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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. [ABSTRACT FROM AUTHOR]

Objective: To investigate whether a single nucleotide polymorphism (SNP) in the mitochondrial gene for NADH dehydrogenase 2 (mt-Nd2) can modulate susceptibility to type 1 diabetes in NOD mice.Research Design and Methods: NOD/ShiLtJ mice conplastic for the alloxan resistant (ALR)/Lt-derived mt-Nd2(a) allele (NOD.mt(ALR)) were created and compared with standard NOD (carrying the mt-Nd2(c) allele) for susceptibility to spontaneous autoimmune diabetes, or to diabetes elicited by reciprocal adoptive splenic leukocyte transfers, as well as by adoptive transfer of diabetogenic T-cell clones. β-Cell lines derived from either the NOD (NIT-1) or the NOD.mt(ALR) (NIT-4) were also created to compare their susceptibility to cytolysis by diabetogenic CD8(+) T-cells in vitro.Results: NOD mice differing at this single SNP developed spontaneous or adoptively transferred diabetes at comparable rates and percentages. However, conplastic mice with the mt-Nd2(a) allele exhibited resistance to transfer of diabetes by the CD4(+) T-cell clone BDC 2.5 as well as the CD8(+) AI4 T-cell clones from T-cell receptor transgenic animals. NIT-4 cells with mt-Nd2(a) were also more resistant to AI4-mediated destruction in vitro than NIT-1 cells.Conclusions: Conplastic introduction into NOD mice of a variant mt-Nd2 allele alone was not sufficient to prevent spontaneous autoimmune diabetes. Subtle nonhematopoietic type 1 diabetes resistance was observed during adoptive transfer experiments with T-cell clones. This study confirms that genetic polymorphisms in mitochondria can modulate β-cell sensitivity to autoimmune T-cell effectors. [ABSTRACT FROM AUTHOR]

Objective: Macrophages play an important role in the pathogenesis of insulin resistance via the production of proinflammatory cytokines. Our goal is to decipher the molecular linkage between proinflammatory cytokine production and insulin resistance in macrophages.Research Design and Methods: We determined cytokine profiles in cultured macrophages and identified interleukin (IL)-1β gene as a potential target of FoxO1, a key transcription factor that mediates insulin action on gene expression. We studied the mechanism by which FoxO1 mediates insulin-dependent regulation of IL-1β expression in cultured macrophages and correlated FoxO1 activity in peritoneal macrophages with IL-1β production profiles in mice with low-grade inflammation or insulin resistance.Results: FoxO1 selectively promoted IL-1β production in cultured macrophages. This effect correlated with the ability of FoxO1 to bind and enhance IL-1β promoter activity. Mutations of the FoxO1 binding site within the IL-1β promoter abolished FoxO1 induction of IL-1β expression. Macrophages from insulin-resistant obese db/db mice or lipopolysaccharide-inflicted mice were associated with increased FoxO1 production, correlating with elevated levels of IL-1β mRNA in macrophages and IL-1 protein in plasma. In nonstimulated macrophages, FoxO1 remained inert with benign effects on IL-1β expression. In response to inflammatory stimuli, FoxO1 activity was augmented because of an impaired ability of insulin to phosphorylate FoxO1 and promote its nuclear exclusion. This effect along with nuclear factor-κB acted to stimulate IL-1β production in activated macrophages.Conclusions: FoxO1 signaling through nuclear factor-κB plays an important role in coupling proinflammatory cytokine production to insulin resistance in obesity and diabetes. [ABSTRACT FROM AUTHOR]

IDDM results from the destruction of pancreatic beta-cells by autoreactive T-cells that appear to avoid deletion early in development, possibly due to improper interaction with antigen-presenting cells (APCs) resident in the thymus or periphery. In the nonobese diabetic (NOD) mouse, there exists a defect in APC function characterized by its failure to fully mature upon stimulation. The NOD mouse thus provides an excellent model for the investigation of APC dysfunction and development and how these relate to the incidence of autoimmune diabetes. We initiated studies of APC function in the NOD mouse with respect to antigen processing and presentation, using a well-characterized antigen hen egg lysozyme (HEL) and comparing it with the closely related, major histocompatibility complex (MHC) (I-Ag7) identical, diabetes-resistant mouse strain NOR. Proliferation assays comparing NOD and NOR HEL-specific T-cells demonstrated that the T-cell proliferation response of the NOD mouse to both native and denatured forms of the antigen is lower than that of NOR. When crisscross proliferation experiments were conducted using purified T-cells and irradiated spleen cells as APCs from both strains, the results demonstrated that the defect in proliferation resided in the APC compartment of activation. The levels of intracellular glutathione (GSH) were compared in splenic macrophages from NOD and NOR mice; it was found that on antigenic stimulation, NOR macrophages produced significantly more intracellular GSH than did NOD macrophages, even under hyperglycemic (50 mmol/l glucose) conditions. The lower amount of GSH seen in the NOD may result in less efficient processing of antigen, and subsequently, lower levels of T-cell activation. [ABSTRACT FROM AUTHOR]

