Browsing by Subject "Immunity, Innate"
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Item Enhancement of the An1 Population by BAFF: Potential Role in Autoimmune Disease(2010-01-12) Sadat, Eva Lasmin; Scheuermann, Richard H.The cytokine BAFF is a TNF family member found to be essential for the homeostasis of B cells. Numerous studies have shown BAFF to be involved in the pathogenesis of autoimmune diseases, including Systemic Lupus Erythematosus (SLE) and Rheumatoid Arthritis. In an effort to understand the role of BAFF in the pathogenesis of autoimmune diseases, extensive in vitro studies on primary B cells have been performed. A recent study has described the existence of an anergic or silent autoreactive B cell population called An1, present within the wild-type repertoire of B cells in the periphery. Our differentiation studies using primary murine splenic B cells indicate an increase in this An1 population in response to BAFF stimulation. In order to ascertain whether this population is truly anergic, calcium flux assays have been performed. Interestingly, these assays show a decrease in response to BCR signaling in BAFF-treated cultures compared to untreated controls. Additional studies with sorted populations of cells indicate that these An1 cells are emerging mainly from the mature and the T2 populations. Survival studies using Annexin V as an apoptosis marker show that BAFF increases the survival of B cells in each of their different stages affirming the role of BAFF in promoting survival of the An1 cells. Microarray gene expression studies done on splenic B cells treated with or without BAFF show higher expression of a set of genes that have been reported to be upregulated in anergic B cells. Purified B cells from mice injected with BAFF also showed an increase in AA4.1 hi cells, which include the An1 cells. B cells from these mice show a lowered calcium flux upon BCR stimulation indicating anergic properties. These data suggest that BAFF stimulation results in the induction of B cell anergy, both in vitro and in vivo. The induction of anergy in B cells in response to stimulation through the antigen receptor may provide a mechanism by which autoreactive cells can evade deletion. Ultimately, the presence of these anergic B cells in the periphery poses a risk of activation and reversion to autoreactivity thus leading to automimmune disease.Item Functional Prions in Mammalian Innate Immune Signaling(2014-07-07) Cai, Xin; Zinn, Andrew R.; Beutler, Bruce; Chen, Zhijian J.; Goldstein, Joseph L.Pathogens and cellular danger signals activate mammalian cytosolic sensors such as RIG-I and NLRP3 which signal through respective adaptor proteins MAVS and ASC to produce robust innate immune and inflammatory responses. MAVS and ASC harbor N-terminal CARD and PYRIN domains, respectively, essential for their signaling ability. Using the Sup35 based yeast prion assay, we show that CARD and PYRIN function as bona fide prions in yeast when fused to Sup35C. In response to respective upstream sensors RIG-I and NLRP3, both CARD and PYRIN form self-perpetuating, SDS-resistant polymers that are inherited cytoplasmically through multiple cell divisions. Similar to other cases of prion switch, CARD exhibits nucleation- and polymerization-dependent prion conversion in yeast. Likewise, a yeast prion domain (NM) can functionally replace CARD and PYRIN in mammalian innate immune and inflammasome signaling. Mutations in MAVS and ASC that disrupt their prion activities in yeast also abrogate their ability to signal in mammalian cells. Furthermore, fibers of recombinant PYRIN can convert ASC into functional polymers capable of activating caspase-1. Remarkably, homologous domains from a conserved NOD- like receptor (NWD2) and classic prion (HET-s) in fungi can functionally reconstitute signaling of NLRP3 and ASC PYRINs in mammalian cells. These results indicate that prion- like polymerization is a conserved signal transduction mechanism in innate immunity and inflammation.Item Hepatitis C Virus Entry into Hepatocytes and Engagement of Innate Immune Defenses(2011-08-10) Owen, David Matthew; Gale, Michael, Jr.Hepatitis C virus (HCV) infection is a major cause of liver disease and a global health problem with inadequate treatment options. An improved understanding of how HCV exploits and subverts host factors to establish infection should yield potential targets for therapy. This study uses a recently developed cell culture model of HCV infection to examine HCV entry and engagement of innate immune defenses. HCV associates with host apolipoproteins and enters hepatocytes through complex processes involving