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.     

Inflammasome activation causes dual recruitment of NLRC4 and NLRP3 to the same macromolecular complex

Pathogen recognition by nucleotide-binding oligomerization domain-like receptor (NLR) results in the formation of a macromolecular protein complex (inflammasome) that drives protective inflammatory responses in the host. It is thought that the number of inflammasome complexes forming in a cell is determined by the number of NLRs being activated, with each NLR initiating its own inflammasome assembly independent of one another; however, we show here that the important foodborne pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) simultaneously activates at least two NLRs, whereas only a single inflammasome complex is formed in a macrophage. Both nucleotide-binding domain and leucine-rich repeat caspase recruitment domain 4 and nucleotide-binding domain and leucine-rich repeat pyrin domain 3 are simultaneously present in the same inflammasome, where both NLRs are required to drive IL-1β processing within the Salmonella-infected cell and to regulate the bacterial burden in mice. Superresolution imaging of Salmonella-infected macrophages revealed a macromolecular complex with an outer ring of apoptosis-associated speck-like protein containing a caspase activation and recruitment domain and an inner ring of NLRs, with active caspase effectors containing the pro–IL-1β substrate localized internal to the ring structure. Our data reveal the spatial localization of different components of the inflammasome and how different members of the NLR family cooperate to drive robust IL-1β processing during Salmonella infection.Inflammasomes are cytosolic multimeric protein complexes formed in the host cell in response to the detection of pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs). Formation of the inflammasome in response to PAMPs is critical for host defense because it facilitates processing of the proinflammatory cytokines pro–IL-1β and pro–IL-18 into their mature forms (1). The inflammasome also initiates host cell death in the form of pyroptosis, releasing macrophage-resident microbes to be killed by other immune mechanisms (2). The current paradigm is that there are individual, receptor-specific inflammasomes consisting of one nucleotide-binding oligomerization domain-like receptor (NLR; leucine-rich repeat–containing) or PYHIN [pyrin domain and hematopoietic expression, interferon-inducible nature, and nuclear localization (HIN) domain-containing] receptor, the adaptor protein apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (CARD; ASC), and caspase-1 (3). How the protein constituents of the inflammasome are spatially orientated is unclear.Nucleotide-binding domain and leucine-rich repeat caspase recruitment domain 4 (NLRC4) and nucleotide-binding domain and leucine-rich repeat pyrin domain 3 (NLRP3) are the best-characterized inflammasomes, especially with respect to their responses to pathogenic bacteria. The NLRC4 inflammasome is activated primarily by bacteria, including Aeromonas veronii (4), Escherichia coli (5), Listeria monocytogenes (6, 7), Pseudomonas aeruginosa (5), Salmonella enterica serovar Typhimurium (S. Typhimurium) (5, 8–10), and Yersinia species (11). In mouse macrophages, the NLRC4 inflammasome responds to flagellin and type III secretion system-associated needle or rod proteins (5, 8, 9) after their detection by NLR family, apoptosis inhibitory protein (NAIP) 5 or NAIP6 and NAIP1 or NAIP2, respectively (12–15). Phosphorylation of NLRC4 at a single, evolutionarily conserved residue, Ser 533, by PKCδ kinase is required for NLRC4 inflammasome assembly (16). The NLRP3 inflammasome is activated by a large repertoire of DAMPs, including ATP, nigericin, maitotoxin, uric acid crystals, silica, aluminum hydroxide, and muramyl dipeptide (17–20). NLRP3 is also activated by bacterial PAMPs from many species, including Aeromonas species (4, 21), L. monocytogenes (6, 7, 22), Neisseria gonorrhoeae (23), S. Typhimurium (10), Streptococcus pneumoniae (24), and Yersinia species (11). The mechanisms by which NLRC4 and NLRP3 inflammasomes contribute to host defense against bacterial pathogens are emerging; however, little is known about the dynamics governing inflammasome assembly in infections caused by bacteria that activate multiple NLRs, such as S. Typhimurium (10), A. veronii (4), and Yersinia (11).NLRP3 does not have a CARD and requires ASC to interact with the CARD of procaspase-1. This interaction requires a charged interface around Asp27 of the procaspase-1 CARD (25). Whether ASC is also required for the assembly of the NLRC4 inflammasome is less clear. NLRC4 contains a CARD that can interact directly with the CARD of procaspase-1 (26); however, ASC is required for some of the responses driven by NLRC4 (27). Macrophages infected with S. Typhimurium or other pathogens exhibit formation of a distinct cytoplasmic ASC focus or speck, which can be visualized under the microscope and is indicative of inflammasome activation (10, 28, 29). Our laboratory and others have shown that only one ASC speck is formed per cell irrespective of the stimulus used (29–32). However, many bacteria activate two or more NLRs, and it is unclear whether a singular inflammasome is formed at a time or if multiple inflammasomes are formed independent of each other, with each inflammasome containing one member of the NLR family.In this study, we describe the endogenous molecular constituents of the Salmonella-induced inflammasome and their spatial orientation. In cross-section, ASC forms a large external ring with the NLRs and caspases located internally. Critically, NLRC4, NLRP3, caspase-1, and caspase-8 coexist in the same ASC speck to coordinate pro–IL-1β processing. All ASC specks observed contained both NLRC4 and NLRP3. These results suggest that Salmonella infection induces a single inflammasome protein complex containing different NLRs and recruiting multiple caspases to coordinate a multifaceted inflammatory response to infection.     

