Cytokine-induced Stress Research Articles

Pancreatic stellate cells support human pancreatic β-cell viability in vitro and enhance survival of immunoisolated human islets exposed to cytokines

Pancreatic islet transplantation is proposed as a cure for type 1 diabetes mellitus (T1D). Despite its success in optimal regulation of glucose levels, limitations in longevity of islet grafts still require innovative solutions. Inflammatory stress post-transplantation and loss of extracellular matrix attribute to the limited β-cell survival. Pancreatic stellate cells (PSCs), identified as pancreatic-specific stromal cells, have the potential to play a crucial role in preserving islet survival. Our study aimed to determine the effects of PSCs co-cultured with human CM β-cells and human islets under inflammatory stress induced by a cytokine cocktail of IFN-γ, TNF-α and IL-1β. Transwell culture inserts were utilized to assess the paracrine impact of PSCs on β-cells, alongside co-cultures enabling direct interaction between PSCs and human islets. We found that co-culturing PSCs with human CM β-cells and human cadaveric islets had rescuing effects on cytokine-induced stress. Effects were different under normoglycemic and hyperglycemic conditions. PSCs were associated with upregulation of β-cell mitochondrial activity and suppression of inflammatory gene expression. The rescuing effects exist both in indirect and direct co-culture methods. Furthermore, we tested whether PSCs have rescuing effects on human islets in conventional alginate-based microcapsules and in composite microcapsules composed of alginate-pectin collagen type IV, laminin sequence RGD, Nec-1, and amino acid. PSCs partially prevented cytokine-induced stress in both systems, but beneficial effects were stronger in composite capsules. Our findings show novel effects of PSCs on islet health. Islets and PSCs coculturing or co-transplantation might mitigate the inflammation stress and improve islet transplantation outcomes.

ECM-based bioactive microencapsulation significantly improves islet function and graft performance.

Microencapsulation is a promising strategy to prolong the survival and function of transplanted pancreatic islets for diabetes therapy, albeit its translation has been impeded by incoherent graft performance. The use of decellularized ECM has lately gained substantial research momentum due to its innate capacity to augment the function of cells originating from the same tissue type. In the present study, the advantages of both these approaches are leveraged in a porcine pancreatic ECM (pECM)-based microencapsulation platform, thus significantly enhancing murine pancreatic islet performance. pECM-encapsulated islets sustain high insulin secretion levels in vitro, surpassing those of islets encapsulated in conventional alginate microcapsules. Moreover, pECM-encapsulated islet cells proliferate and produce an enriched intra-islet ECM framework, displaying a distinctive structural rearrangement. The beneficial effect of pECM encapsulation is further reinforced by the temporary protection against cytokine-induced cytotoxicity. In-vivo, this platform significantly improves glucose tolerance and achieves glycemic correction in 100% of immunocompetent diabetic mice without any immunosuppression, compared to only 50% mice achieved glycemic correction by alginate encapsulation. Altogether, the results presented herein reveal that pECM-based microencapsulation offers a natural pancreatic niche that can restore the function of isolated pancreatic islets and deliver them safely, avoiding the need for immunosuppression. STATEMENT OF SIGNIFICANCE: Aiming to improve pancreatic islet transplantation outcomes in diabetic patients, we developed a microencapsulation platform based on pancreatic extracellular matrix (pECM). In these microcapsules the islets are entrapped within a pECM hydrogel that mimics the natural pancreatic microenvironment. We show that pECM encapsulation supports the islets' viability and function in culture, and provides temporal protection against cytokine-induced stress. In a diabetic mouse model, pECM encapsulation significantly improved glucose tolerance and achieved glycemic correction without any immunosuppression. These results reveal the potential of pECM encapsulation as a viable treatment for diabetes, providing a solid scientific basis for more advanced preclinical studies.

Protecting islet functional viability using mesenchymal stromal cells.