some combination of CD81, claudin-I, occludin, and scavenger receptor BI. Here I show that HCV forms a complex with very low density lipoprotein (VLDL) within infected hepatocytes and uses this association to support infection through the low density lipoprotein receptor (LDL-R). Blocking experiments demonstrate that beta-VLDL and apolipoprotein E (apoE) can compete with HCV for entry. Knockdown of the LDL-R by treatment with 25-hydroxycholesterol or siRNA ablated ligand uptake and reduced HCV infection of cells, whereas infection was rescued upon cell ectopic LDL-R expression. Analyses of gradient-fractionated HCV demonstrate that apoE is associated with HCV virions exhibiting peak infectivity and dependence upon the LDL-R for cell entry. These results define the LDL-R as a cooperative HCV co-receptor that supports viral entry and infectivity through interaction with apoE ligand present in an infectious HCV/lipoprotein complex comprising the virion. Furthermore, upon entry HCV induces an initial transient activation of interferon regulatory factor-3 (IRF3) which is dependent on retinoic acid inducible gene I (RIG-I) and interferon-beta promoter stimulator-1 (IPS-1). This activation produces an antiviral activity which inhibits HCV entry and replication. HCV NS3/4A protease activity blocks this activation within 48 hours. At later time points post infection HCV activates NF-kappaB in a RIG-I independent manner leading to inflammatory cytokine production. These studies identify 3 potential targets for future HCV therapy: 1) alteration of HCV-lipoprotein interaction to disrupt entry, 2) blockade of NS3/4A protease activity to restore innate antiviral response, and 3) modulation of HCV induced NF-kappaB signaling to downregulate chronic inflammation.Item Host Modulators of the Death Response to Influenza A Infection(2012-07-17) Ward, Samuel Enoch; White, Michael A.Influenza A virus infects 5-20% of the population annually, resulting in ~35,000 deaths and significant morbidity. Current treatments include vaccines and drugs that target viral proteins. However, both of these approaches have limitations, as vaccines require yearly development and the rapid evolution of viral proteins gives rise to drug resistance. In consequence additional intervention strategies, that target host factors required for the viral life cycle, are under investigation. Here I employed arrayed whole-genome siRNA screening strategies to identify cell-autonomous molecular components that are subverted to support H1N1 influenza A virus infection of human mucosal epithelial cells. Integration across relevant public data sets exposed druggable gene products required for epithelial cell infection or required for viral proteins to deflect host cell suicide checkpoint activation. Pharmacological inhibition of representative targets, RGGT and CHEK1, resulted in significant protection against infection of human epithelial cells by the A/WS/33 virus. In addition, chemical inhibition of RGGT partially protected against H5N1 and the 2009 H1N1 pandemic strain. The observations reported here thus contribute to decoding vulnerabilities in the command and control networks specified by influenza virulence factors.Item Immune mechanisms in lung transplatation(2017-04-21) Kaza, VaidehiItem Innate Immune Responses and Viral Changes after Oral Transmission of SIV in Rhesus Macaques(2010-01-12) Durudas, Andre; Sodora, Donald L.Recent HIV vaccine trial failures indicated the need to increase our knowledge with regard to mucosal immune responses after exposure to HIV/SIV. The studies presented here utilized oral transmission of simian immunodeficiency virus (SIV) in Rhesus macaques with the goals of (1) determining associations between innate immune responses at different tissue sites and disease progression and (2) assessing the differences in immune and viral changes that occur after oral administrations with low or high SIV doses. During previous work in the Sodora laboratory, mRNA levels of innate immune responses were assessed at mucosal sites of orally SIV-infected macaques. The studies presented here build upon this work by assessing innate immune modulator transcripts within lymph nodes and peripheral blood. Dividing the SIV+ macaques with regard to their rate of disease progression, rapidly progressing macaques exhibited elevated expression of IFNα, OAS, CXCL9 and CXCL10 mRNA at lymph nodes. In peripheral blood, only expression of OAS and CXCL10 mRNA was associated with faster disease progression. Assessment of macaques orally infected by high or low doses of SIV revealed that high doses, as expected, resulted in transmission