Initial studies in this work explored the role of interleukin-6 (IL6) and leukemia inhibitory factor (LIF) in preimplantation bovine embryos. Neither cytokine affected the total percentage of embryos which developed to the blastocyst stage in vitro. However, supplementation of IL6 increased blastocyst inner cell mass (ICM) cell number without affecting trophectoderm (TE) cell number. Additionally, we found that IL6 activated signal transducer and activator of transcription 3 (STAT3) specifically within ICM cells. LIF, however, did not affect ICM cell number or activate STAT3 in ICM cells, and was not pursued further. This increase in ICM cell number by IL6 was largely comprised of hypoblast (GATA6+:NANOG-) cells, and most IL6-responsive cells in day 9 blastocysts were hypoblast cells (as measured by STAT3 activation). However, some epiblast (NANOG+) cells were also IL6-responsive, and IL6 appeared to initially slow epiblast differentiation. Finally, IL6-treated blastocysts also had increased transcripts of hypoblast/primitive endoderm (PE) markers. These results indicate that IL6 may improve pregnancy retention of IVP embryos by improving yolk sac development, but further work is needed to confirm this theory. Activation of STAT3 by IL6 could be blocked with a chemical Janus kinase 2 (JAK2) inhibitor (AZD1480). JAK2 inhibition from day 5 to 8 resulted in blastocyst ICMs with fewer than 10% the normal cell number, regardless of IL6 supplementation. This indicates that STAT3 is critical for bovine ICM development. Further analysis revealed that inhibition of JAK2/STAT did not prevent ICM formation but disrupted its maintenance. Additionally, we assessed the suitability of zinc sulfate and a bovine embryonic stem cell culture media (TeSR) for improving bovine embryo development in vitro. Zinc sulfate increased day 8 blastocyst total and ICM cell number. Therefore, zinc sulfate appears to improve blastocyst quality. The TeSR medium improved embryo development beyond day 8. In normal synthetic oviduct fluid, blastocysts degenerated after day 8, while blastocysts moved to TeSR had greatly increased cell numbers, and even exhibited PE migration out from the ICM, a phenomenon that has not been reported in vitro. This indicates that extended blastocyst culture is possible with TeSR media. / Doctor of Philosophy / Bovine embryos have been produced in vitro for the purpose of being transferred to recipient cattle to produce a calf since the 1980s. This practice allows cattle breeders to increase the number of offspring from their best females each year, and also allows for more rapid progress in generational genetic improvement. However, only approximately 10% of bovine oocytes survive and produce a calf. This poor efficiency of bovine in vitro embryo production negatively impacts the procedure's widespread use. A significant portion of these embryo losses are likely a result of inadequate in vitro culture conditions, particularly of the embryo culture media, the fluid in which embryos are grown. This media is often called "synthetic oviduct fluid," or SOF, because it is designed to mimic the fluid present in the cow's oviduct, where the embryo would normally reside. However, SOF is much simpler in nature than actual cow oviduct fluid, and this leads to reduced embryonic survival of in vitro produced embryos. Unfortunately, we know very little of what molecules control and promote bovine embryo development. Therefore, one major goal of bovine embryo research is to identify these factors and add them to SOF. The goal of this work was to examine the ability of three molecules, interleukin-6 (IL6), leukemia inhibitory factor (LIF), and zinc sulfate, to increase the number and quality of blastocysts produced through in vitro culture techniques. Additionally, I tested the replacement of SOF with a complex cell culture media, known as TeSR. This medium is more complex than SOF, and therefore should better promote embryo development. This work revealed that IL6, but not LIF, improves in vitro produced (IVP) bovine blastocyst quality. Unfortunately, neither IL6 nor LIF affected the percentage of embryos which survived to the blastocyst stage. However, IL6, but not LIF, increased the number of cells in the inner cell mass (ICM) of the blastocysts. ICM cells are the portion of the embryo which will produce the future calf. IVP bovine embryos are known to have fewer cells than normal, in vivo derived, blastocysts, and this issue is believed to cause some embryonic death after embryo transfer. Therefore, treatment with IL6 may increase the percentage of embryos which will survive after transfer and produce a calf. We also found the addition of zinc sulfate to SOF to benefit embryo quality. None of the concentrations of zinc significantly improved the percentage of embryos which survived to the blastocyst stage, but 2 µM zinc did increase ICM cell number. Like IL6, this may improve embryo survival after transfer. The use of the TeSR media as a replacement for SOF had some benefits. Unfortunately, this media is unusable for producing embryos for transfer to recipients, as we discovered early embryos could not survive in the media. However, blastocyst-stage embryos thrived in it, and could be cultured in vitro for a longer period of time as a result. Therefore, this media will be a useful tool for studying bovine embryo development in vitro, however it is unlikely to benefit calf production. In summary, this work provides evidence that zinc sulfate and IL6 are beneficial additions to SOF. However, future work is needed to determine if embryos produced with these factors are more able to produce a calf. Additionally, we discovered that TeSR is a superior extended blastocyst culture medium.

