Infiltration Of Islets Research Articles

PD-L1+ Lymphocytes Are Associated with CD4+, Foxp3+CD4+, IL17+CD4+ T Cells and Subtypes of Macrophages in Resected Early-Stage Non-Small Cell Lung Cancer.

The non-canonical PD-L1 pathway revealed that programmed-death ligand 1 (PD-L1) expression in immune cells also plays a crucial role in immune response. Moreover, immune cell distribution in a tumour microenvironment (TME) is pivotal for tumour genesis. However, the results remain controversial and further research is needed. Distribution of PD-L1-positive (PD-L1+) tumour-infiltrating lymphocytes in the context of TME was assessed in 72 archival I-III stage surgically resected NSCLC tumour specimens. Predominant PD-L1+ lymphocyte distribution in the tumour stroma, compared to islets, was found (p = 0.01). Higher PD-L1+ lymphocyte infiltration was detected in smokers due to their predominance in the stroma. High PD-L1+ lymphocyte infiltration in tumour stroma was more common in tumours with higher CD4+ T cell infiltration in islets and stroma, Foxp3+CD4+ T cell infiltration in islets and lover M1 macrophage infiltration in the stroma (p = 0.034, p = 0.034, p = 0.005 and p = 0.034 respectively). Meanwhile, high PD-L1+ lymphocyte infiltration in islets was predominantly found in tumours with high levels of IL-17A+CD4+ T cells in islets and Foxp3+CD4+ T cells in islets and stroma (p = 0.032, p = 0.009 and p = 0.034, respectively). Significant correlations between PD-L1+ lymphocytes and tumour-infiltrating CD4+, Foxp3+CD4+, IL-17A+CD4+ T cells and M2 macrophages were found. An analysis of the tumour-immune phenotype revealed a significant association between PD-L1 expression and IL17+CD4+ and Foxp3+CD4+ immune phenotypes. PD-L1+ lymphocytes are associated with the distribution of CD4+, Foxp3+CD4+, IL17A+CD4+ T cells, M1 and M2 macrophages in TME of resected NSCLC.

8090 A Gut Commensal, Parabacteroides distasonis, and Molecular Mimicry in Type 1 Diabetes

Abstract Disclosure: A. Randall: None. Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by the destruction of pancreatic, insulin-producing beta cells by autoreactive T-cells. The trigger(s) of T1D are unclear and genetics alone cannot explain the recent increase in T1D incidence. The gut microbiome composition in T1D patients is altered and thought to play a key role in T1D pathogenesis. Most longitudinal studies have been descriptive. However, a direct cause-and-effect relationship hasn't been established yet. Insulin is a key autoantigen in T1D autoimmunity, and insulin B-chain amino acids 9-23 (insB:9-23) is an important, commonly recognized T-cell epitope. Molecular mimicry is an autoimmunity mechanism where host-like microbial proteins trigger autoimmune responses. We previously identified a peptide mimic of insB:9-23, amino acids 4-18 of hypoxanthine phosphoribosyl transferase (HPRT:4-18) of Parabacteroides distasonis (Girdhar et al., 2022). HPRT:4-18 stimulated insB:9-23-specific human and non-obese diabetic (NOD) mice T-cells. We hypothesize that P. distasonis HPRT:4-18 is required to accelerate diabetes via molecular mimicry of insB:9-23. By orally gavaging NOD mice (n=40 mice/group), we showed that P. distasonis accelerated diabetes onset. Next, we colonized NOD mice with another common gut commensal, Bacteroides fragilis, as a control, and did not observe any effect on islet infiltration. In addition, P. distasonis colonization increased infiltration of immune cells into the islets of germ-free NOD mice (n=6-8 mice/group), while the diabetes incidence experiment in mice is ongoing. P. distasonis-colonized mice had significantly more splenic CD8 and naïve T-cells, dendritic cells and macrophages but fewer FOXP3 cells (n=3-6 mice/group). To determine whether P. distasonis’s effect is specific to the pancreas, we checked intestinal barrier function and intestinal immune cell composition. Disruption of intestinal barrier function has been associated with diabetes. P. distasonis colonization did not alter gut permeability. Furthermore, P. distasonis did not increase intestinal inflammation. Among the intestinal intraepithelial lymphocytes (IELs), there were fewer naïve, central and effector T-cells and B-cells. The findings indicate that P. distasonis does not stimulate an unspecific immune response and its inflammatory effect is specific to the pancreas, supporting our initial hypothesis. Taken together, our results indicate that exposure to P. distasonis in the first years of life has the potential to stimulate molecular mimicry mechanism and cause T1D onset. Reference: Girdhar, K., Huang, Q., Chow, I. T., Vatanen, T., Brady, C., Raisingani, A., . . . Altindis, E. (2022). A gut microbial peptide and molecular mimicry in the pathogenesis of type 1 diabetes. Proc Natl Acad Sci U S A, 119(31), e2120028119. doi:10.1073/pnas.2120028119 Presentation: 6/2/2024

