Gut immune deficits in LEW.1AR1-iddm rats partially overcome by feeding a diabetes-protective diet.

The 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.

Islet infiltration, cytokine expression and beta cell death in the NOD mouse, BB rat, Komeda rat, LEW.1AR1-iddm rat and humans with type 1 diabetes.

AIMS/HYPOTHESIS: Research on the pathogenesis of type 1 diabetes relies heavily on good animal models. The aim of this work was to study the translational value of animal models of type 1 diabetes to the human situation. METHODS: We compared the four major animal models of spontaneous type 1 diabetes, namely the NOD mouse, BioBreeding (BB) rat, Komeda rat and LEW.1AR1-iddm rat, by examining the immunohistochemistry and in situ RT-PCR of immune cell infiltrate and cytokine pattern in pancreatic islets, and by comparing findings with human data. RESULTS: After type 1 diabetes manifestation CD8(+) T cells, CD68(+) macrophages and CD4(+) T cells were observed as the main immune cell types with declining frequency, in infiltrated islets of all diabetic pancreases. IL-1ß and TNF-α were the main proinflammatory cytokines in the immune cell infiltrate in NOD mice, BB rats and LEW.1AR1-iddm rats, as well as in humans. The Komeda rat was the exception, with IFN-γ and TNF-α being the main cytokines. In addition, IL-17 and IL-6 and the anti-inflammatory cytokines IL-4, IL-10 and IL-13 were found in some infiltrating immune cells. Apoptotic as well as proliferating beta cells were observed in infiltrated islets. In healthy pancreases no proinflammatory cytokine expression was observed. CONCLUSIONS/INTERPRETATION: With the exception of the Komeda rat, the animal models mirror very well the situation in humans with type 1 diabetes. Thus animal models of type 1 diabetes can provide meaningful information on the disease processes in the pancreas of patients with type 1 diabetes.

Glucose metabolism and pancreatic defects in spinal muscular atrophy.

OBJECTIVE: Spinal muscular atrophy (SMA) is the number 1 genetic killer of young children. It is caused by mutation or deletion of the survival motor neuron 1 (SMN1) gene. Although SMA is primarily a motor neuron disease, metabolism abnormalities such as metabolic acidosis, abnormal fatty acid metabolism, hyperlipidemia, and hyperglycemia have been reported in SMA patients. We thus initiated an in-depth analysis of glucose metabolism in SMA. METHODS: Glucose metabolism and pancreas development were investigated in the Smn(2B/-) intermediate SMA mouse model and type I SMA patients. RESULTS: Here, we demonstrate in an SMA mouse model a dramatic cell fate imbalance within pancreatic islets, with a predominance of glucagon-producing α cells at the expense of insulin-producing ß cells. These SMA mice display fasting hyperglycemia, hyperglucagonemia, and glucose resistance. We demonstrate similar abnormalities in pancreatic islets from deceased children with the severe infantile form of SMA in association with supportive evidence of glucose intolerance in at least a subset of such children. INTERPRETATION: Our results indicate that defects in glucose metabolism may play an important contributory role in SMA pathogenesis.

Antibodies to the wheat storage globulin Glo-3A in children before and at diagnosis of celiac disease.

