms that activate caspase-1, which leads to the processing and secretion of the proinflammatory cytokines IL-1β and IL-18. Previous studies demonstrated that bacterial RNAs activate the nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3 (NLRP3) inflammasome in both human and murine macrophages. Interestingly, only mRNA, but neither tRNA nor rRNAs, derived from bacteria could activate the murine Nlrp3 inflammasome. Here, we report that all three types of bacterially derived RNA (mRNA, tRNA, and rRNAs) were capable of activating the NLRP3 inflammasome in human macrophages. Bacterial RNA’s 5′-end triphosphate moieties, secondary structure, and double-stranded structure were dispensable; small fragments of bacterial RNA were sufficient to activate the inflammasome. In addition, we also found that 20-guanosine ssRNA can activate the NLRP3 inflammasome in human macrophages but not in murine macrophages. Therefore, human and murine macrophages may have evolved to recognize bacterial cytosolic RNA differently during bacterial infections.The innate immune system is the first line of defense against microbial infections. Germ-line–encoded pattern-recognition receptors (PRRs) of the innate immune system recognize the presence of invariant evolutionarily conserved microbial components called “pathogen-associated molecular patterns” (1–3). In response to microbial infections, PRRs rapidly initiate signal-transduction pathways to induce type 1 IFN production, proinflammatory cytokine production, and inflammasome activation. The inflammasome is a cytosolic large caspase-1–containing multiprotein complex that enables autocatalytic activation of caspase-1. Once caspase-1 is activated, it starts to cleave prointerleukin-1β (pro–IL-1β) and prointerleukin-18 (pro–IL-18) proteolytically into bioactive IL-1β and IL-18 (4–7). The mature forms of IL-1β and IL-18 play roles in a variety of infectious and inflammatory processes.Cytosolic microbial nucleic acids are important activators of the innate immune system against both bacterial and viral infections, which induce type 1-IFN and proinflammatory cytokine responses as well as inflammasome activation. The role of microbial nucleic acids in inflammasome activation has been studied mostly in murine bone marrow-derived dendritic cells (BMDCs) or bone marrow-derived macrophages (BMDMs). AIM2 has been identified as a specific cytosolic dsDNA sensor that directly binds ASC (apoptosis-associated speck-like protein containing a carboxyl-terminal CARD-like domain) and forms inflammasome complexes in human and murine cells (8–11).Viral dsRNA was found to activate the nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3 (NLRP3) inflammasome in human and murine cells (12–15). Several groups have reported that cytosolic bacterial RNA activate the Nlrp3 inflammasome in murine macrophages (13, 16, 17). Our group also has reported that human THP-1–derived macrophages recognize cytosolic bacterial RNA and induce NLRP3 inflammasome activation (12). Bacterial RNA is composed of mRNA, tRNA, and three different sizes of rRNA (23s, 16s, and 5s). Sander et al. (18) reported that, of the different types of Escherichia coli RNA, only E. coli mRNA induced the secretion of IL-1β by murine BMDMs, but E. coli tRNA and E. coli rRNAs did not.We aimed to study (i) whether a variety of cytosolic bacterial RNAs could activate the inflammasome in human myeloid cells and (ii) what types of bacterial RNA activate the inflammasome in human and murine myeloid cells. Here, we demonstrate that a broad spectrum of cytosolic bacterial RNAs strongly induce the cleavage of caspase-1 and the secretion of IL-1β and IL-18 in human macrophages. Human macrophages can sense mRNA, tRNA, rRNAs, and small synthetic ssRNA through NLRP3, but murine macrophages can sense only the mRNA component. Bacterial RNA’s 5′-end triphosphate moieties, secondary structure, and double-stranded structure were dispensable, but small fragments of bacterial RNA were sufficient to activate the inflammasome. These findings suggest that upon bacterial infections the human and murine NLRP3 inflammasomes sense cytosolic bacterial RNAs differently.     

