Inflammasome and Caspase-1 Activity Characterization and Evaluation: An Imaging Flow Cytometer-Based Detection and Assessment of Inflammasome Specks and Caspase-1 Activation.

Inflammasome dysregulation is a hallmark of various inflammatory diseases. Evaluating inflammasome-associated structures (ASC specks) and caspase-1 activity by microscopy is time consuming and limited by small sample size. The current flow cytometric method, time of flight inflammasome evaluation (TOFIE), cannot visualize ASC specks or caspase-1 activity, making colocalization studies of inflammasome components and enzymatic activity impossible. We describe a rapid, high-throughput, single-cell, fluorescence-based image analysis method utilizing the Amnis ImageStreamX instrument that quantitatively and qualitatively characterizes the frequency, area, and cellular distribution of ASC specks and caspase-1 activity in mouse and human cells. Unlike TOFIE, this method differentiates between singular perinuclear specks and false positives. With our technique we also show that the presence of NLRP3 reduces the size of ASC specks, which is further reduced by the presence of active caspase-1. The capacity of our approach to simultaneously detect and quantify ASC specks and caspase-1 activity, both at the population and single-cell level, renders it the most powerful tool available for visualizing and quantifying the impact of mutations on inflammasome assembly and activity.

Interleukin 1α (IL-1α) Promotes Pathogenic Immature Myeloid Cells and IL-1ß Favors Protective Mature Myeloid Cells During Acute Lung Infection.

Bacterial pneumonia is a common risk factor for acute lung injury and sepsis-mediated death, but the mechanisms underlying the overt inflammation and accompanying pathology are unclear. Infiltration of immature myeloid cells and necrotizing inflammation mediate severe pathology and death during pulmonary infection with Francisella tularensis. However, eliciting mature myeloid cells provides protection. Yet, the host factors responsible for this pathologic immature myeloid cell response are unknown. Here, we report that while the influx of both mature and immature myeloid cells is strictly MyD88 dependent, the interleukin 1 (IL-1) receptor mediates an important dual function via its ligands IL-1α and IL-1ß. Although IL-1ß favors the appearance of bacteria-clearing mature myeloid cells, IL-1α contributes to lung infiltration by ineffective and pathologic immature myeloid cells. Finally, IL-1α and IL-1ß are not the sole factors involved, but myeloid cell responses during acute pneumonia were largely unaffected by lung levels of interleukin 10, interleukin 17, CXCL1, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor.

An Immature Myeloid/Myeloid-Suppressor Cell Response Associated with Necrotizing Inflammation Mediates Lethal Pulmonary Tularemia.

Inhalation of Francisella tularensis (Ft) causes acute and fatal pneumonia. The lung cytokine milieu favors exponential Ft replication, but the mechanisms underlying acute pathogenesis and death remain unknown. Evaluation of the sequential and systemic host immune response in pulmonary tularemia reveals that in contrast to overwhelming bacterial burden or cytokine production, an overt innate cellular response to Ft drives tissue pathology and host mortality. Lethal infection with Ft elicits medullary and extra-medullary myelopoiesis supporting recruitment of large numbers of immature myeloid cells and MDSC to the lungs. These cells fail to mature and die, leading to subsequent necrotic lung damage, loss of pulmonary function, and host death that is partially dependent upon immature Ly6G+ cells. Acceleration of this process may account for the rapid lethality seen with Ft SchuS4. In contrast, during sub-lethal infection with Ft LVS the pulmonary cellular response is characterized by a predominance of mature neutrophils and monocytes required for protection, suggesting a required threshold for lethal bacterial infection. Further, eliciting a mature phagocyte response provides transient, but dramatic, innate protection against Ft SchuS4. This study reveals that the nature of the myeloid cell response may be the primary determinant of host mortality versus survival following Francisella infection.

Inflammasome-Independent NLRP3 Restriction of a Protective Early Neutrophil Response to Pulmonary Tularemia.

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-1ß 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-1ß 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-1ß 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.

