Pathogenesis Of Type 1 Diabetes Research Articles

Using GeoMx DSP Spatial Proteomics to Investigate Immune Infiltration of NOD Mouse Islet and Exocrine Compartments

Abstract Purpose Type 1 Diabetes (T1D) pathogenesis involves immune cells infiltrating pancreatic Islets of Langerhans, leading to T cell activation, beta cell destruction, and impaired insulin production. However, infiltration has a heterogenic nature that isn’t described in detail, as not all islets are infiltrated. The aim of this study was to investigate if the observed heterogeneity is coupled to differences in immune and/or dysfunctional status of islets or exocrine cells, and if specific markers could elucidate mechanistic details of T1D pathogenesis. Procedures The GeoMx platform was used to spatially quantify protein levels in pancreatic islets and exocrine tissue in Non-Obese Diabetic (NOD) mice. The protein panel included 17 immune activity markers and nine dysfunction markers. Immunohistochemical (IHC) staining and digital image analysis was used to analyze select marker proteins. Results Use of the GeoMx platform to investigate T1D was shown to be possible, as Granzyme B protein levels were found to be lower in distal islet areas when compared to proximal areas. Smooth Muscle Actin protein levels were higher in exocrine areas proximal to immune-infiltrated islets, when compared to distally located exocrine areas. Findings from GeoMx were however not observed in IHC-stained sections. Conclusions This study demonstrates that investigating T1D is possible with spatial proteomics, as the assays revealed presence of heterogenic islet areas in NOD mice, which may play a role in T1D progression and escape from immune recognition. This study highlights the potential of spatial technologies for elucidating T1D pathogenesis and future treatment strategies.

Role of SIRPG gene in type 1 diabetes and lichen planus.

Type 1 diabetes (T1D) is a form of diabetes caused by pancreatic β-cell destruction and absolute insulin deficiency. Lichen planus (LP) is an idiopathic inflammatory skin disease of unclear etiology. The role of SIRPG gene dysregulation in T1D and LP remains unclear. Mendelian randomization (MR) using matched samples was employed to study causal relationship between T1D and increased risk of LP. T1D-related single nucleotide polymorphism identification was conducted. Datasets GSE156035 for T1D and GSE52130 for LP were obtained from gene expression omnibus. Differentially expressed genes were identified, analyses included weighted gene co-expression network analysis, functional enrichment, gene set enrichment analysis, and protein-protein interaction network construction and analysis. Heatmaps of gene expression levels were generated. Comparative toxicogenomics database was used to identify diseases most relevant to core genes. Inverse variance weighted, MR-Egger, weighted median methods estimated genetic predisposition between T1D and LP, showing consistent positive correlations using both weighted median and inverse variance weighted methods. Horizontal pleiotropy analysis with MR-Egger intercept indicated no evidence of significant directional pleiotropy (P = .70645) for LP. There was no evidence of directional pleiotropy effects between T1D and LP. One hundred eighteen differentially expressed genes were identified. In biological processes, they were mainly enriched in apoptosis, inflammatory response, insulin receptor signaling pathway, glucose metabolism. In cellular components, enrichment was observed in mediator complex and replication fork. In molecular function, they were concentrated in leukotriene receptor activity and helicase activity. Kyoto Encyclopedia of Genes and Genomes analysis revealed enrichment in metabolic pathways, PI3K-Akt signaling pathway, cell cycle, p53 signaling pathway, AGE-RAGE signaling pathway in diabetic complications. Weighted gene co-expression network analysis with a soft threshold power of 4. SIRPG showing high expression in both T1D and LP samples. There is a positive causal relationship between T1D and LP. Comparative toxicogenomics database analysis revealed associations of core genes with metabolic syndrome, lipid metabolism disorders, cardiovascular diseases, immune system diseases, peripheral neuropathic pain, and inflammation. SIRPG is highly expressed in both T1D and LP, providing a new insight into the pathogenesis of T1D and LP.

Shifting the paradigm of type 1 diabetes: a narrative review of disease modifying therapies

A new diagnosis of type 1 diabetes (T1D) may be accompanied by numerous lifelong financial, emotional, and physical challenges, thus advancements in therapies that can delay the onset of clinical disease are crucial. T1D is an autoimmune condition involving destruction of pancreatic beta cells leading to insulin deficiency, hyperglycemia, and long-term insulin dependence. The pathogenesis of T1D is classified into stages, with the first signal being the detection of autoantibodies without any glycemic changes. In the second stage, dysglycemia develops without symptoms, and in stage 3, symptoms of hyperglycemia become apparent, and at this time a clinical diagnosis of T1D is made. As a greater understanding of these stages of T1D have evolved, research efforts have been devoted to delaying the onset of clinical disease. To date, only one medication, teplizumab, has been approved by the Food and Drug Administration (FDA) for the treatment of stage 2 T1D. This narrative review present published trials and ongoing research on disease modifying therapies (DMT) in T1D, the mechanisms of action for each therapy, and the stages of T1D that these interventions are being studied.

