Pathogen-triggered activation of the inflammasome complex leading to caspase-1 activation and IL-1␤ production involves similar sensor proteins between mouse and human. However, the specific sensors used may differ between infectious agents and host species. In mice, Francisella infection leads to seemingly exclusive activation of the Aim2 inflammasome with no apparent role for Nlrp3. Here we examine the IL-1␤ response of human cells to Francisella infection. Francisella strains exhibit differences in IL-1␤ production by influencing induction of IL-1␤ and ASC transcripts. Unexpectedly, our results demonstrate that Francisella activates the NLRP3 inflammasome in human cells. Innate immune responses to pathogens are initiated by recognition of pathogen-associated molecular patterns by both extracellular and intracellular sensors (1-3). Pathogen-associated molecular pattern recognition by members of the Toll-like receptor (TLR) 3 family result in the activation of the NF-B, MAPK, and/or interferon regulatory factor signaling pathways depending on the specific TLR engaged (1, 4 -8). Intracellular receptors of the MAVS/RIG-I family act similarly and are involved in recognition of viral nucleic acids (9, 10). Thus, production of inflammatory cytokines and chemokines such as TNF␣, IL-6, and MCP-1 in response to infection generally follows activation via these receptors. The inflammatory cytokines IL-1␤ and IL-18, however, require processing by caspase-1 to produce their active forms (11). Pathogen-associated molecular patterns and danger-associated molecular patterns activate various members of the nucleotide binding leucine-rich receptor (NLR) family in the cytoplasm, resulting in the assembly of an NLR containing, multiprotein complex (the inflammasome) that recruits and activates caspase-1 leading to IL-1␤ processing (12, 13). Although CARD domain-containing NLRs (e.g. Ipaf) can directly interact with caspase-1, most inflammasomes are assembled by Pyrin domain containing NLRs (NLRPs), which recruit caspase-1 indirectly through the adapter molecule ASC (14).Francisella tularensis is the causative agent of tularemia and a potential bioweapon (15). Pulmonary infection with even a single, virulent F. tularensis bacterium is potentially lethal if untreated (16,17). For humans, the type A strain, SchuS4 (F. tularensis sp. tularensis) results in the highest mortality, whereas neither the attenuated type B live vaccine strain LVS (F. tularensis sp. holarctica) or the U112 strain (Francisella tularensis sp. novicida) are virulent. In mice however, the SchuS4, LVS and U112 strains are lethal (18). Because of its importance in controlling bacterial infection and promoting adaptive immunity, the innate immune response to F. tularensis has been an area of recent interest. In mouse models of tularemia, the macrophage response to F. tularensis LVS is heavily reliant upon TLR2 as TLR2-deficient macrophages fail to produce TNF␣, IL-6, and other NF-B dependent proinflammatory cytokines (19,20). Mouse macrophages infected with U112 ...

Pyrin domain-only proteins (POPs) are recently evolved, primate-specific proteins demonstrated in vitro as negative regulators of inflammatory responses. However, their in vivo function is not understood. Of the four known POPs, only POP2 is reported to regulate NF-κB-dependent transcription and multiple inflammasomes. Here we use a transgenic mouse-expressing POP2 controlled by its endogenous human promotor to study the immunological functions of POP2. Despite having significantly reduced inflammatory cytokine responses to LPS and bacterial infection, POP2 transgenic mice are more resistant to bacterial infection than wild-type mice. In a pulmonary tularaemia model, POP2 enhances IFN-γ production, modulates neutrophil numbers, improves macrophage functions, increases bacterial control and diminishes lung pathology. Thus, unlike other POPs thought to diminish innate protection, POP2 reduces detrimental inflammation while preserving and enhancing protective immunity. Our findings suggest that POP2 acts as a high-order regulator balancing cellular function and inflammation with broad implications for inflammation-associated diseases and therapeutic intervention.

Pseudogenes are duplicated yet defunct copies of functional parent genes. However, some pseudogenes have gained or retained function. In this study we consider a functional role for the NLRP2-related, higher primate specific, processed pseudogene NLRP2P, which is closely related to Pyrin-only protein 2 (POP2/PYDC2), a regulator of NF-κB and the inflammasome. The NLRP2P open reading frame on chromosome X has features consistent with a processed pseudogene (retrotransposon), yet encodes a 45 amino acid, Pyrin-domain related protein. The open reading frame of NLRP2P shares 80% identity with POP2 and is under purifying selection across Old World primates. Although widely expressed, NLRP2P mRNA is upregulated by LPS in human monocytic cells. Functionally, NLRP2P impairs NF-κB p65 transactivation by reducing activating phosphorylation of RelA/p65. Reminiscent of POP2, NLRP2P reduces production of the NF-κB-dependent cytokines TNFα and IL-6 following TLR stimulation. In contrast to POP2, NLRP2P fails to inhibit the ASC-dependent NLRP3 inflammasome. In addition, beyond regulating cytokine production, NLRP2P has a potential role in cell cycle regulation and cell death. Collectively, our findings suggest that NLRP2P is a resurrected processed pseudogene that regulates NF-κB RelA/p65 activity and thus represents the newest member of the POP family, POP4.

