Adipocytes are specialized cells found in vertebrates to ensure survival in terms of adaption to food deficit and abundance. However, their dysfunction accounts for the pathophysiology of metabolic diseases such as T2DM. Preliminary data generated by Mona Löffler suggested that PKD1 is involved in adipocyte function. Here, I show that PKD1 expression and activity is linked to lipid metabolism of murine adipocytes. PKD1 gene expression and activity was reduced in murine white adipose tissue upon fasting, a physiological condition which induces lipolysis. Isoproterenol-stimulated lipolysis in adipose tissue and 3T3-L1 adipocytes reduced PKD1 gene expression. Silencing ATGL in adipocytes inhibited isoproterenol-stimulated lipolysis, however, the β-adrenergic stimulation of ATGL-silenced adipocytes lowered PKD1 expression levels as well. Adipose tissue of obese mice exhibited high PKD1 RNA levels but paradoxically lower protein levels of phosphorylated PKD1-Ser916. However, HFD generated a second PKD1 protein product of low molecular weight in mouse adipose tissue. Furthermore, constitutively active PKD1 predominantly displayed nuclear localization in 3T3-L1 adipocytes containing many fat vacuoles. However, adipocytes overexpressing non-functional PKD1 contained fewer lipid droplets and PKD1-KD was distributed in cytoplasm. Most importantly, deficiency of PKD1 in mouse adipose tissue caused expression of genes involved in adaptive thermogenesis such as UCP-1 and thus generated brown-like phenotype adipocytes. Thus, PKD1 is implicated in adipose tissue function and presents an interesting target for therapeutic approaches in the prevention of obesity and associated diseases. / Adipozyten sind spezialisierte Zellen der Wirbeltiere, die das Überleben durch Anpassung an Nahrungsmangel und Nahrungsüberfluss gewährleisten. Eine Dysfunktion von Adipozyten bedingt jedoch die Pathophysiologie von Stoffwechselerkrankungen wie dem T2DM. Vorläufige Ergebnisse von Mona Löfflers Versuchen zeigten, dass PKD1 in der Funktion von Adipozyten involviert ist. Innerhalb dieser Arbeit konnte dargestellt werden, dass die Expression und Aktivität von PKD1 in murinen Adipozyten an den Lipidmetabolismus gekoppelt ist. Beim Hungern von murinem weißen Fettgewebe, einem physiologischen Zustand, der Lipolyse induziert, war die Genexpression von PKD1 reduziert. Isoproterenol-stimulierte Lipolyse führte ebenfalls zu verminderter Expression von PKD1 in murinen weißen Fettgewebe und 3T3-L1 Adipozyten. In ATGL-silenced Adipozyten war die Isoproterenol-stimulierte Lipolyse zwar inhibiert, allerdings wurde die Genexpression von PKD1 durch die β-adrenerge Stimulation ebenfalls vermindert. Fettgewebe von adipösen Mäusen hingegen wiesen hohe PKD1 RNA Level sowie einen niedrigen Proteingehalt der phosphorylierten Form PKD1-Ser916 auf. Fettreiche Ernährung von Mäusen generierte in Fettgewebe jedoch ein weiteres Produkt von PKD1 mit niedrigem Molekulargewicht im Western Blot. Des Weiteren wurde dargestellt, dass konstitutiv aktives PKD1 in 3T3-L1 Adipozyten vorwiegend nuklear lokalisiert war und diese Adipozyten einen hohen Gehalt von Fettvakuolen aufwiesen. Adipozyten, die funktionsloses PKD1 exprimierten, enthielten wenige Lipidtropfen und PKD1-KD war im Cytoplasma verteilt. Vor allem zeigte diese Arbeit, dass die Deletion von PKD1 spezifisch in murinem Fettgewebe die Expression von Genen wie UCP-1 verursachte, die eine Rolle in adaptiver Thermogenese spielen, und dadurch einen brown-like Phänotypen generierte. Zusammenfassend ist PKD1 in die Funktionen von Adipozyten verwickelt und stellt ein attraktives Ziel für therapeutische Ansätze in der Prävention von Übergewicht und damit assoziierten Erkrankungen dar.

