Secondary exposure to heavy metal in genetically susceptible mice leads to acceleration of autoimmune response.

Exposure to mercury (Hg) and silver (Ag) has been shown to induce autoimmune diseases in genetically susceptible rodents. Here, A.SW mice were initially exposed to HgCl2, AgNO3 or tap water (control) for 3 weeks. After 13 weeks of stoppage, all mice had secondary exposure to 203HgCl2. After secondary exposure, higher and earlier ANoA titers were observed in mice initially exposed to Hg or Ag compared to control. Further, mice initially exposed to Ag showed higher total IgG1 and IgG2a, Whole Body Retention and lymph nodes and spleen accumulation of Hg compared to mice initially exposed to Hg and controls. These findings showed an earlier and stronger immunological response in A.SW mice compared with control, following re-exposure to heavy metals indicating an immunological memory. Additionally, secondary exposure to a different heavy metal may aggravate the effects of exposure of at least one of the metals indicating cross-reactivity.

Intralymphatic glutamic acid decarboxylase administration in type 1 diabetes patients induced a distinctive early immune response in patients with DR3DQ2 haplotype.

GAD-alum given into lymph nodes to Type 1 diabetes (T1D) patients participating in a multicenter, randomized, placebo-controlled double-blind study seemed to have a positive effect for patients with DR3DQ2 haplotype, who showed better preservation of C-peptide than the placebo group. Here we compared the immunomodulatory effect of GAD-alum administered into lymph nodes of patients with T1D versus placebo with focus on patients with DR3DQ2 haplotype. Methods: GAD autoantibodies, GADA subclasses, GAD65-induced cytokine secretion (Luminex panel) and proliferation of peripheral mononuclear cells were analyzed in T1D patients (n=109) who received either three intra-lymphatic injections (one month apart) with 4 µg GAD-alum and oral vitamin D supplementation (2000 IE daily for 120 days), or placebo. Results: Higher GADA, GADA subclasses, GAD65-induced proliferation and cytokine secretion was observed in actively treated patients after the second injection of GAD-alum compared to the placebo group. Following the second injection of GAD-alum, actively treated subjects with DR3DQ2 haplotype had higher GAD65-induced secretion of several cytokine (IL4, IL5, IL7, IL10, IL13, IFNγ, GM-CSF and MIP1ß) and proliferation compared to treated individuals without DR3DQ2. Stratification of samples from GAD-alum treated patients according to C-peptide preservation at 15 months revealed that "good responder" individuals with better preservation of C-peptide secretion, independently of the HLA haplotype, had increased GAD65-induced proliferation and IL13 secretion at 3 months, and a 2,5-fold increase of IL5 and IL10 as compared to "poor responders". The second dose of GAD-alum also induced a more pronounced cytokine secretion in "good responders" with DR3DQ2, compared to few "good responders" without DR3DQ2 haplotype. Conclusion: Patients with DR3DQ2 haplotype had a distinct early cellular immune response to GAD-alum injections into the lymph node, and predominant GAD65-induced IL13 secretion and proliferation that seems to be associated with a better clinical outcome. If confirmed in the ongoing larger randomized double-blind placebo-controlled clinical trial (DIAGNODE-3), including only patients carrying DR3DQ2 haplotype, these results might be used as early surrogate markers for clinical efficacy.

Intralymphatic GAD-Alum (Diamyd®) Improves Glycemic Control in Type 1 Diabetes With HLA DR3-DQ2.

