Blocking Neuronal Signaling to Immune Cells Treats Streptococcal Invasive Infection.

The nervous system, the immune system, and microbial pathogens interact closely at barrier tissues. Here, we find that a bacterial pathogen, Streptococcus pyogenes, hijacks pain and neuronal regulation of the immune response to promote bacterial survival. Necrotizing fasciitis is a life-threatening soft tissue infection in which "pain is out of proportion" to early physical manifestations. We find that S. pyogenes, the leading cause of necrotizing fasciitis, secretes streptolysin S (SLS) to directly activate nociceptor neurons and produce pain during infection. Nociceptors, in turn, release the neuropeptide calcitonin gene-related peptide (CGRP) into infected tissues, which inhibits the recruitment of neutrophils and opsonophagocytic killing of S. pyogenes. Botulinum neurotoxin A and CGRP antagonism block neuron-mediated suppression of host defense, thereby preventing and treating S. pyogenes necrotizing infection. We conclude that targeting the peripheral nervous system and blocking neuro-immune communication is a promising strategy to treat highly invasive bacterial infections. VIDEO ABSTRACT.

Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection.

Programmed cell death contributes to host defense against pathogens. To investigate the relative importance of pyroptosis, necroptosis, and apoptosis during Salmonella infection, we infected mice and macrophages deficient for diverse combinations of caspases-1, -11, -12, and -8 and receptor interacting serine/threonine kinase 3 (RIPK3). Loss of pyroptosis, caspase-8-driven apoptosis, or necroptosis had minor impact on Salmonella control. However, combined deficiency of these cell death pathways caused loss of bacterial control in mice and their macrophages, demonstrating that host defense can employ varying components of several cell death pathways to limit intracellular infections. This flexible use of distinct cell death pathways involved extensive cross-talk between initiators and effectors of pyroptosis and apoptosis, where initiator caspases-1 and -8 also functioned as executioners when all known effectors of cell death were absent. These findings uncover a highly coordinated and flexible cell death system with in-built fail-safe processes that protect the host from intracellular infections.

The TWIK2 Potassium Efflux Channel in Macrophages Mediates NLRP3 Inflammasome-Induced Inflammation.

Potassium (K+) efflux across the plasma membrane is thought to be an essential mechanism for ATP-induced NLRP3 inflammasome activation, yet the identity of the efflux channel has remained elusive. Here we identified the two-pore domain K+ channel (K2P) TWIK2 as the K+ efflux channel triggering NLRP3 inflammasome activation. Deletion of Kcnk6 (encoding TWIK2) prevented NLRP3 activation in macrophages and suppressed sepsis-induced lung inflammation. Adoptive transfer of Kcnk6-/- macrophages into mouse airways after macrophage depletion also prevented inflammatory lung injury. The K+ efflux channel TWIK2 in macrophages has a fundamental role in activating the NLRP3 inflammasome and consequently mediates inflammation, pointing to TWIK2 as a potential target for anti-inflammatory therapies.

AIM2 inflammasome surveillance of DNA damage shapes neurodevelopment.

Neurodevelopment is characterized by rapid rates of neural cell proliferation and differentiation followed by massive cell death in which more than half of all recently generated brain cells are pruned back. Large amounts of DNA damage, cellular debris, and by-products of cellular stress are generated during these neurodevelopmental events, all of which can potentially activate immune signalling. How the immune response to this collateral damage influences brain maturation and function remains unknown. Here we show that the AIM2 inflammasome contributes to normal brain development and that disruption of this immune sensor of genotoxic stress leads to behavioural abnormalities. During infection, activation of the AIM2 inflammasome in response to double-stranded DNA damage triggers the production of cytokines as well as a gasdermin-D-mediated form of cell death known as pyroptosis1-4. We observe pronounced AIM2 inflammasome activation in neurodevelopment and find that defects in this sensor of DNA damage result in anxiety-related behaviours in mice. Furthermore, we show that the AIM2 inflammasome contributes to central nervous system (CNS) homeostasis specifically through its regulation of gasdermin-D, and not via its involvement in the production of the cytokines IL-1 and/or IL-18. Consistent with a role for this sensor of genomic stress in the purging of genetically compromised CNS cells, we find that defective AIM2 inflammasome signalling results in decreased neural cell death both in response to DNA damage-inducing agents and during neurodevelopment. Moreover, mutations in AIM2 lead to excessive accumulation of DNA damage in neurons as well as an increase in the number of neurons that incorporate into the adult brain. Our findings identify the inflammasome as a crucial player in establishing a properly formed CNS through its role in the removal of genetically compromised cells.