TNF-α-dependent hematopoiesis following Bcl11b deletion in T cells restricts metastatic melanoma.

Using several tumor models, we demonstrate that mice deficient in Bcl11b in T cells, although having reduced numbers of T cells in the peripheral lymphoid organs, developed significantly less tumors compared with wild-type mice. Bcl11b(-/-) CD4(+) T cells, with elevated TNF-α levels, but not the Bcl11b(-/-) CD8(+) T cells, were required for the reduced tumor burden, as were NK1.1(+) cells, found in increased numbers in Bcl11b(F/F)/CD4-Cre mice. Among NK1.1(+) cells, the NK cell population was predominant in number and was the only population displaying elevated granzyme B levels and increased degranulation, although not increased proliferation. Although the number of myeloid-derived suppressor cells was increased in the lungs with metastatic tumors of Bcl11b(F/F)/CD4-Cre mice, their arginase-1 levels were severely reduced. The increase in NK cell and myeloid-derived suppressor cell numbers was associated with increased bone marrow and splenic hematopoiesis. Finally, the reduced tumor burden, increased numbers of NK cells in the lung, and increased hematopoiesis in Bcl11b(F/F)/CD4-Cre mice were all dependent on TNF-α. Moreover, TNF-α treatment of wild-type mice also reduced the tumor burden and increased hematopoiesis and the numbers and activity of NK cells in the lung. In vitro treatment with TNF-α of lineage-negative hematopoietic progenitors increased NK and myeloid differentiation, further supporting a role of TNF-α in promoting hematopoiesis. These studies reveal a novel role for TNF-α in the antitumor immune response, specifically in stimulating hematopoiesis and increasing the numbers and activity of NK cells.

Differential transmembrane domain GXXXG motif pairing impacts major histocompatibility complex (MHC) class II structure.

Major histocompatibility complex (MHC) class II molecules exhibit conformational heterogeneity, which influences their ability to stimulate CD4 T cells and drive immune responses. Previous studies suggest a role for the transmembrane domain of the class II αß heterodimer in determining molecular structure and function. Our previous studies identified an MHC class II conformer that is marked by the Ia.2 epitope. These Ia.2(+) class II conformers are lipid raft-associated and able to drive both tyrosine kinase signaling and efficient antigen presentation to CD4 T cells. Here, we establish that the Ia.2(+) I-A(k) conformer is formed early in the class II biosynthetic pathway and that differential pairing of highly conserved transmembrane domain GXXXG dimerization motifs is responsible for formation of Ia.2(+) versus Ia.2(-) I-A(k) class II conformers and controlling lipid raft partitioning. These findings provide a molecular explanation for the formation of two distinct MHC class II conformers that differ in their inherent ability to signal and drive robust T cell activation, providing new insight into the role of MHC class II in regulating antigen-presenting cell-T cell interactions critical to the initiation and control of multiple aspects of the immune response.

Repression of inflammasome by Francisella tularensis during early stages of infection.

Francisella tularensis is an important human pathogen responsible for causing tularemia. F. tularensis has long been developed as a biological weapon and is now classified as a category A agent by the Centers for Disease Control because of its possible use as a bioterror agent. F. tularensis represses inflammasome; a cytosolic multi-protein complex that activates caspase-1 to produce proinflammatory cytokines IL-1ß and IL-18. However, the Francisella factors and the mechanisms through which F. tularensis mediates these suppressive effects remain relatively unknown. Utilizing a mutant of F. tularensis in FTL_0325 gene, this study investigated the mechanisms of inflammasome repression by F. tularensis. We demonstrate that muted IL-1ß and IL-18 responses generated in macrophages infected with F. tularensis live vaccine strain (LVS) or the virulent SchuS4 strain are due to a predominant suppressive effect on TLR2-dependent signal 1. Our results also demonstrate that FTL_0325 of F. tularensis impacts proIL-1ß expression as early as 2 h post-infection and delays activation of AIM2 and NLRP3-inflammasomes in a TLR2-dependent fashion. An enhanced activation of caspase-1 and IL-1ß observed in FTL_0325 mutant-infected macrophages at 24 h post-infection was independent of both AIM2 and NLRP3. Furthermore, F. tularensis LVS delayed pyroptotic cell death of the infected macrophages in an FTL_0325-dependent manner during the early stages of infection. In vivo studies in mice revealed that suppression of IL-1ß by FTL_0325 early during infection facilitates the establishment of a fulminate infection by F. tularensis. Collectively, this study provides evidence that F. tularensis LVS represses inflammasome activation and that F. tularensis-encoded FTL_0325 mediates this effect.