The tight junctions (TJ), which are located in the apical region between epithelial and endothelial cells, regulate the paracellular diffusion of ions and small molecules and play an important role in maintaining cell polarity, cell-cell integrity, and permeability. In the lung, epithelial cells are attached by TJ structures. They provide a permeable barrier and cell communication. The loss of barrier integrity, which is maintained by the expression of claudins (Cldn), results in cellular permibilization and leads to paracellular diffusion of solutes and harmful molecules. There are 27 known Cldn homologous members in mice and human. Cldn6 is mostly expressed in embryonic stem cells and associated with the programing of epithelial cells during embryo development and lung morphogenesis. In order to test the hypothesis that Cldn6 expression affects lung morphogenesis, we analyzed the expression pattern of Cldn6 during lung ontogenesis to examine cell-specific expression pattern of Cldn6 during each embryonic period in the mouse lung. Also, we assessed transcriptional regulators and control mechanisms that precisely influence Cldn6 expression in pulmonary cells. We discovered that Cldn6 is an important tight junctional component expressed by pulmonary epithelium during lung organogenesis. We found that normal down-regulation of Cldn6 as development proceeds influences differentiation associated with the transition between the embryonic to the alveolar stage. Conditional gain-of-function and loss-of-function experiments in animal models prove to be the most beneficial tool in deciphering the impact of Cldn in organ formation and maintenance. We generated a conditional transgenic mouse that provides the opportunity to genetically up-regulate Cldn6 in distal lung. Our transgenic mouse showed a delay in lung development and down-regulation of transcriptional factors. Cldn6 is both temporally and spatially controlled in the developing lung and its regulation is maintained by critical transcriptional control networks managed by TTF-1. In lung diseases, altered Cldn expression leads to diseases such as COPD, asthma, and ARDS. The tight junctional proteins are differentially regulated by tobacco smoke exposure and Cldn6 is potentially involved as neighboring epithelial cells respond to tobacco smoke. We exposed adult mice to controlled doses of second hand smoke during four days and A-549 cells to 10% CSE for 6 hours. We discovered that mice lungs respond by down-regulating Cldn6 basal levels and impair barrier function. These results reveal that midgestational up-regulation of Cldn6 and its marked down-regulation as development proceeds illustrate the notion that Cldn6 function is important during early programming stages of lung morphogenesis.

Food allergy is a potentially life-threatening disease affecting up to 10% of individuals in Western countries. Clinical reactivity to food allergens is primarily mediated by immunoglobulin (Ig) E, with symptoms ranging from mild urticaria to anaphylaxis. Currently, food allergy remains a disease without a cure. Oral immunotherapy (OIT), which involves consuming small amounts of allergen, remains an experimental treatment in Canada, although has been approved by the Food and Drug Administration (FDA) in the United States for treatment of peanut allergy. While efficacious to induce desensitization, OIT is accompanied by a significant rate of adverse effects. Sublingual immunotherapy (SLIT) is a novel route of treatment for food allergy, where small amounts of allergen are placed under the tongue and held for 2-3 minutes. In contrast to OIT, SLIT offers not only treatment efficacy but also promises an excellent safety profile. The first objective of this thesis was to first develop a SLIT regimen in murine models of food allergy where sensitization is carried out either epicutaneously or intragastrically. Secondly, we investigated the efficacy of SLIT in modulating the clinical and humoral responses in prophylactic and semi-therapeutic settings. In the prophylactic setting, where SLIT was administered prior to sensitizing allergen exposures, SLIT-treated mice were completely protected from allergic sensitization including absent production of serum ovalbumin-specific IgE. In the semi-therapeutic setting, where SLIT was administered to mice primed to develop food allergy, it produced a partial protection against food-induced clinical reactivity. This was associated with lower levels of IgE production in comparison to non-treated, allergic mice. Together, this work provides both an optimized SLIT protocol, as well as evidence on the efficacy of SLIT in the treatment of food allergy in murine models. These findings will aid future work investigating the cellular and molecular mechanisms underlying SLIT-induced protection. / Thesis / Master of Science (MSc) / Food allergy is a potentially life-threatening disease which is primarily mediated by IgE antibodies. Strict allergen avoidance and use of rescue epinephrine upon accidental allergen exposure remain the standard of care. Oral immunotherapy, where individuals ingest small amounts of allergen, is currently the experimental treatment of reference to induce clinical tolerance; however, it is accompanied by a significant rate of adverse reactions. In contrast, sublingual immunotherapy (SLIT), which is less efficacious, upholds a superior safety profile. The primary objective of this thesis was to investigate the impact of SLIT in inducing clinical and immunological changes in murine models of food allergy. We demonstrated that when administered prophylactically, SLIT prevents mice from undergoing anaphylaxis. When administered to sensitized mice in a pre-allergic state, SLIT was protective against severe clinical reactivity after challenge. In conclusion, the work presented here establishes a useful platform to investigate the mechanisms underlying SLIT-mediated protection.

