Publications by authors named "Richard J Ulevitch"

38 Publications

Immunoediting role for major vault protein in apoptotic signaling induced by bacterial -acyl homoserine lactones.

Proc Natl Acad Sci U S A 2021 Mar;118(12)

Department of Chemistry and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Be'er Sheva 8410501, Israel;

The major vault protein (MVP) mediates diverse cellular responses, including cancer cell resistance to chemotherapy and protection against inflammatory responses to Here, we report the use of photoactive probes to identify MVP as a target of the -(3-oxo-dodecanoyl) homoserine lactone (C12), a quorum sensing signal of certain proteobacteria including A treatment of normal and cancer cells with C12 or other -acyl homoserine lactones (AHLs) results in rapid translocation of MVP into lipid raft (LR) membrane fractions. Like AHLs, inflammatory stimuli also induce LR-localization of MVP, but the C12 stimulation reprograms (functionalizes) bioactivity of the plasma membrane by recruiting death receptors, their apoptotic adaptors, and caspase-8 into LR. These functionalized membranes control AHL-induced signaling processes, in that MVP adjusts the protein kinase p38 pathway to attenuate programmed cell death. Since MVP is the structural core of large particles termed vaults, our findings suggest a mechanism in which MVP vaults act as sentinels that fine-tune inflammation-activated processes such as apoptotic signaling mediated by immunosurveillance cytokines including tumor necrosis factor-related apoptosis inducing ligand (TRAIL).
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http://dx.doi.org/10.1073/pnas.2012529118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8000436PMC
March 2021

Lack of Both Nucleotide-Binding Oligomerization Domain-Containing Proteins 1 and 2 Primes T Cells for Activation-Induced Cell Death.

J Immunol 2017 08 26;199(3):1196-1205. Epub 2017 Jun 26.

Division of Nephrology and Hypertension, Department of Medicine, University of California, San Diego, La Jolla, CA 92093; and

Nucleotide-binding oligomerization domain (Nod)-containing proteins Nod1 and Nod2 play important roles in the innate immune response to pathogenic microbes, but mounting data suggest these pattern recognition receptors might also play key roles in adaptive immune responses. Targeting Nod1 and Nod2 signaling pathways in T cells is likely to provide a new strategy to modify inflammation in a variety of disease states, particularly those that depend on Ag-induced T cell activation. To better understand how Nod1 and Nod2 proteins contribute to adaptive immunity, this study investigated their role in alloantigen-induced T cell activation and asked whether their absence might impact in vivo alloresponses using a severe acute graft versus host disease model. The study provided several important observations. We found that the simultaneous absence of Nod1 and Nod2 primed T cells for activation-induced cell death. T cells from mice rapidly underwent cell death upon exposure to alloantigen. The T cells had sustained p53 expression that was associated with downregulation of its negative regulator MDM2. In vivo, mice transplanted with an inoculum containing T cells were protected from severe graft versus host disease. The results show that the simultaneous absence of Nod1 and Nod2 is associated with accelerated T cell death upon alloantigen encounter, suggesting these proteins might provide new targets to ameliorate T cell responses in a variety of inflammatory states, including those associated with bone marrow or solid organ transplantation.
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http://dx.doi.org/10.4049/jimmunol.1600667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5600491PMC
August 2017

NLRP3 activation and mitosis are mutually exclusive events coordinated by NEK7, a new inflammasome component.

Nat Immunol 2016 Mar 7;17(3):250-8. Epub 2015 Dec 7.

Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

The NLRP3 inflammasome responds to microbes and danger signals by processing and activating proinflammatory cytokines, including interleukin 1β (IL-1β) and IL-18. We found here that activation of the NLRP3 inflammasome was restricted to interphase of the cell cycle by NEK7, a serine-threonine kinase previously linked to mitosis. Activation of the NLRP3 inflammasome required NEK7, which bound to the leucine-rich repeat domain of NLRP3 in a kinase-independent manner downstream of the induction of mitochondrial reactive oxygen species (ROS). This interaction was necessary for the formation of a complex containing NLRP3 and the adaptor ASC, oligomerization of ASC and activation of caspase-1. NEK7 promoted the NLRP3-dependent cellular inflammatory response to intraperitoneal challenge with monosodium urate and the development of experimental autoimmune encephalitis in mice. Our findings suggest that NEK7 serves as a cellular switch that enforces mutual exclusivity of the inflammasome response and cell division.
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http://dx.doi.org/10.1038/ni.3333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4862588PMC
March 2016

The costimulatory immunogen LPS induces the B-Cell clones that infiltrate transplanted human kidneys.

Proc Natl Acad Sci U S A 2012 Apr 6;109(16):6036-41. Epub 2012 Apr 6.