Leveraging pre-trained machine learning models for islet quantification in type 1 diabetes

Human islets display a high degree of heterogeneity in terms of size, number, architecture, and endocrine cell-type compositions. An ever-increasing number of immunohistochemistry-stained whole slide images (WSIs) are available through the online pathology database of the Network for Pancreatic Organ donors with Diabetes (nPOD) program at the University of Florida (UF). We aimed to develop an enhanced machine learning-assisted WSI analysis workflow to utilize the nPOD resource for analysis of endocrine cell heterogeneity in the natural history of type 1 diabetes (T1D) in comparison to donors without diabetes. To maximize usability, the user-friendly open-source software QuPath was selected for the main interface. The WSI data were analyzed with two pre-trained machine learning models (i.e., Segment Anything Model (SAM) and QuPath's pixel classifier), using the UF high-performance-computing cluster, HiPerGator. SAM was used to define precise endocrine cell and cell grouping boundaries (with an average quality score of 0.91 per slide), and the artificial neural network-based pixel classifier was applied to segment areas of insulin- or glucagon-stained cytoplasmic regions within each endocrine cell. An additional script was developed to automatically count CD3+ cells inside and within 20 μm of each islet perimeter to quantify the number of islets with inflammation (i.e., CD3+ T-cell infiltration). Proof-of-concept analysis was performed to test the developed workflow in 12 subjects using 24 slides. This open-source machine learning-assisted workflow enables rapid and high throughput determinations of endocrine cells, whether as single cells or within groups, across hundreds of slides. It is expected that use of this workflow will accelerate our understanding of endocrine cell and islet heterogeneity in the context of T1D endotypes and pathogenesis.

Elevated levels of plasma inactive stromal cell derived factor-1a predict long-term adverse outcomes in diabetic patients with coronary artery disease

Abstract Background Because of the significantly increased cardiovascular risk by complicating type 2 diabetes (T2D) in patients with coronary artery disease (CAD), the appropriate risk estimation is crucial in patients with T2D following percutaneous coronary intervention (PCI). Nevertheless, there is no established biomarker which can precisely predict outcomes in this particular population. Although stromal cell derived factor-1α (SDF-1α) has been suggested to play a key role in the pathogenesis of T2D, the prognostic impact of SDF-1α in the secondary prevention of CAD is yet to be elucidated. In particular, the ability of SDF-1α isoforms in outcome prediction in this population has not been assessed. Purpose The aim of present study was to investigate the prognostic implication of three isoforms of SDF-1α, such as total, active, and inactive forms of SDF-1α in diabetic patients following PCI. Methods This single-center retrospective analysis involved consecutive patients with T2D who underwent PCI for the first time between 2008 and 2018 (n=849). Primary and secondary outcome measures were all-cause death and the composite of cardiovascular death, non-fatal myocardial infarction, and ischemic stroke (3P-MACE), respectively. For determining plasma levels of SDF-1α, we measured not only total, but also the active type of SDF-1α by ELISA. Inactive isoform of the SDF-1α was calculated by subtracting the active isoform from total SDF-1α. Participants were divided into two groups according to the median of three forms of plasma SDF-1α levels (total SDF-1α: 2270 pg/mL, active SDF-1α: 686 pg/mL, inactive SDF-1α: 1537 pg/mL) at PCI procedure and the incidence and risk of subsequent endpoints following PCI were assessed. Results Kaplan-Meier analyses revealed increased risk of both all-cause death and 3P-MACE in patients with elevated levels of inactive SDF-1α (Figure 1). Constantly, multivariate Cox hazard analyses adjusted by age, sex, body mass index (>25), acute coronary syndrome, and chronic kidney disease (≥ stage 3) repeatedly indicated the 1 higher log-transformed inactive SDF-1α was significantly associated with increased risk of all-cause death (hazard ratio (HR): 2.21, 95% confidence interval (CI): 1.05–4.67, p=0.04) and 3P-MACE (HR: 2.30, 95% CI: 1.01–5.09, p=0.04). However, neither of total and active SDF-1α reached statistical significance (Figure 2). Furthermore, the addition of inactive SDF-1α to baseline models in multivariate Cox proportional hazard analysis significantly enhanced both the net reclassification improvement (NRI) and the integrated discrimination improvement (IDI) for all-cause death (NRI 0.29, p=0.009; IDI 0.007, p=0.03) and 3P-MACE (NRI 0.38, p=0.001; IDI 0.007, p=0.01) while adding total and active SDF-1α did not improve them. Conclusion In diabetic patients following PCI, elevated levels of plasma inactive SDF-1α, not total and active isoforms, might be a useful indicator of adverse outcomes.

