MafB-dependent neurotransmitter signaling promotes ß cell migration in the developing pancreas.

Hormone secretion from pancreatic islets is essential for glucose homeostasis, and loss or dysfunction of islet cells is a hallmark of type 2 diabetes. Maf transcription factors are crucial for establishing and maintaining adult endocrine cell function. However, during pancreas development, MafB is not only expressed in insulin- and glucagon-producing cells, but also in Neurog3+ endocrine progenitor cells, suggesting additional functions in cell differentiation and islet formation. Here, we report that MafB deficiency impairs ß cell clustering and islet formation, but also coincides with loss of neurotransmitter and axon guidance receptor gene expression. Moreover, the observed loss of nicotinic receptor gene expression in human and mouse ß cells implied that signaling through these receptors contributes to islet cell migration/formation. Inhibition of nicotinic receptor activity resulted in reduced ß cell migration towards autonomic nerves and impaired ß cell clustering. These findings highlight a novel function of MafB in controlling neuronal-directed signaling events required for islet formation.

Transcription factor E2-2 is an essential and specific regulator of plasmacytoid dendritic cell development.

Plasmacytoid dendritic cells (PDCs) represent a unique immune cell type specialized in type I interferon (IFN) secretion in response to viral nucleic acids. The molecular control of PDC lineage specification has been poorly understood. We report that basic helix-loop-helix transcription factor (E protein) E2-2/Tcf4 is preferentially expressed in murine and human PDCs. Constitutive or inducible deletion of murine E2-2 blocked the development of PDCs but not of other lineages and abolished IFN response to unmethylated DNA. Moreover, E2-2 haploinsufficiency in mice and in human Pitt-Hopkins syndrome patients was associated with aberrant expression profile and impaired IFN response of the PDC. E2-2 directly activated multiple PDC-enriched genes, including transcription factors involved in PDC development (SpiB, Irf8) and function (Irf7). These results identify E2-2 as a specific transcriptional regulator of the PDC lineage in mice and humans and reveal a key function of E proteins in the innate immune system.

Recruited fibroblasts reconstitute the peri-islet membrane: a longitudinal imaging study of human islet grafting and revascularisation.

AIMS/HYPOTHESIS: Rapid and adequate islet revascularisation and restoration of the islet-extracellular matrix (ECM) interaction are significant factors influencing islet survival and function of the transplanted islets in individuals with type 1 diabetes. Because the ECM encapsulating the islets is degraded during islet isolation, understanding the process of revascularisation and engraftment after transplantation is essential and needs further investigation. METHODS: Here we apply a longitudinal and high-resolution imaging approach to investigate the dynamics of the pancreatic islet engraftment process up to 11 months after transplantation. Human and mouse islet grafts were inserted into the anterior chamber of the mouse eye, using a NOD.ROSA-tomato.Rag2-/- or B6.ROSA-tomato host allowing the investigation of the expansion of host vs donor cells and the contribution of host cells to aspects such as promoting the encapsulation and vascularisation of the graft. RESULTS: A fibroblast-like stromal cell population of host origin rapidly migrates to ensheath the transplanted islet and aid in the formation of a basement membrane-like structure. Moreover, we show that the vessel network, while reconstituted by host endothelial cells, still retains the overall architecture of the donor islets. CONCLUSIONS/INTERPRETATION: In this transplantation situation the fibroblast-like stromal cells appear to take over as main producers of ECM or act as a scaffold for other ECM-producing cells to reconstitute a peri-islet-like basement membrane. This may have implications for our understanding of long-term graft rejection and for the design of novel strategies to interfere with this process.

TCF4 (E2-2) harbors tumor suppressive functions in SHH medulloblastoma.

