CARD-only proteins regulate in vivo inflammasome responses and ameliorate gout.

Inflammatory responses are crucial for controlling infections and initiating tissue repair. However, excessive and uncontrolled inflammation causes inflammatory disease. Processing and release of the pro-inflammatory cytokines interleukin-1ß (IL-1ß) and IL-18 depend on caspase-1 activation within inflammasomes. Assembly of inflammasomes is initiated upon activation of cytosolic pattern recognition receptors (PRRs), followed by sequential polymerization of pyrin domain (PYD)-containing and caspase recruitment domain (CARD)-containing proteins mediated by homotypic PYD and CARD interactions. Small PYD- or CARD-only proteins (POPs and COPs, respectively) evolved in higher primates to target these crucial interactions to limit inflammation. Here, we show the ability of COPs to regulate inflammasome activation by modulating homotypic CARD-CARD interactions in vitro and in vivo. CARD16, CARD17, and CARD18 displace crucial CARD interactions between caspase-1 proteins through competitive binding and ameliorate uric acid crystal-mediated NLRP3 inflammasome activation and inflammatory disease. COPs therefore represent an important family of inflammasome regulators and ameliorate inflammatory disease.

POP1 inhibits MSU-induced inflammasome activation and ameliorates gout.

Canonical inflammasomes are innate immune protein scaffolds that enable the activation of inflammatory caspase-1, and subsequently the processing and release of interleukin (IL)-1ß, IL-18, and danger signals, as well as the induction of pyroptotic cell death. Inflammasome assembly and activation occurs in response to sensing of infectious, sterile and self-derived molecular patterns by cytosolic pattern recognition receptors, including the Nod-like receptor NLRP3. While these responses are essential for host defense, excessive and uncontrolled NLRP3 inflammasome responses cause and contribute to a wide spectrum of inflammatory diseases, including gout. A key step in NLRP3 inflammasome assembly is the sequentially nucleated polymerization of Pyrin domain (PYD)- and caspase recruitment domain (CARD)-containing inflammasome components. NLRP3 triggers polymerization of the adaptor protein ASC through PYD-PYD interactions, but ASC polymerization then proceeds in a self-perpetuating manner and represents a point of no return, which culminates in the activation of caspase-1 by induced proximity. In humans, small PYD-only proteins (POPs) lacking an effector domain regulate this key process through competitive binding, but limited information exists on their physiological role during health and disease. Here we demonstrate that POP1 expression in macrophages is sufficient to dampen MSU crystal-mediated inflammatory responses in animal models of gout. Whether MSU crystals are administered into a subcutaneous airpouch or into the ankle joint, the presence of POP1 significantly reduces neutrophil infiltration. Also, airpouch exudates have much reduced IL-1ß and ASC, which are typical pro-inflammatory indicators that can also be detected in synovial fluids of gout patients. Exogenous expression of POP1 in mouse and human macrophages also blocks MSU crystal-induced NLRP3 inflammasome assembly, resulting in reduced IL-1ß and IL-18 secretion. Conversely, reduced POP1 expression in human macrophages enhances IL-1ß secretion. We further determined that the mechanism for the POP1-mediated inhibition of NLRP3 inflammasome activation is through its interference with the crucial NLRP3 and ASC interaction within the inflammasome complex. Strikingly, administration of an engineered cell permeable version of POP1 was able to ameliorate MSU crystal-mediated inflammation in vivo, as measured by neutrophil infiltration. Overall, we demonstrate that POP1 may play a crucial role in regulating inflammatory responses in gout.

Liogenys Guérin-Méneville, 1831 (Coleoptera: Scarabaeidae: Melolonthinae: Diplotaxini) from the Chacoan Province and its boundaries: taxonomic overview with four new species.