Departments of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

The mechanism of chronic rejection of transplanted human kidneys is unknown. An understanding of this process is important because, chronic rejection ultimately leads to loss of the kidney allograft in most transplants. One feature of chronic rejection is the infiltration of ectopic B-cell clusters that are clonal into the transplanted kidney. We now show that the antibodies produced by these B-cells react strongly with the core carbohydrate region of LPS. Since LPS is a costimulatory immunogen that can react with both the B-cell receptor (BCR) and the Toll-like receptor 4 (TLR4), these results suggest a mechanism for the selective pressure that leads to clonality of these B-cell clusters and opens the possibility that infection and the attendant exposure to LPS plays a role in the chronic rejection of human kidney transplants. If confirmed by clinical studies, these results suggest that treating patients with signs of chronic rejection with antibiotics may improve kidney allograft survival.
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http://dx.doi.org/10.1073/pnas.1202214109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3341022PMC
April 2012

The use of small molecule probes to study spatially separated stimulus-induced signaling pathways.

Bioorg Med Chem Lett 2012 Mar 14;22(5):2043-5. Epub 2012 Jan 14.

Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, 92037 CA, USA.

Simultaneous activation of signaling pathways requires dynamic assembly of higher-order protein complexes at the cytoplasmic domains of membrane-associated receptors in a stimulus-specific manner. Here, using the paradigm of cellular activation through cytokine and innate immune receptors, we demonstrate the proof-of-principle application of small molecule probes for the dissection of receptor-proximal signaling processes, such as activation of the transcription factor NF-κB and the protein kinase p38.
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http://dx.doi.org/10.1016/j.bmcl.2012.01.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3288742PMC
March 2012

Modulation of mammalian cell processes by bacterial quorum sensing molecules.

Methods Mol Biol 2011 ;692:133-45

Department of Immunology & Microbial Science, The Scripps Research Institute, La Jolla, CA, USA.

Microbial pathogens use a wide repertoire of pathogen-associated molecular patterns (PAMPs) that affect host cell responses through activation of intracellular signaling events in a PAMP-specific manner. Here we describe a set of western blot-based methodologies for the evaluation of biochemical effects specifically induced by N-(3-oxo-acyl) homoserine lactones (3-oxo-AHLs) small molecules secreted by a number of Gram-negative bacteria, including the opportunistic human pathogen Pseudomonas aeruginosa. First, we will highlight the AHL-mediated effects on proapoptotic and stress pathways. Secondly, we will demonstrate that AHLs possess the ability to alter stimulus-induced NF-κB signaling, a key biochemical marker of inflammation and innate immune responses.
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http://dx.doi.org/10.1007/978-1-60761-971-0_10DOI Listing
February 2011

An inflammasome-independent role for epithelial-expressed Nlrp3 in renal ischemia-reperfusion injury.

J Immunol 2010 Nov 20;185(10):6277-85. Epub 2010 Oct 20.

Department of Immunology and Microbial Sciences, The Scripps Research Institute, La Jolla, CA 92037, USA.

Cytoplasmic innate immune receptors are important therapeutic targets for diseases associated with overproduction of proinflammatory cytokines. One cytoplasmic receptor complex, the Nlrp3 inflammasome, responds to an extensive array of molecules associated with cellular stress. Under normal conditions, Nlrp3 is autorepressed, but in the presence of its ligands, it oligomerizes, recruits apoptosis-associated speck-like protein containing a caspase recruitment domain (Asc), and triggers caspase 1 activation and the maturation of proinflammatory cytokines such as IL-1β and IL-18. Because ischemic tissue injury provides a potential source for Nlrp3 ligands, our study compared and contrasted the effects of renal ischemia in wild-type mice and mice deficient in components of the Nlrp3 inflammasome (Nlrp3(-/-) and Asc(-/-) mice). To examine the role of the inflammasome in renal ischemia-reperfusion injury (IRI) we also tested its downstream targets caspase 1, IL-1β, and IL-18. Both Nlrp3 and Asc were highly expressed in renal tubular epithelium of humans and mice, and the absence of Nlrp3, but not Asc or the downstream inflammasome targets, dramatically protected from kidney IRI. We conclude that Nlrp3 contributes to renal IRI by a direct effect on renal tubular epithelium and that this effect is independent of inflammasome-induced proinflammatory cytokine production.
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http://dx.doi.org/10.4049/jimmunol.1002330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3020135PMC
November 2010

Nod1 and nod2 are expressed in human and murine renal tubular epithelial cells and participate in renal ischemia reperfusion injury.

J Immunol 2010 Mar 1;184(5):2297-304. Epub 2010 Feb 1.

Department of Immunology and Microbial Sciences, The Scripps Research Institute, La Jolla, CA 92037, USA.

