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Find video protocols related to scientific articles indexed in Pubmed.
Interruption of Macrophage-Derived IL-27(p28) Production by IL-10 during Sepsis Requires STAT3 but Not SOCS3.
J. Immunol.
PUBLISHED: 10-27-2014
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Severe sepsis and septic shock are leading causes of morbidity and mortality worldwide. Infection-associated inflammation promotes the development and progression of adverse outcomes in sepsis. The effects of heterodimeric IL-27 (p28/EBI3) have been implicated in the natural course of sepsis, whereas the molecular mechanisms underlying the regulation of gene expression and release of IL-27 in sepsis are poorly understood. We studied the events regulating the p28 subunit of IL-27 in endotoxic shock and polymicrobial sepsis following cecal ligation and puncture. Neutralizing Abs to IL-27(p28) improved survival rates, restricted cytokine release, and reduced bacterial burden in C57BL/6 mice during sepsis. Genetic disruption of IL-27 signaling enhanced the respiratory burst of macrophages. Experiments using splenectomized mice or treatment with clodronate liposomes suggested that macrophages in the spleen may be a significant source of IL-27(p28) during sepsis. In cultures of TLR4-activated macrophages, the frequency of F4/80(+)CD11b(+)IL-27(p28)(+) cells was reduced by the addition of IL-10. IL-10 antagonized both MyD88-dependent and TRIF-dependent release of IL-27(p28). Genetic deletion of STAT3 in Tie2-Cre/STAT3flox macrophages completely interrupted the inhibition of IL-27(p28) by IL-10 after TLR4 activation. In contrast, IL-10 remained fully active to suppress IL-27(p28) with deletion of SOCS3 in Tie2-Cre/SOCS3flox macrophages. Blockade of IL-10R by Ab or genetic deficiency of IL-10 resulted in 3-5-fold higher concentrations of IL-27(p28) in endotoxic shock and polymicrobial sepsis. Our studies identify IL-10 as a critical suppressing factor for IL-27(p28) production during infection-associated inflammation. These findings may be helpful for a beneficial manipulation of adverse IL-27(p28) release during sepsis.
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Shigella IpaH7.8 E3 ubiquitin ligase targets glomulin and activates inflammasomes to demolish macrophages.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 09-22-2014
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When nucleotide-binding oligomerization domain-like receptors (NLRs) sense cytosolic-invading bacteria, they induce the formation of inflammasomes and initiate an innate immune response. In quiescent cells, inflammasome activity is tightly regulated to prevent excess inflammation and cell death. Many bacterial pathogens provoke inflammasome activity and induce inflammatory responses, including cell death, by delivering type III secreted effectors, the rod component flagellin, and toxins. Recent studies indicated that Shigella deploy multiple mechanisms to stimulate NLR inflammasomes through type III secretion during infection. Here, we show that Shigella induces rapid macrophage cell death by delivering the invasion plasmid antigen H7.8 (IpaH7.8) enzyme 3 (E3) ubiquitin ligase effector via the type III secretion system, thereby activating the NLR family pyrin domain-containing 3 (NLRP3) and NLR family CARD domain-containing 4 (NLRC4) inflammasomes and caspase-1 and leading to macrophage cell death in an IpaH7.8 E3 ligase-dependent manner. Mice infected with Shigella possessing IpaH7.8, but not with Shigella possessing an IpaH7.8 E3 ligase-null mutant, exhibited enhanced bacterial multiplication. We defined glomulin/flagellar-associated protein 68 (GLMN) as an IpaH7.8 target involved in IpaH7.8 E3 ligase-dependent inflammasome activation. This protein originally was identified through its association with glomuvenous malformations and more recently was described as a member of a Cullin ring ligase inhibitor. Modifying GLMN levels through overexpression or knockdown led to reduced or augmented inflammasome activation, respectively. Macrophages stimulated with lipopolysaccharide/ATP induced GLMN puncta that localized with the active form of caspase-1. Macrophages from GLMN(+/-) mice were more responsive to inflammasome activation than those from GLMN(+/+) mice. Together, these results highlight a unique bacterial adaptation that hijacks inflammasome activation via interactions between IpaH7.8 and GLMN.
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Cytosolic double-stranded RNA activates the NLRP3 inflammasome via MAVS-induced membrane permeabilization and K+ efflux.
J. Immunol.
PUBLISHED: 09-15-2014
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The nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (Nlrp3) inflammasome plays an important role in inflammation by controlling the maturation and secretion of the cytokines IL-1? and IL-18 in response to multiple stimuli including pore-forming toxins, particulate matter, and ATP. Although the pathways activated by the latter stimuli lead to a decrease in intracellular K(+) concentration, which is required for inflammasome activation, the mechanism by which microbial RNA activates Nlrp3, remains poorly understood. In this study, we found that cytosolic poly(I:C), but not total RNA from healthy macrophages, macrophages undergoing pyroptosis, or mitochondrial RNA, induces caspase-1 activation and IL-1? release through the Nlrp3 inflammasome. Experiments with macrophages deficient in Tlr3, Myd88, or Trif, indicate that poly(I:C) induces Nlrp3 activation independently of TLR signaling. Further analyses revealed that the cytosolic sensors Rig-I and melanoma differentiation-associated gene 5 act redundantly via the common adaptor mitochondrial antiviral signaling (Mavs) to induce Nlrp3 activation in response to poly(I:C), but not ATP or nigericin. Mechanistically, Mavs triggered membrane permeabilization and K(+) efflux independently of the inflammasome which were required for poly(I:C)-induced Nlrp3 activation. We conclude that poly (I:C) activates the inflammasome through an Mavs-dependent surveillance pathway that converges into a common K(+) lowering step in the cytosol that is essential for the induction of Nlrp3 activation.
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A Genome-wide Small Interfering RNA (siRNA) Screen Reveals Nuclear Factor-?B (NF-?B)-independent Regulators of NOD2-induced Interleukin-8 (IL-8) Secretion.
J. Biol. Chem.
PUBLISHED: 08-28-2014
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NOD2 encodes an intracellular multidomain pattern recognition receptor that is the strongest known genetic risk factor in the pathogenesis of Crohn disease (CD), a chronic relapsing inflammatory disorder of the intestinal tract. NOD2 functions as a sensor for bacterial cell wall components and activates proinflammatory and antimicrobial signaling pathways. Here, using a genome-wide small interfering RNA (siRNA) screen, we identify numerous genes that regulate secretion of the proinflammatory cytokine IL-8 in response to NOD2 activation. Moreover, many of the identified IL-8 regulators are linked by protein-protein interactions, revealing subnetworks of highly connected IL-8 regulators implicated in processes such as vesicle formation, mRNA stability, and protein ubiquitination and trafficking. A TNF? counterscreen to induce IL-8 secretion in an NOD2-independent manner reveals that the majority of the identified regulators affect IL-8 secretion irrespective of the initiating stimuli. Using immortalized macrophages, we validate the ubiquitin protease, USP8, and the endosomal sorting protein, VPS28, as negative regulators of NOD2-induced cytokine secretion. Interestingly, several genes that affect NOD2-induced IL-8 secretion are present in loci associated with CD risk by genome-wide association studies, supporting a role for the NOD2/IL-8 pathway, and not just NOD2, in the pathogenesis of CD. Overall, this screen provides a valuable resource in the advancement of our understanding of the genes that regulate the secretion of IL-8.
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TACI deficiency enhances antibody avidity and clearance of an intestinal pathogen.
J. Clin. Invest.
PUBLISHED: 08-21-2014
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The transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) controls differentiation of long-lived plasma cells, and almost 10% of individuals with common variable immunodeficiency (CVID) express either the C104R or A181E variants of TACI. These variants impair TACI function, and TACI-deficient mice exhibit a CVID-like disease. However, 1%-2% of normal individuals harbor the C140R or A181E TACI variants and have no outward signs of CVID, and it is not clear why TACI deficiency in this group does not cause disease. Here, we determined that TACI-deficient mice have low baseline levels of Ig in the blood but retain the ability to mutate Ig-associated genes that encode antigen-specific antibodies. The antigen-specific antibodies in TACI-deficient mice were produced in bursts and had higher avidity than those of WT animals. Moreover, mice lacking TACI were able to clear Citrobacter rodentium, a model pathogen for severe human enteritis, more rapidly than did WT mice. These findings suggest that the high prevalence of TACI deficiency in humans might reflect enhanced host defense against enteritis, which is more severe in those with acquired or inherited immunodeficiencies.
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Peptidoglycan recognition protein 3 and Nod2 synergistically protect mice from dextran sodium sulfate-induced colitis.
J. Immunol.
PUBLISHED: 08-11-2014
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Aberrant immune response and changes in the gut microflora are the main causes of inflammatory bowel disease (IBD). Peptidoglycan recognition proteins (Pglyrp1, Pglyrp2, Pglyrp3, and Pglyrp4) are bactericidal innate immunity proteins that maintain normal gut microbiome, protect against experimental colitis, and are associated with IBD in humans. Nucleotide-binding oligomerization domain 2 (Nod2) is an intracellular bacterial sensor and may be required for maintaining normal gut microbiome. Mutations in Nod2 are strongly associated with Crohn's disease, but the causative mechanism is not understood, and the role of Nod2 in ulcerative colitis is not known. Because IBD is likely caused by variable multiple mutations in different individuals, in this study, we examined the combined role of Pglyrp3 and Nod2 in the development of experimental colitis in mice. We demonstrate that a combined deficiency of Pglyrp3 and Nod2 results in higher sensitivity to dextran sodium sulfate-induced colitis compared with a single deficiency. Pglyrp3(-/-)Nod2(-/-) mice had decreased survival and higher loss of body weight, increased intestinal bleeding, higher apoptosis of colonic mucosa, elevated expression of cytokines and chemokines, altered gut microbiome, and increased levels of ATP in the colon. Increased sensitivity to dextran sodium sulfate-induced colitis in Pglyrp3(-/-)Nod2(-/-) mice depended on increased apoptosis of intestinal epithelium, changed gut microflora, and elevated ATP. Pglyrp3 deficiency contributed colitis-predisposing intestinal microflora and increased intestinal ATP, whereas Nod2 deficiency contributed higher apoptosis and responsiveness to increased level of ATP. In summary, Pglyrp3 and Nod2 are both required for maintaining gut homeostasis and protection against colitis, but their protective mechanisms differ.
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IKK? negatively regulates ASC-dependent inflammasome activation.
