Classical and alternative nuclear factor-kappaB in epithelium: impacts in allergic airway disease and avenues for redox regulation

Nuclear Factor kappaB (NF-êB) is a transcription factor whose activation is increased in settings of allergic asthma. At least two parallel NF-êB pathways exist: the classical pathway, which plays a role in inflammation and cell survival, and the alternative pathway, which regulates lymphoid cell development and organogenesis. The classical NF-êB pathway regulates inflammatory responses derived from lung epithelial cells; however, the role of the alternative pathway in lung epithelial cells remains unclear. We demonstrate that both classical and alternative NF-êB are activated in lung epithelial cells in response to multiple pro-inflammatory agonists, and siRNA-mediated knockdown of alternative NF-êB proteins largely attenuates pro-inflammatory cytokine production. Furthermore, simultaneous activation of both pathways leads to cooperative increases in pro-inflammatory responses, indicating a potential role for both classical and alternative NF-êB in the regulation of epithelial-derived pro-inflammatory responses. NF-êB activation in the epithelium modulates allergic inflammation in mouse models of allergic airway disease, however, its role in the context of an allergen relevant to human asthma remains unknown. In order to address the impact of inhibition of NF-êB in the epithelium in vivo, we utilized a House Dust Mite (HDM)-induced model of allergic airway disease. We demonstrate that HDM exposure activates classical and alternative NF-êB in both murine lung epithelium and human bronchial epithelial cells. Furthermore, following exposure to HDM, airway hyperresponsiveness, neutrophilic inflammation, and remodeling are attenuated in transgenic CC10-NF-êBSR (airway epithelial specific inhibitor of classical and alternative NF-êB) mice in comparison to wild type mice. Our data also demonstrate that specific knockdown of the alternative NF-êB protein, RelB, in the lung partially protects against HDM-induced pro-inflammatory responses, indicating that both classical and alternative NF-êB are important in HDM-induced responses. NF-êB proteins are modified by the redox-dependent post-translational modification, S-glutathionylation, under conditions of oxidative stress. S-glutathionylation of IKKâ, an upstream kinase in the NF-êB pathway, is known to decrease its catalytic activity; however, it is unknown how S-glutathionylation of IKKâ occurs. GSTP is an enzyme that catalyzes protein S-glutathionylation under conditions of oxidative stress and has been associated with the development of allergic asthma. We aimed to determine whether GSTP regulates NF-êB signaling, S-glutathionylation of IKKâ, and pro-inflammatory cytokine production. We demonstrate that siRNA-mediated knockdown of GSTP modulates NF-êB activation, NF-êB transcriptional activity, and pro-inflammatory cytokine production in response to LPS, a component of a bacterial cell wall. Furthermore, we demonstrate that GSTP associates with IKKâ in response to agonist stimulation and dampens IKKâ-induced pro-inflammatory cytokine production, surprisingly, independent of its catalytic activity. We also show that GSTP associates with other proteins of the NF-êB pathway, indicating a potential dual mechanism for repression of NF-êB-induced signaling. These studies collectively demonstrate that classical and alternative NF-êB contribute to epithelial-derived inflammatory responses, and GSTP may be a novel target by which NF-êB can be regulated

Sub-Emetic Toxicity of Bacillus cereus Toxin Cereulide on Cultured Human Enterocyte-Like Caco-2 Cells.

Cereulide (CER) intoxication occurs at relatively high doses of 8 µg/kg body weight. Recent research demonstrated a wide prevalence of low concentrations of cereulide in rice and pasta dishes. However, the impact of an exposure to low doses of cereulide was never studied before. In this research, we investigate the effect of low concentrations of cereulide on the behavior of intestinal cells using Caco-2 cell line. The MTT (mitochondrial 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) and the SRB (Sulforhodamine B) reactions were used to measure the mitochondrial activity and the measurement of cellular protein content, respectively. Both assays showed that differentiated Caco-2 cells were sensitive to low concentrations of CER (in MTT reaction 1 ng/mL after 3 days of treatment ; in SRB reaction 0.125 ng/mL after 3 days of treatment). Cell counts revealed that cells were released from the differentiated monolayer at 0.5 ng/mL of cereulide. Additionally, 0.5 and 2 ng/mL of CER increased lactate presence in the cell culture medium. Proteomic data showed that cereulide at concentration of 1 ng/mL led towards a significant decrease in energy managing and H2O2 detoxification proteins and to an increase in cell death markers. This is amongst first reports to describe the influence of sub-emetic concentrations of cereulide on a differentiated intestinal monolayer model showing that low doses may induce an altered enterocyte metabolism and membrane integrity