Loss of NLRP6 expression increases the severity of intestinal injury after syngeneic hematopoietic stem cell transplantation.

NLRP6 plays a crucial role in maintaining intestinal homeostasis by regulating the interaction between the intestinal mucosa and the microbiota. However, the impact of NLRP6 deficiency on intestinal damage following hematopoietic stem cell transplantation (HSCT) remains poorly understood. In this study, we established a syngeneic HSCT mouse model using C57BL/6 mice as donors and NLRP6-/- or C57BL/6 mice as recipients. Our findings revealed that NLRP6 deficiency had minimal influence on peripheral blood cell counts and splenic immune cell proportions in transplanted mice. However, it exacerbated pathological changes in the small intestine on day 14 post-transplantation, accompanied by increased proportions of macrophages, dendritic cells, and neutrophils. Furthermore, the NLRP6 deficiency resulted in elevated expression of MPO and CD11b, while reducing the levels mature caspase-1 and mature IL-1ß in the intestine. Moreover, the NLRP6 deficiency disturbed the expression of apoptosis-related molecules and decreased the tight junction protein occludin. Notably, recipient mice with NLRP6 deficiency exhibited lower mRNA expression levels of antimicrobial genes, such as Reg3γ and Pla2g2a. The short-term increase in inflammatory cell infiltration caused by NLRP6 deficiency was associated with intestinal damage, increased apoptosis, reduced expression of antimicrobial peptides, and impaired intestinal repair. Taken together, our findings demonstrate that the loss of NLRP6 exacerbates post-transplantation intestinal damage in recipient mice.

The Nlrp3 Inflammasome Suppresses Colorectal Cancer Metastatic Growth in the Liver by Promoting Natural Killer Cell Tumoricidal Activity.

The crosstalk between inflammation and tumorigenesis is now clearly established. However, how inflammation is elicited in the metastatic environment and the corresponding contribution of innate immunity pathways in suppressing tumor growth at secondary sites are poorly understood. Here, we show that mice deficient in Nlrp3 inflammasome components had exacerbated liver colorectal cancer metastatic growth, which was mediated by impaired interleukin-18 (IL-18) signaling. Control of tumor growth was independent of differential cancer cell colonization or proliferation, intestinal microbiota effects, or tumoricidal activity by the adaptive immune system. Instead, the inflammasome-IL-18 pathway impacted maturation of hepatic NK cells, surface expression of the death ligand FasL, and capacity to kill FasL-sensitive tumors. Our results define a regulatory signaling circuit within the innate immune system linking inflammasome activation to effective NK-cell-mediated tumor attack required to suppress colorectal cancer growth in the liver.

Aquaporin-3 is involved in NLRP3-inflammasome activation contributing to the setting of inflammatory response.

Inflammasomes are large immune multiprotein complexes that tightly regulate the production of the pro-inflammatory cytokines, being dependent on cell regulatory volume mechanisms. Aquaporins (AQPs) are protein channels that facilitate the transport of water and glycerol (aquaglyceroporins) through membranes, essential for cell volume regulation. Although these membrane proteins are highly expressed in monocytes and macrophages, their role in the inflammatory process is still unclear. Here, we investigated the role of aquaglyceroporin AQP3 in NLRP3-inflammasome activation by complementary approaches based either on shRNA silencing or on AQP3 selective inhibition. The latter has been achieved using a reported potent gold-based inhibitor, Auphen. AQP3 inhibition or silencing partially blocked LPS-priming and decreased production of IL-6, proIL-1ß, and TNF-α, suggesting the possible involvement of AQP3 in macrophage priming by Toll-like receptor 4 engagement. Moreover, AQP3-dependent cell reswelling increased IL-1ß release through caspase-1 activation. NLRP3-inflammasome activation induced by reswelling, nigericin, and ATP was also blocked when AQP3 was inhibited or silenced. Altogether, these data point towards AQPs as potential players in the setting of the inflammatory response.

Insights into the Role of Innate Immunity in Cervicovaginal Papillomavirus Infection from Studies Using Gene-Deficient Mice.

