Defining the Physiological and Molecular Role of Pellino3 in TLR3 Signalling

Innate immunity utilises a series of pathogen associated molecular pattern receptors such as the Toll-like receptors (TLRs) to detect and respond to invading microorganisms. The different TLRs play a pivotal role in the first line of defence by recognising the type of pathogenic threat encountered and by responding in kind. TLR pathogen interaction triggers the production of a number of pro-inflammatory cytokines and anti-viral mediators to initiate the process of microbial elimination. However, if left unregulated, such immunostimulatory and cytotoxic factors have the potential to cause grievous harm to the host and can facilitate and propagate autoimmune disease. Therefore, the TLR signalling response is under the control of stringent regulatory mechanisms to prevent self-harm. In this thesis, the generation and characterisation of a genetically deficient Pellino3 mouse is described. Pellino3 is a member of the highly evolutionary conserved Pellino family of E3 ubiquitin ligases. Using this model, the physiological role of Pellino3 in TLR signalling is delineated. Peli3-deficient mice display no obvious abnormalities in cytokine production in response to pathogenic bacterial and fungal ligands. Pellino3 functions as a specific regulator of anti-viral type 1 interferon production in response to double stranded viral RNA recognition by TLR3. Pellino3-deficient mice are more resistant to the pathogenic and lethal effects of encephalomycarditis virus (EMCV) infection. Pellino3 functions in a novel auto-regulatory mechanism to specifically prevent excessive TLR3 induced expression of type 1 IFNs but leaves pro-inflammatory cytokine production intact. TLR3 signalling induces Pellino3 expression which in turn interacts with TRAF6 and facilitates its polyubiquitination. This modification of TRAF6 suppresses its ability to bind and ubiquitinate a key anti-viral transcription factor IRF7, thereby down regulating the type 1 IFN response. The findings outlined in this thesis define for the first time, a physiological role for Pellino3

Mal Mediates TLR-Induced Activation of CREB and Expression of IL-10

TLRs initiate immune responses by direct detection of molecular motifs that distinguish invading microbes from host cells. Five intracellular adaptor proteins, each containing a Toll/IL-1R (TIR) domain, are used by TLRs and play key roles in dictating gene expression patterns that are tailored to the invader. Such gene expression is mediated by transcription factors, and although TIR adaptor-induced activation of NF-kB and the IFN regulatory factors have been intensively studied, there is a dearth of information on the role of TIR adaptors in regulating CREB. In this paper, we describe a role for the TIR adaptor Mal in enhancing activation of CREB. Mal-deficient murine bone marrow-derived macrophages show a loss in responsiveness to TLR2 and TLR4 ligands with respect to activation of CREB. Mal-deficient cells also fail to express the CREB-responsive genes IL-10 and cyclooxygenase 2 in response to Pam2Cys-Ser-(Lys)4 and LPS. We reveal that Mal-mediated activation of CREB is dependent on Pellino3 and TNFR-associated factor 6, because CREB activation is greatly diminished in Pellino3 knockdown cells and TNFRassociated factor 6-deficient cells. We also demonstrate the importance of p38 MAPK in this pathway with the p38 inhibitor SB203580 abolishing activation of CREB in murine macrophages. MAPK-activated protein kinase 2 (MK2), a substrate for p38 MAPK, is the likely downstream mediator of p38 MAPK in this pathway, because Mal is shown to activate MK2 and inhibition of MK2 decreases TLR4-induced activation of CREB. Overall, these studies demonstrate a new role for Mal as a key upstream regulator of CREB and as a contributor to the expression of both pro- and anti-inflammatory gen

Mal Mediates TLR-Induced Activation of CREB and Expression of IL-10

TLRs initiate immune responses by direct detection of molecular motifs that distinguish invading microbes from host cells. Five intracellular adaptor proteins, each containing a Toll/IL-1R (TIR) domain, are used by TLRs and play key roles in dictating gene expression patterns that are tailored to the invader. Such gene expression is mediated by transcription factors, and although TIR adaptor-induced activation of NF-kB and the IFN regulatory factors have been intensively studied, there is a dearth of information on the role of TIR adaptors in regulating CREB. In this paper, we describe a role for the TIR adaptor Mal in enhancing activation of CREB. Mal-deficient murine bone marrow-derived macrophages show a loss in responsiveness to TLR2 and TLR4 ligands with respect to activation of CREB. Mal-deficient cells also fail to express the CREB-responsive genes IL-10 and cyclooxygenase 2 in response to Pam2Cys-Ser-(Lys)4 and LPS. We reveal that Mal-mediated activation of CREB is dependent on Pellino3 and TNFR-associated factor 6, because CREB activation is greatly diminished in Pellino3 knockdown cells and TNFRassociated factor 6-deficient cells. We also demonstrate the importance of p38 MAPK in this pathway with the p38 inhibitor SB203580 abolishing activation of CREB in murine macrophages. MAPK-activated protein kinase 2 (MK2), a substrate for p38 MAPK, is the likely downstream mediator of p38 MAPK in this pathway, because Mal is shown to activate MK2 and inhibition of MK2 decreases TLR4-induced activation of CREB. Overall, these studies demonstrate a new role for Mal as a key upstream regulator of CREB and as a contributor to the expression of both pro- and anti-inflammatory gen

