Autoimmune Insulitis Research Articles

Failure to replicate the diabetes alleviating effect of a maternal gluten-free diet in non-obese diabetic mice.

Type 1 diabetes (T1D) is an autoimmune disease with an unexplained rising incidence for which environmental factors like gluten may play a role. Previously, we showed that a gluten-free (GF) diet provided strictly in utero reduces the autoimmune diabetes incidence in Non-Obese Diabetic (NOD) mice compared to a gluten-containing standard (STD) diet. The current study was initiated to elucidate possible mechanisms behind the diabetes-alleviating effect of the same diet intervention. NOD mice received either a GF Altromin diet or a STD Altromin diet during pregnancy. Female offspring from both groups were fed a STD diet throughout life and their diabetes incidence was recorded for 200 days. The following parameters were measured in 13-week-old female offspring: insulitis degree, glucose and insulin tolerance, and plasma insulin autoantibody titer. The diet intervention showed no reduction in autoimmune diabetes incidence, insulitis degree, glucose nor insulin tolerance and plasma insulin autoantibody titer. In conclusion, this study could not replicate the previously observed diabetes alleviative effects of a maternal gluten-free diet in NOD mouse offspring and could therefore not further elucidate potential mechanisms.

Prevalence of Positivity for Diabetes-Associated Autoantibodies in Individuals with Type 2 Diabetes and Their Further Characterisation: Cross-sectional Study from Slovakia.

Individuals initially diagnosed with type 2 diabetes (T2D) might exhibit positivity for diabetes-associated autoantibodies (DAA +). We investigated the prevalence of DAA positivity in a group of individuals with T2D who were referred to a tertiary diabetes centre within a pre-specified period of time. We aimed to identify characteristics linked with DAA positivity by comparing DAA + individuals with their DAA-negative counterparts. This was a cross-sectional study into which all T2D patients referred to the NationalInstitute of Endocrinology and Diabetology, Ľubochňa, Slovakia, between 1 January and 30 June 2016 were included. Data on > 70 participants' characteristics, including antibodies against glutamic acid decarboxylase (anti-GAD65), insulinoma-associated antigen IA-2 (IA-2A) and insulin (IAA), were collected. Six hundred and ninety-two individuals (387, 55.6% female) with a median (range) age of 62 (24-83) years, HbA1c of 8.9 (5.0-15.7)% [74 (31-148mmol/mol)] and diabetes duration of 13.0 (0-42) years were analysed. One hundred and forty-five (145/692, 21.0%) tested positive for at least one DAA; 136/692 (19.7%) were positive for anti-GAD65, 21/692 (3.0%) were positive for IA-2A and 9/692 (1.3%) were positive for IAA. Only 84.9% of the DAA + individuals aged > 30years at the time of diabetes diagnosis met the current diagnostic criteria for latent autoimmune diabetes of adults (LADA). DAA + differed from DAA- individuals in multiple characteristics, including the incidence of hypoglycaemia. Several pathological processes linked with distinct types of diabetes can develop in parallel, including insulin resistance and autoimmune insulitis. In this single-centre cross-sectional study from Slovakia, we report a higher than previously published prevalence of DAA positivity in agroup ofindividualswith a formal diagnosis of T2D.

Newly-onset Autoimmune Diabetes Mellitus Triggered by COVID 19 Infection: A Case-based Review.

The devastating global pandemic Coronavirus disease 2019 (COVID 19) isolated in China in January 2020 is responsible for an outbreak of pneumonia and other multisystemic complications. The clinical picture of the infection has extreme variability: it goes from asymptomatic patients or mild forms with fever, cough, fatigue and loss of smell and taste to severe cases ending up in the intensive care unit (ICU). This is due to a possible cytokine storm that may lead to multiorgan failure, septic shock, or thrombosis. Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV -2), which is the virus that causes COVID 19, binds to angiotensin-converting enzyme 2 (ACE2) receptors, which are expressed in key metabolic organs and tissues, including pancreatic beta cells, adipose tissue, the small intestine and the kidneys. Therefore it is possible to state that newly-onset diabetes is triggered by COVID 19 infection. Although many hypotheses have clarified the potential diabetogenic effect of COVID 19, a few observations were reported during this pandemic. Two male patients admitted to us with devastating hyperglycemia symptoms were diagnosed with type 1/autoimmune diabetes mellitus within 3 months following COVID 19 infection. Autoantibodies and decreased C peptide levels were detected in these patients. We speculated that several mechanisms might trigger autoimmune insulitis and pancreatic beta-cell destruction by COVID 19 infection. We aim to raise awareness of the possible link between SARS-CoV-2 and newly onset type 1 diabetes mellitus. Further studies are needed to determine a more definitive link between the two clinical entities.

