E2-2 Dependent Plasmacytoid Dendritic Cells Control Autoimmune Diabetes

Autoimmune diabetes is a consequence of immune-cell infiltration and destruction of pancreatic β-cells in the islets of Langerhans. We analyzed the cellular composition of the insulitic lesions in the autoimmune-prone non-obese diabetic (NOD) mouse and observed a peak in recruitment of plasmacytoid dendritic cells (pDCs) to NOD islets around 8–9 weeks of age. This peak coincides with increased spontaneous expression of type-1-IFN response genes and CpG1585 induced production of IFN-α from NOD islets. The transcription factor E2-2 is specifically required for the maturation of pDCs, and we show that knocking out E2-2 conditionally in CD11c+ cells leads to a reduced recruitment of pDCs to pancreatic islets and reduced CpG1585 induced production of IFN-α during insulitis. As a consequence, insulitis has a less aggressive expression profile of the Th1 cytokine IFN-γ and a markedly reduced diabetes incidence. Collectively, these observations demonstrate a disease-promoting role of E2-2 dependent pDCs in the pancreas during autoimmune diabetes in the NOD mouse

Single cell RNA-seq reveals profound transcriptional similarity between Barrett's oesophagus and oesophageal submucosal glands

Barrett’s oesophagus is a precursor of oesophageal adenocarcinoma. In this common condition, squamous epithelium in the oesophagus is replaced by columnar epithelium in response to acid reflux. Barrett’s oesophagus is highly heterogeneous and its relationships to normal tissues are unclear. Here we investigate the cellular complexity of Barrett’s oesophagus and the upper gastrointestinal tract using RNA-sequencing of single cells from multiple biopsies from six patients with Barrett’s oesophagus and two patients without oesophageal pathology. We find that cell populations in Barrett’s oesophagus, marked by LEFTY1 and OLFM4, exhibit a profound transcriptional overlap with oesophageal submucosal gland cells, but not with gastric or duodenal cells. Additionally, SPINK4 and ITLN1 mark cells that precede morphologically identifiable goblet cells in colon and Barrett’s oesophagus, potentially aiding the identification of metaplasia. Our findings reveal striking transcriptional relationships between normal tissue populations and cells in a premalignant condition, with implications for clinical practice

Deficiency in plasmacytoid dendritic cells and type I interferon signalling prevents diet-induced obesity and insulin resistance in mice

Aims/hypothesis: Obesity is associated with glucose intolerance and insulin resistance and is closely linked to the increasing prevalence of type 2 diabetes. In mouse models of diet-induced obesity (DIO) and type 2 diabetes, an increased fat intake results in adipose tissue expansion and the secretion of proinflammatory cytokines. The innate immune system not only plays a crucial role in obesity-associated chronic low-grade inflammation but it is also proposed to play a role in modulating energy metabolism. However, little is known about how the modulation of metabolism by the immune system may promote increased adiposity in the early stages of increased dietary intake. Here we aimed to define the role of type I IFNs in DIO and insulin resistance. Methods: Mice lacking the receptor for IFN-α (IFNAR−/−) and deficient in plasmacytoid dendritic cells (pDCs) (B6.E2-2fl/fl.Itgax-cre) were fed a diet with a high fat content or normal chow. The mice were analysed in vivo and in vitro using cellular, biochemical and molecular approaches. Results: We found that the development of obesity was inhibited by an inability to respond to type I IFNs. Furthermore, the development of obesity and insulin resistance in this model was associated with pDC recruitment to the fatty tissues and liver of obese mice (a 4.3-fold and 2.7-fold increase, respectively). Finally, we demonstrated that the depletion of pDCs protects mice from DIO and from developing obesity-associated metabolic complications. Conclusions/interpretation: Our results provide genetic evidence that pDCs, via type I IFNs, regulate energy metabolism and promote the development of obesity

E2-2 Dependent Plasmacytoid Dendritic Cells Control Autoimmune Diabetes.

Autoimmune diabetes is a consequence of immune-cell infiltration and destruction of pancreatic β-cells in the islets of Langerhans. We analyzed the cellular composition of the insulitic lesions in the autoimmune-prone non-obese diabetic (NOD) mouse and observed a peak in recruitment of plasmacytoid dendritic cells (pDCs) to NOD islets around 8-9 weeks of age. This peak coincides with increased spontaneous expression of type-1-IFN response genes and CpG1585 induced production of IFN-α from NOD islets. The transcription factor E2-2 is specifically required for the maturation of pDCs, and we show that knocking out E2-2 conditionally in CD11c+ cells leads to a reduced recruitment of pDCs to pancreatic islets and reduced CpG1585 induced production of IFN-α during insulitis. As a consequence, insulitis has a less aggressive expression profile of the Th1 cytokine IFN-γ and a markedly reduced diabetes incidence. Collectively, these observations demonstrate a disease-promoting role of E2-2 dependent pDCs in the pancreas during autoimmune diabetes in the NOD mouse

