Psoriasis is an immune mediated inflammatory disease which affects 2-3% of the world’s population. Over the last decade, psoriasis has been acknowledged as an independent risk factor for atherosclerosis. The precise mechanism or mechanisms of the heightened risk is widely speculated. Endothelial cells and macrophages are central players in the immunopathological development of both diseases. Interleukin-36 cytokines (IL-36) have been heavily implicated in psoriasis immunopathology. Significant upregulation of epidermal IL-36 is a recognised characteristic of psoriatic skin inflammation. IL-36 induces inflammatory responses in dendritic cells, fibroblasts and epithelial cells. While vascular alterations are a hallmark of psoriatic lesions and dermal endothelial cells are well known to play a critical role in dermal inflammation, the effects of IL-36 on endothelial cells have not been defined. We report that endothelial cells including dermal microvascular cells express a functionally active IL-36 receptor. Adhesion molecules VCAM-1 and ICAM-1 are upregulated following IL-36γ stimulation, and this is reversed in the presence of the endogenous IL-36 receptor antagonist. IL-36γ-stimulated endothelial cells secrete the proinflammatory chemokines IL-8, CCL2 and CCL20. Chemotaxis assays showed increased migration of T-cells following IL-36γ stimulation of endothelial cells. Both resident and infiltrating inflammatory myeloid cells contribute to the immunopathology of psoriasis by promoting the IL-23/IL-17 axis. We show that IL-36γ induces the production of psoriasis-associated cytokines from macrophages (IL-23, TNFα) and that this response is enhanced in macrophages from psoriasis patients. This effect is specific for IL-36γ and could not be mimicked by other IL-1 family cytokines such as IL-1α. Furthermore, IL-36γ stimulated macrophages potently activated endothelial cells as illustrated by ICAM-1(CD54) upregulation, and led to increased adherence of monocytes, effects that were markedly more pronounced for psoriatic macrophages. Interestingly, regardless of stimulus, monocytes isolated from psoriasis patients showed increased adherence to both the stimulated and unstimulated endothelium when compared to monocytes from healthy individuals. Collectively, these findings add to the growing evidence for IL-36γ having roles in psoriatic responses, by enhancing endothelium directed leukocyte infiltration into the skin and strengthening the IL-23/IL-17 pathway. Our findings also point to a cellular response which could potentially support cardiovascular comorbidities in psoriasis.

Epithelial cells are the first line of defense against invading pathogens and external threats in the environment. Keratinocytes, often not perceived of as immune cells, release cytokines in response to infection or injury to signal danger to neighboring cells and recruit effector leukocytes to prevent further damage to the host. IL-1 and IL-36 cytokines are a group of closely related proteins that share similarities in structure and function and have been shown to play key roles in inflammatory responses of epithelial tissues. While IL-1, consisting of IL-1α and IL-1β, have been widely studied and recognized as pinnacle cytokines in a variety of inflammatory responses, relatively little is understood about IL-36 cytokines since their discovery more than 15 years ago, and how they differ from their better-known IL-1 relatives. IL-36 cytokines, consisting of IL-36α, IL-36β, and IL-36γ, signal through the same receptor, IL-36R, which is expressed most abundantly on epithelial cells. IL-36 proteins garnered attention when it was discovered that a missense mutation in the gene encoding the naturally occurring receptor antagonist, IL-36Ra, was associated with the deadly form of psoriasis, generalized pustular psoriasis (GPP). This disease is characterized by episodic flares of keratinocyte hyperproliferation leading to red scaly lesions all over the body, excessive neutrophil recruitment to the epidermis resulting in pustule formation, and severe fever. Our data presented here demonstrate that IL-36α, but not IL-36β or IL-36γ is critical for the psoriatic phenotype, including epidermal thickening and neutrophil recruitment, generated during a murine model of psoriasis induced by the drug Imiquimod. Furthermore, IL-36α was found to induce IL-1α expression and vice versa through a signaling feedback loop which perpetuated disease. These data provide insight into mechanisms whereby IL-36 signaling can lead to excessive inflammatory effects in patients with pre-existing regulation deficiencies, which can lead to acute flares of disease. Beyond their association with disease, IL-1 has been shown to contribute to anti-bacterial and anti-viral responses of the immune system by upregulating inflammatory signals and chemoattractants. Herpes Simplex Virus-1 (HSV-1) is a human pathogen that has developed several strategies to manipulate