Interferon-α promotes MHC I antigen presentation of islet βcells through STAT1-IRF7 pathway in type 1 diabetes.

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease. Increased incidence of T1D was reported in patients receiving IFN-α treatment. However, the exact mechanisms of IFN-α that facilitate the pathogenesis of T1D are not fully understood. To explore the mechanism of IFN-α on the immune system and islets, non-obese diabetic (NOD) mice were injected with IFN-α and the progression of autoimmune insulitis was assessed by haematoxylin and eosin (HE) staining, immunohistochemical and flow cytometry analysis. Transcriptional profiling of islets treated with IFN-α was explored by RNA-seq. IFN-α induced antigen presentation was evaluated by qRT-PCR, western blot and immunofluorescence, and key transcription factors were inhibited by small interfering RNAs (siRNAs). Our data show that IFN-α contributed to the progression of autoimmune insulitis in NOD mice by promoting the proliferation of CD8+ T cells. IFN-α upregulated antigen presentation related genes MHC I, TAP1, B2M, PSMB8, NLRC5 and transcriptional regulator STAT1, STAT2, IRF7 at a time and dose-dependent manner. The silence of STAT1 or STAT2 both weakened IFN-α-induced increase of antigen presenting related molecules. IRF7 was also merely influenced by STAT1 silence. The knockdown of IRF7 decreased the IFN-α induced expressions of TAP1, PSMB8 and MHC I and prevented the expression of STAT2 but not STAT1. Our study demonstrated that STAT1-IRF7-MHC I complex axis were crucial for IFN-α signalling in islets, and created positive feedback through IRF7-STAT2 cascade amplifying signals which accelerated the process of T1D.

Crosstalk between Macrophages and Pancreatic β-Cells in Islet Development, Homeostasis and Disease.

Macrophages are highly heterogeneous and plastic immune cells with peculiar characteristics dependent on their origin and microenvironment. Following pathogen infection or damage, circulating monocytes can be recruited in different tissues where they differentiate into macrophages. Stimuli present in the surrounding milieu induce the polarisation of macrophages towards a pro-inflammatory or anti-inflammatory profile, mediating inflammatory or homeostatic responses, respectively. However, macrophages can also derive from embryonic hematopoietic precursors and reside in specific tissues, actively participating in the development and the homeostasis in physiological conditions. Pancreatic islet resident macrophages are present from the prenatal stages onwards and show specific surface markers and functions. They localise in close proximity to β-cells, being exquisite sensors of their secretory ability and viability. Over the years, the crucial role of macrophages in β-cell differentiation and homeostasis has been highlighted. In addition, macrophages are emerging as central players in the initiation of autoimmune insulitis in type 1 diabetes and in the low-grade chronic inflammation characteristic of obesity and type 2 diabetes pathogenesis. The present work reviews the current knowledge in the field, with a particular focus on the mechanisms of communication between β-cells and macrophages that have been described so far.

The JAK1 Selective Inhibitor ABT 317 Blocks Signaling Through Interferon-γ and Common γ Chain Cytokine Receptors to Reverse Autoimmune Diabetes in NOD Mice.