AIM2 and NLRC4 inflammasomes contribute with ASC to acute brain injury independently of NLRP3

Inflammation that contributes to acute cerebrovascular disease is driven by the proinflammatory cytokine interleukin-1 and is known to exacerbate resulting injury. The activity of interleukin-1 is regulated by multimolecular protein complexes called inflammasomes. There are multiple potential inflammasomes activated in diverse diseases, yet the nature of the inflammasomes involved in brain injury is currently unknown. Here, using a rodent model of stroke, we show that the NLRC4 (NLR family, CARD domain containing 4) and AIM2 (absent in melanoma 2) inflammasomes contribute to brain injury. We also show that acute ischemic brain injury is regulated by mechanisms that require ASC (apoptosis-associated speck-like protein containing a CARD), a common adaptor protein for several inflammasomes, and that the NLRP3 (NLR family, pyrin domain containing 3) inflammasome is not involved in this process. These discoveries identify the NLRC4 and AIM2 inflammasomes as potential therapeutic targets for stroke and provide new insights into how the inflammatory response is regulated after an acute injury to the brain.Proinflammatory cytokines of the interleukin-1 (IL-1) family are critical regulators of host responses to infection and orchestrate damaging inflammatory responses that occur during disease (1). One of the main mediators of damaging sterile inflammation is IL-1β, which is implicated in the etiology of many major diseases, including acute cerebrovascular disease (2). Acute cerebrovascular disease presents as a range of conditions, including devastating injuries such as subarachnoid hemorrhage (SAH) and ischemic stroke, which account for up to 10% of mortality worldwide and are the leading cause of morbidity (2). Treatments for acute stroke are limited to thrombolysis for up to 10% of all strokes, antiplatelet agents, and stroke unit care. Thus, treatment of acute cerebrovascular disease remains an area of unmet clinical need. Understanding the mechanisms regulating production of IL-1β during ischemic brain injury may lead to the identification of new therapeutic targets.IL-1β is produced by many cells, most commonly those of macrophage lineage, as a pro–IL-1β precursor. Pro–IL-1β is expressed in response to pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) that bind to pattern recognition receptors (PRRs) to up-regulate proinflammatory gene expression (3, 4). PAMPs are motifs carried by pathogens, such as bacterial endotoxin (or LPS), and DAMPs are commonly endogenous molecules released by necrosis. Pro–IL-1β is inactive and remains intracellular until a further PAMP or DAMP stimulation activates cytosolic PRRs, often of the nucleotide-binding domain and leucine-rich repeat containing receptor (NLR) family, to form large multiprotein complexes called inflammasomes (5). These complexes consist of the PRR, procaspase-1, and, depending upon the PRR, an adaptor protein called ASC, that interact via CARD and pyrin homology-binding domains (5). When the PRR senses PAMPs or DAMPs, it recruits ASC, which in turn recruits caspase-1, causing its activation. Caspase-1 then processes pro–IL-1β to a mature form that is rapidly secreted from the cell (5). The activation of caspase-1 can also cause cell death (6).A number of inflammasome-forming PRRs have been identified, including NLR family, pyrin domain containing 1 (NLRP1); NLRP3; NLRP6; NLRP7; NLRP12; NLR family, CARD domain containing 4 (NLRC4); AIM 2 (absent in melanoma 2); IFI16; and RIG-I (5). Of these inflammasomes identified to date, the best characterized, and most strongly associated with sterile inflammation, is formed by NLRP3 (7). Indeed, there are now several studies suggesting that NLRP3 inflammasomes contribute to ischemic brain injury (8, 9). However, the picture is more complicated. NLRP1 inflammasomes have been implicated in several models of brain injury (6, 10, 11), as have AIM2 inflammasomes, which are suggested to mediate pyroptotic neuronal cell death (12). There is also evidence supporting a role for caspase-1 in brain injury (13), with a selective caspase-1 inhibitor, VRT-018858, a nonpeptide, active metabolite of the prodrug pralnacasan, showing marked protection in a rat model of stroke (14). However, data for the related caspase-1 inhibitor VRT-043198 suggest that it is also an effective inhibitor of caspase-4 (15), a human ortholog of caspase-11. Caspase-11 is also implicated in ischemic brain injury (16, 17), and given that we now also know that the original caspase-1^−/− mouse is also deficient in caspase-11 (18), it is clear that caspase-11 could have a role in ischemic brain injury. Our aim here was to elucidate which inflammasomes contribute to ischemic brain injury, using mice in which specific inflammasome components are deleted (^−/−).     