In this review, we explore the complex interplay between the immune system and pancreatic β cells in the context of type 1 diabetes (T1D). While T1D is predominantly considered a T-cell-mediated autoimmune disease, the inability of human leukocyte antigen (HLA)-risk alleles alone to explain disease development suggests a role for β cells in initiating and/or propagating disease. This review delves into the vulnerability of β cells, emphasizing their susceptibility to endoplasmic reticulum (ER) stress and protein modifications, which may give rise to neoantigens. Additionally, we discuss the role of viral infections as contributors to T1D onset, and of genetic factors with dual impacts on the immune system and β cells. A greater understanding of the interplay between environmental triggers, autoimmunity, and the β cell will not only lead to insight as to why the islet β cells are specifically targeted by the immune system in T1D but may also reveal potential novel therapeutic interventions., (Copyright © 2024 Cold Spring Harbor Laboratory Press; all rights reserved.)

Hepatic inflammation is culpable for the evolution of asymptomatic steatosis to nonalcoholic steatohepatitis (NASH). Hepatic inflammation results from abnormal macrophage activation. We found that FoxO1 links overnutrition to hepatic inflammation by regulating macrophage polarization and activation. FoxO1 was upregulated in hepatic macrophages, correlating with hepatic inflammation, steatosis, and fibrosis in mice and patients with NASH. Myeloid cell conditional FoxO1 knockout skewed macrophage polarization from proinflammatory M1 to the antiinflammatory M2 phenotype, accompanied by a reduction in macrophage infiltration in liver. These effects mitigated overnutrition-induced hepatic inflammation and insulin resistance, contributing to improved hepatic metabolism and increased energy expenditure in myeloid cell FoxO1-knockout mice on a high-fat diet. When fed a NASH-inducing diet, myeloid cell FoxO1-knockout mice were protected from developing NASH, culminating in a reduction in hepatic inflammation, steatosis, and fibrosis. Mechanistically, FoxO1 counteracts Stat6 to skew macrophage polarization from M2 toward the M1 signature to perpetuate hepatic inflammation in NASH. FoxO1 appears to be a pivotal mediator of macrophage activation in response to overnutrition and a therapeutic target for ameliorating hepatic inflammation to stem the disease progression from benign steatosis to NASH.

T cells are strongly regulated by oxidizing environments and amino acid restriction. How T cells reprogram metabolism to adapt to these extracellular stress situations is not well understood. Here, we show that oxidizing environments and amino acid starvation induce ATF4 in CD4 + T cells. We also demonstrate that Atf4-deficient CD4 + T cells have defects in redox homeostasis, proliferation, differentiation, and cytokine production. We further reveal that ATF4 regulates a coordinated gene network that drives amino acid intake, mTORC1 activation, protein translation, and an anabolic program for de novo synthesis of amino acids and glutathione. ATF4 also promotes catabolic glycolysis and glutaminolysis and oxidative phosphorylation and thereby provides precursors and energy for anabolic pathways. ATF4-deficient mice mount reduced Th1 but elevated Th17 immune responses and develop more severe experimental allergic encephalomyelitis (EAE). Our study demonstrates that ATF4 is critical for CD4 + T cell-mediated immune responses through driving metabolic adaptation., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Pathophysiological conditions that lead to the release of the prototypic damage-associated molecular pattern molecule high mobility group box 1 (HMGB1) also result in activation of poly(ADP-ribose) polymerase 1 (PARP1; now known as ADP-ribosyl transferase 1 [ARTD1]). Persistent activation of PARP1 promotes energy failure and cell death. The role of poly(ADP-ribosyl)ation in HMGB1 release has been explored previously; however, PARP1 is a versatile enzyme and performs several other functions including cross-talk with another nicotinamide adenine dinucleotide- (NAD(+)) dependent member of the Class III histone deacetylases (HDACs), sirtuin-1 (SIRT1). Previously, it has been shown that the hyperacetylation of HMGB1 is a seminal event prior to its secretion, a process that also is dependent on HDACs. Therefore, in this study, we seek to determine if PARP1 inhibition alters LPS-mediated HMGB1 hyperacetylation and subsequent secretion due to its effect on SIRT1. We demonstrate in an in vitro model that LPS treatment leads to hyperacetylated HMGB1 with concomitant reduction in nuclear HDAC activity. Treatment with PARP1 inhibitors mitigates the LPS-mediated reduction in nuclear HDAC activity and decreases HMGB1 acetylation. By utilizing an NAD(+)-based mechanism, PARP1 inhibition increases the activity of SIRT1. Consequently, there is an increased nuclear retention and decreased extracellular secretion of HMGB1. We also demonstrate that PARP1 physically interacts with SIRT1. Further confirmation of this data was obtained in a murine model of sepsis, that is, administration of PJ-34, a specific PARP1 inhibitor, led to decreased serum HMGB1 concentrations in mice subjected to cecal ligation and puncture (CLP) as compared with untreated mice. In conclusion, our study provides new insights in understanding the molecular mechanisms of HMGB1 secretion in sepsis.