elements of the immune system to avoid detection by the host. One such mechanism is the prevention of activation and release of IL-1β from infected cells thereby blocking its pro-inflammatory responses. Our data show that keratinocytes infected with HSV-1 actively release IL-1α to alert danger to neighboring cells to circumvent this blockage of IL-1β signaling. This release of IL-1α initiates recruitment of leukocytes to early HSV-1 microinfection sites resulting in increased protection against disease, as evident by the increased mortality rate of mice deficient in the IL-1 receptor, IL-1R1. This study, for the first time in vivo, demonstrates the ability of IL-1α to act as an alarmin to initiate an immune response to combat infection. The role of IL-36 cytokines during viral infections has been less defined than that of IL-1. Several studies have shown the upregulation of IL-36 expression during viral infections in epithelial tissues, such as HSV-1 and Influenza, yet a direct link has not been established between these proteins and anti-viral responses. Our research presented within this thesis show that IL-36β, but not IL-36α nor IL-36γ, provides protection against the lethal outcome of cutaneous HSV-1 infection, as demonstrated by IL-36β knockout mice dying earlier and more often than wild type mice. Surprisingly, while previous reports have found IL-36 cytokines to be capable of activating the adaptive immune system, our results found no significant differences in development of HSV-1 specific antibodies or CD8+ T cell development between wild type and IL-36β knockout mice. Furthermore, we found no significant differences in viral copy numbers at infection sites between the two groups. Although our data show that IL-36β clearly plays a critical role in controlling the outcome of HSV-1 infection, further studies are necessary to define the mechanisms behind this protection. The final section of this thesis focuses on the endogenous nature of IL-36 cytokines, specifically IL-36γ, and their potential processing. IL-36 cytokines were originally believed to be synthesized as full-length fully active proteins; however, large concentrations of the recombinant proteins were required to elicit cellular responses in vitro. Since then, studies have shown that IL-36 cytokines gained up to 1000-fold increases in reactivity following processing at very specific N-terminal locations of each individual cytokine, however this processing has never been shown to occur in vivo. These studies were recently expanded when neutrophil proteases were found to be responsible for processing of these proteins in vitro. Data presented here show, for the first time, that IL-36γ may be endogenously processed by neutrophils in wounded murine skin in vivo, yet, the amino acid processing site appears to be different from that predicted. Although further studies are required to fully characterize the nature of this processing, these data provide valuable insight into the natural mechanisms involved in the potential activation of these cytokines. Taken together, the research presented within this thesis sheds light on the mechanisms whereby IL-1 and IL-36 cytokines enhance immunological defenses against potential threats, and yet, can contribute to disease if unregulated. Furthermore, these studies demonstrate the evolutionary advantage of producing multiple cytokines that appear to have redundant roles within the body, yet can provide multiple levels of protection to the host. This knowledge contributes to our overall understanding of these proteins and their contribution to immunological systems within the body. / Microbiology and Immunology

IL-36 gamma is a proinflamatory cytokine which belongs to the IL-1 family of cytokines. It is expressed in the skin and by epithelial cells (ECs) lining lung and gut tissue. We used human 3-D organotypic cells, that recapitulate either in vivo human vaginal or cervical tissue, to explore the possible role of IL-36 gamma in host defense against pathogens in the human female reproductive tract (FRT). EC were exposed to compounds derived from virus or bacterial sources and induction and regulation of IL-36 gamma and its receptor was determined. Polyinosinic-polycytidylic acid (poly I:C), flagellin, and synthetic lipoprotein (FSL-1) significantly induced expression of IL-36 gamma in a dose-dependent manner, and appeared to be TLR-dependent. Recombinant IL-36 gamma treatment resulted in self amplification of IL-36 gamma and its receptor (IL-36R) via increased gene expression, and promoted other inflammatory signaling pathways. This is the first report to demonstrate that the IL-36 receptor and IL-36 gamma are present in the human FRT EC and that they are differentially induced by microbial products at this site. We conclude that IL-36 gamma is a driver for epithelial and immune activation following microbial insult and, as such, may play a critical role in host defense in the FRT.