OBJECTIVE: Celiac disease (CD) is an autoimmune disease triggered by exposure to gluten-containing foods. IgA autoantibodies to tissue transglutaminase (TTG) are elevated in CD, but little is known about the gastrointestinal state before the appearance of TTG. Antibodies to wheat storage globulin Glo-3A have been studied in type 1 diabetes, and may be a marker of altered mucosal barrier and/or immune function. In the present study, we investigated antibody responses to Glo-3A in CD. PATIENTS AND METHODS: In the Diabetes Autoimmunity Study in the Young, children were studied prospectively from birth for the appearance of TTG and CD. Fifty cases of CD were frequency matched with 50 controls on age (of TTG seroconversion in the case), sex, ethnicity, presence of a first-degree relative with type 1 diabetes mellitus, and human leukocyte antigen -DR3 genotype. In cases and controls, IgG antibodies to Glo-3A were analyzed in a blinded manner in the sample collected at the time of seroconversion to TTG positivity (or the matched sample in controls) and in all of the previous samples since birth (mean 4.5 samples). The association between Glo-3A antibody levels and CD case status was explored using t tests at the TTG-positive visit and when Glo-3A levels were highest, and mixed modeling to describe Glo-3A over time. RESULTS: At the time of first elevated TTG (mean 4.9 years), patients with CD had higher Glo-3A antibody levels than controls (13.3 ± 17.2 vs 7.6 ± 11.7, P = 0.005). In both cases and controls, Glo-3A antibodies appear to peak at a mean age of 2.9 years, before mean age of initial TTG seroconversion. The peak Glo-3A antibody levels were higher in cases than controls (25.5 ± 21.8 vs 14.9 ± 18.3 P = 0.0007). Using mixed modeling to account for multiple visits per person, cases had higher levels of Glo-3A antibodies than controls at all ages from birth to TTG seroconversion (ß = 0.53, P = 0.002). CONCLUSIONS: Compared with controls, CD cases have higher Glo-3A antibody responses in the beginning years, before initial detection of TTG.

Identification and characterization of a heme periplasmic-binding protein in Haemophilus ducreyi.

Haemophilus ducreyi, a gram-negative and heme-dependent bacterium, is the causative agent of chancroid, a genital ulcer sexually transmitted infection. Heme acquisition in H. ducreyi proceeds via a receptor mediated process in which the initial event involves binding of hemoglobin and heme to their cognate outer membrane proteins, HgbA and TdhA, respectively. Following this specific interaction, the fate of the periplasmic deposited heme is unclear. Using protein expression profiling of the H. ducreyi periplasmic proteome, a periplasmic-binding protein, termed hHbp, was identified whose expression was enhanced under heme-limited conditions. The gene encoding this protein was situated in a locus displaying genetic characteristics of an ABC transporter. The purified protein bound heme in a dose-dependent and saturable manner and this binding was specifically competitively inhibited by heme. The hhbp gene functionally complemented an Escherichia coli heme uptake mutant. Expression of the heme periplasmic-binding protein was detected in a limited survey of H. ducreyi and H. influenzae clinical strains. These results indicate that the passage of heme into the cytoplasm of H. ducreyi involves a heme dedicated ABC transporter.

Antibodies from a patient with type 1 diabetes and celiac disease bind to macrophages that express the scavenger receptor CD163.

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.

Peripherin-reactive antibodies in mouse, rabbit, and human blood.

Type 1 diabetes (T1D) is an autoimmune disorder that results from the destruction of insulin-producing beta-cells in the islets of Langerhans. To date, autoimmune T-cell response and antibody reactivity to more than 20 autoantigens have been linked to this disease. Some studies have described the intermediate filament protein peripherin (PRPH) as an autoantigen associated with T1D in non-obese diabetic (NOD) mice. We evaluated immune reactivity of mouse and rabbit sera and human plasma to a 58 kDa protein expressed in RIN-m5F rat insulinoma cells. The protein was isolated using 2-DE and identified by mass spectrometry as PRPH. Antibodies from healthy humans and T1D patients, CD-1 mice, C57BL/6 mice, NOR (non-obese diabetes resistant) mice, and NOD mice reacted with PRPH on Western blots. However, antibody response to PRPH was stronger in NOD than non-autoimmune prone C57BL/6 mice. We conclude that immune reactivity to PRPH is not exclusively associated with NOD mice or human patients with T1D. Furthermore, the frequent occurrence of PRPH-reactive antibodies in mouse and human blood suggests that binding may be non-specific or could reflect the presence of natural autoantibodies against PRPH. These findings point to the need for a re-evaluation of PRPH as a T1D autoantigen in NOD mice and raise the question of the physiological relevance of such widespread immune reactivity against this peripheral nervous system protein.

Enhanced islet expansion by beta-cell proliferation in young diabetes-prone rats fed a protective diet.

Type 1 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-1, 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.

Identification of three wheat globulin genes by screening a Triticum aestivum BAC genomic library with cDNA from a diabetes-associated globulin.