The induction and function of the anti-inflammatory fate of TH17 cells

TH17 cells exemplify environmental immune adaptation: they can acquire both a pathogenic and an anti-inflammatory fate. However, it is not known whether the anti-inflammatory fate is merely a vestigial trait, or whether it serves to preserve the integrity of the host tissues. Here we show that the capacity of TH17 cells to acquire an anti-inflammatory fate is necessary to sustain immunological tolerance, yet it impairs immune protection against S. aureus. Additionally, we find that TGF-β signalling via Smad3/Smad4 is sufficient for the expression of the anti-inflammatory cytokine, IL-10, in TH17 cells. Our data thus indicate a key function of TH17 cell plasticity in maintaining immune homeostasis, and dissect the molecular mechanisms explaining the functional flexibility of TH17 cells with regard to environmental changes.Fil: Xu, Hao. University of Yale. School of Medicine; Estados UnidosFil: Agalioti, Theodora. University Medical Center Hamburg-Eppendorf; AlemaniaFil: Zhao, Jun. University of Yale. School of Medicine; Estados UnidosFil: Steglich, Babett. University Medical Center Hamburg-Eppendorf; AlemaniaFil: Wahib, Ramez. University Medical Center Hamburg-Eppendorf; AlemaniaFil: Amezcua Vesely, Maria Carolina. University of Yale. School of Medicine; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Bielecki, Piotr. University of Yale. School of Medicine; Estados UnidosFil: Bailis, Will. University of Yale. School of Medicine; Estados UnidosFil: Jackson, Ruaidhri. University of Yale. School of Medicine; Estados UnidosFil: Perez, Daniel. University Medical Center Hamburg-Eppendorf; AlemaniaFil: Izbicki, Jakob. University Medical Center Hamburg-Eppendorf; AlemaniaFil: Licona-Limón, Paula. University of Yale. School of Medicine; Estados UnidosFil: Kaartinen, Vesa. University Medical Center Hamburg-Eppendorf; AlemaniaFil: Geginat, Jens. University Medical Center Hamburg-Eppendorf; AlemaniaFil: Esplugues, Enric. University of Yale. School of Medicine; Estados UnidosFil: Tolosa, Eva. University of Yale. School of Medicine; Estados UnidosFil: Huber, Samuel. University of Yale. School of Medicine; Estados UnidosFil: Flavell, Richard A.. University of Yale. School of Medicine; Estados UnidosFil: Gagliani, Nicola. University Medical Center Hamburg-Eppendorf; Alemani

Defining the Physiological and Molecular Role of Pellino3 in TLR3 Signalling

Innate immunity utilises a series of pathogen associated molecular pattern receptors such as the Toll-like receptors (TLRs) to detect and respond to invading microorganisms. The different TLRs play a pivotal role in the first line of defence by recognising the type of pathogenic threat encountered and by responding in kind. TLR pathogen interaction triggers the production of a number of pro-inflammatory cytokines and anti-viral mediators to initiate the process of microbial elimination. However, if left unregulated, such immunostimulatory and cytotoxic factors have the potential to cause grievous harm to the host and can facilitate and propagate autoimmune disease. Therefore, the TLR signalling response is under the control of stringent regulatory mechanisms to prevent self-harm. In this thesis, the generation and characterisation of a genetically deficient Pellino3 mouse is described. Pellino3 is a member of the highly evolutionary conserved Pellino family of E3 ubiquitin ligases. Using this model, the physiological role of Pellino3 in TLR signalling is delineated. Peli3-deficient mice display no obvious abnormalities in cytokine production in response to pathogenic bacterial and fungal ligands. Pellino3 functions as a specific regulator of anti-viral type 1 interferon production in response to double stranded viral RNA recognition by TLR3. Pellino3-deficient mice are more resistant to the pathogenic and lethal effects of encephalomycarditis virus (EMCV) infection. Pellino3 functions in a novel auto-regulatory mechanism to specifically prevent excessive TLR3 induced expression of type 1 IFNs but leaves pro-inflammatory cytokine production intact. TLR3 signalling induces Pellino3 expression which in turn interacts with TRAF6 and facilitates its polyubiquitination. This modification of TRAF6 suppresses its ability to bind and ubiquitinate a key anti-viral transcription factor IRF7, thereby down regulating the type 1 IFN response. The findings outlined in this thesis define for the first time, a physiological role for Pellino3