La tuberculose est encore aujourd’hui un problème de santé majeur et l’augmentation des cas de tuberculoses résistantes au niveau mondial, associé au dépistage et diagnostic insuffisants, suggère une éradication totale encore lointaine. La compréhension des relations hôte-pathogène est essentielle pour permettre d’établir de nouvelles stratégies thérapeutiques. Ces travaux de thèse ont mis en exergue la contribution limitée de la voie de signalisation IL-36, cytokine de la famille de l’IL-1, au cours de l’infection mycobactérienne. Ces données permettent d’envisager sérieusement l’IL-36 en tant que cible thérapeutique pour le psoriasis sans risquer la réactivation de tuberculose latente chez les patients. De plus, nous avons également démontré le rôle différentiel de la voie de signalisation TNF au sein de populations cellulaires spécifiques lors de l’infection par M. tuberculosis, et nos résultats apportent des connaissances solides pour envisager des stratégies immuno-modulatoires qui pourraient constituer l’avenir des traitements antituberculeux. D’autre part, nous avons caractérisé un modèle murin humanisé pour une étude facilitée des anticorps anti-TNF humains actuels. L’utilisation de ce modèle pourrait permettre, à termes, d’identifier et de valider de nouveaux candidats d’anticorps anti-TNF de deuxième génération. Enfin, nous avons montré que des anticorps neutralisants l’IL-17 ne perturbent pas la réponse immunitaire à la tuberculose, contrairement aux anticorps anti-TNF, et que l’absence de l’IL-17 n’est pas compensée par l’IL-22 puisque des animaux déficients pour ces deux voies de signalisation sont capables de contrôler l’infection. / Tuberculosis remains a major health problem in the world nowadays. The increasing incidence of resistant tuberculosis is associated with a poor diagnosis, reflecting important difficulties for total eradication. Understand host-pathogen interactions is crucial to establish new therapeutic strategies. This work first shows the limited contribution of IL-36 pathway during mycobacterial infection. These results suggest that IL-36 could be targeted for the treatment of psoriasis without a high risk of tuberculosis reactivation. We then demonstrate the differential role of TNF pathway among myeloid or lymphoid cells during M. tuberculosis infection, and our data support the development of immunomodulatory strategies to boost host immune response, thus helping to clear the infection. Moreover, we characterize a humanized murine model allowing the study of new anti-TNF candidates in the context of M. tuberculosis infection. Finally, we show that antibodies targeting IL-17 does not dampen host control of M. tuberculosis infection, unlike anti-TNF, and that IL-22 does not compensate absence of IL-17 for this control.

