Rev. diss. Univ. of Hawaii.

Inflammation plays a key role during pathological hypertrophy and heart failure. Whilst the roles of pro-inflammatory cytokines are relatively well understood, little is known about the anti-inflammatory cytokines in the heart. Interleukin-10 (IL-10) is a major anti-inflammatory cytokine that is expressed in the heart and may play a crucial role during cardiac remodelling. IL-10 exerts its function by binding to the IL-10 receptor (IL-10R). The primary aim of the PhD study was to investigate the effects of the ubiquitous ablation of IL-10R1 gene during pressure overload induced hypertrophy and to characterise the downstream pathway regulated by IL-10R1 in the heart following pressure overload. The second aim was to investigate the effects of cell specific ablation of IL-10R1 in both the macrophages and cardiomyocytes during pressure overload induced hypertrophy and to identify the specific site where IL-10R1 regulates hypertrophy in the heart. During this study three mouse lines were used: IL-10R1 global knockout (IL-10R1-/-), IL-10R1 macrophage-specific knockout (IL-10R1mKO) and IL-10R1 cardiomyocyte-specific knockout (IL-10R1cKO).Mice with systemic ablation of IL-10 receptor1 (IL-10R1-/-) displayed a significant increase in hypertrophy following two weeks of transverse aortic constriction (TAC) as indicated by heart weight/tibia length ratio (HW/TL). This was accompanied by a significant increase in cardiomyocyte surface area as well as expression of hypertrophic markers such as brain natriuretic peptide (BNP) and Atrial natriuretic peptide (ANP). The IL-10R1-/- mice also had a significant increase in cardiac fibrosis when compared to the WT TAC littermates. Importantly, ejection fraction (EF) and fractional shortening (FS) were significantly reduced in IL-10R1-/- mice compared with WT littermates following TAC. The STAT3 pathway is known as the major downstream signalling pathway regulated by the IL-10R via the activation of the JAK1/STAT3 pathway.  Western blot analysis showed that activation of the STAT3 signalling pathway was significantly reduced in IL-10R1-/- mice following TAC, indicating the possible involvement of this pathway. Furthermore, expression of STAT3 target genes: suppressor of cytokine signalling (SOCS3), tissue inhibitor of metalloproteinases3 (TIMP-3) and heme oxygenase (HO-1) were downregulated in the IL-10R1-/- mice following TAC. Overall the data obtain from the IL-10R1-/- mice indicate that IL-10R1 signalling plays a protective role in reducing pathological hypertrophy in the heart. Interestingly, IL-10R1mKO mice showed no difference in the hypertrophic response following TAC. Analysis of cardiac function and STAT3 activation also showed no difference between IL-10R1mKO and WT controls. This indicated that the protective effects of IL-10R1 mediated signalling during cardiac pressure overload was unlikely due to the effects in residential macrophages. In contrast, IL-10R1cKO mice displayed an elevated hypertrophic response, reduction of cardiac function and less STAT3 activation after TAC. This phenotype resembled those of IL-10R1 global knockout mice. In conclusion, this PhD study has shown that IL-10R1 mediated signalling in the heart is important in controlling pressure-overload hypertrophy. Using cell-specific knockout mice I have shown that IL-10R signalling in cardiomyocytes and not in macrophages is important in this process. These results will open a new insight in targeting IL-10 receptor in the treatment of myocardial hypertrophy in future.