Identification of a novel Francisella tularensis factor required for intramacrophage survival and subversion of innate immune response.

Francisella tularensis, the causative agent of tularemia, is one of the deadliest agents of biological warfare and bioterrorism. Extremely high virulence of this bacterium is associated with its ability to dampen or subvert host innate immune response. The objectives of this study were to identify factors and understand the mechanisms of host innate immune evasion by F. tularensis. We identified and explored the pathogenic role of a mutant interrupted at gene locus FTL_0325, which encodes an OmpA-like protein. Our results establish a pathogenic role of FTL_0325 and its ortholog FTT0831c in the virulent F. tularensis SchuS4 strain in intramacrophage survival and suppression of proinflammatory cytokine responses. This study provides mechanistic evidence that the suppressive effects on innate immune responses are due specifically to these proteins and that FTL_0325 and FTT0831c mediate immune subversion by interfering with NF-κB signaling. Furthermore, FTT0831c inhibits NF-κB activity primarily by preventing the nuclear translocation of p65 subunit. Collectively, this study reports a novel F. tularensis factor that is required for innate immune subversion caused by this deadly bacterium.

The Ia.2 epitope defines a subset of lipid raft-resident MHC class II molecules crucial to effective antigen presentation.

Previous work established that binding of the 11-5.2 anti-I-A(k) mAb, which recognizes the Ia.2 epitope on I-A(k) class II molecules, elicits MHC class II signaling, whereas binding of two other anti-I-A(k) mAbs that recognize the Ia.17 epitope fail to elicit signaling. Using a biochemical approach, we establish that the Ia.2 epitope recognized by the widely used 11-5.2 mAb defines a subset of cell surface I-A(k) molecules predominantly found within membrane lipid rafts. Functional studies demonstrate that the Ia.2-bearing subset of I-A(k) class II molecules is critically necessary for effective B cell-T cell interactions, especially at low Ag doses, a finding consistent with published studies on the role of raft-resident class II molecules in CD4 T cell activation. Interestingly, B cells expressing recombinant I-A(k) class II molecules possessing a ß-chain-tethered hen egg lysosome peptide lack the Ia.2 epitope and fail to partition into lipid rafts. Moreover, cells expressing Ia.2(-) tethered peptide-class II molecules are severely impaired in their ability to present both tethered peptide or peptide derived from exogenous Ag to CD4 T cells. These results establish the Ia.2 epitope as defining a lipid raft-resident MHC class II conformer vital to the initiation of MHC class II-restricted B cell-T cell interactions.

Flow Imaging of the Inflammasome: Evaluating ASC Speck Characteristics and Caspase-1 Activity.

Examining inflammasome-associated speck structures is one of the most preferred and easiest ways to evaluate inflammasome activation. Microscopy-based evaluation of specks is preferable, but this approach is time-consuming and limited to small sample sizes. Speck-containing cells can also be quantitated by a flow cytometric method, time of flight inflammasome evaluation (TOFIE). However, TOFIE cannot perform single-cell analysis such as simultaneously visualizing ASC specks and caspase-1 activity, their location, and physical characteristics. Here we describe the application of an imaging flow cytometry-based approach that overcomes these limitations. Inflammasome and Caspase-1 Activity Characterization and Evaluation (ICCE) is a high-throughput, single-cell, rapid image analysis utilizing the Amnis ImageStream X instrument with over 99.5% accuracy. ICCE quantitatively and qualitatively characterizes the frequency, area, and cellular distribution of ASC specks and caspase-1 activity in mouse and human cells.