abstract: An estimated 267 million women worldwide are HSV-2 seropositive, including roughly 20% of reproductive-aged American women. HSV-2 is a neurotropic virus that establishes a persistent, life-long infection that increases risk for STI acquisition in individuals. The vaginal epithelium represents a critical first line of defense against infection, and during acute infection, underlying immune mechanisms in the epithelium may be critical to protect against disease pathogenesis. The recently identified pro-inflammatory cytokine IL-36gamma has been shown to be expressed at mucosal epithelia, including the female reproductive tract (FRT) and may be an important factor in host defense. Although IL-36gamma has been shown to be induced in the FRT after exposure to microbial products, the contributions of IL-36gamma to host defense mechanisms in response to this clinically relevant STI pathogen are not well understood. This dissertation describes the regulation of IL-36gamma in the FRT and explores its contribution to the host response against genital HSV-2 infection. To test the hypothesis that IL-36gamma is a key regulator of mucosal inflammation and immunity in the FRT, hormonal regulation of IL-36gamma in the FRT was investigated using estrogen- and progesterone-conditioned mice. From this preliminary study, it was shown that progesterone dampens IL36G expression relative to estrogen and may potentially increase susceptibility to infection. Next, the impact of IL-36gamma treatment on HSV-2 infection and replication in human 3-D vaginal epithelial cells was explored. In parallel, the impact of intravaginal IL-36gamma delivery on HSV-2 disease pathogenesis was evaluated using a lethal murine challenge model. IL-36gamma pre-treatment significantly limited HSV-2 replication in vitro and in vivo and was associated with transient neutrophil infiltration that corresponded with decreased disease severity and increased survival in mice. Last, the requirement for IL-36gamma in host defense was investigated utilizing IL-36gamma-/- mice in a lethal HSV-2 murine challenge model. Following infection, IL-36gamma-/- mice exhibited significantly impaired neutrophil recruitment, decreased overall survival time, and significantly increased viral neuroinvasion relative to wild type mice. Collectively, these data indicate that IL-36gamma is a crucial regulator of HSV-2-induced neutrophil infiltration and appears to function in a previously uncharacterized manner to limit viral neuroinvasion in genital HSV-2 disease pathogenesis. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2019

Psoriasis is an immune mediated inflammatory disease which affects 2-3% of the world’s population. Over the last decade, psoriasis has been acknowledged as an independent risk factor for atherosclerosis. The precise mechanism or mechanisms of the heightened risk is widely speculated. Endothelial cells and macrophages are central players in the immunopathological development of both diseases. Interleukin-36 cytokines (IL-36) have been heavily implicated in psoriasis immunopathology. Significant upregulation of epidermal IL-36 is a recognised characteristic of psoriatic skin inflammation. IL-36 induces inflammatory responses in dendritic cells, fibroblasts and epithelial cells. While vascular alterations are a hallmark of psoriatic lesions and dermal endothelial cells are well known to play a critical role in dermal inflammation, the effects of IL-36 on endothelial cells have not been defined. We report that endothelial cells including dermal microvascular cells express a functionally active IL-36 receptor. Adhesion molecules VCAM-1 and ICAM-1 are upregulated following IL-36γ stimulation, and this is reversed in the presence of the endogenous IL-36 receptor antagonist. IL-36γ-stimulated endothelial cells secrete the proinflammatory chemokines IL-8, CCL2 and CCL20. Chemotaxis assays showed increased migration of T-cells following IL-36γ stimulation of endothelial cells. Both resident and infiltrating inflammatory myeloid cells contribute to the immunopathology of psoriasis by promoting the IL-23/IL-17 axis. We show that IL-36γ induces the production of psoriasis-associated cytokines from macrophages (IL-23, TNFα) and that this response is enhanced in macrophages from psoriasis patients. This effect is specific for IL-36γ and could not be mimicked by other IL-1 family cytokines such as IL-1α. Furthermore, IL-36γ stimulated macrophages potently activated endothelial cells as illustrated by ICAM-1(CD54) upregulation, and led to increased adherence of monocytes, effects that were markedly more pronounced for psoriatic macrophages. Interestingly, regardless of stimulus, monocytes isolated from psoriasis patients showed increased adherence to both the stimulated and unstimulated endothelium when compared to monocytes from healthy individuals. Collectively, these findings add to the growing evidence for IL-36γ having roles in psoriatic responses, by enhancing endothelium directed leukocyte infiltration into the skin and strengthening the IL-23/IL-17 pathway. Our findings also point to a cellular response which could potentially support cardiovascular comorbidities in psoriasis. / University of Bradford and the Centre for Skin Sciences