Cytokines that signal through the JAK-STAT pathway, such as interferon-γ (IFN-γ) and common γ chain cytokines, contribute to the destruction of insulin-secreting β cells by CD8+ T cells in type 1 diabetes (T1D). We previously showed that JAK1/JAK2 inhibitors reversed autoimmune insulitis in non-obese diabetic (NOD) mice and also blocked IFN-γ mediated MHC class I upregulation on β cells. Blocking interferons on their own does not prevent diabetes in knockout NOD mice, so we tested whether JAK inhibitor action on signaling downstream of common γ chain cytokines, including IL-2, IL-7 IL-15, and IL-21, may also affect the progression of diabetes in NOD mice. Common γ chain cytokines activate JAK1 and JAK3 to regulate T cell proliferation. We used a JAK1-selective inhibitor, ABT 317, to better understand the specific role of JAK1 signaling in autoimmune diabetes. ABT 317 reduced IL-21, IL-2, IL-15 and IL-7 signaling in T cells and IFN-γ signaling in β cells, but ABT 317 did not affect GM-CSF signaling in granulocytes. When given in vivo to NOD mice, ABT 317 reduced CD8+ T cell proliferation as well as the number of KLRG+ effector and CD44hiCD62Llo effector memory CD8+ T cells in spleen. ABT 317 also prevented MHC class I upregulation on β cells. Newly diagnosed diabetes was reversed in 94% NOD mice treated twice daily with ABT 317 while still on treatment at 40 days and 44% remained normoglycemic after a further 60 days from discontinuing the drug. Our results indicate that ABT 317 blocks common γ chain cytokines in lymphocytes and interferons in lymphocytes and β cells and are thus more effective against diabetes pathogenesis than IFN-γ receptor deficiency alone. Our studies suggest use of this class of drug for the treatment of type 1 diabetes.

289-OR: JAK Inhibitors Block Interferon and Common Gamma Chain Cytokine Signaling to Reverse Diabetes in NOD Mice

Cytokines that signal through the JAK-STAT pathway, such as interferon-γ (IFN-γ) and common γ chain cytokines, play an important role in facilitating the destruction of insulin-secreting β cells by CD8+ T cells in type 1 diabetes. We previously showed that inhibiting JAK1/JAK2 reversed autoimmune insulitis in non-obese diabetic (NOD) mice and also blocked IFN-γ mediated MHC class I upregulation on β cells. Blocking interferons on their own did not prevent diabetes in knockout NOD mice, so we tested whether JAK inhibitor action on signaling downstream of common γ chain family cytokines, including IL-2, IL-21, IL-7 and IL-15, may also affect progression of diabetes in NOD mice. Common γ chain cytokines activate JAK1 and JAK3 to regulate T cell proliferation. We compared JAK1-selective inhibitors with our previous data with JAK1/JAK2 inhibition. JAK1-selective inhibitors reduced IL-21, IL-2 and IL-7 signaling in T cells (p<0.05) and IFN-γ signaling in β cells, but did not affect GM-CSF signaling in granulocytes. When given in vivo to NOD mice, JAK1-selective inhibitors reduced CD8+ T cell proliferation (Ki-67) in islets, reduced immune cell infiltration into islets and prevented MHC class I upregulation on β cells as measured by flow cytometry. Newly diagnosed diabetes was reversed in 94% NOD mice treated twice daily with JAK1-selective inhibitors. Our results indicate that JAK inhibitors block common γ chain cytokines in lymphocytes and interferons in lymphocytes and β cells and are thus more effective against diabetes pathogenesis than IFNγ receptor deficiency alone. Our studies pave the way for use of this class of drug in clinical trials for type 1 diabetes. Disclosure G. Jhala: None. T. Ge: None. B. Krishnamurthy: None. T. Kay: None. H.E. Thomas: None. Funding National Health and Medical Research Council of Australia (GNT1145507); JDRF (1-SRA-2018-599-S-B)

1747-P: 3D Imaging and Quantification of Pancreatic Immune Cell Infiltration during Emergence and Progression of Spontaneous Autoimmune Diabetes in Nonobese Diabetic (NOD) Mice