Islet transplantation is an emerging treatment for type 1 diabetes which offers the prospect of physiological control of blood glucose and reductions in acute hypoglycaemic episodes. However, current protocols are limited by a rapid decline in islet functional viability during the isolation process, culture period, and post‐transplantation. Much of this can be attributed to the deleterious effects of hypoxic and cytokine stressors on β cells. One experimental strategy to improve the functional viability of islets is coculture or cotransplantation with mesenchymal stromal cells (MSCs). Numerous studies have shown that MSCs have the capacity to improve islet survival and insulin secretory function, and the mechanisms of these effects are becoming increasingly well understood. In this review, we will focus on recent studies demonstrating the capacity for MSCs to protect islets from hypoxia‐ and cytokine‐induced stress. Islets exposed to acute hypoxia (1%‐2% O2) or to inflammatory cytokines (including IFN‐γ, TNF‐α, and IL‐B) in vitro undergo apoptosis and a rapid decline in glucose‐stimulated insulin secretion. Coculture of islets with MSCs, or with MSC‐conditioned medium, protects from these deleterious effects, primarily with secreted factors. These protective effects are distinct from the immunomodulatory and structural support MSCs provide when cotransplanted with islets. Recent studies suggest that MSCs may support secretory function by the physical transfer of functional mitochondria, particularly to metabolically compromised β cells. Understanding how MSCs respond to stressed islets will facilitate the development of MSC secretome based, cell‐free approaches to supporting islet graft function during transplantation by protecting or repairing β cells.

Paracrine regulation and improvement of β-cell function by thioredoxin

The failure of insulin-producing β-cells is the underlying cause of hyperglycemia in diabetes mellitus. β-cell decay has been linked to hypoxia, chronic inflammation, and oxidative stress. Thioredoxin (Trx) proteins are major actors in redox signaling and essential for signal transduction and the cellular stress response. We have analyzed the cytosolic, mitochondrial, and extracellular Trx system proteins in hypoxic and cytokine-induced stress using β-cell culture, isolated pancreatic islets, and pancreatic islet transplantation modelling low oxygen supply.Protein levels of cytosolic Trx1 and Trx reductase (TrxR) 1 significantly decreased, while mitochondrial Trx2 and TrxR2 increased upon hypoxia and reoxygenation. Interestingly, Trx1 was secreted by β-cells during hypoxia. Moreover, murine and human pancreatic islet grafts released Trx1 upon glucose stimulation. Survival of transplanted islets was substantially impaired by the TrxR inhibitor auranofin.Since a release was prominent upon hypoxia, putative paracrine effects of Trx1 on β-cells were examined. In fact, exogenously added recombinant hTrx1 mitigated apoptosis and preserved glucose sensitivity in pancreatic islets subjected to hypoxia and inflammatory stimuli, dependent on its redox activity. Human subjects were studied, demonstrating a transient increase in extracellular Trx1 in serum after glucose challenge. This increase correlated with better pancreatic islet function. Moreover, hTrx1 inhibited the migration of primary murine macrophages.In conclusion, our study offers evidence for paracrine functions of extracellular Trx1 that improve the survival and function of pancreatic β-cells.

2137-P: Cholecystokinin Protects Mouse Pancreatic Beta Cells against Cytokine Insult through the Cholecystokinin A Receptor

