Introduction
Defensins are small molecules (2–5 kDa) that play an important role in the innate and adaptive defence system. They belong to a large family of antimicrobial peptides of ancient conservation. Humans express two subfamilies, Alpha- and Beta-defensins which differ in the length of cysteine peptide segments and pairing of the cysteines by disulphide bonds [
1‐
3]. The Alpha family is most often regulated on the secretory level by release from granules in neutrophils or Paneth cells upon a trigger in the environment, while the Beta family is regulated transcriptionally [
1,
4,
5]. Epithelial cells in various tissues consistently express certain Beta-defensins, such as hbD-1 [
5,
6]. Inflammation or exposure to bacterial toxins in the environment can lead to modulation and increase of Beta-defensins [
6,
7].
Defensin families differ in structure and regulation, they also differ in their antimicrobial action and modulation of the immune system. Beta-defensins have antimicrobial, anti-viral and chemotactic properties [
8] and are considered to have a role in the adaptive immune response. The antimicrobial effect has predominantly been shown against gram negative bacteria, such as
Escherichia coli [
9]. Alpha-defensins exhibit potent antimicrobial activity against both gram-negative and gram-positive bacteria by membrane disruption. Additionally, they have been shown to inhibit the adhesion of enveloped viruses to host cells and prevent uncoating of capsid viruses [
5]. Thus far, there has been no description of a link to the adaptive system for the Alpha-defensin family.
Defensins can also drive an inflammatory process into a chronic pathological state involving other inflammatory mediators and the adaptive immune system [
10]. Psoriasis and Crohn's disease are two clinically significant examples where altered defensin expression plays an important role [
11].
Although defensins have been extensively researched in the skin, gut, and oral cavity, their function in the pancreas remains largely unknown [
12,
13]. Several studies describe ongoing inflammation of the pancreas in subjects with recent onset Type 1 diabetes [
14‐
17]. There is also an association between bacterial infections during infancy and a high risk of developing islet-autoantibodies and Type 1 diabetes [
18]. Translocation of bacteria and viruses from the duodenum to the pancreas could trigger the activation of innate inflammatory immune response [
17,
19]. The inflammatory response driven by infectious agents has been suggested as triggers for Type 1 diabetes [
20,
21]. The aim of this study was to characterize the expression of different defensins in non-diabetic organ donors of different ages as well as in donors with Type 1 diabetes with different disease duration to examine a tentative role in Type 1 diabetes.
Materials and methods
Ethics
The research work conducted using human tissue followed guidelines outlined in the Declaration of Helsinki. Pancreatic tissue was procured from organ donors, and consent to use it for research purposes was obtained from the next of kin either verbally by the attending physician or from an online database. Procedures were fully documented according to Swedish law and regional standards. The present study utilized pancreatic samples from patients who were part of the DiViD study, approved by The Norwegian Governments Regional Ethics Committee. Prior to participation, patients were provided comprehensive oral and written information from the diabetologist and surgeon separately.
Human pancreatic samples
Biopsies from 41 human pancreases were included in the study, divided into five different groups. One donor died at onset, four living patients (DiViD-study) [
22], 13 organ donors with longstanding Type 1 diabetes, 19 non-diabetic organ donors and four organ donors under five years of age (Table
1). The adult non-diabetic individuals were matched to the subjects with Type 1 diabetes for age, sex and BMI. Three parts of the pancreas, head, body and tail, was chosen from each donor where this was possible.