L’endométriose est une pathologie chronique, bénigne, caractérisée par la présence de tissu endométrial (glande et stroma) en dehors de l’utérus. La forte prévalence de cette maladie, sa symptomatologie invalidante et son coût annuel considérable en font un véritable enjeu de santé publique. Le traitement de l’endométriose comprend un volet chirurgical, potentiellement délabrant, et un volet médical, basé sur des produits anti-gonadotropes en première intention. L’endométriose est d’origine multifactorielle. Sa physiopathologie demeure mal connue. Alors que le reflux menstruel via les trompes semble être le primum movens, plusieurs étapes sont nécessaires à la formation des lésions d’endométriose : l’adhésion et l’implantation des cellules endométriales au mésothélium péritonéal, la prolifération cellulaire soutenue par un phénomène d’angiogenèse et enfin, la réaction inflammatoire. Ces étapes sont dépendantes de facteurs génétiques, immunologiques et environnementaux. L’inflammation joue un rôle clef dans la pathogénie de l’endométriose. Nous avons montré dans un premier temps, à l’aide d’une double approche de PCR quantitative et d’immunohistochimie, la surexpression du récepteur de la LH (LHCGR) dans les tissus endometriosiques ectopiques. A l’aide d’un modèle in vitro, nous avons démontré que la stimulation du LHCGR par l’hCG activait les MAPK (avec une augmentation du rapport pERK/ERK), exerçait une effet prolifératif et enfin, induisait la surexpression de nombreux gènes cibles : CYP19A1, NR5A1, INSL3, VEGFA et PTGS2. Ensuite, nous avons étudié les principaux acteurs de la voie des prostaglandines. Nous avons montré une nette perturbation de cette voie en faveur d’une augmentation de l’inflammation avec une surexpression de la PTGS2 ainsi que des récepteurs des prostaglandines PTGER2, 3 et 4 dans l’endomètre des patientes endométriosiques. Nous avons ensuite étudié la voie des sphingosines en analysant l’expression de ses acteurs clef (SPHK1-2, SGPP1-2, SGPL1, SPHAKAP, S1PR1-5). Nous avons mis en évidence pour la première fois l’existence d’une profonde dérégulation de l’expression des enzymes et des récepteurs de cette voie en faveur d’une diminution du catabolisme du Sphingosine-1phosphate. Cette perturbation est à l’origine de la réaction inflammatoire qui participe à l’entretien de la prolifération et de la croissance des cellules endométriosiques. Dans un deuxième temps, nous avons exploré le retentissement systémique de l’endométriose à l’aide du modèle d’une interleukine pro-inflammatoire et fibrosante, l’IL-33, et de deux interleukines anti-inflammatoires l’IL-19 et l’IL-22. L’IL-33 est significativement plus élevée dans le sérum des patientes endométriosiques en particulier en cas d’endométriose profonde. Nous avons également mis en évidence l’existence d’une corrélation significative avec le nombre et la sévérité des lésions profondes. En opposition, les interleukines anti-inflammatoires IL-19 et IL-22 sont significativement diminuées dans le sérum des patientes endométriosiques. En conclusion, nous avons montré l’existence d’une perturbation des voies inflammatoires : la voie de la PTGS2 et de celle des sphingosines ainsi que le rôle pro-inflammatoire du LCGHR. Nous avons également mis en évidence le déséquilibre de la balance des cytokines systémiques inflammatoires et anti-inflammatoires dans l’endométriose. Ainsi, ces médiateurs de l’inflammation pourraient être considérés comme de potentiels marqueurs évolutifs de l’endométriose. Leur utilisation pourrait permettre d’effectuer un diagnostic plus précoce, et d’envisager de nouvelles thérapeutiques ciblées. / Pas de résumé en anglais

IL-12, an immunoregulatory cytokine, plays a key role in the development of cell-mediated immune responses. However, very little is known about the regulation and induction of the other members of this family, particularly IL-23 and IL-27. The regulation of these cytokines was studied in the human primary monocytes and monocyte-derived macrophages (MDMs) as they play a key role in innate and adaptive immune responses. THP-1 promonocytic cells were employed as a model system to confirm the results obtained with monocytes and MDMs. Two stimuli IFN-γ and LPS were used as both are strong inducers of IL-12 family cytokines. My results show that IFN-γ induced the production of IL-12/23p40 and IL-23p19 mRNA as well as IL-12p40 and IL-23 proteins in primary human monocytes isolated by positive selection. IFN-γ-induced IL-23 and IL-12/23p40 expression was positively regulated by the p38 mitogen-activated protein kinases (MAPK), independent of the Janus kinase (Jak)/signal transducers and activators of transcription (STAT) signaling. In contrast, IL-12 and IL-23 were negatively regulated by the Jak/STAT, phosphoinositide-3 kinase (PI3K) and the c-Jun-N-terminal kinase (JNK) MAPKs in IFN-γ-stimulated monocytes. LPS significantly stimulated IL-23p19 and IL-12/23p40 mRNA expression as well as IL-12/23p40 and IL-23 protein production in THP-1 cells, while IFN-γ stimulation alone did not affect IL-23 mRNA or protein levels. THP-1 cells were pre-treated with ERK, JNK or p38 MAPK inhibitors and then stimulated with LPS. LPS-induced IL-12p40 and IL-23 proteins were positively regulated by the p38 and JNK MAPKs and PI3K, whereas LPS-induced IL-23p19 mRNA expression was negatively regulated by these kinases. These results were confirmed using siRNA in LPS-stimulated THP-1 cells. My results also show that IFN-γ/LPS-induced IL-23 expression is not regulated through MAPK or PI3K signaling pathways in human MDMs. My results also show for the first time that IFN-γ alone without any second stimulus induced IL-27p28 gene expression and IL-27 protein production in human monocytic cells. I investigated the signalling pathways governing the regulation of IL-27 protein and its subunit IL-27p28 following stimulation with IFN-γ in primary human monocytic cells. IFN-γ-mediated IL-27 protein, but not IL-27p28 gene expression was positively regulated by JNK MAPK and PI3K, independent of JAK/STAT signaling in primary human monocytes. I also investigated the signalling pathways governing the regulation of IL-27 and its α subunit, IL-27p28 following stimulation with IFN-γ alone or IFN-γ-primed LPS-stimulated macrophages (IFN-γ/LPS) and THP-1 cells. A differential regulation of IL-27p28 and IL-27 in response to stimulation by either IFN-γ or IFN-γ/LPS was observed. IFN-γ- and IFN-γ/LPS induced IL-27 expression was positively regulated by the JNK, p38 MAPK and PI3K, independent of Jak/STAT signaling in human MDMs and THP-1 cells. Taken together, my results show that IL-23 induction is differentially regulated by different pathways in response to different stimuli, whereas IL-27 expression is regulated by JNK, p38 MAPK and PI3K regardless in the stimulus in human myeloid cells. These results may provide additional strategies aimed at targeting disease, autoimmune disorders and cancer.

