Separate Gut Plasma Cell Populations Produce Auto-Antibodies against Transglutaminase 2 and Transglutaminase 3 in Dermatitis Herpetiformis.

Dermatitis herpetiformis (DH) is an inflammatory skin disorder often considered as an extra intestinal manifestation of celiac disease (CeD). Hallmarks of CeD and DH are auto-antibodies to transglutaminase 2 (TG2) and transglutaminase 3 (TG3), respectively. DH patients have auto-antibodies reactive with both transglutaminase enzymes. Here it is reported that in DH both gut plasma cells and serum auto-antibodies are specific for either TG2 or TG3 with no TG2-TG3 cross reactivity. By generating monoclonal antibodies from TG3-specific duodenal plasma cells of DH patients, three conformational epitope groups are defined. Both TG2-specific and TG3-specific gut plasma cells have few immunoglobulin (Ig) mutations, and the two transglutaminase-reactive populations show distinct selection of certain heavy and light chain V-genes. Mass spectrometry analysis of TG3-specific serum IgA corroborates preferential usage of IGHV2-5 in combination with IGKV4-1. Collectively, these results demonstrate parallel induction of anti-TG2 and anti-TG3 auto-antibody responses involving separate B-cell populations in DH patients.

Antibody Responses to Transglutaminase 3 in Dermatitis Herpetiformis: Lessons from Celiac Disease.

Dermatitis herpetiformis (DH) is the skin manifestation of celiac disease, presenting with a blistering rash typically on the knees, elbows, buttocks and scalp. In both DH and celiac disease, exposure to dietary gluten triggers a cascade of events resulting in the production of autoantibodies against the transglutaminase (TG) enzyme, mainly TG2 but often also TG3. The latter is considered to be the primary autoantigen in DH. The dynamics of the development of the TG2-targeted autoimmune response have been studied in depth in celiac disease, but the immunological process underlying DH pathophysiology is incompletely understood. Part of this process is the occurrence of granular deposits of IgA and TG3 in the perilesional skin. While this serves as the primary diagnostic finding in DH, the role of these immunocomplexes in the pathogenesis is unknown. Intriguingly, even though gluten-intolerance likely develops initially in a similar manner in both DH and celiac disease, after the onset of the disease, its manifestations differ widely.

Quantitative genome-scale metabolic modeling of human CD4+ T cell differentiation reveals subset-specific regulation of glycosphingolipid pathways.

T cell activation, proliferation, and differentiation involve metabolic reprogramming resulting from the interplay of genes, proteins, and metabolites. Here, we aim to understand the metabolic pathways involved in the activation and functional differentiation of human CD4+ T cell subsets (T helper [Th]1, Th2, Th17, and induced regulatory T [iTreg] cells). Here, we combine genome-scale metabolic modeling, gene expression data, and targeted and non-targeted lipidomics experiments, together with in vitro gene knockdown experiments, and show that human CD4+ T cells undergo specific metabolic changes during activation and functional differentiation. In addition, we confirm the importance of ceramide and glycosphingolipid biosynthesis pathways in Th17 differentiation and effector functions. Through in vitro gene knockdown experiments, we substantiate the requirement of serine palmitoyltransferase (SPT), a de novo sphingolipid pathway in the expression of proinflammatory cytokines (interleukin [IL]-17A and IL17F) by Th17 cells. Our findings provide a comprehensive resource for selective manipulation of CD4+ T cells under disease conditions characterized by an imbalance of Th17/natural Treg (nTreg) cells.

Missing Insight Into T and B Cell Responses in Dermatitis Herpetiformis.

Dermatitis herpetiformis is a cutaneous form of celiac disease manifesting as an itching rash typically on the elbows, knees and buttocks. It is driven by the ingestion of gluten-containing cereals and characterized by granular deposits of immunoglobulin A in the papillary dermis. These antibodies target transglutaminase (TG) 3 and in the majority of patients they are also found in circulation. The circulating antibodies disappear and skin symptoms resolve as a result of gluten-free diet but the cutaneous anti-TG3 IgA deposits may persist for several years. In dermatitis herpetiformis, plasma cells secreting antibodies against TG3 are located in the intestinal mucosa similarly to those producing TG2 antibodies characteristic for celiac disease. In fact, both TG2- and TG3-specific plasma cells and gluten responsive T cells are found in dermatitis herpetiformis patients but the interplay between these cell populations is unknown. The small bowel mucosal damage in celiac disease is believed to be mediated by co-operation of cytotoxic intraepithelial T cells and the inflammatory milieu contributed by gluten-reactive CD4+ T cells, whereas the skin lesions in dermatitis herpetiformis appear to be devoid of gluten reactive T cells. Thus, how celiac disease-type intestinal T and B cell responses develop into an autoimmune condition affecting the skin is still incompletely understood. Finally, the skin and small bowel lesions may reappear upon reintroduction of gluten in patients treated with gluten-free diet but virtually nothing is known about the long-lived B cell and memory T cell populations activating in response to dietary gluten in dermatitis herpetiformis.

