Investigation of toll-like receptor tolerance in the regulation of inflammation

Inflammation is a complex and highly organised immune response to microbes and tissue injury. Recognition of noxious stimuli by pathogen recognition receptor families including Toll-like receptors results in the expression of hundreds of genes that encode cytokines, chemokines, antimicrobials and regulators of inflammation. Regulation of TLR activation responses is controlled by TLR tolerance which induces a global change in the cellular transcriptional expression profile resulting in gene specific suppression and induction of transcription. In this thesis the plasticity of TLR receptor tolerance is investigated using an in vivo, transcriptomics and functional approach to determine the plasticity of TLR tolerance in the regulation of inflammation. Firstly, using mice deficient in the negative regulator of TLR gene transcription, Bcl-3 (Bcl-3-/-) in a model of intestinal inflammation, we investigated the role of Bcl-3 in the regulation of intestinal inflammatory responses. Our data revealed a novel role for Bcl-3 in the regulation of epithelial cell proliferation and regeneration during intestinal inflammation. Furthermore this data revealed that increased Bcl-3 expression contributes to the development of inflammatory bowel disease (IBD). Secondly, we demonstrate that lipopolysaccharide tolerance is transient and recovery from LPS tolerance results in polarisation of macrophages to a previously un-described hybrid state (RM). In addition, we identified that RM cells have a unique transcriptional profile with suppression and induction of genes specific to this polarisation state. Furthermore, using a functional approach to characterise the outcomes of TLR tolerance plasticity, we demonstrate that cytokine transcription is uncoupled from cytokine secretion in macrophages following recovery from LPS tolerance. Here we demonstrate a novel mechanism of regulation of TLR tolerance through suppression of cytokine secretion in macrophages. We show that TNF-α is alternatively trafficked towards a degradative intracellular compartment. These studies demonstrate that TLR tolerance is a complex immunological response with the plasticity of this state playing an important role in the regulation of inflammation.

An investigation into the role of Bcl-3 in toll-like receptor signalling

Through the recognition of potentially harmful stimuli, Toll-like receptors (TLRs) initiate the innate immune response and induce the expression of hundreds of immune and pro-inflammatory genes. TLRs are critical in mounting a defence against invading pathogens however, strict control of TLR signalling is vital to prevent host damage from excessive or prolonged immune activation. In this thesis the role of the IκB protein Bcl (B-cell lymphoma)-3 in the regulation of TLR signalling is investigated. Bcl3-/- mice and cells are hyper responsive to TLR stimulation and are defective in LPS tolerance. Bcl-3 interacts with and blocks the ubiquitination of homodimers of the NF-κB subunit, p50. Through stabilisation of inhibitory p50 homodimers, Bcl-3 negatively regulates NF-κB dependent inflammatory gene transcription following TLR activation. Firstly, we investigated the nature of the interaction between Bcl-3 and p50 and using peptide array technology. Key amino acids required for the formation of the p50:Bcl-3 immunosuppressor complex were identified. Furthermore, we demonstrate for the first time that interaction between Bcl-3 and p50 is necessary and sufficient for the anti-inflammatory properties of Bcl-3. Using the data generated from peptide array analysis we then generated cell permeable peptides designed to mimic Bcl-3 function and stabilise p50 homodimers. These Bcl-3 derived peptides are potent inhibitors of NF-κB dependent transcription activity in vitro and provide a solid basis for the development of novel gene-specific approaches in the treatment of inflammatory diseases. Secondly, we demonstrate that Bcl-3 mediated regulation of TLR signalling is not limited to NF-κB and identify the MAK3K Tumour Progression Locus (Tpl)-2 as a new binding partner of Bcl-3. Our data establishes role for Bcl-3 as a negative regulator of the MAPK-ERK pathway.

