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Pyroptosis is rapidly emerging as a mechanism of anti-microbial host defense, and of extracellular release of the inflammasome-dependent cytokines interleukin (IL)-1β and IL-18, which contributes to autoinflammatory pathology. Caspases 1, 4, 5 and 11 trigger this regulated form of necrosis by cleaving the pyroptosis effector gasdermin D (GSDMD), causing its pore-forming amino-terminal domain to oligomerize and perforate the plasma membrane. However, the subcellular events that precede pyroptotic cell lysis are ill defined. In this study, we triggered primary macrophages to undergo pyroptosis from three inflammasome types and recorded their dynamics and morphology using high-resolution live-cell spinning disk confocal laser microscopy. Based on quantitative analysis of single-cell subcellular events, we propose a model of pyroptotic cell disintegration that is initiated by opening of GSDMD-dependent ion channels or pores that are more restrictive than recently proposed GSDMD pores, followed by osmotic cell swelling, commitment of mitochondria and other membrane-bound organelles prior to sudden rupture of the plasma membrane and full permeability to intracellular proteins. This study provides a dynamic framework for understanding cellular changes that occur during pyroptosis, and charts a chronological sequence of GSDMD-mediated subcellular events that define pyroptotic cell death at the single-cell level.

Although spontaneous protein crystallization is a rare event in vivo, Charcot-Leyden crystals (CLCs) consisting of galectin-10 (Gal10) protein are frequently observed in eosinophilic diseases, such as asthma. We found that CLCs derived from patients showed crystal packing and Gal10 structure identical to those of Gal10 crystals grown in vitro. When administered to the airways, crystalline Gal10 stimulated innate and adaptive immunity and acted as a type 2 adjuvant. By contrast, a soluble Gal10 mutein was inert. Antibodies directed against key epitopes of the CLC crystallization interface dissolved preexisting CLCs in patient-derived mucus within hours and reversed crystal-driven inflammation, goblet-cell metaplasia, immunoglobulin E (IgE) synthesis, and bronchial hyperreactivity (BHR) in a humanized mouse model of asthma. Thus, protein crystals may promote hallmark features of asthma and are targetable by crystal-dissolving antibodies.

The porcine reproductive and respiratory syndrome virus (PRRSV) shows a restricted tropism for subsets of porcine macrophages in vivo. To date, two PRRSV receptors have been identified on primary macrophages, heparan sulphate for binding and sialoadhesin for binding and internalization. However, additional factors are needed because the expression of both receptors in non-permissive cells results in virus internalization but not in virus uncoating and productive infection. Recently, CD163 was described as a PRRSV receptor on Marc-145 cells that renders non-permissive cells susceptible to PRRSV. Therefore, the potential role of CD163 in PRRSV entry in macrophages and its potential interplay with sialoadhesin were studied. Incubation of macrophages at 37 6C with either sialoadhesin-or CD163-specific antibodies reduced PRRSV infection by up to 75 %, while infection was completely blocked by a combination of both antibodies. When incubated at 4 6C, only sialoadhesin-and not CD163-specific antibodies reduced PRRSV infection. In addition, confocal analysis of PRRSV entry in non-permissive cells expressing only sialoadhesin showed PRRSV internalization but no uncoating. In contrast, when both sialoadhesin and CD163 were expressed, PRRSV was uncoated upon internalization, resulting in productive infection. Virus internalization was not observed when only CD163 was expressed; although, cells became productively infected. Thus, sialoadhesin is confirmed as a PRRSV internalization receptor and CD163 is shown to be involved in PRRSV entry, probably during uncoating. Co-expression of recombinant sialoadhesin and CD163 in non-permissive cells increased virus production 10-100 times compared with cells expressing only CD163, sustaining the requirement of both for efficient PRRSV infection. INTRODUCTIONA 'mystery swine disease' appeared in the 1980s and has been present ever since in the pig industry, causing important economical losses worldwide (Neumann et al., 2005). The causative agent, designated porcine reproductive and respiratory syndrome virus (PRRSV), was isolated in the Netherlands in 1991 and shortly after in the USA (Collins et al., 1992;Neumann et al., 2005;Wensvoort et al., 1991). PRRSV, a small enveloped positive-stranded RNA virus, is classified in the order Nidovirales, family Arteriviridae, genus Arterivirus together with equine arteritis virus, lactate dehydrogenase-elavating virus and simian hemorrhagic fever virus based on similar morphology, genomic organization, replication strategy and protein composition (Cavanagh, 1997). In addition, they share a very narrow host tropism and a marked preference for cells of the monocyte-macrophage lineage. More specifically, in vivo, PRRSV infects subpopulations of differentiated macrophages, with alveolar macrophages being the major target cells during acute infection (Duan et al., 1997b; Teifke et al., 2001). In vitro, PRRSV replicates in primary cultures of alveolar macrophages and to some extent in peripheral blood monocytes, with pre-treatment of alpha interfero...