Cholecystokinin (CCK) is an incretin-like hormone that is also produced by pancreatic β-cells under conditions of stress and obesity. In the past, we have established a role for CCK in protection from β-cell apoptosis. However, the specific CCK receptor responsible for this protective effect was unknown. Both Cckar and Cckbr mRNA are expressed in mouse islet, although Cckbr expression is very low. Incubation of WT mouse pancreatic islets with CCK-8 (100nM) increases the transcript levels of Cckar but does not impact Cckbr expression. Conversely, 24-hour treatment with proinflammatory cytokine cocktail decreases CCKAR transcript levels but again does not modulate Cckbr expression. Therefore, we hypothesized that the CCKA receptor was the most likely mediator of CCK signaling in the β-cell. We used isolated islets from mice with germline knockout of CCK receptors to determine which receptor was required for the pro-survival effects of CCK on the β-cell. Using imaging flow cytometry of dissociated islet cells stained for annexin V and propidium iodide (PI) we find that islet cells from CCKBR knockout mice have a 19.3% (p<0.005) reduction in cytokine-mediated cell death when treated with CCK-8 (100 nM), similar to the protection seen in wild type islets. This protective effect is lost in the CCKAR and double knockout mouse islets. We also confirm this finding using insulin and TUNEL immunohistochemistry to specifically identify β-cell apoptosis. In CCKBR knockout mouse islets, CCK-8 (100 nM) reduces the % TUNEL positive β-cells from 12.98% to 4.39% (p<0.05) after cytokine exposure, similar to protection seen in WT mouse islets (10.47% to 4.48%). Again, CCK-8 does not protect β-cells from cytokine-induced apoptosis in CCKAR knockout or double CCKR null mouse islets. Taken together, CCKAR, but not CCKBR, is required for CCK-mediated protection of pancreatic β-cells from cytokine-induced stress. Disclosure H. Kim: None. S. Sacotte: None. R.A. Williams: None. A.H. de Souza: None. J. Han: None. J.T. Bartosiak: None. G.H. Yang: None. D.B. Davis: None. Funding National Institutes of Health

The Role of the Antioxidant Protein DJ-1 in Type 2 Diabetes Mellitus.

Type 2 diabetes mellitus (T2DM) is a worldwide escalating health disorder resulting from insulin resistance and functional loss of insulin-producing beta cells that finally cause chronically elevated blood glucose concentrations. Here we review the role of ubiquitously expressed antioxidant protein DJ-1 in the pathogenesis of T2DM. In beta cells, DJ-1 protects against oxidative stress, endoplasmic reticulum stress, and streptozotocin- and cytokine-induced stress and preserves beta cell viability and insulin secretion. In skeletal muscle, DJ-1 controls energy metabolism and efficient fuel utilization, whereas in adipose tissue a role in adipogenesis and obesity-induced inflammation has been reported. This suggests that DJ-1 plays multiple roles in many cell types under metabolically challenging conditions as seen in obesity, insulin resistance, and T2DM.

Distinct activation of primary human BDCA1(+) dendritic cells upon interaction with stressed or infected β cells.

Derailment of immune responses can lead to autoimmune type 1 diabetes, and this can be accelerated or even induced by local stress caused by inflammation or infection. Dendritic cells (DCs) shape both innate and adaptive immune responses. Here, we report on the responses of naturally occurring human myeloid BDCA1(+) DCs towards differentially stressed pancreatic β cells. Our data show that BDCA1(+) DCs in human pancreas-draining lymph node (pdLN) suspensions and blood-derived BDCA1(+) DCs both effectively engulf β cells, thus mimicking physiological conditions. Upon uptake of enterovirus-infected, but not mock-infected cells, BDCA1(+) DCs induced interferon (IFN)-α/β responses, co-stimulatory molecules and proinflammatory cytokines and chemokines. Notably, induction of stress in β cells by ultraviolet irradiation, culture in serum-free medium or cytokine-induced stress did not provoke strong DC activation, despite efficient phagocytosis. DC activation correlated with the amount of virus used to infect β cells and required RNA within virally infected cells. DCs encountering enterovirus-infected β cells, but not those incubated with mock-infected or stressed β cells, suppressed T helper type 2 (Th2) cytokines and variably induced IFN-γ in allogeneic mixed lymphocyte reaction (MLR). Thus, stressed β cells have little effect on human BDCA1(+) DC activation and function, while enterovirus-infected β cells impact these cells significantly, which could help to explain their role in development of autoimmune diabetes in individuals at risk.

