Dysbiosis, Tolerance, and Development of Autoimmune Diseases

The pathogenesis of autoimmune diseases (AIDS) is not only attributed to genetic susceptibility, but also to environmental factors, among which, those disturbing gut microbiota have attracted increasing attention lately. Healthy gut microbiota has beneficial effects on the development and activity of the immune system, playing a central role in peripheric tolerance. Compositional and functional changes in gut microbiota were reported in various AIDS, and increasing evidence suggests that disturbed gut microbiota contributes to their immunopathogenesis. Thyroid and intestinal diseases prevalently coexist—for instance, Hashimoto’s thyroiditis and Graves’ disease are the most common autoimmune thyroid diseases and often co-occur with celiac disease. This association can be at least explained by increased intestinal permeability, allowing antigens to cross the barrier more easily and activate the immune system. The passage of microbial antigens into the internal environment may break the self-tolerance, generating the production of autoantibodies and/or autoreactive T cells. In this chapter, we briefly present the roles of intestinal microbiota in human physiology, with a focus on the role of microbiota in immune tolerance

Dysbiosis in the Development of Type I Diabetes and Associated Complications: From Mechanisms to Targeted Gut Microbes Manipulation Therapies

Publication history: Accepted - 8 March 2021; Published online - 9 March 2021Globally, we are facing a worrying increase in type 1 diabetes mellitus (T1DM) incidence, with onset at younger age shedding light on the need to better understand the mechanisms of disease and step-up prevention. Given its implication in immune system development and regulation of metabolism, there is no surprise that the gut microbiota is a possible culprit behind T1DM pathogenesis. Additionally, microbiota manipulation by probiotics, prebiotics, dietary factors and microbiota transplantation can all modulate early host–microbiota interactions by enabling beneficial microbes with protective potential for individuals with T1DM or at high risk of developing T1DM. In this review, we discuss the challenges and perspectives of translating microbiome data into clinical practice. Nevertheless, this progress will only be possible if we focus our interest on developing numerous longitudinal, multicenter, interventional and double-blind randomized clinical trials to confirm their efficacy and safety of these therapeutic approachesThis research was funded by UEFISCDI, project ID PN-III-P1-1.1-PD-2019-0499, grant number 224/2021

Mechanisms of chronic complications of diabetes with focus on mitochondria and oxygen sensing

Chronic complications of diabetes represent a major medical and economical concern. It is an imperative need to establish the pathogenic mechanisms that contribute to development of chronic complications in order to design new therapeutic approaches. Several pathogenic pathways are activated in diabetes and have been proposed to be responsible for the development of long-term complications of diabetes including an overproduction of reactive oxygen species (ROS) by the mitochondria electron-transport chain, suggested to be a common mechanism for all the others. Hypoxia directly and through induction of ROS has been recently suggested to have an important role in the development of chronic complications of diabetes. Adaptive responses of cells to hypoxia are mediated by the hypoxia-inducible factor-1 (HIF-1), which is an ubiquitary heterodimeric transcription factor, regulated by oxygen at the degradation level of its alpha subunit. The oxygen regulation of HIF is complex and involves a family of hydroxylases (HIF hydroxylases), that need Fe2+ or alpha-ketoglutarate as cofactors and regulates both HIF stability and transactivation in hypoxia. Under hypoxic conditions, HIF-1alpha is stabilized against degradation and upregulates a series of genes (more than 70) involved in essential processes i.e. angiogenesis, glycolytic energy metabolism, cell proliferation and survival. ROS, produced in excess both in hyperglycemia and hypoxia can interact with different macromolecules including DNA. The close proximity of the mitochondrial DNA (mtDNA) to the ROS-generating sites makes mtDNA more vulnerable to oxidative damage. Different cellular specific antioxidants mechanisms are available but they are not always able to fully protect DNA from the oxidative damage. Beside mtDNA mutations that contribute to the maternal inherited diabetes (0.5-1%) several somatic mtDNA point mutations and deletions (classically associated with aging) have been described in patients with diabetes even though not consistent. A drawback of these studies is the heterogeneous genetic background of the patients with diabetes coupled with the lack of reliable information about the duration of the exposure to high glucose levels. The direct influence of chronic hyperglycaemia and/or hypoxia upon mtDNA stability and repair is not clear. Hyperglycemia impairs HIF-1alpha stability and function and it has been suggested that by this it contributes to the development of chronic complications of diabetes (wound healing, coronary heart disease etc). We have therefore hypothesized in the first paper that the defect in wound healing present in diabetes is a result of an inhibition of HIF-1 activity. We could first demonstrate that the repression of HIF in hyperglycemia is complex and implies not only the stability of the HIF-1alpha in hypoxia but also the transactivation of both N-terminal transactivation domain (NTAD) and C-terminal transactivation domain (CTAD). Furthermore we show that by blocking HIF-1alpha hydroxylation through chemical inhibition it is possible to reverse the negative regulatory effect of hyperglycemia on HIF-1 alpha both in vitro and in vivo in a mouse model (db/db) of diabetic wound. Local HIF-1 alpha induction was able to improve several processes essential for wound healing i.e. granulation, vascularisation, epidermal regeneration, and recruitment of endothelial precursors cells (EPC). Stabilisation of HIF-1alpha was necessary and sufficient for promoting wound healing in a diabetic environment. Thus, it is important to develop specific hydroxylase inhibitors as therapeutic agents for chronic diabetes wounds. In the second paper we investigated by long amplification QPCR the stability of mitochondrial DNA against ROS overproduction in diabetic condition. By using human dermal fibroblasts (HDF) we were able to confirm an increase of ROS production by hyperglycemia alone or in combination with hypoxia. However, mtDNA damage was observed only when the cells were exposed to both hyperglycemia and hypoxia, confirming the pathogenic role of the combination of these factors. The mtDNA damage was mediated through excess production of ROS by mitochondria as far as mtDNA was fully protected when the cells were treated with inhibitors of the electron transport chain. We have further studied by long amplification QPCR the effect of diabetes on mtDNA lesions in db/db mouse that is an inbred model of type 2 diabetes. We investigated the incidence of mtDNA lesions in heart and kidney of two groups of db/db mice: young prediabetic mice (before developing diabetes) and old diabetic mice (34 weeks) and compared with nondiabetic mice (heterozygotes) of the same age. Unexpectedly, the old diabetic mice had a lower incidence of mtDNA lesions in both tissues studied, compared with old non-diabetic and young prediabetic animals even though the tissues are exposed to an excess ROS (as shown by increased protein nitrosylation). This was explained by an increase in the antioxidant capacity (expression of Mn Superoxide Dismutase (SOD2) and Catalase) and of the mitochondrial base excision repair (mtBER) activity in old diabetic animals. In conclusion, we have shown that hypoxia plays together with hyperglycemia an important role in chronic complications of diabetes by activating several pathogenic pathways. Some mechanisms are fully compensated (i.e. antioxidant defence and reparatory capacity of mtDNA) while others (HIF repression) need to be addressed therapeutically in order to be able to improve the outcome