A taxonomic revision of the Liogenys Guérin-Méneville, 1831 (Coleoptera: Scarabaeidae: Melolonthinae: Diplotaxini) from the Chacoan Biogeographical Province is presented. Liogenys now includes 92 species, including four new species described here: L. neoforcipata Cherman, new species; L. foveata Cherman, new species; L. isotarsis Cherman, new species; and L. truncata Cherman, new species; and the female of L. tarsalis Moser is described for the first time. Six new synonymies are proposed: L. denticulata Moser, 1918 is a new synonym of L. denticeps Blanchard, 1851; L. ophtalmica Frey, 1973 is a new synonym of L. bidenticeps Moser, 1919; L. mendozana incisa Frey, 1969 is a new synonym of L. mendozana Moser, 1918; L. flavicollis Blanchard, 1851 and L. fulvescens Blanchard, 1851 are new synonyms of L. pallens Blanchard, 1851; and L. densicollis Moser, 1921 is a new synonym of L. opacicollis Fairmaire, 1892. Liogenys cribricollis Moser, 1921 species status is revalidated from its synonymy with L. densicollis. A neotype is designated for Liogenys mendozana incisa Frey, 1969, as well as lectotypes for: L. bruchi Moser, 1924; L. cribricollis, L. denticulata, L. denticeps, L. fulvescens, L. latitarsis Moser, 1918; L. mendozana Moser, 1918; L. obscura Blanchard, 1851; L. opacicollis; and L. pallens. Redescriptions and/or diagnoses and updated geographical distributions are provided for 16 species. Six species previously known only from Argentina have their distribution expanded to Bolivia (L. mendozana; L. opacicollis; L. rectangula Frey, 1969), Paraguay (L. nigrofusca Moser, 1918; L. pallens), or to both of these countries (L. latitarsis).

TLR4-dependent fibroblast activation drives persistent organ fibrosis in skin and lung.

Persistent fibrosis in multiple organs is the hallmark of systemic sclerosis (SSc). Recent genetic and genomic studies implicate TLRs and their damage-associated molecular pattern (DAMP) endogenous ligands in fibrosis. To test the hypothesis that TLR4 and its coreceptor myeloid differentiation 2 (MD2) drive fibrosis persistence, we measured MD2/TLR4 signaling in tissues from patients with fibrotic SSc, and we examined the impact of MD2 targeting using a potentially novel small molecule. Levels of MD2 and TLR4, and a TLR4-responsive gene signature, were enhanced in SSc skin biopsies. We developed a small molecule that selectively blocks MD2, which is uniquely required for TLR4 signaling. Targeting MD2/TLR4 abrogated inducible and constitutive myofibroblast transformation and matrix remodeling in fibroblast monolayers, as well as in 3-D scleroderma skin equivalents and human skin explants. Moreover, the selective TLR4 inhibitor prevented organ fibrosis in several preclinical disease models and mouse strains, and it reversed preexisting fibrosis. Fibroblast-specific deletion of TLR4 in mice afforded substantial protection from skin and lung fibrosis. By comparing experimentally generated fibroblast TLR4 gene signatures with SSc skin biopsy gene expression datasets, we identified a subset of SSc patients displaying an activated TLR4 signature. Together, results from these human and mouse studies implicate MD2/TLR4-dependent fibroblast activation as a key driver of persistent organ fibrosis. The results suggest that SSc patients with high TLR4 activity might show optimal therapeutic response to selective inhibitors of MD2/TLR4 complex formation.

A taxonomic revision of Liogenys occurring in Brazil with an interactive key and remarks on New World Diplotaxini (Coleoptera, Melolonthidae).

Liogenys Guérin-Méneville, 1831 is the major genus of Neotropical Diplotaxini, with 78 species distributed from Panama to southern Argentina and Chile, except for Ecuador. Due to the large numbers of both described and undescribed species, as well as its agricultural importance, mainly of those in Brazil, Liogenys was redefined and redescribed. Nine new species are described: L. cavifrons Cherman, sp. n., L. femella Cherman, sp. n., L. piauiensis Cherman, sp. n., L. rotundicollis Cherman, sp. n., L. pseudosanctaecrucis Cherman, sp. n., L. grossii Cherman, sp. n., L. pseudospiniventris Cherman, sp. n., L. sulcoventris Cherman, sp. n., and L. freyi Cherman, sp. n. All the new species are Brazilian, except for the last one, which is Argentinian. Twenty-three Brazilian species are redescribed and illustrated. Five new synonyms are proposed, and 19 lectotypes are designated. New geographical distribution records for 19 species are presented, as well as a key to New World Diplotaxini and Brazilian species of Liogenys.

