Lee Hopkins

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

Significance The nucleotide-binding oligomerization domain-like receptor (NLR) family members, NLRC4 and NLRP3, activate the inflammasome to provide host defenses against infection. The precise molecular constituents of an inflammasome are unknown; however, it is believed that receptor-specific complexes containing apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) and caspase-1 are formed. Here, we used confocal and superresolution microscopy to show that in macrophages infected with Salmonella Typhimurium, a pathogen that activates two distinct NLRs, ASC forms an outer ring-like structure that comprises NLRC4, NLRP3, caspase-1, caspase-8, and pro–IL-1β within the same macromolecular complex. These results suggest that the inflammasome is a highly dynamic macromolecular protein complex capable of recruiting different NLRs and effectors to coordinate inflammasome responses to infection. 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.

Salmonella Infection Induces Recruitment of Caspase-8 to the Inflammasome To Modulate IL-1β Production

Nucleotide-binding oligomerization domain–like receptors (NLRs) detect pathogens and danger-associated signals within the cell. Salmonella enterica serovar Typhimurium, an intracellular pathogen, activates caspase-1 required for the processing of the proinflammatory cytokines, pro–IL-1β and pro–IL-18, and pyroptosis. In this study, we show that Salmonella infection induces the formation of an apoptosis-associated specklike protein containing a CARD (ASC)–Caspase-8–Caspase-1 inflammasome in macrophages. Caspase-8 and caspase-1 are recruited to the ASC focus independently of one other. Salmonella infection initiates caspase-8 proteolysis in a manner dependent on NLRC4 and ASC, but not NLRP3, caspase-1 or caspase-11. Caspase-8 primarily mediates the synthesis of pro-IL-1β, but is dispensable for Salmonella-induced cell death. Overall, our findings highlight that the ASC inflammasome can recruit different members of the caspase family to induce distinct effector functions in response to Salmonella infection.

Proteinase K-sensitive disease-associated ovine prion protein revealed by conformation-dependent immunoassay

PrPSc [abnormal disease-specific conformation of PrP (prion-related protein)] accumulates in prion-affected individuals in the form of amorphous aggregates. Limited proteolysis of PrPSc results in a protease-resistant core of PrPSc of molecular mass of 27-30 kDa (PrP27-30). Aggregated forms of PrP co-purify with prion infectivity, although infectivity does not always correlate with the presence of PrP27-30. This suggests that discrimination between PrPC (normal cellular PrP) and PrPSc by proteolysis may underestimate the repertoire and quantity of PrPSc subtypes. We have developed a CDI (conformation-dependent immunoassay) utilizing time-resolved fluorescence to study the conformers of disease-associated PrP in natural cases of sheep scrapie, without using PK (proteinase K) treatment to discriminate between PrPC and PrPSc. The capture-detector CDI utilizes N-terminal- and C-terminal-specific anti-PrP monoclonal antibodies that recognize regions of the prion protein differentially buried or exposed depending on the extent of denaturation of the molecule. PrPSc was precipitated from scrapie-infected brain stem and cerebellum tissue following sarkosyl extraction, with or without the use of sodium phosphotungstic acid, and native and denatured PrPSc detected by CDI. PrPSc was detectable in brain tissue from homozygous VRQ (V136 R154 Q171) and ARQ (A136 R154 Q171) scrapie-infected sheep brains. The highest levels of PrPSc were found in homozygous VRQ scrapie-infected brains. The quantity of PrPSc was significantly reduced, up to 90% in some cases, when samples were treated with PK prior to the CDI. Collectively, our results show that the level of PrPSc in brain samples from cases of natural scrapie display genotypic differences and that a significant amount of this material is PK-sensitive.

Allergens as Immunomodulatory Proteins: The Cat Dander Protein Fel d 1 Enhances TLR Activation by Lipid Ligands

Allergic responses can be triggered by structurally diverse allergens. Most allergens are proteins, yet extensive research has not revealed how they initiate the allergic response and why the myriad of other inhaled proteins do not. Among these allergens, the cat secretoglobulin protein Fel d 1 is a major allergen and is responsible for severe allergic responses. In this study, we show that similar to the mite dust allergen Der p 2, Fel d 1 substantially enhances signaling through the innate receptors TLR4 and TLR2. In contrast to Der p 2, however, Fel d 1 does not act by mimicking the TLR4 coreceptor MD2 and is not able to bind stably to the TLR4/MD2 complex in vitro. Fel d 1 does, however, bind to the TLR4 agonist LPS, suggesting that a lipid transfer mechanism may be involved in the Fel d 1 enhancement of TLR signaling. We also show that the dog allergen Can f 6, a member of a distinct class of lipocalin allergens, has very similar properties to Fel d 1. We propose that Fel d 1 and Can f 6 belong to a group of allergen immunomodulatory proteins that enhance innate immune signaling and promote airway hypersensitivity reactions in diseases such as asthma.