Table 1
Detailed list of antibodies used
Anti α-1 (Abcam) | – | 1:200 | Human spleen |
Anti β-1 Abcam) | M11-14b-D10 | 1:100 | – |
Anti β-2 (Abcam) | – | 1:500 | Human tonsil |
Anti β-3 (Abcam) | – | 1:200 | – |
Anti-Cathelicidin (Abcam) | – | 1:1000 | Human spleen |
Anti-GP2 (Abcam) | GP2/1712 | 1:200 | Human pancreas |
Anti-Neutrophil-4 (Abcam) | – | 1:50 | Human spleen |
Anti-REG3A (Abcam) | – | 1:100 | Human duodenum |
Anti-CD45 (Agilent) | 2B11 + PD7/26 | 1:75 | Human spleen |
Anti-CD68 (Agilent) | KP1 | 1:50 | Human spleen |
Anti-CD3 (Abcam) | 265-3kl | 1:50 | Human spleen |
Anti-NES (Invitrogen) | F7.2.3B | 1:25 | Human spleen |
The organs were procured within the Nordic Network for Clinical Islet Transplantation.
Immunohistochemistry
Formalin-fixed and paraffin-embedded tissue were cut into 6 µm sections, consecutive sections were processed and labeled using a standard immunoperoxidase technique. All antigens were unmasked by heat-induced epitope retrieval using pH 6.0 or pH 9.0 according to recommendations by the manufacturer. Primary antibodies specific for CD45, insulin, synapthofysin and eight different defensin molecules were used (Table
2). Bound antibodies were visualized using Dako EnVision or EnVision DuoFLEX Doublestain system (Agilent, California, USA) and diaminobenzidine-based substrate (Agilent, California, USA). Sections were counterstained with hematoxylin, dehydrated, mounted and analyzed by light microscope (Leica, Germany). Positive controls were running in parallel with each defensin and isotype mAbs were used as negative controls. For double staining, Cathelicidin and immune cells antibodies toward CD45, CD68, NES and CD3 were used and visualized by immunofluorescence. Confocal microscope Zeiss LSM700 (Zeiss,Germany
) and software Zen black 3.0 SR (Zeiss, Germany) was used to analyze the slides.
Table 2
Donor characterization
Number of subjects | 23 | 1 | 4 | 13 |
Duration of Type-1 diabetes (weeks) | N/A | 0 | 3–9 | > 200 |
Age | 34.5 ± 21.9 | 29 | 28.5 ± 4.8 | 44.8 ± 20.4 |
Female | 10 | – | 2 | 7 |
Male | 13 | 1 | 2 | 7 |
Body mass index (kg/m2) | 24.3 ± 5.22 | 24.2 | 22.8 ± 2.2 | 25.7 ± 5.12 |
Hba1c | 37.5 ± 3.5 | 95 | 72.75 ± 4.32 | 73 ± 25 |
Analysis and statistical analysis
All slides were analyzed by two independent investigators blinded with regard to donor type. The analysis was performed as a semi-quantitative method using a standard four scale IHC score system (0,1,2,3). The IHC score was set by combining three parameters. (1) staining intensity, (2) proportion of stained pancreatic area, and (3) staining pattern. The staining was evaluated with regard to cytoplasmic and nucleic expression of the various defensins in exocrine and endocrine pancreas, as well as in blood vessels, connective tissue, ducts and adipose tissue of the pancreas. Immune cells were analyzed by comparing overlay of staining between Cathelicidin and immune cell staining.
Statistical analysis
A mean IHC score for each donor was calculated from the examined sections.
The mean IHC score for each subject was used in the box-plot figures and statistical analysis. Statistical significance between groups were calculated by performing a Kruskal–Wallis analysis followed by Dunns multiple comparisons. The significance level was < 0.05 by using Graphpad prism 9 software.
Discussion
Herein we report a markedly reduced expression of central defensins belonging to different families in both exocrine and endocrine tissue of the pancreas in subjects with Type 1 diabetes as compared to non-diabetic controls. Moreover, we found a positive correlation in non-diabetic subjects between inflammation in both endocrine and exocrine tissue and expression of several of the defensins. Notably, this correlation was markedly reduced or even absent in subjects with Type 1 diabetes. Among the three beta-defensins expression of Beta-1 is of most importance against bacterial infections [
23]. Beta defensins also modulate the adaptive immune response by promoting chemotactic activities for immature dendritic cells and memory T-cells [
7,
24]. Expression of Beta-1 was reduced in subjects with Type 1 diabetes compared to non-diabetic adult controls. Glucose homeostasis seems to have an important role in controlling expression of Beta-1 [
23,
25]. However, no difference in expression of Beta-1 could be observed between the Type 1 diabetes groups suggesting a reduced expression of Beta-1 in subjects with Type 1 diabetes per se.