The PYRIN domain-only protein POP2 inhibits inflammasome priming and activation.

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)-1ß 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.

Effect of different diets on biology, reproductive variables and life and fertility tables of Harmonia axyridis (Pallas) (Coleoptera, Coccinellidae)

ABSTRACT The biology, reproductive variables and population growth indicators of Harmonia axyridis (Pallas, 1773) (Coleoptera: Coccinellidae) fed on three diets, namely Cinara atlantica (Wilson, 1919) (Hemiptera: Aphididae), Brevicoryne brassicae (Linnaeus, 1758) (Hemiptera: Aphididae), and frozen eggs of Anagasta kuehniella (Zeller, 1879) (Lepidoptera: Pyralidae), were evaluated. With all three diets, birth rate was higher than mortality, resulting in positive rm values and thus indicating population growth. Under the conditions used in the experiments, H. axyridis was able to survive, develop and reproduce normally. This demonstrates that are different kind of food that can be essential for supporting the reproduction of some species of Coccinellidae, but not with the same optimization of preferred prey.

ASC-particle-induced Peritonitis.

In response to pathogen infection and tissue damage, inflammasome sensors such as NLRP3 and AIM2 are activated, which triggers PYRIN domain (PYD)-mediated ASC nucleation, followed by self-perpetuating ASC polymerization, which ultimately culminates in caspase-1 activation, interleukin (IL)-1ß and IL-18 processing and release and pyroptosis (Ratsimandresy et al., 2013; Cai et al., 2014). Inflammasomes release not only cytokines, but also the polymeric ASC danger particles (pASC) by pyroptosis, which perpetuate and propagate inflammasome responses to bystander cells to engage cell intrinsic ASC and caspase-1 (Baroja-Mazo et al., 2014; Franklin et al., 2014). In this protocol we describe intraperitoneal injection of polymeric ASC particles as a danger signal and measure neutrophil infiltration and levels of the pro-inflammatory cytokine IL-1ß by ELISA in the peritoneal lavage (de Almeida et al., 2015).

In vivo Analysis of Neutrophil Infiltration during LPS-induced Peritonitis.

Bacterial lipopolysaccharide (LPS) is present in the outer membrane of Gram-negative bacteria and functions as pathogen-associated molecular pattern (PAMP) (Whitfield and Trent, 2014). LPS therefore is a potent activator of inflammatory responses leading to cytokine release and neutrophils recruitment. The lipid A moiety of LPS activates the complex consisting of the LPS binding protein (LBP), CD14, MD-2 and Toll-like receptor 4 (TLR4) and the non-canonical inflammasome-linked caspases-4, 5 and 11, which in turn activate the canonical NLRP3 inflammasome (Shi et al., 2014; Hagar et al., 2013; Kayagaki et al., 2013; Hoshino et al., 1999; Poltorak, 1998; Nagai et al., 2002; Park et al., 2009; Ratsimandresy et al., 2013). In particular, the cytokine interleukin (IL)-1ß produced in response to inflammasome activation has a crucial role in neutrophil recruitment through promoting neutrophil adhesion and migration (McDonald et al., 2010).This protocol allows studying of inflammatory response induced by LPS that affect neutrophil infiltration by tracking myeloperoxidase (MPO) activity in vivo (de Almeida et al., 2015).

The PYRIN Domain-only Protein POP1 Inhibits Inflammasome Assembly and Ameliorates Inflammatory Disease.