Interleukin-6 and suPAR are associated with diastolic function in type 1 diabetes: The Thousand & 1 Study

Abstract Background Heart failure (HF) is a common complication in individuals with type 1 diabetes (T1D). Inflammation is an important contributor to the pathogenesis and progression of both T1D and HF – especially HF with preserved ejection fraction (HFpEF). Diastolic dysfunction is a hallmark of HFpEF. Despite this, the association between diastolic function and inflammatory biomarkers is not well-known in individuals with T1D. Methods The Thousand & 1 study of individuals with T1D without known heart disease was studied. Diastolic parameters, including E/e’, was obtained by echocardiography and associated with Interleukin-6 (IL-6), soluble urokinase-type plasminogen activator receptor (suPAR) and high-sensitivity C-reactive protein (hsCRP). These inflammatory biomarkers were analysed as continuous variables and dichotomized in above/below 3 pg/mL (IL-6), 4 ng/mL (suPAR), and 4 mg/L (hsCRP). In multivariable regression analysis, adjustments were made for age, gender, body mass index, duration of diabetes, HbA1c, systolic blood pressure, left ventricular ejection fraction, glomerular filtration rate, albuminuria status, use of statins, tobacco use and physical activity levels. In an additional analyse, we also adjusted for N-terminal pro–B-type natriuretic peptide (NT-proBNP). Results We included 1,014 individuals (52% male, mean age 50±15). IL-6 was elevated in 207 individuals, suPAR in 182 individuals, and hsCRP in 863 individuals. Mean E/e’ was 7.6 and mean left ventricular ejection fraction was 58±5%. In fully adjusted models, inflammatory biomarkers were significantly associated with diastolic function. Thus, individuals with IL-6 > 3 pg/mL had 0.6 (95% confidence intervals: 0.2 to 1.0), P=0.001) higher E/e’ compared to individuals with IL-6 < 3 pg/mL. For every doubling of suPAR levels, E/e’ increased by 0.5 (0.2 to 0.9, P=0.003). For every doubling of hsCRP levels, E/e’ increased by 0.1 (0.02 to 0.2, P=0.022). Individuals with elevated levels of all three biomarkers had 1.9 (0.5 to 3.2, P=0.006) higher E/e’ compared to low levels of all three biomarkers. Including NTproBNP to the model did essentially not change the estimates. Conclusions Firstly, in T1D, elevated levels of IL-6, suPAR and hsCRP are associated with diastolic function assessed by E/e’, independent of traditional risk factors including NT-proBNP. Secondly, IL-6, suPAR and hsCRP may contain information about diastolic function in T1D which is not detected by NT-proBNP.Figure 1Figure 2

High expression of CNOT6L contributes to the negative development of type 2 diabetes.

Type 2 diabetes (T2D) is a chronic metabolic disorder characterized by reduced responsiveness of body cells to insulin, leading to elevated blood sugar levels. CNOT6L is involved in glucose metabolism, insulin secretion regulation, pancreatic beta-cell proliferation, and apoptosis. These functions may be closely related to the pathogenesis of T2D. However, the exact molecular mechanisms linking CNOT6L to T2D remain unclear. Therefore, this study aims to elucidate the role of CNOT6L in T2D. The T2D datasets GSE163980 and GSE26168 profiles were downloaded from the Gene Expression Omnibusdatabase generated by GPL20115 and GPL6883.The R package limma was used to screen differentially expressed genes (DEGs). A weighted gene co-expression network analysis was performed. Construction and analysis of the protein-protein interaction (PPI) network, functional enrichment analysis, gene set enrichment analysis, and comparative toxicogenomics database (CTD) analysis were performed. Target Scan was used to screen miRNAs that regulate central DEGs. The results were verified by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), western blotting (WB), and blood glucose measurements in mice. A total of 1951 DEGs were identified. GO and KEGG enrichment analysis revealed that differentially expressed genes were mainly enriched in the insulin signaling pathway, ECM-receptor interaction, and PPAR signaling pathway. Metascape analysis indicated enrichment primarily in the cAMP signaling pathway and enzyme-linked receptor protein signaling pathway. WGCNA analysis yielded 50 intersecting genes. PPI network construction and algorithm identification identified two core genes (CNOT6L and GRIN2B), among which CNOT6L gene was associated with multiple miRNAs. CTD analysis revealed associations of core genes with type 2 diabetes, diabetic complications, dyslipidemia, hyperglycemia, and inflammation. WB and RT-qPCR results showed that in different pathways, CNOT6L protein and mRNA levels were upregulated in type 2 diabetes. CNOT6L is highly expressed in type 2 diabetes mellitus, and can cause diabetes complications, inflammation and other physiological processes by regulating miRNA, PPAR and other related signaling pathways, with poor prognosis. CNOT6L can be used as a potential therapeutic target for type 2 diabetes.

Loss of atrial natriuretic peptide signaling causes insulin resistance, mitochondrial dysfunction, and low endurance capacity.