Genotype-phenotype analyses continue to be the primary goal for genome analyses in livestock and poultry breeding. Essential to accomplish this goal is the need to identify variation at the genomic level. To test the hypothesis that DNA sequence variations in inflammatory response genes are associated with phenotypic differences in the heritage turkey, the primary objective of this project was to search for single nucleotide polymorphisms (SNPs) in candidate inflammatory response genes. A minor objective was to develop a system for inducing inflammatory response in the turkey using a microbe-based lipopolysaccharide (LPS), an approach previously described for the chicken. A total of 18 SNPs was identified in the three genes screened in this project: Interleukin 6 (IL6) and 8 (IL8), and NLRX1. Mortality data from the LPS challenge were not significantly different among the strains. Further gene expression analyses will be part of future work. The SNP data represent the first extensive analyses of candidate inflammatory response genes in the turkey. Combined with the protocols developed for inflammation assessment in the turkey the SNPs described here will be valuable resources for future inflammation:genotype evaluation in the turkey / MS / Though progress has been made in the genome analyses of the turkey, Meleagris gallopavo, our understanding of the genotype: phenotype relationships continue to lag those of agriculturally important animal species. Among the phenotypes for which genetic understanding can be useful is inflammation, a complex trait that is influence by many interdependent response mechanisms. These mechanisms, regarding differences across heritage turkeys, has been mildly investigated. Since Nucleotide Polymorphism (SNP) screening is a common method used to comprehend the robust effects these differences have on genotype and phenotype. Here, we report initial investigations in our lab of the genetics of inflammation in the turkey using comparative information from the chicken NOD like receptor X1 (NLRX1), turkey interleukin 6 (IL6), and Interleukin 8 (IL8). These genes were screened for nucleotide variants that may be informative for future studies that will investigate the turkey’s response to Salmonella derived lipopolysaccharide that can induce inflammation. The rationale for selecting these three genes is that IL8, IL6, and NLRX1 have pro inflammatory and/or anti-inflammatory functions that respond to maintain homeostasis. Primers were designed and investigated using DNA from Broad Breasted White (BBW), and Broad Breasted Bronze (BBB), Blue slate (SL), Spanish Black (SBL), Midget White (MW) and Royal Palm (RP). The birds were also challenged with 1.5 mg/kg Lipopolysaccharide (LPS) intra-abdominally to collect tissue post LPS challenge. Tissues was collected from the thymus, spleen, and bursa of fabricius: organs identified to effect inflammation. A total of 2,239 bp for IL8, 2,439 bp for IL 6, and 572 bp for NLRX1 were screened for SNPs. SNP analysis revealed 16 SNPs in the inflammatory response genes mentioned.