Impact of metabolic perturbation on ovarian function

Metabolic perturbations including hyperinsulinemia that are induced during obesity and heat stress in humans and production animals are allied with several health hallmarks and impaired fertility. This dissertation research focused on charactering the impact of changes to central metabolism on ovarian function. We hypothesized that hyperinsulinemia induced during central metabolic perturbations, alters ovarian insulin-mediated PI3K signaling, negatively impacting ovarian folliculogenesis, steroidogenesis and xenobiotic biotransformation. To test this hypothesis, mRNA and protein expression profiles of insulin, PI3K, steroidogenic, inflammatory and chemical metabolism members were quantified using qRT-PCR, Western blotting or immunohistochemistry techniques using three models of hyperinsulinemia: 1) high fat diet (HFD)-induced obesity, 2) a transgenic mouse model of progressive obesity and 3) a porcine model of hyperinsulinemia. Overall, our data demonstrates that the ovarian insulin-KITLG-KIT-AKT signaling pathway is active and upregulated during central metabolic alterations. Perturbations to ovarian insulin-KITLG-KIT-AKT signaling pathway are likely to impact 1) follicle activation, oocyte viability and recruitment, 2) steroid hormone biosynthesis, and 3) xenobiotic biotransformation, potentially accelerating susceptibility to chemical exposure. All of these scenarios could lead to impairment of ovarian function, and may at least partially explain why female fecundity is compromised during altered metabolic states

Impact of metabolic perturbation on ovarian function

Metabolic perturbations including hyperinsulinemia that are induced during obesity and heat stress in humans and production animals are allied with several health hallmarks and impaired fertility. This dissertation research focused on charactering the impact of changes to central metabolism on ovarian function. We hypothesized that hyperinsulinemia induced during central metabolic perturbations, alters ovarian insulin-mediated PI3K signaling, negatively impacting ovarian folliculogenesis, steroidogenesis and xenobiotic biotransformation. To test this hypothesis, mRNA and protein expression profiles of insulin, PI3K, steroidogenic, inflammatory and chemical metabolism members were quantified using qRT-PCR, Western blotting or immunohistochemistry techniques using three models of hyperinsulinemia: 1) high fat diet (HFD)-induced obesity, 2) a transgenic mouse model of progressive obesity and 3) a porcine model of hyperinsulinemia. Overall, our data demonstrates that the ovarian insulin-KITLG-KIT-AKT signaling pathway is active and upregulated during central metabolic alterations. Perturbations to ovarian insulin-KITLG-KIT-AKT signaling pathway are likely to impact 1) follicle activation, oocyte viability and recruitment, 2) steroid hormone biosynthesis, and 3) xenobiotic biotransformation, potentially accelerating susceptibility to chemical exposure. All of these scenarios could lead to impairment of ovarian function, and may at least partially explain why female fecundity is compromised during altered metabolic states

DNA metüülimise roll kokaiini poolt põhjustatud käitumusliku sensitisatsiooni kujunemises