Background: The receptor for advanced glycation end-products (RAGE) has been suggested to modulate lung injury in models of acute pulmonary inflammation. To study this further, model systems utilizing wild type and RAGE knockout (KO) mice were used to determine the role of RAGE signaling in lipopolysaccharide (LPS) and E. coli induced acute pulmonary inflammation. The effect of intraperitoneal (i.p.) and intratracheal (i.t.) administration of mouse soluble RAGE on E. coli injury was also investigated., Methodology/principal Findings: C57BL/6 wild type and RAGE KO mice received an i.t. instillation of LPS, E. coli, or vehicle control. Some groups also received i.p. or i.t. administration of mouse soluble RAGE. After 24 hours, the role of RAGE expression on inflammation was assessed by comparing responses in wild type and RAGE KO. RAGE protein levels decreased in wild type lung homogenates after treatment with either LPS or bacteria. In addition, soluble RAGE and HMGB1 increased in the BALF after E. coli instillation. RAGE KO mice challenged with LPS had the same degree of inflammation as wild type mice. However, when challenged with E. coli, RAGE KO mice had significantly less inflammation when compared to wild type mice. Most cytokine levels were lower in the BALF of RAGE KO mice compared to wild type mice after E. coli injury, while only monocyte chemotactic protein-1, MCP-1, was lower after LPS challenge. Neither i.p. nor i.t. administration of mouse soluble RAGE attenuated the severity of E. coli injury in wild type mice., Conclusions/significance: Lack of RAGE in the lung does not protect against LPS induced acute pulmonary inflammation, but attenuates injury following live E. coli challenge. These findings suggest that RAGE mediates responses to E. coli-associated pathogen-associated molecular pattern molecules other than LPS or other bacterial specific signaling responses. Soluble RAGE treatment had no effect on inflammation.

Sustained oxidative stress is a known sequel to focal cerebral ischemia. This study examined the effects of treatment with a single dose or sustained infusion of the redox-modulating MnPorphyrin Mn(III)TDE-2-ImP(5+) on outcome from middle cerebral artery occlusion (MCAO) in the rat. Normothermic rats were subjected to 90 min MCAO followed by 90 min reperfusion and then were treated with a single intracerebroventricular dose of Mn(III)TDE-2-ImP(5+). Neurologic and histologic outcomes were assessed at 1 or 8 weeks postischemia. A single dose of Mn(III)TDE-2-ImP(5+) caused a dose-dependent improvement in histologic and neurologic outcome when assessed 1 week postischemia. Mn(III)TDE-2-ImP(5+) afforded preservation of brain aconitase activity at 5.5 h after reperfusion onset, consistent with its known antioxidant properties. Mn(III)TDE-2-ImP(5+) also attenuated postischemic NF-kappaB activation. Evidence for effects on cerebral infarct size and neurologic function had completely dissipated when rats were allowed to survive for 8 weeks postischemia. In contrast, a 1-week continuous intracerebroventricular Mn(III)TDE-2-ImP(5+) infusion caused persistent and substantive reduction in both cerebral infarct size and neurologic deficit at 8 weeks postischemia. Pharmacologic modulation of postischemic oxidative stress is likely to require sustained intervention for enduring efficacy in improving neurologic and histologic outcome from a transient focal ischemic insult.

