It is unknown whether there is a gene signature in pancreas which is associated with type 1 diabetes (T1D). We performed partial pancreatectomies on 30-day preinsulitic, diabetes-prone BioBreeding (BBdp) rats to prospectively identify factors involved in early prediabetes. Microarrays of the biopsies revealed downregulation of endoplasmic reticulum (ER) stress, metabolism and apoptosis. Based on these results, additional investigations compared gene expression in control (BBc) and BBdp rats age ~8, 30 and 60 days using RT-qPCR. Neonates had increased ER stress gene expression in pancreas. This was associated with decreased insulin, cleaved caspase-3 and Ins1 whereas Gcg and Pcsk2 were increased. The increase in ER stress was not sustained at 30 days and decreased by 60 days. In parallel, the liver gene profile showed a similar signature in neonates but with an early decrease of the unfolded protein response (UPR) at 30 days. This suggested that changes in the liver precede those in the pancreas. Tnf and Il1b expression was increased in BBdp pancreas in association with increased caspase-1, cleaved caspase-3 and decreased proinsulin area. Glucagon area was increased in both 30-day and 60-day BBdp rats. Increased colocalization of BIP and proinsulin was observed at 60 days in the pancreas, suggesting insulin-related ER dysfunction. We propose that dysregulated metabolism leads to ER stress in neonatal rats long before insulitis, creating a microenvironment in both pancreas and liver that promotes autoimmunity.

Immunoregulatory and regenerative processes are activated in the pancreas during the development of type 1 diabetes (T1D) but are insufficient to prevent the disease. We hypothesized that the induction of cytoprotective heme oxygenase-1 (HO-1) by cobalt protophoryrin (CoPP) would prevent T1D by promoting anti-inflammatory and pro-repair processes. Diabetes-prone BioBreeding rats received ip CoPP or saline twice per week for 3 weeks, starting at 30 days and were monitored for T1D. Immunohistochemistry, confocal microscopy, quantitative RT-PCR, and microarrays were used to evaluate postinjection pancreatic changes at 51 days, when islet inflammation is first visible. T1D was prevented in CoPP-treated rats (29% vs 73%). Pancreatic Hmox1 was up-regulated along with islet-associated CD68(+)HO-1(+) cells, which were also observed in a striking peri-lobular interstitial infiltrate. Most interstitial cells expressed the mesenchymal marker vimentin and the hematopoietic marker CD34. Spindle-shaped, CD34(+)vimentin(+) cells coexpressed collagen V, characteristic of fibrocytes. M2 macrophage factors Krüppel-like factor 4, CD163, and CD206 were expressed by interstitial cells, consistent with pancreatic upregulation of several M2-associated genes. CoPP upregulated islet-regenerating REG genes and increased neogenic REG3β(+) and insulin(+) clusters. Thus, short-term induction of HO-1 promoted a protective M2-like milieu in the pancreas and recruited mesenchymal cells, M2 macrophages, and fibrocytes that imparted immunoregulatory and pro-repair effects, preventing T1D.

Chronic Disease Program (M.H., G.-S.W., C.P., J.A.C., J.A.N., A.S., F.W.S.), Ottawa Hospital Research Institute, Ottawa, Ontario, Canada K1H 8L6 and Departments of Biochemistry, Microbiology, and Immunology (M.H., C.P., A.J.M., J.A.N., F.W.S.) and Medicine (F.W.S.), University of Ottawa, Ottawa, Ontario, Canada K1H 8L6; Food Directorate (A.J.M.), Health Products and Food Branch, Health Canada, Ottawa, Ontario, Canada K1A 0K9