The TCF4 gene encodes for the basic helix-loop-helix transcription factor 4 (TCF4), which plays an important role in the development of the central nervous system (CNS). Haploinsufficiency of TCF4 was found to cause Pitt-Hopkins syndrome (PTHS), a severe neurodevelopmental disorder. Recently, the screening of a large cohort of medulloblastoma (MB), a highly aggressive embryonal brain tumor, revealed almost 20% of adult patients with MB of the Sonic hedgehog (SHH) subtype carrying somatic TCF4 mutations. Interestingly, many of these mutations have previously been detected as germline mutations in patients with PTHS. We show here that overexpression of wild-type TCF4 in vitro significantly suppresses cell proliferation in MB cells, whereas mutant TCF4 proteins do not to the same extent. Furthermore, RNA sequencing revealed significant upregulation of multiple well-known tumor suppressors upon expression of wild-type TCF4. In vivo, a prenatal knockout of Tcf4 in mice caused a significant increase in apoptosis accompanied by a decreased proliferation and failed migration of cerebellar granule neuron precursor cells (CGNP), which are thought to be the cells of origin for SHH MB. In contrast, postnatal in vitro and in vivo knockouts of Tcf4 with and without an additional constitutive activation of the SHH pathway led to significantly increased proliferation of CGNP or MB cells. Finally, publicly available data from human MB show that relatively low expression levels of TCF4 significantly correlate with a worse clinical outcome. These results not only point to time-specific roles of Tcf4 during cerebellar development but also suggest a functional linkage between TCF4 mutations and the formation of SHH MB, proposing that TCF4 acts as a tumor suppressor during postnatal stages of cerebellar development.

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.E2-2 fl/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.

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

AIMS/HYPOTHESIS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS/INTERPRETATION: 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.

Association of CD247 (CD3ζ) gene polymorphisms with T1D and AITD in the population of northern Sweden.

BACKGROUND: T1D and AITD are autoimmune disorders commonly occurring in the same family and even in the same individual. The genetic contribution to these disorders is complex making uncovering of susceptibility genes very challenging. The general aim of this study was to identify loci and genes contributing to T1D/AITD susceptibility. Our strategy was to perform linkage and association studies in the relatively genetically homogenous population of northern Sweden. We performed a GWLS to find genomic regions linked to T1D/AITD in families from northern Sweden and we performed an association study in the families to test for association between T1D/AITD and variants in previously published candidate genes as well as a novel candidate gene, CD247. METHODS: DNA prepared from 459 individuals was used to perform a linkage and an association study. The ABI PRISM Linkage Mapping Set v2.5MD10 was employed for an initial 10-cM GWLS, and additional markers were added for fine mapping. Merlin was used for linkage calculations. For the association analysis, a GoldenGate Custom Panel from Illumina containing 79 SNPs of interest was used and FBAT was used for association calculations. RESULTS: Our study revealed linkage to two previously identified chromosomal regions, 4q25 and 6p22, as well as to a novel chromosomal region, 1q23. The association study replicated association to PTPN22, HLA-DRB1, INS, IFIH1, CTLA4 and C12orf30. Evidence in favor of association was also found for SNPs in the novel susceptibility gene CD247. CONCLUSIONS: Several risk loci for T1D/AITD identified in published association studies were replicated in a family material, of modest size, from northern Sweden. This provides evidence that these loci confer disease susceptibility in this population and emphasizes that small to intermediate sized family studies in this population can be used in a cost-effective manner for the search of genes involved in complex diseases. The linkage study revealed a chromosomal region in which a novel T1D/AITD susceptibility gene, CD247, is located. The association study showed association between T1D/AITD and several variants in this gene. These results suggests that common susceptibility genes act in concert with variants of CD247 to generate genetic risk for T1D/AITD in this population.

CD4(+) type II NKT cells mediate ICOS and programmed death-1-dependent regulation of type 1 diabetes.