AIMS: Residual beta cell function in type 1 diabetes (T1D) is associated with lower risk of complications. Autoantigen therapy with GAD-alum (Diamyd) given in 3 intralymphatic injections with oral vitamin D has shown promising results in persons with T1D carrying the human leukocyte antigen (HLA) DR3-DQ2 haplotype in the phase 2b trial DIAGNODE-2. We aimed to explore the efficacy of intralymphatic GAD-alum on blood glucose recorded by continuous glucose monitoring (CGM). METHODS: DIAGNODE-2 (NCT03345004) was a multicenter, randomized, placebo-controlled, double-blind trial of 109 recent-onset T1D patients aged 12 to 24 years with GAD65 antibodies and fasting C-peptide > 0.12 nmol/L, which randomized patients to 3 intralymphatic injections of 4 µg GAD-alum and oral vitamin D, or placebo. We report results for exploratory endpoints assessed by 14-day CGM at months 0, 6, and 15. Treatment arms were compared by mixed-effects models for repeated measures adjusting for baseline values. RESULTS: We included 98 patients with CGM recordings of sufficient quality (DR3-DQ2-positive patients: 27 GAD-alum-treated and 15 placebo-treated). In DR3-DQ2-positive patients, percent of time in range (TIR, 3.9-10 mmol/L) declined less between baseline and month 15 in GAD-alum-treated compared with placebo-treated patients (-5.1% and -16.7%, respectively; P = 0.0075), with reduced time > 13.9 mmol/L (P = 0.0036), and significant benefits on the glucose management indicator (P = 0.0025). No differences were detected for hypoglycemia. GAD-alum compared to placebo lowered the increase in glycemic variability (standard deviation) observed in both groups (P = 0.0219). Change in C-peptide was correlated with the change in TIR. CONCLUSIONS: Intralymphatic GAD-alum improves glycemic control in recently diagnosed T1D patients carrying HLA DR3-DQ2.

Intra-lymphatic administration of GAD-alum in type 1 diabetes: long-term follow-up and effect of a late booster dose (the DIAGNODE Extension trial).

AIM: To evaluate the long-term effect of intra-lymphatic administration of GAD-alum and a booster dose 2.5 years after the first intervention (DIAGNODE Extension study) in patients with recent-onset type 1 diabetes. METHODS: DIAGNODE-1: Samples were collected from 12 patients after 30 months who had received 3 injections of 4 µg GAD-alum into a lymph node with one-month interval. DIAGNODE Extension study: First in human, a fourth booster dose of autoantigen (GAD-alum) was given to 3 patients at 31.5 months, who were followed for another 12 months. C-peptide was measured during mixed meal tolerance tests (MMTTs). GADA, IA-2A, GADA subclasses, GAD65-induced cytokines, PBMCs proliferation and T cells markers were analyzed. RESULTS: After 30-month treatment, efficacy was still seen in 8/12 patients (good responders, GR). Partial remission (IDAA1c < 9) had decreased compared to 15 months, but did not differ from baseline, and HbA1c remained stable. GAD65-specific immune responses induced by the treatment started to wane after 30 months, and most changes observed at 15 months were undetectable. GADA subclasses IgG2, IgG3 and IgG4 were predominant in the GR along with IgG1. A fourth intra-lymphatic GAD-alum dose to three patients after 31.5 months gave no adverse events. In all three patients, C-peptide seemed to increase the first 6 months, and thereafter, C-peptide, HbA1c, insulin requirement and IDAA1c remained stable. CONCLUSION: The effect of intra-lymphatic injections of GAD-alum had decreased after 30 months. Good responders showed a specific immune response. Administration of a fourth booster dose after 31.5 months was safe, and there was no decline in C-peptide observed during the 12-month follow-up.

Antiviral Function of NKEF against VHSV in Rainbow Trout.

Natural killer enhancing factor (NKEF) belongs to the peroxiredoxin family of proteins, a group of antioxidants that has been extensively studied in mammals. Recently, we identified NKEF in the immunoprecipitated proteome of rainbow trout red blood cells (RBCs) exposed to viral hemorrhagic septicemia virus (VHSV). In the present study, we evaluated the role of NKEF in the antiviral response of rainbow trout against VHSV by examining the expression profile of NKEF in VHSV-exposed RBCs and rainbow trout gonad-2 (RTG-2) cell line. We found an in vitro correlation between decreased VHSV replication and increased NKEF expression after RBCs were exposed to VHSV, however this was not found in RTG-2 cells where the infection highly increased and nkef transcripts remained almost unchanged. In addition, siRNA silencing of the nkef gene in rainbow trout RBCs and RTG-2 cells resulted in increased VHSV replication. We also found a correlation between nkef gene silencing and a decrease in the expression of genes related to type 1 interferon (IFN1) pathway. These findings indicated that NKEF is involved in the antiviral mechanisms of rainbow trout RBCs against VHSV and thus support its antiviral role and implication in the modulation of their immune response. Finally, overexpression of NKEF in an EPC cell line significantly reduced VHSV infectivity and was coupled to an increment in IFN1-related genes. In conclusion, NKEF may be a potential target for new therapeutic strategies against viral infections.