Nat Commun
PUBLISHED: 07-17-2014
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The inflammasomes are multiprotein complexes that activate caspase-1 in response to infections and stress, resulting in the secretion of pro-inflammatory cytokines. Here we report that I?B kinase ? (IKK?) is a critical negative regulator of apoptosis-associated specklike protein containing a C-terminal caspase-activation-andrecruitment (CARD) domain (ASC)-dependent inflammasomes. IKK? controls the inflammasome at the level of the adaptor ASC, which interacts with IKK? in the nucleus of resting macrophages in an IKK? kinase-dependent manner. Loss of IKK? kinase activity results in inflammasome hyperactivation. Mechanistically, the downstream nuclear effector IKK-related kinase (IKKi) facilitates translocation of ASC from the nucleus to the perinuclear area during inflammasome activation. ASC remains under the control of IKK? in the perinuclear area following translocation of the ASC/IKK? complex. Signal 2 of NLRP3 activation leads to inhibition of IKK? kinase activity through the recruitment of PP2A, allowing ASC to participate in NLRP3 inflammasome assembly. Taken together, these findings reveal a IKKi-IKK?-ASC axis that serves as a common regulatory mechanism for ASC-dependent inflammasomes.
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A comparative study of the effect of ?-, ?-, and ?-cyclodextrins as stabilizing agents in the synthesis of silver nanoparticles using a green chemistry method.
Mater Sci Eng C Mater Biol Appl
PUBLISHED: 07-10-2014
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This paper describes the effect of different types of cyclodextrins (CDs) in the synthesis of silver nanoparticles (Ag-NPs), using an easy green chemistry method. The Ag-NPs were obtained using an aqueous silver nitrate solution (AgNO3) with ?-, ?-, or ?-CDs (aqueous solutions) as stabilizing agents, employing the chemical reduction method with citric acid as a reducing agent. A comparative study was done to determine which cyclodextrin (CD) was the best stabilizing agent, and we found out that ?-CD was the best due to the number of glucopyranose units in its structure. The formation of the Ag-NPs was demonstrated by analysis of UV-vis spectroscopy, atomic force microscopy (AFM), scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS) and transmission electron microscopy (TEM). SEM-EDS showed the formation of a cluster with a significant amount of silver, for ?-CD-Ag-NPs, spherical agglomerates can be observed. However, for ?-, ?-CD, the agglomerates do not have a specific form, but their appearance is porous. TEM analysis shows spherical nanoparticles in shape and size between ~0.5 to 7 nm. The clear lattice fringes in TEM images and the typical selected area electron diffraction (SAED) pattern, showed that the Ag-NPs obtained were highly crystalline with a face cubic center structure (FCC).
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Interleukin-22 Regulates the Complement System to Promote Resistance against Pathobionts after Pathogen-Induced Intestinal Damage.
Immunity
PUBLISHED: 04-23-2014
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Pathobionts play a critical role in disease development, but the immune mechanisms against pathobionts remain poorly understood. Here, we report a critical role for interleukin-22 (IL-22) in systemic protection against bacterial pathobionts that translocate into the circulation after infection with the pathogen Clostridium difficile. Infection with C. difficile induced IL-22, and infected Il22(-/-) mice harbored high numbers of pathobionts in extraintestinal organs despite comparable pathogen load and intestinal damage in mutant and wild-type mice. Pathobionts exhibited increased resistant against complement-mediated phagocytosis, and their intravenous administration resulted in high animal mortality. Selective removal of translocated commensals rescued Il22(-/-) mice, and IL-22 administration enhanced the elimination of pathobionts. Mechanistically, IL-22 augmented bacterial phagocytosis by increasing the expression and bacterial binding of complement C3. Our study demonstrates an unexpected role for IL-22 in controlling the elimination of pathobionts that enter the systemic circulation through the regulation of the complement system.
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Nod2 and Rip2 contribute to innate immune responses in mouse neutrophils.
Immunology
PUBLISHED: 04-15-2014
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Nod-like receptors are a family of innate immune receptors that link cytosolic sensing of microbial and danger stimuli to the activation of immune responses. Two Nod-like receptor family members, Nod1 and Nod2, recognize bacterial peptidoglycan and activate immune responses via nuclear factor-?B (NF-?B) and mitogen-activated protein kinase (MAPK). The function of Nod1 and Nod2 has been largely studied in macrophages, but the role of these receptors in other innate immune cells remains unclear. In this study, we examined the function of Nod1 and Nod2 in innate immune responses of neutrophils. Mice were injected intraperitoneally with thioglycollate, and then peritoneal neutrophils were isolated 4 hr after injection. Tri-DAP and muramyl-dipeptide (MDP) were used as Nod1 and Nod2 agonists, respectively. The level of cytokines [interleukin-6 (IL-6) and tumour necrosis factor-? (TNF-?)] and chemokines (CXCL1 and CCL2) was increased by MDP, but not Tri-DAP in wild-type (WT) neutrophils. Increased production of cytokines and chemokines with MDP was abolished in Nod2- and Rip2-deficient neutrophils. MDP also induced the activation of NF-?B and MAPK in WT neutrophils, but not in Nod2- and Rip2-deficient cells. Flow cytometry analysis showed that L-selectin shedding was induced by MDP in WT neutrophils, but not in Nod2- and Rip2-deficient cells. MDP and Toll-like receptor (TLR) agonists (Pam3 CSK4 and lipopolysaccharide) exerted synergistic effects on the production of IL-6 and CXCL1 in neutrophils. Moreover, Nod2 and TLR4 cooperated to produce IL-6, TNF-?, CXCL1 and CCL2 in neutrophils in response to Gram-negative bacteria. Our findings suggest that the Nod2-Rip2 axis may contribute to the innate immune response of neutrophils against bacterial infection.
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Redundant and Cooperative Interactions between TLR5 and NLRC4 in Protective Lung Mucosal Immunity against Pseudomonas aeruginosa.
J Innate Immun
PUBLISHED: 03-27-2014
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Flagellin is the major structural component of flagella expressed by Pseudomonas aeruginosa (PA) and other bacteria. This protein has been shown to activate the Toll-like receptor TLR5 and the Nod-like receptor Nlrc4/Ipaf, culminating in the expression of innate cytokines and antimicrobial molecules. In this study, we tested the hypothesis that TLR5 and Nlrc4 in combination are required for maximal protective lung innate mucosal immunity against PA. To test this hypothesis, we compared innate immune responses in wild-type (WT) C57B6 mice challenged with PA intratracheally to those observed in mice genetically deficient in TLR5 (TLR5(-/-)) or Nlrc4 (Nlrc4(-/-)) alone or in combination (TLR5/Nlrc4(-/-)). As compared to WT, TLR5(-/-) and Nlrc4(-/-) mice, we observed a significant increase in mortality in TLR5/Nlrc4(-/-) mice, which was associated with a >5,000-fold increase in lung PA colony-forming units and systemic bacterial dissemination. The increased mortality observed in double-deficient mice was not attributable to differences in lung leukocyte influx or lung injury responses. Levels of biologically active IL-1? and IL-18 were reduced in the bronchoalveolar lavage fluid from PA-infected Nlrc4(-/-) and TLR5/Nlrc4(-/-) but not TLR5(-/-) mice, indicating the requirement for Nlrc4-dependent caspase-1 activation. Similarly, decreased production of biologically active IL-1? and activation of caspase-1 was observed in PA-stimulated pulmonary macrophages isolated from Nlrc4(-/-) and TLR5/Nlrc4(-/-) but not TLR5(-/-) mice, whereas the expression of iNOS and the production of NO were significantly reduced in cells from double-mutant but not single-mutant mice. Collectively, our findings indicate that TLR5 and Nlrc4 have both unique and redundant roles in lung antibacterial mucosal immunity, and the absence of both pathogen recognition receptors results in an increase in susceptibility to invasive lung infection. © 2014 S. Karger AG, Basel.
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Infection mobilizes hematopoietic stem cells through cooperative NOD-like receptor and Toll-like receptor signaling.
Cell Host Microbe
PUBLISHED: 03-13-2014
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Adult hematopoietic stem cells (HSCs) are maintained in specialized niches within the bone marrow under steady-state conditions and mobilize for extramedullary hematopoiesis during periods of stress such as bacterial infections. However, the underlying mechanisms are unclear. We show that systemic infection of mice with Escherichia coli, commonly associated with bacteremia in humans, mobilizes functional HSCs to the spleen. Accumulation of splenic HSCs (CD150+CD48-Lin(-/low)Sca1+cKit+) was diminished in TLR4-deficient and RIPK2-deficient mice, implicating TLRs and cytosolic NOD1/NOD2 signaling in the process. Accordingly, dual stimulation of NOD1 and TLR4 in radio-resistant cells alone was sufficient to mobilize HSCs, while TLR4 expression on HSCs was dispensable. Mechanistically, TLR4 and NOD1 synergistically induced granulocyte colony-stimulating factor (G-CSF), which was required for extramedullary HSC accumulation. Mobilized HSCs and progenitor cells gave rise to neutrophils and monocytes and contributed to limiting secondary infection.
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In vivo mapping of a protective linear neutralizing epitope at the N-terminus of alpha hemolysin from Staphylococcus aureus.
Mol. Immunol.
PUBLISHED: 02-25-2014
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Staphylococcus aureus is responsible for a large and diverse burden of human disease associated with significant morbidity and mortality. The dynamic challenge of this pathogen is exemplified by the emergence of highly virulent community-associated methicillin-resistant S. aureus strain USA300, which threatens both healthy and vulnerable individuals and constitutes a public health imperative in the United States. Though S. aureus employs many virulence factors that enable infectivity and evasion of host defenses, evidence suggests that the increased production of alpha hemolysin may be a critical contributor to the increased virulence of USA300. To enable and inform immunological targeting of alpha hemolysin, we sought to precisely map a neutralizing epitope that we hypothesized existed in the N-terminal domain. Using an in vivo mapping strategy employing peptide immunogens and an optimized in vitro toxin neutralization assay, we identified a linear neutralizing determinant in the N-terminal 19 amino acids of alpha hemolysin. Affinity purified rabbit antibody against this neutralizing epitope was shown to be highly effective at mitigating dermonecrosis in inbred and outbred mice challenged with USA300. To our knowledge, this is the first description of a linear neutralizing epitope in alpha hemolysin, and the delineation of this determinant should inform and facilitate the rational design and development of an efficacious, epitope-focused or multivalent vaccine against S. aureus.
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Shigella type III secretion protein MxiI is recognized by Naip2 to induce Nlrc4 inflammasome activation independently of Pkc?.
PLoS Pathog.