BACKGROUND: Exposure to dietary wheat proteins in genetically susceptible individuals has been associated with increased risk for the development of Type 1 diabetes (T1D). Recently, a wheat protein encoded by cDNA WP5212 has been shown to be antigenic in mice, rats and humans with autoimmune T1D. To investigate the genomic origin of the identified wheat protein cDNA, a hexaploid wheat genomic library from Glenlea cultivar was screened. RESULTS: Three unique wheat globulin genes, Glo-3A, Glo3-B and Glo-3C, were identified. We describe the genomic structure of these genes and their expression pattern in wheat seeds. The Glo-3A gene shared 99% identity with the cDNA of WP5212 at the nucleotide and deduced amino acid level, indicating that we have identified the gene(s) encoding wheat protein WP5212. Southern analysis revealed the presence of multiple copies of Glo-3-like sequences in all wheat samples, including hexaploid, tetraploid and diploid species wheat seed. Aleurone and embryo tissue specificity of WP5212 gene expression, suggested by promoter region analysis, which demonstrated an absence of endosperm specific cis elements, was confirmed by immunofluorescence microscopy using anti-WP5212 antibodies. CONCLUSION: Taken together, the results indicate that a diverse group of globulins exists in wheat, some of which could be associated with the pathogenesis of T1D in some susceptible individuals. These data expand our knowledge of specific wheat globulins and will enable further elucidation of their role in wheat biology and human health.

Epigenetics: deciphering how environmental factors may modify autoimmune type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease that has increased two- to threefold over the past half century by as yet unknown means. It is generally accepted that T1D is the result of gene-environment interactions, but such rapid increases in incidence are not explained by Mendelian inheritance. There have been numerous advances in our knowledge of the pathogenesis of T1D. Indeed, there has been a large number of genes identified that contribute to risk for this disease and several environmental factors have been proposed. The complexity of such interactions is yet to be understood for any major chronic disease. Epigenetic regulation is one way to explain the rapid increase in incidence and could be a central mechanism by which environmental factors influence development of diabetes. However, there is remarkably little known about the contribution of epigenetics to T1D pathogenesis. Here we speculate on various candidate processes and molecules of the immune and endocrine systems that could modify risk for T1D through epigenetic regulation.

Changes in insulin, glucagon and ER stress precede immune activation in type 1 diabetes.

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.

Increased islet neogenesis without increased islet mass precedes autoimmune attack in diabetes-prone rats.

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 beta-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 beta-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 beta-cell mass. In addition, the number and area of very large islets was low at 45 days. The frequency of proliferating beta-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.

Differences of pancreatic expression of 7B2 between C57BL/6J and C3H/HeJ mice and genetic polymorphisms at its locus (Sgne1).

C57BL/6 (B6) mice develop glucose intolerance with age, whereas C3H/He (C3H) mice do not. In this study, we examined whether this differential glucose homeostasis was associated with differences of proteolytic activation of pancreatic prohormones. Radioimmunoassays showed comparable levels of fasting plasma insulin between the two strains but a significantly lower glucagon level in B6 mice. Pulse-chase analysis of glucagon biosynthesis in isolated pancreatic islets revealed that proglucagon was less efficiently processed in B6 mice. Because proprotein convertase (PC)2 and its 7B2 helper protein are required for this processing, we quantified islet mRNA levels by RT-PCR and protein levels by immunoblotting. The levels of proPC2 mRNA were similar between the two strains, but B6 protein extracts contained less of the mature PC2. In contrast, 7B2 mRNA and protein levels were both significantly lower in B6 pancreas. Sequencing of the 7B2 gene promoter and cDNA in the two strains revealed seven single nucleotide polymorphisms and one dinucleotide insertion/deletion in the cDNA as well as a single nucleotide polymorphism and two insertions/deletions in the promoter. Differential expression of 7B2 may contribute to the difference between B6 and C3H mice not only in glucagon production and secretion but also in glucose tolerance.

Where genes meet environment-integrating the role of gut luminal contents, immunity and pancreas in type 1 diabetes.