7967 Ruxolitinib Prevents Immune-Checkpoint Inhibitor-Related Diabetes Mellitus Via Inhibition Of Pathogenic IL-21 And IFNγ-Producing CD4+ T Cells

Abstract Disclosure: N.L. Huang: None. J. Ortega: None. S.J. Wang: None. L.N. Scott: None. K. Kimbrell: None. E. McCarthy: None. J. Olay: None. J.N. Nguyen: None. K. Kim: None. J. Lee: None. M.G. Lechner: None. The use of immune checkpoint inhibitor (ICI) therapy has revolutionized the treatment landscape for many advanced cancers by leveraging immune activation through the blockade of checkpoint proteins, including programmed death protein-1 (PD-1). Unfortunately, autoimmune attack of healthy tissue can occur in up to 60% of patients. ICI-induced Type 1 diabetes mellitus (ICI-T1DM) is a rare but life-threatening adverse event during ICI therapy that leads to rapid destruction of pancreatic islets, hyperglycemia, and lifetime dependence on insulin. While cytotoxic CD8+ T cells are recognized as important players in ICI-T1DM, the role for CD4+ helper T cells in disease pathogenesis is less well understood. Using a mouse model of ICI-autoimmunity, in which male and female NOD mice treated with anti-PD-1 (10mg/kg/day, i.p., twice weekly) develop multi-organ autoimmune infiltrates including insulitis and DM, we identified interleukin-21 (IL-21) and interferon gamma (IFNγ) CD4+ T cells as key contributors to ICI-T1DM. Antibody depletion of CD4+ T cells in NOD mice significantly delayed the onset of ICI-T1DM. Immunophenotyping of pancreatic islet infiltrates in ICI-treated mice revealed increased IL-21+ IFNγ+ CD4+ T follicular helper (Tfh; ICOS+ PD1+ CXCR5+) and T peripheral helper (Tph; ICOS+PD1+ CXCR5- CXCR6+) cells. In vitro, IL-21 increased effector CD8+ T cell function and IFNγ promoted production of T cell-attracting chemokines CXCL9, CXCL10, and CXCL16 by macrophages. Given the potential role for IL-21 and IFNγ to amplify ICI-DM progression, we sought to target these signaling pathways as potential interventions. Genetic inhibition of IL-21 or IFNγ signaling in NOD mice reduced insulitis and DM in anti-PD-1 treated mice. Considering that IL-21 and IFNγ act through JAK/STAT signaling in immune cells, we proposed to block this pathway to prevent ICI-DM using ruxolitinib, a clinically available JAK1/2 inhibitor. Indeed, ruxolitinib blocked STAT3 and STAT1 phosphorylation in mouse splenocytes in response to IL-21 or IFNγ, respectively, and completely protected ICI-treated NOD mice from DM. Finally, in a syngeneic tumor model, use of ruxolitinib after initiation of anti-PD-1 therapy did not abrogate ICI anti-tumor effects. Taken together, these data suggest that IL-21 and IFNγ producing CD4+ T cells are important drivers of ICI-T1DM, and that the use of JAK inhibitors may be a promising clinical intervention to reduce ICI-T1DM toxicities in patients. Presentation: 6/2/2024

AnnoSpat annotates cell types and quantifies cellular arrangements from spatial proteomics

Cellular composition and anatomical organization influence normal and aberrant organ functions. Emerging spatial single-cell proteomic assays such as Image Mass Cytometry (IMC) and Co-Detection by Indexing (CODEX) have facilitated the study of cellular composition and organization by enabling high-throughput measurement of cells and their localization directly in intact tissues. However, annotation of cell types and quantification of their relative localization in tissues remain challenging. To address these unmet needs for atlas-scale datasets like Human Pancreas Analysis Program (HPAP), we develop AnnoSpat (Annotator and Spatial Pattern Finder) that uses neural network and point process algorithms to automatically identify cell types and quantify cell-cell proximity relationships. Our study of data from IMC and CODEX shows the higher performance of AnnoSpat in rapid and accurate annotation of cell types compared to alternative approaches. Moreover, the application of AnnoSpat to type 1 diabetic, non-diabetic autoantibody-positive, and non-diabetic organ donor cohorts recapitulates known islet pathobiology and shows differential dynamics of pancreatic polypeptide (PP) cell abundance and CD8+ T cells infiltration in islets during type 1 diabetes progression.