Intralymphatic Glutamic Acid Decarboxylase With Vitamin D Supplementation in Recent-Onset Type 1 Diabetes: A Double-Blind, Randomized, Placebo-Controlled Phase IIb Trial.

OBJECTIVE: To evaluate the efficacy of aluminum-formulated intralymphatic glutamic acid decarboxylase (GAD-alum) therapy combined with vitamin D supplementation in preserving endogenous insulin secretion in all patients with type 1 diabetes (T1D) or in a genetically prespecified subgroup. RESEARCH DESIGN AND METHODS: In a multicenter, randomized, placebo-controlled, double-blind trial, 109 patients aged 12-24 years (mean ± SD 16.4 ± 4.1) with a diabetes duration of 7-193 days (88.8 ± 51.4), elevated serum GAD65 autoantibodies, and a fasting serum C-peptide >0.12 nmol/L were recruited. Participants were randomized to receive either three intralymphatic injections (1 month apart) with 4 µg GAD-alum and oral vitamin D (2,000 IE daily for 120 days) or placebo. The primary outcome was the change in stimulated serum C-peptide (mean area under the curve [AUC] after a mixed-meal tolerance test) between baseline and 15 months. RESULTS: Primary end point was not met in the full analysis set (treatment effect ratio 1.091 [CI 0.845-1.408]; P = 0.5009). However, GAD-alum-treated patients carrying HLA DR3-DQ2 (n = 29; defined as DRB1*03, DQB1*02:01) showed greater preservation of C-peptide AUC (treatment effect ratio 1.557 [CI 1.126-2.153]; P = 0.0078) after 15 months compared with individuals receiving placebo with the same genotype (n = 17). Several secondary end points showed supporting trends, and a positive effect was seen in partial remission (insulin dose-adjusted HbA1c ≤9; P = 0.0310). Minor transient injection site reactions were reported. CONCLUSION: Intralymphatic administration of GAD-alum is a simple, well-tolerated treatment that together with vitamin D supplementation seems to preserve C-peptide in patients with recent-onset T1D carrying HLA DR3-DQ2. This constitutes a disease-modifying treatment for T1D with a precision medicine approach.

Integrated Transcriptomic and Proteomic Analysis of Red Blood Cells from Rainbow Trout Challenged with VHSV Point Towards Novel Immunomodulant Targets.

Teleost red blood cells (RBCs) are nucleated and therefore can propagate cellular responses to exogenous stimuli. RBCs can mount an immune response against a variety of fish viruses, including the viral septicemia hemorrhagic virus (VHSV), which is one of the most prevalent fish viruses resulting in aquaculture losses. In this work, RBCs from blood and head kidney samples of rainbow trout challenged with VHSV were analyzed via transcriptomic and proteomic analyses. We detected an overrepresentation of differentially expressed genes (DEGs) related to the type I interferon response and signaling in RBCs from the head kidney and related to complement activation in RBCs from blood. Antigen processing and presentation of peptide antigen was overrepresented in RBCs from both tissues. DEGs shared by both tissues showed an opposite expression profile. In summary, this work has demonstrated that teleost RBCs can modulate the immune response during an in vivo viral infection, thus implicating RBCs as cell targets for the development of novel immunomodulants.

Potential Role of Rainbow Trout Erythrocytes as Mediators in the Immune Response Induced by a DNA Vaccine in Fish.

In recent years, fish nucleated red blood cells (RBCs) have been implicated in the response against viral infections. We have demonstrated that rainbow trout RBCs can express the antigen encoded by a DNA vaccine against viral hemorrhagic septicemia virus (VHSV) and mount an immune response to the antigen in vitro. In this manuscript, we show, for the first time, the role of RBCs in the immune response triggered by DNA immunization of rainbow trout with glycoprotein G of VHSV (GVHSV). Transcriptomic and proteomic profiles of RBCs revealed genes and proteins involved in antigen processing and presentation of exogenous peptide antigen via MHC class I, the Fc receptor signaling pathway, the autophagy pathway, and the activation of the innate immune response, among others. On the other hand, GVHSV-transfected RBCs induce specific antibodies against VHSV in the serum of rainbow trout which shows that RBCs expressing a DNA vaccine are able to elicit a humoral response. These results open a new direction in the research of vaccination strategies for fish since rainbow trout RBCs actively participate in the innate and adaptive immune response in DNA vaccination. Based on our findings, we suggest the use of RBCs as target cells or carriers for the future design of novel vaccine strategies.