We demonstrate that female C57BL/6J mice are susceptible to a transient lower genital tract infection with MmuPV1 mouse papillomavirus and display focal histopathological abnormalities resembling those of human papillomavirus (HPV) infection. We took advantage of strains of genetically deficient mice to study in vivo the role of innate immune signaling in the control of papillomavirus. At 4 months, we sacrificed MmuPV1-infected mice and measured viral 757/3139 spliced transcripts by TaqMan reverse transcription-PCR (RT-PCR), localization of infection by RNAscope in situ hybridization, and histopathological abnormities by hematoxylin and eosin (H&E) staining. Among mice deficient in receptors for pathogen-associated molecular patterns, MyD88-/- and STING-/- mice had 1,350 and 80 copies of spliced transcripts/µg RNA, respectively, while no viral expression was detected in MAVS-/- and Ripk2-/- mice. Mice deficient in an adaptor molecule, STAT1-/-, for interferon signaling had 46,000 copies/µg RNA. Among mice with targeted deficiencies in the inflammatory response, interleukin-1 receptor knockout (IL-1R-/-) and caspase-1-/- mice had 350 and 30 copies/µg RNA, respectively. Among mice deficient in chemokine receptors, CCR6-/- mice had 120 copies/µg RNA, while CXCR2-/- and CXCR3-/- mice were negative. RNAscope confirmed focal infection in MyD88-/-, STAT1-/-, and CCR6-/- mice but was negative for other gene-deficient mice. Histological abnormalities were seen only in the latter mice. Our findings and the literature support a working model of innate immunity to papillomaviruses involving the activation of a MyD88-dependent pathway and IL-1 receptor signaling, control of viral replication by interferon-stimulated genes, and clearance of virus-transformed dysplastic cells by the action of the CCR6/CCL20 axis.IMPORTANCE Papillomaviruses infect stratified squamous epithelia, and the viral life cycle is linked to epithelial differentiation. Additionally, changes occur in viral and host gene expression, and immune cells are activated to modulate the infectious process. In vitro studies with keratinocytes cannot fully model the complex viral and host responses and do not reflect the contribution of local and migrating immune cells. We show that female C57BL/6J mice are susceptible to a transient papillomavirus cervicovaginal infection, and mice deficient in select genes involved in innate immune responses are susceptible to persistent infection with variable manifestations of histopathological abnormalities. The results of our studies support a working model of innate immunity to papillomaviruses, and the model provides a framework for more in-depth studies. A better understanding of mechanisms of early viral clearance and the development of approaches to induce clearance will be important for cancer prevention and the treatment of HPV-related diseases.

Inflammatory effect of Bothropstoxin-I from Bothrops jararacussu venom mediated by NLRP3 inflammasome involves ATP and P2X7 receptor.

Muscle tissue damage is one of the local effects described in bothropic envenomations. Bothropstoxin-I (BthTX-I), from Bothrops jararacussu venom, is a K49-phospholipase A2 (PLA2) that induces a massive muscle tissue injury, and, consequently, local inflammatory reaction. The NLRP3 inflammasome is a sensor that triggers inflammation by activating caspase 1 and releasing interleukin (IL)-1ß and/or inducing pyroptotic cell death in response to tissue damage. We, therefore, aimed to address activation of NLRP3 inflammasome by BthTX-I-associated injury and the mechanism involved in this process. Intramuscular injection of BthTX-I results in infiltration of neutrophils and macrophages in gastrocnemius muscle, which is reduced in NLRP3- and Caspase-1-deficient mice. The in vitro IL-1ß production induced by BthTX-I in peritoneal macrophages (PMs) requires caspase 1/11, ASC and NLRP3 and is dependent on adenosine 5'-triphosphate (ATP)-induced K+ efflux and P2X7 receptor (P2X7R). BthTX-I induces a dramatic release of ATP from C2C12 myotubes, therefore representing the major mechanism for P2X7R-dependent inflammasome activation in macrophages. A similar result was obtained when human monocyte-derived macrophages (HMDMs) were treated with BthTX-I. These findings demonstrated the inflammatory effect of BthTX-I on muscle tissue, pointing out a role for the ATP released by damaged cells for the NLRP3 activation on macrophages, contributing to the understanding of the microenvironment of the tissue damage of the Bothrops envenomation.

Caspase-11 promotes the fusion of phagosomes harboring pathogenic bacteria with lysosomes by modulating actin polymerization.