PUBLISHED: 02-01-2014
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Recognition of intracellular pathogenic bacteria by members of the nucleotide-binding domain and leucine-rich repeat containing (NLR) family triggers immune responses against bacterial infection. A major response induced by several Gram-negative bacteria is the activation of caspase-1 via the Nlrc4 inflammasome. Upon activation, caspase-1 regulates the processing of proIL-1? and proIL-18 leading to the release of mature IL-1? and IL-18, and induction of pyroptosis. The activation of the Nlrc4 inflammasome requires the presence of an intact type III or IV secretion system that mediates the translocation of small amounts of flagellin or PrgJ-like rod proteins into the host cytosol to induce Nlrc4 activation. Using the Salmonella system, it was shown that Naip2 and Naip5 link flagellin and the rod protein PrgJ, respectively, to Nlrc4. Furthermore, phosphorylation of Nlrc4 at Ser533 by Pkc? was found to be critical for the activation of the Nlrc4 inflammasome. Here, we show that Naip2 recognizes the Shigella T3SS inner rod protein MxiI and induces Nlrc4 inflammasome activation. The expression of MxiI in primary macrophages was sufficient to induce pyroptosis and IL-1? release, which were prevented in macrophages deficient in Nlrc4. In the presence of MxiI or Shigella infection, MxiI associated with Naip2, and Naip2 interacted with Nlrc4. siRNA-mediated knockdown of Naip2, but not Naip5, inhibited Shigella-induced caspase-1 activation, IL-1? maturation and Asc pyroptosome formation. Notably, the Pkc? kinase was dispensable for caspase-1 activation and secretion of IL-1? induced by Shigella or Salmonella infection. These results indicate that activation of caspase-1 by Shigella is triggered by the rod protein MxiI that interacts with Naip2 to induce activation of the Nlrc4 inflammasome independently of the Pkc? kinase.
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Regulation of the immune system by the resident intestinal bacteria.
Gastroenterology
PUBLISHED: 01-27-2014
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The microbiota is an important factor in the development of the immune response. The interaction between the gastrointestinal tract and resident microbiota is well balanced in healthy individuals, but its breakdown can lead to intestinal and extraintestinal disease. We review current knowledge about the mechanisms that regulate the interaction between the immune system and the microbiota, focusing on the role of resident intestinal bacteria in the development of immune responses. We also discuss mechanisms that prevent immune responses against resident bacteria, and how the indigenous bacteria stimulate the immune system to protect against commensal pathobionts and exogenous pathogens. Unraveling the complex interactions between resident intestinal bacteria and the immune system could improve our understanding of disease pathogenesis and lead to new therapeutic approaches.
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Gut dysbiosis promotes M2 macrophage polarization and allergic airway inflammation via fungi-induced PGE?.
Cell Host Microbe
PUBLISHED: 01-21-2014
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Although imbalances in gut microbiota composition, or "dysbiosis," are associated with many diseases, the effects of gut dysbiosis on host systemic physiology are less well characterized. We report that gut dysbiosis induced by antibiotic (Abx) treatment promotes allergic airway inflammation by shifting macrophage polarization in the lung toward the alternatively activated M2 phenotype. Adoptive transfer of alveolar macrophages derived from Abx-treated mice was sufficient to increase allergic airway inflammation. Abx treatment resulted in the overgrowth of a commensal fungal Candida species in the gut and increased plasma concentrations of prostaglandin E? (PGE?), which induced M2 macrophage polarization in the lung. Suppression of PGE? synthesis by the cyclooxygenase inhibitors aspirin and celecoxib suppressed M2 macrophage polarization and decreased allergic airway inflammatory cell infiltration in Abx-treated mice. Thus, Abx treatment can cause overgrowth of particular fungal species in the gut and promote M2 macrophage activation at distant sites to influence systemic responses including allergic inflammation.
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Escherichia coli isolates from inflammatory bowel diseases patients survive in macrophages and activate NLRP3 inflammasome.
Int. J. Med. Microbiol.
PUBLISHED: 01-14-2014
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Crohn's disease (CD) is a multifactorial pathology associated with the presence of adherent-invasive Escherichia coli (AIEC) and NLRP3 polymorphic variants. The presence of intracellular E. coli in other intestinal pathologies (OIP) and the role of NLRP3-inflammasome in the immune response activated by these bacteria have not been investigated. In this study, we sought to characterize intracellular strains isolated from patients with CD, ulcerative colitis (UC) and OIP, and analyze NLRP3-inflammasome role in the immune response and bactericidal activity induced in macrophages exposed to invasive bacteria. For this, intracellular E. coli isolation from ileal biopsies, using gentamicin-protection assay, revealed a prevalence and CFU/biopsy of E. coli higher in biopsies from CD, UC and OIP patients than in controls. To characterize bacterial isolates, pulsed-field gel electrophoresis (PFGE) patterns, virulence genes, serogroup and phylogenetic group were analyzed. We found out that bacteria isolated from a given patient were closely related and shared virulence factors; however, strains from different patients were genetically heterogeneous. AIEC characteristics in isolated strains, such as invasive and replicative properties, were assessed in epithelial cells and macrophages, respectively. Some strains from CD and UC demonstrated AIEC properties, but not strains from OIP. Furthermore, the role of NLRP3 in pro-inflammatory cytokines production and bacterial elimination was determined in macrophages. E. coli strains induced IL-1? through NLRP3-dependent mechanism; however, their elimination by macrophages was independent of NLRP3. Invasiveness of intracellular E. coli strains into the intestinal mucosa and IL-1? production may contribute to CD and UC pathogenesis.
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Temporal trend, clinicopathologic and sociodemographic characterization of age at diagnosis of breast cancer among US women diagnosed from 1990 to 2009.
Springerplus
PUBLISHED: 01-01-2014
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This paper investigates the distribution of age at diagnosis of female breast cancer and its association with temporal trend, clinicopathologic and sociodemographic variables in the presence of two latent clusters that are directly unobservable. Such clusters help to identify two subpopulations of either young or old patients whose etiologies are thought to be different. A large sample drawn from registry data from the National Cancer Institute's Surveillance, Epidemiology, and End Results program from 1990 to 2009 was analyzed using a two-component Gaussian mixture model. Evidence of a steady delay of age at diagnosis and an increasing proportion of young patients being diagnosed during the 20-year period was found. Histopathologic effects indicate that duct and lobular carcinomas differ significantly in regard to subpopulation membership, which confirms that they represent different etiologies. While the presence of estrogen receptor status in the model overlaps the effects of other important variables it is highly correlated with, it is found that the grade, extension and size of the tumor along with lymph node involvement status, race and marital status are important predictors of age at diagnosis. The results highlight the significant impacts that such features can have on breast cancer control efforts, and point to the importance of ensuring that medical decision making should use them along with an indicator of the age subpopulation a patient may belong to.
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3,4-Methylenedioxy-?-nitrostyrene inhibits NLRP3 activation by blocking assembly of the inflammasome.
J. Biol. Chem.
PUBLISHED: 11-21-2013
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The NLRP3 inflammasome is a critical component of the innate immune system. NLRP3 activation is induced by diverse stimuli associated with bacterial infection or tissue damage, but its inappropriate activation is involved in the pathogenesis of inherited and acquired inflammatory diseases. However, the mechanism by which NLRP3 is activated remains poorly understood. In this study, we explored the role of kinases in NLRP3 inflammasome activation by screening a kinase inhibitor library and identified 3,4-Methylenedioxy-?-nitrostyrene (MNS) as an inhibitor for NLRP3 inflammasome activation. Notably, MNS did not affect the activation of the NLRC4 or AIM2 inflammasome. Mechanistically, MNS specifically prevented NLRP3-mediated ASC speckle formation and oligomerization without blocking potassium efflux induced by NLRP3 agonists. Surprisingly, Syk kinase, the reported target of MNS, did not mediate the inhibitory activity of MNS on NLRP3 inflammasome activation. We also found that the nitrovinyl group of MNS is essential for the inhibitory activity of MNS. Immunoprecipitation, mass spectrometry and mutation studies suggest that both the NOD domain and the LRR domain of NLRP3 were the intracellular targets of MNS. Administration of MNS also inhibited NLRP3 ATPase activity in vitro, suggesting that MNS blocks the NLRP3 inflammasome by directly targeting NLRP3 or NLRP3-associated complexes. These studies identified a novel chemical probe for studying the molecular mechanism of NLRP3 inflammasome activation which may advance the development of novel strategies to treat diseases associated with abnormal activation of NLRP3 inflammasome.
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Gut Microbiota Protects against Gastrointestinal Tumorigenesis Caused by Epithelial Injury.
Cancer Res.
PUBLISHED: 10-28-2013
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Inflammation is a critical player in the development of both colitis-associated and sporadic colon cancers. Several studies suggest that the microbiota contribute to inflammation and tumorigenesis; however, studies to understand the role of the microbiota in colon tumor development in germ-free (GF) mice are limited. We therefore studied the effects of the microbiota on the development of inflammation and tumors in GF and conventionally raised specific pathogen-free (SPF) mice treated with azoxymethane (AOM) and dextran sulfate sodium (DSS). We discovered that GF mice developed significantly more and larger tumors compared with that in SPF mice after AOM and DSS treatment despite the lack of early acute inflammation in response to chemically induced injury by DSS. Although the extent of intestinal epithelial damage and apoptosis was not significantly different in GF and SPF mice, there was a delay in intestinal epithelial repair to DSS-induced injury in GF mice resulting in a late onset of proinflammatory and protumorigenic responses and increased epithelial proliferation and microadenoma formation. Recolonization of GF mice with commensal bacteria or administration of lipopolysaccharide reduced tumorigenesis. Thus, although commensal bacteria are capable of driving chronic inflammation and tumorigenesis, the gut microbiota also have important roles in limiting chemically induced injury and proliferative responses that lead to tumor development. Cancer Res; 73(24); 7199-210. ©2013 AACR.
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Staphylococcus ?-toxin induces allergic skin disease by activating mast cells.
Nature
PUBLISHED: 09-12-2013
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Atopic dermatitis is a chronic inflammatory skin disease that affects 15-30% of children and approximately 5% of adults in industrialized countries. Although the pathogenesis of atopic dermatitis is not fully understood, the disease is mediated by an abnormal immunoglobulin-E immune response in the setting of skin barrier dysfunction. Mast cells contribute to immunoglobulin-E-mediated allergic disorders including atopic dermatitis. Upon activation, mast cells release their membrane-bound cytosolic granules leading to the release of several molecules that are important in the pathogenesis of atopic dermatitis and host defence. More than 90% of patients with atopic dermatitis are colonized with Staphylococcus aureus in the lesional skin whereas most healthy individuals do not harbour the pathogen. Several staphylococcal exotoxins can act as superantigens and/or antigens in models of atopic dermatitis. However, the role of these staphylococcal exotoxins in disease pathogenesis remains unclear. Here we report that culture supernatants of S. aureus contain potent mast-cell degranulation activity. Biochemical analysis identified ?-toxin as the mast cell degranulation-inducing factor produced by S. aureus. Mast cell degranulation induced by ?-toxin depended on phosphoinositide 3-kinase and calcium (Ca(2+)) influx; however, unlike that mediated by immunoglobulin-E crosslinking, it did not require the spleen tyrosine kinase. In addition, immunoglobulin-E enhanced ?-toxin-induced mast cell degranulation in the absence of antigen. Furthermore, S. aureus isolates recovered from patients with atopic dermatitis produced large amounts of ?-toxin. Skin colonization with S. aureus, but not a mutant deficient in ?-toxin, promoted immunoglobulin-E and interleukin-4 production, as well as inflammatory skin disease. Furthermore, enhancement of immunoglobulin-E production and dermatitis by ?-toxin was abrogated in Kit(W-sh/W-sh) mast-cell-deficient mice and restored by mast cell reconstitution. These studies identify ?-toxin as a potent inducer of mast cell degranulation and suggest a mechanistic link between S. aureus colonization and allergic skin disease.