Nucleotide-binding oligomerization domain (Nod) 1 and Nod2 are members of a family of intracellular innate sensors that participate in innate immune responses to pathogens and molecules released during the course of tissue injury, including injury induced by ischemia. Ischemic injury to the kidney is characterized by renal tubular epithelial apoptosis and inflammation. Among the best studied intracellular innate immune receptors known to contribute to apoptosis and inflammation are Nod1 and Nod2. Our study compared and contrasted the effects of renal ischemia in wild-type mice and mice deficient in Nod1, Nod2, Nod(1 x 2), and in their downstream signaling molecule receptor-interacting protein 2. We found that Nod1 and Nod2 were present in renal tubular epithelial cells in both mouse and human kidneys and that the absence of these receptors in mice resulted in protection from kidney ischemia reperfusion injury. Significant protection from kidney injury was seen with a deficiency of Nod2 and receptor-interacting protein 2, and the simultaneous deficiency of Nod1 and Nod2 provided even greater protection. We conclude that the intracellular sensors Nod1 and Nod2 play an important role in the pathogenesis of acute ischemic injury of the kidney, although possibly through different mechanisms.
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http://dx.doi.org/10.4049/jimmunol.0903065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3020136PMC
March 2010

Modulation of gene expression via disruption of NF-kappaB signaling by a bacterial small molecule.

Science 2008 Jul 19;321(5886):259-63. Epub 2008 Jun 19.

Department of Immunology and Microbial Sciences, Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

The control of innate immune responses through activation of the nuclear transcription factor NF-kappaB is essential for the elimination of invading microbial pathogens. We showed that the bacterial N-(3-oxo-dodecanoyl) homoserine lactone (C12) selectively impairs the regulation of NF-kappaB functions in activated mammalian cells. The consequence is specific repression of stimulus-mediated induction of NF-kappaB-responsive genes encoding inflammatory cytokines and other immune regulators. These findings uncover a strategy by which C12-producing opportunistic pathogens, such as Pseudomonas aeruginosa, attenuate the innate immune system to establish and maintain local persistent infection in humans, for example, in cystic fibrosis patients.
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http://dx.doi.org/10.1126/science.1156499DOI Listing
July 2008

The quorum quenching antibody RS2-1G9 protects macrophages from the cytotoxic effects of the Pseudomonas aeruginosa quorum sensing signalling molecule N-3-oxo-dodecanoyl-homoserine lactone.

Mol Immunol 2008 May 4;45(9):2710-4. Epub 2008 Mar 4.

Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, USA.

The Gram-negative bacterium Pseudomonas aeruginosa, an opportunistic human pathogen, uses acyl-homoserine lactone-based quorum sensing systems to control its pathogenicity. One of its quorum sensing factors, N-3-oxo-dodecanoyl-homoserine lactone, has been shown not only to mediate bacterial quorum sensing but also to exert cytotoxic effects on mammalian cells. The monoclonal antibody RS2-1G9 generated against a 3-oxo-dodecanoyl-homoserine lactone analogue hapten was able to protect murine bone marrow-derived macrophages from the cytotoxic effects and also prevented the activation of the mitogen-activated protein kinase p38. These data demonstrate that an immunopharmacotherapeutic approach to combat P. aeruginosa infections might be a viable therapeutic option as the monoclonal antibody RS2-1G9 can readily sequester bacterial N-3-oxo-dodecanoyl-homoserine lactone molecules, thus interfering with their biological effects in prokaryotic and eukaryotic systems.
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http://dx.doi.org/10.1016/j.molimm.2008.01.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2359578PMC
May 2008

Infection control by antibody disruption of bacterial quorum sensing signaling.

Chem Biol 2007 Oct;14(10):1119-27

Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

Quorum sensing (QS) is the process through which bacteria communicate utilizing small diffusible molecules termed autoinducers. It has been demonstrated that QS controls a plethora of microbial processes including the expression of virulence factors. Here we report an immunopharmacotherapeutic approach for the attenuation of QS in the Gram-positive human pathogen Staphylococcus aureus. An anti-autoinducer monoclonal antibody, AP4-24H11, was elicited against a rationally designed hapten, and efficiently inhibited QS in vitro through the sequestration of the autoinducing peptide (AIP)-4 produced by S. aureus RN4850. Importantly, AP4-24H11 suppressed S. aureus pathogenicity in an abscess formation mouse model in vivo and provided complete protection against a lethal S. aureus challenge. These findings provide a strong foundation for further investigations of immunopharmacotherapy for the treatment of bacterial infections in which QS controls the expression of virulence factors.
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http://dx.doi.org/10.1016/j.chembiol.2007.08.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2088803PMC
October 2007

Genetic analysis of host responses in sepsis.