Tsoi Lo Ming. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 99-109). / Abstracts in English and Chinese. / Acknowledgements --- p.I / Abstract --- p.II / Abstract in Chinese --- p.III / List of Abbreviations --- p.IV / List of Figures --- p.V / List of Tables --- p.VI / Table of Contents --- p.VII / Chapter Chapter 1 : --- Introduction / Chapter 1.1. --- Apolipoprotein and Lipoprotein Metabolism --- p.1 / Chapter 1.2. --- Molecular Information of ApoE --- p.2 / Chapter 1.3. --- Tissue Distribution of ApoE --- p.2 / Chapter 1.4. --- Functions of ApoE --- p.4 / Chapter 1.5. --- Genetic Polymorphism of ApoE --- p.7 / Chapter 1.6. --- Protein Structure and Characteristics of ApoE Isoforms --- p.9 / Chapter 1.7. --- Plasma and Cellular Expression Level of ApoE Isoforms --- p.12 / Chapter 1.8. --- Association between ApoE Isoforms and Plasma Lipid Profiles --- p.13 / Chapter 1.9. --- ApoE Polymorphisms and Pathophysiological Conditions / Chapter 1.9.1. --- Type III Hyperlipoproteinemia (Type III HLP) --- p.14 / Chapter 1.9.2. --- Alzheimer's Disease --- p.15 / Chapter 1.9.3. --- Atherosclerosis / Chapter 1.9.3.1. --- Atherosclerosis - An Inflammatory Process --- p.15 / Chapter 1.9.3.2. --- Role of ApoE in Atherosclerosis --- p.18 / Chapter (a) --- Functions Associated to Lipid Metabolism --- p.19 / Chapter (b) --- Functions Independent to Lipid Metabolism --- p.20 / Chapter 1.9.3.3. --- TNF-α and IL-6 in Atherosclerosis --- p.25 / Chapter 1.10. --- Macrophage Cytokine Expression and MAPKs / Chapter 1.10.1. --- Organization of MAPKs Signaling Pathway --- p.26 / Chapter 1.10.2. --- Lipopolysaccharide and MAPKs in Macrophage Cytokine Expression --- p.28 / Chapter 1.10.3. --- Regulation of Macrophage Cytokine Expression / Chapter 1.10.3.1. --- ERK1/2 and p38 MAPK Pathway --- p.30 / Chapter 1.10.3.2. --- Arachidonic Acid Metabolism --- p.30 / Chapter 1.11. --- Aim and Hypothesis --- p.31 / Chapter Chapter 2 : --- Materials and Methods / Materials / Chapter 2.1 --- Culture of ApoE-isoform-expressing J774A.1 Macrophage Cell Line --- p.32 / Chapter 2.2 --- RNA Extraction and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.33 / Chapter 2.3 --- Protein Extraction and Quantification --- p.37 / Chapter 2.4 --- Enzyme-linked Immunosorbent Assay (ELISA) --- p.38 / Chapter 2.5 --- Western Blotting --- p.39 / Chapter 2.6 --- LPS Treatment --- p.42 / Chapter 2.7 --- MAPK Inhibitor Experiment --- p.43 / Methods / Chapter 2.8 --- Study on the Effect of Endogenously Expressed ApoE Isoforms on Macrophage Cytokine Secretion / Chapter 2.8.1. --- Establishment of ApoE-isoform-expressing Macrophages --- p.44 / Chapter 2.8.2. --- Semi-quantification of ApoE mRNA Level by RT-PCR / Chapter 1) --- Isolation of Total RNA --- p.45 / Chapter 2) --- RT-PCR --- p.46 / Chapter 2.8.3. --- Determination of ApoE Protein Expression Level by ELISA and Western Blot --- p.47 / Chapter 1) --- Quantification of Total Proteins --- p.48 / Chapter 2) --- ELISA --- p.48 / Chapter 3) --- Western Blot --- p.49 / Chapter 2.8.4. --- LPS Treatment --- p.51 / Chapter 2.8.5. --- MEK1/2 Inhibitor Experiment --- p.53 / Chapter 2.8.6. --- p38 Inhibitor Experiment --- p.54 / Chapter 2.9 --- Study on the Effect of Exogenous ApoE Isoform on Macrophage Cytokine Secretion --- p.55 / Chapter 2.10 --- Statistical Analysis --- p.55 / Chapter Chapter 3: --- Results / Changes of Inflammatory Properties Associated with Endogenous ApoE Isoform Expression in Macrophages / Chapter 3.1 --- Characterization of ApoE-isoform-expressing Macrophages --- p.56 / Chapter 3.1.1. --- Cell Lines with Stable Expression of ApoE Isoforms --- p.56 / Chapter 3.2 --- Cell Morphology Study --- p.58 / Chapter 3.3 --- Changes