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Human Umbilical Cord Blood Mesenchymal Stem Cells Reduce Colitis in Mice by Activating NOD2 Signaling to COX2.
Gastroenterology
PUBLISHED: 08-14-2013
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Decreased levels or function of nucleotide-binding oligomerization domain 2 (NOD2) are associated with Crohns disease. NOD2 regulates intestinal inflammation, and also is expressed by human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs), to regulate their differentiation. We investigated whether NOD2 is required for the anti-inflammatory activities of MSCs in mice with colitis.
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The Cag pathogenicity island and interaction between TLR2/NOD2 and NLRP3 regulate IL-1? production in Helicobacter pylori infected dendritic cells.
Eur. J. Immunol.
PUBLISHED: 05-31-2013
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Helicobacter pylori colonization of the stomach affects about half of the world population and is associated with the development of gastritis, ulcers, and cancer. Polymorphisms in the IL1B gene are linked to an increased risk of H. pylori associated cancer, but the bacterial and host factors that regulate interleukin (IL)-1? production in response to H. pylori infection remain unknown. Using murine BM-derived DCs, we show that the bacterial virulence factors cytotoxin-associated genes pathogenicity island and CagL, but not vacuolating cytotoxin A or CagA, regulate the induction of pro-IL-1? and the production of mature IL-1? in response to H. pylori infection. We further show that the host receptors, Toll-like receptor 2 (TLR2) and nucleotide-binding oligomerization domain 2 (NOD2), but not NOD1, are required for induction of pro-IL-1? and NOD-like receptor pyrin domain containing 3 (NLRP3) in H. pylori infected DCs. In contrast, NLRP3 and the adaptor ASC were essential for the activation of caspase-1, processing of pro-IL-1? into IL-1?, and IL-1? secretion. Finally, we show that mice deficient in caspase-1, IL-1?, and IL-1 receptor, but not NLRP3, are impaired in the clearance of CagA-positive H. pylori from the stomach when compared with WT mice. These studies identify bacterial cag pathogenicity island and the cooperative interaction among host innate receptors TLR2, NOD2, and NLRP3 as important regulators of IL-1? production in H. pylori infected DCs.
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K? efflux is the common trigger of NLRP3 inflammasome activation by bacterial toxins and particulate matter.
Immunity
PUBLISHED: 05-20-2013
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The NLRP3 inflammasome is an important component of the innate immune system. However, its mechanism of activation remains largely unknown. We show that NLRP3 activators including bacterial pore-forming toxins, nigericin, ATP, and particulate matter caused mitochondrial perturbation or the opening of a large membrane pore, but this was not required for NLRP3 activation. Furthermore, reactive oxygen species generation or a change in cell volume was not necessary for NLRP3 activation. Instead, the only common activity induced by all NLRP3 agonists was the permeation of the cell membrane to K? and Na?. Notably, reduction of the intracellular K? concentration was sufficient to activate NLRP3, whereas an increase in intracellular Na? modulated but was not strictly required for inflammasome activation. These results provide a unifying model for the activation of the NLRP3 inflammasome in which a drop in cytosolic K? is the common step that is necessary and sufficient for caspase-1 activation.
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Multiple effects of dendritic cell depletion on murine norovirus infection.
J. Gen. Virol.
PUBLISHED: 05-01-2013
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Dendritic cells (DCs) are permissive to murine norovirus (MNV) infection in vitro and in vivo. However, their roles during infection in vivo are not well defined. To determine the role of DCs during infection, conventional DCs were depleted from CD11c-DTR mice and infected with a persistent MNV strain. Viral titres in the intestine and secondary lymphoid organs were determined at early time points during infection, and anti-MNV antibody responses were analysed later during infection. Depletion of conventional DCs resulted in increased viral loads in intestinal tissues, impaired generation of antibody responses, and a failure of MNV to efficiently infect lymphoid tissues. These data suggest that DCs play multiple roles in MNV pathogenesis, in both innate immunity and the efficient generation of adaptive immune responses against MNV, as well as by promoting the dissemination of MNV to secondary lymphoid tissues. This is the first study to probe the roles of DCs in controlling and/or facilitating a norovirus infection in vivo and provides the basis for further studies aimed at defining mechanisms by which DCs control MNV replication and promote viral dissemination.
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Role of the gut microbiota in immunity and inflammatory disease.
Nat. Rev. Immunol.
PUBLISHED: 04-27-2013
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The mammalian intestine is colonized by trillions of microorganisms, most of which are bacteria that have co-evolved with the host in a symbiotic relationship. The collection of microbial populations that reside on and in the host is commonly referred to as the microbiota. A principal function of the microbiota is to protect the intestine against colonization by exogenous pathogens and potentially harmful indigenous microorganisms via several mechanisms, which include direct competition for limited nutrients and the modulation of host immune responses. Conversely, pathogens have developed strategies to promote their replication in the presence of competing microbiota. Breakdown of the normal microbial community increases the risk of pathogen infection, the overgrowth of harmful pathobionts and inflammatory disease. Understanding the interaction of the microbiota with pathogens and the host might provide new insights into the pathogenesis of disease, as well as novel avenues for preventing and treating intestinal and systemic disorders.
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Induction of bone loss by pathobiont-mediated Nod1 signaling in the oral cavity.
Cell Host Microbe
PUBLISHED: 03-15-2013
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Periodontitis is a common disease that is characterized by resorption of the alveolar bone and mediated by commensal bacteria that trigger host immune responses and bone destruction through unidentified mechanisms. We report that Nod1, an innate intracellular host receptor for bacterial peptidoglycan-related molecules, is critical for commensal-induced periodontitis in a mouse model. Mice lacking Nod1 exhibit reduced bone resorption as well as impaired recruitment of neutrophils to gingival tissues and osteoclasts to the alveolar bone, which mediate tissue and bone destruction. Further analysis showed that accumulation of a Nod1-stimulating commensal bacterium, NI1060, at gingival sites was sufficient to induce neutrophil recruitment and bone resorption. Genomic sequencing revealed that NI1060 is a mouse-specific bacterium that is related to bacteria associated with the development of aggressive periodontitis in humans. These findings provide insight into commensal-host interactions contributing to periodontitis and identify a potential target for preventing this common oral disease.
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A deficiency in the autophagy gene Atg16L1 enhances resistance to enteric bacterial infection.
Cell Host Microbe
PUBLISHED: 03-07-2013
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Polymorphisms in the essential autophagy gene Atg16L1 have been linked with susceptibility to Crohns disease, a major type of inflammatory bowel disease (IBD). Although the inability to control intestinal bacteria is thought to underlie IBD, the role of Atg16L1 during extracellular intestinal bacterial infections has not been sufficiently examined and compared to the function of other IBD susceptibility genes, such as Nod2, which encodes a cytosolic bacterial sensor. We find that Atg16L1 mutant mice are resistant to intestinal disease induced by the model bacterial pathogen Citrobacter rodentium. An Atg16L1 deficiency alters the intestinal environment to mediate an enhanced immune response that is dependent on monocytic cells, but this hyperimmune phenotype and its protective effects are lost in Atg16L1/Nod2 double-mutant mice. These results reveal an immunosuppressive function of Atg16L1 and suggest that gene variants affecting the autophagy pathway may have been evolutionarily maintained to protect against certain life-threatening infections.
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Reducing the need for central dual-energy X-ray absorptiometry in postmenopausal women: efficacy of a clinical algorithm including peripheral densitometry.
Calcif. Tissue Int.
PUBLISHED: 03-06-2013
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We evaluated the efficacy of a triage approach based on a combination of osteoporosis risk-assessment tools plus peripheral densitometry to identify low bone density accurately enough to be useful for clinical decision making in postmenopausal women. We conducted a cross-sectional diagnostic study in postmenopausal Caucasian women from primary and tertiary care. All women underwent dual-energy X-ray absorptiometric (DXA) measurement at the hip and lumbar spine and were categorized as osteoporotic or not. Additionally, patients had a nondominant heel densitometry performed with a PIXI densitometer. Four osteoporosis risk scores were tested: SCORE, ORAI, OST, and OSIRIS. All measurements were cross-blinded. We estimated the area under the curve (AUC) to predict the DXA results of 16 combinations of PIXI plus risk scores. A formula including the best combination was derived from a regression model and its predictability estimated. We included 505 women, in whom the prevalence of osteoporosis was 20 %, similar in both settings. The best algorithm was a combination of PIXI + OST + SCORE with an AUC of 0.826 (95 % CI 0.782-0.869). The proposed formula is Risk = (-12) × [PIXI + (-5)] × [OST + (-2)] × SCORE and showed little bias in the estimation (0.0016). If the formula had been implemented and the intermediate risk cutoff set at -5 to 20, the system would have saved 4,606.34 in the study year. The formula proposed, derived from previously validated risk scores plus a peripheral bone density measurement, can be used reliably in primary care to avoid unnecessary central DXA measurements in postmenopausal women.
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Control of pathogens and pathobionts by the gut microbiota.
Nat. Immunol.
PUBLISHED: 02-27-2013
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A dense resident microbial community in the gut, referred as the commensal microbiota, coevolved with the host and is essential for many host physiological processes that include enhancement of the intestinal epithelial barrier, development of the immune system and acquisition of nutrients. A major function of the microbiota is protection against colonization by pathogens and overgrowth of indigenous pathobionts that can result from the disruption of the healthy microbial community. The mechanisms that regulate the ability of the microbiota to restrain pathogen growth are complex and include competitive metabolic interactions, localization to intestinal niches and induction of host immune responses. Pathogens, in turn, have evolved strategies to escape from commensal-mediated resistance to colonization. Thus, the interplay between commensals and pathogens or indigenous pathobionts is critical for controlling infection and disease. Understanding pathogen-commensal interactions may lead to new therapeutic approaches to treating infectious diseases.
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Acute toxicity of arsenic under different temperatures and salinity conditions on the white shrimp Litopenaeus vannamei.