Curr Infect Dis Rep 2007 Oct;3(5):419-26

Department of Immunology, The Scripps Research Institute, 10550 N. Torrey Pines Road, 92037, La Jolla, CA, USA,

During much of the past century, the microbe itself stood at the heart of microbial pathogenesis. Little thought was devoted to the host per se, though it was granted that differences in susceptibility to certain infections did exist between individuals, and between different ethnic groups. During the past 20 years, extraordinary strides in our grasp of mammalian genetics have made the host side of the equation far more approachable. A restricted collection of genes now presents itself as the likely repository for genetic differences that foretell susceptibility to infectious disease. The Toll-like receptors, of which 10 are presently known to exist in humans, offer an excellent example of this genetic reductionism, in that they embody the afferent component of the innate immune system, and strongly influence the containment of an infection from its earliest stages. The Toll-like receptors were identified as the culmination of a long and relentless inquiry into the yet-unsolved clinical problem of sepsis.
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http://dx.doi.org/10.1007/s11908-007-1009-9DOI Listing
October 2007

MDP-induced interleukin-1beta processing requires Nod2 and CIAS1/NALP3.

J Leukoc Biol 2007 Jul 2;82(1):177-83. Epub 2007 Apr 2.

Department of Immunology, The Scripps Research Institute, La Jolla, California, USA.

Nucleotide-binding oligomerization domain (Nod)2 is a sensor of muramyl dipeptides (MDP) derived from bacterial peptidoglycan. Nod2 also plays a role in some autoinflammatory diseases. Cold-induced autoinflammatory syndrome 1 (CIAS1)/NACHT domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NALP3) has been suggested to be sufficient for MDP-dependent release of mature IL-1beta, but the role of Nod2 in this process is unclear. Using mice bearing selective gene deletions, we provide in vitro and in vivo data showing that MDP-induced IL-1beta release requires Nod2 and CIAS1/NALP3 as well as receptor-interacting protein-2 (Rip2), apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), and caspase-1. In contrast, MDP-dependent IL-6 production only requires Nod2 and Rip2. Together, our data provide a new understanding of this important pathway of IL-1beta production and allow for further studies of the role of these proteins within the broader context of inflammatory disease.
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http://dx.doi.org/10.1189/jlb.1006627DOI Listing
July 2007

The subunit CSN6 of the COP9 signalosome is cleaved during apoptosis.

J Biol Chem 2007 Apr 2;282(17):12557-65. Epub 2007 Mar 2.

Department of Immunology, Scripps Research Institute, La Jolla, California 92037, USA.

The COP9 signalosome is a large multiprotein complex that consists of eight subunits termed CSN1-CSN8. The diverse functions of the COP9 complex include regulation of several important intracellular pathways, including the ubiquitin/proteasome system, DNA repair, cell cycle, developmental changes, and some aspects of immune responses. Nod1 is also thought to be an important cytoplasmic receptor involved in innate immune responses. It detects specific motifs of bacterial peptidoglycan, and this results in activation of multiple signaling pathways and changes in cell function. In this report, we performed a yeast two-hybrid screening and discovered that Nod1 interacts with several components of the COP9 signalosome through its CARD domain. Moreover, we observed that activation of the Nod1 apoptotic pathway leads to specific cleavage of the subunit CSN6. This cleavage is concomitant with caspase processing and generates a short amino-terminal peptide of 3 kDa. A complete inhibition of this cleavage was achieved in the presence of the broad spectrum pharmacological inhibitor of apoptosis, Z-VAD. Furthermore, overexpression of CLARP, a specific caspase 8 inhibitor, completely blocked cleavage of CSN6. Taken together, these results suggest a critical role of caspase 8 in the processing of CSN6. Moreover, these findings suggest that CSN6 cleavage may result in modifications of functions of the COP9 complex that are involved in apoptosis.
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http://dx.doi.org/10.1074/jbc.M609587200DOI Listing
April 2007

Cleavage of p65/RelA of the NF-kappaB pathway by Chlamydia.

Proc Natl Acad Sci U S A 2007 Feb 14;104(8):2933-8. Epub 2007 Feb 14.

Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

Chlamydia trachomatis is a bacterial pathogen that infects the eyes and urogenital tract. Ocular infection by this organism is the leading cause of preventable blindness worldwide. The infection is also a leading cause of sexually transmitted disease in the United States. As obligate intracellular pathogens, chlamydiae have evolved sophisticated, yet undefined, mechanisms to maintain a favorable habitat for intracellular growth while avoiding harm to the host. We show here that chlamydiae have the ability to interfere with the NF-kappaB pathway of host inflammatory response. We found that Chlamydia infection did not promote IkappaBalpha degradation, a prerequisite for NF-kappaB nuclear translocation/activation, nor induce p65/RelA nuclear redistribution. Instead, it caused p65 cleavage into an N terminus-derived p40 fragment and a p22 of the C terminus. The activity was specific because no protein cleavage or degradation of NF-kappaB pathway components was detected. Moreover, murine p65 protein was resistant to cleavage by both human and mouse biovars. The chlamydial protein that selectively cleaved p65 was identified as a tail-specific protease (CT441). Importantly, expression of either this protease or the p40 cleavage product could block NF-kappaB activation. A hallmark of chlamydial STD is its asymptomatic nature, although inflammatory cellular response and chronic inflammation are among the underlying mechanisms. The data presented here demonstrate that chlamydiae have the ability to convert a regulatory molecule of host inflammatory response to a dominant negative inhibitor of the same pathway potentially to minimize inflammation.
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http://dx.doi.org/10.1073/pnas.0608393104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1815284PMC
February 2007

N-(3-oxo-acyl)homoserine lactones signal cell activation through a mechanism distinct from the canonical pathogen-associated molecular pattern recognition receptor pathways.