of IL-6 and TNF-α Secretion Associated with Endogenous ApoE Isoforms Expression / Chapter 3.3.1. --- In the Presence of Lipoproteins --- p.60 / Chapter 3.3.2. --- Serum/Lipoprotein-independent Effects of ApoE Isoforms --- p.63 / Chapter 3.4 --- The Effects of Endogenous ApoE Isoform Expression on the Activities of MAPK Signaling Pathways / Chapter 3.4.1. --- Study on the Activation Status and Expression of MAPKs --- p.66 / Chapter 1) --- ERK1/2 MAPK Pathway --- p.66 / Chapter 2) --- p38 MAPK Pathway --- p.69 / Chapter 3.4.2. --- IL-6 and TNF-a Secretion Among ApoE Isoforms in the Presence of MEK1/2 mhibitor --- p.72 / Chapter 3.4.3. --- IL-6 and TNF-α Secretion Among ApoE Isoforms in the Presence of p38 Inhibitor --- p.75 / Chapter Chapter 4 : --- Discussions / Chapter 4.1. --- Mouse Peritoneal Macrophage Cell Line J774A.1 as Cell Model --- p.79 / Chapter 4.2. --- Inflammatory Properties Associated with Endogenous ApoE Isoform Expression in Macrophages / Chapter 4.2.1. --- Expression Level of ApoE Isoform Transgenes in Mouse Peritoneal Macrophages --- p.80 / Chapter 4.2.2. --- Macrophage Activation by LPS --- p.81 / Chapter 4.2.3. --- Effect of Endogenous ApoE Isoform Expression on Cytokine Secretion and Signal Transduction in Macrophages --- p.82 / Chapter 4.3. --- Conclusions and Future Prospects / Chapter 4.3.1. --- Conclusions --- p.90 / Chapter 4.3.2. --- Future Prospects --- p.91 / Chapter Chapter 5 : --- Appendices / Chapter 5.1 --- Changes of Inflammatory Properties of Macrophages Supplemented with Exogenous ApoE Isoforms / Chapter 5.1.1. --- Changes of IL-6 and TNF-a Secretion in Macrophages Supplemented with Exogenous ApoE Isoforms --- p.92 / Chapter 5.1.2. --- Changes of Signal Transduction in Macrophages Supplemented with Exogenous ApoE Isoforms / Chapter 5.1.2.1. --- Study on the Activation Status and Expression of MAPKs / Chapter 1) --- ERK1/2 MAPK Pathway --- p.95 / Chapter 2) --- p38 MAPK Pathway --- p.97 / Chapter Chapter 6: --- Bibliography --- p.99

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Adipöse Patienten und Typ-2-Diabetiker zeigen ein erhöhtes Risiko für Harnwegsinfekte. Die Ursache der höheren Prävalenz ist noch nicht nachhaltig geklärt. Bekannt ist, dass Typ-2-Diabetiker erhöhte Konzentrationen freier Fettsäuren im Blut aufweisen. Der veränderte Fettstoffwechsel könnte neben bakteriellen Ursachen ein möglicher Grund für abakterielle Entzündungsreaktionen der Harnblase sein. Zur Prüfung dieser Hypothese wurden zeit- und konzentrationsabhängig kultivierte humane Detrusormyozyten im Vergleich zur Lipopolysaccharid (LPS) induzierten Entzündungsreaktion mit Palmitat stimuliert. Es wurde geprüft, ob eine autokrine und/oder endokrine Regulation des IL-6-Signalwegs vorliegt. Im Fokus standen insbesondere die IL-6- und MCP-1-Expression und deren möglichen regulatorischen Proteine gp80, gp130, NF-κB, STAT3, SOCS3 und MEK1. Die Stimulationsversuche mit LPS und Palmitat zeigen einen differenten zeit- und konzentrationsabhängigen Effekt auf die IL-6- und MCP-1-Expression in den humanen Detrusormyozyten. LPS und Palmitat induzieren eine zeitabhängige autokrine Regulation der IL-6-Signalkaskade über phosphoryliertes STAT3 und Feedback-mechanismen via SOCS3. Sowohl LPS als auch Palmitat bewirken über 48h eine mögliche endokrine Regulation des IL-6-Signalwegs. Zusammenfassend zeigt die Palmitatstimulation zeit- und konzentrationsabhängig einen stärkeren Effekt auf die IL-6-Signalwirkung als die Stimulation mit LPS.:Abkürzungsverzeichnis 1. Hintergrund und Ziel der Arbeit 2. Methoden 3. Ergebnisse 3.1 Struktur und Funktion der suburothelialen Myofibroblasten 3.2 Entzündungsmechanismen der Harnblase 3.3 Einfluss von