Uncoupling of Pyrin-only protein 2 (POP2)-mediated dual regulation of NF-κB and the inflammasome.

Activation of transcription factor NF-κB and inflammasome-directed caspase-1 cleavage of IL-1ß are key processes in the inflammatory response to pathogen or host-derived signals. Pyrin-only proteins (POPs) are restricted to Old World monkeys, apes, and humans and have previously been shown to impair inflammasome assembly and/or NF-κB p65 transcriptional activity in transfected epithelial cells. However, the biological role of POP2 and the molecular basis for its observed functions are not well understood. In this report we demonstrate that POP2 regulates TNFα and IL-1ß responses in human monocytic THP-1 cells and in stable transfectants of mouse J774A.1 macrophages. Deletion analysis of POP2 revealed that the first α-helix (residues 1-19) is necessary and sufficient for both inflammasome and NF-κB inhibitory functions. Further, key acidic residues Glu(6), Asp(8), and Glu(16), believed critical for Pyrin/Pyrin domain interaction, are important for inflammasome inhibition. Moreover, these mutations did not reduce the effect of POP2 upon NF-κB, indicating that the inflammasome and NF-κB inhibitory properties of POP2 can be uncoupled mechanistically. Collectively, these data demonstrate that POP2 acts as a regulator of inflammatory signals and exerts its two known functions through distinct modalities employed by its first α-helix.

Francisella tularensis reveals a disparity between human and mouse NLRP3 inflammasome activation.

Pathogen-triggered activation of the inflammasome complex leading to caspase-1 activation and IL-1ß 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-1ß response of human cells to Francisella infection. Francisella strains exhibit differences in IL-1ß production by influencing induction of IL-1ß and ASC transcripts. Unexpectedly, our results demonstrate that Francisella activates the NLRP3 inflammasome in human cells. Francisella infection of THP-1 cells elicits IL-1ß production, which is reduced by siRNA targeting of NLRP3. Moreover, in reconstituted 293T cells, Francisella triggers assembly of the NLRP3 inflammasome complex. In addition, inhibitors of reactive oxygen species, cathepsin B, and K(+) efflux pathways, known to specifically influence NLRP3, substantially but not completely impair the Francisella-elicited IL-1ß response, suggesting the involvement of another inflammasome pathway. Finally, shRNA targeting of NLRP3 and AIM2 reveals that both pathways contribute to the inflammasome response. Together these results establish NLRP3 as a cytosolic sensor for Francisella in human cells, a role not observed in mouse.

CIITA enhances HIV-1 attachment to CD4+ T cells leading to enhanced infection and cell depletion.

Activated CD4(+) T cells are more susceptible to HIV infection than resting T cells; the reason for this remains unresolved. Induction of CIITA and subsequent expression of the MHC class II isotype HLA-DR are hallmarks of CD4(+) T cell activation; therefore, we investigated the role of CIITA expression in T cells during HIV infection. CIITA-expressing SupT1 cells display enhanced virion attachment in a gp160/CD4-dependent manner, which results in increased HIV infection, virus release, and T cell depletion. Although increased attachment and infection of T cells correlated with HLA-DR surface expression, Ab blocking, transient expression of HLA-DR without CIITA, and short hairpin RNA knockdown demonstrate that HLA-DR does not directly enhance susceptibility of CIITA-expressing cells to HIV infection. Further analysis of the remaining MHC class II isotypes, HLA-DP and HLA-DQ, MHC class I isotypes, HLA-A, HLA-B, and HLA-C, and the class II Ag presentation genes, invariant chain and HLA-DM, demonstrate that these proteins likely do not contribute to CIITA enhancement of HIV infection. Finally, we demonstrate that in activated primary CD4(+) T cells as HLA-DR/CIITA expression increases there is a corresponding increase in virion attachment. Overall, this work suggests that induction of CIITA expression upon CD4(+) T cell activation contributes to enhanced attachment, infection, virus release, and cell death through an undefined CIITA transcription product that may serve as a new antiviral target.