Excessive proliferation of vascular smooth muscle cells (VSMCs) is one of the primary lesions in atherosclerosis development during diabetes. High glucose triggers VSMC proliferation and initiates activation of the transcription factor nuclear factor (NF)-kappaB. Recently, clinical studies have demonstrated that replacement therapy with C-peptide, a cleavage product of insulin, to type 1 diabetic (T1D) patients is beneficial on a variety of diabetes-associated vascular complications. However, the mechanisms underlying the beneficial activity of C-peptide on the vasculature in conditions of hyperglycemia are largely unknown. The effects of C-peptide on the proliferation of human umbilical artery smooth muscle cell (UASMC) and aortic smooth muscle cell (AoSMC) lines cultured under high glucose for 48 h were tested. To gain insights on potential intracellular signaling pathways affected by C-peptide, we analyzed NF-kappaB activation in VSMCs since this pathway represents a key mechanism for the accelerated vascular disease observed in diabetes. High glucose conditions (25 mmol/L) stimulated NF-kappaB-dependent VSMC proliferation since the addition of two NF-kappaB-specific inhibitors, BAY11-7082 and PDTC, prevented proliferation. C-peptide at the physiological concentrations of 0.5 and 1 nmol/L decreased high glucose-induced proliferation of VSMCs that was accompanied by decreased phosphorylation of IkappaB and reduced NF-kappaB nuclear translocation. These results suggest that in conditions of hyperglycemia C-peptide reduces proliferation of VSMCs and NF-kappaB nuclear translocation. In patients with T1D, physiological C-peptide levels may exert beneficial effects on the vasculature that, under high glucose conditions, is subject to progressive dysfunction.

Recent studies demonstrated that engagement of cytotoxic T lymphocyte antigen 4 (CTLA-4)/(CD152) generates an inhibitory signal to T cells which arrests an on-going immune response. Since aberrant CD152 activity is thought to contribute to autoimmunity, we examined the effect of CD152-mediated inhibitory signals on the response to self and foreign antigens in autoimmune, diabetes-prone NOD and non-autoimmune BALB/c mice. The interaction of CD152 with its ligand B7 was prevented by treating the mice with anti-CD152 blocking antibody, before and following immunization of the mice with the self-antigen, syngeneic islet cells, or the foreign antigen, key-hole limpet hemocyanin (KLH). CD152 blockade in BALB/c mice stimulated a robust islet-specific T cell-mediated immune response compared to control antibody-treated mice. The augmentation of T cell responses in BALB/c mice was consistent with the role proposed for CD152 as a down-regulator of T cell activation responses. Furthermore, CD152 blockade unmasked islet cell specific autoreactive T cells in the non-autoimmune BALB/c mouse. Conversely, CD152 blockade in NOD mice failed to regulate islet-specific auto-reactive T cell responses. However, CD152 blockade enhanced the T cell response to the exogenous, foreign antigen KLH in both non-autoimmune BALB/c and autoimmune NOD mice. Collectively, these results suggest that there is not a global defect in CD152-mediated regulation of peripheral T cell immune responses in NOD autoimmune mice but rather, a defect specific to T cells recognizing self antigen., (Copyright 2000 Academic Press.)

A whole cell Renibacterium salmoninarum vaccine was developed using 37 degrees C heat treated cells that were subsequently formalin fixed; this treatment reduced bacterial hydrophobicity and cell associated p57. Coho salmon Oncorhynchus kisutch were immunized with the p57- vaccine by either a combination of intraperitoneal (i.p.) and intramuscular (i.m.) injections or per os. In the first experiment, i.p./i.m. vaccination of coho salmon with p57- cells in Freund's Incomplete Adjuvant (FIA) conferred a statistically significant increase in mean time to death after the salmon were i.p. challenged with 4.1 x 10(6) colony forming units (cfu) of R. salmoninarum. There was no significant difference in response between fish immunized with R. salmoninarum cell surface extract in FIA and those immunized with extracellular protein (ECP) concentrated from culture supernatant in FIA. The i.p. challenge dose resulted in complete mortality of all fish by Day 43. In a second experiment, fish were orally vaccinated with p57- R. salmoninarum cells encased in a pH protected, enteric-coated antigen microsphere (ECAM). Fish were bath challenged with 4.2 x 10(6) cfu ml-1 on Day 0 and sampled at time points of 0 (pre-challenge), 50, 90, or 150 d immersion challenge. Vaccine efficacy was determined by monitoring the elaboration of p57 in the kidneys of vaccinated and control fish. Fish vaccinated orally demonstrated a significantly lower concentration of p57 (p < 0.01) at Day 150 post challenge compared to fish receiving ECAMs alone. Fish receiving p57 cells without ECAM coating also showed a significantly lower p57 level (p < 0.03) versus control. In contrast, fish injected intraperitoneally with the p57- cells or fish fed p57+ R. salmoninarum cells in ECAMs demonstrated no significant difference (p > 0.05) versus controls. In summary, these studies suggest the preliminary efficacy of 37 degrees C treatment of R. salmoninarum cells as an oral bacterial kidney disease vaccine.