Palmitat auf die IL-6 and MCP-1 Expression humaner Detrusormyozyten - Link zwischen Stoffwechsel und immunologischer Reaktion der Harnblasenmuskulatur 3.4 IL-6 - JAK/STAT - Signalweg in den Detrusormyozyten 3.5 Schlussfolgerungen 4. Literaturverzeichnis 5. Publikationen 5.1 Struktur und Funktion der suburothelialen Myofibroblasten in der humanen Harnblase unter normalen und pathologischen Bedingungen 5.2 Regulation von Interleukin-6 durch Lipopolysaccharid-Stimulation in kultivierten Detrusormyozyten 5.3 Palmitate induced IL-6 and MCP-1 expression in human bladder smooth muscle cells provides a link between diabetes and urinary tract infections 6. Zusammenfassung der Arbeiten Anlagen / Background: Urinary tract infections (UTI) are more frequent in type-2 diabetes mellitus patients than in subjects with normal glucose metabolism. The mechanisms underlying this higher prevalence of UTI are unknown. However, cytokine levels are altered in diabetic patients and may thus contribute to the development of UTI. Increased levels of free fatty acids (FFA), as observed in obese patients, can induce IL-6 production in various cell types. Therefore we studied the effects of the free fatty acid palmitate and bacterial lipopolysaccharide (LPS) on interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) expression and secretion in cultured human bladder smooth muscle cells (hBSMC). Methodology/Principal Findings: Biopsies were taken from patients undergoing cystectomy due to bladder cancer. Palmitate or LPS stimulated hBSMC were analysed for the production and secretion of the IL-6, gp80, gp80soluble, gp130, MCP-1, pSTAT3, SOCS3, NF-kB and SHP2 by quantitative PCR, ELISA, Western blotting, and confocal immunofluorescence. In signal transduction inhibition experiments we evaluated the involvement of NF-kB and MEK1 in IL-6 and MCP-1 regulation. Palmitate upregulates IL-6 mRNA expression and secretion via NF-kB dependent pathways in a concentration- and timedependent manner. MCP-1 was moderately upregulated by palmitate but was strongly upregulated by LPS involving NF-kB and MEK1 dependent pathways. Soluble IL-6 receptor (gp80soluble) was downregulated by palmitate and LPS, while membrane-bound gp80 was moderately upregulated. LPS increased SOCS3 and SHP2, whereas palmitate only induced SOCS3. Secondary finding: most of the IL-6 is secreted. Conclusions/Significance: Bacterial infection (LPS) or metabolic alterations (palmitate) have distinct effects on IL-6 expression in hBSMC, (i) short term LPS induced autocrine JAK/STAT signaling and (ii) long-term endocrine regulation of IL-6 by palmitate. Induction of IL-6 in human bladder smooth muscle cells by fatty acids may represent a pathogenetic factor underlying the higher frequency and persistence of urinary tract infections in patients with metabolic diseases.:Abkürzungsverzeichnis 1. Hintergrund und Ziel der Arbeit 2. Methoden 3. Ergebnisse 3.1 Struktur und Funktion der suburothelialen Myofibroblasten 3.2 Entzündungsmechanismen der Harnblase 3.3 Einfluss von Palmitat auf die IL-6 and MCP-1 Expression humaner Detrusormyozyten - Link zwischen Stoffwechsel und immunologischer Reaktion der Harnblasenmuskulatur 3.4 IL-6 - JAK/STAT - Signalweg in den Detrusormyozyten 3.5 Schlussfolgerungen 4. Literaturverzeichnis 5. Publikationen 5.1 Struktur und Funktion der suburothelialen Myofibroblasten in der humanen Harnblase unter normalen und pathologischen Bedingungen 5.2 Regulation von Interleukin-6 durch Lipopolysaccharid-Stimulation in kultivierten Detrusormyozyten 5.3 Palmitate induced IL-6 and MCP-1 expression in human bladder smooth muscle cells provides a link between diabetes and urinary tract infections 6. Zusammenfassung der Arbeiten Anlagen