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Ravimsõltuvust iseloomustab vastupandamatu tung tarbida psühhotroopseid aineid hoolimata sellega kaasuvatest tõsistest kõrvaltoimetest. Uuringud on näidanud, et ravimsõltuvus kujuneb välja järk-järgult ja sõltuvusega seotud neurobioloogilised muutused võivad jääda püsima ka pärast psühhotroopsete ainete tarvitamise lõppu. Korduv psühhostimulaatori (nt. kokaiini) manustamine põhjustab katseloomadel suurenenud käitumusliku vastuse. Sellist fenomeni nimetatakse psühhomotoorseks ehk käitumuslikuks sensitisatsiooniks ning see modelleerib adiktiivset käitumist ja psühhostimulaatorite psühhootilisi komplikatsioone inimesel. Kuna käitumuslikule sensitisatsioonile on iseloomulikud püsivad muutused katseloomade käitumises, siis arvatakse, et osaliselt on selle põhjuseks lühi- ja pikaajalised geeniekspressiooni muutused, mis omakorda mõjutavad närviimpulsi ülekannet, sünapsite moodustamist ja närviringide funktsioneerimist. Mitmed uuringud viitavad, et epigeneetilised mehhanismid, eriti geeni promootori piirkonna DNA metüülimine, mille korral liidetakse DNA metüültransferaasi (DNMT) vahendusel metüülrühm tsütosiin-guanosiin (CpG) dinukleotiidsele järjestusele, on seotud pikaajaliste geeniekspressiooni muutustega. Käesoleva töö eesmärgiks oli uurida DNA metüülimise rolli kokaiini poolt põhjustatud käitumusliku sensitisatsiooni kujunemises hiirtel ja rottidel. Töö tulemused näitasid, et kokaiini manustamine suurendas dünaamiliselt Dnmt3a ja Dnmt3b ekspressiooni täiskasvanud hiirte naalduvas tuumas (nucleus accumbens) ja hipokampuses; põhjustas valitud markergeenide promootori piirkonnas nii DNA metüülimist kui ka demetüülimist ning DNMT inhibiitori, zebulariini, manustamine normaliseeris hüpermetüülitud geeni transkriptsiooni täiskasvanud hiirte naalduvas tuumas ja pidurdas käitumusliku sensitisatsiooni teket. Samuti leidsime, et keskkonnategurid, nagu metüülrühma doonor S-adenosüülmetioniin (SAM) ja varajases elueas kogetud stress, võivad DNA metüülimise kaudu soodustada psühhostimulaatoritest tingitud ravimsõltuvuse teket nii hiirtel kui ka rottidel.Drug addiction is a chronic relapsing disorder characterised by a pattern of compulsive drug seeking and taking behaviour despite severe adverse consequences. Prolonged abuse of drugs, such as psychostimulants, may contribute to behavioural abnormalities that can last for months or even years after discontinuing drug consumption. Repeated administration of psychostimulants (such as cocaine) induces an enhanced behavioural response to subsequent drug exposure, a phenomenon known as psychomotor or behavioural sensitisation. Psychostimulant-induced behavioural sensitisation in rodents provides a model for addictive behaviours, such as those associated with craving and relapse, and for the psychotic complications of psychostimulant abuse. Behavioural sensitisation is remarkably persistent phenomenon. In rodents, it can persist from months to years after drug treatment is discontinued. Persistent behavioural sensitisation indicates that drug-induced short- and long-term changes in gene expression may be involved. Accumulating data suggest that epigenetic mechanisms, such as DNA methylation (catalysed by DNA methyltransferases - DNMTs), are critical regulators of persistent gene expression changes and may be related to behavioural disorders. The aim of this study was to investigate the role of DNA methylation in the development of cocaine-induced behavioural sensitisation in mice and rats. Our data demonstrated that cocaine treatment caused a dynamic increase in Dnmt3a and Dnmt3b expression levels in the nucleus accumbens (NAc) and hippocampus of adult mice; induced both DNA methylation/demethylation in the promoter regions of the selected genes; and intracerebroventricular treatment with the DNMT inhibitor zebularine normalised hypermethylated gene transcription in the NAc of adult mice and delayed the development of cocaine-induced behavioural sensitisation. We also found that environmental factors, such as methyl group donor S-adenosylmethionine (SAM) and early life stress, may promote, via DNA methylation, the development of psychostimulant-induced drug addiction in mice and rats

Effect of Tff3 Deficiency and ER Stress in the Liver

Endoplasmic reticulum (ER) stress, a cellular condition caused by the accumulation of unfolded proteins inside the ER, has been recognized as a major pathological mechanism in a variety of conditions, including cancer, metabolic and neurodegenerative diseases. Trefoil factor family (TFFs) peptides are present in different epithelial organs, blood supply, neural tissues, as well as in the liver, and their deficiency has been linked to the ER function. Complete ablation of Tff3 expression is observed in steatosis, and as the most prominent change in the early phase of diabetes in multigenic mouse models of diabesity. To elucidate the role of Tff3 deficiency on different pathologically relevant pathways, we have developed a new congenic mouse model Tff3-/-/C57BL6/N from a mixed background strain (C57BL6/N /SV129) by using a speed congenics approach. Acute ER stress was evoked by tunicamycin treatment, and mice were sacrificed after 24 h. Afterwards the effect of Tff3 deficiency was evaluated with regard to the expression of relevant oxidative and ER stress genes, relevant proinflammatory cytokines/chemokines, and the global protein content. The most dramatic change was noticed at the level of inflammation-related genes, while markers for unfolded protein response were not significantly affected. Ultrastructural analysis confirmed that the size of lipid vacuoles was affected as well. Since the liver acts as an important metabolic and immunological organ, the influence of Tff3 deficiency and physiological function possibly reflects on the whole organism

Role of spleen tyrosine kinase in tumor necrosis factor like weak inhibitor of apoptosis induced dopaminergic neuronal cell death and microglial activation in invitro and in vivo models of Parkinson disease