ROLE OF MULTIPLE TOLL-LIKE RECEPTOR ACTIVATION IN REGULATING CYTOTOXIC T CELL PRIMING TO LYMPOCYTIC CHORIOMENINGITIS VIRUS INFECTIONS

Foreign pathogens are recognized by toll-like receptors (TLR), present on various immune cells such as professional antigen-presenting cells (pAPCs). On recognition of its ligand, these receptors activate pAPCs, which may in turn influence naïve CD8+ T cell activation and affect their abilities to clear viral infection. However, how TLR ligands (TLR-L) can regulate CD8+ T cell responses have not been fully elucidated. This thesis will focus on examining how the presence of components from foreign pathogens, e.g. viral or bacterial infection, can contribute to shaping host immunity during concurrent viral infections. Since nitric oxide (NO), an innate effector immune molecule, was recently suggested to regulate proteasome activity; we sought to examine if NO can influence MHC-I antigen presentation during viral infections. The data in this section of the thesis provides evidence that combined TLR engagement can alter the presentation of certain CD8+ epitopes due to NO-induced inhibition in proteasome activity. Taken together, the data demonstrate that TLR ligation can influence the adaptive immune response due to induction of specific innate effector molecules such as NO. Next, the influence of combined TLR engagement on CD8+ T cell immunodominance hierarchies during viral infections was examined. In this section, we established that dual TLR2 and TLR3 stimulation alters immunodominance hierarchies of LCMV epitopes as a result of reduced uptake of cell-associated antigens and reduced cross-presentation of NP396 consequently suppressing NP396-specific CD8+ T cell responses. These findings are significant as they highlight a new role for TLR ligands in regulating anti-viral CD8+ T cell responses through impairing cross-presentation of cell-associated antigens depending on the type of TLR present in the environment during infections. Finally, we addressed TLR ligand induced type I interferon production and the signalling pathways that regulate them in two different mouse macrophage populations – those derived from the spleen or bone marrow. In this study, we observed that concomitant TLR2 stimulation blocked the induction of type I IFN induced by TLR4 in bone marrow-derived macrophages, but not spleen-derived macrophages in SOCS3-dependent manner. Taken together, the data presented in this thesis have defined new facets of how anti-viral responses are regulated by TLR activation, especially if multiple receptors are engaged simultaneously.

Altered Toll-like Receptor 2-mediated Endotoxin Tolerance Is Related to Diminished Interferon β Production

Induction of endotoxin tolerance leads to a reduced inflammatory response after repeated challenge by LPS and is important for resolution of inflammation and prevention of tissue damage. Enterobacterial LPS is recognized by the TLR4 signaling complex, whereas LPS of some non-enterobacterial organisms is capable of signaling independently of TLR4 utilizing TLR2-mediated signal transduction instead. In this study we report that Porphyromonas gingivalis LPS, a TLR2 agonist, fails to induce a fully endotoxin tolerant state in a human monocytic cell line (THP-1) and mouse bone marrow-derived macrophages. In contrast to significantly decreased production of human IL-8 and TNF-alpha and, in mice, keratinocyte-derived cytokine (KC), macrophage inflammatory protein-2 (MIP-2), and TNF-alpha after repeated challenge with Escherichia coli LPS, cells repeatedly exposed to P. gingivalis LPS responded by producing less TNF-alpha but sustained elevated secretion of IL-8, KC, and MIP-2. Furthermore, in endotoxin-tolerant cells, production of IL-8 is controlled at the signaling level and correlates well with NF-kappa B activation, whereas TNF-alpha expression is blocked at the gene transcription level. Interferon beta plays an important role in attenuation of chemokine expression in endotoxin-tolerized cells as shown in interferon regulatory factor-3 knock-out mice. In addition, human gingival fibroblasts, commonly known not to display LPS tolerance, were found to be tolerant to repeated challenge by LPS if pretreated with interferon beta. The data suggest that the inability of the LPS-TLR2 complex to induce full endotoxin tolerance in monocytes/macrophages is related to diminished production of interferon beta and may partly explain the involvement of these LPS isoforms in the pathogenesis of chronic inflammatory diseases.

Expression of Toll-like receptor 2 is up-regulated in monocytes from patients with chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is characterised by pulmonary and systemic inflammation which flare-up during episodes of acute exacerbation (AECOPD). Given the role of Toll-like receptors (TLRs) in the induction of inflammatory responses we investigated the involvement of TLRs in COPD pathogenesis.