Fish Red Blood Cells Modulate Immune Genes in Response to Bacterial Inclusion Bodies Made of TNFα and a G-VHSV Fragment.

Fish Red-Blood Cells (RBCs) are nucleated cells that can modulate the expression of different sets of genes in response to stimuli, playing an active role in the homeostasis of the fish immune system. Nowadays, vaccination is one of the main ways to control and prevent viral diseases in aquaculture and the development of novel vaccination approaches is a focal point in fish vaccinology. One of the strategies that has recently emerged is the use of nanostructured recombinant proteins. Nanostructured cytokines have already been shown to immunostimulate and protect fish against bacterial infections. To explore the role of RBCs in the immune response to two nanostructured recombinant proteins, TNFα and a G-VHSV protein fragment, we performed different in vitro and in vivo studies. We show for the first time that rainbow trout RBCs are able to endocytose nanostructured TNFα and G-VHSV protein fragment in vitro, despite not being phagocytic cells, and in response to nanostructured TNFα and G-VHSV fragment, the expression of different immune genes could be modulated.

IFIT5 Participates in the Antiviral Mechanisms of Rainbow Trout Red Blood Cells.

Viral hemorrhagic septicemia virus (VHSV) infection appears to be halted in rainbow trout nucleated red blood cells (RBCs). Diverse mechanisms are thought to be related to the antiviral immune response of rainbow trout RBCs to VHSV. However, the specific rainbow trout RBC proteins that interact directly with VHSV are still unknown. In an attempt to identify VHSV-RBC protein interactions, we characterized the immunoprecipitated (IP) proteome of RBCs exposed to VHSV using an antibody against the N protein of VHSV. The IP proteomic characterization identified 31 proteins by mass spectrometry analysis. Among them, we identified interferon-induced protein with tetratricopeptide repeats 5 (IFIT5), a protein belonging to a family of proteins that are induced after the production of type I interferon. Importantly, IFIT5 has been implicated in the antiviral immune response. We confirmed the participation of IFIT5 in the rainbow trout RBC antiviral response by examining the expression profile of IFIT5 in RBCs after VHSV exposure at transcriptional and protein levels. We detected a correlation between the highest IFIT5 expression levels and the decline in VHSV replication at 6 h post-exposure. In addition, silencing ifit5 resulted in a significant increase in VHSV replication in RBCs. Moreover, an increase in VHSV replication was observed in RBCs when the IFIT5 RNA-binding pocket cavity was modulated by using a natural compound from the SuperNatural II database. We performed a proximity ligation assay and detected a significant increase in positive cells among VHSV-exposed RBCs compared to unexposed RBCs, indicating protein-protein colocalization between IFIT5 and the glycoprotein G of VHSV. In summary, these results suggest a possible role of IFIT5 in the antiviral response of RBCs against VHSV.

Rainbow Trout Red Blood Cells Exposed to Viral Hemorrhagic Septicemia Virus Up-Regulate Antigen-Processing Mechanisms and MHC I&II, CD86, and CD83 Antigen-presenting Cell Markers.

Nucleated teleost red blood cells (RBCs) are known to express molecules from the major histocompatibility complex and peptide-generating processes such as autophagy and proteasomes, but the role of RBCs in antigen presentation of viruses have not been studied yet. In this study, RBCs exposed ex vivo to viral hemorrhagic septicemia virus (VHSV) were evaluated by means of transcriptomic and proteomic approaches. Genes and proteins related to antigen presentation molecules, proteasome degradation, and autophagy were up-regulated. VHSV induced accumulation of ubiquitinated proteins in ex vivo VHSV-exposed RBCs and showed at the same time a decrease of proteasome activity. Furthermore, induction of autophagy was detected by evaluating LC3 protein levels. Sequestosome-1/p62 underwent degradation early after VHSV exposure, and it may be a link between ubiquitination and autophagy activation. Inhibition of autophagosome degradation with niclosamide resulted in intracellular detection of N protein of VHSV (NVHSV) and p62 accumulation. In addition, antigen presentation cell markers, such as major histocompatibility complex (MHC) class I & II, CD83, and CD86, increased at the transcriptional and translational level in rainbow trout RBCs exposed to VHSV. In summary, we show that nucleated rainbow trout RBCs can degrade VHSV while displaying an antigen-presenting cell (APC)-like profile.