Recent evolution of the NF-κB and inflammasome regulating protein POP2 in primates.

BACKGROUND: Pyrin-only protein 2 (POP2) is a small human protein comprised solely of a pyrin domain that inhibits NF-κB p65/RelA and blocks the formation of functional IL-1ß processing inflammasomes. Pyrin proteins are abundant in mammals and several, like POP2, have been linked to activation or regulation of inflammatory processes. Because POP2 knockout mice would help probe the biological role of inflammatory regulation, we thus considered whether POP2 is common in the mammalian lineage. RESULTS: BLAST searches revealed that POP2 is absent from the available genomes of not only mice and rats, but those of other domestic mammals and New World monkeys as well. POP2 is however present in the genome of the primate species most closely related to humans including Pan troglodytes (chimpanzees), Macaca mulatta (rhesus macaques) and others. Interestingly, chimpanzee POP2 is identical to human POP2 (huPOP2) at both the DNA and protein level. Macaque POP2 (mqPOP2), although highly conserved is not identical to the human sequence; however, both functions of the human protein are retained. Further, POP2 appears to have arisen in the mammalian genome relatively recently (~25 mya) and likely derived from retrogene insertion of NLRP2. CONCLUSION: Our findings support the hypothesis that the NLR loci of mammals, encoding proteins involved in innate and adaptive immunity as well as mammalian development, have been subject to recent and strong selective pressures. Since POP2 is capable of regulating signaling events and processes linked to innate immunity and inflammation, its presence in the genomes of hominids and Old World primates further suggests that additional regulation of these signals is important in these species.

The ASC Speck and NLRP3 Inflammasome Function Are Spatially and Temporally Distinct.

Although considered the ternary inflammasome structure, whether the singular, perinuclear NLRP3:ASC speck is synonymous with the NLRP3 inflammasome is unclear. Herein, we report that the NLRP3:ASC speck is not required for nigericin-induced inflammasome activation but facilitates and maximizes IL-1ß processing. Furthermore, the NLRP3 agonists H2O2 and MSU elicited IL-1ß maturation without inducing specks. Notably, caspase-1 activity is spatially distinct from the speck, occurring at multiple cytoplasmic sites. Additionally, caspase-1 activity negatively regulates speck frequency and speck size, while speck numbers and IL-1ß processing are negatively correlated, cyclical and can be uncoupled by NLRP3 mutations or inhibiting microtubule polymerization. Finally, when specks are present, caspase-1 is likely activated after leaving the speck structure. Thus, the speck is not the NLRP3 inflammasome itself, but is instead a dynamic structure which may amplify the NLRP3 response to weak stimuli by facilitating the formation and release of small NLRP3:ASC complexes which in turn activate caspase-1.

NLRP3 Sensing of Diverse Inflammatory Stimuli Requires Distinct Structural Features.

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.

Class II MHC cytoplasmic domain-mediated signaling in B cells: A tail of two signals.

In addition to their role in antigen presentation, class II MHC molecules also transmit signals to B lymphocytes. Class II MHC-mediated signals initiate a range of events in B cells, including induction of cell surface proteins, initiation of cell-cycle progression/proliferation, activation of or protection from apoptosis, and antigen-dependent plasma cell differentiation. Although various transmembrane signaling proteins associate with class II MHC molecules, the class II MHC cytoplasmic domains are essential for signals leading to increased intracellular cAMP and activation of protein kinase C (PKC). Although truncation and mutagenesis studies have provided considerable information about the cytoplasmic domain sequences required, how class II MHC molecules elicit cAMP and PKC activation is not known. Further, appropriate T-dependent B cell responses require intact cAMP and PKC signaling, but the extent to which class II MHC signals are involved is also unknown. This review details our current knowledge of class II MHC cytoplasmic domain signaling in B cells with an emphasis on the likely importance of class II MHC signals for T-dependent antibody responses.

Pyrin-only protein 2 limits inflammation but improves protection against bacteria.

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.