Beta cell specific cannabinoid 1 receptor deletion counteracts progression to hyperglycemia in non-obese diabetic mice

ObjectiveType 1 diabetes (T1D) occurs because of islet infiltration by autoreactive immune cells leading to destruction of beta cells and it is becoming evident that beta cell dysfunction partakes in this process. We previously reported that genetic deletion and pharmacological antagonism of the cannabinoid 1 receptor (CB1) in mice improves insulin synthesis and secretion, upregulates glucose sensing machinery, favors beta cell survival by reducing apoptosis, and enhances beta cell proliferation. Moreover, beta cell specific deletion of CB1 protected mice fed a high fat high sugar diet against islet inflammation and beta cell dysfunction. Therefore, we hypothesized that it would mitigate the dysfunction of beta cells in the precipitating events leading to T1D. MethodsWe genetically deleted CB1 specifically from beta cells in non-obese diabetic (NOD; NOD RIP Cre+ Cnr1fl/fl) mice. We evaluated female NOD RIP Cre+ Cnr1fl/fl mice and their NOD RIP Cre−Cnr1fl/fl and NOD RIP Cre+ Cnr1Wt/Wt littermates for onset of hyperglycemia over 26 weeks. We also examined islet morphology, islet infiltration by immune cells and beta cell function and proliferation. ResultsBeta cell specific deletion of CB1 in NOD mice significantly reduced the incidence of hyperglycemia by preserving beta cell function and mass. Deletion also prevented beta cell apoptosis and aggressive insulitis in NOD RIP Cre+ Cnr1fl/fl mice compared to wild-type littermates. NOD RIP Cre+ Cnr1fl/fl islets maintained normal morphology with no evidence of beta cell dedifferentiation or appearance of extra islet beta cells, indicating that protection from autoimmunity is inherent to genetic deletion of beta cell CB1. Pancreatic lymph node Treg cells were significantly higher in NOD RIP Cre+ Cnr1fl/flvs NOD RIP Cre−Cnr1fl/fl. ConclusionsCollectively these data demonstrate how protection of beta cells from metabolic stress during the active phase of T1D can ameliorate destructive insulitis and provides evidence for CB1 as a potential pharmacologic target in T1D.

Mapping of a hybrid insulin peptide in the inflamed islet β-cells from NOD mice.

There is accumulating evidence that pathogenic T cells in T1D recognize epitopes formed by post-translational modifications of β-cell antigens, including hybrid insulin peptides (HIPs). The ligands for several CD4 T-cell clones derived from the NOD mouse are HIPs composed of a fragment of proinsulin joined to peptides from endogenous β-cell granule proteins. The diabetogenic T-cell clone BDC-6.9 reacts to a fragment of C-peptide fused to a cleavage product of pro-islet amyloid polypeptide (6.9HIP). In this study, we used a monoclonal antibody (MAb) to the 6.9HIP to determine when and where HIP antigens are present in NOD islets during disease progression and with which immune cells they associate. Immunogold labeling of the 6.9HIP MAb and organelle-specific markers for electron microscopy were employed to map the subcellular compartment(s) in which the HIP is localized within β-cells. While the insulin B9-23 peptide was present in nearly all islets, the 6.9HIP MAb stained infiltrated islets only in NOD mice at advanced stages of T1D development. Islets co-stained with the 6.9HIP MAb and antibodies to mark insulin, macrophages, and dendritic cells indicate that 6.9HIP co-localizes within insulin-positive β-cells as well as intra-islet antigen-presenting cells (APCs). In electron micrographs, the 6.9HIP co-localized with granule structures containing insulin alone or both insulin and LAMP1 within β-cells. Exposing NOD islets to the endoplasmic reticulum (ER) stress inducer tunicamycin significantly increased levels of 6.9HIP in subcellular fractions containing crinosomes and dense-core granules (DCGs). This work demonstrates that the 6.9HIP can be visualized in the infiltrated islets and suggests that intra-islet APCs may acquire and present HIP antigens within islets.

Immunomodulation of streptozotocin induced Type 1 diabetes mellitus in mouse model by Macrophage migration inhibitory factor-2 (MIF-2) homologue of human lymphatic filarial parasite, Wuchereria bancrofti

Helminth parasites modulate the host immune system to ensure a long-lasting asymptomatic form of infection generally, mediated by the secretion of immunomodulatory molecules and one such molecule is a homologue of human host cytokine, Macrophage migratory Inhibitory Factor (hMIF). In this study, we sought to understand the role of homologue of hMIF from the lymphatic filarial parasite, Wuchereria bancrofti (Wba-MIF2), in the immunomodulation of the Streptozotocin (STZ)-induced Type1 Diabetes Mellitus (T1DM) animal model. Full-length recombinant Wba-MIF2 was expressed and found to have both oxidoreductase and tautomerase activities. Wba-MIF2 recombinant protein was treated to STZ induced T1DM animals, and after 5 weeks pro-inflammatory (IL-1, IL-2, IL-6, TNF-α, IFN-γ) and anti-inflammatory (IL-4, IL-10) cytokines and gene expressions were determined in sera samples and spleen respectively. Pro-inflammatory and anti-inflammatory cytokine levels were significantly (p<0.05) up-regulated and down-regulated respectively, in the STZ-T1DM animals, as compared to treated groups. Histopathology showed macrophage infiltration and greater damage of islets of beta cells in the pancreatic tissue of STZ-T1DM animals, than Wba-MIF2 treated STZ-T1DM animals. The present study clearly showed the potential of Wba-MIF2 as an immunomodulatory molecule, which could modulate the host immune system in the STZ-T1DM mice model from a pro-inflammatory to anti-inflammatory milieu.