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.     

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.     

Mitochondrial protein mitofusin 2 is required for NLRP3 inflammasome activation after RNA virus infection

Nod-like receptor family, pyrin domain-containing 3 (NLRP3), is involved in the early stages of the inflammatory response by sensing cellular damage or distress due to viral or bacterial infection. Activation of NLRP3 triggers its assembly into a multimolecular protein complex, termed “NLRP3 inflammasome.” This event leads to the activation of the downstream molecule caspase-1 that cleaves the precursor forms of proinflammatory cytokines, such as interleukin 1 beta (IL-1β) and IL-18, and initiates the immune response. Recent studies indicate that the reactive oxygen species produced by mitochondrial respiration is critical for the activation of the NLRP3 inflammasome by monosodium urate, alum, and ATP. However, the precise mechanism by which RNA viruses activate the NLRP3 inflammasome is not well understood. Here, we show that loss of mitochondrial membrane potential [ΔΨ(m)] dramatically reduced IL-1β secretion after infection with influenza, measles, or encephalomyocarditis virus (EMCV). Reduced IL-1β secretion was also observed following overexpression of the mitochondrial inner membrane protein, uncoupling protein-2, which induces mitochondrial proton leakage and dissipates ΔΨ(m). ΔΨ(m) was required for association between the NLRP3 and mitofusin 2, a mediator of mitochondrial fusion, after infection with influenza virus or EMCV. Importantly, the knockdown of mitofusin 2 significantly reduced the secretion of IL-1β after infection with influenza virus or EMCV. Our results provide insight into the roles of mitochondria in NLRP3 inflammasome activation.Nod-like receptor family, pyrin domain-containing 3 (NLRP3) can be activated by a wide variety of stimuli, such as endogenous danger signals from damaged cells, bacterial components, environmental irritants, and DNA and RNA viruses (1). It forms a multiprotein complex called the NLRP3 inflammasome, which includes an adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and procaspase-1. The NLRP3 inflammasome-mediated cytokine release requires two signaling pathways (2). The first signal is induced by Toll-like receptors (TLRs), interleukin 1 receptor (IL-1R), or tumor necrosis factor receptor, and leads to the synthesis of inactive NLRP3, pro–IL-1β, and pro–IL-18 in the cytosol. The second signal is triggered by NLRP3 agonists, which induce the activation of caspase-1. Caspase-1 catalyzes the proteolytic processing of pro–IL-1β and pro–IL-18, and their conversion to mature forms, and stimulates their secretion across the plasma membrane (1). These inflammasome-dependent cytokines play a key role in the induction of adaptive immunity and the initiation of tissue healing after influenza virus infection (3–5). Migration of dendritic cells (DCs) to the draining lymph nodes and priming of CD8 T cells during influenza virus infection require IL-1R signaling in respiratory DCs (6). By contrast, chronic activation of the NLRP3 inflammasome has been linked to many inflammatory diseases (7, 8). Therefore, increasing the number of studies dedicated to the investigation of the molecular mechanisms of NLRP3 inflammasome activation will be crucial for improving our understanding of the pathogenesis of infectious and autoinflammatory diseases.Mitochondria are compartmentalized by two membrane bilayers (outer and inner membranes) and are involved in a wide variety of functions in eukaryotic cells. Within the past decade, novel functions of mitochondria have been discovered demonstrating their crucial role in innate antiviral immunity in vertebrates (9). A direct link between mitochondria and innate immunity was first highlighted with the finding that an adaptor protein, mitochondrial antiviral signaling (MAVS; also known as IPS-1, VISA, or Cardif) (10–13), triggered retinoic acid-inducible gene 1 (RIG-I) and melanoma differentiation-associated protein 5-mediated type I interferon (IFN) induction. In addition to their role in antiviral immunity, mitochondria also function as a platform for the activation of the NLRP3 inflammasome by producing mitochondrial reactive oxygen species (mROS) (14, 15). In this context, NLRP3 agonists trigger the generation of mROS from damaged mitochondria, resulting in the dissociation of thioredoxin (TRX) from TRX-interacting protein, which associates with NLRP3 to facilitate inflammasome formation (16). Furthermore, cytosolic mitochondrial DNA (mtDNA) released from damaged mitochondria has been reported to activate the NLRP3 inflammasome (17) and absent in melanoma 2 inflammasome (15), recently identified as a cytoplasmic DNA sensor for the inflammasome (18–21). Although mitochondria are essential for host-cell defense, the mechanism of their involvement in the activation of the NLRP3 inflammasome is still unclear. In the present study, we demonstrate that the mitofusin 2 (Mfn2) is required for the full activation of the NLRP3 inflammasomes in macrophages.     