The Alpha defensins are part of a major interplay between healthy gut homeostasis, microbiota and innate immune system. The defensin resides in granules which are released when bacteria is present [
1]. Decreased expression of Alpha-defensins cause an imbalance of this intricate system which has been considered as a trigger for inflammatory events and has been reported in Crohn´s disease [
26]. Similarly, decreased expression of Alpha-1 defensin in subjects with Type 1 diabetes was found.
Children and subjects with Type 1 diabetes also had a marked reduction in the expression of Cathelicidin when compared with non-diabetic adult controls. With increasing age and therefore longer duration of Type 1 diabetes the expression pattern changed. In the oldest subjects with the longest disease duration expression showed a similar staining pattern as non-diabetic adults. Expression of Cathelicidin was also found in macrophages and neutrophilic granulocytes scattered in the exocrine pancreas especially in the Type 1 diabetes subjects. T-cells in the insulitic lesions in subjects with recent onset Type 1 diabetes were negative. These findings are in agreement with previous reports on expression of Cathelicidin in granules of neutrophils and macrophages [
27]. Cathelicidin is not a defensin per se, but it is classified as an antimicrobial peptide and has a wide range of immunomodulatory effects [
28]. Expression of Cathelicidin is dependent on vitamin-D [
29]. Several studies have implied a correlation between induced risk of Type 1 diabetes and vitamin-D deficiency [
30,
31]. Vitamin-D also plays an important role in beta-cell function and development [
29,
30,
32,
33].
The defensin REG3A showed a slightly lower expression in children and subjects with recent onset Type 1 diabetes when compared with non-diabetic adults. Notably, subjects with longstanding Type 1 diabetes on the other hand had a slightly higher expression than non-diabetic adults. REG3A has been reported in several studies to have a protective role for beta-cells when overexpressed [
34‐
36]. REG3A has also been shown to be altered by mild hyperglycemia [
37]. Even if no statistically significant differences could be found, the findings presented support the view that islets in subjects with Type 1 diabetes suffer from a relative deficiency of REG3A [
34‐
37].
Defensins have an important role in regulating inflammation and acquired immunity. In non-diabetic donors a correlation between pancreatic inflammation and expression of Beta-1, Alpha-1, Cathelicidin andREG3A were found. Inflammation in donors with Type 1 diabetes was more intense and affected larger areas of the exocrine pancreas when compared with that observed in non-diabetic subjects. Even so, expression of defensins remained low. Obtained findings implies that there could be a disturbance in how the innate immune system responds in individuals with Type 1 diabetes, tentatively causing prolonged local inflammation with negative effects on exocrine and endocrine homeostasis. Indeed a patchy inflammation affecting both the exocrine and endocrine pancreas has frequently been reported in subjects with Type 1 diabetes [
14‐
17].
Possible limitations of our study are the relatively low numbers of biopsies available from subjects with newly diagnosed Type 1 diabetes. However, it is well established that this type of biopsies remains rare. Another point of consideration is that the IHC staining allows detailed analysis of the cellular expression pattern of a specific protein, but it is not optimal in quantifying the exact amount of protein. However, for proteins with posttranslational modifications, e.g. defensins, alternative quantitative techniques such as in situ hybridization are not applicable.
Collectively, presented findings implicate a disturbance in the innate immune response in subjects with Type 1 diabetes. Reduced or even lack of expression of defensins could cause prolonged and exaggerated inflammation and dysregulation of the bridge to the adaptive immune responses [
7,
24]. A similar reduced expression of defensins has been seen in other inflammatory diseases [
4,
7]. The findings presented support an important role for defensins in Type 1 diabetes and further studies on the role of the innate immune system in Type 1 diabetes is needed.
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