Inflammasomes are multiprotein complexes that include members of the NLR (nucleotide-binding domain leucine-rich repeat containing) family and caspase-1. Once bacterial molecules are sensed within the macrophage, the inflammasome is assembled, mediating the activation of caspase-1. Caspase-11 mediates caspase-1 activation in response to lipopolysaccharide and bacterial toxins, and yet its role during bacterial infection is unknown. Here, we demonstrated that caspase-11 was dispensable for caspase-1 activation in response to Legionella, Salmonella, Francisella, and Listeria. We also determined that active mouse caspase-11 was required for restriction of L. pneumophila infection. Similarly, human caspase-4 and caspase-5, homologs of mouse caspase-11, cooperated to restrict L. pneumophila infection in human macrophages. Caspase-11 promoted the fusion of the L. pneumophila vacuole with lysosomes by modulating actin polymerization through cofilin. However, caspase-11 was dispensable for the fusion of lysosomes with phagosomes containing nonpathogenic bacteria, uncovering a fundamental difference in the trafficking of phagosomes according to their cargo.

TLR2 and caspase-1 signaling are critical for bacterial containment but not clearance during craniotomy-associated biofilm infection.

BACKGROUND: A craniotomy is required to access the brain for tumor resection or epilepsy treatment, and despite precautionary measures, infectious complications occur at a frequency of 1-3%. Approximately half of craniotomy infections are caused by Staphylococcus aureus (S. aureus) that forms a biofilm on the bone flap, which is recalcitrant to antibiotics. Our prior work in a mouse model of S. aureus craniotomy infection revealed a critical role for myeloid differentiation factor 88 (MyD88) in bacterial containment and pro-inflammatory mediator production. Since numerous receptors utilize MyD88 as a signaling adaptor, the current study examined the importance of Toll-like receptor 2 (TLR2) and TLR9 based on their ability sense S. aureus ligands, namely lipoproteins and CpG DNA motifs, respectively. We also examined the role of caspase-1 based on its known association with TLR signaling to promote IL-1ß release. METHODS: A mouse model of craniotomy-associated biofilm infection was used to investigate the role of TLR2, TLR9, and caspase-1 in disease progression. Wild type (WT), TLR2 knockout (KO), TLR9 KO, and caspase-1 KO mice were examined at various intervals post-infection to quantify bacterial burden, leukocyte recruitment, and inflammatory mediator production in the galea, brain, and bone flap. In addition, the role of TLR2-dependent signaling during microglial/macrophage crosstalk with myeloid-derived suppressor cells (MDSCs) was examined. RESULTS: TLR2, but not TLR9, was important for preventing S. aureus outgrowth during craniotomy infection, as revealed by the elevated bacterial burden in the brain, galea, and bone flap of TLR2 KO mice concomitant with global reductions in pro-inflammatory mediator production compared to WT animals. Co-culture of MDSCs with microglia or macrophages, to model interactions in the brain vs. galea, respectively, also revealed a critical role for TLR2 in triggering pro-inflammatory mediator production. Similar to TLR2, caspase-1 KO animals also displayed increased S. aureus titers coincident with reduced pro-inflammatory mediator release, suggestive of pathway cooperativity. Treatment of caspase-1 KO mice with IL-1ß microparticles significantly reduced S. aureus burden in the brain and galea compared to empty microparticles, confirming the critical role of IL-1ß in limiting S. aureus outgrowth during craniotomy infection. CONCLUSIONS: These results demonstrate the existence of an initial anti-bacterial response that depends on both TLR2 and caspase-1 in controlling S. aureus growth; however, neither pathway is effective at clearing infection in the WT setting, since craniotomy infection persists when both molecules are present.

Type I interferon inhibits interleukin-1 production and inflammasome activation.

Type I interferon (IFN) is a common therapy for autoimmune and inflammatory disorders, yet the mechanisms of action are largely unknown. Here we showed that type I IFN inhibited interleukin-1 (IL-1) production through two distinct mechanisms. Type I IFN signaling, via the STAT1 transcription factor, repressed the activity of the NLRP1 and NLRP3 inflammasomes, thereby suppressing caspase-1-dependent IL-1ß maturation. In addition, type I IFN induced IL-10 in a STAT1-dependent manner; autocrine IL-10 then signaled via STAT3 to reduce the abundance of pro-IL-1α and pro-IL-1ß. In vivo, poly(I:C)-induced type I IFN diminished IL-1ß production in response to alum and Candida albicans, thus increasing susceptibility to this fungal pathogen. Importantly, monocytes from multiple sclerosis patients undergoing IFN-ß treatment produced substantially less IL-1ß than monocytes derived from healthy donors. Our findings may thus explain the effectiveness of type I IFN in the treatment of inflammatory diseases but also the observed "weakening" of the immune system after viral infection.