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.     

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.     

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.     

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.     

Inflammasome activation leads to Caspase-1–dependent mitochondrial damage and block of mitophagy

Inflammasomes are intracellular sensors that couple detection of pathogens and cellular stress to activation of Caspase-1, and consequent IL-1β and IL-18 maturation and pyroptotic cell death. Here, we show that the absent in melanoma 2 (AIM2) and nucleotide-binding oligomerization domain-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasomes trigger Caspase-1–dependent mitochondrial damage. Caspase-1 activates multiple pathways to precipitate mitochondrial disassembly, resulting in mitochondrial reactive oxygen species (ROS) production, dissipation of mitochondrial membrane potential, mitochondrial permeabilization, and fragmentation of the mitochondrial network. Moreover, Caspase-1 inhibits mitophagy to amplify mitochondrial damage, mediated in part by cleavage of the key mitophagy regulator Parkin. In the absence of Parkin activity, increased mitochondrial damage augments pyroptosis, as indicated by enhanced plasma membrane permeabilization and release of danger-associated molecular patterns (DAMPs). Therefore, like other initiator caspases, Caspase-1 activation by inflammasomes results in mitochondrial damage.Inflammasomes are cytosolic complexes that mediate Caspase-1 activation in response to pathogen infection and cellular stress (1, 2). They consist of a regulatory subunit, which couples stimulus recognition to complex assembly; Caspase-1, the effector subunit; and the adaptor protein Asc. The best-characterized inflammasomes include the AIM2 inflammasome, which detects cytosolic DNA during bacterial and viral infection, and the NLRP3 inflammasome, which is activated by many stimuli in a variety of settings including infection and metabolic inflammation. Although not entirely clear, one plausible model of NLRP3 inflammasome activation is the generation of some mitochondria-associated signal by mitochondrial destabilization (3, 4). Recruitment of Caspase-1 into the inflammasome complex leads to its activation, autoprocessing, and subsequent substrate cleavage.The prototypical inflammasome-mediated functions are IL-1β and IL-18 maturation and induction of pyroptosis (1). Additionally, inflammasomes control other processes like unconventional secretion of intracellular proteins (5), such as DAMPs like high mobility group box 1 (HMGB1) (6), and regulation of autophagy (7, 8). These examples suggest the existence of additional inflammasome effector activities that are likely to vary in a context-dependent manner. Interestingly, a recent report indicated that activation of the NLRP3 inflammasome by extracellular ATP leads to NLRP3-dependent dissipation of the mitochondrial membrane potential (9), but subsequent studies proposed that such mitochondrial damage is solely a trigger of inflammasome activation (10, 11) because it occurs normally in the absence of the NLRP3 inflammasome (11). Thus, the relationship between NLRP3 inflammasome activation and mitochondrial damage remains unclear.Pyroptosis is a Caspase-1–mediated, proinflammatory form of cell death. It occurs during infection by many intracellular pathogens where it can critically eliminate an intracellular replication niche (12), as well as other settings (13, 14), but experimental demonstration of its physiological role is hampered by the lack of mechanistic insights into its regulation. Pyroptosis shares some features with necrosis (such as loss of plasma-membrane integrity and release of intracellular contents) and others with apoptosis (including DNA fragmentation and nuclear condensation) (12). Mitochondrial damage critically underlies apoptosis mediated by initiator caspases like Caspase-8 and Caspase-9. Upon activation by death receptors, Caspase-8 cleavage of the protein Bid precipitates mitochondrial outer membrane permeabilization (MOMP), resulting in dissipation of the membrane potential, disruption of mitochondrial function, and release of apoptosis-promoting factors from the intermembrane space (15). MOMP can also lead to mitochondrial permeability transition (MPT), or breach of integrity of the inner membrane caused by opening of an inner membrane pore, which further amplifies mitochondrial damage (16). Whether mitochondrial damage contributes to pyroptosis and in general how pyroptosis is regulated, including the relevant substrates, are not clear.     