The immunomodulatory quinoline-3-carboxamide paquinimod reverses established fibrosis in a novel mouse model for liver fibrosis

Quinoline-3-carboxamides (Q substances) are small molecule compounds with anti-inflammatory properties. In this study, we used one of these substances, Paquinimod, to treat a novel model for chronic liver inflammation and liver fibrosis, the NOD-Inflammation Fibrosis (N-IF) mouse. We show that treatment of N-IF mice significantly reduced inflammation and resulted in the regression of fibrosis, even when the treatment was initiated after onset of disease. The reduced disease phenotype was associated with a systemic decrease in the number and reduced activation of disease-promoting transgenic natural killer T (NKT)-II cells and their type 2-cytokine expression profile. Paquinimod treatment also led to a reduction of CD115+ Ly6Chi monocytes and CD11b+ F4/80+ CD206+ macrophages

Global and 3D spatial assessment of neuroinflammation in rodent models of Multiple Sclerosis.

Multiple Sclerosis (MS) is a progressive autoimmune inflammatory and demyelinating disease of the central nervous system (CNS). T cells play a key role in the progression of neuroinflammation in MS and also in the experimental autoimmune encephalomyelitis (EAE) animal models for the disease. A technology for quantitative and 3 dimensional (3D) spatial assessment of inflammation in this and other CNS inflammatory conditions is much needed. Here we present a procedure for 3D spatial assessment and global quantification of the development of neuroinflammation based on Optical Projection Tomography (OPT). Applying this approach to the analysis of rodent models of MS, we provide global quantitative data of the major inflammatory component as a function of the clinical course. Our data demonstrates a strong correlation between the development and progression of neuroinflammation and clinical disease in several mouse and a rat model of MS refining the information regarding the spatial dynamics of the inflammatory component in EAE. This method provides a powerful tool to investigate the effect of environmental and genetic forces and for assessing the therapeutic effects of drug therapy in animal models of MS and other neuroinflammatory/neurodegenerative disorders

Imaging dynamics of CD11c+ cells and Foxp3+ cells in progressive autoimmune insulitis in the NOD mouse model of type 1 diabetes

The aim of this study was to visualise the dynamics and interactions of the cells involved in autoimmune-driven inflammation in type 1 diabetes. We adopted the anterior chamber of the eye (ACE) transplantation model to perform non-invasive imaging of leucocytes infiltrating the endocrine pancreas during initiation and progression of insulitis in the NOD mouse. Individual, ACE-transplanted islets of Langerhans were longitudinally and repetitively imaged by stereomicroscopy and two-photon microscopy to follow fluorescently labelled leucocyte subsets. We demonstrate that, in spite of the immune privileged status of the eye, the ACE-transplanted islets develop infiltration and beta cell destruction, recapitulating the autoimmune insulitis of the pancreas, and exemplify this by analysing reporter cell populations expressing green fluorescent protein under the Cd11c or Foxp3 promoters. We also provide evidence that differences in morphological appearance of subpopulations of infiltrating leucocytes can be correlated to their distinct dynamic behaviour. Together, these findings demonstrate that the kinetics and dynamics of these key cellular components of autoimmune diabetes can be elucidated using this imaging platform for single cell resolution, non-invasive and repetitive monitoring of the individual islets of Langerhans during the natural development of autoimmune diabetes

Impaired recruitment of pDCs to pancreatic islets alters cytokine profile of insulitis and prevents diabetes development.