Emergence of multiple prion strains from single isolates of ovine scrapie

The infectious agent associated with prion diseases such as ovine scrapie shows strain diversity. Ovine prion strains have typically been identified by their transmission properties in wild-type mice. However, strain typing of ovine scrapie isolates in wild-type mice may not reveal properties of the infectious prion agent as they exist in the original host. This could be circumvented if ovine scrapie isolates are passaged in ovine prion protein (PrP)-transgenic mice. This study used incubation time, lesion profile, immunohistochemistry of the disease-associated PrP (PrP(Sc)) and molecular profile to compare the range of ovine prion strains that emerged from sheep scrapie isolates following serial passage in wild-type and ovine PrP transgenic mice. It was found that a diverse range of ovine prion strains emerged from homozygous ARQ and VRQ scrapie isolates passaged in wild-type and ovine PrP transgenic mice. However, strain-specific PrP(Sc) deposition and PrP27-30 molecular profile patterns were identified in ovine PrP transgenic mice that were not detected in wild-type mice. Significantly, it was established that the individual mouse brain selected for transmission during prion strain typing had a significant influence on strain definition. Serial passage of short- and long-incubation-time animals from the same group of scrapie-inoculated mice revealed different prion strain phenotypes. These observations are consistent with the possibility that some scrapie isolates contain more than one prion strain.

Mitochondrial damage contributes to Pseudomonas aeruginosa activation of the inflammasome and is downregulated by autophagy

The nucleotide-binding domain, leucine-rich repeat containing family caspase recruitment domain containing 4 (NLRC4) inflammasome can be activated by pathogenic bacteria via products translocated through the microbial type III secretion apparatus (T3SS). Recent work has shown that activation of the NLRP3 inflammasome is downregulated by autophagy, but the influence of autophagy on NLRC4 activation is unclear. We set out to determine how autophagy might influence this process, using the bacterium Pseudomonas aeruginosa, which activates the NLRC4 inflammasome via its T3SS. Infection resulted in T3SS-dependent mitochondrial damage with increased production of reactive oxygen intermediates and release of mitochondrial DNA. Inhibiting mitochondrial reactive oxygen release or degrading intracellular mitochondrial DNA abrogated NLRC4 inflammasome activation. Moreover, macrophages lacking mitochondria failed to activate NLRC4 following infection. Removal of damaged mitochondria by autophagy significantly attenuated NLRC4 inflammasome activation. Mitochondrial DNA bound specifically to NLRC4 immunoprecipitates and transfection of mitochondrial DNA directly activated the NLRC4 inflammasome; oxidation of the DNA enhanced this effect. Manipulation of autophagy altered the degree of inflammasome activation and inflammation in an in vivo model of P. aeruginosa infection. Our results reveal a novel mechanism contributing to NLRC4 activation by P. aeruginosa via mitochondrial damage and release of mitochondrial DNA triggered by the bacterial T3SS that is downregulated by autophagy.

Molecular and Transmission Characteristics of Primary-Passaged Ovine Scrapie Isolates in Conventional and Ovine PrP Transgenic Mice

ABSTRACT A more complete assessment of ovine prion strain diversity will be achieved by complementing biological strain typing in conventional and ovine PrP transgenic mice with a biochemical analysis of the resultant PrPSc. This will provide a correlation between ovine prion strain phenotype and the molecular nature of different PrP conformers associated with particular prion strains. Here, we have compared the molecular and transmission characteristics of ovine ARQ/ARQ and VRQ/VRQ scrapie isolates following primary passage in tg338 (VRQ) and tg59 (ARQ) ovine PrP transgenic mice and the conventional mouse lines C57BL/6 (Prnpa), RIII (Prnpa), and VM (Prnpb). Our data show that these different genotypes of scrapie isolates display similar incubation periods of >350 days in conventional and tg59 mice. Facilitated transmission of sheep scrapie isolates occurred in tg338 mice, with incubation times reduced to 64 days for VRQ/VRQ inocula and to ≤210 days for ARQ/ARQ samples. Distinct genotype-specific lesion profiles were seen in the brains of conventional and tg59 mice with prion disease, which was accompanied by the accumulation of more conformationally stable PrPSc, following inoculation with ARQ/ARQ compared to VRQ/VRQ scrapie isolates. In contrast, the lesion profiles, quantities, and stability of PrPSc induced by the same inocula in tg338 mice were more similar than in the other mouse lines. Our data show that primary transmission of different genotypes of ovine prions is associated with the formation of different conformers of PrPSc with distinct molecular properties and provide the basis of a molecular approach to identify the true diversity of ovine prion strains.