The non-obese diabetic (NOD) mouse develops diabetes as the result of spontaneous autoimmune insulitis and is widely used as a translational polygenic model of type 1 diabetes. Disease progression in the NOD mouse model is slow, involving a long prodromal phase of pancreatic islet infiltration before autoimmune β-cell destruction and manifestation of diabetic hyperglycemia, making the NOD mouse ideal for probing the effectiveness of potential disease prevention therapies. Improved understanding of the spatial dynamics of immune cell infiltration of the pancreas during the emergence and natural progression of autoimmune diabetes would permit further resolution of drug treatment effects in this model. The present study therefore aimed to image the spontaneous development and progression of autoimmune diabetes dynamically and at 3-dimensional cellular resolution in the NOD mouse. Female NOD mice were terminated before diabetes onset, at 14 and 18 weeks of age, and pancreata were isolated and divided into two alternating samples consisting of either the head and tail. One half was stained for insulin and CD45 using iDISCO whole-mount immunohistochemistry and subsequently scanned using a light sheet microscope. Islet infiltration was quantified using an in-house developed algorithm. The other half of each pancreas was simultaneously evaluated by flow cytometry to discriminate and quantify immune cells of lymphoid and myeloid lineage. Infiltrating CD45-positive leukocytes progressively increased in abundance over time, however, with highly variable distribution in the pancreas. Flow cytometry confirmed extensive leukocyte infiltration and revealed predominant changes in T-cell profiles. In conclusion, 3D imaging allows for visualization and unbiased quantitative analysis of the unsynchronized spreading of insulitis into different parts of the pancreas in NOD mice. Disclosure B.B. Boland: Employee; Self; Gubra, MedImmune. A. Manresa-Arraut: None. R.V. Grônlund: None. U. Roostalu: Employee; Self; Gubra. J. Hecksher-Sørensen: Employee; Self; Gubra. Stock/Shareholder; Self; Novo Nordisk A/S. N. Vrang: Board Member; Self; Gubra. Stock/Shareholder; Self; Gubra. J. Jelsing: Stock/Shareholder; Self; Gubra. L.N. Fink: Employee; Self; Gubra. Stock/Shareholder; Self; Novo Nordisk A/S.

The eye as a novel imaging site in diabetes research.

Diabetes develops due to deficient functional β cell mass, insulin resistance, or both. Yet, various challenges in understanding the mechanisms underlying diabetes development in vivo remain to be overcome owing to the lack of appropriate intravital imaging technologies. To meet these challenges, we have exploited the anterior chamber of the eye (ACE) as a novel imaging site to understand diabetes basics and clinics in vivo. We have developed a technology platform transplanting pancreatic islets into the ACE where they later on can be imaged non-invasively for long time. It turns out that the ACE serves as an optimal imaging site and provides implanted islets with an oxygen-rich milieu and an immune-privileged niche where they undergo optimal engraftment, rich vascularization and dense innervation, preserve organotypic features and live with satisfactory viability and functionality. The ACE technology has led to a series of significant observations. It enables in vivo microscopy of islet cytoarchitecture, function and viability in the physiological context and intravital imaging of a variety of pathological events such as autoimmune insulitis, defects in β cell function and mass and insulin resistance during diabetes development in a real-time manner. Furthermore, application of the ACE technology in humanized mice and non-human primates verifies translational and clinical values of the technology. In this article, we describe the ACE technology in detail, review accumulated knowledge gained by means of the ACE technology and delineate prospective avenues for the ACE technology.

Extracellular matrix and the maintenance and loss of peripheral immune tolerance in autoimmune insulitis.

There is a growing appreciation that the extracellular matrix (ECM) contributes to both the maintenance of immune tolerance in healthy tissues and to its loss at sites of autoimmunity. Here, we review recent literature on the role of ECM and particularly the glycosaminoglycans hyaluronan and heparan sulfate in the development of autoimmune, type 1 diabetes (T1D). Data from transplant models suggest that healthy islets are embedded within an intact ECM that supports beta-cell homeostasis and provides physical and immunoregulatory barriers against immune infiltration. However, studies of human insulitis as well as the non-obese diabetic (NOD) and DORmO mouse models of T1D indicate that autoimmune insulitis is associated with the degradation of basement membrane structures, the catabolism of the islet interstitium, and the accumulation of a hyaluronan-rich, pro-inflammatory ECM. Moreover, in these models of autoimmune diabetes, either the pharmacologic inhibition of heparan sulfate catabolism, the reduction of hyaluronan synthesis, or the targeting of the pathways that sense these ECM changes can all prevent beta-cell destruction. Together these data support an emerging paradigm that in healthy islets the local ECM contributes to both immune tolerance and beta-cell homeostasis while in chronic inflammation the islet ECM is permissive to immune infiltration and beta-cell destruction. Therapies that support ECM-mediated 'barrier tolerance' may have potential as adjunctive agents in combination regimens designed to prevent or treat autoimmunity.