Biol Trace Elem Res
PUBLISHED: 02-20-2013
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The aim of this study was to determine acute toxicity in the post larvae of the white shrimp Litopenaeus vannamei after 96 h of exposure to dissolved arsenic under three different temperatures and salinity conditions. Recent reports have shown an increase in the presence of this metalloid in coastal waters, estuaries, and lagoons along the Mexican coast. The white shrimp stands out for its adaptability to temperature and salinity changes and for being the main product for many commercial fisheries; it has the highest volume of oceanic capture and production in Mexican shrimp farms. Lethal concentrations (LC50-96 h) were obtained at nine different combinations (3?×?3 combinations in total) of temperature (20, 25, and 30 °C) and salinity (17, 25, and 33) showing mean LC50-96 h values (±standard error) of 9.13?±?0.76, 9.17?±?0.56, and 6.23?±?0.57 mgAs?L(-1)(at 20 °C and 17, 25, and 33 salinity); 12.29?±?2.09, 8.70?±?0.82, and 8.03?±?0.59 mgAs?L(-1) (at 25 °C and 17, 25, and 33 salinity); and 7.84?±?1.30, 8.49?±?1.40, and 7.54?±?0.51 mgAs?L(-1) (at 30 °C and 17, 25, and 33 salinity), respectively. No significant differences were observed for the optimal temperature and isosmotic point of maintenance (25 °C-S 25) for the species, with respect to the other experimental conditions tested, except for at 20 °C-S 33, which was the most toxic. Toxicity under 20 °C-S 33 conditions was also higher than 25 °C-S 17 and 20 °C (S 17 or 25). The least toxic condition was 25 °C-S 17. All this suggests that the toxic effect of arsenic is not affected by temperature changes; it depends on the osmoregulatory pattern developed by the shrimp, either hyperosmotic at low salinity or hiposmotic at high salinity, as observed at least on the extreme salinity conditions here tested (17 and 33). However, further studies testing salinities near the isosmotic point (between 20 and 30 salinities) are needed to clarify these mechanisms.
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Role of the gut microbiota in the development and function of lymphoid cells.
J. Immunol.
PUBLISHED: 02-05-2013
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Mammals are colonized by large numbers of microorganisms, including trillions of bacteria, most of which live in the intestinal tract. These indigenous microorganisms that inhabit the body of humans and animals are referred collectively to as the microbiota. Accumulating evidence indicates that the microbiota regulates the development and/or function of different types of immune cells in the intestine. For example, the microbiota drives homeostatic, pathogenic, and regulatory T cell immune responses that contribute to tissue homeostasis, but also can promote disease. The gut microbes also facilitate IgA responses, which in turn regulate the composition and function of the gut microbiota. Thus, the reciprocal regulation of the gut microbiota and the host immune system may influence the balance between homeostasis and disease in the intestine.
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Understanding the microbiota in the midst of Renaissance architecture and olive groves.
Nat. Immunol.
PUBLISHED: 01-22-2013
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Leading scientists working on the microbiome gathered in an October 2012 meeting in Baeza, Spain, to discuss recent advances in the understanding of the role of the microbiota in immunity, pathogen colonization, metabolism and disease.
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A genome-wide siRNA screen reveals positive and negative regulators of the NOD2 and NF-?B signaling pathways.
Sci Signal
PUBLISHED: 01-17-2013
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The cytoplasmic receptor NOD2 (nucleotide-binding oligomerization domain 2) senses peptidoglycan fragments and triggers host defense pathways, including activation of nuclear factor ?B (NF-?B) signaling, which lead to inflammatory immune responses. Dysregulation of NOD2 signaling is associated with inflammatory diseases, such as Crohns disease and Blau syndrome. We used a genome-wide small interfering RNA screen to identify regulators of the NOD2 signaling pathway. Several genes associated with Crohns disease risk were identified in the screen. A comparison of candidates from this screen with other "omics" data sets revealed interconnected networks of genes implicated in NF-?B signaling, thus supporting a role for NOD2 and NF-?B pathways in the pathogenesis of Crohns disease. Many of these regulators were validated in secondary assays, such as measurement of interleukin-8 secretion, which is partially dependent on NF-?B. Knockdown of putative regulators in human embryonic kidney 293 cells followed by stimulation with tumor necrosis factor-? revealed that most of the genes identified were general regulators of NF-?B signaling. Overall, the genes identified here provide a resource to facilitate the elucidation of the molecular mechanisms that regulate NOD2- and NF-?B-mediated inflammation.
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The protein kinase PKR is critical for LPS-induced iNOS production but dispensable for inflammasome activation in macrophages.
Eur. J. Immunol.
PUBLISHED: 01-08-2013
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Inflammasomes are multi-protein platforms that drive the activation of caspase-1 leading to the processing and secretion of biologically active IL-1? and IL-18. Different inflammasomes including NOD-like receptor (NLR) family pyrin domain-containing 3 (NLRP3), NLR caspase-recruitment domain-containing 4 (NLRC4) and absent in melanoma 2 (AIM2) are activated and assembled in response to distinct microbial or endogenous stimuli. However, the mechanisms by which upstream stimuli trigger inflammasome activation remain poorly understood. Double-stranded RNA-activated protein kinase (PKR), a protein kinase activated by viral infection, has been recently shown to be required for the activation of the inflammasomes. Using macrophages from two different mouse strains deficient in PKR, we found that PKR is important for the induction of the inducible nitric oxide synthase (iNOS). However, PKR was dispensable for caspase-1 activation, processing of pro-IL-1?/IL-18 and secretion of IL-1? induced by stimuli that trigger the activation of NLRP3, NLRC4 and AIM2. These results indicate that PKR is not required for inflammasome activation in macrophages.
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Innate immune recognition of flagellin limits systemic persistence of Brucella.
Cell. Microbiol.
PUBLISHED: 01-07-2013
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Brucella are facultative intracellular bacteria that cause chronic infections by limiting innate immune recognition. It is currently unknown whether Brucella?FliC flagellin, the monomeric subunit of flagellar filament, is sensed by the host during infection. Here, we used two mutants of Brucella melitensis, either lacking or overexpressing flagellin, to show that FliC hinders bacterial replication in vivo. The use of cells and mice genetically deficient for different components of inflammasomes suggested that FliC was a target of the cytosolic innate immune receptor NLRC4?in vivo but not in macrophages in vitro where the response to FliC was nevertheless dependent on the cytosolic adaptor ASC, therefore suggesting a new pathway of cytosolic flagellin sensing. However, our work also suggested that the lack of TLR5 activity of Brucella flagellin and the regulation of its synthesis and/or delivery into host cells are both part of the stealthy strategy of Brucella towards the innate immune system. Nevertheless, as a flagellin-deficient mutant of B.?melitensis wasfound to cause histologically demonstrable injuries in the spleen of infected mice, we suggested that recognition of FliC plays a role in the immunological stand-off between Brucella and its host, which is characterized by a persistent infection with limited inflammatory pathology.
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NOD2-mediated dysbiosis predisposes mice to transmissible colitis and colorectal cancer.
J. Clin. Invest.
PUBLISHED: 01-02-2013
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Instability in the composition of gut bacterial communities (dysbiosis) has been linked to common human intestinal disorders, such as Crohns disease and colorectal cancer. Here, we show that dysbiosis caused by Nod2 deficiency gives rise to a reversible, communicable risk of colitis and colitis-associated carcinogenesis in mice. Loss of either Nod2 or RIP2 resulted in a proinflammatory microenvironment that enhanced epithelial dysplasia following chemically induced injury. The condition could be improved by treatment with antibiotics or an anti-interleukin-6 receptor-neutralizing antibody. Genotype-dependent disease risk was communicable via maternally transmitted microbiota in both Nod2-deficient and WT hosts. Furthermore, reciprocal microbiota transplantation reduced disease risk in Nod2-deficient mice and led to long-term changes in intestinal microbial communities. Conversely, disease risk was enhanced in WT hosts that were recolonized with dysbiotic fecal microbiota from Nod2-deficient mice. Thus, we demonstrated that licensing of dysbiotic microbiota is a critical component of disease risk. Our results demonstrate that NOD2 has an unexpected role in shaping a protective assembly of gut bacterial communities and suggest that manipulation of dysbiosis is a potential therapeutic approach in the treatment of human intestinal disorders.
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Interleukin-10 ablation promotes tumor development, growth, and metastasis.
Cancer Res.
PUBLISHED: 11-28-2011
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Interleukin-10 (IL-10) is a broadly acting immune inhibitory cytokine that is generally thought to support tumor growth. Here we challenge this view with evidence that genetic ablation of IL-10 in the mouse significantly heightens sensitivity to chemical carcinogenesis, growth of transplanted tumors, and formation of metastases. Tumor growth in IL-10-deficient (IL-10(-/-)) mice was associated with an increased level of myeloid-derived suppressor cells (MDSC) and CD4(+)Foxp3(+) regulatory T (Treg) cells in both the tumor microenvironment and the tumor-draining lymph nodes. IL-10(-/-) MDSCs express high levels of MHC and IL-1, and they efficiently induced formation of Treg cells. IL-1 signaling blockade reduced tumor growth mediated by IL-10 deficiency, associated with a partial rescue of tumor infiltration and function of effector T cells and a decrease in tumor angiogenesis and tumor infiltration by Treg cells. Taken together, our findings establish that endogenous IL-10 inhibits inflammatory cytokine production and hampers the development of Treg cells and MDSCs, two key components of the immunosuppressive tumor microenvironment, thereby inhibiting tumor development, growth, and metastasis.
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TLR2 and RIP2 pathways mediate autophagy of Listeria monocytogenes via extracellular signal-regulated kinase (ERK) activation.
J. Biol. Chem.
PUBLISHED: 10-27-2011
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Listeria monocytogenes is a facultative intracellular pathogen that invades both phagocytic and non-phagocytic cells. Recent studies have shown that L. monocytogenes infection activates the autophagy pathway. However, the innate immune receptors involved and the downstream signaling pathways remain unknown. Here, we show that macrophages deficient in the TLR2 and NOD/RIP2 pathway display defective autophagy induction in response to L. monocytogenes. Inefficient autophagy in Tlr2(-/-) and Nod2(-/-) macrophages led to a defect in bacteria colocalization with the autophagosomal marker GFP-LC3. Consequently, macrophages lacking TLR2 and NOD2 were found to be more susceptible to L. monocytogenes infection, as were the Rip2(-/-) mice. Tlr2(-/-) and Nod2(-/-) cells showed perturbed NF-?B and ERK signaling. However, autophagy against L. monocytogenes was dependent selectively on the ERK pathway. In agreement, wild-type cells treated with a pharmacological inhibitor of ERK or ERK-deficient cells displayed inefficient autophagy activation in response to L. monocytogenes. Accordingly, fewer bacteria were targeted to the autophagosomes and, consequently, higher bacterial growth was observed in cells deficient in the ERK signaling pathway. These findings thus demonstrate that TLR2 and NOD proteins, acting via the downstream ERK pathway, are crucial to autophagy activation and provide a mechanistic link between innate immune receptors and induction of autophagy against cytoplasm-invading microbes, such as L. monocytogenes.
