Investigating the regulation and enzymatic function of human ecsit in innate immune signalling.

Toll-like receptor (TLR) signalling represents the first line of defence against infection. TLRs respond to recognition of pathogens by activating transcription factors such as NFκB and the interferon regulatory factors (IRF)s to induce pro-inflammatory cytokines and IFNs. Whilst many of the components of the TLR signal transduction pathways have been identified, a full understanding of these complex regulatory systems remains to be delineated. This thesis probes the molecular, cellular and physiological roles of a protein termed evolutionary conserved signalling intermediate in toll (ECSIT) in innate immune signalling pathways. ECSIT was initially described as a TRAF6 interacting protein that bridged the TLR signalling intermediate TRAF6 to MEKK1 and downstream activation of NFκB. Subsequent data revealed an important developmental role for ECSIT in the BMP signalling pathway based on the embryonic lethal phenotype in the ECSIT knockout mouse. Other studies have indicated an important role for ECSIT in bacterial killing during infection via the production of mitochondrial reactive oxygen species (mROS). To date no information on the human orthologue of ECSIT (hECSIT) has been available. In this study we describe novel functional roles for hECSIT in TLR signalling. Through use of knockdown studies and a humanised animal model we demonstrate that hECSIT negatively regulates innate immune signalling pathways despite its 80% sequence homology to mECSIT. We show that phosphorylation, ubiquitination and processing of hECSIT leads to an inhibitory C-terminal form that contains both E3 ligase and DUB activity. We also demonstrate that hECSIT can independently target and regulate the ubiquitination of TRAF6, TRAF3 and RIP1 in TLR4/IL-1R, TLR3 and TNF-α signalling, respectively. These findings identify hECSIT as a novel regulator of innate immune signalling and highlight a sophisticated evolutionary difference between two highly conserved proteins

Investigating the regulation and enzymatic function of human ecsit in innate immune signalling.

Toll-like receptor (TLR) signalling represents the first line of defence against infection. TLRs respond to recognition of pathogens by activating transcription factors such as NFκB and the interferon regulatory factors (IRF)s to induce pro-inflammatory cytokines and IFNs. Whilst many of the components of the TLR signal transduction pathways have been identified, a full understanding of these complex regulatory systems remains to be delineated. This thesis probes the molecular, cellular and physiological roles of a protein termed evolutionary conserved signalling intermediate in toll (ECSIT) in innate immune signalling pathways. ECSIT was initially described as a TRAF6 interacting protein that bridged the TLR signalling intermediate TRAF6 to MEKK1 and downstream activation of NFκB. Subsequent data revealed an important developmental role for ECSIT in the BMP signalling pathway based on the embryonic lethal phenotype in the ECSIT knockout mouse. Other studies have indicated an important role for ECSIT in bacterial killing during infection via the production of mitochondrial reactive oxygen species (mROS). To date no information on the human orthologue of ECSIT (hECSIT) has been available. In this study we describe novel functional roles for hECSIT in TLR signalling. Through use of knockdown studies and a humanised animal model we demonstrate that hECSIT negatively regulates innate immune signalling pathways despite its 80% sequence homology to mECSIT. We show that phosphorylation, ubiquitination and processing of hECSIT leads to an inhibitory C-terminal form that contains both E3 ligase and DUB activity. We also demonstrate that hECSIT can independently target and regulate the ubiquitination of TRAF6, TRAF3 and RIP1 in TLR4/IL-1R, TLR3 and TNF-α signalling, respectively. These findings identify hECSIT as a novel regulator of innate immune signalling and highlight a sophisticated evolutionary difference between two highly conserved proteins

Investigating the regulation and enzymatic function of human ecsit in innate immune signalling.

Toll-like receptor (TLR) signalling represents the first line of defence against infection. TLRs respond to recognition of pathogens by activating transcription factors such as NFκB and the interferon regulatory factors (IRF)s to induce pro-inflammatory cytokines and IFNs. Whilst many of the components of the TLR signal transduction pathways have been identified, a full understanding of these complex regulatory systems remains to be delineated. This thesis probes the molecular, cellular and physiological roles of a protein termed evolutionary conserved signalling intermediate in toll (ECSIT) in innate immune signalling pathways. ECSIT was initially described as a TRAF6 interacting protein that bridged the TLR signalling intermediate TRAF6 to MEKK1 and downstream activation of NFκB. Subsequent data revealed an important developmental role for ECSIT in the BMP signalling pathway based on the embryonic lethal phenotype in the ECSIT knockout mouse. Other studies have indicated an important role for ECSIT in bacterial killing during infection via the production of mitochondrial reactive oxygen species (mROS). To date no information on the human orthologue of ECSIT (hECSIT) has been available. In this study we describe novel functional roles for hECSIT in TLR signalling. Through use of knockdown studies and a humanised animal model we demonstrate that hECSIT negatively regulates innate immune signalling pathways despite its 80% sequence homology to mECSIT. We show that phosphorylation, ubiquitination and processing of hECSIT leads to an inhibitory C-terminal form that contains both E3 ligase and DUB activity. We also demonstrate that hECSIT can independently target and regulate the ubiquitination of TRAF6, TRAF3 and RIP1 in TLR4/IL-1R, TLR3 and TNF-α signalling, respectively. These findings identify hECSIT as a novel regulator of innate immune signalling and highlight a sophisticated evolutionary difference between two highly conserved proteins