of more virions, and viral replication exhibited slightly faster kinetics early after transmission. Furthermore, high dose macaques exhibited higher levels of the anti-viral factors IFNα and OAS in tissues. However, other innate and adaptive immune responses were comparable between macaques infected by high or low doses. Also, mRNA expression of immune modulators in peripheral blood was similar between the two groups, with only expression of OAS and CXCL10 transcripts being upregulated. These findings indicate that expression of these two immune modulators is preferentially upregulated during faster disease progression and is independent of viral dose. This suggests that OAS and CXCL10 transcript levels could potentially be used as diagnostic markers of AIDS progression. Taken together, these studies indicate that multiple tissue compartments need to be assessed to obtain a complete understanding of immune and viral factors in SIV/HIV disease. A more complete knowledge of the mucosal and systemic immune responses prior to, during and following HIV transmission is likely to lead to new approaches for the development of novel vaccines and therapies to combat HIV infection/disease.Item The Ligand and Function of the RegIII Family of Bactericidal C-Type Lectins(2006-08-11) Cash, Heather Lynn; Hooper, Lora V.Beginning at birth, the intestines of humans and other mammals are colonized with a diverse society of resident bacteria that play a crucial role in host nutrient metabolism. To maintain this commensal relationship, resident microbes must be prevented from crossing the intestinal epithelium into host tissues where they can cause inflammation and sepsis. The innate immune system plays a crucial role in preventing bacterial incursions across gut epithelial surfaces. Mucosal epithelial cells produce a variety of secreted antimicrobial proteins that help to prevent bacterial attachment and encroachment at epithelial surfaces. Among these, Paneth cells are specialized small intestinal epithelial cells that have been shown to produce and secrete antimicrobial proteins and peptides. To gain new insights into the adaptation of mucosal surfaces to microbial challenges, the Hooper lab has used DNA microarrays to screen for Paneth cell genes whose expression is modulated by intestinal microbes. This screen revealed that expression of two C-type lectins, RegIIIbeta and RegIIIgamma , is strongly induced following intestinal colonization with resident microbes. Two features suggested that members of the RegIII family may have microbicidal functions. First, they are C-type lectin family members. Other C-type lectins, including the mannose binding lectin, have well-characterized innate immune functions and play critical roles in microbial killing by recruiting complement. Second, I have shown that the murine RegIII lectins localize to intestinal crypt cells, including Paneth cell secretory granules, and that they bind to luminal bacteria harvested from intestinal conditions. Based on these observations, we hypothesized that this family of proteins may perform an innate immune function, specifically antimicrobial defense. The studies reported in this thesis characterize a family of C-type lectins. Specifically, we determined that these proteins interact with peptidoglycan by binding with high affinity to its glycan structure, representing a unique blend of peptidoglycan recognition and lectin function. Additionally, we have demonstrated that this binding results in the specific disruption of the Gram positive bacterial cell wall, where peptidoglycan is exposed, which is the first example of a family of directly bactericidal C-type lectins. We also present evidence for the regulation of these bactericidal proteins by colonization with an intestinal microflora. Therefore, the research presented in this thesis elucidates the function of three members of the RegIII family, in both mice and humans.Item Liquid-Liquid Phase Separations in Innate Immune DNA Sensing and NF-κB Signaling Pathways(August 2021) Du, Mingjian; Liu, Yi; Chen, Zhijian J.; Beutler, Bruce; O'Donnell, Kathryn A.The binding of DNA to cyclic GMP-AMP synthase (cGAS) leads to the production of the secondary messenger cyclic GMP-AMP (cGAMP), which activates innate immune responses. We have shown that DNA binding to cGAS robustly induced the formation of liquidlike droplets in which cGAS was activated. The disordered and positively charged cGAS N terminus enhanced cGAS-DNA phase separation by increasing the valencies of DNA binding. Long DNA was more efficient in promoting cGAS liquid phase separation and cGAS enzyme activity than short