TLR-induced PAI-2 expression suppresses IL-1β processing via increasing autophagy and NLRP3 degradation

The NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome, a multiprotein complex, triggers caspase-1 activation and maturation of the proinflammatory cytokines IL-1β and IL-18 upon sensing a wide range of pathogen- and damage-associated molecules. Dysregulation of NLRP3 inflammasome activity contributes to the pathogenesis of many diseases, but its regulation remains poorly defined. Here we show that depletion of plasminogen activator inhibitor type 2 (PAI-2), a serine protease inhibitor, resulted in NLRP3- and ASC (apoptosis-associated Speck-like protein containing a C-terminal caspase recruitment domain)‐dependent caspase-1 activation and IL-1β secretion in macrophages upon Toll-like receptor 2 (TLR2) and TLR4 engagement. TLR2 or TLR4 agonist induced PAI-2 expression, which subsequently stabilized the autophagic protein Beclin 1 to promote autophagy, resulting in decreases in mitochondrial reactive oxygen species, NLRP3 protein level, and pro–IL-1β processing. Likewise, overexpressing Beclin 1 in PAI-2–deficient cells rescued the suppression of NLRP3 activation in response to LPS. Together, our data identify a tier of TLR signaling in controlling NLRP3 inflammasome activation and reveal a cell-autonomous mechanism which inversely regulates TLR- or Escherichia coli-induced mitochondrial dysfunction, oxidative stress, and IL-1β–driven inflammation.Innate immunity, the first line of host defense against pathogen infection, is composed of diverse germ line-encoded pattern-recognition receptors, such as Toll-like receptors (TLRs) and NOD-like receptors (NLRs), that recognize pathogen-associated molecular patterns (PAMPs) from pathogens or danger-associated molecular patterns from damaged tissue (1, 2). TLRs recognize a variety of PAMPs from microbes to induce autophagy and cytokine production for host defense against microbial infections. Inflammasomes, multiple protein complexes containing NLR proteins or AIM2, mediate caspase-1 activation leading to the processing of the proinflammatory cytokines IL-1β and IL-18 (3). The inflammasome/caspase-1 complexes also may target additional effector molecules to regulate diverse physiological functions, such as pyroptosis and tissue repair (4). Among the identified inflammasomes, the NLRP3 inflammasome has been studied extensively and has been shown to be activated by a large variety of activators that share no structural similarity (2). For this reason, it has been suggested that the NLRP3 inflammasome is activated through a secondary mediator, such as potassium (K⁺) efflux, reactive oxygen species (ROS), or lysosomal proteases (1). The inflammasomes play a critical role in the clearance of microbial pathogens and tissue repair (2, 5). However, dysregulation of inflammasome activation has been associated with a variety of human diseases, including autoinflammatory diseases, metabolic disorders, and cancer (3, 6).Autophagy, an evolutionarily conserved cellular catabolic process, facilitates the recycling of damaged proteins and organelles (7). Increasing evidence indicates that autophagy is involved in the regulation of immune responses and inflammation (7). Macrophages treated with an autophagy inhibitor or with the deletion of several autophagic components, including Atg16L1, Beclin 1, and LC3, induced greater caspase-1 activation and IL-1β secretion in response to LPS or LPS plus an NLRP3 agonist (8, 9). These data strongly suggest that the NLRP3 inflammasome activity is negatively regulated by autophagy, but the underlying mechanism is poorly understood.Plasminogen activator inhibitor type 2 (PAI-2), a serine proteinase inhibitor (SERPIN), originally was identified as an inhibitor of the urokinase-type plasminogen activator (uPA) involved in cellular invasion and tissue remodeling (10). Recently, PAI-2 has been associated with newly identified uPA-independent biological functions, probably through targeting an as yet uncharacterized intracellular molecule (11). In addition, PAI-2 is one of the major molecules up-regulated in macrophages in response to TLR4 activators or inflammatory mediators, suggesting its function in the regulation of innate immunity (12, 13).Macrophages treated with LPS alone do not release mature IL-1β and IL-18 unless accompanied by a second stimulus, such as ATP or crystals (8, 14). LPS activates TLR4 to induce the synthesis of pro–IL-1β and the inflammasome component NLRP3 via IκB kinase (IKK)/NF-κB activation; a second stimulus is required for inflammasome assembly and caspase-1 activation to cleave pro–IL-1β and pro–IL-18 to their mature forms. Nevertheless, previous work showed that LPS alone is sufficient to induce mature IL-1β production in IKKβ-deficient macrophages because of enhanced pro–IL-1β processing (15). Additionally, LPS-induced PAI-2 expression is blunted in IKKβ-deficient macrophages, and reintroduction of PAI-2 blocks IL-1β maturation in a caspase-1–dependent manner, suggesting that PAI-2 inhibits pro–IL-1β processing upon LPS stimulation; however, the underlying mechanism is unknown.Here, we show that depletion of PAI-2 in macrophages induces caspase-1 activation and IL-1β production in response to TLR agonists and Escherichia coli with no need of a second stimulus. TLR engagement induced PAI-2 expression and enhanced association of PAI-2 with Beclin 1, leading to an increase in autophagy, which then caused reduced mitochondrial ROS (mROS) and increased NLRP3 degradation, resulting in decreased IL-1β maturation. Inflammatory cytokines and cellular ROS play vital roles in innate immunity, but prolonged and excess production of these mediators can be detrimental. Our results suggest that PAI-2 is a cell-autonomous mechanism that counteracts the detrimental effects caused by TLR2/4- and E. coli-triggered cellular stress by reducing ROS production and the inflammasome activation, thereby resulting in less inflammation and tissue damage.     