Rainbow Trout Erythrocytes ex vivo Transfection With a DNA Vaccine Encoding VHSV Glycoprotein G Induces an Antiviral Immune Response.

Fish red blood cells (RBCs), are integral in several biologic processes relevant to immunity, such as pathogen recognition, pathogen binding and clearance, and production of effector molecules and cytokines. So far, one of the best strategies to control and prevent viral diseases in aquaculture is DNA immunization. DNA vaccines (based on the rhabdoviral glycoprotein G [gpG] gene) have been shown to be effective against fish rhabdoviruses. However, more knowledge about the immune response triggered by DNA immunization is necessary to develop novel and more effective strategies. In this study, we investigated the role of fish RBCs in immune responses induced by DNA vaccines. We show for the first time that rainbow trout RBCs express gpG of viral hemorrhagic septicaemia virus (VHSV) (GVHSV) when transfected with the DNA vaccine ex vivo and modulate the expression of immune genes and proteins. Functional network analysis of transcriptome profiling of RBCs expressing GVHSV revealed changes in gene expression related to G-protein coupled receptor (GPCR)-downstream signaling, complement activation, and RAR related orphan receptor α (RORA). Proteomic profile functional network analysis of GVHSV-transfected RBCs revealed proteins involved in the detoxification of reactive oxygen species, interferon-stimulated gene 15 (ISG15) antiviral mechanisms, antigen presentation of exogenous peptides, and the proteasome. Conditioned medium of GVHSV-transfected RBCs conferred antiviral protection and induced ifn1 and mx gene expression in RTG-2 cells infected with VHSV. In summary, rainbow trout nucleated RBCs could be actively participating in the regulation of the fish immune response to GVHSV DNA vaccine, and thus may represent a possible carrier cells for the development of new vaccine approaches.

In Silico Functional Networks Identified in Fish Nucleated Red Blood Cells by Means of Transcriptomic and Proteomic Profiling.

Nucleated red blood cells (RBCs) of fish have, in the last decade, been implicated in several immune-related functions, such as antiviral response, phagocytosis or cytokine-mediated signaling. RNA-sequencing (RNA-seq) and label-free shotgun proteomic analyses were carried out for in silico functional pathway profiling of rainbow trout RBCs. For RNA-seq, a de novo assembly was conducted, in order to create a transcriptome database for RBCs. For proteome profiling, we developed a proteomic method that combined: (a) fractionation into cytosolic and membrane fractions, (b) hemoglobin removal of the cytosolic fraction, (c) protein digestion, and (d) a novel step with pH reversed-phase peptide fractionation and final Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC ESI-MS/MS) analysis of each fraction. Combined transcriptome- and proteome- sequencing data identified, in silico, novel and striking immune functional networks for rainbow trout nucleated RBCs, which are mainly linked to innate and adaptive immunity. Functional pathways related to regulation of hematopoietic cell differentiation, antigen presentation via major histocompatibility complex class II (MHCII), leukocyte differentiation and regulation of leukocyte activation were identified. These preliminary findings further implicate nucleated RBCs in immune function, such as antigen presentation and leukocyte activation.

Shape-Shifted Red Blood Cells: A Novel Red Blood Cell Stage?

