Lipopolysaccharide (LPS) of Gram-negative bacteria can elicit a strong immune response. Although extracellular LPS is sensed by TLR4 at the cell surface and triggers a transcriptional response, ...cytosolic LPS binds and activates non-canonical inflammasome caspases, resulting in pyroptotic cell death, as well as canonical NLRP3 inflammasome-dependent cytokine release. Contrary to the highly regulated multiprotein platform required for caspase-1 activation in the canonical inflammasomes, the non-canonical mouse caspase-11 and the orthologous human caspase-4 function simultaneously as innate sensors and effectors, and their regulation is unclear. Here we show that the oxidized phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (oxPAPC) inhibits the non-canonical inflammasome in macrophages, but not in dendritic cells. Aside from a TLR4 antagonistic role, oxPAPC binds directly to caspase-4 and caspase-11, competes with LPS binding, and consequently inhibits LPS-induced pyroptosis, IL-1β release and septic shock. Therefore, oxPAPC and its derivatives might provide a basis for therapies that target non-canonical inflammasomes during Gram-negative bacterial sepsis.
Inflammasomes are protein platforms linking recognition of microbe, pathogen-associated and damage-associated molecular patterns by cytosolic sensory proteins to caspase-1 activation. Caspase-1 ...promotes pyroptotic cell death and the maturation and secretion of interleukin (IL)-1β and IL-18, which trigger inflammatory responses to clear infections and initiate wound-healing; however, excessive responses cause inflammatory disease. Inflammasome assembly requires the PYRIN domain (PYD)-containing adaptor ASC, and depends on PYD-PYD interactions. Here we show that the PYD-only protein POP2 inhibits inflammasome assembly by binding to ASC and interfering with the recruitment of ASC to upstream sensors, which prevents caspase-1 activation and cytokine release. POP2 also impairs macrophage priming by inhibiting the activation of non-canonical IκB kinase ɛ and IκBα, and consequently protects from excessive inflammation and acute shock in vivo. Our findings advance our understanding of the complex regulatory mechanisms that maintain a balanced inflammatory response and highlight important differences between individual POP members.
Human papilloma virus-like particles (HPV VLP) serve as the basis of the current licensed vaccines for HPV. We have previously shown that encapsidation of DNA expressing the model antigen M/M2 from ...respiratory syncytial virus (RSV) in HPV pseudovirions (PsV) is immunogenic when delivered intravaginally. Because the HPV capsids confer tropism for basal epithelium, they represent attractive carriers for vaccination targeted to the skin using microneedles. In this study we asked: 1) whether HPV16 VLP administered by microneedles could induce protective immune responses to HPV16 and 2) whether HPV16 PsV-encapsidated plasmids delivered by microneedles could elicit immune responses to both HPV and the antigen delivered by the transgene. Mice immunized with HPV16 VLP coated microneedles generated robust neutralizing antibody responses and were protected from HPV16 challenge. Microneedle arrays coated with HPV16-M/M2 or HPV16-F protein (genes of RSV) were then tested and dose-dependent HPV and F-specific antibody responses were detected post-immunization, and M/M2-specific T-cell responses were detected post RSV challenge, respectively. HPV16 PsV-F immunized mice were fully protected from challenge with HPV16 PsV and had reduced RSV viral load in lung and nose upon intranasal RSV challenge. In summary, HPV16 PsV-encapsidated DNA delivered by microneedles induced neutralizing antibody responses against HPV and primed for antibody and T-cell responses to RSV antigens encoded by the encapsidated plasmids. Although the immunogenicity of the DNA component was just above the dose response threshold, the HPV-specific immunity was robust. Taken together, these data suggest microneedle delivery of lyophilized HPV PsV could provide a practical, thermostable combined vaccine approach that could be developed for clinical evaluation.