The rise in new cases of type 1 diabetes (T1D) in genetically susceptible individuals over the past half century has been attributed to numerous environmental "triggers" or promoters such as enteroviruses, diet, and most recently, gut bacteria. No single cause has been identified in humans, likely because there are several pathways by which one can develop T1D. There is renewed attention to the role of the gut and its immune system in T1D pathogenesis based largely on recent animal studies demonstrating that altering the gut microbiota affects diabetes incidence. Although T1D patients display dysbiosis in the gut microbiome, it is unclear whether this is cause or effect. The heart of this question involves several moving parts including numerous risk genes, diet, viruses, gut microbiota, timing, and loss of immune tolerance to ß-cells. Most clinical trials have addressed only one aspect of this puzzle using some form of immune suppression, without much success. The key location where our genes meet and deal with the environment is the gastrointestinal tract. The influence of all of its major contents, including microbes, diet, and immune system, must be understood as part of the integrative biology of T1D before we can develop durable means of preventing, treating, or curing this disease. In the present review, we expand our previous gut-centric model based on recent developments in the field.

Oral exposure to diabetes-promoting food or immunomodulators in neonates alters gut cytokines and diabetes.

Disease development in diabetes-prone BB rats is modified by the type of diet fed after weaning. The aim of this investigation was to determine whether exposure during the first week of life to antigens from a known diabetes-promoting diet (NIH-07) could modify diabetes incidence and, if so, to what extent this occurs via alterations in systemic T-cell reactivity, gut cytokines, or islet infiltration. Diabetes-prone BB (BBdp) rats were hand-fed twice daily between age 4 and 7 days with vehicle, a hydrolyzed casein (HC)-based infant formula, Pregestimil (PG), PG + cereal-based NIH-07 diet, PG + lipopolysaccharides (LPS) or PG + LPS + silica. After weaning, they were fed either an NIH-07 diet or a semipurified HC (diabetes-retardant) diet until 150 days. In separate studies, 5-day-old BBdp rat pups were administered the aforementioned treatments, and expression of intestinal mRNA for gamma-interferon (IFN-gamma) or transforming growth factor-beta (TGF-beta) was quantified using reverse transcriptase-polymerase chain reaction. The effect of early oral treatment with NIH-07 or PG on systemic T-cell reactivity was evaluated using footpad swelling delayed-type hypersensitivity (DTH) and the popliteal lymph node assay. Oral exposure of neonates to a complex mixture of antigens from the diabetes-promoting diet delayed onset of diabetes (79 vs. 88 days) and prevented disease in approximately one-third of animals. A similar protective effect was seen for neonatal exposure to wheat gluten in animals subsequently weaned onto a semipurified wheat gluten diet. By contrast, LPS-treated neonates displayed more severe insulitis and developed diabetes at an increased rate, which was significantly suppressed by co-administration of silica particles. The protective effect of early exposure to diabetogenic diets was not associated with significant reduction of islet infiltration, and there was no impact on the DTH response to food antigens. However, whereas diabetes-resistant BBc rats developed systemic tolerance to NIH-07 antigens fed chronically, BBdp rats did not. The lack of effect of the early oral antigen regimen on the DTH reaction in the footpad, a classic Th1-mediated reaction, suggests little effect on systemic T-cell reactivity. However, local effects were observed in the small intestine. Oral exposure to diabetes-promoting food antigens or LPS downregulated the Th1 cytokine IFN-gamma and decreased the IFN-gamma/TGF-beta ratio. Thus, oral exposure to diabetes-promoting food antigens and immune modulators in neonates can modify diabetes expression in association with changes in local cytokine balance in the gut.

Quantitative and qualitative alterations of intestinal mucins in BioBreeding rats.

In diabetes-prone BioBreeding rats, an enteropathy often precedes the onset of auto-immune insulitis. The present study draws attention to quantitative and qualitative alterations of intestinal mucins in this animal model of type 1 diabetes mellitus. Male and female diabetes-resistant (BBc) and diabetes-prone (BBdp) BioBreeding rats fed, from one to two weeks after weaning onwards, either a plant-based diabetes-promoting diet (NTP) or a hydrolysed casein diabetes-protective diet (HC), were sacrificed at 11-14 weeks of age. Proteins and total mucins, as well as acid and neutral mucins, were measured in a segment of the intestinal tract, located 25-30 cm below the pylorus. No significant difference between BBc and BBdp rats was found when fed the HC diet. However, the NTP diet lowered both total and neutral mucins, whilst failing to affect significantly acidic mucins. The effects of the NTP diet were more pronounced in BBdp rats than in BBc rats. It is speculated that the quantitative and qualitative changes evoked by the NTP diet in BBdp rats may play a role in the alteration of gut permeability found under the same experimental conditions.