The poxvirus protein A52R targets Toll-like receptor signaling complexes to suppress host defense

Toll-like receptors (TLRs) are crucial in the innate immune response to pathogens, in that they recognize and respond to pathogen associated molecular patterns, which leads to activation of intracellular signaling pathways and altered gene expression. Vaccinia virus (VV), the poxvirus used to vaccinate against smallpox, encodes proteins that antagonize important components of host antiviral defense. Here we show that the VV protein A52R blocks the activation of the transcription factor nuclear factor kappa B (NF-kappa B) by multiple TLRs, including TLR3, a recently identified receptor for viral RNA. A52R associates with both interleukin 1 receptor-associated kinase 2 (IRAK2) and tumor necrosis factor receptor-associated factor 6 (TRAF6), two key proteins important in TLR signal transduction. Further, A52R could disrupt signaling complexes containing these proteins. A virus deletion mutant lacking the A52R gene was attenuated compared with wild-type and revertant controls in a murine intranasal model of infection. This study reveals a novel mechanism used by VV to suppress the host immunity. We demonstrate viral disabling of TLRs, providing further evidence for an important role for this family of receptors in the antiviral response.

Epstein-Barr Virus Large Tegument Protein BPLF1 Contributes to Innate Immune Evasion through Interference with Toll-Like Receptor Signaling

Viral infection triggers an early host response through activation of pattern recognition receptors, including Toll-like receptors (TLR). TLR signaling cascades induce production of type I interferons and proinflammatory cytokines involved in establishing an anti-viral state as well as in orchestrating ensuing adaptive immunity. To allow infection, replication, and persistence, (herpes)viruses employ ingenious strategies to evade host immunity. The human gamma-herpesvirus Epstein-Barr virus (EBV) is a large, enveloped DNA virus persistently carried by more than 90% of adults worldwide. It is the causative agent of infectious mononucleosis and is associated with several malignant tumors. EBV activates TLRs, including TLR2, TLR3, and TLR9. Interestingly, both the expression of and signaling by TLRs is attenuated during productive EBV infection. Ubiquitination plays an important role in regulating TLR signaling and is controlled by ubiquitin ligases and deubiquitinases (DUBs). The EBV genome encodes three proteins reported to exert in vitro deubiquitinase activity. Using active site-directed probes, we show that one of these putative DUBs, the conserved herpesvirus large tegument protein BPLF1, acts as a functional DUB in EBV-producing B cells. The BPLF1 enzyme is expressed during the late phase of lytic EBV infection and is incorporated into viral particles. The N-terminal part of the large BPLF1 protein contains the catalytic site for DUB activity and suppresses TLR-mediated activation of NF-κB at, or downstream of, the TRAF6 signaling intermediate. A catalytically inactive mutant of this EBV protein did not reduce NF-κB activation, indicating that DUB activity is essential for attenuating TLR signal transduction. Our combined results show that EBV employs deubiquitination of signaling intermediates in the TLR cascade as a mechanism to counteract innate anti-viral immunity of infected hosts.

Orphan receptor IL-17RD regulates Toll-like receptor signalling via SEFIR/TIR interactions

Receptor families of the innate immune response engage in 'cross-talk' to tailor optimal immune responses against invading pathogens. However, these responses are subject to multiple levels of regulation to keep in check aberrant inflammatory signals. Here, we describe a role for the orphan receptor interleukin-17 receptor D (IL-17RD) in negatively regulating Toll-like receptor (TLR)-induced responses. Deficiency of IL-17RD expression in cells leads to enhanced pro-inflammatory signalling and gene expression in response to TLR stimulation, and Il17rd(-/-) mice are more susceptible to TLR-induced septic shock. We demonstrate that the intracellular Sef/IL-17R (SEFIR) domain of IL-17RD targets TIR adaptor proteins to inhibit TLR downstream signalling thus revealing a paradigm involving cross-regulation of members of the IL-17R and TLR families.