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The purpose of these studies was to determine if an immunomodulator was present in caprine serum. Controlled studies demonstrated that CSF-I, material fractionated from caprine serum possessed an immunomodulatory compound. Caprine serum was further fractionated into it?s peptidic components and a small contaminant of immunoglobulin G and albumin (Caprine serum fraction - immunomodulator 2, or CSF-I2). This was refined to a three peptidic isolate collectively identified as tri-peptidic immunostimulant or TPI. CSF-I2 does not possess antibacterial capabilities (as typically characteristic of a cationic peptide or defensin), does not contain a level of endotoxin sufficient to promote a pyrogenic response, and its functional ability to improve animal survival after an infectious challenge does not reside with molecular weight components greater than 10 kilodaltons, effectively excluding the immunoglobins, albumin, cytokines, and collectins. CSF-I2 was able to significantly reduce the mortality observed in chickens (from 80% to 13%) infected with Pasteurella multocida (Willeford et al., 2000), in mice (from 83% to 13.3%) infected with Salmonella typhimurium, and in canines (from 50% to 9.8%) diagnosed with parvovirus. CSF-I2 may well prove to provide prophylactic and therapeutic health benefits to humans. CSF-I2 may effectively combat pathogenesis when used as either an adjunct with conventional therapy (e.g., antibiotics) or when provided as the primary medicant.

No description available.

No description available.

Conventional Dendritic cells (cDCs), a specialized group of immunological sentinels with tree-like or dendritic shapes, are critical for recognition of danger signals, presentation of antigens and control of a spectrum of innate and adaptive immune responses. Type I interferons (IFNs), as important danger signals, activate cDCs through the canonical type I IFN receptor signaling. Type I IFNs are the first line of host defense against viral infection by up-regulating IFN-stimulated genes (ISGs). However, there are circumstances in which the silencing of excessive type I IFNs could be beneficial to the host, such as IFN-dependent autoimmune diseases, gene therapy that uses viral vectors and transplantation. The role of type I IFNs in DC development, activation and antigen presentation function remains to be completely investigated. In this dissertation, we studied the regulation of Type I IFNs in murine DCs, both cDCs and plasmacytoid DCs (pDCs), and specifically we investigated the role of two molecules, Signal Transducer and Activator of Transcription 2 (STAT2) and Three prime Repair EXonuclease 1 (Trex1), in DC biology. Our research furthers our understanding of DC development, activation and function, and provides important data for the therapeutic application of modified DCs to induce immunological tolerance in gene therapy, IFN-dependent autoimmune diseases and transplantation. STAT2 is a nuclear transcription factor downstream of type I IFN receptor-mediated signaling, the role of which has been mostly explored in antiviral responses mediated by type I IFNs. However, the involvement of STAT2 in cDC activation and function such as cross-presentation remains hitherto unclear. We report that STAT2 is essential for murine cDC activation upon TLR3, -4, -7 and -9 stimulation. In the absence of STAT2, cDCs displayed impaired up-regulation of type I IFN response (costimulatory molecules and type I IFN-stimulated genes), and reduced inflammatory cytokine production when stimulated with TLR ligands. STAT2 was required in all of the DC responses to exogenous IFNα, suggesting that the canonical STAT1-STAT2 heterodimers are the major signaling transducers downstream of type I IFNs in DCs. Of interest, LPS-induced TNFα and IL6 production were reduced in STAT2-/- DCs but not in IFNAR1-/- DCs, suggesting a novel STAT2-dependent pathway mediated by LPS, bypassing type I IFN-receptor signaling. STAT2-deficient cDCs showed impaired cross-presentation leading to decreased CD8+ T cell response both in vitro and CTL killing in vivo, indicating that STAT2 is essential for TLR-induced cross-presentation. These results demonstrate that STAT2 is critical in the regulation of TLR-induced DC activation and cross-presentation, suggesting an essential role for STAT2 in anti-viral and anti-tumor immune responses. We also propose a novel regulatory function of STAT2 in the LPS response independent of type I IFN receptor signaling. Trex1 mutations are associated with a spectrum of type I IFN-dependent autoimmune diseases such as Aicardi-Goutières syndrome and systemic lupus erythematosus. Trex1 plays an essential role in preventing accumulation of excessive cytoplasmic DNA, avoiding cell-intrinsic innate DNA sensor activation and suppressing activation of both type I IFN-stimulated and IFN-independent antiviral genes. Trex1 also helps HIV escape cytoplasmic detection by DNA sensors. However, regulation of Trex1 in DC biology is lacking. We report that murine cDCs have high constitutive expression of Trex1 in vitro and in vivo in the spleen. In resting bone marrow-derived cDCs, type I IFNs up-regulate Trex1 expression via the canonical IFN receptor signaling pathway (STAT1-, STAT2-dependent). DC activation induced by TLR3, -4, -7 and -9 ligands also augments Trex1 expression through autocrine IFNß production and triggering of the IFN signaling pathway, while TLR4 ligand LPS also stimulates an early expression of Trex1 through an IFN-independent NFΚB-dependent signaling pathway. Furthermore, retroviral infection also induces Trex1 up-regulation in cDCs, as we found that a gene therapy HIV-1-based lentiviral vector induces significant Trex1 expression, suggesting that Trex1 may affect local and systemic administration of gene therapy vehicles. Our data indicate that Trex1 is induced in cDCs during activation upon IFN- and TLR- stimulation through the canonical IFN signaling pathway, and suggest that Trex1 may play a role in cDC activation during infection and autoimmunity. Finally, these results suggest that HIV-like viruses may up-regulate Trex1 to increase their ability to escape immunosurveillance. In order to dissect the role of Trex1 in DC functions, we compared DCs from Trex1-/- and wild-type mice. We report that Trex1 deficiency reduces absolute number of pDCs in BM but not in spleen of male over female mice. Furthermore, Trex1 deficiency preferentially represses Flt3L-induced DC development both in vitro and in vivo but not GM-CSF-dependent DC development, suggesting that Trex1 plays an indispensable role in Flt3L-induced DC development and GM-CSF may compensate the effect of Trex1 deficiency. This defect is only limited to male Trex1-/- DCs, and mimics the effect of mTOR inhibition. Furthermore, although Flt3L-induced Trex1-/- DCs expressed a type I IFN signature, they also exhibited decreased pDC development markers, indicating Trex1 regulates pDC development at the transcriptional level. Thus, we propose a novel and essential role of Trex1 in Flt3L-induced DC development, and the effect of Trex1 regulation is gender-dependent. Together, these findings enhance our understanding of regulatory roles of Type I IFNs in DC development, activation and function, supporting the beneficial role of STAT2/type I IFN axis in TLR-induced DC activation and cross-presentation. Our study in Trex1 reveals Trex1 induction by viral infection, type I IFNs and TLRs in DCs, and a new role of Trex1 in early development of Flt3L-induced DCs in a gender-dependent manner, whereby a balance between type I IFNs and Trex1 is important for DC activation and hemostasis. / Microbiology and Immunology

