Investigating the Biological Mechanisms underlying the Initiation of Autoimmunity against Beta Cell Antigens in Type 1 Diabetes

Type 1 diabetes (T1D) is an autoimmune disease characterized by circulating autoantibodies, lymphocytic infiltration of pancreatic islets of Langerhans, and cell-specific destruction of beta cells, leading to insulin deficiency and symptomatic hyperglycemia. Some of the major target autoantigens in T1D are intracellular proteins, such as GAD65, IA-2 and proinsulin, whose initial encounter with the immune system is poorly understood. Here we describe a method for culturing monolayers of primary pancreatic islet cells in vitro, which enables detailed observation of proteins and sub-cellular processes in primary human and rat beta cells by super-resolution and live-cell light microscopy. This technical advance is broadly applicable to obtaining novel biological insights as demonstrated by our identification of a mechanism to control proliferation in primary beta cells through growth and disassembly of primary cilia. This protocol also allowed us to identify two novel interrelated mechanisms for how beta cell proteins, such as the antigen GAD65, can become target of autoimmunity and initiate T1D. A triggering factor that these two pathways have in common is represented by endoplasmic reticulum (ER) stress, to which pancreatic islet beta cells are particularly susceptible, and can be induced by inflammatory factors such as Th1 cytokines. In the first mechanism, we show that ER stress in beta cells perturbs the palmitoylation cycle controlling GAD65 endomembrane distribution, resulting in aberrant accumulation of the palmitoylated form in Golgi membranes. The palmitoylated form has heightened immunogenicity, exhibiting increased uptake by antigen presenting cells (APCs) and T cell stimulation compared to the non-palmitoylated form. Similar accumulation of GAD65 in Golgi membranes is observed in human beta cells in pancreatic sections from GAD65 autoantibody positive individuals, who have not yet progressed to clinical onset of T1D, and T1D patients with residual beta cell mass and ongoing T cell infiltration of islets. Thus, aberrant accumulation of immunogenic GAD65 in Golgi membranes facilitates inappropriate presentation to the immune system following release from stressed and/or damaged beta cells, triggering autoimmunity. In the second mechanism, we present that, following a pathway originating from the Golgi, both rat and human pancreatic islets release the intracellular beta cell autoantigens GAD65, IA-2 and proinsulin in a subtype of extracellular vesicles called exosomes. Islet-exosomes are then taken up by and can activate APCs including dendritic cells. Accordingly, anchoring of GAD65 to exosome-mimetic liposomes strongly boosts antigen presentation and T cell activation in the context of the human T1D susceptibility haplotype HLA-DR4. Furthermore, cytokine-induced ER stress enhances exosome secretion by beta cells, induces exosomal release of the immunostimulatory chaperones calreticulin, Gp96 and ORP150 and increases exosomal stimulation of APCs. Therefore, stress-induced exosomal release of intracellular autoantigens and immunostimulatory chaperones may play a role in initiation of autoimmune responses in T1D.

Aberrant Accumulation of the Diabetes Autoantigen GAD65 in Golgi Membranes in Conditions of ER Stress and Autoimmunity

Steinunn Baekkeskov Hanahan, Chiara Cianciaruso, Jeffrey Alan Hubbell, Miriella Carmela Pasquier, Edward Allen Phelps

Pancreatic islet β-cells are particularly susceptible to endoplasmic reticulum (ER) stress, which is implicated in β-cell dysfunction and loss during the pathogenesis of type 1 diabetes (T1D). The peripheral membrane protein GAD65 is an autoantigen in human T1D. GAD65 synthesizes γ-aminobutyric acid, an important autocrine and paracrine signaling molecule and a survival factor in islets. We show that ER stress in primary β-cells perturbs the palmitoylation cycle controlling GAD65 endomembrane distribution, resulting in aberrant accumulation of the palmitoylated form in trans-Golgi membranes. The palmitoylated form has heightened immunogenicity, exhibiting increased uptake by antigen-presenting cells and T-cell stimulation compared with the nonpalmitoylated form. Similar accumulation of GAD65 in Golgi membranes is observed in human β-cells in pancreatic sections from GAD65 autoantibody-positive individuals who have not yet progressed to clinical onset of T1D and from patients with T1D with residual β-cell mass and ongoing T-cell infiltration of islets. We propose that aberrant accumulation of immunogenic GAD65 in Golgi membranes facilitates inappropriate presentation to the immune system after release from stressed and/or damaged β-cells, triggering autoimmunity.

