Pancreas Whole Tissue Transcriptomics Highlights the Role of the Exocrine Pancreas in Patients With Recently Diagnosed Type 1 Diabetes

Although type 1 diabetes (T1D) is primarily a disease of the pancreatic beta-cells, understanding of the disease-associated alterations in the whole pancreas could be important for the improved treatment or the prevention of the disease. We have characterized the whole-pancreas gene expression of patients with recently diagnosed T1D from the Diabetes Virus Detection (DiViD) study and non-diabetic controls. Furthermore, another parallel dataset of the whole pancreas and an additional dataset from the laser-captured pancreatic islets of the DiViD patients and non-diabetic organ donors were analyzed together with the original dataset to confirm the results and to get further insights into the potential disease-associated differences between the exocrine and the endocrine pancreas. First, higher expression of the core acinar cell genes, encoding for digestive enzymes, was detected in the whole pancreas of the DiViD patients when compared to non-diabetic controls. Second, In the pancreatic islets, upregulation of immune and inflammation related genes was observed in the DiViD patients when compared to non-diabetic controls, in line with earlier publications, while an opposite trend was observed for several immune and inflammation related genes at the whole pancreas tissue level. Third, strong downregulation of the regenerating gene family (REG) genes, linked to pancreatic islet growth and regeneration, was observed in the exocrine acinar cell dominated whole-pancreas data of the DiViD patients when compared with the non-diabetic controls. Fourth, analysis of unique features in the transcriptomes of each DiViD patient compared with the other DiViD patients, revealed elevated expression of central antiviral immune response genes in the whole-pancreas samples, but not in the pancreatic islets, of one DiViD patient. This difference in the extent of antiviral gene expression suggests different statuses of infection in the pancreas at the time of sampling between the DiViD patients, who were all enterovirus VP1+ in the islets by immunohistochemistry based on earlier studies. The observed features, indicating differences in the function, status and interplay between the exocrine and the endocrine pancreas of recent onset T1D patients, highlight the importance of studying both compartments for better understanding of the molecular mechanisms of T1D.


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Additional details regarding the data analysis of the preprocessed datasets All data analyses of the preprocessed datasets were performed using the R statistical programming software environment version 3.6.0 [14].

Comparison of the DiViD cases
To compare the expression of genes between the DiViD cases, the gene expression in each dataset was z-score standardized. Furthermore, to allow for the comparison of expression patterns between cases over the examined different datasets, only the DiViD cases (not the varying non-diabetic organ donor controls) were used in the z-score standardization within each dataset. Thus, the z-score standardized expression for gene i in sample j in any examined dataset was defined as: where is the expression of gene i in sample j, is the averaged expression of gene i over the DiViD cases and is the standard deviation of gene i over the DiViD cases.

Cell type proportion estimation
Following the recommendations in the CIBERSORT software, the quantile normalization option was disabled for the RNASeq dataset. Measurements from the acinar, alpha, beta, delta, and ductal pancreatic cell types of 20-40 year old individuals were utilized, excluding cells labeled as low quality.

Functional enrichment analysis
For Figure 3A, the expression of the differentially expressed genes in the whole pancreas transcriptome 1 and the pancreatic islet transcriptome involved in the terms GO:0006955 (immune response) and GO:0006954 (inflammatory response) were explored and compared.
To determine the subcellular localization of gene products in the terms GO:0006955 (immune response) and GO:0006954 (inflammatory response) for Figure 3B, Ingenuity Pathway Analysis (IPA) (QIAGEN Inc.) [15] was applied. The interactions, expression and subcellular localization among the immune and inflammatory response gene products for Figure 3B were visualized using Cytoscape version 3.7.2 [16]. Genes   80  70  60  50  40  30  20  10  0  10  20  30 regulation of cellular component organization regulation of cell differentiation response to oxygen−containing compound regulation of tube size cellular ion homeostasis positive regulation of cellular component organization cellular metal ion homeostasis cytokine−mediated signaling pathway regulation of blood vessel size rhythmic process regulation of cell proliferation immune response regulation of cell death aging cation homeostasis response to mechanical stimulus positive regulation of cell death ion homeostasis platelet degranulation regulation of programmed cell death angiogenesis inorganic ion h +omeostasis positive regulation of response to stimulus regulation of apoptotic process metal ion homeostasis leukocyte migration regulation of cell migration positive regulation of multicellular organismal process blood vessel morphogenesis collagen catabolic process digestion vasculature development regulation of multicellular organismal development blood vessel development regulation of transport response to cytokine regulation of locomotion biological adhesion cell adhesion multicellular organism metabolic process enzyme linked receptor protein signaling pathway collagen metabolic process circulatory system development cardiovascular system development regulation of wound healing cellular response to growth factor stimulus cellular response to endogenous stimulus multicellular organism catabolic process regulation of cell motility regulation of cellular component movement cell surface receptor signaling pathway regulation of response to wounding response to growth factor movement of cell or subcellular component negative regulation of multicellular organismal process response to endogenous stimulus multicellular organismal macromolecule metabolic process locomotion localization of cell cell motility cell migration wound healing acute−phase response response to wounding response to external stimulus cellular response to organic substance defense response response to organic substance cellular response to chemical stimulus acute inflammatory response inflammatory response extracellular structure organization extracellular matrix organization Supplementary Figure 3. Related to Figure 4A. A signature of 45 genes was discovered differentiating DiViD case 6 from the other DiViD patients and also the non-diabetic organ donor controls. The Z-score standardized expression of these genes is shown in the whole pancreas transcriptome 1.