Dietary modulation of the microbiome affects autoinflammatory disease.

The incidences of chronic inflammatory disorders have increased considerably over the past three decades. Recent shifts in dietary consumption may have contributed importantly to this surge, but how dietary consumption modulates inflammatory disease is poorly defined. Pstpip2(cmo) mice, which express a homozygous Leu98Pro missense mutation in the Pombe Cdc15 homology family protein PSTPIP2 (proline-serine-threonine phosphatase interacting protein 2), spontaneously develop osteomyelitis that resembles chronic recurrent multifocal osteomyelitis in humans. Recent reports demonstrated a crucial role for interleukin-1ß (IL-1ß) in osteomyelitis, but deletion of the inflammasome components caspase-1 and NLRP3 failed to rescue Pstpip2(cmo) mice from inflammatory bone disease. Thus, the upstream mechanisms controlling IL-1ß production in Pstpip2(cmo) mice remain to be identified. In addition, the environmental factors driving IL-1ß-dependent inflammatory bone erosion are unknown. Here we show that the intestinal microbiota of diseased Pstpip2(cmo) mice was characterized by an outgrowth of Prevotella. Notably, Pstpip2(cmo) mice that were fed a diet rich in fat and cholesterol maintained a normal body weight, but were markedly protected against inflammatory bone disease and bone erosion. Diet-induced protection against osteomyelitis was accompanied by marked reductions in intestinal Prevotella levels and significantly reduced pro-IL-1ß expression in distant neutrophils. Furthermore, pro-IL-1ß expression was also decreased in Pstpip2(cmo) mice treated with antibiotics, and in wild-type mice that were kept under germ-free conditions. We further demonstrate that combined deletion of caspases 1 and 8 was required for protection against IL-1ß-dependent inflammatory bone disease, whereas the deletion of either caspase alone or of elastase or neutrophil proteinase 3 failed to prevent inflammatory disease. Collectively, this work reveals diet-associated changes in the intestinal microbiome as a crucial factor regulating inflammasome- and caspase-8-mediated maturation of IL-1ß and osteomyelitis in Pstpip2(cmo) mice.

Negative regulation of the NLRP3 inflammasome by A20 protects against arthritis.

Rheumatoid arthritis is a chronic autoinflammatory disease that affects 1-2% of the world's population and is characterized by widespread joint inflammation. Interleukin-1 is an important mediator of cartilage destruction in rheumatic diseases, but our understanding of the upstream mechanisms leading to production of interleukin-1ß in rheumatoid arthritis is limited by the absence of suitable mouse models of the disease in which inflammasomes contribute to pathology. Myeloid-cell-specific deletion of the rheumatoid arthritis susceptibility gene A20/Tnfaip3 in mice (A20(myel-KO) mice) triggers a spontaneous erosive polyarthritis that resembles rheumatoid arthritis in patients. Rheumatoid arthritis in A20(myel-KO) mice is not rescued by deletion of tumour necrosis factor receptor 1 (ref. 2). Here we show, however, that it crucially relies on the Nlrp3 inflammasome and interleukin-1 receptor signalling. Macrophages lacking A20 have increased basal and lipopolysaccharide-induced expression levels of the inflammasome adaptor Nlrp3 and proIL-1ß. As a result, A20-deficiency in macrophages significantly enhances Nlrp3 inflammasome-mediated caspase-1 activation, pyroptosis and interleukin-1ß secretion by soluble and crystalline Nlrp3 stimuli. In contrast, activation of the Nlrc4 and AIM2 inflammasomes is not altered. Importantly, increased Nlrp3 inflammasome activation contributes to the pathology of rheumatoid arthritis in vivo, because deletion of Nlrp3, caspase-1 and the interleukin-1 receptor markedly protects against rheumatoid-arthritis-associated inflammation and cartilage destruction in A20(myel-KO) mice. These results reveal A20 as a novel negative regulator of Nlrp3 inflammasome activation, and describe A20(myel-KO) mice as the first experimental model to study the role of inflammasomes in the pathology of rheumatoid arthritis.