Amer Diabetes Assoc2016

Primary human and rat beta cells release the intracellular autoantigens GAD65, IA-2 and proinsulin in exosomes together with cytokine-induced enhancers of immunity

Steinunn Baekkeskov Hanahan, Chiara Cianciaruso, Michele De Palma, Davide Demurtas, Romain Hamelin, Sachiko Hirosue, Jeffrey Alan Hubbell, Miriella Carmela Pasquier, Edward Allen Phelps, Melody Swartz

The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1D), are intracellular membrane proteins, whose initial encounter with the immune system is poorly understood. Here we propose a new model for how these proteins can initiate autoimmunity. We found that rat and human pancreatic islets release the intracellular β-cell autoantigens in human T1D, GAD65, IA-2 and proinsulin, in exosomes, which are taken up by and activate dendritic cells. Accordingly, anchoring of GAD65 to exosome-mimetic liposomes strongly boosted antigen presentation and T cell activation in the context of the human type 1 diabetes susceptibility haplotype HLA-DR4. Cytokine-induced ER-stress enhanced exosome secretion by β-cells, induced exosomal release of the immunostimulatory chaperones calreticulin, Gp96 and ORP150 and increased exosomal stimulation of antigen presenting cells. We propose that stress-induced exosomal release of intracellular autoantigens and immunostimulatory chaperones may play a role in initiation of autoimmune responses in T1D.

American Diabetes Association2017

Elucidating the mechanisms of paclitaxel-loaded-nanoparticle-induced immune response

Efthymia Vokali

Dentritic cells (DC) are the most potent antigen-presenting cells and play a key role in the induction of effective immune responses, suggesting a novel target for anti-tumor immunotherapy. Paclitaxel (PXL) is a widely used chemotherapeutic, initially characterized as a mitotic inhibitor, that has been shown to enhance maturation and function of DCs as well as up-regulate the production of inflammatory cytokines. Thus, modulation of DC function by the approved-for-human-use PXL could constitute a novel approach for reshaping immune responses against a tumor. For targeted delivery of PXL to DCs in the lymph nodes (LNs), where they are present in high concentrations and apt for antigen uptake, poly(propylene) sulfide core nanopatricles (NPs) developed by our laboratory have been used. PXL loaded-NP (PXL-NP) treatment induces massive immune cell infiltration into the draining LN 24h after intradermal injection of PXL-NPs in mice, suggesting that PXL-NP treatment induces a local and robust immune response (Thomas et al., in preparation). We hypothesized that this effect stems directly from the action of PXL-NPs on LN-resident DCs. We demonstrate that PXL-NPs are able to induce maturation and cytokine production in murine and human DCs in vitro, which may explain the in vivo observations of robust cell recruitment to the draining LN and generation of adaptive immune responses. Furthermore, the striking cell infiltration in the LN upon PXL-NP treatment was abrogated in complement protein 3 (C3) knockout mice in vivo (Thomas et al. in preparation). Interestingly, we show that the presence of C3 regulates IL-12p40 expression and, in turn, plays a crucial role in the activation of mature, functional DCs in response to PXL-NPs. Thus, the absence of functional DCs in case of C3 deficiency might account for the hindered infiltration. Together, this data suggests that PXL-NPs may effectively direct the immunomodulatory action of PXL to LN-resident DCs to alter immune responses.

2011

Elucidating the mechanisms of paclitaxel-loaded-nanoparticle-induced immune response

Efthymia Vokali

2011