In response to infections and tissue damage, ASC-containing inflammasome protein complexes are assembled that promote caspase-1 activation, IL-1ß and IL-18 processing and release, pyroptosis, and the release of ASC particles. However, excessive or persistent activation of the inflammasome causes inflammatory diseases. Therefore, a well-balanced inflammasome response is crucial for the maintenance of homeostasis. We show that the PYD-only protein POP1 inhibited ASC-dependent inflammasome assembly by preventing inflammasome nucleation, and consequently interfered with caspase-1 activation, IL-1ß and IL-18 release, pyroptosis, and the release of ASC particles. There is no mouse ortholog for POP1, but transgenic expression of human POP1 in monocytes, macrophages, and dendritic cells protected mice from systemic inflammation triggered by molecular PAMPs, inflammasome component NLRP3 mutation, and ASC danger particles. POP1 expression was regulated by TLR and IL-1R signaling, and we propose that POP1 provides a regulatory feedback loop that shuts down excessive inflammatory responses and thereby prevents systemic inflammation.

Wing Morphometry and Acoustic Signals in Sterile and Wild Males: Implications for Mating Success in Ceratitis capitata.

The sterile insect technique (SIT) is widely utilized in the biological control of fruit flies of the family Tephritidae, particularly against the Mediterranean fruit fly. This study investigated the interaction between mating success and morphometric variation in the wings and the production of acoustic signals among three male groups of Ceratitis capitata (Wiedemann): (1) wild males, (2) irradiated with Co-60 (steriles), and (3) irradiated (steriles) and treated with ginger oil. The canonical variate analysis discriminated two groups (males irradiated and males wild), based on the morphological shape of the wings. Among males that emit buzz signals, wild males obtained copulation more frequently than males in Groups 2 and 3. The individuals of Group 3 achieved more matings than those in Group 2. Wild males displayed lower pulse duration, higher intervals between pulses, and higher dominant frequency. Regarding the reproductive success, the morphological differences in the wings' shape between accepted and nonaccepted males are higher in wild males than in the irradiated ones. The present results can be useful in programs using the sterile insect technique for biological control of C. capitata.

The PYRIN domain-only protein POP3 inhibits ALR inflammasomes and regulates responses to infection with DNA viruses.

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.

NLRP7 and related inflammasome activating pattern recognition receptors and their function in host defense and disease.

Host defense requires the maturation and release of the pro-inflammatory cytokines interleukin (IL)-1ß and IL-18 and the induction of pyroptotic cell death, which depends on the activation of inflammatory Caspases within inflammasomes by innate immune cells. Several cytosolic pattern recognition receptors (PRRs) have been implicated in this process in response to infectious and sterile agonists. Here we summarize the current knowledge on inflammasome-organizing PRRs, emphasizing the recently described NLRP7, and their implications in human disease.

An NLRP7-containing inflammasome mediates recognition of microbial lipopeptides in human macrophages.

Cytosolic pathogen- and damage-associated molecular patterns are sensed by pattern recognition receptors, including members of the nucleotide-binding domain and leucine-rich repeat-containing gene family (NLR), which cause inflammasome assembly and caspase-1 activation to promote maturation and release of the inflammatory cytokines interleukin-1ß (IL-1ß) and IL-18 and induction of pyroptosis. However, the contribution of most of the NLRs to innate immunity, host defense, and inflammasome activation and their specific agonists are still unknown. Here we describe identification and characterization of an NLRP7 inflammasome in human macrophages, which is induced in response to microbial acylated lipopeptides. Activation of NLRP7 promoted ASC-dependent caspase-1 activation, IL-1ß and IL-18 maturation, and restriction of intracellular bacterial replication, but not caspase-1-independent secretion of the proinflammatory cytokines IL-6 and tumor necrosis factor-α. Our study therefore increases our currently limited understanding of NLR activation, inflammasome assembly, and maturation of IL-1ß and IL-18 in human macrophages.

Scanning electron micrographs of Oryzophagus oryzae (Coleoptera, Curculionidae), plastron structure and swimming behavior.