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Cytokines tumor necrosis factor-? and interferon-? induce pancreatic ?-cell apoptosis through STAT1-mediated Bim protein activation.
J. Biol. Chem.
PUBLISHED: 09-21-2011
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Type 1 diabetes is characterized by local inflammation (insulitis) in the pancreatic islets causing ?-cell loss. The mitochondrial pathway of apoptosis is regulated by the balance and interaction between Bcl-2 members. Here we clarify the molecular mechanism of ?-cell death triggered by the pro-inflammatory cytokines tumor necrosis factor (TNF)-? and interferon (IFN)-?. The combination of TNF-? + IFN-? induced DP5, p53 up-regulated modulator of apoptosis (PUMA), and Bim expression in human islets and rodent ?-cells. DP5 and PUMA inactivation by RNA interference partially protected against TNF-? + IFN-?-induced ?-cell apoptosis. DP5 knock-out mice had increased ?-cell area, and isolated islets from these mice were resistant to cytokine exposure. Bim expression was transcriptionally regulated by STAT1, and its activation triggered cleavage of caspases. Silencing of Bim protected rodent and human ?-cells to a large extent against TNF-? + IFN-?, indicating a major role of this BH3-only activator protein in the mechanism of apoptosis. Our data support a highly regulated and context-dependent modulation of specific Bcl-2 members controlling the mitochondrial pathway of ?-cell apoptosis during insulitis.
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Both exogenous commensal and endogenous self antigens stimulate T cell proliferation under lymphopenic conditions.
Cell. Immunol.
PUBLISHED: 08-18-2011
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Within lymphopenic recipients, naïve T cells undergo proliferation that is induced by homeostatic mechanisms. Earlier studies have demonstrated that commensal antigens play a key role in inducing the proliferation. However, a relative contribution of endogenous self antigens in this process has not been formally investigated. In this study, we utilized a pharmacologic inhibitor that blocks T cell egress from the lymphoid tissues, antibiotics, and germ-free animals to examine the role of commensal and self antigens. The results suggest that T cell proliferation under lymphopenic conditions is a heterogeneous process triggered by both exogenous commensal and endogenous self antigens.
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Cutting edge: Crohns disease-associated Nod2 mutation limits production of proinflammatory cytokines to protect the host from Enterococcus faecalis-induced lethality.
J. Immunol.
PUBLISHED: 08-17-2011
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Nucleotide-binding oligomerization domain 2 (Nod2) mutations including L1007fsinsC are associated with the development of Crohns disease (CD). These CD-associated Nod2 mutations are common in healthy white populations, suggesting that they may confer some protective function, but experimental evidence is lacking. Using a mouse strain that expresses Nod2(2939iCstop), the equivalent of the L1007fsinsC mutation, we found that macrophages homozygous for Nod2(2939iCstop) are impaired in the recognition of muramyl dipeptide and Enterococcus faecalis, a commensal bacterium that is a common cause of sepsis-associated lethality in humans. Notably, Nod2 deficiency and homozygocity for Nod2(2939iCstop) were associated with reduced production of TNF-? and IL-6 and lethality after systemic infection with E. faecalis despite normal bacteria loads. Consistently, inhibition of TNF-? signaling protected wild-type mice from E. faecalis-induced lethality. These results suggest that the same Nod2 mutation can increase the susceptibility to CD, but also protect the host from systemic infection by a common enteric bacterium.
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Inflammasomes in intestinal inflammation and cancer.
Gastroenterology
PUBLISHED: 08-03-2011
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Inflammasomes are multi-protein complexes that mediate activation of caspase-1, which promotes secretion of the proinflammatory cytokines interleukin-1? and interleukin-18 and pyroptosis, a form of phagocyte cell death induced by bacterial pathogens. Members of the Nod-like receptor family (including Nlrp1, Nlrp3, and Nlrc4), the DNA sensor Aim2, the adaptor apoptosis-associated speck-like protein (ASC), and pro-caspase-1 are important components of inflammasomes. Stimulation with specific microbial and endogenous molecules leads to inflammasome assembly and caspase-1 activation. Inflammasomes are believed to mediate host defense against microbial pathogens and tissue homeostasis within the intestine, and their dysregulation might contribute to inflammatory diseases and intestinal cancer. Improving our understanding of inflammasome signaling pathways could provide insights into the pathogenesis of many gastrointestinal disorders and the development of therapeutic targets and approaches to treat diseases such as inflammatory bowel diseases and gastrointestinal cancers.
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Cutting edge: reactive oxygen species inhibitors block priming, but not activation, of the NLRP3 inflammasome.
J. Immunol.
PUBLISHED: 06-15-2011
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A common denominator among the multiple damage-inducing agents that ultimately lead to activation of NLRP3 has not yet been identified. Recently, production of reactive oxygen species (ROS) has been suggested to act as a common event upstream of the NLRP3 inflammasome machinery. Because de novo translation of NLRP3 is an essential step in the activation of NLRP3, we investigated the role of substances that inhibit either ROS production or its oxidative activity. Although we observe that NLRP3 inflammasome activation is unique among other known inflammasomes in its sensitivity to ROS inhibition, we have found that this phenomenon is attributable to the fact that NLRP3 strictly requires priming by a proinflammatory signal, a step that is blocked by ROS inhibitors. Although these data do not exclude a general role for ROS production in the process of NLRP3-triggered inflammation, they would put ROS upstream of NLRP3 induction, but not activation.
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A new twist on the PYRIN Mediterranean coast.
Immunity
PUBLISHED: 05-28-2011
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Familial Mediterranean fever is caused by mutations of the PYRIN protein. Chae et al. (2011) provide evidence for a ASC protein-dependent pathway of caspase-1 activation in which gain-of-function PYRIN mutations lead to IL-1? cytokine overproduction and inflammatory disease.
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Mechanism of impaired NLRP3 inflammasome priming by monophosphoryl lipid A.
Sci Signal
PUBLISHED: 05-05-2011
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Monophosphoryl lipid A (MLA), a nontoxic derivative of the endotoxin lipopolysaccharide (LPS), has been approved in the United States for use as a vaccine adjuvant. LPS and MLA are ligands of Toll-like receptor 4 (TLR4), and it has been unclear why LPS triggers toxic inflammation, whereas MLA generates safe and effective immunostimulation. Signaling downstream of TLR4 is mediated by the adaptor proteins TRIF [Toll-interleukin-1 (IL-1) receptor (TIR) domain-containing adaptor-inducing interferon-?], which is required for adaptive immune outcomes, and MyD88 (myeloid differentiation marker 88), which is responsible for many proinflammatory effects. Two models have provided nonexclusive explanations for the differential effects of LPS and MLA. According to the first model, MLA fails to induce maturation of the proinflammatory cytokine IL-1? because it fails to activate caspase-1, which is required for the conversion of pro-IL-1? into its bioactive form. The second model suggests that MLA triggers unequal engagement of both of the signaling adaptor pathways of TLR4, such that signaling mediated by TRIF is largely intact, whereas signaling mediated by MyD88 is incomplete. We show that the TRIF-biased signaling that is characteristic of low-toxicity MLA explains its failure to activate caspase-1. Defective induction of NLRP3, which depends on MyD88, led to decreased assembly of components of the IL-1?-activating inflammasome required for the activation of preformed, inactive procaspase-1. In addition, we elucidated the contributions of MyD88 and TRIF to priming of the NLRP3 inflammasome and demonstrated that TRIF-biased TLR4 activation by MLA was responsible for the defective production of mature IL-1?.
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A functional role for Nlrp6 in intestinal inflammation and tumorigenesis.
J. Immunol.
PUBLISHED: 05-04-2011
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The nucleotide-binding oligomerization domain-like receptor (NLR) family member, Nlrp6, has been implicated in inflammasome signaling to activate caspase-1, which is essential for the production of mature IL-1? and IL-18. However, a function for Nlrp6 in vivo has never been demonstrated. Due to the relative high expression of Nlrp6 in intestinal tissue, we hypothesized that Nlrp6 has a role in intestinal homeostasis. Indeed, Nlrp6-deficient mice are more susceptible to chemically induced colitis as well as colitis-induced tumorigenesis than wild-type (WT) mice. Nlrp6-deficient mice exhibited significantly more inflammation within the colon than WT mice after dextran sulfate sodium treatment. Their inability to resolve inflammation and repair damaged epithelium as efficiently as WT mice resulted in prolonged increases in epithelial proliferative activity that likely underlie the increased propensity for tumors in these mice during chronic inflammation. We further show that the activity of Nlrp6 in hematopoietic cells is critical for protection against inflammation-related colon tumorigenesis. This study highlights the importance of NLR function in maintaining intestinal homeostasis to prevent the development of aberrant inflammation and tumor development within the colon.
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Viral infection augments Nod1/2 signaling to potentiate lethality associated with secondary bacterial infections.
Cell Host Microbe
PUBLISHED: 04-01-2011
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Secondary bacterial infection is a common sequela to viral infection and is associated with increased lethality and morbidity. However, the underlying mechanisms remain poorly understood. We show that the TLR3/MDA5 agonist poly I:C or viral infection dramatically augments signaling via the NLRs Nod1 and Nod2 and enhances the production of proinflammatory cytokines. Enhanced Nod1 and Nod2 signaling by poly I:C required the TLR3/MDA5 adaptors TRIF and IPS-1 and was mediated by type I IFNs. Mechanistically, poly I:C or IFN-? induced the expression of Nod1, Nod2, and the Nod-signaling adaptor Rip2. Systemic administration of poly I:C or IFN-? or infection with murine norovirus-1 promoted inflammation and lethality in mice superinfected with E. coli, which was independent of bacterial burden but attenuated in the absence of Nod1/Nod2 or Rip2. Thus, crosstalk between type I IFNs and Nod1/Nod2 signaling promotes bacterial recognition, but induces harmful effects in the virally infected host.
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Enteric glia are multipotent in culture but primarily form glia in the adult rodent gut.