Exchange protein directly activated by cAMP plays a critical role in bacterial invasion during fatal rickettsioses

Rickettsiae are responsible for some of the most devastating human infections. A high infectivity and severe illness after inhalation make some rickettsiae bioterrorism threats. We report that deletion of the exchange protein directly activated by cAMP (Epac) gene, Epac1, in mice protects them from an ordinarily lethal dose of rickettsiae. Inhibition of Epac1 suppresses bacterial adhesion and invasion. Most importantly, pharmacological inhibition of Epac1 in vivo using an Epac-specific small-molecule inhibitor, ESI-09, completely recapitulates the Epac1 knockout phenotype. ESI-09 treatment dramatically decreases the morbidity and mortality associated with fatal spotted fever rickettsiosis. Our results demonstrate that Epac1-mediated signaling represents a mechanism for host–pathogen interactions and that Epac1 is a potential target for the prevention and treatment of fatal rickettsioses.Rickettsiae are responsible for some of the most devastating human infections (1–4). It has been forecasted that temperature increases attributable to global climate change will lead to more widespread distribution of rickettsioses (5). These tick-borne diseases are caused by obligately intracellular bacteria of the genus Rickettsia, including Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF) in the United States and Latin America (2, 3), and Rickettsia conorii, the causative agent of Mediterranean spotted fever endemic to southern Europe, North Africa, and India (6). A high infectivity and severe illness after inhalation make some rickettsiae (including Rickettsia prowazekii, R. rickettsii, Rickettsia typhi, and R. conorii) bioterrorism threats (7). Although the majority of rickettsial infections can be controlled by appropriate broad-spectrum antibiotic therapy if diagnosed early, up to 20% of misdiagnosed or untreated (1, 3) and 5% of treated RMSF cases (8) result in a fatal outcome caused by acute disseminated vascular endothelial infection and damage (9). Fatality rates as high as 32% have been reported in hospitalized patients diagnosed with Mediterranean spotted fever (10). In addition, strains of R. prowazekii resistant to tetracycline and chloramphenicol have been developed in laboratories (11). Disseminated endothelial infection and endothelial barrier disruption with increased microvascular permeability are the central features of SFG rickettsioses (1, 2, 9). The molecular mechanisms involved in rickettsial infection remain incompletely elucidated (9, 12). A comprehensive understanding of rickettsial pathogenesis and the development of novel mechanism-based treatment are urgently needed.Living organisms use intricate signaling networks for sensing and responding to changes in the external environment. cAMP, a ubiquitous second messenger, is an important molecular switch that translates environmental signals into regulatory effects in cells (13). As such, a number of microbial pathogens have evolved a set of diverse virulence-enhancing strategies that exploit the cAMP-signaling pathways of their hosts (14). The intracellular functions of cAMP are predominantly mediated by the classic cAMP receptor, protein kinase A (PKA), and the more recently discovered exchange protein directly activated by cAMP (Epac) (15). Thus, far, two isoforms, Epac1 and Epac2, have been identified in humans (16, 17). Epac proteins function by responding to increased intracellular cAMP levels and activating the Ras superfamily small GTPases Ras-proximate 1 and 2 (Rap1 and Rap2). Accumulating evidence demonstrates that the cAMP/Epac1 signaling axis plays key regulatory roles in controlling various cellular functions in endothelial cells in vitro, including cell adhesion (18–21), exocytosis (22), tissue plasminogen activator expression (23), suppressor of cytokine signaling 3 (SOCS-3) induction (24–27), microtubule dynamics (28, 29), cell–cell junctions, and permeability and barrier functions (30–37). Considering the critical importance of endothelial cells in rickettsioses, we examined the functional roles of Epac1 in rickettsial pathogenesis in vivo, taking advantage of the recently generated Epac1 knockout mouse (38) and Epac-specific inhibitors (39, 40) generated from our laboratory. Our studies demonstrate that Epac1 plays a key role in rickettsial infection and represents a therapeutic target for fatal rickettsioses.     