CD44 variant inhibits insulin secretion in pancreatic \u03b2 cells by attenuating LAT1-mediated amino acid uptake

CD44 variant (CD44v) contributes to cancer stemness by stabilizing the xCT subunit of system xc(−) and thereby promoting its glutamate-cystine antiporter activity. CD44 has also been implicated in autoimmune insulitis and inflammation in diabetic islets, but whether CD44v regulates insulin secretion has remained unclear. Here we show that CD44v inhibits insulin secretion by attenuating amino acid transport mediated by the L-type amino acid transporter LAT1. CD44v expression level was inversely related to insulin content in islets of normal and diabetic model mice. Knockdown of CD44 increased insulin secretion, the intracellular insulin level, and the transport of neutral amino acids mediated by LAT1 in Min6 cells. Attenuation of the uptake of neutral amino acids with a LAT inhibitor reduced insulin secretion and insulin content in Min6 cells, whereas overexpression of LAT1 increased insulin secretion. Moreover, inhibition of LAT1 prevented the increase in insulin secretion and content induced by CD44 depletion in Min6 cells. Our results thus implicate CD44v in the regulation of insulin secretion and reveal that amino acid transport is rate limiting for such secretion. They further suggest that amino acid transport mediated by LAT1 is a potential therapeutic target for diabetes.

Hyaluronan content governs tissue stiffness in pancreatic islet inflammation

We have identified a novel role for hyaluronan (HA), an extracellular matrix polymer, in governing the mechanical properties of inflamed tissues. We recently reported that insulitis in type 1 diabetes of mice and humans is preceded by intraislet accumulation of HA, a highly hygroscopic polymer. Using the double transgenic DO11.10 × RIPmOVA (DORmO) mouse model of type 1 diabetes, we asked whether autoimmune insulitis was associated with changes in the stiffness of islets. To measure islet stiffness, we used atomic force microscopy (AFM) and developed a novel "bed of nails"-like approach that uses quartz glass nanopillars to anchor islets, solving a long-standing problem of keeping tissue-scale objects immobilized while performing AFM. We measured stiffness via AFM nanoindentation with a spherical indenter and found that insulitis made islets mechanically soft compared with controls. Conversely, treatment with 4-methylumbelliferone, a small-molecule inhibitor of HA synthesis, reduced HA accumulation, diminished swelling, and restored basal tissue stiffness. These results indicate that HA content governs the mechanical properties of islets. In hydrogels with variable HA content, we confirmed that increased HA leads to mechanically softer hydrogels, consistent with our model. In light of recent reports that the insulin production of islets is mechanosensitive, these findings open up an exciting new avenue of research into the fundamental mechanisms by which inflammation impacts local cellular responses.

Effect of eicosapentaenoic acid and docosahexaenoic acid supplementation on C-peptide preservation in pregnant women with type-1 diabetes: randomized placebo controlled clinical trial.

Type-1 diabetes mellitus (T1DM) is caused by autoimmune insulitis. There are evidences that pregnancy and n-3 fatty acids exhibit suppressive effect on human inflammatory system. Ninety pregnant women with T1DM were included in the prospective randomized placebo controlled clinical trial. Forty-seven of them were put on standard diabetic diet enriched with EPA and DHA twice a day (EPA 120 mg and DHA 616 mg; Study group) and 43 pregnant diabetic women were on standard diabetic diet with placebo (Control group). Duration of T1DM in all participants was between 5 to 30 years. Blood samples were analyzed from all pregnant women for fasting C-peptide (FC-peptide), fasting plasma glucose (FPG) and HbA1c in each trimester throughout pregnancy and after delivery. Umbilical vein blood was analyzed for fetal C-peptide level, glucose concentration and insulin resistance. In the Study group FC-peptide concentration raised from 59.6±103.9 pmol/l in first trimester, to 67.7±101.3 pmol/l in the second trimester and to 95.1±152.7 pmol/l in the third trimester. Comparing the FC-peptide values during first and third trimester a statistically significant increase in third trimester was found (P<0.001). In the Control group FC-peptide concentration ranged from 41.7±91.6 pmol/l in the first trimester to 41.2±70.9 mmol/l in the second trimester while in the third trimester it reached 52.4±95.3 pmol/l. Comparing the FC-peptide values during first and third trimester the statistical difference was not significant. Combining of LC n-3 PUFAs and pregnancy yields immunological tolerance and stimulates the production of endogenous insulin in women with T1DM.