Mouse-Adapted Ovine Scrapie Prion Strains Are Characterized by Different Conformers of PrPSc

ABSTRACT The agent responsible for prion disease may exist in different forms, commonly referred to as strains, with each carrying the specific information that determines its own distinct biological properties, such as incubation period and lesion profile. Biological strain typing of ovine scrapie isolates by serial passage in conventional mice has shown some diversity in ovine prion strains. However, this biological diversity remains poorly supported by biochemical prion strain typing. The protein-only hypothesis predicts that variation between different prion strains in the same host is manifest in different conformations adopted by PrPSc. Here we have investigated the molecular properties of PrPSc associated with two principal Prnpa mouse-adapted ovine scrapie strains, namely, RML and ME7, in order to establish biochemical prion strain typing strategies that may subsequently be used to discriminate field cases of mouse-passaged ovine scrapie isolates. We used a conformation-dependent immunoassay and a conformational stability assay, together with Western blot analysis, to demonstrate that RML and ME7 PrPSc proteins show distinct biochemical and physicochemical properties. Although RML and ME7 PrPSc proteins showed similar resistance to proteolytic digestion, they differed in their glycoform profiles and levels of proteinase K (PK)-sensitive and PK-resistant isoforms. In addition, the PK-resistant core (PrP27-30) of ME7 was conformationally more stable following exposure to guanidine hydrochloride or Sarkosyl than was RML PrP27-30. Our data show that mouse-adapted ovine scrapie strains can be discriminated by their distinct conformers of PrPSc, which provides a basis to investigate their diversity at the molecular level.

The stability and aggregation of ovine prion protein associated with classical and atypical scrapie correlates with the ease of unwinding of helix-2.

Susceptibility to scrapie disease in sheep, the archetypal prion disease, correlates with polymorphisms within the ovine PrP (prion-related protein) gene. The VRQ (Val136Arg154Gln171) and AL141RQ (Ala136Leu141Arg154Gln171) allelic variants are associated with classical scrapie, whereas the ARR (Ala136Arg154Arg171), AF141RQ (Ala136Phe141Arg154Gln171) and AHQ (Ala136His154Gln171) allelic variants are associated with atypical scrapie. Recent studies have suggested that there are differences in the stability of PrPSc (abnormal disease-specific conformation of PrP) associated with these different forms of scrapie. To address which structural features of ovine PrP may contribute to this difference, in the present study we have investigated the conformational stability and susceptibility to aggregation of allelic variants of ovine PrP associated with classical or atypical scrapie. We find that the melting temperature of ovine recombinant VRQ and AL141RQ PrP is higher than that of AF141RQ, AHQ and ARR. In addition, monoclonal-antibody studies show that the region around helix-1 of VRQ and AL141RQ is less accessible compared with other ovine PrP allelic variants. Furthermore, the extent of both the structural change to copper-ion-treatment and denaturant-induced aggregation was reduced in PrP associated with atypical scrapie compared with PrP associated with classical scrapie. Through the use of molecular dynamics simulations we have found that these biochemical and biophysical properties of ovine PrP correlate with the ease of unwinding of helix-2 and a concurrent conformational change of the helix-2-helix-3 loop. These results reveal significant differences in the overall stability and potential for aggregation of different allelic variants of ovine PrP and consequently have implications for the differences in stability of PrPSc associated with classical and atypical scrapie.

Ovine plasma prion protein levels show genotypic variation detected by C-terminal epitopes not exposed in cell-surface PrPC

Ovine PBMCs (peripheral blood mononuclear cells) express PrP(C) [cellular PrP (prion-related protein)] and have the potential to harbour and release disease-associated forms of PrP during scrapie in sheep. Cell-surface PrP(C) expression by PBMCs, together with plasma PrP(C) levels, may contribute to the regulatory mechanisms that determine susceptibility and resistance to natural scrapie in sheep. Here, we have correlated cell-surface PrP(C) expression on normal ovine PBMCs by FACS with the presence of PrP(C) in plasma measured by capture-detector immunoassay. FACS showed similar levels of cell-surface PrP(C) on homozygous ARR (Ala136-Arg154-Arg171), ARQ (Ala136-Arg154-Gln171) and VRQ (Val136-Arg154-Gln171) PBMCs. Cell-surface ovine PrP(C) showed modulation of N-terminal epitopes, which was more evident on homozygous ARR cells. Ovine plasma PrP(C) levels showed genotypic variation and the protein displayed C-terminal epitopes not available in cell-surface PrP(C). Homozygous VRQ sheep showed the highest plasma PrP(C) level and homozygous ARR animals the lowest. For comparison, similar analyses were performed on normal bovine PBMCs and plasma. PrP(C) levels in bovine plasma were approx. 4-fold higher than ovine homozygous ARQ plasma despite similar levels of PBMC cell-surface PrP(C) expression. Immunoassays using C-terminal-specific anti-PrP monoclonal antibodies as capture and detector reagents revealed the highest level of PrP(C) in both ovine and bovine plasma, whilst lower levels were detected using N-terminal-specific monoclonal antibody FH11 as the capture reagent. This suggested that a proportion of plasma PrP(C) was N-terminally truncated. Our results indicate that the increased susceptibility to natural scrapie displayed by homozygous VRQ sheep correlates with a higher level of plasma PrP(C).