J. Clin. Invest.
PUBLISHED: 03-25-2011
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It is unclear whether neurogenesis occurs in the adult mammalian enteric nervous system (ENS). Neural crest-derived cells capable of forming multilineage colonies in culture, and neurons and glia upon transplantation into chick embryos, persist throughout adult life in the mammalian ENS. In this study we sought to determine the physiological function of these cells. We discovered that these cells could be identified based on CD49b expression and that they had characteristics of enteric glia, including p75, GFAP, S100B, and SOX10 expression. To test whether new neurons or glia arise in the adult gut under physiological conditions, we marked dividing progenitors with a thymidine analog in rodents under steady-state conditions, or during aging, pregnancy, dietary changes, hyperglycemia, or exercise. We also tested gut injuries including inflammation, irradiation, benzalkonium chloride treatment, partial gut stenosis, and glial ablation. We readily observed neurogenesis in a neurogenic region of the central nervous system, but not reproducibly in the adult ENS. Lineage tracing of glial cells with GFAP-Cre and GFAP-CreERT2 also detected little or no adult ENS neurogenesis. Neurogenesis in the adult gut is therefore very limited under the conditions we studied. In contrast, ENS gliogenesis was readily observed under steady-state conditions and after injury. Adult enteric glia thus have the potential to form neurons and glia in culture but are fated to form mainly glia under physiological conditions and after the injuries we studied.
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Nucleotide-binding oligomerization domain 1 mediates recognition of Clostridium difficile and induces neutrophil recruitment and protection against the pathogen.
J. Immunol.
PUBLISHED: 03-16-2011
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Clostridium difficile is a Gram-positive obligate anaerobic pathogen that causes pseudomembranous colitis in antibiotics-treated individuals. However, host immune protective mechanisms against C. difficile are largely unknown. In this study, we show that C. difficile possesses potent stimulatory activity for nucleotide-binding oligomerization domain 1 (Nod1), an intracellular pattern recognition molecule that senses bacterial peptidoglycan-related molecules. Nod1(-/-), but not Nod2(-/-), mice exhibited increased lethality in response to C. difficile intestinal infection despite comparable levels of intestinal damage and epithelial permeability in Nod1(-/-) and control mice. The enhanced lethality was accompanied by impaired C. difficile clearance, increased bacterial translocation, and elevated levels of endotoxin and IL-1? in the serum of Nod1(-/-) mice. Histological and flow cytometric analyses revealed that Nod1(-/-) mice had defective recruitment of neutrophils, but not macrophages, to the intestine after C. difficile infection. The reduced recruitment of neutrophils correlated with impaired production of CXCL1, but not CCL2, XCL1, and other cytokines/chemokines, in infected Nod1(-/-) mice. The influx of neutrophils also was reduced when C. difficile was administered i.p., suggesting that Nod1 directly recognizes C. difficile to induce the recruitment of neutrophils to the infected site. These results indicate that Nod1 regulates host susceptibility to C. difficile and suggest that Nod1-mediated neutrophil recruitment is an important immune response against the enteric pathogen.
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Mutation of the transcription factor LEAFY COTYLEDON 2 alters the chemical composition of Arabidopsis seeds, decreasing oil and protein content, while maintaining high levels of starch and sucrose in mature seeds.
J. Plant Physiol.
PUBLISHED: 01-27-2011
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The transcription factor LEAFY COTYLEDON 2 (LEC2; At1g28300) is preferentially expressed in developing seeds of Arabidopsis. Detailed biochemical analysis of a loss-of-function lec2 mutant was carried out in seeds 6-21 days after flowering (DAF). In comparison to wild type controls, lec2 seeds had 15% less protein and 30% less oil, but accumulated 140% more sucrose and >5-fold more starch. We also quantified biomass and carbohydrates in the seed coat and embryo. The lec2 mutant had smaller seeds and an altered proportion of dry weight (bigger seed coat and smaller embryos). Mutant plants produced less mature seeds per silique and the harvest index was reduced. Soluble sugars (glucose, fructose and sucrose) was accumulated in the seed coat of the lec2 mutant, whereas the opposite effect was observed in the embryos (decrease in comparison to wild type). The rate of starch synthesis increased during early development, whereas the rate of starch degradation was diminished during late development, leading to higher residual starch in mature seed of the mutant. Starch accumulated in both seed coat and embryo. Homozygous mutant plants produced seeds that could germinate well if they were harvested immaturely, whereas seeds that became dry during maturity lost their germination efficiency very rapidly. We conclude that the LEC2 transcription factor not only controls cotyledon identity and morphology as previously reported, but also alters: (1) the delivery of photosynthates from the seed coat to the embryo (sink strength), (2) carbon partitioning towards different storage compounds (oil, proteins and carbohydrates), (3) the rate of starch synthesis and degradation in developing seeds and (4) germination capacity of dry seeds.
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The ever-expanding function of NOD2: autophagy, viral recognition, and T cell activation.
Trends Immunol.
PUBLISHED: 01-19-2011
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The identification of several families of innate pattern recognition receptors has greatly enhanced our understanding of the host innate immune response against a variety of pathogens. One such family of innate receptors is the nucleotide-binding domain and leucine rich repeat containing receptors (NLRs). NOD2 has been characterized as a cytosolic sensor of bacteria peptidoglycan (PGN). For almost 10 years, NOD2 was assigned with the function of mediating the RICK- and nuclear factor-?B induced proinflammatory response triggered by PGN. Recent studies have extended the biological activity of NOD2 to include the induction of autophagy and antiviral responses, as well as mediating direct T cell activation. Here, we highlight and discuss these new findings in the context of immune activation and pathogen detection.
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The Nod2 sensor promotes intestinal pathogen eradication via the chemokine CCL2-dependent recruitment of inflammatory monocytes.
Immunity
PUBLISHED: 01-18-2011
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The intracellular sensor Nod2 is activated in response to bacteria, and the impairment of this response is linked to Crohns disease. However, the function of Nod2 in host defense remains poorly understood. We found that Nod2-/- mice exhibited impaired intestinal clearance of Citrobacter rodentium, an enteric bacterium that models human infection by pathogenic Escherichia coli. The increased bacterial burden was preceded by reduced CCL2 chemokine production, inflammatory monocyte recruitment, and Th1 cell responses in the intestine. Colonic stromal cells, but not epithelial cells or resident CD11b+ phagocytic cells, produced CCL2 in response to C. rodentium in a Nod2-dependent manner. Unlike resident phagocytic cells, inflammatory monocytes produced IL-12, a cytokine that induces adaptive immunity required for pathogen clearance. Adoptive transfer of Ly6C(hi) monocytes restored the clearance of the pathogen in infected Ccr2-/- mice. Thus, Nod2 mediates CCL2-CCR2-dependent recruitment of inflammatory monocytes, which is important in promoting bacterial eradication in the intestine.
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Dectin-1 and NOD2 mediate cathepsin activation in zymosan-induced arthritis in mice.
Inflamm. Res.
PUBLISHED: 01-14-2011
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Activation of pattern recognition receptors (PRR) may contribute to arthritis. Here, we elucidated the role of NOD2, a genetic cause of inflammatory arthritis, and several other PRR in a murine model of inflammatory arthritis.
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NOD2 deficiency results in increased susceptibility to peptidoglycan-induced uveitis in mice.
Invest. Ophthalmol. Vis. Sci.
PUBLISHED: 01-01-2011
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The innate immune receptor NOD2 is a genetic cause of uveitis (Blau syndrome). Intriguingly, in the intestine where polymorphisms of NOD2 predispose to Crohns disease, NOD2 reportedly suppresses inflammation triggered by the bacterial cell wall component, peptidoglycan (PGN). Whether NOD2 exerts a similar capacity in the regulation of ocular inflammation to PGN has not been explored.
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NOD2 controls the nature of the inflammatory response and subsequent fate of Mycobacterium tuberculosis and M. bovis BCG in human macrophages.
Cell. Microbiol.
PUBLISHED: 11-24-2010
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Mycobacterium tuberculosis (M.tb), which causes tuberculosis, is a host-adapted intracellular pathogen of macrophages. Intracellular pattern recognition receptors in macrophages such as nucleotide-binding oligomerization domain (NOD) proteins regulate pro-inflammatory cytokine production. NOD2-mediated signalling pathways in response to M.tb have been studied primarily in mouse models and cell lines but not in primary human macrophages. Thus we sought to determine the role of NOD2 in regulating cytokine production and growth of virulent M.tb and attenuated Mycobacterium bovis BCG (BCG) in human macrophages. We examined NOD2 expression during monocyte differentiation and observed a marked increase in NOD2 transcript and protein following 2-3 days in culture. Pre-treatment of human monocyte-derived and alveolar macrophages with the NOD2 ligand muramyl dipeptide enhanced production of TNF-? and IL-1? in response to M.tb and BCG in a RIP2-dependent fashion. The NOD2-mediated cytokine response was significantly reduced following knock-down of NOD2 expression by using small interfering RNA (siRNA) in human macrophages. Finally, NOD2 controlled the growth of both M.tb and BCG in human macrophages, whereas controlling only BCG growth in murine macrophages. Together, our results provide evidence that NOD2 is an important intracellular receptor in regulating the host response to M.tb and BCG infection in human macrophages.
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Sterile inflammation: sensing and reacting to damage.
Nat. Rev. Immunol.
PUBLISHED: 11-19-2010
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Over the past several decades, much has been revealed about the nature of the host innate immune response to microorganisms, with the identification of pattern recognition receptors (PRRs) and pathogen-associated molecular patterns, which are the conserved microbial motifs sensed by these receptors. It is now apparent that these same PRRs can also be activated by non-microbial signals, many of which are considered as damage-associated molecular patterns. The sterile inflammation that ensues either resolves the initial insult or leads to disease. Here, we review the triggers and receptor pathways that result in sterile inflammation and its impact on human health.
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Dysregulation of p38 and MKP-1 in response to NOD1/TLR4 stimulation in sarcoid bronchoalveolar cells.
Am. J. Respir. Crit. Care Med.
PUBLISHED: 09-17-2010
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Sarcoidosis is a systemic inflammatory disorder characterized by distinct up-regulation of Th1 cytokines, such as tumor necrosis factor (TNF)-? and IL-12. The mechanism underlying this up-regulation remains unclear. Recognition of microbial moieties through Toll-like or Nod-like receptors evokes sequential activation of mitogen-activated protein kinases (MAPKs), which plays a role in Th1-immune response.
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Respiratory virus-induced TLR7 activation controls IL-17-associated increased mucus via IL-23 regulation.
J. Immunol.
PUBLISHED: 07-12-2010
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The response to respiratory syncytial virus (RSV), negative strand ssRNA virus, depends upon the ability to recognize specific pathogen-associated targets. In the current study, the role of TLR7 that recognizes ssRNA was examined. Using TLR7(-/-) mice, we found that the response to RSV infection in the lung was more pathogenic as assessed by significant increases in inflammation and mucus production. Although there appeared to be no effect of TLR7 deficiency on type I IFN, the pathology was associated with an alteration in T cell responses with increases in mucogenic cytokines IL-4, IL-13, and IL-17. Examination of dendritic cells from TLR7(-/-) animals indicated a preferential activation of IL-23 (a Th17-promoting cytokine) and a decrease in IL-12 production. Neutralization of IL-17 in the TLR7(-/-) mice resulted in a significant decrease in the mucogenic response in the lungs of the RSV-infected mice. Thus, without TLR7-mediated responses, an altered immune environment ensued with a significant effect on airway epithelial cell remodeling and goblet cell hyper/metaplasia, leading to increased mucus production.