The morphological structures that permit Oryzophagus oryzae aquatic activities and swimming behavior were studied and compared with various weevils and other relevant species. The use of scanning electron microscopy facilitated the recognition of three different hydrofuge scales and sensilla. Based on the microscopic observations of behavior, morphological evidence, and comparisons with other curculionid species, it was supported that the gas exchange in O. oryzae adults relies on a subelytral air store maintained by hydrofuge scales and a ribbed margin on the adult elytra. The plastron structure is identical to Lissorhoptrus oryzophilus supporting the application of similar control measures for both species.

Microglial p38α MAPK is a key regulator of proinflammatory cytokine up-regulation induced by toll-like receptor (TLR) ligands or beta-amyloid (Aß).

BACKGROUND: Overproduction of proinflammatory cytokines from activated microglia has been implicated as an important contributor to pathophysiology progression in both acute and chronic neurodegenerative diseases. Therefore, it is critical to elucidate intracellular signaling pathways that are significant contributors to cytokine overproduction in microglia exposed to specific stressors, especially pathways amenable to drug interventions. The serine/threonine protein kinase p38α MAPK is a key enzyme in the parallel and convergent intracellular signaling pathways involved in stressor-induced production of IL-1ß and TNFα in peripheral tissues, and is a drug development target for peripheral inflammatory diseases. However, much less is known about the quantitative importance of microglial p38α MAPK in stressor-induced cytokine overproduction, or the potential of microglial p38α MAPK to be a druggable target for CNS disorders. Therefore, we examined the contribution of microglial p38αMAPK to cytokine up-regulation, with a focus on the potential to suppress the cytokine increase by inhibition of the kinase with pharmacological or genetic approaches. METHODS: The microglial cytokine response to TLR ligands 2/3/4/7/8/9 or to Aß1-42 was tested in the presence of a CNS-penetrant p38α MAPK inhibitor, MW01-2-069A-SRM. Primary microglia from mice genetically deficient in p38α MAPK were used to further establish a linkage between microglia p38α MAPK and cytokine overproduction. The in vivo significance was determined by p38α MAPK inhibitor treatment in a LPS-induced model of acute neuroinflammation. RESULTS: Increased IL-1ß and TNFα production by the BV-2 microglial cell line and by primary microglia cultures was inhibited in a concentration-dependent manner by the p38α MAPK-targeted inhibitor. Cellular target engagement was demonstrated by the accompanying decrease in the phosphorylation state of two p38α MAPK protein substrates, MK2 and MSK1. Consistent with the pharmacological findings, microglia from p38α-deficient mice showed a diminished cytokine response to LPS. Further, oral administration of the inhibitor blocked the increase of IL-1ß in the cerebral cortex of mice stressed by intraperitoneal injection of LPS. CONCLUSION: The p38α MAPK pathway is an important contributor to the increased microglial production of proinflammatory cytokines induced by diverse stressors. The results also indicate the feasibility of targeting p38α MAPK to modulate CNS proinflammatory cytokine overproduction.

S100B secretion in acute brain slices: modulation by extracellular levels of Ca(2+) and K (+).

Hippocampal slices have been widely used to investigate electrophysiological and metabolic neuronal parameters, as well as parameters of astroglial activity including protein phosphorylation and glutamate uptake. S100B is an astroglial-derived protein, which extracellularly plays a neurotrophic activity during development and excitotoxic insult. Herein, we characterized S100B secretion in acute hippocampal slices exposed to different concentrations of K(+) and Ca(2+) in the extracellular medium. Absence of Ca(2+) and/or low K(+) (0.2 mM KCl) caused an increase in S100B secretion, possibly by mobilization of internal stores of Ca(2+). In contrast, high K(+) (30 mM KCl) or calcium channel blockers caused a decrease in S100B secretion. This study suggests that exposure of acute hippocampal slices to low- and high-K(+) could be used as an assay to evaluate astrocyte activity by S100B secretion: positively regulated by low K(+) (possibly involving mobilization of internal stores of Ca(2+)) and negatively regulated by high-K(+) (likely secondary to influx of K(+)).