<p>(<b>A</b>) Flow cytometry analysis of leukocytes (FVD⁻CD45⁺) from handpicked pancreatic islets of fl/fl cre⁻ (black square) or fl/fl cre⁺ (open square) mice at indicated age. Cell types include pDCs (CD3⁻CD19⁻BST2⁺CD11c^intB220⁺), inflammatory DCs (CD3⁻CD19⁻BST2⁻CD11c^hiMHC-II⁺), B-cells (CD3⁻ B220⁺CD19⁺), T-cells (CD3⁺B220⁻CD19⁻). Number of cells per mouse (mean ± sem, <i>n</i> = 5–15 mice, 2–5 independent experiments). * p<0.05. (<b>B</b>) Representative dot plots of FVD⁻CD45⁺CD3⁻CD19⁻ islet cells with the percentage of pDC and inflammatory DC subsets of total CD45⁺ cells indicated (mean ± sem, <i>n</i> = 5–15 mice, 2–5 independent experiments) (<b>C</b>) IFN-α levels in supernatants from cultured fl/fl cre⁻ (black bars) or fl/fl cre⁺ (open bars) islets or cultured single cell suspensions from PaLN at 8–17 weeks of age after 40h or 24h, respectively ± TLR9 ligand CpG₁₅₈₅ is assessed by ELISA (mean ± sem, <i>n</i> = 22–24 mice, 5–6 independent experiments for islets and <i>n</i> = 13–14 mice, 4–5 independent experiments for PaLN). nd = not detected. * p<0.05. (<b>D</b>) Expression of IFN-γ is assessed by qPCR of RNA from handpicked islets of fl/fl cre⁻ (black square) and fl/fl cre⁺ (open square) mice at 3->22 weeks of age (mean ± sem, <i>n</i> = 3–9 mice, 3 independent experiments). ** p<0.005, * p<0.05 (<b>E</b>) Incidence of diabetes in fl/fl cre⁻ (black square) and fl/fl cre⁺ (open square) (<i>n</i> = 36–32). *** p<0.0001. Incidence of diabetes in +/fl cre⁺ (black circle) and +/fl cre⁻ (open circle) (<i>n</i> = 45–42). ** p = 0.0014.</p

Conditional knockout of E2-2 blocks pDC development in NOD mice.

<p>(<b>A</b>) Flow cytometry analysis of BM and spleen from 8–12 weeks old NODwt (black bars), NOD.<i>E2-2</i>^<i>fl/fl</i>-<i>CD11c</i>.<i>cre</i>⁻ (fl/fl cre⁻, striped bars) and NOD.<i>E2-2</i>^<i>fl/fl</i>-<i>CD11c</i>.<i>cre</i>⁺ (fl/fl cre⁺, open bars) mice. Data is normalized to the level of the respective cell type in NODwt. For the pDC population the total cell number in spleen is also shown. Cell subsets include pDCs (CD3⁻CD19⁻BST2⁺CD11c^intB220⁺), DCs (CD3⁻CD19⁻BST2⁻CD11c^hiMHC-II⁺), T-cells (CD3⁺B220⁻CD19⁻), B-cells (CD3⁻B220⁺CD19⁺), granulocytes (Gran) (CD3⁻CD19⁻CD11b⁺SSC^hi) and macrophages/monocytes (Mϕ/mono) (CD3⁻CD19⁻F4/80⁺CD11b⁺MHC-II⁺) (mean ± sem, <i>n</i> = 9–10 mice, 3 independent experiments). * p<0.05, ** p<0.005, *** p<0.0001. (<b>B</b>) Representative dot plots of FVD⁻CD45⁺ islet cells with percentage of pDC and inflammatory DC subsets indicated (mean ± sem, <i>n</i> = 9–10 mice, 3 independent experiments).</p

IFN-α-secreting pDCs peak in the pancreatic islets of NOD mice at 8–9 weeks.

<p>(<b>A</b>) Infiltrating leukocytes (FVD⁻CD45⁺) from pancreatic islets were analyzed by flow cytometry for total number per mouse of T-cells (CD3⁺B220⁻CD19⁻), B-cells (CD3⁻B220⁺CD19⁺), pDCs (CD3⁻CD19⁻BST2⁺CD11c^intB220⁺), inflammatory DCs (CD3⁻CD19⁻BST2⁻CD11c^hiMHC-II⁺) and macrophages (CD3⁻CD19⁻F4/80⁺CD11b⁺MHC-II⁺) from 3 to >23 weeks of age in NOD and B6 mice (mean ± sem, <i>n</i> = 4–21 mice, 2–5 independent experiments). * p<0.05, ** p<0.005, *** p<0.0005 compared to 4-week-old B6. (<b>B</b>) Representative dot plots of FVD⁻CD45⁺CD3⁻CD19⁻ islet cells with the percentage of pDC and inflammatory DC subsets of total CD45⁺ cells indicated (mean ± sem, <i>n</i> = 4–21 mice, 2–5 independent experiments). (<b>C</b>) Expression of interferon response genes IRF7 and ISG15 is assessed by qPCR of RNA from handpicked islets of NOD (black bars) or B6 (open bars) mice at 3 and >8 weeks (<i>n</i> = 6–11 mice, 3 independent experiments). * p<0.05, ** p<0.005. (<b>D</b>) IFN-α levels in supernatants from cultured NOD islets at 4–17 weeks of age after 40h ± TLR9 ligand CpG₁₅₈₅ is assessed by ELISA (mean ± sem, <i>n</i> = 6–23 mice, 6–9 independent experiments). nd = not detected. *** p<0.0005 compared to “No” stimulation at same age.</p