HLA-DQ8 Supports Development of Insulitis Mediated by Insulin-Reactive Human TCR-Transgenic T Cells in Nonobese Diabetic Mice.

In an effort to improve HLA-"humanized" mouse models for type 1 diabetes (T1D) therapy development, we previously generated directly in the NOD strain CRISPR/Cas9-mediated deletions of various combinations of murine MHC genes. These new models improved upon previously available platforms by retaining β2-microglobulin functionality in FcRn and nonclassical MHC class I formation. As proof of concept, we generated H2-Db/H2-Kd double knockout NOD mice expressing human HLA-A*0201 or HLA-B*3906 class I variants that both supported autoreactive diabetogenic CD8+ T cell responses. In this follow-up work, we now describe the creation of 10 new NOD-based mouse models expressing various combinations of HLA genes with and without chimeric transgenic human TCRs reactive to proinsulin/insulin. The new TCR-transgenic models develop differing levels of insulitis mediated by HLA-DQ8-restricted insulin-reactive T cells. Additionally, these transgenic T cells can transfer insulitis to newly developed NSG mice lacking classical murine MHC molecules, but expressing HLA-DQ8. These new models can be used to test potential therapeutics for a possible capacity to reduce islet infiltration or change the phenotype of T cells expressing type 1 diabetes patient-derived β cell autoantigen-specific TCRs.

252-LB: Dectin-2 Deficiency Promotes Proinflammatory Cytokine Release from Macrophages and Impairs Glucose-Stimulated Insulin Secretion

Pancreatic islet inflammation plays an important role in the pathogenesis of diabetes mellitus. Islet macrophages, which have emerged as a key player in islet inflammation, express Dectin-2 (D2), one of the C-type lectin receptors recognizing α-mannans, while endocrine cells such as α-cells and β-cells lack the expression of D2. To elucidate the role of D2 in glucose metabolism and β-cell function, we analyzed the phenotype of D2 knockout (D2KO) mice. D2KO mice exhibited significantly impaired glucose tolerance compared to wild-type (WT) mice, although there were no significant differences in insulin sensitivity. Glucose-stimulated insulin secretion (GSIS) was significantly reduced in isolated islets from D2KO mice compared to WT mice. Comprehensive analysis of RNA-seq in isolated islets from D2KO and WT mice revealed that D2 deficiency induced immune cell infiltration and inflammation in pancreatic islets. Furthermore, RNA-seq analysis in peritoneal macrophages from D2KO and WT mice showed that macrophages from D2KO mice acquired an inflammatory phenotype and exhibited enhanced NF-κB signaling compared to those from WT mice. Next, to determine whether humoral factors secreted from macrophages affect GSIS, we examined GSIS using the culture supernatants of macrophages from D2KO and WT mice. The culture supernatants of macrophages from D2KO mice suppressed GSIS compared to WT mice. Furthermore, the multiplex cytokine assay revealed that IL-1α and IL-6 were significantly increased in the culture supernatants from macrophages of D2KO mice. Finally, we found that the stimulation of IL-1α and IL-6 reduced GSIS in β-cells. These findings suggest that macrophages from D2KO mice suppress GSIS by increasing IL-1α and IL-6 production. This study provides new insights into the mechanism by which D2 is involved in the pathogenesis of pancreatic islet inflammation. Disclosure M. Fujita: None. T. Miyazawa: None. K. Uchida: None. N. Uchida: None. T. Hatayama: None. S. Nakamura: None. Y. Takeichi: None. Y. Miyachi: None. Y. Ogawa: None.