Repurposed JAK1/JAK2 Inhibitor Reverses Established Autoimmune Insulitis in NOD Mice.

Recent advances in immunotherapeutics have not yet changed the routine management of autoimmune type 1 diabetes. There is an opportunity to repurpose therapeutics used to treat other diseases to treat type 1 diabetes, especially when there is evidence for overlapping mechanisms. Janus kinase (JAK) 1/JAK2 inhibitors are in development or clinical use for indications including rheumatoid arthritis. There is good evidence for activation of the JAK1/JAK2 and signal transducer and activator of transcription (STAT) 1 pathway in human type 1 diabetes and in mouse models, especially in β-cells. We tested the hypothesis that using these drugs to block the JAK-STAT pathway would prevent autoimmune diabetes. The JAK1/JAK2 inhibitor AZD1480 blocked the effect of cytokines on mouse and human β-cells by inhibiting MHC class I upregulation. This prevented the direct interaction between CD8+ T cells and β-cells, and reduced immune cell infiltration into islets. NOD mice treated with AZD1480 were protected from autoimmune diabetes, and diabetes was reversed in newly diagnosed NOD mice. This provides mechanistic groundwork for repurposing clinically approved JAK1/JAK2 inhibitors for type 1 diabetes.

Longitudinal three-dimensional visualisation of autoimmune diabetes by functional optical coherence imaging.

It is generally accepted that structural and functional quantitative imaging of individual islets would be beneficial to elucidate the pathogenesis of type 1 diabetes. We here introduce functional optical coherence imaging (FOCI) for fast, label-free monitoring of beta cell destruction and associated alterations of islet vascularisation. NOD mouse and human islets transplanted into the anterior chamber of the eye (ACE) were imaged with FOCI, in which the optical contrast of FOCI is based on intrinsic variations of the index of refraction resulting in a faster tomographic acquisition. In addition, the phase sensitivity allows simultaneous label-free acquisition of vascularisation. We demonstrate that FOCI allows longitudinal quantification of progressive autoimmune insulitis, including the three-dimensional quantification of beta cell volume, inflammation and vascularisation. The substantially increased backscattering of islets is dominated by the insulin-zinc nanocrystals in the beta cell granules. This translates into a high specificity for the functional beta cell volume of islets. Applying FOCI to a spontaneous mouse model of type 1 diabetes, we quantify the modifications of the pancreatic microvasculature accompanying the progression of diabetes and reveal a strong correlation between increasing insulitis and density of the vascular network of the islet. FOCI provides a novel imaging technique for investigating functional and structural diabetes-induced alterations of the islets. The label-free detection of beta cell volume and infiltration together with vascularisation offers a unique extension to study ACE-transplanted human islets. These results are contributing to a deeper understanding of human islet transplant rejection and label-free in vivo monitoring of drug efficacy.

11-Keto-β-Boswellic Acids Prevent Development of Autoimmune Reactions, Insulitis and Reduce Hyperglycemia During Induction of Multiple Low-Dose Streptozotocin (MLD-STZ) Diabetes in Mice.