Type 2 diabetes (T2D) and obesity are strongly associated with low natriuretic peptide (NP) plasma levels and a down-regulation of NP guanylyl cyclase receptor-A (GCA) in skeletal muscle and adipose tissue. However, no study has so far provided evidence for a causal link between atrial NP (ANP)/GCA deficiency and T2D pathogenesis. Here, we show that both systemic and skeletal muscle ANP/GCA deficiencies in mice promote metabolic disturbances and prediabetes. Skeletal muscle insulin resistance is further associated with altered mitochondrial function and impaired endurance running capacity. ANP/GCA-deficient mice exhibit increased proton leak and reduced content of mitochondrial oxidative phosphorylation proteins. We further show that GCA is related to several metabolic traits in T2D and positively correlates with markers of oxidative capacity in human skeletal muscle. Together, these results indicate that ANP/GCA signaling controls muscle mitochondrial integrity and oxidative capacity in vivo and plays a causal role in the development of prediabetes.

Exploring lipodystrophy gene expression in adipocytes: unveiling insights into the pathogenesis of insulin resistance, type 2 diabetes, and clustering diseases (metabolic syndrome) in Asian Indians.

Studying the molecular mechanisms of lipodystrophy can provide valuable insights into the pathophysiology of insulin resistance (IR), type 2 diabetes (T2D), and other clustering diseases [metabolic syndrome (MetS)] and its underlying adipocentric disease (MetS disease). A high-confidence lipodystrophy gene panel comprising 50 genes was created, and their expressions were measured in the visceral and subcutaneous (both peripheral and abdominal) adipose depots of MetS and non-MetS individuals at a tertiary care medical facility. Most lipodystrophy genes showed significant downregulation in MetS individuals compared to non-MetS individuals in both subcutaneous and visceral depots. In the abdominal compartment, all the genes showed relatively higher expression in visceral depot as compared to their subcutaneous counterpart, and this difference narrowed with increasing severity of MetS. Their expression level shows an inverse correlation with T2D, MetS, and HOMA-IR and with other T2D-related intermediate traits. Results also demonstrated that individualization of MetS patients could be done based on adipose tissue expression of just 12 genes. Adipose tissue expression of lipodystrophy genes shows an association with MetS and its intermediate phenotypic traits. Mutations of these genes are known to cause congenital lipodystrophy syndromes, whereas their altered expression in adipose tissue contributes to the pathogenesis of IR, T2D, and MetS.

Inflammatory Trajectory of Type 2 Diabetes: Novel Opportunities for Early and Late Treatment.

Low-grade inflammation (LGI) represents a key driver of type 2 diabetes (T2D) and its associated cardiovascular diseases (CVDs). Indeed, inflammatory markers such as hs-CRP and IL-6 predict the development of T2D and its complications, suggesting that LGI already increases before T2D diagnosis and remains elevated even after treatment. Overnutrition, unhealthy diets, physical inactivity, obesity, and aging are all recognized triggers of LGI, promoting insulin resistance and sustaining the pathogenesis of T2D. Once developed, and even before frank appearance, people with T2D undergo a pathological metabolic remodeling, with an alteration of multiple CVD risk factors, i.e., glycemia, lipids, blood pressure, and renal function. In turn, such variables foster a range of inflammatory pathways and mechanisms, e.g., immune cell stimulation, the accrual of senescent cells, long-lasting epigenetic changes, and trained immunity, which are held to chronically fuel LGI at the systemic and tissue levels. Targeting of CVD risk factors partially ameliorates LGI. However, some long-lasting inflammatory pathways are unaffected by common therapies, and LGI burden is still increased in many T2D patients, a phenomenon possibly underlying the residual inflammatory risk (i.e., having hs-CRP > 2 mg/dL despite optimal LDL cholesterol control). On the other hand, selected disease-modifying drugs, e.g., GLP-1RA, seem to also act on the pathogenesis of T2D, curbing the inflammatory trajectory of the disease and possibly preventing it if introduced early. In addition, selected trials demonstrated the potential of canonical anti-inflammatory therapies in reducing the rate of CVDs in patients with this condition or at high risk for it, many of whom had T2D. Since colchicine, an inhibitor of immune cell activation, is now approved for the prevention of CVDs, it might be worth exploring a possible therapeutic paradigm to identify subjects with T2D and an increased LGI burden to treat them with this drug. Upcoming studies will reveal whether disease-modifying drugs reverse early T2D by suppressing sources of LGI and whether colchicine has a broad benefit in people with this condition.