NLRP3 deficiency protects from type 1 diabetes through the regulation of chemotaxis into the pancreatic islets

Studies in animal models and human subjects have shown that both innate and adaptive immunity contribute to the pathogenesis of type 1 diabetes (T1D). Whereas the role of TLR signaling pathways in T1D has been extensively studied, the contribution of the nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing protein (NLRP) 3 inflammasome pathway remains to be explored. In this study, we report that NLRP3 plays an important role in the development of T1D in the nonobese diabetic (NOD) mouse model. NLRP3 deficiency not only affected T-cell activation and Th1 differentiation, but also modulated pathogenic T-cell migration to the pancreatic islet. The presence of NLRP3 is critical for the expression of the chemokine receptors CCR5 and CXCR3 on T cells. More importantly, NLRP3 ablation reduced the expression of chemokine genes CCL5 and CXCL10 on pancreatic islet cells in an IRF-1–dependent manner. Our results suggest that molecules involved in chemotaxis, accompanied by the activation of the NLRP3 inflammasome, may be effective targets for the treatment of T1D.Type 1 diabetes (T1D) is a T-cell–mediated autoimmune disease characterized by the destruction of insulin-producing pancreatic beta cells in genetically predisposed individuals. Studies in animal models and human subjects have shown that both innate and adaptive immunity play a role in disease pathogenesis. Strategies targeting either T or B cells have shown some efficacy in T1D in both animal and human studies (1–4). Recently, the role of innate immunity in T1D has been increasingly appreciated. We, and others, have demonstrated that Toll-like receptor (TLR) signaling pathways are essential for the development of T1D. Nonobese diabetic (NOD) mice deficient in TLR2, TLR9, or MyD88 showed delayed disease development or were protected from diabetes (5–9). However, the development of autoimmune diabetes was accelerated in TLR4^−/− NOD mice (5–7, 10). Whereas the role of TLR signaling has been intensively studied, the contribution of the nucleotide binding domain-like receptor (NLR) signaling pathway to the pathogenesis of T1D remains to be explored.Nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing protein (NLRP) 3 is a NLR family member, together with ASC and caspase-1, forms protein complexes that are responsible for the innate immune response to pathogens and/or “danger” signals (11). Increasing evidence indicates that the NLRP3 inflammasome plays an important role in obesity and type 2 diabetes (12–14). However, little is known about the role of NLRP3 in autoimmune diabetes. Whereas the inflammasome has been extensively studied in the control of infection, only recently has the role of the NLRP inflammasome in autoimmune disease been recognized. Polymorphisms in inflammasome genes are involved in the predisposition to systemic lupus erythematosus (15). NLRP3 deficiency dramatically delayed the course and reduced severity of experimental autoimmune encephalomyelitis by suppression of Th1 and Th17 responses (16). Mice deficient in ASC, the adaptor protein of the NLRP3 inflammasome pathway, were also less susceptible to collagen-induced arthritis (17). Nevertheless, the role of the inflammasome pathway in the pathogenesis of T1D is unclear. Although caspase-1 or IL-1β deficiency did not protect NOD mice from T1D (18, 19), IL-1 blockade showed a synergistic protective effect when combined with anti-CD3 therapy for T1D in a mouse model (20). Interestingly, recent genetic association studies suggested that polymorphisms in inflammasome genes might be involved in the predisposition to T1D. A coding polymorphism in NLRP1 was demonstrated to confer susceptibility to T1D (21). Furthermore, two single-nucleotide polymorphisms in NLRP3 were identified in a separate association study as a predisposing factor for T1D (22).Thus, we generated NLRP3-deficient (NLRP3^−/− or KO) NOD mice to understand the role of NLRP3 in the pathogenesis of T1D. Here, we show that NOD mice deficient in NLRP3 were protected from T1D development. Mechanistic studies suggested that the expression of NLRP3, in both hematopoietic and nonhematopoietic cells, was important for diabetes development. Whereas NLRP3 deficiency in the hematopoietic compartment reduced the diabetogenicity of immune cells, its ablation in nonhematopoietic cells, particularly in the pancreatic islets, compromised the migration of immune cells into the target tissue. Destruction of beta cells was reduced via the down-regulation of chemokine gene expression in the pancreatic islets leading to protection from diabetes.