The extent and magnitude of islet T cell infiltration as powerful tools to define the progression to type 1 diabetes

Aims/hypothesisInsulitis is not present in all islets, and it is elusive in humans. Although earlier studies focused on islets that fulfilled certain criteria (e.g. ≥15 CD45+ cells or ≥6 CD3+ cells), there is a fundamental lack of understanding of the infiltration dynamics in terms of its magnitude (i.e. how much) and extent (i.e. where). Here, we aimed to perform an in-depth characterisation of T cell infiltration by investigating islets with moderate (1–5 CD3+ cells) and high (≥6 CD3+ cells) infiltration in individuals with and without type 1 diabetes.MethodsPancreatic tissue sections from 15 non-diabetic, eight double autoantibody-positive and ten type 1 diabetic (0–2 years of disease duration) organ donors were obtained from the Network for Pancreatic Organ Donors with Diabetes, and stained for insulin, glucagon, CD3 and CD8 by immunofluorescence. T cell infiltration was quantified in a total of 8661 islets using the software QuPath. The percentage of infiltrated islets and islet T cell density were calculated. To help standardise the analysis of T cell infiltration, we used cell density data to develop a new T cell density threshold capable of differentiating non-diabetic and type 1 diabetic donors.ResultsOur analysis revealed that 17.1% of islets in non-diabetic donors, 33% of islets in autoantibody-positive and 32.5% of islets in type 1 diabetic donors were infiltrated by 1 to 5 CD3+ cells. Islets infiltrated by ≥6 CD3+ cells were rare in non-diabetic donors (0.4%) but could be found in autoantibody-positive (4.5%) and type 1 diabetic donors (8.2%). CD8+ and CD8− populations followed similar patterns. Likewise, T cell density was significantly higher in the islets of autoantibody-positive donors (55.4 CD3+ cells/mm2) and type 1 diabetic donors (74.8 CD3+ cells/mm2) compared with non-diabetic individuals (17.3 CD3+ cells/mm2), which was accompanied by higher exocrine T cell density in type 1 diabetic individuals. Furthermore, we showed that the analysis of a minimum of 30 islets and the use of a reference mean value for T cell density of 30 CD3+ cells/mm2 (the 30–30 rule) can differentiate between non-diabetic and type 1 diabetic donors with high specificity and sensitivity. In addition, it can classify autoantibody-positive individuals as non-diabetic or type 1 diabetic-like.Conclusions/interpretationOur data indicates that the proportion of infiltrated islets and T cell density change dramatically during the course of type 1 diabetes, and these changes can be already observed in double autoantibody-positive individuals. This suggests that, as disease progresses, T cell infiltration extends throughout the pancreas, reaching the islets and exocrine compartment. While it predominantly targets insulin-containing islets, large accumulations of cells are rare. Our study fulfils the need to further understand T cell infiltration, not only after diagnosis but also in individuals with diabetes-related autoantibodies. Furthermore, the development and application of new analytical tools based on T cell infiltration, like the 30–30 rule, will allow us to correlate islet infiltration with demographic and clinical variables with the aim of identifying individuals at the very early stages of the disease.Graphical abstract

Insulitis in Human Type 1 Diabetic Pancreas: From Stem Cell Grafting to Islet Organoids for a Successful Cell-Based Therapy.

Type 1 diabetes (T1D) is an autoimmune disease with immune cells' islet infiltration (called "insulitis"), which leads to beta cell loss. Despite being the critical element of T1D occurrence and pathogenesis, insulitis is often present in a limited percentage of islets, also at diagnosis. Therefore, it is needed to define reproducible methods to detect insulitis and beta-cell decline, to allow accurate and early diagnosis and to monitor therapy. However, this goal is still far due to the morphological aspect of islet microvasculature, which is rather dense and rich, and is considerably rearranged during insulitis. More studies on microvasculature are required to understand if contrast-enhanced ultrasound sonography measurements of pancreatic blood-flow dynamics may provide a clinically deployable predictive marker to predict disease progression and therapeutic reversal in pre-symptomatic T1D patients. Therefore, it is needed to clarify the relation between insulitis and the dynamics of β cell loss and with coexisting mechanisms of dysfunction, according to clinical stage, as well as the micro vessels' dynamics and microvasculature reorganization. Moreover, the ideal cell-based therapy of T1D should start from an early diagnosis allowing a sufficient isolation of specific Procr+ progenitors, followed by the generation and expansion of islet organoids, which could be transplanted coupled to an immune-regulatory therapy which will permit the maintenance of pancreatic islets and an effective and long-lasting insulitis reversal.