Impact of COVID-19 on the Microbiome and Inflammatory Status of Type 2 Diabetes Patients

The severe acute respiratory syndrome–related coronavirus 2 (SARS-CoV-2) pandemic has advanced our understanding of the host–microbiome–virus interplay. Several studies in various geographical regions report that SARS-CoV-2 infection disrupts the intestinal microbiota, allowing pathogenic bacteria such as Enterobacteriaceae to thrive, and triggering more severe disease outcomes. Here, we profile the microbiota of 30 individuals, 15 healthy controls and 15 type 2 diabetes (T2D) patients, before and after coronavirus disease 2019 (COVID-19). Despite similar viral loads in both patients and controls, SARS-CoV-2 infection led to exacerbated microbiome changes in T2D patients, characterized by higher levels of Enterobacteriaceae, loss of butyrate producers and an enrichment in fungi such as Candida spp. and Aspergillus spp. Several members of the microbiota were associated with more severe clinical and inflammatory (IL-8 and IL-17) parameters. Future studies to delineate the connection between cytokine release and microbiota disturbances will enhance our understanding of whether these microbial shifts directly impact the cytokine storm in COVID-19 patients or whether they are consecutive to the critical disease

Snapshot into the Type-2-Diabetes-Associated Microbiome of a Romanian Cohort

The prevalence of type 2 diabetes mellitus (T2D) is alarmingly increasing worldwide, urgently calling for a better understanding of the underlying mechanisms in order to step up prevention and improve therapeutic approaches. It is becoming evident that the gut microbiota seem to have an endless capacity to impact T2D. In this study, we profile the gut microbiome patterns in T2D patients from Romania, by using quantitative Real-Time PCR and next generation sequencing. We enrolled a total of 150 individuals (105 T2D patients, 50 of them without metformin treatment and 45 healthy volunteers). The levels of potentially beneficial butyrate-producing bacteria were significantly reduced, while potentially pathogenic microorganisms such as Enterobacteriaceae and Fusobacterium were enriched in T2D patients. We evaluated the correlation between clinical parameters and gut microbiota and identified the genera Bacteroides, Alistipes, Dialister, Bilophila and Sutterella as possible detrimental factors in T2D. Our findings suggest that the gut microbiota may be a potential target in novel approaches to halt the development of T2D-associated complications

Mono-epoxy-tocotrienol-α enhances wound healing in diabetic mice and stimulates in vitro angiogenesis and cell migration

Diabetes mellitus is characterized by hyperglycemia and capillary hypoxia that causes excessive production of free radicals and impaired antioxidant defense, resulting in oxidative stress and diabetes complications such as impaired wound healing. We have previously shown that modified forms of tocotrienols possess beneficial effects on the biosynthesis of the mevalonate pathway lipids including increase in mitochondrial CoQ. The aim of this study is to investigate the effects of mono-epoxy-tocotrienol-α on in vitro and in vivo wound healing models as well as its effects on mitochondrial function. Gene profiling analysis and gene expression studies on HepG2 cells and human dermal fibroblasts were performed by microarray and qPCR, respectively. In vitro wound healing using human fibroblasts was studied by scratch assay and in vitro angiogenesis using human dermal microvascular endothelial cells was studied by the tube formation assay. In vivo wound healing was performed in the diabetic db/db mouse model. For the study of mitochondrial functions and oxygen consumption rate Seahorse XF-24 was employed. In vitro, significant increase in wound closure and cell migration (p < 0.05) both in normal and high glucose and in endothelial tube formation (angiogenesis) (p < 0.005) were observed. Microarray profiling analysis showed a 20-fold increase of KIF26A gene expression and 11-fold decrease of lanosterol synthase expression. Expression analysis by qPCR showed significant increase of the growth factors VEGFA and PDGFB. The epoxidated compound induced a significantly higher basal and reserve mitochondrial capacity in both HDF and HepG2 cells. Additionally, in vivo wound healing in db/db mice, demonstrated a small but significant enhancement on wound healing upon local application of the compound compared to treatment with vehicle alone. Mono-epoxy-tocotrienol-α seems to possess beneficial effects on wound healing by increasing the expression of genes involved in cell growth, motility and angiogenes as well as on mitochondrial function