Inhibitors of Ribosome Rescue Arrest Growth of Francisella tularensis at All Stages of Intracellular Replication.

Bacteria require at least one pathway to rescue ribosomes stalled at the ends of mRNAs. The primary pathway for ribosome rescue is trans-translation, which is conserved in >99% of sequenced bacterial genomes. Some species also have backup systems, such as ArfA or ArfB, which can rescue ribosomes in the absence of sufficient trans-translation activity. Small-molecule inhibitors of ribosome rescue have broad-spectrum antimicrobial activity against bacteria grown in liquid culture. These compounds were tested against the tier 1 select agent Francisella tularensis to determine if they can limit bacterial proliferation during infection of eukaryotic cells. The inhibitors KKL-10 and KKL-40 exhibited exceptional antimicrobial activity against both attenuated and fully virulent strains of F. tularensis in vitro and during ex vivo infection. Addition of KKL-10 or KKL-40 to macrophages or liver cells at any time after infection by F. tularensis prevented further bacterial proliferation. When macrophages were stimulated with the proinflammatory cytokine gamma interferon before being infected by F. tularensis, addition of KKL-10 or KKL-40 reduced intracellular bacteria by >99%, indicating that the combination of cytokine-induced stress and a nonfunctional ribosome rescue pathway is fatal to F. tularensis Neither KKL-10 nor KKL-40 was cytotoxic to eukaryotic cells in culture. These results demonstrate that ribosome rescue is required for F. tularensis growth at all stages of its infection cycle and suggest that KKL-10 and KKL-40 are good lead compounds for antibiotic development.

Autophagy is required for stem cell mobilization by G-CSF

AbstractGranulocyte colony-stimulating factor (G-CSF) is widely used clinically to prevent neutropenia after cytotoxic chemotherapy and to mobilize hematopoietic stem cells (HSCs) for transplantation. Autophagy, a process of cytoplasmic component recycling, maintains cellular homeostasis and protects the cell during periods of metabolic stress or nutrient deprivation. We have observed that G-CSF activates autophagy in neutrophils and HSCs from both mouse and human donors. Furthermore, G-CSF–induced neutrophil and HSC mobilization is impaired in the absence of autophagy. In contrast, autophagy is dispensable for direct HSC mobilization in response to the CXCR4 antagonist AMD3100. Altogether, these data demonstrate an important role for G-CSF in invoking autophagy within hematopoietic and myeloid cells and suggest that this pathway is critical for ensuring cell survival in response to clinically relevant cytokine-induced stress. These findings have direct relevance to HSC transplantation and the increasing clinical use of agents that modulate autophagy.

Roles for Peroxisome Proliferator-activated Receptor γ (PPARγ) and PPARγ Coactivators 1α and 1β in Regulating Response of White and Brown Adipocytes to Hypoxia

Obese white adipose tissue is hypoxic but is incapable of inducing compensatory angiogenesis. Brown adipose tissue is highly vascularized, facilitating delivery of nutrients to brown adipocytes for heat production. In this study, we investigated the mechanisms by which white and brown adipocytes respond to hypoxia. Brown adipocytes produced lower amounts of hypoxia-inducible factor 1α (HIF-1α) than white adipocytes in response to low O(2) but induced higher levels of hypoxia-associated genes. The response of white adipocytes to hypoxia required HIF-1α, but its presence alone was incapable of inducing target gene expression under normoxic conditions. In addition to the HIF-1α targets, hypoxia also induced many inflammatory genes. Exposure of white adipocytes to a peroxisome proliferator-activated receptor γ (PPARγ) ligand (troglitazone) attenuated induction of these genes but enhanced expression of the HIF-1α targets. Knockdown of PPARγ in mature white adipocytes prevented the usual robust induction of HIF-1α targets in response to hypoxia. Similarly, knockdown of PPARγ coactivator (PGC) 1β in PGC-1α-deficient brown adipocytes eliminated their response to hypoxia. These data demonstrate that the response of white adipocytes requires HIF-1α but also depends on PPARγ in white cells and the PPARγ cofactors PGC-1α and PGC-1β in brown cells.