Exenatide regulates Th17/Treg balance via PI3K/Akt/FoxO1 pathway in db/db mice

BackgroundThe T helper 17 (Th17)/T regulatory (Treg) cell imbalance is involved in the course of obesity and type 2 diabetes mellitus (T2DM). In the current study, the exact role of glucagon-like peptide-1 receptor agonist (GLP-1RA) exenatide on regulating the Th17/Treg balance and the underlying molecular mechanisms are investigated in obese diabetic mice model.MethodsMetabolic parameters were monitored in db/db mice treated with/without exenatide during 8-week study period. The frequencies of Th17 and Treg cells from peripheral blood and pancreas in db/db mice were assessed. The phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/Forkhead box O1 (FoxO1) pathway in Th17 and Treg cells from the spleens of male C57BL/6J mice was detected by western blotting. In addition, the expression of glucagon-like peptide-1 receptor (GLP-1R) in peripheral blood mononuclear cells (PBMCs) of male C57BL/6J mice was analyzed.ResultsExenatide treatment improved β-cell function and insulitis in addition to glucose, insulin sensitivity and weight. Increased Th17 and decreased Treg cells in peripheral blood were present as diabetes progressed while exenatide corrected this imbalance. Progressive IL-17 + T cell infiltration of pancreatic islets was alleviated by exenatide intervention. In vitro study showed no significant difference in the level of GLP-1R expression in PBMCs between control and palmitate (PA) groups. In addition, PA could promote Th17 but suppress Treg differentiation along with down-regulating the phosphorylation of PI3K/Akt/FoxO1, which was reversed by exenatide intervention. FoxO1 inhibitor AS1842856 could abrogate all these effects of exenatide against lipid stress.ConclusionsExenatide could restore systemic Th17/Treg balance via regulating FoxO1 pathway with the progression of diabetes in db/db mice. The protection of pancreatic β-cell function may be partially mediated by inhibiting Th17 cell infiltration into pancreatic islets, and the resultant alleviation of islet inflammation.

Nitazoxanide, Ivermectin, and Artemether effects against cryptosporidiosis in diabetic mice: parasitological, histopathological, and chemical studies.

Human cryptosporidiosis is one of the most significant causes of water borne epidemics of diarrhea worldwide. It is extremely important in immunocompromised hosts and malnourished children as it could cause severe life-threatening diarrhea. Despite the global burden of the disease, there are only few available therapies against cryptosporidiosis. Diabetes mellitus is a common metabolic disorder that impair both the innate and adaptive immune responses of the patient. This study aimed to test the effect of Nitazoxanide, Ivermectin, and Artemether against cryptosporidiosis in diabetic mice. Sixty white albino mice were categorized into 6 groups; 10 mice each: GI: normal non-infected non-treated (healthy- control), GII-GVI (diabetic groups), GII: non-infected non treated (diabetic control), GIII: infected non treated (infected control), GIV: infected and treated with Nitazoxanide (NTZ), GV: infected and treated with Ivermectin (IVC), GVI: infected and treated with Artemether (ART). Parasitological, histopathological, and chemical examinations were done to evaluate the effect of NTZ, IVC, and ART against cryptosporidiosis in diabetic mice. Parasitological examination revealed maximum reduction of oocyst shedding in GVI, while histopathological examination showed the least pathologic changes in GV with mild vascular wall fibrosis and moderate lymphocytic infiltration of islets of Langerhans. Measurement of blood glucose level showed the best results with GIV. Nitazoxanide is effective against cryptosporidiosis in diabetic patients with minimal hyperglycemia, Artemether is especially effective in reducing the oocyst shedding in stool, whereas Ivermectin is associated with the least pathological changes in pancreatic islets of Langerhans.Graphical abstract

Insulin B-chain hybrid peptides are agonists for T cells reactive to insulin B:9-23 in autoimmune diabetes.

Insulin is considered to be a key antigenic target of T cells in Type 1 Diabetes (T1D) and autoimmune diabetes in the NOD mouse with particular focus on the B-chain amino acid sequence B:9-23 as the primary epitope. Our lab previously discovered that hybrid insulin peptides (HIPs), comprised of insulin C-peptide fragments fused to other β-cell granule peptides, are ligands for several pathogenic CD4 T cell clones derived from NOD mice and for autoreactive CD4 T cells from T1D patients. A subset of CD4 T cell clones from our panel react to insulin and B:9-23 but only at high concentrations of antigen. We hypothesized that HIPs might also be formed from insulin B-chain sequences covalently bound to other endogenously cleaved ß-cell proteins. We report here on the identification of a B-chain HIP, termed the 6.3HIP, containing a fragment of B:9-23 joined to an endogenously processed peptide of ProSAAS, as a strong neo-epitope for the insulin-reactive CD4 T cell clone BDC-6.3. Using an I-Ag7 tetramer loaded with the 6.3HIP, we demonstrate that T cells reactive to this B-chain HIP can be readily detected in NOD mouse islet infiltrates. This work suggests that some portion of autoreactive T cells stimulated by insulin B:9-23 may be responding to B-chain HIPs as peptide ligands.

