The complex etiology of type 1 diabetes (T1D) is the outcome of failures in regulating immunity in combination with beta cell perturbations. Mitochondrial dysfunction in beta cells and immune cells ...may be involved in T1D pathogenesis. Mitochondrial energy production is essential for the major task of beta cells (the secretion of insulin in response to glucose). Mitochondria are a major site of reactive oxygen species (ROS) production. Under immune attack, mitochondrial ROS (mtROS) participate in beta cell damage. Similarly, T cell fate during immune responses is tightly regulated by mitochondrial physiology, morphology, and metabolism. Production of mtROS is essential for signaling in antigen-specific T cell activation. Mitochondrial dysfunction in T cells has been noted as a feature of some human autoimmune diseases. Recent Advances: Preclinical and clinical studies indicate that mitochondrial dysfunction in beta cells sensitizes these cells to immune-mediated destruction via direct or indirect mechanisms. Sensitivity of beta cells to mtROS is associated with genetic T1D risk loci in human and the T1D-prone nonobese diabetic (NOD) mouse. Mitochondrial dysfunction and altered metabolism have also been observed in immune cells of NOD mice and patients with T1D. This immune cell mitochondrial dysfunction has been linked to deleterious functional changes.
It remains unclear how mitochondria control T cell receptor signaling and downstream events, including calcium flux and activation of transcription factors during autoimmunity.
Mechanistic studies are needed to investigate the mitochondrial pathways involved in autoimmunity, including T1D. These studies should seek to identify the role of mitochondria in regulating innate and adaptive immune cell activity and beta cell failure.
IL-12 and IL-18 synergize to promote TH1 responses and have been implicated as accelerators of autoimmune pathogenesis in type 1 diabetes (T1D). We investigated the influence of these cytokines on ...immune cells involved in human T1D progression: natural killer (NK) cells, regulatory T cells (Tregs), and cytotoxic T lymphocytes (CTL). NK cells from T1D patients exhibited higher surface CD226 versus controls and lower CD25 compared to first-degree relatives and controls. Changes in NK cell phenotype towards terminal differentiation were associated with cytomegalovirus (CMV) seropositivity, while possession of IL18RAP, IFIH1, and IL2RA T1D-risk variants impacted NK cell activation as evaluated by immuno-expression quantitative trait loci (eQTL) analyses. IL-12 and IL-18 stimulated NK cells from healthy donors exhibited enhanced specific killing of myelogenous K562 target cells. Moreover, activated NK cells increased expression of NKG2A, NKG2D, CD226, TIGIT and CD25, which enabled competition for IL-2 upon co-culture with Tregs, resulting in Treg downregulation of FOXP3, production of IFNγ, and loss of suppressive function. We generated islet-autoreactive CTL “avatars”, which upon exposure to IL-12 and IL-18, upregulated IFNγ and Granzyme-B leading to increased lymphocytotoxicity of a human β-cell line in vitro. These results support a model for T1D pathogenesis wherein IL-12 and IL-18 synergistically enhance CTL and NK cell cytotoxic activity and disrupt immunoregulation by Tregs.
Working Model Summarizing the Hypothesized Contributions of Elevated IL-12 and IL-18 Levels Toward Failure in Immunoregulation and T1D Pathogenesis. In immune homeostasis (left), regulatory T cells (Treg) suppress activation and function of CD8+T cells, CD4+T cells and NK cells via various mechanisms including competition for IL-2. In settings of increased genetic risk for T1D, exposure to some environmental trigger(s) compound genetic defects to induce a break in tolerance (right), during which time IL-12 and IL-18 levels are elevated and NK cells upregulate CD25. This allows for direct competition with Tregs for IL-2, resulting in decreased Treg IL-2 signaling, STAT5 phosphorylation (pSTAT5), and FOXP3 expression, ultimately abrogating suppression. We hypothesize that this, together with enhanced production of cytokines and cytolytic proteins by CD4+conventional T cells and CD8+cytotoxic T cells, leads to augmented lysis of β-cells (right). Display omitted
•IL-12 & IL-18 enhance NK cell and antigen specific CD8 T cell killing.•Regulatory T cells (Tregs) lose suppressive capacity and produce IFN-γ.•Type 1 Diabetes candidate genes permit CD25 upregulation on NK cells.•NK cells exhibit altered receptor balance and are able to compete with Tregs for IL-2 in vitro.