12362 Pathophysiology From Oxidative Stress Effectively Monitored In A Pre-clinical Model Of Type 2 Diabetes (T2D) With Point-of-care Microscale Magnetic Resonance (µNMR)

Abstract Disclosure: A.T. Alves: ; Co-Founder. ; Self. ; LarmorBio. S.S. Thamarath: ; Co-Founder. ; Self. ; LarmorBio. S. Fjordside: Employee; Self; Novo Nordisk. S. Sassower, MS.EE: ; Co-Founder. ; Self. ; LarmorBio. J. Han: None. L. Bouchard: Advisory Board Member; Self; LarmorBio. R. Rohr, BS.EE: ; Co-Founder, CEO. ; Self. ; LarmorBio. A.P. Chambers: Employee; Self; Novo Nordisk. The declining age of onset of T2D is a vital factor influencing its prospective burden. An early manifestation of the disease leads to an extended duration, which amplifies and accelerates the risk of micro and macrovascular complications. Oxidative stress is a major driver in the pathogenesis of T2D. Early detection and prevention of oxidative stress overload can improve and potentially modify the long-term harmful outcomes of metabolic diseases, including chronic hyperglycaemia.A novel µNMR assay was developed to monitor oxidative stress from minimally invasive plasma samples at the point of care. This approach is highly sensitive to redox changes in the blood microenvironment, including increased ferric iron (Fe3+), protein oxidation, and lipid peroxidation[1]. We hypothesized that oxidative stress would increase significantly faster in the disease group, enabling disease progression monitoring throughout the study and near real-time subject risk stratification based on the core pathophysiology of the diabetic phenotype.This 15-week longitudinal study (6th - 21st week of age) quantified oxidative stress in db/db and db/+ mice (n = 15 per group), accompanied by HbA1c, blood glucose (BG), and body weight (BW). The assay detailed separation (P ≤ 0.01) between the db/db and db/+ mice from the onset of the diabetic phenotype of db/db at 10 weeks of age (mean HbA1c = 6.5%). The oxidative stress monitored continued to increase in the subsequent weeks, where the HbA1c of db/db mice reached maximum (mean HbA1c = 8.8%). The definition of good (HbA1c ≤ 8) and poor (HbA1c > 8) glycaemic control was defined where sub-stratification (P ≤ 0.05) was achieved. Furthermore, the assay highlights a positive correlation with BW (ρ+/db = 0.52, P ≤ 0.0001, ρdb/db = 0.57, P ≤ 0.0001). In the two groups of mice, a positive correlation of oxidative stress with HbA1c (ρ = 0.58, P ≤ 0.0001) and BG (ρ = 0.47, P ≤ 0.0001) was reported. During the 15-week study, the control db/+ group exhibited a 13% mean increase in oxidative stress. In contrast, the db/db group demonstrated a cumulative 20% increase. Notably, considering the diabetic phenotype of the db/db mice, we quantified oxidative stress attributed to aging at 13%.In view of changing diabetes demographics, a multimodal approach of targeted sub-phenotype treatment combined with advanced metabolic monitoring may provide holistic, clinically relevant information to reduce the micro and macrovascular burden of diabetes. In summary, the µNMR assay is an effective method for real-time monitoring of oxidative stress assessment in metabolic disease research and opens new possibilities for cardiometabolic risk assessment and targeted interventions.(1) Peng, W.K. et.al., npj Aging Mech Dis 2020, 6, 11 Presentation: 6/1/2024

8681 Preoteogenomic Identification Of Novel Translated And Regulatory Open Reading Frames In Human Beta-Cells

Abstract Disclosure: K. Walters: None. R.C. Guiterrez: None. S. Sakhar: None. A. Baldwin: None. E.S. Nakayasu: None. H. Russ: None. N. Mukherjee: None. Preoteogenomic Identification of Novel Translated and Regulatory Open Reading Frames in Human Beta-cells The discovery of novel autoreactive T cells that target β cells during type 1 diabetes (T1D) has been challenging due to our limited knowledge in autoreactive β cell antigens. Most of these antigens are derived from canonical open reading frames (ORFs) of protein coding genes, such as preproinsulin and chromogranin A. However, a defective ribosomal product (DRiP) was recently identified from the insulin mRNA (INS-DRiP). This antigen is the result of translation from a novel or unannotated open reading frames (nuORFs). nuORFs have a range of functions from regulating translation to encoding novel small bioactive peptides to producing neoantigens. In this study, we use a transcriptome-wide approach to identify nuORFs in human β cells in an unbiased manner. We performed ribosome profiling (ribo-seq) in stem cell derived β cells (sBCs). Human pluripotent stem cells were differentiated into sBCs expressing a flag-tagged ribosomal protein (RPL22-Flag) driven by the rat insulin promoter. We performed IP-ribo-seq on sBCs enriching for RPL22-Flag containing ribosomes. These data exhibited high-resolution 3-nucleotide periodicity consistent with translating codons and identified sequencing reads consistent with translation of the INS-DRiP. We performed de novo ORF-calling using two software using complementary statistical approaches (RiboCode and ORFQuant). We identified over 12,000 canonical annotated ORFs of which ∼85% were had protein level support from matched proteomics confirming the high specificity of our approach. Of nearly 1000 nuORFs identified in sBCs, over half had protein level support in sBCs and cadaveric human islets. We found that 149 nuORFs were associated with Type-1 Diabetes (T1D) genes, including a novel upstream ORF (uORF) in TYK2, which had the strongest association with T1D and is a promising therapeutic target to halt T1D progression. Using reporter assay, we show that the novel uORF in TYK2 is a translational activator and warrants further investigation. Finally, our data represents a valuable database for the search of novel autoreactive antigens in β cells and accelerate the discovery of novel autoreactive T cells implicated in T1D pathogenesis. Presentation: 6/2/2024