DPP8 and DPP9 inhibition induces pro-caspase-1-dependent monocyte and macrophage pyroptosis.

Val-boroPro (Talabostat, PT-100), a nonselective inhibitor of post-proline cleaving serine proteases, stimulates mammalian immune systems through an unknown mechanism of action. Despite this lack of mechanistic understanding, Val-boroPro has attracted substantial interest as a potential anticancer agent, reaching phase 3 trials in humans. Here we show that Val-boroPro stimulates the immune system by triggering a proinflammatory form of cell death in monocytes and macrophages known as pyroptosis. We demonstrate that the inhibition of two serine proteases, DPP8 and DPP9, activates the pro-protein form of caspase-1 independent of the inflammasome adaptor ASC. Activated pro-caspase-1 does not efficiently process itself or IL-1ß but does cleave and activate gasdermin D to induce pyroptosis. Mice lacking caspase-1 do not show immune stimulation after treatment with Val-boroPro. Our data identify what is to our knowledge the first small molecule that induces pyroptosis and reveals a new checkpoint that controls the activation of the innate immune system.

Interleukin-18 in intestinal inflammation: friend and foe?

Genetic studies and other experimental data have linked inflammatory bowel diseases with inflammasome activation. In this issue of Immunity, Zaki et al. (2010) and Dupaul-Chicoine et al. (2010) provide a detailed characterization of the regulatory task of the inflammasome in intestinal epithelial cells.

Control of intestinal homeostasis, colitis, and colitis-associated colorectal cancer by the inflammatory caspases.

Inflammatory caspases are essential effectors of inflammation and cell death. Here, we investigated their roles in colitis and colorectal cancer and report a bimodal regulation of intestinal homeostasis, inflammation and tumorigenesis by caspases-1 and -12. Casp1(-/-) mice exhibited defects in mucosal tissue repair and succumbed rapidly after dextran sulfate sodium administration. This phenotype was rescued by administration of exogenous interleukin-18 and was partially reproduced in mice deficient in the inflammasome adaptor ASC. Casp12(-/-) mice, in which the inflammasome is derepressed, were resistant to acute colitis and showed signs of enhanced repair. Together with their increased inflammatory response, the enhanced repair response of Casp12(-/-) mice rendered them more susceptible to colorectal cancer induced by azoxymethane (AOM)+DSS. Taken together, our results indicate that the inflammatory caspases are critical in the induction of inflammation in the gut after injury, which is necessary for tissue repair and maintenance of immune tolerance.

Ablation of caspase-1 protects against TBI-induced pyroptosis in vitro and in vivo.

BACKGROUND: Traumatic brain injury (TBI) is a critical public health and socioeconomic problem throughout the world. Inflammation-induced secondary injury is one of the vital pathogenic parameters of TBI. Molecular signaling cascades of pyroptosis, a specific type of cellular necrosis, are key drivers of TBI-induced inflammation. METHODS: In this study, mice with genetically ablated caspase-1 (caspase-1-/-) were subjected to controlled cortical impact injury in vivo, and primary neuron deficient in caspase-1 through siRNA knockdown and pharmacologic inhibition was stimulated by mechanical scratch, equiaxial stretch, and LPS/ATP in vitro. We evaluated the effects of caspase-1 deficiency on neurological deficits, inflammatory factors, histopathology, cell apoptosis, and pyroptosis. RESULTS: During the acute post-injury period (0-48 h) in vivo, motor deficits, anti-inflammatory cytokines (TGF-ß and IL-10), pro-inflammatory cytokines (IFN-γ, IL-1ß, and IL-18), and blood lactate dehydrogenase (LDH), as well as pyroptosis-related proteins (caspase-1, caspase-1 fragments, caspase-11 and GSDMD), were increased. Caspase-1 was activated in the cortex of TBI mice. Inflammatory activation was more profound in injured wild-type mice than in caspase-1-/- mice. In vitro, mechanical scratch, equiaxial stretch, and LPS/ATP-induced neuron pyroptosis, apoptosis, LDH release, and increased expression of inflammatory factors. The effects of mechanical and inflammatory stress were reduced through inhibition of caspase-1 activity through siRNA knockdown and pharmacologic inhibition. CONCLUSION: Collectively, these data demonstrate that pyroptosis is involved in neuroinflammation and neuronal injury after TBI, and ablation of caspase-1 inhibits TBI-induced pyroptosis. Our findings suggest that caspase-1 may be a potential target for TBI therapy.