Introduktion: Psoriasis är en kronisk inflammatorisk systemsjukdom som drabbar främst hud och leder.  På grund av mycket snabbt tillväxt av hudceller ger psoriasis utslag på huden.  En av de vanligaste typerna av psoriasis är plack-psoriasis som kännetecknas av större än 0,5 cm i diameter runda fjällande hudutslag som kliar. Mekanismen som ligger bakom plack-psoriasis medieras med hjälp av T-celler och dendritiska celler som är en viktig del av immunsystemet. Stimulering av dessa immunceller leder till frisättning av följande proinflammatoriska cytokiner såsom IL-17, IFN-γ, TNF samt IL-22. Dessa cytokiner utövar sin effekt på keratinocyter genom att öka psoriatisk inflammation. Flera biologiska läkemedel utvecklas nu för behandling av plack-psoriasis. I detta arbete har fokus lagts på två av dem, sekukinumab som är en IL-17A hämmare och ustekinumab, som är en IL-12 och IL-23 hämmare. Syfte: Syftet med arbetet är att undersöka effektivitet av sekukinumab vid behandling av plack-psoriasis. Vidare undersöks även hur behandling av plack-psoriasis med sekukinumab skiljer sig från behandling med ustekinumab gällande minskning av plack-psoriasis. Metod: Detta arbete är en litteraturstudie som inkluderar fem vetenskapliga artiklar som baserades på randomiserade kontrollerade studier som sponsrades av läkemedelsföretag.  Resultat: Resultatet av de fem studier visar att behandling med hjälp av sekukinumab 300 mg och 150 mg var mer effektiv än behandling med ustekinumab 45/90 mg, enligt PASI 75, PASI 90 och PASI 100. Diskussion: Alla fem studierna var dubbel-blinda randomiserade kontrollerade studier vilket leder till säkra och tillförlitliga resultat. Däremot var samtliga studier sponsrade av läkemedelsföretag vilket kan leda till att positiv effekt beskrivs i större utsträckning för att kunna sälja sin produkt.  Slutsats: Sekukinumab 300 mg och 150 mg är signifikant mer effektiv än placebo och signifikant mer effektiv än ustekinumab 45/90 mg vid behandling av medelsvår och svår plack-psoriasis. / Introduction: Psoriasis is a chronic inflammatory systemic disease that mainly affects the skin and joints. Due to the very rapid growth of skin cells, psoriasis causes rashes on the skin. One of the most common types of psoriasis is plaque psoriasis which is characterized by larger than 0.5 cm in diameter round scaly skin rash that itches. The mechanism behind plaque psoriasis is mediated by T cells and dendritic cells, which are an important part of the immune system. Stimulation of these immune cells leads to the release of the following proinflammatory cytokines such as IL-17, IFN-γ, TNF and IL-22. These cytokines exert their effect on keratinocytes by increasing psoriatic inflammation.  Several biological drugs are now being developed for the treatment of plaque psoriasis. In this study, the focus has been on two of them, secukinumab which is an IL-17A inhibitor and ustekinumab, which is an IL-12 and IL-23 inhibitor. Purpose: The purpose of this study is to investigate the efficacy of secukinumab in the treatment of plaque psoriasis. Furthermore, it is also investigated if the treatment of plaque psoriasis with secukinumab differs from treatment with ustekinumab regarding reduction of plaque psoriasis. Method: This work is a literature study that includes five scientific articles based on randomized controlled trials sponsored by pharmaceutical companies.  Results: The results of the five studies show that treatment with secukinumab 300 mg and 150 mg was more effective than treatment with ustekinumab 45/90 mg, according to PASI 75, PASI 90 and PASI 100. Discussion: All five studies were double-blind randomized controlled trials leading to safe and reliable results. On the other hand, all studies were sponsored by pharmaceutical companies, which may lead to a positive effect being described to a greater extent in order to be able to sell their product. Conclusion: Secukinumab 300 mg and 150 mg are significantly more effective than placebo and significantly more effective than ustekinumab 45/90 mg in the treatment of moderate to severe plaque psoriasis.