8074 Investigating Viral Infections, Systemic Inflammation, and their Complex Interplay in Type 1 Diabetes Pathogenesis

Abstract Disclosure: K. Girdhar: None. E. Elko: None. A. Pina: None. M. Atkinson: None. J. Ludvigsson: None. J. Ladner: None. E. Altindis: None. Type 1 diabetes (T1D) is an autoimmune disorder characterized by the immune-mediated destruction of insulin-producing beta cells. The etiological factors triggering the immunogenicity of endogenous β-cell antigens remain a focal point of investigation. Viral infections, particularly enteroviruses, coxsackieviruses, and rotaviruses, have been proposed as potential contributors to T1D onset and progression. However, the causal relationship between viral infections and T1D pathogenesis remains elusive. To address this, we conducted an in-depth analysis of viral infection histories of T1D patients and pediatric T1D progressors compared to the controls. To this end, we employed highly multiplexed serology using PepSeq sequencing, an in vitro platform designed for conducting proteomic assays against customizable targets via DNA-barcoded peptides. Utilizing samples from the All Babies in Sweden (ABIS) study (n=58) and the TrialNet study (n=116 new-onset T1D, n=25 established T1D, n=128 controls), we assessed seropositivity against 79 different human viruses. Contrary to previous reports, no significant differences were identified in viral infection histories between T1D individuals and controls. Subsequent analyses of enriched Peptide Count and Viral Homology Search (VHS) species provided further confirmation of the absence of distinctions in T1D progression. Next, we determined whether there are any differences in the markers of systematic inflammation in spite of their similar viral history profile. Therefore, we employed Luminex assays to investigate cytokine profiles in established T1D individuals (n=25). While we could not identify any differences when we compared T1D patients to controls, we identified notable sex-specific differences. Anti-inflammatory cytokines (IL-4, IL-13, IL-22) were lower in female T1D patients compared to female controls. Likewise, female T1D patients had higher pro-inflammatory cytokines (IL-17E, TNF-β). On the other hand, male T1D patients exhibited increased levels of epidermal growth factor and platelet-derived growth factor compared to male controls, with IL-22 uniquely elevated. Our findings challenge the direct association between viral infections and T1D, highlighting the importance of investigating alternative factors. Moreover, the observed sex-specific cytokine alterations underscore the complex interplay between immune responses and T1D, emphasizing the need for sex-specific therapeutic strategies. This research contributes valuable insights into the intricate dynamics of viral infections, systemic inflammation, and their potential roles in T1D pathogenesis. Presentation: 6/2/2024

8090 A Gut Commensal, Parabacteroides distasonis, and Molecular Mimicry in Type 1 Diabetes

Abstract Disclosure: A. Randall: None. Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by the destruction of pancreatic, insulin-producing beta cells by autoreactive T-cells. The trigger(s) of T1D are unclear and genetics alone cannot explain the recent increase in T1D incidence. The gut microbiome composition in T1D patients is altered and thought to play a key role in T1D pathogenesis. Most longitudinal studies have been descriptive. However, a direct cause-and-effect relationship hasn't been established yet. Insulin is a key autoantigen in T1D autoimmunity, and insulin B-chain amino acids 9-23 (insB:9-23) is an important, commonly recognized T-cell epitope. Molecular mimicry is an autoimmunity mechanism where host-like microbial proteins trigger autoimmune responses. We previously identified a peptide mimic of insB:9-23, amino acids 4-18 of hypoxanthine phosphoribosyl transferase (HPRT:4-18) of Parabacteroides distasonis (Girdhar et al., 2022). HPRT:4-18 stimulated insB:9-23-specific human and non-obese diabetic (NOD) mice T-cells. We hypothesize that P. distasonis HPRT:4-18 is required to accelerate diabetes via molecular mimicry of insB:9-23. By orally gavaging NOD mice (n=40 mice/group), we showed that P. distasonis accelerated diabetes onset. Next, we colonized NOD mice with another common gut commensal, Bacteroides fragilis, as a control, and did not observe any effect on islet infiltration. In addition, P. distasonis colonization increased infiltration of immune cells into the islets of germ-free NOD mice (n=6-8 mice/group), while the diabetes incidence experiment in mice is ongoing. P. distasonis-colonized mice had significantly more splenic CD8 and naïve T-cells, dendritic cells and macrophages but fewer FOXP3 cells (n=3-6 mice/group). To determine whether P. distasonis’s effect is specific to the pancreas, we checked intestinal barrier function and intestinal immune cell composition. Disruption of intestinal barrier function has been associated with diabetes. P. distasonis colonization did not alter gut permeability. Furthermore, P. distasonis did not increase intestinal inflammation. Among the intestinal intraepithelial lymphocytes (IELs), there were fewer naïve, central and effector T-cells and B-cells. The findings indicate that P. distasonis does not stimulate an unspecific immune response and its inflammatory effect is specific to the pancreas, supporting our initial hypothesis. Taken together, our results indicate that exposure to P. distasonis in the first years of life has the potential to stimulate molecular mimicry mechanism and cause T1D onset. Reference: Girdhar, K., Huang, Q., Chow, I. T., Vatanen, T., Brady, C., Raisingani, A., . . . Altindis, E. (2022). A gut microbial peptide and molecular mimicry in the pathogenesis of type 1 diabetes. Proc Natl Acad Sci U S A, 119(31), e2120028119. doi:10.1073/pnas.2120028119 Presentation: 6/2/2024