Primitive nucleated erythroid cells in the bloodstream have long been suggested to be more similar to nucleated red cells of fish, amphibians, and birds than the red cells of fetal and adult mammals. Rainbow trout Ficoll-purified red blood cells (RBCs) cultured in vitro undergo morphological changes, especially when exposed to stress, and enter a new cell stage that we have coined shape-shifted RBCs (shRBCs). We have characterized these shRBCs using transmission electron microscopy (TEM) micrographs, Wright⁻Giemsa staining, cell marker immunostaining, and transcriptomic and proteomic evaluation. shRBCs showed reduced density of the cytoplasm, hemoglobin loss, decondensed chromatin in the nucleus, and striking expression of the B lymphocyte molecular marker IgM. In addition, shRBCs shared some features of mammalian primitive pyrenocytes (extruded nucleus surrounded by a thin rim of cytoplasm and phosphatidylserine (PS) exposure on cell surface). These shRBCs were transiently observed in heat-stressed rainbow trout bloodstream for three days. Functional network analysis of combined transcriptomic and proteomic studies resulted in the identification of proteins involved in pathways related to the regulation of cell morphogenesis involved in differentiation, cellular response to stress, and immune system process. In addition, shRBCs increased interleukin 8 (IL8), interleukin 1 β (IL1β), interferon É£ (IFNÉ£), and natural killer enhancing factor (NKEF) protein production in response to viral hemorrhagic septicemia virus (VHSV). In conclusion, shRBCs may represent a novel cell stage that participates in roles related to immune response mediation, homeostasis, and the differentiation and development of blood cells.

Infectious pancreatic necrosis virus triggers antiviral immune response in rainbow trout red blood cells, despite not being infective.

Background: Some fish viruses, such as piscine orthoreovirus and infectious salmon anemia virus, target red blood cells (RBCs), highly replicate inside them and induce an immune response. However, the implications of RBCs in the context of birnavirus infection (i.e, infectious pancreatic necrosis virus (IPNV)) have not yet been studied. Methods:Ex vivo trout RBCs were obtained from peripheral blood, ficoll purified and exposed to IPNV in order to analyze infectivity and induced immune response using RT-qPCR, immune fluorescence imaging, flow cytometry and western-blotting techniques. Results: IPNV could not infect RBCs; however, IPNV-exposed RBCs increased the expression of the INF1-related genes ifn-1, pkr and mx genes. Moreover, conditioned media from IPNV-exposed RBCs conferred protection against IPNV infection in CHSE-214 fish cell line. Conclusions: Trout RBCs could trigger an antiviral immune response against IPNV infection despite not being infected. Fish RBCs could be considered mediators of the antiviral response and therefore targets of novel DNA vaccines and new strategies against fish viral infections. Further research is ongoing to completely understand the molecular mechanism that triggers this immune response in trout RBCs.

Identification of diverse defense mechanisms in rainbow trout red blood cells in response to halted replication of VHS virus.

Background: It has been described that fish nucleated red blood cells (RBCs) generate a wide variety of immune-related gene transcripts when viruses highly replicate inside them and are their main target cell. The immune response and mechanisms of fish RBCs against viruses targeting other cells or tissues has not yet been explored and is the objective of our study. Methods: Trout RBCs were obtained from peripheral blood, ficoll purified and exposed to Viral Haemorrhagic Septicaemia virus (VHSV). Immune response was evaluated by means of RT-qPCR, flow cytometry, immunofluorescence and isobaric tag for relative and absolute quantification (iTRAQ) protein profiling Results: VHSV N gene transcripts incremented early postexposure and were drastically decreased after 6 hours postexposure (hpe). The expression of the type I interferon ( ifn1) gene was significantly downregulated at early postexposure (3 hpe), together with a gradual downregulation of interferon-inducible mx and pkr genes until 72 hpe. Type I IFN protein was downregulated and interferon-inducible Mx protein was maintained at basal levels. Co-culture assays of RBCs with TSS (stromal cell line from spleen) revealed the IFN crosstalk between both cell types. On the other hand, anti-microbial peptide ß-defensin 1 and neutrophil chemotactic factor interleukin 8 were slightly upregulated in VHSV-exposed RBCs Isobaric tag for relative and absolute quantification (iTRAQ) revealed that VHSV exposure can induce a global protein downregulation in trout RBCs, mainly related to RNA stability and proteasome pathways. The antioxidant/antiviral response is also suggested to be involved in the response of trout RBCs to VHSV. Conclusions: A variety of mechanisms are proposed to be implicated in the antiviral response of trout RBCs against VHSV halted infection. Ongoing research is focused on understanding the mechanisms in detail. To our knowledge, this is the first report that implicates fish RBCs in the antiviral response against viruses not targeting RBCs.