Familial Mediterranean fever (FMF) is the most common monogenic autoinflammatory disease worldwide. It is caused by mutations in the inflammasome adaptor Pyrin, but how FMF mutations alter signaling in FMF patients is unknown. Herein, we establish Clostridium difficile and its enterotoxin A (TcdA) as Pyrin-activating agents and show that wild-type and FMF Pyrin are differentially controlled by microtubules. Diverse microtubule assembly inhibitors prevented Pyrin-mediated caspase-1 activation and secretion of IL-1β and IL-18 from mouse macrophages and human peripheral blood mononuclear cells (PBMCs). Remarkably, Pyrin inflammasome activation persisted upon microtubule disassembly in PBMCs of FMF patients but not in cells of patients afflicted with other autoinflammatory diseases. We further demonstrate that microtubules control Pyrin activation downstream of Pyrin dephosphorylation and that FMF mutations enable microtubule-independent assembly of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) micrometer-sized perinuclear structures (specks). The discovery that Pyrin mutations remove the obligatory requirement for microtubules in inflammasome activation provides a conceptual framework for understanding FMF and enables immunological screening of FMF mutations.

Inflammasomes promote interleukin (IL)-1β secretion and pyroptosis. Kanneganti et al. now show that the pyroptosis effector gasdermin D (GSDMD) is required for systemic IL-1β secretion and autoinflammatory pathology in a mouse model of Familial Mediterranean Fever (FMF), suggesting GSDMD inhibitors as potential antiinflammatory treatments.

Porcine reproductive and respiratory syndrome virus (PRRSV) emerged in the late 1980s and rapidly became one of the most significant viral pathogens in the swine industry. In vivo, the virus shows a very narrow cell tropism and targets specific subsets of porcine macrophages. The entry of PRRSV into its host cell is the first crucial step in infection and has been the focus of many fundamental studies. This review provides a comprehensive overview of the current knowledge on PRRSV entry into the porcine macrophage, covering virus binding, internalization and genome release, and integrates these findings into a general model of the entry process. IntroductionPorcine reproductive and respiratory syndrome (PRRS) severely affects swine populations worldwide. The aetiological agent, the PRRS virus (PRRSV), is an RNA virus that is classified within the family Arteriviridae, order Nidovirales (Cavanagh, 1997). PRRSV causes considerable production losses as infection with the virus can result in severe reproductive and respiratory disorders in pigs (Neumann et al., 2005). With the recent outbreaks of highly virulent variants of the virus in China (Li et al., 2007;Tian et al., 2007;Zhou et al., 2008), interest in PRRSV has increased greatly and the demand for safe and effective vaccines for PRRSV control is higher than ever.The PRRSV virion consists of a nucleocapsid, composed of a positive-strand RNA genome (±15 kb) and the nucleocapsid protein (N), which is surrounded by a lipid bilayer envelope Dea et al., 1995;Mardassi et al., 1994;Meulenberg et al., 1993;Spilman et al., 2009;Wensvoort et al., 1992). The viral envelope contains six structural proteins: the small envelope protein E, the membrane protein M and the N-glycosylated glycoproteins GP 2 (or GP 2a ), GP 3 , GP 4 and GP 5 . M, N and GP 5 are the major structural proteins of PRRSV, while E, GP 2 , GP 3 and GP 4 are minor virion components. The M and GP 5 proteins occur as disulfide-linked heterodimers in the envelope, while the minor structural proteins E, GP 2 , GP 3 and GP 4 appear to associate via non-covalent interactions (Mardassi et al., 1995(Mardassi et al., , 1996Meulenberg et al., 1993Meulenberg et al., , 1995van Nieuwstadt et al., 1996; Wissink et al., 2005;Wu et al., 2001). In addition, recent data suggest that interactions exist between major and minor envelope proteins (Das et al., 2010).Since the discovery of PRRSV, many studies have been performed to gain insight into the biology of this important pathogen. This review reflects on two decades of research on the initial steps of the PRRSV replication cycle, covering virus attachment, internalization and disassembly. A general model of PRRSV entry into the porcine macrophage is proposed and delineates where further research is necessary. PRRSV cell tropismLike other members of the family Arteriviridae, PRRSV has a very narrow cell tropism. In vivo, the virus shows a preference for cells of the monocyte/macrophage lineage and infects specific subsets of differentiated macrophages in lungs, lymphoid ...

SummaryThe caspase activation and recruitment domain (CARD)-based inflammasome sensors NLRP1b and NLRC4 induce caspase-1-dependent pyroptosis independent of the inflammasome adaptor ASC. Here, we show that NLRP1b and NLRC4 trigger caspase-8-mediated apoptosis as an alternative cell death program in caspase-1−/− macrophages and intestinal epithelial organoids (IECs). The caspase-8 adaptor FADD was recruited to ASC specks, which served as cytosolic platforms for caspase-8 activation and NLRP1b/NLRC4-induced apoptosis. We further found that caspase-1 protease activity dominated over scaffolding functions in suppressing caspase-8 activation and induction of apoptosis of macrophages and IECs. Moreover, TLR-induced c-FLIP expression inhibited caspase-8-mediated apoptosis downstream of ASC speck assembly, but did not affect pyroptosis induction by NLRP1b and NLRC4. Moreover, unlike during pyroptosis, NLRP1b- and NLRC4-elicited apoptosis retained alarmins and the inflammasome-matured cytokines interleukin 1β (IL-1β) and IL-18 intracellularly. This work identifies critical mechanisms regulating apoptosis induction by the inflammasome sensors NLRP1b and NLRC4 and suggests converting pyroptosis into apoptosis as a paradigm for suppressing inflammation.