Cathelicidin antimicrobial peptide (CAMP) is a naturally occurring secreted peptide that is expressed in several organs with pleiotropic roles in immunomodulation, wound healing, and cell growth. We previously demonstrated that gut Camp expression is upregulated when type 1 diabetes-prone rats are protected from diabetes development. Unexpectedly, we have also identified novel CAMP expression in the pancreatic β-cells of rats, mice, and humans. CAMP was present even in sterile rat embryo islets, germ-free adult rat islets, and neogenic tubular complexes. Camp gene expression was downregulated in young BBdp rat islets before the onset of insulitis compared with control BBc rats. CAMP treatment of dispersed islets resulted in a significant increase in intracellular calcium mobilization, an effect that was both delayed and blunted in the absence of extracellular calcium. Additionally, CAMP treatment promoted insulin and glucagon secretion from isolated rat islets. Thus, CAMP is a promoter of islet paracrine signaling that enhances islet function and glucoregulation. Finally, daily treatment with the CAMP/LL-37 peptide in vivo in BBdp rats resulted in enhanced β-cell neogenesis and upregulation of potentially beneficial gut microbes. In particular, CAMP/LL-37 treatment shifted the abundance of specific bacterial populations, mitigating the gut dysbiosis observed in the BBdp rat. Taken together, these findings indicate a novel functional role for CAMP/LL-37 in islet biology and modification of gut microbiota.

SummaryThe gut immune system and its modification by diet have been implicated in the pathogenesis of type 1 diabetes (T1D). Therefore, we investigated gut immune status in non‐diabetes‐prone LEW.1AR1 and diabetes‐prone LEW.1AR1‐iddm rats and evaluated the effect of a low antigen, hydrolysed casein (HC)‐based diet on gut immunity and T1D. Rats were weaned onto a cereal‐based or HC‐based diet and monitored for T1D. Strain and dietary effects on immune homeostasis were assessed in non‐diabetic rats (50–60 days old) and rats with recent‐onset diabetes using flow cytometry and immunohistochemistry. Immune gene expression was analysed in mesenteric lymph nodes (MLN) and jejunum using quantitative RT‐PCR and PCR arrays. T1D was prevented in LEW.1AR1‐iddm rats by feeding an HC diet. Diabetic LEW.1AR1‐iddm rats had fewer lymphoid tissue T cells compared with LEW.1AR1 rats. The percentage of CD4+ Foxp3+ regulatory T (Treg) cells was decreased in pancreatic lymph nodes (PLN) of diabetic rats. The jejunum of 50‐day LEW.1AR1‐iddm rats contained fewer CD3+ T cells, CD163+ M2 macrophages and Foxp3+ Treg cells. Ifng expression was increased in MLN and Foxp3 expression was decreased in the jejunum of LEW.1AR1‐iddm rats; Ifng/Il4 was decreased in jejunum of LEW.1AR1‐iddm rats fed HC. PCR arrays revealed decreased expression of M2‐associated macrophage factors in 50‐day LEW.1AR1‐iddm rats. Wheat peptides stimulated T‐cell proliferation and activation in MLN and PLN cells from diabetic LEW.1AR1‐iddm rats. LEW.1AR1‐iddm rats displayed gut immune cell deficits and decreased immunoregulatory capacity, which were partially corrected in animals fed a low antigen, protective HC diet consistent with other models of T1D.

We are exposed to millions of microbial and dietary antigens via the gastrointestinal tract, which likely play a key role in type 1 diabetes (T1D). We differentiated the effects of these two major environmental factors on gut immunity and T1D. Diabetes-prone BioBreeding (BBdp) rats were housed in specific pathogen-free (SPF) or germ-free (GF) conditions and weaned onto diabetes-promoting cereal diets or a protective low-antigen hydrolyzed casein (HC) diet, and T1D incidence was monitored. Fecal microbiota 16S rRNA genes, immune cell distribution, and gene expression in the jejunum were analyzed. T1D was highest in cereal-SPF (65%) and cereal-GF rats (53%) but inhibited and delayed in HC-fed counterparts. Nearly all HC-GF rats remained diabetes-free, whereas HC-fed SPF rats were less protected (7 vs. 29%). Bacterial communities differed in SPF rats fed cereal compared with HC. Cereal-SPF rats displayed increased gut CD3(+) and CD8α(+) lymphocytes, ratio of Ifng to Il4 mRNA, and Lck expression, indicating T-cell activation. The ratio of CD3(+) T cells expressing the Treg marker Foxp3(+) was highest in HC-GF and lowest in cereal-SPF rats. Resident CD163(+) M2 macrophages were increased in HC-protected rats. The cathelicidin antimicrobial peptide (Camp) gene was upregulated in the jejunum of HC diet-protected rats, and CAMP(+) cells colocalized with CD163. A cereal diet was a stronger promoter of T1D than gut microbes in association with impaired gut immune homeostasis.