Type 1 diabetes (T1D) is a chronic autoimmune disease that results from T cell-mediated destruction of pancreatic ß cells. CD1d-restricted NKT lymphocytes have the ability to regulate immunity, including autoimmunity. We previously demonstrated that CD1d-restricted type II NKT cells, which carry diverse TCRs, prevented T1D in the NOD mouse model for the human disease. In this study, we show that CD4(+) 24αß type II NKT cells, but not CD4/CD8 double-negative NKT cells, were sufficient to downregulate diabetogenic CD4(+) BDC2.5 NOD T cells in adoptive transfer experiments. CD4(+) 24αß NKT cells exhibited a memory phenotype including high ICOS expression, increased cytokine production, and limited display of NK cell markers, compared with double-negative 24αß NKT cells. Blocking of ICOS or the programmed death-1/programmed death ligand 1 pathway was shown to abolish the regulation that occurred in the pancreas draining lymph nodes. To our knowledge, these results provide for the first time cellular and molecular information on how type II CD1d-restricted NKT cells regulate T1D.

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

AIMS/HYPOTHESIS: The aim of this study was to visualise the dynamics and interactions of the cells involved in autoimmune-driven inflammation in type 1 diabetes. METHODS: 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. RESULTS: 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. CONCLUSIONS/INTERPRETATION: 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.

Variation in the Cd3 zeta (Cd247) gene correlates with altered T cell activation and is associated with autoimmune diabetes.

Tuning of TCR-mediated activation was demonstrated to be critical for lineage fate in T cell development, as well as in the control of autoimmunity. In this study, we identify a novel diabetes susceptibility gene, Idd28, in the NOD mouse and provide evidence that Cd3zeta (Cd247) constitutes a prime candidate gene for this locus. Moreover, we show that the allele of the Cd3zeta gene expressed in NOD and DBA/2 mouse strains confers lower levels of T cell activation compared with the allele expressed by C57BL/6 (B6), BALB/c, and C3H/HeJ mice. These results support a model in which the development of autoimmune diabetes is dependent on a TCR signal mediated by a less-efficient NOD allele of the Cd3zeta gene.

Distinct phenotype and function of NK cells in the pancreas of nonobese diabetic mice.

Little is known about target organ-infiltrating NK cells in type 1 diabetes and other autoimmune diseases. In this study, we identified NK cells with a unique phenotype in the pancreas of NOD mice. Pancreatic NK cells, localized to the endocrine and exocrine parts, were present before T cells during disease development and did not require T cells for their infiltration. Furthermore, NK cells, or NK cell precursors, from the spleen could traffic to the pancreas, where they displayed the pancreatic phenotype. Pancreatic NK cells from other mouse strains shared phenotypic characteristics with pancreatic NK cells from NOD mice, but displayed less surface killer cell lectin-like receptor G1, a marker for mature NK cells that have undergone proliferation, and also did not proliferate to the same extent. A subset of NOD mouse pancreatic NK cells produced IFN-gamma spontaneously, suggesting ongoing effector responses. However, most NOD mouse pancreatic NK cells were hyporesponsive compared with spleen NK cells, as reflected by diminished cytokine secretion and a lower capacity to degranulate. Interestingly, such hyporesponsiveness was not seen in pancreatic NK cells from the nonautoimmune strain C57BL/6, suggesting that this feature is not a general property of pancreatic NK cells. Based on our data, we propose that NK cells are sentinel cells in a normal pancreas. We further speculate that during inflammation, pancreatic NK cells initially mediate proinflammatory effector functions, potentially contributing to organ-specific autoimmunity, but later become hyporesponsive because of exhaustion or regulation.

Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber.

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology have been developed and utilized in vivo, they are limited in terms of allowing single cell resolution and continuous longitudinal recordings. Because of the transparency of the cornea, the anterior chamber of the eye (ACE) in mice is well suited to study human and mouse pancreatic islet cell biology. Here is a description of how this approach can be used to perform continuous longitudinal recordings of grafting and revascularization of individual human islet grafts. Human islet grafts are inserted into the ACE, using NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mice as recipients. This allows for the investigation of the expansion of recipient versus donor cells and the contribution of recipient cells in promoting the encapsulation and vascularization of the graft. Further, a step-by-step approach for image analysis and quantification of the islet volume or segmented vasculature and islet capsule forming recipient cells is outlined.

NKT cells promote both type 1 and type 2 inflammatory responses in a mouse model of liver fibrosis.