J Biol Chem 2006 Sep 7;281(39):28822-30. Epub 2006 Aug 7.

Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA.

Innate immune system receptors function as sensors of infection and trigger the immune responses through ligand-specific signaling pathways. These ligands are pathogen-associated products, such as components of bacterial walls and viral nuclear acids. A common response to such ligands is the activation of mitogen-activated protein kinase p38, whereas double-stranded viral RNA additionally induces the phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha). Here we have shown that p38 and eIF2alpha phosphorylation represent two biochemical markers of the effects induced by N-(3-oxo-acyl)homoserine lactones, the secreted products of a number of Gram-negative bacteria, including the human opportunistic pathogen Pseudomonas aeruginosa. Furthermore, N-(3-oxo-dodecanoyl)homoserine lactone induced distension of mitochondria and the endoplasmic reticulum as well as c-jun gene transcription. These effects occurred in a wide variety of cell types including alveolar macrophages and bronchial epithelial cells, requiring the structural integrity of the lactone ring motif and its natural stereochemistry. These findings suggest that N-(3-oxo-acyl)homoserine lactones might be recognized by receptors of the innate immune system. However, we provide evidence that N-(3-oxo-dodecanoyl)homoserine lactone-mediated signaling does not require the presence of the canonical innate immune system receptors, Toll-like receptors, or two members of the NLR/Nod/Caterpillar family, Nod1 and Nod2. These data offer a new understanding of the effects of N-(3-oxo-dodecanoyl)homoserine lactone on host cells and its role in persistent airway infections caused by P. aeruginosa.
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http://dx.doi.org/10.1074/jbc.M606613200DOI Listing
September 2006

Enhanced bacterial clearance and sepsis resistance in caspase-12-deficient mice.

Nature 2006 Apr;440(7087):1064-8

Department of Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121, USA.

Caspases function in both apoptosis and inflammatory cytokine processing and thereby have a role in resistance to sepsis. Here we describe a novel role for a caspase in dampening responses to bacterial infection. We show that in mice, gene-targeted deletion of caspase-12 renders animals resistant to peritonitis and septic shock. The resulting survival advantage was conferred by the ability of the caspase-12-deficient mice to clear bacterial infection more efficiently than wild-type littermates. Caspase-12 dampened the production of the pro-inflammatory cytokines interleukin (IL)-1beta, IL-18 (interferon (IFN)-gamma inducing factor) and IFN-gamma, but not tumour-necrosis factor-alpha and IL-6, in response to various bacterial components that stimulate Toll-like receptor and NOD pathways. The IFN-gamma pathway was crucial in mediating survival of septic caspase-12-deficient mice, because administration of neutralizing antibodies to IFN-gamma receptors ablated the survival advantage that otherwise occurred in these animals. Mechanistically, caspase-12 associated with caspase-1 and inhibited its activity. Notably, the protease function of caspase-12 was not necessary for this effect, as the catalytically inactive caspase-12 mutant Cys299Ala also inhibited caspase-1 and IL-1beta production to the same extent as wild-type caspase-12. In this regard, caspase-12 seems to be the cFLIP counterpart for regulating the inflammatory branch of the caspase cascade. In mice, caspase-12 deficiency confers resistance to sepsis and its presence exerts a dominant-negative suppressive effect on caspase-1, resulting in enhanced vulnerability to bacterial infection and septic mortality.
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http://dx.doi.org/10.1038/nature04656DOI Listing
April 2006

NF-kappa B-inducing kinase regulates selected gene expression in the Nod2 signaling pathway.

Infect Immun 2006 Apr;74(4):2121-7

Department of Immunology, IMM-12, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA.

The innate immune system surveys the extra- and intracellular environment for the presence of microbes. Among the intracellular sensors is a protein known as Nod2, a cytosolic protein containing a leucine-rich repeat domain. Nod2 is believed to play a role in determining host responses to invasive bacteria. A key element in upregulating host defense involves activation of the NF-kappaB pathway. It has been suggested through indirect studies that NF-kappaB-inducing kinase, or NIK, may be involved in Nod2 signaling. Here we have used macrophages derived from primary explants of bone marrow from wild-type mice and mice that either bear a mutation in NIK, rendering it inactive, or are derived from NIK-/- mice, in which the NIK gene has been deleted. We show that NIK binds to Nod2 and mediates induction of specific changes induced by the specific Nod2 activator, muramyl dipeptide, and that the role of NIK occurs in settings where both the Nod2 and TLR4 pathways are activated by their respective agonists. Specifically, we have linked NIK to the induction of the B-cell chemoattractant known as BLC and suggest that this chemokine may play a role in processes initiated by Nod2 activation that lead to improved host defense.
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http://dx.doi.org/10.1128/IAI.74.4.2121-2127.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1418900PMC
April 2006

TLR2 is required for the altered transcription of p75NGF receptors in gram positive infection.