DNA. Moreover, free zinc ions enhanced cGAS enzyme activity both in vitro and in cells by promoting cGAS-DNA phase separation. These results demonstrated that the DNA-induced phase transition of cGAS promotes cGAMP production and innate immune signaling. Beyond cGAS-DNA phase separation, we sought to determine whether protein liquid-liquid phase separation is a ubiquitous mechanism across immune signaling pathways. NF-kappa-B essential modulator (NEMO), also known as IKBKG, is essential for the activation of IκB kinase (IKK) complex in NF-κB signaling, including Interleukin-1 (IL-1β), Tumor Necrosis Factor (TNFα) and Toll-like receptors (TLR) pathways. NEMO activates IKK complex by binding to polyubiquitin chains. Here we show that Lys63(K63)-linked or linear(M1)-linked polyubiquitin chains binding to NEMO robustly induced the formation of liquidlike droplets in which IKK was activated both in vitro and in cells. Both NEMO ubiquitin binding (NUB) domain and zinc finger (ZF) domain of NEMO contributed the multivalencies for binding to polyubiquitin chains. Long polyubiquitin chains were more efficient in promoting NEMO phase separation than short polyubiquitin chains. These results demonstrated that polyubiquitin chains induced phase transition of NEMO to promote IKK complex activation and NF-κB signaling.Item Molecular Mechanisms Underlying Innate Immune Kinase TBK1-Driven Oncogenic Transformation(2013-04-16) Ou, Yi-Hung 1977-; Lum, Lawrence; White, Michael A.; Cobb, Melanie H.; Minna, John D.An essential kinase in innate immune signaling, TBK1 couples pathogen surveillance to induction of host defense mechanisms. The pathological activation of TBK1 in cancer can overcome programmed cell death cues, enabling cells to survive oncogenic stress. The mechanistic basis of TBK1 prosurvival signaling, however, has been enigmatic. Here we show that TBK1 directly activates AKT by phosphorylation of the canonical activation loop and hydrophobic motif sites independently of PDK1 and mTORC2. A population of AKT is bound to components of the exocyst complex. Upon mitogen stimulation, triggering of the innate immune response, re-exposure to glucose, or oncogene activation, TBK1 is recruited to the exocyst, where it activates AKT. In cells lacking TBK1, insulin activates AKT normally, but AKT activation by these exocyst-dependent mechanisms is impaired. Discovery and characterization of a 6-aminopyrazolopyrimidine derivative, as a selective low nanomolar TBK1 inhibitor, indicates this regulatory arm can be pharmacologically perturbed independently of canonical PI3K/PDK1 signaling. Thus, AKT is a direct TBK1 substrate that connects TBK1 to prosurvival signaling. Additionally, biochemical and cell biological evidence indicates critical roles of TBK1 and its analog IKKε in the amino acid-dependent activation of mTORC1. TBK1 and IKKε are activated by amino acids and both proteins interact with mTORC1. In TBK1 and/or IKKε-deficient cells, mTORC1 activation by amino acids is impaired. Of note, we also discovered a set of TBK1 substrates and interacting proteins participating in amino acid-dependent mTORC1 signaling. In conclusion, our results suggest that TBK1 not only supports physiological and oncogenic activation of AKT, but also plays a central role in the regulation of mTORC1 activation in response to amino acids. In addition, our studies reveal novel mTORC1 components and provide new insights into the regulation of the mTORC1 signaling network.Item The Pathogenic Cascade of Acanthamoeba Keratitis(2006-05-15) Clarke, Daniel William; Niederkorn, Jerry Y.Acanthamoeba keratitis is a blinding infection of the cornea caused by the ubiquitous, free-living amoeba, Acanthamoeba. The pathogenic cascade of Acanthamoeba keratitis is a sequential process that begins with adherence of trophozoites to the corneal epithelium and culminates in the destruction of the corneal epithelium and the dissolution of the corneal stroma. This work examined the pathophysiology and immunobiology of Acanthamoeba keratitis. First, we explored possible mechanisms to explain why A. castellanii remains restricted to the cornea and rarely produces intraocular infections. One hypothesis proposed that trophozoites cannot penetrate Descemet's membrane and the corneal endothelium to enter the anterior chamber. However, amoebae utilized a mannose-induced serine protease to penetrate Descemet's membrane within 24 hr of in vitro culture. The second hypothesis proposed that the trophozoites can enter the anterior chamber; however, the aqueous humor contains factors that either induce encystment or kill the amoebae. Injection of amoebae into