Cathelicidin Antimicrobial Peptide: A Novel Regulator of Islet Function, Islet Regeneration, and Selected Gut Bacteria.

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 ß-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 ß-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.

Heme Oxygenase-1 Induction Prevents Autoimmune Diabetes in Association With Pancreatic Recruitment of M2-Like Macrophages, Mesenchymal Cells, and Fibrocytes.

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 REG3ß(+) 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.

Variable effects of gender and Western diet on lipid and glucose homeostasis in aged PCSK9-deficient C57BL/6 mice CSK9PC57BL/6.

BACKGROUND: Proprotein convertase subtilisin/kexin-type 9 (PCSK9) downregulates clearance of plasma cholesterol by liver. Its inactivation increases this clearance, reducing cardiovascular risk. However, a lack of PCSK9 could also lead to cholesterol accumulation in pancreatic islet beta cells, impairing insulin secretion. We reported earlier that 4-month-old male PCSK9-deficient (KO) C57BL/6 mice were hyperglycemic and insulin-insufficient relative to their wild-type (WT) counterparts. Here, we examined how gender and diet affect lipid and glucose homeostasis in these mice at 8 months of age. METHODS: After being fed a normal diet or a Western diet for over 6 months, KO mice were compared with same-gender WT mice for fasting plasma levels of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), glucose, and insulin; for glucose disposal and glucose-stimulated insulin secretion (GSIS); and for pancreatic islet morphology. RESULTS: A. Females: On normal diet, KO mice showed lower plasma TC, HDL-C, and LDL-C, higher plasma glucose, and normal glucose disposal despite impaired GSIS. On Western diet, they showed comparable plasma TC and HDL-C, but lower LDL-C, higher plasma glucose, and normal glucose disposal despite impaired GSIS. B. Males: On normal and Western diets, KO mice showed lower plasma TC, HDL-C, and LDL-C, similarly elevated plasma glucose, glucose intolerance, and impaired GSIS. C. Both: KO mice on either diet showed pancreatic islet dysmorphism, with larger, possibly immature secretory granules. CONCLUSIONS: Lower LDL-C and impaired GSIS are two major phenotypes in aged PCSK9-deficient C57BL/6 mice. These phenotypes are modulated by gender and diet.

T-bet/GATA-3 ratio as a measure of the Th1/Th2 cytokine profile in mixed cell populations: predominant role of GATA-3.

The differentiation of naive T-helper (Th) cells towards Th1 or Th2 cells is regulated by the transcription factors T-box expressed in T-cells (T-bet) and GATA-binding protein-3 (GATA-3). In the present study, the gene expression of T-bet and GATA-3 was measured by semi-quantitive reverse transcription polymerase chain reaction (RT-PCR) in Th1 and Th2 cells derived from purified splenic CD4+ T cells from DO11.10/Rag2(-/-) transgenic mice and control BioBreeding (BBc) Wistar rat splenic T cells stimulated under Th1 or Th2 conditions. In both sets of experiments, changes in the ratio of expression of T-bet and GATA-3 reflected changes in the Th1-specific cytokine interferon-gamma (IFN-gamma) and Th2-specific cytokine interleukin (IL)-4. T-bet gene expression was not maintained in fully polarized rat Th1 cells whereas GATA-3 gene expression was maintained in long-term polarized rat Th2 cells, indicating that maintenance of Th1/Th2 status occurred more as a result of altered GATA-3 mRNA expression than T-bet. These transcription factors are up-regulated in several cells that produce type 1 and type 2 cytokines and can be analyzed readily by RT-PCR using total RNA isolated from mixed cell populations or cultured splenocytes thereby providing a surrogate marker of Th1/Th2 cytokine balance under a variety of conditions.