Defining the Physiological and Molecular Role of Pellino3 in TLR3 Signalling

Innate immunity utilises a series of pathogen associated molecular pattern receptors such as the Toll-like receptors (TLRs) to detect and respond to invading microorganisms. The different TLRs play a pivotal role in the first line of defence by recognising the type of pathogenic threat encountered and by responding in kind. TLR pathogen interaction triggers the production of a number of pro-inflammatory cytokines and anti-viral mediators to initiate the process of microbial elimination. However, if left unregulated, such immunostimulatory and cytotoxic factors have the potential to cause grievous harm to the host and can facilitate and propagate autoimmune disease. Therefore, the TLR signalling response is under the control of stringent regulatory mechanisms to prevent self-harm. In this thesis, the generation and characterisation of a genetically deficient Pellino3 mouse is described. Pellino3 is a member of the highly evolutionary conserved Pellino family of E3 ubiquitin ligases. Using this model, the physiological role of Pellino3 in TLR signalling is delineated. Peli3-deficient mice display no obvious abnormalities in cytokine production in response to pathogenic bacterial and fungal ligands. Pellino3 functions as a specific regulator of anti-viral type 1 interferon production in response to double stranded viral RNA recognition by TLR3. Pellino3-deficient mice are more resistant to the pathogenic and lethal effects of encephalomycarditis virus (EMCV) infection. Pellino3 functions in a novel auto-regulatory mechanism to specifically prevent excessive TLR3 induced expression of type 1 IFNs but leaves pro-inflammatory cytokine production intact. TLR3 signalling induces Pellino3 expression which in turn interacts with TRAF6 and facilitates its polyubiquitination. This modification of TRAF6 suppresses its ability to bind and ubiquitinate a key anti-viral transcription factor IRF7, thereby down regulating the type 1 IFN response. The findings outlined in this thesis define for the first time, a physiological role for Pellino3

Defining the Physiological and Molecular Role of Pellino3 in TLR3 Signalling

Innate immunity utilises a series of pathogen associated molecular pattern receptors such as the Toll-like receptors (TLRs) to detect and respond to invading microorganisms. The different TLRs play a pivotal role in the first line of defence by recognising the type of pathogenic threat encountered and by responding in kind. TLR pathogen interaction triggers the production of a number of pro-inflammatory cytokines and anti-viral mediators to initiate the process of microbial elimination. However, if left unregulated, such immunostimulatory and cytotoxic factors have the potential to cause grievous harm to the host and can facilitate and propagate autoimmune disease. Therefore, the TLR signalling response is under the control of stringent regulatory mechanisms to prevent self-harm. In this thesis, the generation and characterisation of a genetically deficient Pellino3 mouse is described. Pellino3 is a member of the highly evolutionary conserved Pellino family of E3 ubiquitin ligases. Using this model, the physiological role of Pellino3 in TLR signalling is delineated. Peli3-deficient mice display no obvious abnormalities in cytokine production in response to pathogenic bacterial and fungal ligands. Pellino3 functions as a specific regulator of anti-viral type 1 interferon production in response to double stranded viral RNA recognition by TLR3. Pellino3-deficient mice are more resistant to the pathogenic and lethal effects of encephalomycarditis virus (EMCV) infection. Pellino3 functions in a novel auto-regulatory mechanism to specifically prevent excessive TLR3 induced expression of type 1 IFNs but leaves pro-inflammatory cytokine production intact. TLR3 signalling induces Pellino3 expression which in turn interacts with TRAF6 and facilitates its polyubiquitination. This modification of TRAF6 suppresses its ability to bind and ubiquitinate a key anti-viral transcription factor IRF7, thereby down regulating the type 1 IFN response. The findings outlined in this thesis define for the first time, a physiological role for Pellino3

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

Mal Mediates TLR-Induced Activation of CREB and Expression of IL-10

TLRs initiate immune responses by direct detection of molecular motifs that distinguish invading microbes from host cells. Five intracellular adaptor proteins, each containing a Toll/IL-1R (TIR) domain, are used by TLRs and play key roles in dictating gene expression patterns that are tailored to the invader. Such gene expression is mediated by transcription factors, and although TIR adaptor-induced activation of NF-kB and the IFN regulatory factors have been intensively studied, there is a dearth of information on the role of TIR adaptors in regulating CREB. In this paper, we describe a role for the TIR adaptor Mal in enhancing activation of CREB. Mal-deficient murine bone marrow-derived macrophages show a loss in responsiveness to TLR2 and TLR4 ligands with respect to activation of CREB. Mal-deficient cells also fail to express the CREB-responsive genes IL-10 and cyclooxygenase 2 in response to Pam2Cys-Ser-(Lys)4 and LPS. We reveal that Mal-mediated activation of CREB is dependent on Pellino3 and TNFR-associated factor 6, because CREB activation is greatly diminished in Pellino3 knockdown cells and TNFRassociated factor 6-deficient cells. We also demonstrate the importance of p38 MAPK in this pathway with the p38 inhibitor SB203580 abolishing activation of CREB in murine macrophages. MAPK-activated protein kinase 2 (MK2), a substrate for p38 MAPK, is the likely downstream mediator of p38 MAPK in this pathway, because Mal is shown to activate MK2 and inhibition of MK2 decreases TLR4-induced activation of CREB. Overall, these studies demonstrate a new role for Mal as a key upstream regulator of CREB and as a contributor to the expression of both pro- and anti-inflammatory gen