Sterile liver inflammation and fibrosis are associated with many liver disorders of different etiologies. Both type 1 and type 2 inflammatory responses have been reported to contribute to liver pathology. However, the mechanisms controlling the balance between these responses are largely unknown. Natural killer T (NKT) cells can be activated to rapidly secrete cytokines and chemokines associated with both type 1 and type 2 inflammatory responses. As these proteins have been reported to accumulate in different types of sterile liver inflammation, we hypothesized that these cells may play a role in this pathological process. We have found that a transgenic NKT (tgNKT) cell population produced in the immunodeficient 2,4αßNOD.Rag2-/- mice, but not in 2,4αßNOD.Rag2+/- control mice, promoted a type 1 inflammatory response with engagement of the NOD-, LRR- and pyrin domain-containing protein-3 (NLRP3) inflammasome. The induction of the type 1 inflammatory response was followed by an altered cytokine profile of the tgNKT cell population with a biased production of anti-inflammatory/profibrotic cytokines and development of liver fibrosis. These findings illustrate how the plasticity of NKT cells modulates the inflammatory response, suggesting a key role for the NKT cell population in the control of sterile liver inflammation.

A role for E2-2 at the DN3 stage of early thymopoiesis.

Roles for the E-proteins E2A and HEB during T lymphocyte development have been well established. Based on our previous observations of counter selection against T cells lacking E2-2, it seemed reasonable to assume that there would be a function also for E2-2 in thymocyte development. Aiming at assigning such a role for E2-2, we analyzed the expression of E2-2, E2A, HEB as well as Id mRNA during T cell development. Interestingly, whereas all three E-proteins were expressed during early thymocyte development, significant expression beyond the DP stage was detected only for E2A. Among the Id proteins, Id2 displayed a prominent expression exclusively in DN1, whereas Id3 showed some expression in DN1, followed by a down regulation and then a prominent induction, peaking in the DP stage. E2-2 was expressed during the DN stages, as well as in the DP stage, suggesting that E2-2 operates in concert with the other E-proteins during early thymocyte development. We found that E2-2 null thymocytes displayed a partial block at the DN3 stage of development, as well as a reduced expression of pre-T alpha, known to be regulated also by E2A and HEB. The fact that E2-2 deficient thymocytes develop without gross abnormalities is likely to stem from redundancy due to the co-expression of E2A and HEB.

The Concerted Action of E2-2 and HEB Is Critical for Early Lymphoid Specification.

The apparition of adaptive immunity in Gnathostomata correlates with the expansion of the E-protein family to encompass E2-2, HEB, and E2A. Within the family, E2-2 and HEB are more closely evolutionarily related but their concerted action in hematopoiesis remains to be explored. Here we show that the combined disruption of E2-2 and HEB results in failure to express the early lymphoid program in Common lymphoid precursors (CLPs) and a near complete block in B-cell development. In the thymus, Early T-cell progenitors (ETPs) were reduced and T-cell development perturbed, resulting in reduced CD4 T- and increased γδ T-cell numbers. In contrast, hematopoietic stem cells (HSCs), erythro-myeloid progenitors, and innate immune cells were unaffected showing that E2-2 and HEB are dispensable for the ancestral hematopoietic lineages. Taken together, this E-protein dependence suggests that the appearance of the full Gnathostomata E-protein repertoire was critical to reinforce the gene regulatory circuits that drove the emergence and expansion of the lineages constituting humoral immunity.

Loss of MafA and MafB expression promotes islet inflammation.