the anterior chamber induced a robust neutrophil infiltrate, which was associated with complete clearance by day 15 post anterior chamber injection. This indicates that neutrophils of the innate immune apparatus are important in preventing Acanthamoeba keratitis from progressing to become an intraocular infection. Previous reports have shown that intracorneal instillation of sterile latex beads results in resistance to Acanthamoeba keratitis and mitigation of corneal inflammation. This study examined the mechanisms that could be responsible for the latex bead protective effect. Latex bead treatment induced a significant increase in the infiltration of macrophages into the corneas that peaked at day 4 of infection. Additionally, depletion of conjunctival macrophages with the macrophagicidal drug, clodronate, eliminated the latex bead protective effect, providing further evidence that macrophages are crucial in resistance to Acanthamoeba keratitis. With the exception of mucosal IgA antibody, the adaptive immune apparatus is not typically effective against Acanthamoeba keratitis. The results presented here provide evidence that neutrophils and macrophages of the innate immune response are crucial in resistance to Acanthamoeba keratitis. Collectively, these results suggest that recruitment and/or activation of the innate immune apparatus may lead to resolution of disease in Acanthamoeba keratitis patients.Item Regulation of the cGAS-STING Pathway in Health and Disease(2018-11-27) Pokatayev, Vladislav Andreyevich; van Oers, Nicolai S. C.; Conrad, Nicholas; Chen, Zhijian J.; Yan, NanThe innate immune system senses non-self or altered-self molecular structures through pattern recognition receptors in order to eliminate pathogens or damaged cells, and restore an organism to its basal physiology. Nearly all nucleated cell types can sense intracellular viral nucleic acids. These sensors detect either viral RNA through RIG-I like receptors or DNA through the cGAS-STING signaling pathway. Antiviral immune pathways are vital for resolution of viral infections; however, their dysregulation may give rise to various immune-mediated diseases. The neuro-inflammatory autoimmune disease Aicardi-Goutières Syndrome (AGS) develops from mutations in genes encoding several nucleic acid processing proteins, including RNase H2. Defective RNase H2 may induce accumulation of self-nucleic acid species which trigger chronic inflammation leading to AGS pathology. We created a knock-in mouse model with an RNase H2 AGS mutation in a highly conserved residue of the catalytic subunit, Rnaseh2aG37S/G37S (G37S), the most severe Rnaseh2a mutation categorized as it abolishes nuclease activity to less than 10% of WT RNase H2, to understand disease pathology. Importantly, I found that the G37S mutation induces a cellular anti-viral state, and an increased expression of interferon-stimulated genes dependent on the cGAS-STING signaling pathway. G37S homozygotes are perinatal lethal, and ablation of STING in G37S mice results in partial rescue of the perinatal lethality and complete rescue of the immune phenotype. This study motivates inhibitors of the cGAS-STING pathway in the goal of resolving Rnaseh2a-mediated AGS. As my previous work implicates STING in the development of AGS, I performed a genetic screen to identify novel regulators of this protein. I discovered that TOLLIP, a protein previously identified as a regulator of extracellular Toll-like receptor pathways, can function as a positive regulator of the cGAS-STING pathway. TOLLIP antagonizes STING protein degradation through a regulatory pathway controlled by the protein IRE1α. In Tollip-/- cells, IRE1α is activated and induces lysomal-mediated degradation of STING. Chronic activation of this degradative pathway blunts the cellular response to cGAS or STING agonists. These findings have implications in vivo, as deleting Tollip in a mouse model for AGS, the Trex1-/- mouse, can rescue symptoms of the disease. These findings have clinical importance, as novel therapeutics against TOLLIP can be developed to treat auto-inflammation caused by dysregulation of the cGAS-STING signaling pathway.Item Regulatory Mechanisms in Innate Immunity(2018-07-16) Ren, Junyao; Olson, Eric N.; Chen, Zhijian J.; Hooper, Lora V.; Beutler, BruceInnate immunity is the frontline for the host to defend against infections. This process entails the cooperation among pathogen recognition receptors, adaptor proteins, kinases, and transcription factors that elicit the production of effector cytokines. As an important transcription factor, IRF5 was known to be essential for the host cytokine production