Neurochem Res 2006 Feb 3;31(2):297-301. Epub 2006 Mar 3.

The Harold L. Dorris Neurological Research Center, Department of Neuropharmacology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.

Neuroimmune interactions play a decisive role in neuronal cell survival and cell death during neuronal injury, oxidative and free radical stress. In neurons, NGF occupancy of p75 neurotrophin receptor (p75(NTR)) has been shown to promote neuronal apoptosis, while occupancy of tropomyosin receptor kinase A (TrkA) promotes survival of injured neurons. In macrophages, recent results suggest that NGF via TrkA mediates resistance to cell death through the interaction with TLR2. We have investigated the transcriptional regulation of TrkA, p75(NTR) and their ligand nerve growth factor beta (NGFbeta) upon stimulation with the TLR2 ligand Staphylococcus aureus in the spleen of C57BL/6 mice, TLR2 (-/-) and p75(NTR) (-/-) mice. S. aureus challenge (i.p.) resulted in a significant increase in NGFbeta mRNA levels in C57BL/6 (100%), TLR2 (-/-) (300%) and p75(NTR) (-/-) mice (355%). TrkA mRNA levels were upregulated only in p75(NTR) (-/-) mice (87%) whereas in TLR2 (-/-) mice they remained unchanged and even decreased in C57BL/6 mice (46%). p75(NTR) mRNA was increased in spleen of C57BL/6 mice (60%) whereas the levels in TLR2 (-/-) mice remained almost unchanged. Finally, TLR2 mRNA was upregulated by 350% in C57BL/6 mice and by 283% in p75(NTR) (-/-) mice. These data suggest that in splenocytes signaling via TLR2 is required for Gram positive infection mediated alteration of neurotrophin receptor expression as observed in an in vivo infection model with transgenic mice. This observation provides a link between Gram-positive infection and neurotrophic responses, which may be important in preserving neurons at sites of the infection.
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http://dx.doi.org/10.1007/s11064-005-9020-8DOI Listing
February 2006

Kdo2-Lipid A of Escherichia coli, a defined endotoxin that activates macrophages via TLR-4.

J Lipid Res 2006 May 14;47(5):1097-111. Epub 2006 Feb 14.

Department of Biochemistry, Duke University Medical Center, Durham, NC, USA.

The LIPID MAPS Consortium (www.lipidmaps.org) is developing comprehensive procedures for identifying all lipids of the macrophage, following activation by endotoxin. The goal is to quantify temporal and spatial changes in lipids that occur with cellular metabolism and to develop bioinformatic approaches that establish dynamic lipid networks. To achieve these aims, an endotoxin of the highest possible analytical specification is crucial. We now report a large-scale preparation of 3-deoxy-D-manno-octulosonic acid (Kdo)(2)-Lipid A, a nearly homogeneous Re lipopolysaccharide (LPS) sub-structure with endotoxin activity equal to LPS. Kdo(2)-Lipid A was extracted from 2 kg cell paste of a heptose-deficient Escherichia coli mutant. It was purified by chromatography on silica, DEAE-cellulose, and C18 reverse-phase resin. Structure and purity were evaluated by electrospray ionization/mass spectrometry, liquid chromatography/mass spectrometry and (1)H-NMR. Its bioactivity was compared with LPS in RAW 264.7 cells and bone marrow macrophages from wild-type and toll-like receptor 4 (TLR-4)-deficient mice. Cytokine and eicosanoid production, in conjunction with gene expression profiling, were employed as readouts. Kdo(2)-Lipid A is comparable to LPS by these criteria. Its activity is reduced by >10(3) in cells from TLR-4-deficient mice. The purity of Kdo(2)-Lipid A should facilitate structural analysis of complexes with receptors like TLR-4/MD2.
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http://dx.doi.org/10.1194/jlr.M600027-JLR200DOI Listing
May 2006

Nod1-dependent control of tumor growth.

Proc Natl Acad Sci U S A 2006 Feb 30;103(6):1840-5. Epub 2006 Jan 30.

Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

Nod1, a cytosolic protein that senses meso-diaminopimelic acid-containing ligands derived from peptidoglycan, plays a role in host responses to invasive bacteria. Here we describe a function for Nod1, whereby it controls tumor formation. Cell lines derived from the human breast cancer epithelial cell line MCF-7 were used in a severe combined immune deficiency (SCID) mouse xenograft model to characterize a pathway linking Nod1 to the growth of estrogen-sensitive tumors. In MCF-7 cells, the absence of Nod1 correlates with tumor growth, an increased sensitivity to estrogen-induced cell proliferation, and a failure to undergo Nod1-dependent apoptosis. Conversely, overexpression of Nod1 in MCF-7 cells results in inhibition of estrogen-dependent tumor growth and reduction of estrogen-induced proliferative responses in vitro.
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http://dx.doi.org/10.1073/pnas.0509228103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1413646PMC
February 2006

Limiting inflammatory responses during activation of innate immunity.

Nat Immunol 2005 Dec;6(12):1198-205

The Key Laboratory of The Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen 361005, China.

The idea of the importance of mounting an inflammatory response for effective immunity is supported by a multiplicity of experimental data. It is also well understood that resolution of inflammation is essential for maintaining the balance between health and disease. When the normal regulatory mechanisms are disturbed, the potential for developing chronic inflammatory diseases is increased. Inflammation is a key element in the response of the innate immune system to a variety of challenges, including those provided by bacterial and viral infection as well as by damaged or dying host cells. Here we review elements of innate immunity that lead to inflammation and some of the host mechanisms that allow for the resolution of the inflammatory responses.
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http://dx.doi.org/10.1038/ni1274DOI Listing
December 2005

A synthetic TLR4 antagonist has anti-inflammatory effects in two murine models of inflammatory bowel disease.

J Immunol 2005 May;174(10):6416-23

Corixa Corporation, Seattle, WA 98101, USA.

Current evidence indicates that the chronic inflammation observed in the intestines of patients with inflammatory bowel disease is due to an aberrant immune response to enteric flora. We have developed a lipid A-mimetic, CRX-526, which has antagonistic activity for TLR4 and can block the interaction of LPS with the immune system. CRX-526 can prevent the expression of proinflammatory genes stimulated by LPS in vitro. This antagonist activity of CRX-526 is directly related to its structure, particularly secondary fatty acyl chain length. In vivo, CRX-526 treatment blocks the ability of LPS to induce TNF-alpha release. Importantly, treatment with CRX-526 inhibits the development of moderate-to-severe disease in two mouse models of colonic inflammation: the dextran sodium sulfate model and multidrug resistance gene 1a-deficient mice. By blocking the interaction between enteric bacteria and the innate immune system, CRX-526 may be an effective therapeutic molecule for inflammatory bowel disease.
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http://dx.doi.org/10.4049/jimmunol.174.10.6416DOI Listing
May 2005

Innate immune responses during infection.

Vaccine 2004 Dec;22 Suppl 1:S25-30

Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

The innate immune system senses bacteria in the environment and defends against infection. Here we will discuss two types of sensor protein families. The plasma membrane receptors that comprise the Toll-like receptor (TLRs) family and the intracellular proteins termed NOD1 and NOD2. These proteins directly bind bacterial products such as lipopolysaccharides (LPS), peptidoglycan fragments, bacterial DNA, and receptor binding leads to intracellular signaling and gene expression. TLR signaling involves members of the MyD88 family of adaptor proteins. In contrast NOD1 or NOD2 utilize pathways that do not depend on the MyD88 family members.
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http://dx.doi.org/10.1016/j.vaccine.2004.08.013DOI Listing
December 2004

Therapeutics targeting the innate immune system.

Nat Rev Immunol 2004 Jul;4(7):512-20

Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA.

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http://dx.doi.org/10.1038/nri1396DOI Listing
July 2004

Triad3A, an E3 ubiquitin-protein ligase regulating Toll-like receptors.

Nat Immunol 2004 May 25;5(5):495-502. Epub 2004 Apr 25.

Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.

Activation of Toll-like receptors (TLRs) results in a proinflammatory response needed to combat infection. Thus, limiting TLR signaling is essential for preventing a protective response from causing injury to the host. Here we describe how a RING finger protein, Triad3A, acts as an E3 ubiquitin-protein ligase and enhances ubiquitination and proteolytic degradation of some TLRs. Triad3A overexpression promoted substantial degradation of TLR4 and TLR9 with a concomitant decrease in signaling, but did not affect TLR2 expression or signaling. Conversely, a reduction in endogenous Triad3A by small interfering RNA increased TLR expression and enhanced TLR activation. Thus, ubiquitination by Triad3A represents one pathway by which the intensity and duration of TLR signaling is controlled.
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http://dx.doi.org/10.1038/ni1066DOI Listing
May 2004

Targeted deletion of BMK1/ERK5 in adult mice perturbs vascular integrity and leads to endothelial failure.

J Clin Invest 2004 Apr;113(8):1138-48

Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA.