Investigating the regulation and enzymatic function of human ecsit in innate immune signalling.

Toll-like receptor (TLR) signalling represents the first line of defence against infection. TLRs respond to recognition of pathogens by activating transcription factors such as NFκB and the interferon regulatory factors (IRF)s to induce pro-inflammatory cytokines and IFNs. Whilst many of the components of the TLR signal transduction pathways have been identified, a full understanding of these complex regulatory systems remains to be delineated. This thesis probes the molecular, cellular and physiological roles of a protein termed evolutionary conserved signalling intermediate in toll (ECSIT) in innate immune signalling pathways. ECSIT was initially described as a TRAF6 interacting protein that bridged the TLR signalling intermediate TRAF6 to MEKK1 and downstream activation of NFκB. Subsequent data revealed an important developmental role for ECSIT in the BMP signalling pathway based on the embryonic lethal phenotype in the ECSIT knockout mouse. Other studies have indicated an important role for ECSIT in bacterial killing during infection via the production of mitochondrial reactive oxygen species (mROS). To date no information on the human orthologue of ECSIT (hECSIT) has been available. In this study we describe novel functional roles for hECSIT in TLR signalling. Through use of knockdown studies and a humanised animal model we demonstrate that hECSIT negatively regulates innate immune signalling pathways despite its 80% sequence homology to mECSIT. We show that phosphorylation, ubiquitination and processing of hECSIT leads to an inhibitory C-terminal form that contains both E3 ligase and DUB activity. We also demonstrate that hECSIT can independently target and regulate the ubiquitination of TRAF6, TRAF3 and RIP1 in TLR4/IL-1R, TLR3 and TNF-α signalling, respectively. These findings identify hECSIT as a novel regulator of innate immune signalling and highlight a sophisticated evolutionary difference between two highly conserved proteins

Molecular and physiological roles of Pellino E3 ubiquitin ligases in immunity

The sensing of foreign agents by the innate and adaptive immune system triggers complex signal transduction cascades that culminate in expression of gene patterns that facilitate host protection from the invading agent. Post‐translational modification of intracellular signaling proteins in these pathways is a key regulatory mechanism with ubiquitination being one of the important processes that controls levels and activities of signaling molecules. E3 ubiquitin ligases are the determining enzymes in dictating the ubiquitination status of individual proteins. Among these hundred E3 ubiquitin ligases are a family of Pellino proteins that are emerging to be important players in immunity and beyond. Herein, we review the roles of the Pellino E3 ubiquitin ligases in innate and adaptive immunity. We discuss their early discovery and characterization and how this has been aided by the highly conserved nature of innate immune signaling across evolution. We describe the molecular roles of Pellino proteins in immune signaling with particular emphasis on their involvement in pathogen recognition receptor (PRR) signaling. The growing appreciation of the importance of Pellino proteins in a wide range of immune‐mediated diseases are also evaluated

The E3 ubiquitin ligase Pellino2 mediates priming of the NLRP3 inflammasome

The NLRP3 inflammasome is important for inducing IL-1β and IL-18 inflammatory responses. Here the authors show, by generating and characterizing Peli2 deficient mice and immune cells, that an E3 ubiquitin ligase Pellino2 promotes inflammasome priming by inducing NLRP3 ubiquitination and by targeting IRAK1

Identification of the Flagellin Glycosylation System in Burkholderia cenocepacia and the Contribution of Glycosylated Flagellin to Evasion of Human Innate Immune Responses