MicroRNA-34a and promoter methylation contribute to peroxisome proliferator-activated receptor gamma gene expression in patients with type 2 diabetes

AimsThis study aimed to investigate the roles of DNA methylation and miR-34a in the regulation of peroxisome proliferator-activated receptor gamma (PPARγ) in patients with type 2 diabetes (T2D). MethodsWe investigated the methylation status of four regions of the PPARγ promoter and PPARγ expression in a panel of 84 T2D patients using methylation-specific PCR (MSP) and RT-qPCR, respectively. Moreover, we quantified DNA methyltransferases (DNMTs) expression and global DNA methylation levels by RT-qPCR and ELISA, respectively. We measured the expression levels of miR-34a and protein expression of PPARγ by stem-loop RT-qPCR and ELISA, respectively. ResultsWe found significant DNA hypermethylation in the R2 and R3 regions of the PPARγ promoter in people with diabetes. Functionally, this was associated with a significant reduction in PPARγ expression. In addition, we observed a significant increase in 5-methylcytosine levels in people with diabetes. A marked increase in circulating miR-34a in the early stages of T2D (up to 10 years) and a significant decrease in circulating miR-34a with increasing diabetes duration from 10 years after the onset of diabetes. Interestingly, upregulation of DNA methyltransferases 1 (DNMT1), DNMT3A, and DNMT3B was observed in people with diabetes, and the average expression of DNMTs was negatively correlated with circulating miR-34a levels. In contrast, the serum protein level of PPARγ, a direct target of miR-34a, increased considerably with diabetes duration and showed a negative correlation with circulating miR-34a, cholesterol, triglyceride, and low-density lipoprotein. ConclusionPPARγ promoter hypermethylation and miR-34a upregulation are associated with T2D pathogenesis through PPARγ dysregulation.

High glucose induces DNA methyltransferase 1 dependent epigenetic reprogramming of the endothelial exosome proteome in type 2 diabetes

In response to hyperglycemia, endothelial cells (ECs) release exosomes with altered protein content and contribute to paracrine signalling, subsequently leading to vascular dysfunction in type 2 diabetes (T2D). High glucose reprograms DNA methylation patterns in various cell/tissue types, including ECs, resulting in pathologically relevant changes in cellular and extracellular proteome. However, DNA methylation-based proteome reprogramming in endothelial exosomes and associated pathological implications in T2D are not known. Hence, in the present study, we used Human umbilical vein endothelial cells (HUVECs), High Fat Diet (HFD) induced diabetic mice (C57BL/6) and clinical models to understand epigenetic basis of exosome proteome regulation in T2D pathogenesis . Exosomes were isolated by size exclusion chromatography and subjected to tandem mass tag (TMT) labelled quantitative proteomics and bioinformatics analysis. Immunoblotting was performed to validate exosome protein signature in clinically characterized individuals with T2D. We observed ECs cultured in high glucose and aortic ECs from HFD mouse expressed elevated DNA methyltransferase1 (DNMT1) levels. Quantitative proteomics of exosomes isolated from ECs treated with high glucose and overexpressing DNMT1 showed significant alterations in both protein levels and post translational modifications which were aligned to T2D associated vascular functions. Based on ontology and gene-function-disease interaction analysis, differentially expressed exosome proteins such as Thrombospondin1, Pentraxin3 and Cystatin C related to vascular complications were significantly increased in HUVECs treated with high glucose and HFD animals and T2D individuals with higher levels of glycated hemoglobin. These proteins were reduced upon treatment with 5-Aza-2’-deoxycytidine. Our study shows epigenetic regulation of exosome proteome in T2D associated vascular complications.

Integrative Analyses of Mitophagy-Related Genes and Mechanisms Associated with Type 2 Diabetes in Muscle Tissue.

Type 2 diabetes (T2D) represents the most prevalent metabolic condition that is primarily distinguished by a range of metabolic imbalances, including hyperglycemia, hyperlipidemia, and insulin resistance (IR). Currently, mitophagy has become increasingly recognized as an important process involved in the pathogenesis and progression of T2D. Therefore, it is very important to explore the role of mitochondrial damage and autophagy-related genes in T2D. This study investigated the role of mitophagy in the development of T2D, and 12 MRHGs associated with T2D were identified using bioinformatic analysis and machine learning methods. Our findings provide the first insight into mitophagy-related genes and their mechanisms in T2D. This study aimed to investigate possible molecular targets for therapy and the underlying mechanisms involved in T2D. This information might be useful to further elucidate the pathogenesis of T2D-related diseases and identify more optimal therapeutic approaches.