Big mitogen-activated protein kinase 1 (BMK1), also known as ERK5, is a member of the MAPK family. Genetic ablation of BMK1 in mice leads to embryonic lethality, precluding the exploration of pathophysiological roles of BMK1 in adult mice. We generated a BMK1 conditional mutation in mice in which disruption of the BMK1 gene is under the control of the inducible Mx1-Cre transgene. Ablation of BMK1 in adult mice led to lethality within 2-4 weeks after the induction of Cre recombinase. Physiological analysis showed that the blood vessels became abnormally leaky after deletion of the BMK1 gene. Histological analysis revealed that, after BMK1 ablation, hemorrhages occurred in multiple organs in which endothelial cells lining the blood vessels became round, irregularly aligned, and, eventually, apoptotic. In vitro removal of BMK1 protein also led to the death of endothelial cells partially due to the deregulation of transcriptional factor MEF2C, which is a direct substrate of BMK1. Additionally, endothelial-specific BMK1-KO leads to cardiovascular defects identical to that of global BMK1-KO mutants, whereas, surprisingly, mice lacking BMK1 in cardiomyocytes developed to term without any apparent defects. Taken together, the data provide direct genetic evidence that the BMK1 pathway is critical for endothelial function and for maintaining blood vessel integrity.
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http://dx.doi.org/10.1172/JCI19890DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC385403PMC
April 2004

MKK3 mitogen-activated protein kinase pathway mediates carbon monoxide-induced protection against oxidant-induced lung injury.

Am J Pathol 2003 Dec;163(6):2555-63

Section of Pulmonary and Critical Care Medicine, Yale University School of Medicine, New Haven, Connecticut, USA.

The stress-inducible gene heme oxygenase (HO-1) has previously been shown to provide cytoprotection against oxidative stress. The mechanism(s) by which HO-1 provides this cytoprotection is poorly understood. We demonstrate here that carbon monoxide (CO), a byproduct released during the degradation of heme by HO, plays a major role in mediating the cytoprotection against oxidant-induced lung injury. We show in vitro that CO protects cultured epithelial cells from hyperoxic damage. By using dominant negative mutants and mice deficient in the genes for the various MAP kinases, we demonstrate that the cytoprotective effects of CO are mediated by selective activation of the MKK3/p38 beta protein MAP kinase pathway. In vivo, our experiments demonstrate that CO at a low concentration protects the lungs, extends the survival of the animals, and exerts potent anti-inflammatory effects with reduced inflammatory cell influx into the lungs and marked attenuation in the expression of pro-inflammatory cytokines.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1892354PMC
http://dx.doi.org/10.1016/S0002-9440(10)63610-3DOI Listing
December 2003

Structure-activity relationship of synthetic toll-like receptor 4 agonists.

J Biol Chem 2004 Feb 21;279(6):4440-9. Epub 2003 Oct 21.

Corixa Corporation, Seattle, Washington 98104, USA.

Important questions remain regarding the impact of variations in the structure of the lipid A portion of lipopolysaccharide on activation of cells via the Toll-like receptor 4 complex. We have studied a series of synthetic lipid A mimetic compounds known as aminoalkyl glucosaminide phosphates in which the length of the secondary acyl chain has been systematically varied. Using transcriptional profiling of human monocytes and responses of Toll-like receptor 4 complex cell transfectants, we demonstrate a clear dependence of length on secondary acyl chain on Toll-like receptor 4 activation. Compounds with secondary acyl chains less than eight carbons in length have dramatically reduced activity, and substitutions of the left-sided secondary acyl chain had the most important effect on the Toll-like receptor 4 agonist activity of these molecules. The structure-function relationships of these compounds assessed via the induction of chemokines and cytokines following in vivo administration closely mirrored those seen with cell-based studies. This novel set of synthetic lipid A mimetics will be useful for Toll-like receptor 4-based investigations and may have clinical utility as stand-alone immunomodulators.
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http://dx.doi.org/10.1074/jbc.M310760200DOI Listing
February 2004

Regulation of receptor-dependent activation of the innate immune response.

J Infect Dis 2003 Jun;187 Suppl 2:S351-5

Department of Immunology, Scripps Research Institute, La Jolla, California 92037, USA.

In the United States, >750,000 patients annually are thought to be at high risk for developing septic shock, with mortality rates reaching 60%. Thus, huge societal and financial costs are associated with this syndrome. Because of the high incidence and poor prognosis of septic shock, basic research has focused on the innate immune system for >2 decades. The pathophysiology of severe sepsis/shock is exceedingly complex, but there is little doubt that infection often progresses from systemic inflammatory response to severe sepsis and shock. Infection is the primary event in this sequence. There is evidence that the severity of the systemic reaction to infection (severe sepsis) is strongly influenced by the intensity of the inflammatory process at the infection sites. Efforts to understand the molecular mechanisms involved in recognition of bacterial products by members of the Toll-like receptor family are described, as well as some events that occur after receptor ligand binding that lead to new gene activation.
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http://dx.doi.org/10.1086/374605DOI Listing
June 2003