Hepatitis B virus (HBV) vaccination is recommended for individuals with hepatitis C virus (HCV) infection given their shared risk factors and increased liver-related morbidity and mortality upon super-infection. Vaccine responses in this setting are often blunted, with poor response rates to HBV vaccinations in chronically HCV-infected individuals compared to healthy subjects. In this study, we investigated the role of T cell immunoglobulin mucin domain-3 (Tim-3)-mediated immune regulation in HBV vaccine responses during HCV infection. We found that Tim-3, a marker for T cell exhaustion, was over-expressed on monocytes, leading to a differential regulation of IL-12/IL-23 production which in turn TH17 cell accumulation, in HCV-infected HBV vaccine non-responders compared to HCV-infected HBV vaccine responders or healthy subjects (HS). Importantly, ex vivo blockade of Tim-3 signaling corrected the imbalance of IL-12/IL-23 as well as the IL-17 bias observed in HBV vaccine non-responders during HCV infection. These results suggest that Tim-3-mediated dysregulation of innate to adaptive immune responses is involved in HBV vaccine failure in individuals with chronic HCV infection, raising the possibility that blocking this negative signaling pathway might improve the success rate of HBV immunization in the setting of chronic viral infection.

Hepatitis B virus (HBV) vaccination is recommended for individuals with hepatitis C virus (HCV) infection given their shared risk factors and increased liver-related morbidity and mortality upon super-infection. Vaccine responses in this setting are often blunted, with poor response rates to HBV vaccinations in chronically HCV-infected individuals compared to healthy subjects. In this study, we investigated the role of T cell immunoglobulin mucin domain-3 (Tim-3)-mediated immune regulation in HBV vaccine responses during HCV infection. We found that Tim-3, a marker for T cell exhaustion, was over-expressed on monocytes, leading to a differential regulation of IL-12/IL-23 production which in turn TH17 cell accumulation, in HCV-infected HBV vaccine non-responders compared to HCV-infected HBV vaccine responders or healthy subjects (HS). Importantly, ex vivo blockade of Tim-3 signaling corrected the imbalance of IL-12/IL-23 as well as the IL-17 bias observed in HBV vaccine non-responders during HCV infection. These results suggest that Tim-3-mediated dysregulation of innate to adaptive immune responses is involved in HBV vaccine failure in individuals with chronic HCV infection, raising the possibility that blocking this negative signaling pathway might improve the success rate of HBV immunization in the setting of chronic viral infection.

LPS pretreatment of human pro-monocytic THP-1 cells induces tolerance to secondary LPS stimulation with reduced TNFα production. However, secondary stimulation with heat-killed Staphylococcus aureus (HKSa) induces priming as evidenced by augmented TNFα production. The pro-inflammatory cytokine, IFNγ, also abolishes suppression of TNFα in LPS tolerance. The effect of LPS tolerance on HKSa and IFNγ-induced inflammatory mediator production is not well defined. We hypothesized that LPS, HKSa and IFNγ differentially regulate pro-inflammatory mediators and chemokine production in LPS-induced tolerance. THP-1 cells were pretreated for 24h with LPS (100ng/ml) or LPS (100ng/ml)+IFNγ (1μg/ml). Cells were subsequently stimulated with LPS or HKSa (10μg/ml) for 24h. The production of the cytokines TNFα, IL-6, IL-1β, and GMCSF and the chemokine IL-8 were measured in supernatants. LPS and HKSa stimulated TNFα (3070±711pg/ml and 217±9pg/ml, respectively) and IL-6 (237±8.9pg/ml and 56.2±2.9pg/ml, p<0.05, n=3, respectively) in control cells compared to basal levels (<25pg/ml). LPS induced tolerance to secondary LPS stimulation as evidenced by a 90% (p<0.05, n=3) reduction in TNFα. However, LPS pretreatment induced priming to HKSa as demonstrated by increased TNFα (2.7 fold, from 217 to 580pg/ml, p<0.05, n=3). In contrast to suppressed TNFα, IL-6 production was augmented to secondary LPS stimulation (9 fold, from 237 to 2076pg/ml, p<0.01, n=3) and also primed to HKSa stimulation (62 fold, from 56 to 3470pg/ml, p<0.01, n=3). LPS induced IL-8 production and to a lesser extent IL-1β and GMCSF. LPS pretreatment did not affect secondary LPS stimulated IL-8 or IL-1β, although HKSa stimulation augmented both mediators. In addition, IFNγ pretreatment reversed LPS tolerance as evidenced by increased TNFα levels while IL-6, IL-1β, and GMCSF levels were further augmented. However, IL-8 production was not affected by IFNγ. These data support our hypothesis of differential regulation of cytokines and chemokines in gram-negative- and gram-positive-induced inflammatory events. Such changes may have implications in the pathogenesis of polymicrobial sepsis.