Indiana University-Purdue University Indianapolis (IUPUI) / Type-2-diabetes (T2D) is a progressive metabolic disease characterized by insulin resistance and β-cell dysfunction leading to persistent hyperglycemia. It is a multisystem disease that causes deterioration of multiple organ systems and obesity. Of interest, T2D affects the urinary system and is the leading cause of kidney disease. Both T2D and chronic kidney negatively impacts the skeletal system and increases fracture incidence in patients. Therefore, it is important to establish an animal model that captures the complex multiorgan effects that is common in T2D. In this study, we characterized the metabolic phenotype of the KK/Ay mouse model, a polygenic mutation model of T2D. We concluded that KK/Ay mice closely mimic T2D and are hyperglycemic, hyperinsulinemic and insulin resistant. KK/Ay mice have also had worsened kidney function as supported by elevated levels of blood urea nitrogen, phosphorous, creatinine, and calcium in plasma exhibiting the kidney’s inefficiency in clearing waste from the body. Even though we were able to confirm a metabolic phenotype for T2D and diabetic nephropathy, the skeletal effects of the disease were minimal and major differences in bone physiology were driven by sex differences. This study offered valuable insight into preliminary endpoints for the KK/Ay mouse mode that will decide the direction for future use of this model. We plan to use older mice in future studies to allow a longer time for skeletal effects to more prominently manifest.

No / Here, we studied how epithelial energy metabolism impacts overall skin development by selectively deleting intraepithelial mtDNA in mice by ablating a key maintenance factor (TfamEKO), which induces loss of function of the electron transport chain (ETC). Quantitative (immuno)histomorphometry demonstrated that TfamEKO mice showed significantly reduced hair follicle (HF) density and morphogenesis, fewer intrafollicular keratin15+ epithelial progenitor cells, increased apoptosis, and reduced proliferation. TfamEKO mice also displayed premature entry into (aborted) HF cycling by apoptosis-driven HF regression (catagen). Ultrastructurally, TfamEKO mice exhibited severe HF dystrophy, pigmentary abnormalities, and telogen-like condensed dermal papillae. Epithelial HF progenitor cell differentiation (Plet1, Lrig1 Lef1, and β-catenin), sebaceous gland development (adipophilin, Scd1, and oil red), and key mediators/markers of epithelial–mesenchymal interactions during skin morphogenesis (NCAM, versican, and alkaline phosphatase) were all severely altered in TfamEKO mice. Moreover, the number of mast cells, major histocompatibility complex class II+, or CD11b+ immunocytes in the skin mesenchyme was increased, and essentially no subcutis developed. Therefore, in contrast to their epidermal counterparts, pilosebaceous unit stem cells depend on a functional ETC. Most importantly, our findings point toward a frontier in skin biology: the coupling of HF keratinocyte mitochondrial function with the epithelial–mesenchymal interactions that drive overall development of the skin and its appendages.