Burkholderia cenocepacia is an opportunistic pathogen threatening patients with cystic fibrosis. Flagella are required for biofilm formation, as well as adhesion to and invasion of epithelial cells. Recognition of flagellin via the Toll-like receptor 5 (TLR5) contributes to exacerbate B. cenocepacia-induced lung epithelial inflammatory responses. In this study, we report that B. cenocepacia flagellin is glycosylated on at least 10 different sites with a single sugar, 4,6-dideoxy-4-(3-hydroxybutanoylamino)-d-glucose. We have identified key genes that are required for flagellin glycosylation, including a predicted glycosyltransferase gene that is linked to the flagellin biosynthesis cluster and a putative acetyltransferase gene located within the O-antigen lipopolysaccharide cluster. Another O-antigen cluster gene, rmlB, which is required for flagellin glycan and O-antigen biosynthesis, was essential for bacterial viability, uncovering a novel target against Burkholderia infections. Using glycosylated and nonglycosylated purified flagellin and a cell reporter system to assess TLR5-mediated responses, we also show that the presence of glycan in flagellin significantly impairs the inflammatory response of epithelial cells. We therefore suggest that flagellin glycosylation reduces recognition of flagellin by host TLR5, providing an evasive strategy to infecting bacteria

The E3 ubiquitin ligase Pellino3 protects against obesity-induced inflammation and insulin resistance

SummaryDiet-induced obesity can induce low-level inflammation and insulin resistance. Interleukin-1β (IL-1β) is one of the key proinflammatory cytokines that contributes to the generation of insulin resistance and diabetes, but the mechanisms that regulate obesity-driven inflammation are ill defined. Here we found reduced expression of the E3 ubiquitin ligase Pellino3 in human abdominal adipose tissue from obese subjects and in adipose tissue of mice fed a high-fat diet and showing signs of insulin resistance. Pellino3-deficient mice demonstrated exacerbated high-fat-diet-induced inflammation, IL-1β expression, and insulin resistance. Mechanistically, Pellino3 negatively regulated TNF receptor associated 6 (TRAF6)-mediated ubiquitination and stabilization of hypoxia-inducible factor 1α (HIF1α), resulting in reduced HIF1α-induced expression of IL-1β. Our studies identify a regulatory mechanism controlling diet-induced insulin resistance by highlighting a critical role for Pellino3 in regulating IL-1β expression with implications for diseases like type 2 diabetes

Secretory Leucoprotease Inhibitor (SLPI) Promotes Survival during Acute Pseudomonas aeruginosa Infection by Suppression of Inflammation Rather Than Microbial Killing

Secretory leucoprotease inhibitor (SLPI) has multifaceted functions, including inhibition of protease activity, antimicrobial functions, and anti-inflammatory properties. In this study, we show that SLPI plays a role in controlling pulmonary Pseudomonas aeruginosa infection. Mice lacking SLPI were highly susceptible to P. aeruginosa infection, however there was no difference in bacterial burden. Utilising a model of P. aeruginosa LPS-induced lung inflammation, human recombinant SLPI (hrSLPI) administered intraperitoneally suppressed the recruitment of inflammatory cells in the bronchoalveolar lavage fluid (BALF) and resulted in reduced BALF and serum levels of inflammatory cytokines and chemokines. This anti-inflammatory effect of hrSLPI was similarly demonstrated in a systemic inflammation model induced by intraperitoneal injection of LPS from various bacteria or lipoteichoic acid, highlighting the broad anti-inflammatory properties of hrSLPI. Moreover, in bone-marrow-derived macrophages, hrSLPI reduced LPS-induced phosphorylation of p-IkB-α, p-IKK-α/β, p-P38, demonstrating that the anti-inflammatory effect of hrSLPI was due to the inhibition of the NFκB and MAPK pathways. In conclusion, administration of hrSLPI attenuates excessive inflammatory responses and is therefore, a promising strategy to target inflammatory diseases such as acute respiratory distress syndrome or sepsis and could potentially be used to augment antibiotic treatment

Succination inactivates gasdermin D and blocks pyroptosis

Activated macrophages undergo a metabolic switch to aerobic glycolysis accumulating Krebs cycle intermediates that alter transcription of immune response genes. Here we extend these observations by defining fumarate as an inhibitor of pyroptotic cell death. We found that dimethyl fumarate (DMF) delivered to cells or endogenous fumarate reacts with gasdermin D (GSDMD) at critical cysteine residues to form S-(2-succinyl)-cysteine. GSDMD succination prevents its interaction with caspases, limiting its processing, oligomerization, and capacity to induce cell death. In mice, the administration of DMF protects against LPS shock and alleviates familial Mediterranean fever and experimental autoimmune encephalitis (EAE) by targeting GSDMD. Collectively, these findings identify GSDMD as a target of fumarate and reveal a mechanism of action for fumarate-based therapeutics including DMF used to treat multiple sclerosis