Astroglial and cognitive effects of chronic cerebral hypoperfusion in the rat.

The permanent occlusion of common carotid arteries (2VO) causes a significant reduction of cerebral blood flow (hypoperfusion) in rats and constitutes a well established experimental model to investigate neuronal damage and cognitive impairment that occurs in human ageing and Alzheimer's disease. In the present study, we evaluated two astroglial proteins--S100B and glial fibrillary acidic protein (GFAP)--in cerebral cortex and hippocampus tissue, glutamate uptake and glutamine synthetase activity in hippocampus tissue, as well as S100B in cerebrospinal fluid. Cognition, as assessed by reference and working spatial memory protocols, was also investigated. Adult male Wistar rats were submitted to 10 weeks of chronic cerebral hypoperfusion by the 2VO method. A significant increase of S100B and GFAP in hippocampus tissue was observed, as well a significant decrease in glutamate uptake. Interestingly, we observed a decrease in S100B in cerebrospinal fluid. As for the cognitive outcome, there was an impairment of both reference and working spatial memory in the water maze; positive correlation between cognitive impairment and glutamate uptake decrease was evidenced in hypoperfused rats. These data support the hypothesis that astrocytes play a crucial role in the mechanisms of experimental neurodegeneration and that hippocampal pathology arising after chronic hypoperfusion gives rise to memory deficits.

The p38alpha mitogen-activated protein kinase as a central nervous system drug discovery target.

Protein kinases are critical modulators of a variety of cellular signal transduction pathways, and abnormal phosphorylation events can be a cause or contributor to disease progression in a variety of disorders. This has led to the emergence of protein kinases as an important new class of drug targets for small molecule therapeutics. A serine/threonine protein kinase, p38alpha mitogen-activated protein kinase (MAPK), is an established therapeutic target for peripheral inflammatory disorders because of its critical role in regulation of proinflammatory cytokine production. There is increasing evidence that p38alpha MAPK is also an important regulator of proinflammatory cytokine levels in the central nervous system, raising the possibility that the kinase may be a drug discovery target for central nervous system disorders where cytokine overproduction contributes to disease progression. Development of bioavailable, central nervous system-penetrant p38alpha MAPK inhibitors provides the required foundation for drug discovery campaigns targeting p38alpha MAPK in neurodegenerative disorders.

Resveratrol protects against oxidative injury induced by H2O2 in acute hippocampal slice preparations from Wistar rats.

There is a current interest in dietary compounds (such as trans-resveratrol) that can inhibit or reverse oxidative stress, the common pathway for a variety of brain disorders, including Alzheimer's disease and stroke. The objective of the present study was to investigate the effects of resveratrol, under conditions of oxidative stress induced by H(2)O(2), on acute hippocampal slices from Wistar rats. Here, we evaluated cell viability, extracellular lactate, glutathione content, ERK(MAPK) activity, glutamate uptake and S100B secretion. Resveratrol did not change the decrease in lactate levels and in cell viability (by MTT assay) induced by 1mM H(2)O(2), but prevented the increase in cell permeability to Trypan blue induced by H(2)O(2). Moreover, resveratrol per se increased total glutathione levels and prevented the decrease in glutathione induced by 1mM H(2)O(2). The reduction of S100B secretion induced by H(2)O(2) was not changed by resveratrol. Glutamate uptake was decreased in the presence of 1mM H(2)O(2) and this effect was not prevented by resveratrol. There was also a significant activation of ERK1/2 by 1mM H(2)O(2) and resveratrol was able to completely prevent this activation, leading to activity values lower than control levels. The impairments in astrocyte activities, induced by H(2)O(2), confirmed the importance of these cells as targets for therapeutic strategy in brain disorders involving oxidative stress. This study reinforces the protective role of resveratrol and indicates some possible molecular sites of activity of this compound on glial cells, in the acute damage of brain tissue during oxidative stress.