Lymphocyte infiltration in histologic subtypes of epithelial ovarian carcinoma and its effect on survival (229)

Lymphocyte infiltration in histologic subtypes of epithelial ovarian carcinoma and its effect on survival (229)

XAF1 overexpression exacerbates diabetes by promoting pancreatic β-cell apoptosis

AimsPancreatic β-cell apoptosis may be involved in the onset and progression of type 2 diabetes mellitus, although its mechanism remains unclear. We previously demonstrated that macrophage-derived interferon (IFN) β induced X-linked inhibitor of apoptosis–associated factor 1 (XAF1) expression in β-cells and accelerated β-cell apoptosis in vitro. Here, we explored the effects of XAF1 on β-cell function and progression of diabetes in vivo.MethodsPancreatic β-cell-selective XAF1 overexpressing (Xaf1 Tg) mice were generated. Xaf1 Tg mice and their wild-type (WT) littermates were fed either a normal diet or a 40% or 60% high-fat diet (HFD). The effects of β-cell XAF1 on β-cell apoptosis and exacerbation of diabetes were investigated.ResultsPalmitic acid induced IFNβ expression in macrophages, and HFD intake promoted macrophage infiltration in pancreatic islets, both of which cooperatively upregulated XAF1 expression in mouse islets. Furthermore, HFD-fed Xaf1 Tg mice demonstrated increased β-cell apoptosis, lowered insulin expression, and impaired glucose tolerance compared with WT mice fed the same diet. These effects were more pronounced in the 60%HFD group than in the 40%HFD group.ConclusionsPancreatic β-cell XAF1 expression was enhanced via HFD-induced, macrophage-derived IFNβ, which promoted β-cell apoptosis and led to a reduction in insulin secretion and progression of diabetes. To our knowledge, this is the first report to demonstrate an association between pancreatic β-cell XAF1 overexpression and exacerbation of diabetes, thus providing insight into the mechanism of β-cell mass reduction in diabetes.

Spontaneous Myocarditis in Mice Predisposed to Autoimmune Disease: Including Vaccination-Induced Onset

Nonobese diabetic (NOD)/ShiLtJ mice, such as biobreeding rats, are used as an animal model for type 1 diabetes. Diabetes develops in NOD mice as a result of insulitis, a leukocytic infiltrate of the pancreatic islets. The onset of diabetes is associated with moderate glycosuria and nonfasting hyperglycemia. Previously, in NOD/ShiLtJ mice spontaneously developing type 1 diabetes, the possible involvement of decreased expression of nuclear factor-kappa B1 (NF-κB1) (also known as p50) in the development of type 1 diabetes was investigated. In response to these arguments, NOD mice with inconsistent NF-κB1 expression were established. Surprisingly, the majority of NOD Nfκb1 homozygote mice were found to die by the eighth week of life because of severe myocarditis. The incidence of spontaneous myocarditis in mice was slightly higher in males than in females. Furthermore, insulitis was observed in all NOD Nfκb1 heterozygote mice as early as 4 months of age. Additionally, in NOD Nfκb1 heterozygote mice, myocarditis with an increase in cTnT levels due to influenza or hepatitis B virus vaccination was observed with no significant gender difference. However, myocarditis was not observed with the two types of human papillomavirus vaccination. The results of immunological assays and histopathological examinations indicated that vaccination could induce myocarditis in genetically modified mice. In this study, we report that NOD Nfκb1 heterozygote mice can be used for investigating the risk of myocarditis development after vaccination.

1322-P: Role of Macrophage NOD1 in Fat-Induced ß-Cell Dysfunction

In individuals with obesity, free fatty acids (FFA) are chronically raised resulting in ß-cell dysfunction, leading to type 2 diabetes (T2D) . Recent data implicate the role of inflammation in developing ß-cell dysfunction, which is in part mediated by nucleotide-oligomerization domain 1 (NOD1) , an intracellular pattern recognition receptor. NOD1 is activated by peptidoglycans of bacterial cell walls; however, in vitro studies have documented that NOD1 can also be activated by saturated FFA. Previous data from our lab have shown that palmitate-induced ß-cell dysfunction was prevented in whole body and ß-cell specific NOD1 null mice. We have seen earlier reports on increased macrophage infiltration in islets after elevation in circulating palmitate, and much higher expression of NOD1 in macrophages than ß-cells. Hence, we hypothesized that macrophage NOD1 receptors play a pivotal role in FFA-induced ß-cell dysfunction. We have generated myeloid specific NOD1 null mice to target macrophage NOD1 receptors by crossing NOD1 floxed mice with Lyz2 Cre+ mice. Both knockouts and their controls (floxed and Cre) were infused for 48 h with ethylpalmitate (hydrolyzed to palmitate and ethanol in plasma) , to achieve elevated circulating FFA in a non-toxic manner. Ethanol vehicle was infused as control treatment. To assess in vivo ß-cell function, hyperglycemic clamp was performed following the 48 h infusion, and the disposition index (DI) was calculated. Ethylpalmitate resulted in elevated plasma FFA compared to vehicle infusion. Ethylpalmitate caused marked reduction in DI in control mice, while knockout mice displayed no significant changes in DI between ethylpalmitate and vehicle infused groups. These results suggest that macrophage NOD1 plays a causal role in palmitate-induced ß-cell dysfunction in vivo. Overall, our data reveal NOD1 as a key factor and a potential therapeutic target for obesity-associated diabetes. Disclosure S.Rahman: None. J.Yung: None. Y.Zhang: None. A.Giacca: None.