Tweak and TNF family members are novel regulators of acute and chronic inflammation. Tweak / Fn14 interaction appears to be involved in angiogenesis, inflammation, proliferation, migration, cytokine production, cytotoxicity and apoptosis. Thus, TWEAK can induce both cell death and proliferation. Recently, TWEAK has been shown to be associated with neurodegenerative effects in an MPTP mouse model of PD. Despite numerous studies demonstrating TWEAK’s ability to cause cell death in diverse cell types the mechanism(s) by which TWEAK-induce dopaminergic cell death remain poorly defined. Therefore in an attempt to better understand the molecular basis of TWEAK-induced dopaminergic neurotoxicity we evaluated the apoptotic effect of TWEAK and associated molecular mechanisms using N27 dopaminergic neuronal cells. TWEAK-induced a time dependent increase in ROS generation, mitochondrial dysfunction, caspase activation, and NFkB activation. Additionally, a concurrent activation of SYK and proteolytic cleavage of PKC delta was evidenced in TWEAK treated cells. In contrast a marked down regulation of p-GSK 3b (Ser 9) and Akt activity was evidenced in TWEAK treated cells. Intriguingly, inhibition of SYK activity via R708 attenuated TWEAK-induced loss of dopaminergic cell viability. Likewise, SN50, NFkB inhibitor and Quercetin, a ubiquitous bioactive plant flavonoid attenuated TWEAK-induced apoptotic cell death further highlighting the pivotal role of NFkB and mitochondria dependent oxidative stress signaling events in the mechanism of dopaminergic neurodegeneration. Taken together, our studies demonstrate the involvement SYK/NFkB signaling axis and mitochondrial dysfunction in TWEAK-induced apoptotic cell death. In the next series of studies we determined the effects of TWEAK on microglia. A growing body of evidence suggests that persistent microglial activation and accompanying oxidative stress may act as co-conspirators in mediating dopaminergic neurodegeneration in the substantia nigra in PD pathogenesis. Previously we demonstrated that PKC delta a redox sensitive kinase is a critical determinant of microglial activation response in response to diverse inflammagens. More recently, SYK, has been implicated in the activation of inflammatory cells in response to infection. Therefore, in the current study we hypothesized that SYK may act as an upstream regulator of NLRP3 inflammasome thereby leading to a heightened microglial activation response in TWEAK stimulated cells. In the present study we systematically elucidated the signaling network underlyingTWEAK-induced microglial activation response. A concentration dependent increase in SYK activation and accompanying increase in kinase activity was evidenced in TWEAK treated cells. Our results with pharmacological inhibitors and siRNA mediated gene silencing revealed the regulatory role of SYK in ER stress response (ERS), NOX2 upregulation, GSK 3β activation as well as autophagolysosomal system (ALS) and mitochondrial dysfunction. Taken together our findings demonstrate a role for SYK signaling network in mediating TWEAK triggered inflammatory response by positively regulating NLRP3 inflammasome activation and ERS in an autophagy dependent manner. We have discovered the pivotal role of SYK in mediating dopaminergic neurodegeneration as well as heightened microglial activation response upon TWEAK treatment. By inhibiting SYK activation we can limit dopaminergic neuronal degeneration as well as microglial activation response in response to diverse inflammagens including TWEAK. Our studies further highlight the therapeutic advantage of targeting SYK for the treatment of inflammation related disorders including PD

The NRF2, Thioredoxin, and Glutathione System in Tumorigenesis and Anticancer Therapies

Cancer remains an elusive, highly complex disease and a global burden. Constant change by acquired mutations and metabolic reprogramming contribute to the high inter- and intratumor heterogeneity of malignant cells, their selective growth advantage, and their resistance to anticancer therapies. In the modern era of integrative biomedicine, realizing that a personalized approach could benefit therapy treatments and patients’ prognosis, we should focus on cancer-driving advantageous modifications. Namely, reactive oxygen species (ROS), known to act as regulators of cellular metabolism and growth, exhibit both negative and positive activities, as do antioxidants with potential anticancer effects. Such complexity of oxidative homeostasis is sometimes overseen in the case of studies evaluating the effects of potential anticancer antioxidants. While cancer cells often produce more ROS due to their increased growth-favoring demands, numerous conventional anticancer therapies exploit this feature to ensure selective cancer cell death triggered by excessive ROS levels, also causing serious side effects. The activation of the cellular NRF2 (nuclear factor erythroid 2 like 2) pathway and induction of cytoprotective genes accompanies an increase in ROS levels. A plethora of specific targets, including those involved in thioredoxin (TRX) and glutathione (GSH) systems, are activated by NRF2. In this paper, we briefly review preclinical research findings on the interrelated roles of the NRF2 pathway and TRX and GSH systems, with focus given to clinical findings and their relevance in carcinogenesis and anticancer treatments