Francisella tularensis (Ft) causes a frequently fatal, acute necrotic pneumonia in humans and animals. Following lethal Ft infection in mice, infiltration of the lungs by predominantly immature myeloid cells and subsequent myeloid cell death drive pathogenesis and host mortality. However, following sub-lethal Ft challenge, more mature myeloid cells are elicited and are protective. In addition, inflammasome-dependent IL-1β and IL-18 are important for protection. As Nlrp3 appears dispensable for resistance to infection with Francisella novicida, we considered its role during infection with the virulent Type A strain SchuS4 and the attenuated Type B live vaccine strain LVS. Here we show that both in vitro macrophage and in vivo IL-1β and IL-18 responses to Ft LVS and SchuS4 involve both the Aim2 and Nlrp3 inflammasomes. However, following lethal infection with Francisella, IL-1r-, Caspase-1/11-, Asc- and Aim2-deficient mice exhibited increased susceptibility as expected, while Nlrp3-deficient mice were more resistant. Despite reduced levels of IL-1β and IL-18, in the absence of Nlrp3, Ft infected mice have dramatically reduced lung pathology, diminished recruitment and death of immature myeloid cells, and reduced bacterial burden in comparison to wildtype and inflammasome-deficient mice. Further, increased numbers of mature neutrophil appear in the lung early during lethal Ft infection in Nlrp3-deficient mice. Finally, Ft infection induces myeloid and lung stromal cell death that in part requires Nlrp3, is necrotic/necroptotic in nature, and drives host mortality. Thus, Nlrp3 mediates an inflammasome-independent process that restricts the appearance of protective mature neutrophils and promotes lethal necrotic lung pathology.

The NLRP3 inflammasome is central to host defense and implicated in various inflammatory diseases and conditions. While the favored paradigm of NLRP3 inflammasome activation stipulates a unifying signal intermediate that de-represses NLRP3, this view has not been tested. Further, structures within NLRP3 required for inflammasome activation are poorly defined. Here we demonstrate that while the NLRP3 LRRs are not auto-repressive and are not required for inflammasome activation by all agonists, distinct sequences within the NLRP3 LRRs positively and negatively modulate inflammasome activation by specific ligands. In addition, elements within the HD1/HD2 "hinge" of NLRP3 and the nucleotide-binding domain have contrasting functions depending upon the specific agonists. Further, while NLRP3 1-432 is minimally sufficient for inflammasome activation by all agonists tested, the pyrin, and linker domains (1-134) function cooperatively and are sufficient for inflammasome activation by certain agonists. Conserved cysteines 8 and 108 appear important for inflammasome activation by sterile, but not infectious insults. Our results define common and agonist-specific regions of NLRP3 that likely mediate ligand-specific responses, discount the hypothesis that NLRP3 inflammasome activation has a unified mechanism, and implicate NLRP3 as an integrator of agonist-specific, inflammasome activating signals.

IgG (mAb)-opsonized, inactivated Francisella tularensis LVS (iFt-mAb) enhances TLR2-dependent IL-6 production by macrophages via Fcγ receptors (FcγR). In mice, vaccination with iFt-mAb provides IgA-dependent protection against lethal challenge with Ft LVS. Because inflammasome maturation of IL-1β is thought important for antibody-mediated immunity, we considered the possibility that iFt-mAb elicits an FcγR-dependent myeloid cell inflammasome response. Herein, we find that iFt-mAb enhances macrophage and dendritic cell IL-1β responses in a TLR2- and FcγR-dependent fashion. Although iFt-mAb complexes bind FcγR and are internalized, sensing of cytosolic DNA by absent in melanoma 2 (AIM2) is not required for the IL-1β response. In contrast, ASC, caspase-1, and NLR family pyrin domain-containing 3 (NLRP3) are indispensable. Further, FcγR-mediated spleen tyrosine kinase (Syk) signaling is required for this NLRP3-dependent IL-1β response, but the alternative IL-1β convertase caspase-8 is insufficient. Finally, iFt-mAb-vaccinated wild-type mice exhibit a significant delay in time to death, but IL-1R1- or Nlrp3-deficient mice vaccinated in this way are not protected and lack appreciable Francisella-specific antibodies. This study demonstrates that FcγR-mediated Syk activation leads to NLRP3 inflammasome-dependent IL-1β production in macrophages and suggests that an Nlrp3- and IL-1R-dependent process contributes to the IgA response important for protection against Ft LVS. These findings extend our understanding of cellular responses to inactivated pathogen-opsonized vaccine, establish FcγR-elicited Syk kinase-mediated NLRP3 inflammasome activation, and provide additional insight toward understanding crosstalk between TLR and FcγR signals.

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Inflammation and innate immune responses are mediated through the activation of NF‐κB‐dependent cytokine transcription and the formation of the IL‐1β converting inflammasome complex. Inflammasome assembly requires pyrin domain (PYD)‐dependent interactions between an NLR and ASC with subsequent recruitment of the IL‐1β converting enzyme caspase‐1. Proteins containing only a PYD can act as modulators of NF‐κB activity and inflammasome functions. We have recently characterized a novel protein, pyrin only protein‐2 (POP2) that inhibits p65‐induced NF‐κB activity and blocks inflammasome formation by disrupting the interaction between ASC and various NLRs. While expressed in macrophages, the molecular basis for POP2 mediated inhibition of NFκB and inflammasome assembly are unknown. We present data that the action of POP2 differs from that of POP1. We have also further refined POP2's mode of action and mapped the functional domain of POP2 required for regulation of NF‐κB activity. Further, the residues involved in NF‐κB inhibition appear to be distinct from those involved in ASC binding and inflammasome inhibition, suggesting that NF‐κB and inflammasome inhibition are independent functions. We propose a model for role of these distinct functions of POP2 in regulating innate immunity.