Maf transcription factors are critical regulators of beta-cell function. We have previously shown that reduced MafA expression in human and mouse islets is associated with a pro-inflammatory gene signature. Here, we investigate if the loss of Maf transcription factors induced autoimmune processes in the pancreas. Transcriptomics analysis showed expression of pro-inflammatory as well as immune cell marker genes. However, clusters of CD4+ T and B220+ B cells were associated primarily with adult MafA-/-MafB+/-, but not MafA-/- islets. MafA expression was detected in the thymus, lymph nodes and bone marrow suggesting a novel role of MafA in regulating immune-cell function. Analysis of pancreatic lymph node cells showed activation of CD4+ T cells, but lack of CD8+ T cell activation which also coincided with an enrichment of naïve CD8+ T cells. Further analysis of T cell marker genes revealed a reduction of T cell receptor signaling gene expression in CD8, but not in CD4+ T cells, which was accompanied with a defect in early T cell receptor signaling in mutant CD8+ T cells. These results suggest that loss of MafA impairs both beta- and T cell function affecting the balance of peripheral immune responses against islet autoantigens, resulting in local inflammation in pancreatic islets.

High-resolution three-dimensional imaging of islet-infiltrate interactions based on optical projection tomography assessments of the intact adult mouse pancreas.

A predicament when assessing the mechanisms underlying the pathogenesis of type-1 diabetes (T1D) has been to maintain simultaneous global and regional information on the loss of insulin-cell mass and the progression of insulitis. We present a procedure for high-resolution 3-D analyses of regions of interest (ROIs), defined on the basis of global assessments of the 3-D distribution, size, and shape of molecularly labeled structures within the full volume of the intact mouse pancreas. We apply a refined protocol for optical projection tomography (OPT)-aided whole pancreas imaging in combination with confocal laser scanning microscopy of site-directed pancreatic microbiopsies. As such, the methodology provides a useful tool for detailed cellular and molecular assessments of the autoimmune insulitis in T1D. It is anticipated that the same approach could be applied to other areas of research where 3-D molecular distributions of both global and regional character is required.

Paquinimod prevents development of diabetes in the non-obese diabetic (NOD) mouse.

Quinoline-3-carboxamides (Q compounds) are immunomodulatory compounds that have shown efficacy both in autoimmune disease and cancer. We have in here investigated the impact of one such compound, paquinimod, on the development of diabetes in the NOD mouse model for type I diabetes (T1D). In cohorts of NOD mice treated with paquinimod between weeks 10 to 20 of age and followed up until 40 weeks of age, we observed dose-dependent reduction in incidence of disease as well as delayed onset of disease. Further, in contrast to untreated controls, the majority of NOD mice treated from 15 weeks of age did not develop diabetes at 30 weeks of age. Importantly, these mice displayed significantly less insulitis, which correlated with selectively reduced number of splenic macrophages and splenic Ly6Chi inflammatory monocytes at end point as compared to untreated controls. Collectively, these results demonstrate that paquinimod treatment can significantly inhibit progression of insulitis to T1D in the NOD mouse. We propose that the effect of paquinimod on disease progression may be related to the reduced number of these myeloid cell populations. Our finding also indicates that this compound could be a candidate for clinical development towards diabetes therapy in humans.

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.

A Role for the Non-Receptor Tyrosine Kinase Abl2/Arg in Experimental Neuroinflammation.

Multiple sclerosis is a neuroinflammatory degenerative disease, caused by activated immune cells infiltrating the CNS. The disease etiology involves both genetic and environmental factors. The mouse genetic locus, Eae27, linked to disease development in the experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis, was studied in order to identify contributing disease susceptibility factors and potential drug targets for multiple sclerosis. Studies of an Eae27 congenic mouse strain, revealed that genetic variation within Eae27 influences EAE development. The Abl2 gene, encoding the non-receptor tyrosine kinase Arg, is located in the 4,1 megabase pair long Eae27 region. The Arg protein plays an important role in cellular regulation and is, in addition, involved in signaling through the B- and T-cell receptors, important for the autoimmune response. The presence of a single nucleotide polymorphism causing an amino acid change in a near actin-interacting domain of Arg, in addition to altered lymphocyte activation in the congenic mice upon immunization with myelin antigen, makes Abl2/Arg a candidate gene for EAE. Here we demonstrate that the non-synonymous SNP does not change Arg's binding affinity for F-actin but suggest a role for Abl kinases in CNS inflammation pathogenesis by showing that pharmacological inhibition of Abl kinases ameliorates EAE, but not experimental arthritis.