Introduction. Les mécanismes physiopathologiques du remodelage vasculaire pulmonaire chez le patient BPCO sont encore mal élucidés. L'inflammation pourrait jouer un rôle déterminant.Objectifs. Déterminer le rôle de l'interleukine 6 (IL6) dans la physiopathologie de l'hypertension pulmonaire (HTP) associée à la BPCO.Méthodes. Nous avons étudié le lien entre l'IL6 et l'HTP sur une population de patients atteints de BPCO, l'effet de l'hypoxie sur le développement d'une HTP chez des souris IL6-/- et l'effet in vitro de l'IL6 sur les cellules endothéliales et les cellules musculaires lisses d'artère pulmonaire humaine.Résultats. Les patients BPCO avec HTP présentaient un taux plasmatique d'IL6 circulante plus élevé. Le génotype GG de l'IL6 était corrélé à un risque plus élevé de développer une HTP. L'IL6 est produite par tous les acteurs cellulaires impliqués dans la physiopathologie du remodelage vasculaire pulmonaire et en particulier la cellule musculaire lisse. Sa synthèse, ainsi que celle de ses récepteurs, est très nettement stimulée par l'hypoxie aigue et chronique. L'IL6 participe probablement à l'entretien de la dysfonction endothéliale, à la migration des cellules musculaires lisses et au recrutement des cellules inflammatoires. Les souris IL6-/- sont partiellement protégées de l'hypertension pulmonaire hypoxique et présentent un moindre recrutement pulmonaire de cellules inflammatoires induit par l'hypoxie. Le taux d'IL6 est corrélé à une longueur télomérique plus courte chez les patients BPCO, témoignant d'un processus de vieillissement biologique accéléré. L'HTP associée à la BPCO ou à l'hypoxie chronique pourrait résulter d'une accentuation du processus de sénescence des cellules vasculaires pulmonaires, favorisé par l'inflammation.Conclusion. L'inflammation et plus particulièrement l'IL6 semblent fortement impliquées dans la physiopathologie du remodelage vasculaire pulmonaire chez le patient BPCO. / Introduction. The pathophysiological mechanisms responsible for pulmonary vascular remodeling in COPD remain poorly understood. Inflammation may play a major role.Objectives. To study the role of interleukin-6 (IL6) in the pathophysiology of pulmonary hypertension associated with COPD.Methods. We studied the relationship between IL6 and PH in a population of patients with COPD, the effect of hypoxia on the development of PH in mice IL6-/- and the effect of IL-6 on endothelial cells and smooth muscle cells of human pulmonary artery in vitro.Results. COPD patients with PH were characaterised by a more pronounced hypoxia and higher plasma levels of circulating IL-6. The GG genotype of IL-6 also correlated with a higher risk of developing PH in these patients. Each of the different cellular elements that promote pulmonary vascular remodeling are known to produce IL-6, with smooth muscle cells known to be a particularly important source of this cytokine In addition, synthesis of IL-6 and its associated receptors is increased in response to acute and chronic hypoxia. It is likely that IL-6 contributes to endothelial dysfunction, the migration and, indirectly, proliferation of smooth muscle cells, and recruitment of inflammatory cells. Indeed, IL6-/- mice are partially protected from hypoxia-associated pulmonary hypertension and demonstrate attenuated hypoxia-induced lung recruitment of inflammatory cells. Levels of IL-6 also correlate with shorter telomere length in patients with COPD, indicating a process of accelerated biological aging. This suggests that pulmonary hypertension secondary to COPD or chronic hypoxia may be due to inflammation-associated acceleration of normal pulmonary vascular cell sénescence.Conclusion. Inflammation in general, and IL-6 in particular, appear to be strongly involved in the pathophysiology of pulmonary vascular remodeling in patients with COPD