Development of a melanoma therapeutic vaccine candidate by the coencapsulation of melanoma antigen peptides and toll-like receptor ligands as adjuvants into polymeric nanoparticles

Tese de doutoramento, Farmácia (Tecnologia Farmacêutica), Universidade de Lisboa, Faculdade de Farmácia, 2014

Control of endosomal Toll-like receptor (TLR) signaling by nanoparticles and applications in cancer therapy

Thesis (Ph.D.)--University of Washington, 2014

La régulation de l’hepcidine à travers les récepteurs Toll-like dans les macrophages

L'interaction entre le système immunitaire et le métabolisme du fer est bien illustrée par l'anémie des maladies chroniques (ACD), qui est fréquemment rencontrée dans les infections chroniques, l'inflammation et le cancer. La majorité des modifications dans les paramètres du fer observées dans l’ACD tient compte des modifications de l’homéostasie du fer, avec la délocalisation du métal de la circulation et les sites de l'érythropoïèse au compartiment de stockage dans les macrophages. Les mécanismes de la réponse hyposidérémique impliquent des cytokines, notamment TNF-alpha et IL-6, qui régulent les niveaux de plusieurs gènes du métabolisme du fer, y compris les transporteurs de fer et de l'hepcidine, un régulateur négatif de l’absorption du fer, ce qui entraîne l'inhibition de l'exportation du fer à travers la ferroportine 1 (FPN1) au niveau de l'intestin et les macrophages. Des études antérieures ont montré que l'IL-6 induit l’expression d’hepcidine dans les hépatocytes, mais il y a très peu de données concernant la façon par laquelle l'hepcidine et la FPN1 sont régulées dans les macrophages. Récemment, nous avons constaté que l'induction de l'hepcidine dans le foie par le lipopolysaccharide (LPS) dépend de la voie de signalisation médiée par le récepteur Toll-like 4 (TLR4). Le but de ce travail est d’identifier les ligands des TLRs capables d'induire l'hepcidine dans les macrophages et de déterminer l’exigence des TLRs dans l’induction de l’hepcidine et le développement d’hyposidérémie. En plus, nous voulons étudier l’effet de l’inflammation causée par les ligands des TLRs sur le taux de fer sérique, la production des cytokines et l'expression de l’hepcidine et de la ferroportine. D’autre part nous voulons étudier l’effet du taux du fer sur la production d’IL-6 macrophagique en réponse à la stimulation par le TLR4. D'abord, pour identifier les ligands des TLRs capables d'induire l'hepcidine dans les macrophages, nous avons traité les macrophages RAW 264.7 et les macrophages péritonéaux de souris (MPMs) avec différents ligands TLRs et on a mesuré l’expression de l'hepcidine par qRT-PCR. Nous avons observé que Pam3CSK4 (Pam), un ligand de TLR2/1; LPS, un ligand de TLR-4 et FSL1 un ligand de TLR2/6 induisent l’expression de l'hepcidine dans les cellules RAW 264.7 et les MPMs, contrairement au polyinosinic: polycytidylic acid (Poly I: C), un ligand de TLR3. De plus, LPS était capable de réprimer l’expression de la ferroportine dans les cellules RAW 264.7. Afin de mieux définir la nécessité des TLRs pour assurer cette expression, nous avons utilisé les souris TLR-2 knock-out et on a établi que l'expression de l'hepcidine dans les macrophages par LPS, Pam ou FSL1 est dépendante du TLR2. En accord avec les expériences in vitro, les études effectuées in vivo ont montré que LPS réprime l’expression de la ferroportine, ainsi que PolyI:C n’est pas capable de stimuler l'expression d'hepcidine hépatique, par contre il était efficace pour déclencher une hyposidérémie. Ensuite, on voulait déterminer la voie de signalisation utilisée dans l’induction de l’hepcidine dans les macrophages. Comme il y deux voies majeures connues pour la signalisation des TLRs : une dépendante et l’autre indépendante de la protéine MyD88, on a étudié l’expression de l’hepcidine dans les MPMs isolés des souris MyD88-/- et nous avons constaté que l'absence de signalisation MyD88 abolit l'induction de l'hepcidine déclenchée par Pam, LPS et FSL1. D’autre part, la stimulation avec du LPS induisait in vivo la production d’IL-6 et de TNF-alpha, et la stimulation d’IL-6 était renforcée in vitro par la présence du fer. Ces observations indiquent que l’expression de HAMP (Hepcidin Antimicrobial Peptide) dans les macrophages peut être régulée par différents TLRs, ce qui suggère que la production d'hepcidine macrophagique fait partie d'une réponse immunitaire activées par les TLRs.