Human Parvovirus Infection of Human Airway Epithelia Induces Pyroptotic Cell Death by Inhibiting Apoptosis.

Human bocavirus 1 (HBoV1) is a human parvovirus that causes acute respiratory tract infections in young children. In this study, we confirmed that, when polarized/well-differentiated human airway epithelia are infected with HBoV1 in vitro, they develop damage characterized by barrier function disruption and cell hypotrophy. Cell death mechanism analyses indicated that the infection induced pyroptotic cell death characterized by caspase-1 activation. Unlike infections with other parvoviruses, HBoV1 infection did not activate the apoptotic or necroptotic cell death pathway. When the NLRP3-ASC-caspase-1 inflammasome-induced pathway was inhibited by short hairpin RNA (shRNA), HBoV1-induced cell death dropped significantly; thus, NLRP3 mediated by ASC appears to be the pattern recognition receptor driving HBoV1 infection-induced pyroptosis. HBoV1 infection induced steady increases in the expression of interleukin 1α (IL-1α) and IL-18. HBoV1 infection was also associated with the marked expression of the antiapoptotic genes BIRC5 and IFI6 When the expression of BIRC5 and/or IFI6 was inhibited by shRNA, the infected cells underwent apoptosis rather than pyroptosis, as indicated by increased cleaved caspase-3 levels and the absence of caspase-1. BIRC5 and/or IFI6 gene inhibition also significantly reduced HBoV1 replication. Thus, HBoV1 infection of human airway epithelial cells activates antiapoptotic proteins that suppress apoptosis and promote pyroptosis. This response may have evolved to confer a replicative advantage, thus allowing HBoV1 to establish a persistent airway epithelial infection. This is the first report of pyroptosis in airway epithelia infected by a respiratory virus.IMPORTANCE Microbial infection of immune cells often induces pyroptosis, which is mediated by a cytosolic protein complex called the inflammasome that senses microbial pathogens and then activates the proinflammatory cytokines IL-1 and IL-18. While virus-infected airway epithelia often activate NLRP3 inflammasomes, studies to date suggest that these viruses kill the airway epithelial cells via the apoptotic or necrotic pathway; involvement of the pyroptosis pathway has not been reported previously. Here, we show for the first time that virus infection of human airway epithelia can also induce pyroptosis. Human bocavirus 1 (HBoV1), a human parvovirus, causes lower respiratory tract infections in young children. This study indicates that HBoV1 kills airway epithelial cells by activating genes that suppress apoptosis and thereby promote pyroptosis. This strategy appears to promote HBoV1 replication and may have evolved to allow HBoV1 to establish persistent infection of human airway epithelia.

NLRP3 (Nucleotide Oligomerization Domain-Like Receptor Family, Pyrin Domain Containing 3)-Caspase-1 Inflammasome Degrades Contractile Proteins: Implications for Aortic Biomechanical Dysfunction and Aneurysm and Dissection Formation.

OBJECTIVE: Increasing evidence suggests that contractile dysfunction in smooth muscle cells (SMCs) plays a critical role in aortic biomechanical dysfunction and aortic aneurysm and dissection (AAD) development. However, the mechanisms underlying SMC contractile dysfunction in sporadic AAD are poorly understood. In this study, we examined the role of the NLRP3 (nucleotide oligomerization domain-like receptor family, pyrin domain containing 3)-caspase-1 inflammasome, a key inflammatory cascade, in SMC contractile dysfunction in AAD. APPROACH AND RESULTS: We observed significant SMC contractile protein degradation in aortas from patients with sporadic thoracic AAD. The contractile protein degradation was associated with activation of the NLRP3-caspase-1 inflammasome cascade. In SMCs, caspase-1 bound and directly cleaved and degraded contractile proteins, leading to contractile dysfunction. Furthermore, Nlrp3 or caspase-1 deficiency in mice significantly reduced angiotensin II-induced contractile protein degradation, biomechanical dysfunction, and AAD formation in both thoracic and abdominal aortas. Finally, blocking this cascade with the inflammasome inhibitor, glyburide (an antidiabetic medication), reduced angiotensin II-induced AAD formation. CONCLUSIONS: Inflammasome-caspase-1-mediated degradation of SMC contractile proteins may contribute to aortic biomechanical dysfunction and AAD development. This cascade may be a therapeutic target in AAD formation. In addition, glyburide may have protective effects against AAD development.