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.     

Macrophage arginase-1 controls bacterial growth and pathology in hypoxic tuberculosis granulomas

Lung granulomas develop upon Mycobacterium tuberculosis (Mtb) infection as a hallmark of human tuberculosis (TB). They are structured aggregates consisting mainly of Mtb-infected and -uninfected macrophages and Mtb-specific T cells. The production of NO by granuloma macrophages expressing nitric oxide synthase-2 (NOS2) via l-arginine and oxygen is a key protective mechanism against mycobacteria. Despite this protection, TB granulomas are often hypoxic, and bacterial killing via NOS2 in these conditions is likely suboptimal. Arginase-1 (Arg1) also metabolizes l-arginine but does not require oxygen as a substrate and has been shown to regulate NOS2 via substrate competition. However, in other infectious diseases in which granulomas occur, such as leishmaniasis and schistosomiasis, Arg1 plays additional roles such as T-cell regulation and tissue repair that are independent of NOS2 suppression. To address whether Arg1 could perform similar functions in hypoxic regions of TB granulomas, we used a TB murine granuloma model in which NOS2 is absent. Abrogation of Arg1 expression in macrophages in this setting resulted in exacerbated lung granuloma pathology and bacterial burden. Arg1 expression in hypoxic granuloma regions correlated with decreased T-cell proliferation, suggesting that Arg1 regulation of T-cell immunity is involved in disease control. Our data argue that Arg1 plays a central role in the control of TB when NOS2 is rendered ineffective by hypoxia.The generic term “granuloma” describes an organized aggregate of immune and other cells formed in response to persistent stimuli of noninfectious or infectious origin (1–3). Granulomas develop in tuberculosis (TB), leprosy, schistosomiasis, and leishmaniasis and function to contain and sometimes destroy the etiologic agent (1–3). The precise role of granulomas in protection against TB remains elusive (1, 4–6). Although Mycobacterium tuberculosis (Mtb)-infected macrophages within granulomas are endowed with antimycobacterial defenses induced by the action of T cells, the long-term persistence of Mtb in the face of strong immune responses suggests that these mechanisms could be both evaded and harnessed by Mtb or that they function inefficiently in the granuloma environment (5, 7–9).Two enzymes associated with key macrophage functions in TB granulomas are nitric oxide synthase-2 (NOS2) and arginase-1 (Arg1), which compete for the same substrate, l-arginine. The predominance of either enzyme spatially influences macrophage activation in different granuloma environments (8, 10–13). In the presence of oxygen, NOS2 metabolizes l-arginine into l-citrulline and nitric oxide (NO), which is associated with killing of intracellular pathogens (10, 13–15). Arg1 hydrolyzes l-arginine, producing urea and l-ornithine, which can be further metabolized to downstream products such as polyamines; Arg1 activity is associated with anti-inflammatory responses (10, 12, 13, 16). Knowledge of the immune and inflammatory functions of NOS2 and Arg1 comes largely from murine studies (12, 13). Production of NO and other reactive nitrogen