74-OR: Adoptively Transferred T Lymphocytes with Reduced iPLA2b Expression Delays Type 1 Diabetes Development in NOD Mice

Type 1 diabetes (T1D) is a T-cell mediated autoimmune disease characterized by the destruction of insulin-producing pancreatic islet β-cells. An understudied area is the role lipid signaling plays in this process. We find that the Ca2+-independent phospholipase A2β (iPLA2β) is induced under a diabetic milieu and mitigation of iPLA2β activity attenuates β-cell death. iPLA2β, a member of the PLA2 family, hydrolyzes the sn-2 substituent from glycerophospholipid substrates to yield a free fatty acid. When the fatty acid is arachidonic acid, it can be metabolized to eicosanoids, many of which are pro-inflammatory. To address our overarching hypothesis that iPLA2β-derived lipids (iDLs) contribute to T1D development, we investigated the role of immune cell-iDLs by utilizing a T-cell adoptive transfer. We isolated CD4+/8+ T-cells from splenocytes prepared from 11-week-old donor female NOD and NOD.iPLA2 β +/- (HET) . These were transferred i.p. to 4-week-old NOD.scid recipient mice. We find that NOD4/8-recipient mice develop T1D between 14-16 weeks of age. In contrast, T1D onset in HET4/8-recipient mice is delayed by 12 weeks and this was associated with better glucose tolerance, compared to NOD4/8 T-cell recipients. This finding is also supported by histology analysis of the pancreatic islet, which revealed less islet infiltration, more islet abundance, and the frequency of CD4 T cells were increased in the pancreas of HET4/8-recipient mice compared to NOD4/8 recipients. Further, lipidomics analyses revealed that HET donor T-cell production of proinflammatory lipids (i.e., PGE2 and DHETs) is decreased, relative to NOD T-cells. These findings suggest that pro-inflammatory iDLs generated by CD4 T-cells are critical contributors T1D development and can be targeted to counter T1D onset. Disclosure T.White: None. C.Chalfant: None. T.P.Dilorenzo: None. S.Ramanadham: None. D.Stephenson: None. Funding Training Program in Cell, Molecular, and Developmental Biology (5T32GM008111-34) NIH/NIDDK (R01DK110292) NIH/NIAID R21 (AI 146743) Cell, Developmental, &amp; Integrative Biology (CDIB) , the University of Alabama at Birmingham

188-OR: Metabolic Blockade of Glycolysis Can Delay the Adoptive Transfer of Type 1 Diabetes

Type 1 diabetes (T1D) is a result of insulin-secreting pancreatic β-cell destruction by autoreactive CD4 and CD8 T cells. There is no cure for T1D, but determining the role of immunometabolism in promoting autoreactive CD4+ T cell effector responses may provide a novel target in delaying T1D. Systemically inhibiting glycolysis with 2-deoxyglucose (2-DG) can decrease autoreactive CD8 T cell islet infiltration. However, the effects of 2-DG on CD4 T cell diabetogenicity is unknown. Proinflammatory Th1 cells mediate β-cell destruction prefer glycolysis for maintaining effector responses, while immunosuppressive Treg cells prefer oxidative phosphorylation. We hypothesize that metabolic inhibition of glycolysis will diminish proinflammatory Th1 cell responses and delay β-cell destruction in T1D. Utilizing 2-DG, we performed an adoptive transfer of diabetogenic CD4+ T cells from NOD.BDC-2.5 or NOD.BDC-6.9 mice into NOD.scid mice. Recipient mice were systemically treated with 30 mM 2-DG in the drinking water beginning 4 days prior to adoptive transfer and monitored for hyperglycemia for 80 days post-transfer. NOD.scid mice treated with 2-DG had a significant delay in diabetes onset when transferred with either BDC-2.5 (n=14, p=0.0284) or BDC-6.9 (n=17, p=0.0249) CD4+ T cells compared to vehicle-treated groups. To define the effects of 2-DG on autoreactive CD4 T cell effector responses, NOD.BDC-6.9 splenocytes were in vitro stimulated with their cognate hybrid insulin peptide (HIP; insulin C-peptide and islet amyloid polypeptide) with or without 2-DG. Flow cytometry analysis revealed a decrease in T cell proliferation, T cell activation, and Th1 lineage commitment as shown by Ki67, CD25, and T-bet expression, respectively. Effector responses were also dampened as IFN-γ synthesis was decreased in our 2-DG treatment group compared to HIP only controls. Future studies will investigate the effects of 2-DG on CD4 T cell chemokine secretion, trafficking into the islet, and cellular metabolic profiles. Disclosure M.D.Chavez: None. R.Mcdowell: None. H.M.Tse: None.