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Activation of the NLRP3 inflammasome by islet amyloid polypeptide provides a mechanism for enhanced IL-1? in type 2 diabetes.
Nat. Immunol.
PUBLISHED: 06-29-2010
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Interleukin 1? (IL-1?) is an important inflammatory mediator of type 2 diabetes. Here we show that oligomers of islet amyloid polypeptide (IAPP), a protein that forms amyloid deposits in the pancreas during type 2 diabetes, triggered the NLRP3 inflammasome and generated mature IL-1?. One therapy for type 2 diabetes, glyburide, suppressed IAPP-mediated IL-1? production in vitro. Processing of IL-1? initiated by IAPP first required priming, a process that involved glucose metabolism and was facilitated by minimally oxidized low-density lipoprotein. Finally, mice transgenic for human IAPP had more IL-1? in pancreatic islets, which localized together with amyloid and macrophages. Our findings identify previously unknown mechanisms in the pathogenesis of type 2 diabetes and treatment of pathology caused by IAPP.
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P-selectin glycoprotein ligand-1 regulates adhesive properties of the endothelium and leukocyte trafficking into adipose tissue.
Circ. Res.
PUBLISHED: 06-17-2010
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Adhesive interactions between endothelial cells and leukocytes affect leukocyte trafficking in adipose tissue. The role of P-selectin glycoprotein ligand-1 (Psgl-1) in this process is unclear.
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AIM2 joins the gang of microbial sensors.
Cell Host Microbe
PUBLISHED: 05-19-2010
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AIM2 was recently implicated in the assembly and activation of the inflammasome in response to eukaryotic and viral dsDNA. Sauer and colleagues (2010) demonstrate that AIM2 is also involved in the activation of the inflammasome in response to the intracellular bacterium Listeria monocytogenes.
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Arabidopsis sucrose synthase 2 and 3 modulate metabolic homeostasis and direct carbon towards starch synthesis in developing seeds.
Planta
PUBLISHED: 04-05-2010
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Two genes encoding sucrose synthase (SUS), namely SUS2 (At5g49190) and SUS3 (At4g02280), are strongly and differentially expressed in Arabidopsis seed. Detailed biochemical analysis was carried out in developing seeds 9-21 days after flowering (DAF) of wild type and two knockouts. SUS2 and SUS3 are not redundant genes since single knockouts show a phenotype in developing seeds. The mutants had 30-50% less SUS activity and therefore accumulated 40% more sucrose and 50% less fructose at 15 DAF. This did not affect the hexose-P pool, but led to 30-70% less starch in embryo and seed coat. Lipids were 55% higher in both mutants at 9-15 DAF. It seems that sucrolysis via SUS is not required for oil or protein synthesis but rather for channeling carbon toward ADP-glucose and starch in seeds. Metabolite profiling with GC-TOF revealed specific downstream changes in primary metabolism as a consequence of signaling or regulatory fine-tuning. While sucrose increased, hexoses and specific amino acids decreased reciprocally. There was a developmental shift regarding an earlier timing of dry weight accumulation, germinative maturity, oil deposition, sugar levels, transient starch buildup, and protein storage. Nevertheless, final seed size and composition were unaltered due to an earlier cessation of growth, thus giving rise to an apparent silent phenotype of mature mutant seeds. We conclude that SUS is important for metabolite homeostasis and timing of seed development, and propose that an altered sucrose/hexose ratio can modify carbon partitioning and the pattern of storage compounds in Arabidopsis.
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Pathogenic Vibrio activate NLRP3 inflammasome via cytotoxins and TLR/nucleotide-binding oligomerization domain-mediated NF-kappa B signaling.
J. Immunol.
PUBLISHED: 03-26-2010
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Vibrio vulnificus and Vibrio cholerae are Gram-negative pathogens that cause serious infectious disease in humans. The beta form of pro-IL-1 is thought to be involved in inflammatory responses and disease development during infection with these pathogens, but the mechanism of beta form of pro-IL-1 production remains poorly defined. In this study, we demonstrate that infection of mouse macrophages with two pathogenic Vibrio triggers the activation of caspase-1 via the NLRP3 inflammasome. Activation of the NLRP3 inflammasome was mediated by hemolysins and multifunctional repeat-in-toxins produced by the pathogenic bacteria. NLRP3 activation in response to V. vulnificus infection required NF-kappaB activation, which was mediated via TLR signaling. V. cholerae-induced NLRP3 activation also required NF-kappaB activation but was independent of TLR stimulation. Studies with purified V. cholerae hemolysin revealed that toxin-stimulated NLRP3 activation was induced by TLR and nucleotide-binding oligomerization domain 1/2 ligand-mediated NF-kappaB activation. Our results identify the NLRP3 inflammasome as a sensor of Vibrio infections through the action of bacterial cytotoxins and differential activation of innate signaling pathways acting upstream of NF-kappaB.
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Inflammasomes as microbial sensors.
Eur. J. Immunol.
PUBLISHED: 03-05-2010
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Members of the Nod-like receptor family and the adaptor ASC assemble into multiprotein platforms, termed inflammasomes, to mediate the activation of caspase-1 and subsequent secretion of IL-1beta and IL-18. Recent studies have identified microbial and endogenous molecules as well as possible mechanisms involved in inflammasome activation.
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Caspase-1 independent IL-1beta production is critical for host resistance to mycobacterium tuberculosis and does not require TLR signaling in vivo.
J. Immunol.
PUBLISHED: 03-03-2010
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To investigate the respective contributions of TLR versus IL-1R mediated signals in MyD88 dependent control of Mycobacterium tuberculosis, we compared the outcome of M. tuberculosis infection in MyD88, TRIF/MyD88, IL-1R1, and IL-1beta-deficient mice. All four strains displayed acute mortality with highly increased pulmonary bacterial burden suggesting a major role for IL-1beta signaling in determining the MyD88 dependent phenotype. Unexpectedly, the infected MyD88 and TRIF/MyD88-deficient mice, rather than being defective in IL-1beta expression, displayed increased cytokine levels relative to wild-type animals. Similarly, infected mice deficient in caspase-1 and ASC, which have critical functions in inflammasome-mediated IL-1beta maturation, showed unimpaired IL-1beta production and importantly, were considerably less susceptible to infection than IL-1beta deficient mice. Together our findings reveal a major role for IL-1beta in host resistance to M. tuberculosis and indicate that during this infection the cytokine can be generated by a mechanism that does not require TLR signaling or caspase-1.
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NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals.
Nature
PUBLISHED: 02-18-2010
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The inflammatory nature of atherosclerosis is well established but the agent(s) that incite inflammation in the artery wall remain largely unknown. Germ-free animals are susceptible to atherosclerosis, suggesting that endogenous substances initiate the inflammation. Mature atherosclerotic lesions contain macroscopic deposits of cholesterol crystals in the necrotic core, but their appearance late in atherogenesis had been thought to disqualify them as primary inflammatory stimuli. However, using a new microscopic technique, we revealed that minute cholesterol crystals are present in early diet-induced atherosclerotic lesions and that their appearance in mice coincides with the first appearance of inflammatory cells. Other crystalline substances can induce inflammation by stimulating the caspase-1-activating NLRP3 (NALP3 or cryopyrin) inflammasome, which results in cleavage and secretion of interleukin (IL)-1 family cytokines. Here we show that cholesterol crystals activate the NLRP3 inflammasome in phagocytes in vitro in a process that involves phagolysosomal damage. Similarly, when injected intraperitoneally, cholesterol crystals induce acute inflammation, which is impaired in mice deficient in components of the NLRP3 inflammasome, cathepsin B, cathepsin L or IL-1 molecules. Moreover, when mice deficient in low-density lipoprotein receptor (LDLR) were bone-marrow transplanted with NLRP3-deficient, ASC (also known as PYCARD)-deficient or IL-1alpha/beta-deficient bone marrow and fed on a high-cholesterol diet, they had markedly decreased early atherosclerosis and inflammasome-dependent IL-18 levels. Minimally modified LDL can lead to cholesterol crystallization concomitant with NLRP3 inflammasome priming and activation in macrophages. Although there is the possibility that oxidized LDL activates the NLRP3 inflammasome in vivo, our results demonstrate that crystalline cholesterol acts as an endogenous danger signal and its deposition in arteries or elsewhere is an early cause rather than a late consequence of inflammation. These findings provide new insights into the pathogenesis of atherosclerosis and indicate new potential molecular targets for the therapy of this disease.
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Immunogenicity of whole-parasite vaccines against Plasmodium falciparum involves malarial hemozoin and host TLR9.
Cell Host Microbe
PUBLISHED: 02-02-2010
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Although whole-parasite vaccine strategies for malaria infection have regained attention, their immunological mechanisms of action remain unclear. We find that immunization of mice with a crude blood stage extract of the malaria parasite Plasmodium falciparum elicits parasite antigen-specific immune responses via Toll-like receptor (TLR) 9 and that the malarial heme-detoxification byproduct, hemozoin (HZ), but not malarial DNA, produces a potent adjuvant effect. Malarial and synthetic (s)HZ bound TLR9 directly to induce conformational changes in the receptor. The adjuvant effect of sHZ depended on its method of synthesis and particle size. Although natural HZ acts as a TLR9 ligand, the adjuvant effects of synthetic HZ are independent of TLR9 or the NLRP3-inflammasome but are dependent on MyD88. The adjuvant function of sHZ was further validated in a canine antiallergen vaccine model. Thus, HZ can influence adaptive immune responses to malaria infection and may have therapeutic value in vaccine adjuvant development.
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Metallothionein-Like Proteins and Energy Reserve Levels after Ni and Pb Exposure in the Pacific White Prawn Penaeus vannamei.
J Toxicol
PUBLISHED: 01-13-2010
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This study analyzed the changes in metallothionein-like proteins (MTLPs) and Energy Reserves (ERs) in hepatopancreas and abdominal muscle of the white prawn Penaeus vannamei. Realistic metal concentration exposure for 10 days to Ni and Pb in solution revealed that juvenile prawns partially induce MTLP in hepatopancreas after Pb exposure. Ni was distributed equally between soluble and insoluble fractions, while Pb was present only in the insoluble fraction, suggesting different detoxification strategy. No changes in lipids and glycogen concentration were detected under these experimental conditions in both tissues analyzed. MTLP could not be considered as a suitable indicator for lead exposure in hepatopancreas.
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