Murine T cells exposed to rapamycin maintain flexibility towards Th1/Tc1 differentiation, thereby indicating that rapamycin promotion of regulatory T cells (Tregs) is conditional. The degree to which ...rapamycin might inhibit human Th1/Tc1 differentiation has not been evaluated. In the presence of rapamycin, T cell costimulation and polarization with IL-12 or IFN-α permitted human CD4+ and CD8+ T cell differentiation towards a Th1/Tc1 phenotype; activation of STAT1 and STAT4 pathways essential for Th1/Tc1 polarity was preserved during mTOR blockade but instead abrogated by PI3 kinase inhibition. Such rapamycin-resistant human Th1/Tc1 cells: (1) were generated through autophagy (increased LC3BII expression; phenotype reversion by autophagy inhibition via 3-MA or siRNA for Beclin1); (2) expressed anti-apoptotic bcl-2 family members (reduced Bax, Bak; increased phospho-Bad); (3) maintained mitochondrial membrane potentials; and (4) displayed reduced apoptosis. In vivo, type I polarized and rapamycin-resistant human T cells caused increased xenogeneic graft-versus-host disease (x-GVHD). Murine recipients of rapamycin-resistant human Th1/Tc1 cells had: (1) persistent T cell engraftment; (2) increased T cell cytokine and cytolytic effector function; and (3) T cell infiltration of skin, gut, and liver. Rapamycin therefore does not impair human T cell capacity for type I differentiation. Rather, rapamycin yields an anti-apoptotic Th1/Tc1 effector phenotype by promoting autophagy.
Intracellular sensing of stress and danger signals initiates inflammatory innate immune responses by triggering inflammasome assembly, caspase-1 activation and pyroptotic cell death as well as the ...release of interleukin 1β (IL-1β), IL-18 and danger signals. NLRP3 broadly senses infectious patterns and sterile danger signals, resulting in the tightly coordinated and regulated assembly of the NLRP3 inflammasome, but the precise mechanisms are incompletely understood. Here, we identified NLRP11 as an essential component of the NLRP3 inflammasome in human macrophages. NLRP11 interacted with NLRP3 and ASC, and deletion of NLRP11 specifically prevented NLRP3 inflammasome activation by preventing inflammasome assembly, NLRP3 and ASC polymerization, caspase-1 activation, pyroptosis and cytokine release but did not affect other inflammasomes. Restored expression of NLRP11, but not NLRP11 lacking the PYRIN domain (PYD), restored inflammasome activation. NLRP11 was also necessary for inflammasome responses driven by NLRP3 mutations that cause cryopyrin-associated periodic syndrome (CAPS). Because NLRP11 is not expressed in mice, our observations emphasize the specific complexity of inflammasome regulation in humans.
The PYRIN domain (PYD) is a protein–protein interaction domain, which belongs to the death domain fold (DDF) superfamily. It is best known for its signaling function in innate immune responses and ...particularly in the assembly of inflammasomes, which are large protein complexes that allow the induced proximity-mediated activation of caspase-1 and subsequently the release of pro-inflammatory cytokines. The molecular mechanism of inflammasome assembly was only recently elucidated and specifically requires PYD oligomerization. Here we discuss the recent advances in our understanding of PYD signaling and its regulation by PYD-only proteins.
The innate immune system responds to infection and tissue damage by activating cytosolic sensory complexes called 'inflammasomes'. Cytosolic DNA is sensed by AIM2-like receptors (ALRs) during ...bacterial and viral infections and in autoimmune diseases. Subsequently, recruitment of the inflammasome adaptor ASC links ALRs to the activation of caspase-1. A controlled immune response is crucial for maintaining homeostasis, but the regulation of ALR inflammasomes is poorly understood. Here we identified the PYRIN domain (PYD)-only protein POP3, which competes with ASC for recruitment to ALRs, as an inhibitor of DNA virus-induced activation of ALR inflammasomes in vivo. Data obtained with a mouse model with macrophage-specific POP3 expression emphasize the importance of the regulation of ALR inflammasomes in monocytes and macrophages.