Type 1 diabetes (T1D) is a disease that arises due to complex immunogenetic mechanisms. Key cell-cell interactions involved in the pathogenesis of T1D are activation of autoreactive T cells by ...dendritic cells (DC), migration of T cells across endothelial cells (EC) lining capillary walls into the islets of Langerhans, interaction of T cells with macrophages in the islets, and killing of β-cells by autoreactive CD8
T cells. Overall, pathogenic cell-cell interactions are likely regulated by the individual's collection of genetic T1D-risk variants. To accurately model the role of genetics, it is essential to build systems to interrogate single candidate genes in isolation during the interactions of cells that are essential for disease development. However, obtaining single-donor matched cells relevant to T1D is a challenge. Sourcing these genetic variants from human induced pluripotent stem cells (iPSC) avoids this limitation. Herein, we have differentiated iPSC from one donor into DC, macrophages, EC, and β-cells. Additionally, we also engineered T cell avatars from the same donor to provide an
platform to study genetic influences on these critical cellular interactions. This proof of concept demonstrates the ability to derive an isogenic system from a single donor to study these relevant cell-cell interactions. Our system constitutes an interdisciplinary approach with a controlled environment that provides a proof-of-concept for future studies to determine the role of disease alleles (e.g.
in regulating cell-cell interactions and cell-specific contributions to the pathogenesis of T1D.
Type 1 diabetes (T1D) has a strong genetic component. The
(
)
locus was identified in crosses of T1D-susceptible NOD mice with the strongly T1D-resistant ALR strain. The NODcALR-(
)/Mx (NOD-
) ...recombinant congenic mouse strain was generated in which NOD mice carry the full
confidence interval. NOD-
mice exhibit almost complete protection from spontaneous T1D and a significant reduction in insulitis. Our goal was to unravel the mode of
-based protection using in vivo and in vitro models. We determined that
did not impact immune cell diabetogenicity or β cell resistance to cytotoxicity in vitro. However, NOD-
mice were highly protected against adoptive transfer of T1D. Transferred CTLs trafficked to the pancreatic lymph node and proliferated to the same extent in NOD and NOD-
mice, yet the accumulation of pathogenic CTLs in the islets was significantly reduced in NOD-
mice, correlating with disease resistance. Pancreatic endothelial cells from NOD-
animals expressed lower levels of adhesion molecules, even in response to inflammatory stimuli. Lower adhesion molecule expression resulted in weaker adherence of T cells to NOD-
endothelium compared with NOD-derived endothelium. Taken together, these results provide evidence that
regulates the ability of β cell-autoreactive T cells to traffic into the pancreatic islets and may represent a new target for pharmaceutical intervention to potentially prevent T1D.
Axonal degeneration is the final common path in many neurological disorders. Subsets of neuropathies involving the sensory neuron are known as hereditary sensory neuropathies (HSNs). Hereditary ...sensory neuropathy type I (HSN-I) is the most common subtype of HSN with autosomal dominant inheritance. It is characterized by the progressive degeneration of the dorsal root ganglion (DRG) with clinical symptom onset between the second or third decade of life. Heterozygous mutations in the serine palmitoyltransferase (SPT) long chain subunit 1 (
SPTLC1
) gene were identified as the pathogenic cause of HSN-I. Ultrastructural analysis of mitochondria from HSN-I patient cells has displayed unique morphological abnormalities that are clustered to the perinucleus where they are wrapped by the endoplasmic reticulum (ER). This investigation defines a small subset of proteins with major alterations in abundance in mitochondria harvested from HSN-I mutant SPTLC1 cells. Using mitochondrial protein isolates from control and patient lymphoblasts, and a combination of 2D gel electrophoresis, immunoblotting and mass spectrometry, we have shown the increased abundance of ubiquinol-cytochrome c reductase core protein 1, an electron transport chain protein, as well as the immunoglobulin, Ig kappa chain C. The regulation of these proteins may provide a new route to understanding the cellular and molecular mechanisms underlying HSN-I.