in response to various ligands and SNPs in IRF5 have been closely related to autoimmune diseases. However, the mechanism by which IRF5 is activated is not well understood. In the first part of this dissertation, I presented evidence that the kinase IKK2 phosphorylates IRF5 on Serine 445, leading to its dimerization and nuclear translocation. cGAMP is the first cyclic di-nucleotide discovered in metazoan. It is produced by the cytosolic DNA sensor cGAS in response to pathogen or self DNA as a second messenger to activate STING. cGAMP has been proven to be very important in anti-viral response and anti-tumor process. In the second part of the dissertation, I used next-generation sequencing techniques and presented that STING is the predominant receptor for cGAMP and innate immune response. As the essential and general DNA sensor, cGAS was purified and identified from cellular cytosols. Upon DNA binding, cGAS utilize ATP and GTP to synthesize cGAMP. However, the regulation of cGAS activity in cells is still poorly understood. Here I presented that certain RNA species that is interferon inducible could inhibit cGAS catalytic activity in vitro and probably regulate cGAS mediated immune response in cells. Besides, I have discovered that during the cell cycle, cGAS is recruited and co-localize with chromosome and in actively dividing cells, cGAS remains in the nucleus. I further presented evidence that certain protein(s) in the nucleus can inhibit cGAS activity thus prevent cGAS from being activated by host DNA in the nucleus.Item The role of inflammation and infection in neurodegeneration(2024-01-12) Beckham, J. DavidItem The Role of the cGAS-Sting Pathway in DNA Vaccination and Autoimmune Disease(2017-07-10) Cheng, Philip R.; Hooper, Lora V.; Zinn, Andrew R.; Wakeland, Edward K.; Chen, Zhijian J.The innate immune system recognizes certain molecular patterns expressed by pathogens via pattern recognition receptors (PRR). As a PRR, cyclic GMP-AMP synthase (cGAS) functions as a cytosolic DNA sensor. Stimulator of Interferon Genes (STING) functions as the downstream adaptor protein. Activation of the cGAS-STING pathway results in proinflammatory cytokine production. Here I show that the cGAS-STING pathway plays dual roles in mediating DNA adjuvant activity and the use of 2'3'-cyclic GMP-AMP (cGAMP) as a vaccine adjuvant in mice, in addition to promoting autoantibody production and autoimmune inflammatory cell accumulation in lupus-prone mice. It is unclear which DNA sensor is responsible for mediating the adjuvant effects of plasmid DNA during the course of DNA vaccination. I show that the cGAS-STING pathway is required for generation of antigen-specific immune responses following DNA-adjuvanted vaccination. Mice vaccinated with influenza antigens co-administered with 2'3'-cGAMP develop robust neutralizing antibody titers, enhanced antigen-specific CD8+ T-cell responses, and are protected against lethal influenza virus challenge. The efficacy of 2'3'-cGAMP as a vaccine adjuvant can be enhanced by liposome-assisted delivery, the use of non-hydrolyzable analogs, or co-administration with CpG-C DNA. Systemic lupus erythematosus (SLE) is a chronic autoimmune disease. The exact etiology of SLE is unclear, but work utilizing mouse models of SLE have shown that the PRRs of the innate immune system contribute to disease pathogenesis. My results show that in C57BL/6J-Faslpr/Faslpr mice, genetic ablation of cGAS or STING significantly decreases antinuclear autoantibody titers as well as a number of autoimmune inflammatory cell populations. These results are dependent on the genetic background of the mice, as genetic ablation of cGAS or STING in B6.MRL/Mp-Faslpr/Faslpr mice or B6.Sle1-Faslpr/Faslpr mice does not recapitulate the phenotype of cGAS-/- or STING gt/gt.C57BL/6J-Faslpr/Faslpr mice. My results provide more insight into the innate immune mechanisms involved in DNA vaccination and show that 2'3'-cGAMP promotes the enhanced development of protective immune responses, thereby demonstrating the potential utility of 2'3'-cGAMP as a molecular adjuvant for vaccines. Furthermore, my results demonstrate that the cGAS-STING pathway contributes to autoimmune disease development in C57BL/6J-Faslpr/Faslpr mice and implicates cGAS or STING as potential therapeutic targets for the treatment of SLE.Item Structural Basis for the Activation of RIG-I/MAVS Antiviral Immune Signaling(2015-04-09) Xu, Hui; Rice, Luke M.; Chen, Zhijian J.; Jiang, Qiu-Xing; Rosen, Michael K.; Liu, QinghuaRetinoic acid inducible gene-I (RIG-I) is a key cytosolic pathogen RNA sensor that activates mitochondrial antiviral signaling