Inflammasomes are intracellular multiprotein signaling complexes that link Pathogen Associated and Danger Associated Molecular Patterns (PAMPs and DAMPs) by Pattern Recognition Receptors (PRRs) to ...the activation of Caspase-1, leading to the secretion of pro-inflammatory cytokines interleukin (IL)-1β and IL-18, and the induction of pyroptosis. Nucleotide-binding oligomerization domain (NOD)-like containing a PYRIN domain (PYD) 3 (NLRP3) is the most well characterized inflammasome forming PRR and recruits the common adaptor protein, apoptosis-associated speck like protein containing a caspase recruitment domain, (ASC), to activate caspase-1 at the mitochondria. Dysfunction of NLRP3 is involved in microbial infections, metabolic and inflammatory diseases, and cryopyrinopathies. Potassium efflux is thought to be a unifying step that is essential for NLRP3 inflammasome activation induced by various stimuli. Despite extensive study of this protein, the exact molecular mechanism leading to NLRP3 activation remains elusive. Here I report the identification of a protein evolved specifically in humans, NLRP11, as an essential modulator for NLRP3 inflammasome activation in human monocytes and macrophages. NLRP11 functions as an NLRP3-binding protein that acts downstream of potassium efflux to regulate NLRP3 oligomerization and activation. In the absence of NLRP11, ASC recruitment, caspase-1 activation, and IL-1β secretion are abrogated. Upon activation, NLRP3-NLRP11 interact and subsequently form a high NLRP3-NLRP11 molecular weight complex. These studies demonstrate that NLRP11 is essential for NLRP3 inflammasome activation and may serve as a potential therapeutic target for various inflammatory disorders.
Specific recognition of DNA by transcription factors is essential for precise gene regulation. In Wingless (Wg) signaling in Drosophila, target gene regulation is controlled by T cell factor (TCF), ...which binds to specific DNA sequences through a high mobility group (HMG) domain 1. However, there is considerable variability in TCF binding sites 2–5, raising the possibility that they are not sufficient for target location. Some isoforms of human TCF contain a domain, termed the C-clamp, that mediates binding to an extended sequence in vitro 6. However, the significance of this extended sequence for the function of Wnt response elements (WREs) is unclear. In this report, we identify a cis-regulatory element that, to our knowledge, was previously unpublished. The element, named the TCF Helper site (Helper site), is essential for the activation of several WREs. This motif greatly augments the ability of TCF binding sites to respond to Wg signaling. Drosophila TCF contains a C-clamp that enhances in vitro binding to TCF-Helper site pairs and is required for transcriptional activation of WREs containing Helper sites. A genome-wide search for clusters of TCF and Helper sites identified two new WREs. Our data suggest that DNA recognition by fly TCF occurs through a bipartite mechanism, involving both the HMG domain and the C-clamp, which enables TCF to locate and activate WREs in the nucleus.
Abstract
The innate immune system responds to infections and tissue damage by activating cytosolic sensory complexes called inflammasomes. Cytosolic DNA is sensed by AIM2-like receptors (ALRs) during ...bacterial and viral infections and in autoimmune diseases. Subsequently, recruitment of the adaptor protein ASC through PYRIN domain (PYD)-PYD interaction, links ALRs to the activation of caspase-1 and subsequent maturation of the proinflammatory cytokines IL-1β IL-18 and induction of pyroptotic cell death. A controlled inflammasome response is crucial for maintaining homeostasis, whereas excessive and uncontrolled cytokine production contributes to autoinflammatory diseases. However, ALR inflammasome regulation is poorly understood. Here, we identified the type I interferon (IFN)-inducible PYD-only protein 3 (POP3) as a binding partner of ALRs. POP3 competes with ASC for recruitment to ALRs and thereby inhibits DNA virus-induced ALR inflammasome activation and IL-18 and IFN-γ-dependent host defense in vivo. A mouse model with macrophage-specific POP3 expression recapitulates global AIM2 deficiency and thus, emphasizes the importance of ALR inflammasome regulation in the monocytic/macrophage lineage. Collectively, we show that POP3 represents one of the type I IFN response factors that regulate human and mouse ALR inflammasomes in macrophages within a type I IFN-regulatory loop.
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