intermediates (RNIs) is a key protective mechanism against Mtb infection in mice because NOS2-deficient (Nos2^−/−) mice infected with Mtb via aerosol and i.v. routes succumb to disease (17–21). Like NOS2, Arg1 is induced in murine and human macrophages upon infection with Mtb, and NOS2 activity in Mtb infection is hampered by Arg1 expression (22–24). Murine studies argue that Arg1 participates in dampening effective immunity against Mtb, because aerosol Mtb-infected mice that are deficient in macrophage-specific Arg1 have lower bacterial loads and smaller cellular infiltrates in the lungs than similarly infected WT mice (22). Moreover, Mtb aerosol infection of transgenic mice that overexpress IL-10 and IL-13 in macrophages results in increased pulmonary expression of Arg1, correlating with increased bacterial loads as compared with WT mice (25, 26). In these mouse models, Arg1 expression was linked to reduced production of RNIs, suggesting that l-arginine depletion by Arg1 suppresses NOS2 activity and, therefore, Mtb killing (22, 25). However, evidence from murine models of parasitic infection argues that Arg1 can have at least two other functions in addition to suppressing NOS2. First, macrophage Arg1 can reduce l-arginine availability to T cells, which are l-arginine auxotrophic, and can restrict local T-cell proliferation (10, 13, 27–29). Arginine restriction is an essential regulatory mechanism to restrain excessive T-cell responses in schistosomiasis granulomas, in which a Th2 response predominates and NOS2 expression is minimal (30). Moreover, depletion of l-arginine by Arg1 during Leishmania infection impairs Th1 responses required for parasite killing and subsequent lesion healing (31, 32). Second, products of l-arginine metabolism by Arg1 can affect parasite fitness directly. In particular, l-ornithine and polyamines promote the proliferation of Leishmania and Plasmodium parasites (33–35) but also directly block helminthic motility (36).Because of the dominant NOS2 response during murine TB, determining the relative contributions of the Arg1 functions that are independent of NOS2 suppression in mice during Mtb infection remains technically challenging.Here we devised a way to dissect the role of Arg1-expressing cells in TB lung granulomas in the absence of NOS2 by using the granuloma model of Nos2^−/− mice dermally infected with Mtb. Nos2^−/− mice dermally infected with Mtb, but not aerosol Mtb-infected Nos2^−/− mice, recapitulate several features of TB granuloma pathology in humans, including hypoxia and caseation in the central regions of lung granulomas (37). We identified Arg1-expressing macrophages in hypoxic TB granulomas in the lungs of dermally Mtb-infected Nos2^−/− mice that were analogous to TB lung granulomas in humans and nonhuman primates (NHPs) (11, 38). We demonstrate a critical role for Arg1-expressing macrophages in maintaining the ability of granulomas to control bacterial replication and to prevent pathology and provide evidence that this role involves spatial modulation of T-cell responses in specific granuloma microenvironments. Hence, our results indicate regulation by Arg1 in granulomas is important for balancing the control of Mtb growth and immunopathology during TB.     