Systemic Effects of White Adipose Tissue Dysregulation and Obesity‐Related Inflammation

Systemic Effects of White Adipose Tissue Dysregulation and Obesity‐Related Inflammation

HMG-CoA Reductase Inhibitors Decrease Hyperglycemia on Animal Model of Systemic Inflammatory Response Syndrome (SIRS)

Systemic Inflammatory Response Syndrome (SIRS) is a pathophysiologic state associated with trauma and major surgery. Inflammatory cytokines plays an important role in the pathogenesis of SIRS. Cytokines induce the activation of the classic hypothalamic-pituitary stress response that leads to increase of secretion of the stress hormone ACTH and hyperglycemia. HMG-CoA reductase inhibitors, such as statins, have been shown to have anti-inflammatory properties. Recent studies in humans indicate that the perioperative use of statins may decrease morbidity and mortality. Here we tested the hypothesis that statins may decrease the cytokine-induced stress response and hyperglycemia in a murine model of SIRS. SIRS was induced with intraperitoneal injection of 0.1 mg lipopolysaccharide (LPS) per mouse. Mice were pretreated with 0.5 mg of simvastatin or lovastatin 18 hours before the administration of LPS, and plasma levels of Interleukin-2 (IL-2), Tumor Necrosis Factor alpha (TNF-?), the stress hormone adrenocorticotrophic hormone (ACTH), and glucose were determined. We observed that pretreatment of mice with statins nearly completely suppressed the LPS-induced cytokine, ACTH and hyperglycemic responses. Conclusion: Statins suppress cytokine production in a murine model of SIRS. This decreased cytokine production may lead to suppression of the SIRS-induced stress response and hyperglycemia. We postulate that statins may have an important role as regulators of the SIRS and stress response induced by surgery and trauma, and that akin to ?-blockers, statins may become part of the therapeutic arsenal aimed to decrease perioperative morbidity and mortality.

Ethanol, oxidative stress, and cytokine-induced liver cell injury

Both clinical findings and results of experiments with animal models of alcoholic hepatitis have shown the importance of cytokine-mediated cell–cell interactions in the onset of ethanol-induced liver damage. Proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1beta (IL-1β), and interleukin-6, are released from Kupffer cells or infiltrating neutrophils and macrophages and elicit defensive responses in parenchymal cells, including activation of apoptosis. Reactive oxygen species (ROS) and reactive nitrogen species (RNS), generated in response to cytokine-induced stress signals in parenchymal cells and also by activation of Kupffer cells and inflammatory cells, further mobilize cellular defense mechanisms. When these defensive responses are overwhelmed cells may die by necrosis, further stimulating inflammatory responses and infiltration of neutrophils. Chronic ethanol intake (i.e., many years of heavy alcohol use in human patients, several weeks or months in experimental animals) enhances the damaging consequences of these events through a variety of mechanisms. The formation of cytokines in the liver is stimulated by increasing circulating levels of endotoxin and by enhancing the responsiveness of Kupffer cells to such stimuli. In addition, ethanol promotes oxidative stress, both by increased formation of ROS and by depletion of oxidative defenses in the cell. Furthermore, liver cells from ethanol-treated animals are more susceptible to the cytotoxic effects of TNF-α and other cytokines than cells from control animals. Mitochondria play a critical role in the apoptotic response, and alterations in mitochondrial function after chronic ethanol treatment may contribute to enhanced cell death by apoptosis or necrosis. How the shift in the balance of cytokine-induced defensive and damage responses in hepatocytes contributes to the liver injury that occurs in alcoholic hepatitis remains poorly characterized and should be a rewarding area for future studies.