The aim of the work was to study whether or not 11-keto-β-boswellic acids prevent induction of autoimmune reactions, insulitis, and hyperglycemia in the model of multiple low-dose streptozotocin (MLD-STZ) diabetes. Using male mice (n = 6) diabetes was induced by daily i.p. injections of 40 mg/kg STZ for 5 days. In a second series together with STZ, daily i. p. injections of 11-keto-β-boswellic acid (KBA) and O-acetyl-11-keto-β-boswellic acid (AKBA) (7.5 and 15.0 mg/kg) were applied for 10 days. Thereafter, pro-and anti-inflammatory cytokines in the blood, histochemistry of pancreatic islets, and blood glucose levels were assayed. Five days after the last injection of STZ, a significant burst of pro-and anti-inflammatory cytokines in the blood, infiltration of lymphocytes (CD3) into pancreatic islets, and appearance of peri-insular apoptotic cells were observed. Plasma glucose increased significantly (124.4 ± 6.65 vs. 240.2 ± 27.36 mg/dl, p <0.05). Simultaneous treatment with KBA and AKBA significantly reduced pro-and anti-inflammatory cytokines (IFN-γ p < 0.01, p < 0.01; IL-1A p < 0.001, p < 0.001; IL-1B p < 0.001, p < 0.001; IL-2 p < 0.001, p < 0.001; IL-6 p < 0.01, p < 0.001; TNF-α p < 0.05, p < 0.001; IL-4 p < 0.01, p < 0.001; IL-10 p < 0.001, p < 0.001) in the blood. No infiltration of lymphocytes into pancreatic islets and appearance of peri-insular cells were detected. Moreover, KBA and AKBA reduced STZ-mediated increase of blood glucose on day 10 to 163.25 ± 16.6 (p < 0.05) and 187.6 ± 19.5 mg/dl (p < 0.05), respectively. In the model of MLD-STZ induced diabetes KBA and AKBA prevent cytokine burst, development of insulitis and reduce increase of blood glucose through "silencing" a forced-up immune reaction.

Inhibition of hyaluronan synthesis restores immune tolerance during autoimmune insulitis (THER5P.919)

Abstract We recently reported that abundant deposits of the extracellular matrix polysaccharide hyaluronan (HA) characterize autoimmune insulitis in human type 1 diabetes (T1D) but the significance of these deposits was unclear. Here, we demonstrate that HA is critical for the pathogenesis of autoimmune diabetes. Using the DO11.10xRIPmOVA (DORmO) mouse model of T1D, we show that HA deposits are temporally and anatomically associated with the development of insulitis. Moreover, treatment with an inhibitor of HA synthesis, 4‑methylumbelliferone (4-MU), halted progression to diabetes even after the onset of insulitis. 4-MU reduced HA accumulation, constrained effector T-cells to non-destructive insulitis, and increased numbers of intra-islet Foxp3+ regulatory T-cells (Treg). Consistent with this, Treg differentiation was inhibited by HA and anti-CD44 antibodies and rescued by 4-MU in an ERK1/2-dependent manner. These data may explain how peripheral immune tolerance is impaired in tissues under autoimmune attack, including islets in T1D. We propose that 4-MU, already an approved drug used to treat biliary spasm, could be repurposed to prevent, and possibly treat, T1D in at-risk individuals.

The Inhibitory G‐protein, Gz, Accelerates the Progression of Insulitis and Hyperglycemia in a Type 1 Diabetes Mouse Model

In type 1 diabetes (T1D), an autoimmune insulitis leads to β‐cell death. The non‐obese diabetic (NOD) mouse model mimics these facets of the human disease. Our laboratory has previously shown that mice deficient in the alpha subunit of the heterotrimeric G protein, Gz (Gαz), were protected from developing chemically induced diabetes via decreased β‐cell apoptosis and increased replication. We aimed to confirm this in the NOD model, as well as delineate the mechanisms of any protection. Between 4‐16 weeks of age, weekly blood glucose levels were monitored in wild‐type and Gαz‐null NOD mice. As compared to wild‐type mice, Gαz‐null mice were completely protected from developing hyperglycemia. Pancreas histologic analysis showed that Gαz‐null pancreata had significantly lower insulitis scores at 16 weeks as compared to wild‐type mice. We hypothesized that decreased immune infiltration increased β‐cell survival. To test our hypothesis, we calculated beta‐cell fractional area, and found it was increased in Gαz‐null NOD mice. New cohorts of mice were sacrificed at 4, 8, and 12 weeks of age. At 4 weeks, immune infiltration was already observed in the wild‐type islets, correlating with apoptotic beta‐cells in islet regions immediately surrounding the invading immune cells. As the first step in delineating mechanism, we showed that islets from Gαz‐null NOD mice have significantly less IL‐1β and IL‐6 cytokine expression; pro‐inflammatory cytokines that have been linked with β‐cell death. Overall, our results further strengthen our claim that Gαz has a protective effect in maintaining β‐cell regeneration and survival, and that the Gαz signaling pathway may be a valid therapeutic target for T1D.