Antibodies against the wheat storage globulin Glo-3A from a patient with both type 1 diabetes (T1D) and celiac disease were enriched to identify potential molecular mimicry between wheat antigens and T1D target tissues. Recombinant Glo-3A was used to enrich anti-Glo-3A immunoglobulin G antibodies from plasma by batch affinity chromatography. Rat jejunum and pancreas, as well as human duodenum and monocytes were probed, and binding was evaluated by immunohistochemistry and confocal microscopy. Glo-3A-enriched antibodies bound to a specific subset of cells in the lamina propria of rat jejunum that co-localized mostly with a marker of resident, alternatively activated CD163-positive (CD163 + ) macrophages. Blood monocytes and macrophage-like cells in human duodenum were also labelled with the enriched antibodies. Blocking studies revealed that binding to CD163 + macrophages was not due to cross-reactivity with anti-Glo-3A antibodies, but rather to non-Glo-3A antibodies co-purified during antibody enrichment. The novel finding of putative autoantibodies against tolerogenic intestinal CD163 + macrophages suggests that regulatory macrophages were targeted in this patient with celiac disease and T1D.

Type I diabetes is inhibited in diabetes-prone BioBreeding (BBdp) rats fed a low-antigen hydrolyzed casein (HC) diet. In cereal-fed BBdp rats, islet expansion is defective accompanied by a futile upregulation of islet neogenesis without increased islet mass, due to a subtle blockage in islet cell cycle. We hypothesized that islet growth is enhanced before insulitis in HC-fed young BBdp rats and that islet neogenesis could be stimulated by a trophic factor, islet neogenesis-associated protein (INGAP). beta-Cell homeostasis was analyzed using immunohistochemistry, morphometry, laser capture microdissection and RT-PCR in BBdp rats fed HC or cereal diets. beta-cell proliferation in small and medium islets, and the number and area fraction of medium and large islets were increased in HC-fed animals. In situ islet cell cycle analysis revealed an increased proportion of proliferating S + G2 cells in medium and large islets of 25-45 day HC-fed rats. Expression of the cell cycle inhibitor, p16(INK4a) correlated with islet size and the percentage of p16(INK4a+) beta-cells increased in HC-fed BBdp rats, likely reflecting an increase in large islet area fraction. In HC-fed rats, extra-islet insulin(+) clusters (EIC), insulin(+) duct cells, large islet area fraction, and beta-cell mass were increased. Neurogenin-3 and Pdx-I, markers of beta-cell progenitors, were increased in EIC of weanling HC-fed rats. Daily injection of INGAP (30-45 days) increased the number of small islets, total islets, and insulin(+) cells in small ducts. Thus, in BBdp rats fed a protective HC diet, beta-cell expansion is enhanced through increased beta-cell proliferation and stimulation of islet neogenesis. J. Cell. Physiol. J. Cell. Physiol. 224: 501-508, 2010. (C) 2010 Wiley-Liss, Inc.

The gastrointestinal tract represents the largest immune interface with the environment. Exposure to large numbers of dietary and microbial antigens requires complex and highly regulated intestinal immune responses by different immune cell types for the maintenance of oral tolerance. Defective immune homeostasis can cause gut barrier dysfunction and breakdown of tolerance, leading to chronic inflammation and autoimmunity. In this review, we summarize the key immune cell populations involved in oral tolerance. We also describe diet-modifiable aspects of gut immunity that alter the intricate balance between inflammatory and tolerogenic immune responses in the gut and contribute to disease development.