Deciphering pre-leukaemic dysregulation caused by mutant C/EBPα at the level of the single cell

Abstract Deciphering pre-leukaemic dysregulation caused by mutant C/EBPα at the level of the single cell Moosa Rashid Qureshi C/EBPα is an ideal candidate to explore pre-leukaemic dysregulation of transcriptional networks because it is a significant player in normal myelopoiesis, it interacts with other transcription factors (TFs) in lineage specification, and the CEBPA gene has been identified as an early mutation in acute myeloid leukaemia (AML). N321D is a mutation affecting the leucine zipper domain of C/EBPα which has previously generated an aggressive AML phenotype in a mouse model. The first objective of my thesis was to construct a novel in vitro cellular model to recapitulate pre-leukaemic changes associated with the CEBPA N321D mutation. The conditionally immortalised murine Hoxb8-FL cell line can differentiate into relatively homogenous populations of functional myeloid, lymphoid and dendritic cells in appropriate cell culture conditions. These cells were transduced with retroviral vectors which expressed either no transgene or “Empty Vector” (EV), the CEBPA wildtype (WT) or N321D mutation. CEBPA N321D-transduced cells had immature morphological appearance in Flt3L differentiation media, proliferated indefinitely, and were characterised by a plasmacytoid dendritic cell (pDC)-like immunophenotype. These cells were capable of long-term engraftment in a mouse model but did not recapitulate leukaemia for several months after transplantation. The second objective was to characterise the transcriptional deregulation associated with the phenotypes demonstrated by EV, CEBPA WT and N321D-expressing cells in the presence of Flt3L. Single cell RNA-seq (scRNA-seq) confirmed phenotypic observations that CEBPA N321D-transduced cells had a pDC precursor-like gene expression signature when placed in Flt3L conditions of differentiation. Single cell analysis also identified transcriptional heterogeneity in the CEBPA N321D compartment at single cell resolution. The third objective was to use ChIP-seq to investigate the effect of the N321D mutation on C/EBPα’s TF binding profile, and correlate this with scRNA-seq data to identify a set of candidate genes (NOTCH2, JAK2, SIRPA, FOS, and others) where gene expression changes were associated with direct binding events by the mutant TF. In addition to characterising the transcriptional effects of the CEBPA mutation, a fourth objective was to update and expand the Göttgens group’s CODEX database of TF-binding and histone modification data for human blood cells, increasing annotation of the genome to 14.83%. This online database now provides significant coverage of the non-coding portion of the genome, and is therefore potentially of interest to the wider haematology research community

The role of oxidative stress in the regulation of dendritic cell function

The archetypal view of the immune system is that it has evolved primarily to distinguish between non-infectious "self' and infectious "non-self'. However, this view has been superseded by the paradigm that the immune system is based on the recognition of a "danger" signal. The key to this signal is thought to be activation of dendritic cells (DC), which is a critical step in the initiation of adaptive immune responses. However, there is little experimental evidence to support the role of "danger" signals in the activation of DC. Perhaps the most noteworthy hypothesis is that oxidative stress may constitute a common denominator pathway for the activation of DC. This hypothesis has yet to be investigated in detail, and is the subject of this thesis. Human DC were differentiated from monocytes by seven days of culture in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4. When supplemented to purified cell populations, non-toxic concentrations of reactive oxygen species (ROS) were unable to induce the maturation of DC. In addition, ROS were found not to represent prerequisite activation signals for DC in vitro. It was hypothesised subsequently that the effects of oxidative stress on DC may be mediated indirectly via the actions of oxidised protein and lipid derivatives. Thus, either advanced oxidation protein products (AOPP) or oxidised low-density lipoproteins (LDL) could be representative of the fundamental mechanism for the identification of damaged-"self ' by DC. AOPP were found to increase the T-cell stimulatory capacity of DC (as assessed by oxidative mitogenesis assays) without inducing their conventional maturation, whilst oxidised LDL were shown to cause both phenotypic and functional maturation of DC. Furthermore, native LDL caused DC aggregation, and oxidised LDL caused DC apoptosis. In conclusion, oxidised derivatives of "self' may play an important contribution to physiological and pathophysiological responses involving DC maturation