Renibacterium salmoninarum is a Gram-positive diplo-bacillus and the causative agent of bacterial kidney disease, a prevalent disease of salmonid fish. Virulent isolates of R. salmoninarum have a hydrophobic cell surface and express the 57-58 kDa protein (p57). Here we have investigated parameters which effect cell hydrophobicity and p57 degradation. Incubation of R. salmoninarum cells at 37 degrees C for > 4 h decreased cell surface hydrophobicity as measured by the salt aggregation assay, and decreased the amount of cell associated p57. Incubation of cells at lower temperatures (22, 17, 4 or -20 degrees C) for up to 16 h did not reduce hydrophobicity or the amount of cell associated p57. Both the loss of cell surface hydrophobicity and the degradation of p57 were inhibited by pre-incubation with the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF). Cell surface hydrophobicity was specifically reconstituted by incubation with extracellular protein (ECP) concentrated from culture supernatant and was correlated with the reassociation of p57 onto the bacterial cell surface as determined by western blot and total protein stain analyses. The ability of p57 to reassociate suggests that the bacterial cell surface is not irreversibly modified by the 37 degrees C treatment and that p57 contributes to the hydrophobic nature of R. salmoninarum. In summary, we describe parameters effecting the removal of the p57 virulence factor and suggest the utility of this modification for generating a whole cell vaccine against bacterial kidney disease.

To investigate host leukocytes recruited to the pancreas by diabetogenic T cells, we administered islet-specific CD4(+) T cell clones to 2-week-old nonobese diabetic (NOD) mice and examined the resulting pancreatic infiltrate by flow cytometry. Two different Vbeta4(+)CD4(+) T cell clones, BDC 2.5 and BDC 6.9, were found to recruit a heterogeneous T cell population as determined by staining with a panel of anti-TCR Vbeta monoclonal antibodies. The majority of the diabetes-initiating, Vbeta4(+) T cell clones migrated to the spleen whereas only 5-8% of the T cell population infiltrating the pancreas was Vbeta4(+). Anti-IL-2 receptor staining indicated that fewer than 10% of the total population of infiltrating lymphocytes within the pancreas were in a highly activated state. We have further found that normal splenic T cells from the NOD mouse proliferate poorly to IL-2 in vitro, yet secrete IFN-gamma in response to IL-2 stimulation. These results suggest that the recruited host T cells in our disease transfer system are not directly pathogenic but, rather, are responding to the small numbers of inflammatory T cell clones by providing cytokines that facilitate the disease process., (Copyright 1998 Academic Press.)

Bacterial kidney disease has consistently resisted attempts to control it by prophylactic immunisation. Although successful vaccines have been produced to a number of Gram-negative fish pathogens, the relatively simple method used in these cases have not been successful with Renibacterium salmoninarum. A more circumspect and thorough knowledge of the biological function of R. salmoninarum antigens must be obtained. Also required is a more precise understanding of the role of regional immunity in effective prophylaxis. Aspects of R. salmoninarum's biology provide a provocative challenge to the vaccinologist. Its residence in, and apparent commandeering of the macrophage, indicate that a vigorous cell-mediated response will probably be required to generate protective immunity. Its most biologically potent secreted product, p57, appears to be an aggressin. Further, p57 has the capability of frustrating immunoprophylaxis by either misdirecting the immune response, or by preventing its induction. Many immunization studies have used injection immunization and challenge protocols. It now appears that alternative routes of immunization which had been considered less protective (i.e. oral immunization) may not only be more efficacious, but may be the only route that does not lead to a misdirected and possibly pathological immune response. Also, the general reliance on serum antibodies as the only means to assess immunity is fraught with difficulties, particularly with pathogens such as R. salmoninarum. Recent advances in the analysis of cellular immunity will be a great aid in the design of future vaccines.