Diabetes incidence is reduced in diabetes-prone BioBreeding (BBdp) rats fed a hydrolyzed casein (HC) diet compared with a standard cereal-based rodent diet such as NTP-2000 (NTP). To further characterize the basis of this protective effect, islet neogenesis, apoptosis and cell proliferation were analyzed using immunohistochemistry, morphometry, Laser Capture Microdissection, and RT-PCR in BBdp rats weaned onto an NTP or HC diet at 23 d and sacrificed at 25, 30, and 45 d. Islet area fraction was greater in medium and large islets of HC-fed rats at 30 d and in large islets only at 45 d. There were more small islets in HC-fed rats both at 30 and 45 d and β-cell mass was significantly greater at 45 d. Cell cycle analysis revealed an increased ratio of S+G2/G0+G1 in HC-fed animals between 25 and 45 d. PDX-1+ clusters (<4 cells) were increased in HC-fed rats, whereas extra-islet insulin+ clusters (EIC) and insulin+ cells in ducts, representative of islet neogenesis, were increased at 45 d. Ngn3 mRNA was higher in EIC of HC-fed rats at 24 d. Glucagon-like peptide-1 receptor protein and mRNA were increased in islets of HC-fed rats. In summary, the protective HC diet increased β-cell mass in diabetes-prone rats through upregulation of islet neogenesis and β-cell proliferation. (GSW and LMK contributed equally; Supported by Canadian Diabetes Association and Canadian Institutes of Health Research)

We reported previously that young BioBreeding diabetes-prone (BBdp) rats display increased neogenic extra-islet insulin+ clusters (EICs, <4 insulin+ cells) without an increase in β-cell mass. Therefore, we investigated the possibility that abnormal islet expansion occurs in BBdp rats before the appearance of islet inflammation. Islet expansion was analyzed in pancreata from 14 to 45 day BBdp and control (BioBreeding control, BBc) rats using immunohistochemistry, morphometry, laser capture microdissection and reverse transcriptase-PCR. mRNA expression for Neurogenin-3, a developmental marker of endocrine progenitors, was three-fold greater in EIC of weanling BBdp and BBc rats compared with islet cells. With increasing age (14–30 days), Neurogenin-3 expression decreased in EIC and increased in islets. In BBdp rats, EIC number and β-cell proliferation within EIC was greater compared with BBc animals; apoptosis did not differ. The area of small and medium islets in BBdp rats was greater than BBc rats between 14 and 30 days, but this did not result in increased total islet area or β-cell mass. In addition, the number and area of very large islets was low at 45 days. The frequency of proliferating β-cells decreased with increasing islet size in BBdp but was constant in BBc rats. Cell cycle analysis of islets revealed more G1 cells and fewer G2 cells in BBdp rats. The ratio of cyclinD2/Cdkn1a, genes that respectively promote or inhibit cell cycle progression, was decreased in BBdp islets. These results suggest that despite increased islet neogenesis, the capacity for islet expansion in diabetes-prone rats is compromised possibly due to decreased proliferative capacity with increasing islet size associated with a partial block at the G1/S cell cycle boundary in islet cells.

Type 1 diabetes results from autoimmune destruction of pancreatic β-cells. β-cell neoformation has been shown to increase in response to islet neogenesis-associated protein (INGAP) in hamsters. Using immunohistochemistry and morphometry, INGAP effects in diabetes-prone BioBreeding (BBdp) rats were investigated. At 23 d, rats were fed a hydrolyzed casein (HC) diet or a cereal-based NTP-2000 diet. Rats were administered saline intraperitoneally (n=7) or 250 μg of INGAP-peptide (n=7) twice daily for two weeks and killed at 45 d. In HC-fed rats, the number of small and total islets was greater in INGAP-treated rats compared with controls (59 ± 17 vs. 37 ± 8 islets/cm2, mean ± SD, p=0.01 and 143 ± 19 vs. 114 ± 24 islets/cm2, p<0.03). In rats given INGAP, insulin+extra-islet clusters, markers of islet neoformation, were more frequent in those fed HC compared with NTP (82 ± 20 vs. 46 ± 8 clusters/cm2, p<0.01). HC-fed rats given INGAP had a higher percentage of BrdU+/insulin+ clusters compared with controls, indicating enhanced proliferation (26.0 ± 7.6 vs. 13.1 ± 2.7%, p<0.001). INGAP-treated rats fed either an HC or NTP diet had a higher percentage of insulin+ cells in small ducts compared with saline controls. These findings demonstrate the presence of INGAP-stimulated β-cell neoformation, some aspects of which were further enhanced in BBdp rats fed a protective HC diet. (Supported by CDA and CIHR; CP and GSW contributed equally).