Rodents are a natural host for the dimorphic pathogenic fungi Coccidioides immitis and Coccidioides posadasii, and mice are a good model for human infection. Humans and rodents both express Dectin-1 and Toll-like receptor 2 (TLR2) on myeloid cells, and those receptors collaborate to maximize the cytokine/chemokine responses to spherules (the tissue form of the fungi) and to formalin-killed spherules (FKS). We showed that Dectin-1 is necessary for resistance to pulmonary coccidioidomycosis, but the importance of TLR2 in vivo is uncertain. Myeloid differentiation factor 88 (MyD88) is the adapter protein for TLR2 and -4, interleukin-1R1 (IL-1R1), and IL-18R1. MyD88/TRIF -/- and MyD88 -/- mice were equally susceptible to C. immitis infection, in contrast to C57BL/6 (B6) controls. Of the four surface receptors, only IL-1R1 was required for resistance to C. immitis, partially explaining the susceptibility of MyD88 -/- mice. We also found that FKS stimulated production of IL-1Ra by bone marrow-derived dendritic cells (BMDCs), independent of MyD88 and Dectin-1. There also was a very high concentration of IL-1Ra in the lungs of infected B6 mice, supporting the potential importance of this regulatory IL-1 family protein in the largely ineffective response of B6 mice to coccidioidomycosis. These results suggest that IL-1R1 signaling is important for defense against C. immitis infection.

Cancer remains an elusive killer due, in part, to the suppression of normal immunologic antitumor responses. Normal host (NH) macrophage (Mϕ) populations have tumoricidal effects such as tumor antigen phagocytosis and presentation, and cytokine production. Tumor-infiltrating Mϕs may evade these activities by dysregulating production of immunostimulatory cytokines (including Interleukin [IL]-1β, IL-18, and tumor necrosis factor-α [TNF-α]), by production of antagonistic factors. The restoration of IL-1β, IL-18, and TNF-α production by Mϕs could re-establish antitumor host immune responses. Previous work in our laboratory suggests that tumor distal (TD) Mϕs produce more IL-1β than NH Mϕs when stimulated with IFN-γ and lipopolysaccharide (LPS). We hypothesize that the presence of immunomodulatory factors like IL-10 and TGF-β dysregulate IL-1β production in tumor proximal (TP) Mϕs. Indeed, IL-1β production was downregulated among in situ TP Mϕs. We have proposed that IL-18, a structural homologue to IL-1β was similarly dysregulated in TD and TP Mϕs. IL-18 was enhanced in both distal and proximal Mϕs. Differences in the functions of these cytokines could account for this dissimilarity. TNF-α, another proinflammatory cytokine, followed the dysregulation pattern of IL-1β in our tumor-burdened hosts (TBH), likely because of the similar functions of these cytokines. Because it is a potential vehicle for immunotherapeutic treatment, paclitaxel's action on the immune response (TAXOL™) was investigated. Paclitaxel is a potent Mϕ activator that upregulates a variety of cytokines in an LPS-like manner. Paclitaxel enhanced TD Mϕ production of IL-1β, IL-18, and TNF-α in an LPS-like manner. Production of IL-1β and TNF-α was reduced in TP Mϕs when treated with paclitaxel; however, IL-18 production was enhanced. This difference could be due to the different functions of IL-1β and IL-18. To determine whether production of these cytokines translates into downstream expression of transcription products, IL-12 and nitric oxide (NO) were assayed. NO was enhanced distally, but paclitaxel treatment failed to enhance NO production. When treated with paclitaxel, IL-12 was produced by NH and TD Mϕs. Collectively, these studies suggest that tumor-induced cytokine imbalances compromise antitumor immunity and paclitaxel may reverse this activity. / Master of Science