Hereditary sensory neuropathy type 1 (HSN-1) is an autosomal dominant neurodegenerative disease caused by missense mutations in the
SPTLC1
gene. The SPTLC1 protein is part of the SPT enzyme which is ...a ubiquitously expressed, critical and thus highly regulated endoplasmic reticulum bound membrane enzyme that maintains sphingolipid concentrations and thus contributes to lipid metabolism, signalling, and membrane structural functions. Lipid droplets are dynamic organelles containing sphingolipids and membrane bound proteins surrounding a core of neutral lipids, and thus mediate the intracellular transport of these specific molecules. Current literature suggests that there are increased numbers of lipid droplets and alterations of lipid metabolism in a variety of other autosomal dominant neurodegenerative diseases, including Alzheimer’s and Parkinson’s disease. This study establishes for the first time, a significant increase in the presence of lipid droplets in HSN-1 patient-derived lymphoblasts, indicating a potential connection between lipid droplets and the pathomechanism of HSN-1. However, the expression of adipophilin (ADFP), which has been implicated in the regulation of lipid metabolism, was not altered in lipid droplets from the HSN-1 patient-derived lymphoblasts. This appears to be the first report of increased lipid body accumulation in a peripheral neuropathy, suggesting a fundamental molecular linkage between a number of neurodegenerative diseases.
A major goal of type 1 diabetes (T1D) research is development of state-of-the-art devices that recreate the human islet microenvironment to help elucidate the molecular and cellular mechanisms of ...beta cell loss due to autoimmunity. We recently reported a novel synthetic biomaterial technology to form hydrogels from a pair of oppositely-charged peptides that co-assemble into a network of interpenetrating β-sheet nanofibers. Here, we explored the potential of this biomaterial, referred to as “CATCH” for Co-Assembly Tags based on Charge complementarity, to serve as a 3D microenvironment for studies of the islet—immune cell interface within the context of human T1D. Supramolecular CATCH hydrogels offer a number of key advantages for engineering islet biomimetic microenvironments: 1) they are fabricated via self-assembly at neutral pH and 37°C; 2) require no chemical reaction to form; and 3) are intrinsically capable of installing folded proteins or peptides via covalent fusion to one of the synthetic peptides sequences. In initial experiments, we established that CATCH hydrogels are cyto-compatible with human beta cells via encapsulation of BetaLox5 cells (βL5), an immortalized human beta cell line, by demonstrating strong viability (Calcein-AM/Ethidium homodimer-1 stain) of encapsulated βL5 at 3 and 24 hours after gelation similar to βL5 seeded on 2D surfaces. CATCH gels also showed promise as a viable platform for interrogating the engagement of autoreactive immune cells with human beta cells. Engineered CD8+ human T cell avatars expressing human T cell receptors for the islet antigen pre-proinsulin co-embedded into CATCH hydrogels with βL5 cells show strong beta cell killing in 3D by T cells over an 18-hour period (assessed via loss of TMRM staining of mitochondrial membrane potential). These initial successes are guiding our development of CATCH hydrogels for further detailed 3D studies of islet—immune cell interactions relevant to human T1D.
Disclosure
M. Becker: None. D. Seroski: None. S. Stimpson: None. C.E. Mathews: None. G. Hudalla: None. E.A. Phelps: None.
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