Inflammasome-independent IL-1β mediates autoinflammatory disease in Pstpip2-deficient mice

Chronic recurrent multifocal osteomyelitis (CRMO) is a human autoinflammatory disorder that primarily affects bone. Missense mutation (L98P) of proline-serine-threonine phosphatase-interacting protein 2 (Pstpip2) in mice leads to a disease that is phenotypically similar to CRMO called chronic multifocal osteomyelitis (cmo). Here we show that deficiency of IL-1RI in cmo mice resulted in a significant reduction in the time to onset of disease as well as the degree of bone pathology. Additionally, the proinflammatory cytokine IL-1β, but not IL-1α, played a critical role in the pathology observed in cmo mice. In contrast, disease in cmo mice was found to be independent of the nucleotide-binding domain, leucine-rich repeat-containing family, pyrin domain-containing 3 (NLRP3) inflammasome as well as caspase-1. Neutrophils, but not bone marrow-derived macrophages, from cmo mice secreted increased IL-1β in response to ATP, silica, and Pseudomonas aeruginosa compared with neutrophils from WT mice. This aberrant neutrophil response was sensitive to inhibition by serine protease inhibitors. These results demonstrate an inflammasome-independent role for IL-1β in disease progression of cmo and implicate neutrophils and neutrophil serine proteases in disease pathogenesis. These data provide a rationale for directly targeting IL-1RI or IL-1β as a therapeutic strategy in CRMO.Chronic recurrent multifocal osteomyelitis (CRMO) is a sterile inflammatory disorder that affects children and presents with bone pain due to sterile osteomyelitis (1). The etiology of the disease is unknown, but it is associated with Crohn disease, inflammatory arthritis, and psoriasis in affected individuals and their close relatives, suggesting a shared pathophysiology and supporting a genetic contribution to disease (2). There are two autosomal recessive disorders that present with neonatal- or infant-onset sterile osteitis that are histologically similar to the bone disease in CRMO and clinically improve with IL-1 blockade (3, 4). Majeed syndrome, caused by mutations in LPIN2, presents with CRMO, congenital dyserythropoietic anemia, and sterile neutrophilic dermatosis (5). Deficiency of the IL-1 receptor antagonist (DIRA) is caused by mutations in IL1RN, which encodes the IL-1 receptor antagonist (3), resulting in dysregulation of the IL-1 pathway. Affected individuals present with neonatal-onset cutaneous pustulosis, marked elevation of inflammatory markers, sterile multifocal osteitis, and periostitis (3).There are two autosomal recessive murine models of CRMO both due to mutations in proline-serine-threonine interacting protein 2 (Pstpip2) (6–8). The mutation (L98P) present in the chronic multifocal osteomyelitis (cmo) model results in no detectable expression of Pstpip2, a protein expressed predominantly in the cells of the myeloid lineage, and leads to disease that resembles human CRMO (6, 9). The development of osteomyelitis in the cmo mouse is hematopoietically driven and develops in the absence of lymphocytes, consistent with an autoinflammatory mechanism of disease (9). Although it is known that cmo mice have a dysregulated innate immune system, it is not clear what inflammatory pathway is critical for disease.Mutations within NLRP3 (also known as NALP3 or cryopyrin) are associated with the autoinflammatory Muckle-Wells syndrome, familial cold autoinflammatory syndrome, and neonatal-onset multisystem inflammatory disease, collectively known as cryopyrin-associated periodic syndrome (10). These NLRP3 mutations result in a constitutively active form of NLRP3 that leads to increased inflammasome activation with a resultant increase in secretion of IL-1β (10). A diverse array of agonists has been identified that are capable of activating NLRP3, which results in the assembly of the NLRP3 inflammasome composed of NLRP3, the adaptor molecule apoptosis-associated speck-like protein containing a CARD (ASC), and caspase-1 (11). The activation of the inflammasome leads to the activation of caspase-1, with the resultant processing of pro-IL-1β and pro-IL-18 to their mature and secreted forms (12). However, the role of the NLRP3 inflammasome in the pathogenesis of cmo remains unknown.Given the evidence that IL-1 is important in the pathogenesis of sterile bone inflammation in humans (3, 4), we investigated the role of IL-1 in the pathogenesis of sterile osteomyelitis in the cmo mouse. Here we demonstrate that deficiency of IL-1RI (interleukin-1 receptor type I) or IL-1β in cmo mice resulted in delayed onset of disease and an attenuation of disease severity. In contrast, disease progression in cmo mice was found to be independent of the NLRP3 inflammasome, and in vitro findings support a role for neutrophil serine proteases in the abnormally increased secretion of IL-1β. Taken together these data demonstrate an inflammasome-independent role for the IL-1 pathway in the disease pathogenesis of cmo.