protein (MAVS) to trigger rapid innate immune responses. Using RNAs of different lengths as model ligands, we showed that RIG-I oligomerized on dsRNA in an ATP hydrolysis-dependent and dsRNA length-dependent manner, which correlated with the strength of type-I interferon (IFN-I) activation. The obtained negative stain EM structure of full-length RIG-I in complex with a 5'ppp stem-loop RNA and the crystal structure of RIG-I/Ub complex elucidated a two-step oligomerization and conformational change of RIG-I for activation. RIG-I oligomers nucleate MAVS through homotypic interaction of the N-terminal caspase activation and recruitment domains (CARDs) and induce the formation of prion-like aggregates. The obtained cryoEM structure of left-handed helical filaments of MAVS CARD revealed specific interfaces between individual CARD subunits that are dictated by a combination of electrostatic and hydrophobic interactions and hydrogen bonding. Point mutations at multiple locations of these interfaces impaired filament formation and antiviral signaling. Super-resolution imaging of virus-infected cells revealed rod-shaped MAVS clusters on mitochondria. These results elucidated the structural mechanism of RIG-I activation by RNA and K63-linked ubiquitin chains as well as the activation of MAVS through polymerization, revealing a highly efficient signaling cascade for viral RNA sensing.Item Systems Biology of Staphylococcus Aureus Infection Ex Vivo and in Vitro(2012-07-09) Banchereau, Romain; Ramilo, OctavioStaphylococcus aureus has emerged as one of the most common community-acquired bacterial infections, with significant morbidity and mortality. Emergence of multidrug resistant strains worldwide, combined with limited treatment options demand novel approaches to further elucidate host-pathogen interactions, and especially host responses to infection. To this end, we leveraged systems biology approaches to better characterize the status of the host immune system during S. aureus infection ex vivo and in vitro. The transcriptional profiles of PBMC and whole blood from patients with community-acquired S. aureus infection were characterized by microarray analysis, and leukocyte population frequencies were measured by polychromatic flow cytometry. To refine our understanding of inflammatory networks involved, an in vitro system of antigen-presenting cell stimulation with various pathogens, including S. aureus as well as other bacteria and viruses, and their components, was used to identify early inflammatory programs induced in innate immune cells. To reduce the dimension and complexity of the data generated, we developed modular frameworks to analyze and interpret the fingerprints obtained from both the ex vivo and in vitro studies. // Overall, the blood transcriptional response to S. aureus infection was characterized by over-expression of innate immunity and hematopoiesis transcriptional programs, and under-expression of adaptive immunity programs. Flow cytometry and standard cell blood count (CBC) revealed an increase in absolute numbers of circulating monocytes, neutrophils and antigen-presenting cells, including dendritic cells and B cells, combined with a decrease in central memory T cells. To identify transcriptional correlates of clinical heterogeneity, we obtained individual fingerprints and derived the molecular distance to health, a numerical score of transcriptional perturbation for each patient. Patient-by-patient analysis without a priori knowledge of clinical diagnoses identified four major transcriptional clusters based on inflammation, erythropoiesis and interferon-induced profiles. Clinical presentation, bacterial dissemination and time between hospitalization and blood sampling were identified as major factors influencing the signature. The framework obtained from in vitro stimulation of monocyte-derived DC helped us refine the characterization of inflammatory programs activated during S. aureus infection. In addition to inflammatory antibacterial programs, S. aureus induced a subset of interferon response modules, also observed in viral infections and autoimmunity, as well as a specific set of modules linked to cell compartmentalization and lipid biosynthesis. Systems biology approaches provide a global and comprehensive assessment of host responses to acute bacterial infections, bringing a new understanding of disease pathogenesis and underlying patient heterogeneity.Item [UT Southwestern Medical Center News](2013-02-15) Wormser, DeborahItem [UT Southwestern Medical Center News](2011-08-09) Wormser, DeborahItem [UT Southwestern Medical Center News](2011-06-08) Wormser, Deborah