Tear Proteomics in Children and Adolescents with Type 1 Diabetes: A Promising Approach to Biomarker Identification of Diabetes Pathogenesis and Complications.

The aim of the current study was to investigate the tear proteome in children and adolescents with type 1 diabetes (T1D) compared to healthy controls, and to identify differences in the tear proteome of children with T1D depending on different characteristics of the disease. Fifty-six children with T1D at least one year after diagnosis, aged 6-17 years old, and fifty-six healthy age- and sex-matched controls were enrolled in this cross-sectional study. The proteomic analysis was based on liquid chromatography tandem mass spectrometry (LC-MS/MS) enabling the identification and quantification of the protein content via Data-Independent Acquisition by Neural Networks (DIA-NN). Data are available via ProteomeXchange with the identifier PXD052994. In total, 3302 proteins were identified from tear samples. Two hundred thirty-nine tear proteins were differentially expressed in children with T1D compared to healthy controls. Most of them were involved in the immune response, tissue homeostasis and inflammation. The presence of diabetic ketoacidosis at diagnosis and the level of glycemic control of children with T1D influenced the tear proteome. Tear proteomics analysis revealed a different proteome pattern in children with T1D compared to healthy controls offering insights on deregulated biological processes underlying the pathogenesis of T1D. Differences within the T1D group could unravel biomarkers for early detection of long-term complications of T1D.

Sex-Specific Cytokine, Chemokine, and Growth Factor Signatures in T1D Patients and Progressors.

While studies have reported altered levels of cytokines in type 1 diabetes (T1D) patients, the results are inconsistent, likely because of variable factors. This study tests the hypothesis that there are sex-based differences in cytokine levels in T1D, prior to and after disease onset. We analyzed 48 blood cytokine, chemokine, and growth factor levels using a multiplex assay. We found only two cytokines, M-CSF and IL-6, with significant differences between T1D patients (n=25) versus controls overall (n=25). However, we identified notable alterations when comparing sex-age-matched controls and T1D samples. Inflammatory cytokines (TNF-α, IL-6, IL-1a), Th2 cytokines (IL-4, IL-13), and chemokines (MIP-1α, RANTES, MIP-3) were lower in female T1D patients compared to female controls, but not in males. IL-22 was lower in female T1D patients compared to female controls, while it was higher in male T1D patients compared to male controls. In contrast, growth factors (EGF, PDGF-AB/BB) were higher in male T1D patients compared to male controls. In T1D progressors (children who developed the disease years after the sample collection, n=16-21), GROa was lower compared to controls in both sexes. Our findings underscore the importance of understanding sex-specific differences in T1D pathogenesis and their implications for developing personalized treatments.

The Effect of Vitamin D Deficiency on Immune-Related Hub Genes: A Network Analysis Associated With Type 1 Diabetes.

Background Type 1 diabetes (T1D) is an autoimmune disorder that results in the destruction of pancreatic beta cells, causing a shortage of insulin secretion. The development of T1D is influenced by both genetic predisposition and environmental factors, such as vitamin D. This vitamin is known for its ability to regulate the immune system and has been associated with a decreased risk of T1D. However, the specific ways in which vitamin D affects immune regulation and the preservation of beta cells in T1D are not yet fully understood. Gaining a better understanding of these interactions is essential for identifying potential targets for preventing and treating T1D. Methods The analysis focused on two Gene Expression Omnibus (GEO) datasets, namely, GSE55098 and GSE50012, to detect differentially expressed genes (DEGs). Enrichr (Ma'ayan Laboratory, New York, NY) was used to perform enrichment analysis for the Gene Ontology (GO) biological process and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The Search Tool for the Retrieval of Interacting Genes 12.0 (STRING) database was used to generate a protein-protein interaction (PPI) network. The Cytoscape 3.10.1 (Cytoscape Team, San Diego, CA) was used to analyze the PPI network and discover the hub genes. Results The DEGs in both datasets were identified using the GEO2R tool, with a particular focus on genes exhibiting contrasting regulations. Enrichment analysis unveiled the participation of these oppositely regulated DEGs in processes relevant to the immune system. Cytoscape analysis of the PPI network revealed five hub genes, MNDA, LILRB2, FPR2, HCK, and FCGR2A, suggesting their potential role in the pathogenesis of T1D and the response to vitamin D. Conclusion The study elucidates the complex interaction between vitamin D metabolism and immune regulation in T1D. The identified hub genes provide important knowledge on the molecular pathways that underlie T1D and have the potential to be targeted for therapeutic intervention. This research underscores the importance of vitamin D in the immune system's modulation and its impact on T1D development.