Type 1 diabetes pathogenesis - Prevention???

Pathogenesis of type 1 diabetes is multi-faceted, including, autoimmunity, genetics and environment. Autoimmunity directed against pancreatic islet cells results in slowly progressive selective beta-cell destruction (“Primary autoimmune insulitis”), culminating over years in clinically manifested insulin-dependent diabetes mellitus (IDDM). Circulating serum autoantibodies directed against the endocrine cells of the islets of Langerhans (Islet cell autoantibodies - ICAb) are an important hallmark of this disease. Assays for islet cell autoantibodies have facilitated the investigation and understanding of several facets in the pathogenesis of autoimmune diabetes. Their applications have extended into clinical practice and have opened new avenues for early preclinical prediction and preventive prophylaxis in IDDM/type 1 DM. Recently, surprisingly, differences in insulin content between T1DM islets, as well as, ‘patchy’ or ‘lobular’ destruction of islets have been described. These unique pathobiological phenomena, suggest that beta cell destruction may not always be inexorable and inevitably complete/total, and thus raise hopes for possible therapeutic interruption of beta cell autoimmunity – destruction and cure of type 1 diabetes. “Recurrent or secondary autoimmune insulitis” refers to the rapid reappearance of islet cell autoantibodies post pancreas transplant, and selective islet beta cell destruction in the grafted pancreas [never forgetting or “anamnestic” beta cell destructive memory], in the absence of any graft pancreas rejection [monozygotic twin to twin transplantation]. The one definite environmental factor is congenital rubella, because of which a subset of children subsequently develop type 1 diabetes. The putative predisposing factors are viruses, gluten and cow's milk. The putative protective factors include gut flora, helminths, viral infections, and Vitamin D. Prevention of T1DM can include: Primary prevention strategies before the development of autoantibodies and Secondary prevention regimens after autoantibody development. Once islet cell autoantibodies have developed, the goal is to establish a therapeutic regimen to preserve at least 90% of the beta cells, and prevent the development of hyperglycaemia. The targets for T1DM reversal should include autoimmunity, beta cell regeneration and protection of beta cell mass. Anti-CD3 teplizumab and anti-CD3 otelixizumab have been shown to provide C-peptide preservation. The unanswered questions in diabetes research include elimination of autoimmune memory responses, reestablishment of immune self-tolerance, and mechanisms of disease initiation.

Stable activity of diabetogenic cells with age in NOD mice: dynamics of reconstitution and adoptive diabetes transfer in immunocompromised mice

The non-obese diabetic (NOD) mouse is a prevalent disease model of type 1 diabetes. Immune aberrations that cause and propagate autoimmune insulitis in these mice are being continually debated, with evidence supporting both dominance of effector cells and insufficiency of suppressor mechanisms. In this study we assessed the behaviour of NOD lymphocytes under extreme expansion conditions using adoptive transfer into immunocompromised NOD.SCID (severe combined immunodeficiency) mice. CD4(+) CD25(+) T cells do not cause islet inflammation, whereas splenocytes and CD4(+) CD25(-) T cells induce pancreatic inflammation and hyperglycaemia in 80-100% of the NOD.SCID recipients. Adoptively transferred effector T cells migrate to the lymphoid organs and pancreas, proliferate, are activated in the target organ in situ and initiate inflammatory insulitis. Reconstitution of all components of the CD4(+) subset emphasizes the plastic capacity of different cell types to adopt effector and suppressor phenotypes. Furthermore, similar immune profiles of diabetic and euglycaemic NOD.SCID recipients demonstrate dissociation between fractional expression of CD25 and FoxP3 and the severity of insulitis. There were no evident and consistent differences in diabetogenic activity and immune reconstituting activity of T cells from pre-diabetic (11weeks) and new onset diabetic NOD females. Similarities in immune phenotypes and variable distribution of effector and suppressor subsets in various stages of inflammation commend caution in interpretation of quantitative and qualitative aberrations as markers of disease severity in adoptive transfer experiments.