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[Truncated abstract] Fibrosis is a feature of many pulmonary conditions, including idiopathic pulmonary fibrosis (IPF), which is characterised by the accumulation of fibroblasts/myofibroblasts and excessive deposition of collagen. IPF is a disease of unknown aetiology that is unresponsive to current therapy and is typically fatal. The inflammatory cytokine interleukin (IL)-6 is elevated in patients with IPF and recent studies have shown that IL-6-induced signalling is altered in lung fibroblasts from patients with IPF. IL-6 belongs to the gp130 cytokine family, which is a group of ten structurally related cytokines, that all require the membrane bound glycoprotein gp130 to activate intracellular signalling pathways. Gp130 activates intracellular signalling through the Shp2-ERK1/2 and STAT1/3 pathways to mediate cellular activities. This thesis tests the hypothesis that gp130-mediated signalling is dysregulated in the development and progression of pulmonary fibrosis. To address this hypothesis, I assessed the role of gp130-mediated signalling in a mouse model of bleomycin-induced lung fibrosis. This thesis utilised two novel gp130 mutant mice strains with directed and enhanced gp130-mediated Shp2-ERK1/2 (gp130¿STAT/¿STAT) or STAT1/3 (gp130757F/757F) signalling. I observed complete protection from fibrosis in gp130¿STAT/¿STAT mice up to 60 days after bleomycin treatment and profound fibrosis in gp130757F/757F mice compared to wt controls. The enhanced fibrosis observed in gp130757F/757F mice was diminished by monoallelic deletion of STAT3 (gp130757F/757F;STAT3+/-), identifying gp130-STAT3 signalling as a novel promoter of lung fibrosis. ... In addition, IL-6/11 activation of gp130-mediated signalling modulated transforming growth factor (TGF)-ß-induced effects on adult fibroblast proliferation and myofibroblast differentiation. Interaction between IL-6/11 and TGF-ß1 on fibroblast proliferation was dependent on both the gp130-ERK1/2 and gp130-STAT1/3 pathways. Loss of either pathway abrogated the effects of IL-6 and IL-11 on TGF-ß1- 4 induced fibroblast proliferation. However, it was clear that gp130-STAT3 signalling inhibited TGF-ß1-induced myofibroblast differentiation of primary lung fibroblasts. The inhibition of myofibroblast differentiation was associated with gp130-STAT3 dependent inhibition of TGF-ß1-induced Smad3 phosphorylation. These results indicate that IL-6 and IL-11 promote myofibroblastic differentiation of lung fibroblasts, while gp130-STAT3 signalling inhibits TGF-ß1-induced Smad3 phosphorylation and myofibroblastic differentiation of lung fibroblasts While the pathogenesis of IPF is unknown, it is believed that excessive collagen deposition, aberrant fibroblast behaviour and an inflammatory response are critical to the progression of this disease. It has been shown here that IL-6 family cytokines mediate the development and progression of bleomycin-induced lung fibrosis by increasing collagen synthesis, fibroblast proliferation, myofibroblast differentiation and inflammation through gp130-STAT3 signalling. This thesis has demonstrated that differential activation of cytoplasmic signalling pathways by a membrane bound receptor can have a profound effect on pulmonary responses to injury. Furthermore, this thesis is the first study to identify the gp130-STAT3 pathway as a therapeutic target in the treatment of IPF.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2010. / Härtill 5 uppsatser.