The use of peripheral blood mononuclear cells in celiac disease diagnosis and treatment.

Introduction: Celiac disease is characterized by an abnormal immune activation driven by the ingestion of gluten from wheat, barley, and rye. Gluten-specific CD4+ T cells play an important role in disease pathogenesis and are detectable among peripheral blood mononuclear cells (PBMCs). Areas covered: This review summarizes the use of celiac disease patient PBMCs in clinical applications focusing on their exploitation in the development of diagnostic approaches and novel drugs to replace or complement gluten-free diet. Expert opinion: The most used PBMC-based methods applied in celiac disease research include ELISpot and HLA-DQ:gluten tetramer technology. ELISpot has been utilized particularly in research aiming to develop a celiac disease vaccine and in studies addressing the toxicity of different grains in celiac disease. HLA-DQ:gluten tetramer technology on the other hand initially focused on improving current diagnostics but in combination with additional markers it is also a useful outcome measure in clinical trials to monitor the efficacy of drug candidates. In addition, the technology serves well in the more detailed characterization of celiac disease-specific T cells, thereby possibly revealing novel therapeutic targets. Future studies may also reveal clinical applications for PBMC microRNAs and/or dendritic cells or monocytes present among PBMCs.

Metabolic alterations in immune cells associate with progression to type 1 diabetes.

AIMS/HYPOTHESIS: Previous metabolomics studies suggest that type 1 diabetes is preceded by specific metabolic disturbances. The aim of this study was to investigate whether distinct metabolic patterns occur in peripheral blood mononuclear cells (PBMCs) of children who later develop pancreatic beta cell autoimmunity or overt type 1 diabetes. METHODS: In a longitudinal cohort setting, PBMC metabolomic analysis was applied in children who (1) progressed to type 1 diabetes (PT1D, n = 34), (2) seroconverted to ≥1 islet autoantibody without progressing to type 1 diabetes (P1Ab, n = 27) or (3) remained autoantibody negative during follow-up (CTRL, n = 10). RESULTS: During the first year of life, levels of most lipids and polar metabolites were lower in the PT1D and P1Ab groups compared with the CTRL group. Pathway over-representation analysis suggested alanine, aspartate, glutamate, glycerophospholipid and sphingolipid metabolism were over-represented in PT1D. Genome-scale metabolic models of PBMCs during type 1 diabetes progression were developed by using publicly available transcriptomics data and constrained with metabolomics data from our study. Metabolic modelling confirmed altered ceramide pathways, known to play an important role in immune regulation, as specifically associated with type 1 diabetes progression. CONCLUSIONS/INTERPRETATION: Our data suggest that systemic dysregulation of lipid metabolism, as observed in plasma, may impact the metabolism and function of immune cells during progression to overt type 1 diabetes. DATA AVAILABILITY: The GEMs for PBMCs have been submitted to BioModels (www.ebi.ac.uk/biomodels/), under accession number MODEL1905270001. The metabolomics datasets and the clinical metadata generated in this study were submitted to MetaboLights (https://www.ebi.ac.uk/metabolights/), under accession number MTBLS1015.

Regulation of growth in Drosophila melanogaster: the roles of mitochondrial metabolism.

Mitochondrial functions are often considered purely from the standpoint of catabolism, but in growing cells they are mainly dedicated to anabolic processes, and can have a profound impact on the rate of growth. The Drosophila larva, which increases in body mass ∼200-fold over the course of ∼3 days at 25°C, provides an excellent model to study the underlying regulatory machinery that connects mitochondrial metabolic capacity to growth. In this review, we will focus on several key aspects of this machinery: nutrient sensing, endocrine control of feeding and nutrient mobilization, metabolic signalling, protein synthesis regulation and pathways of steroid biosynthesis and activity. In all these aspects, mitochondria appear to play a crucial role.

Mitochondrial dysfunction generates a growth-restraining signal linked to pyruvate in Drosophila larvae.

The Drosophila bang-sensitive mutant tko25t, manifesting a global deficiency in oxidative phosphorylation due to a mitochondrial protein synthesis defect, exhibits a pronounced delay in larval development. We previously identified a number of metabolic abnormalities in tko25t larvae, including elevated pyruvate and lactate, and found the larval gut to be a crucial tissue for the regulation of larval growth in the mutant. Here we established that expression of wild-type tko in any of several other tissues of tko25t also partially alleviates developmental delay. The effects appeared to be additive, whilst knockdown of tko in a variety of specific tissues phenocopied tko25t, producing developmental delay and bang-sensitivity. These findings imply the existence of a systemic signal regulating growth in response to mitochondrial dysfunction. Drugs and RNAi-targeted on pyruvate metabolism interacted with tko25t in ways that implicated pyruvate or one of its metabolic derivatives in playing a central role in generating such a signal. RNA-seq revealed that dietary pyruvate-induced changes in transcript representation were mostly non-coherent with those produced by tko25t or high-sugar, consistent with the idea that growth regulation operates primarily at the translational and/or metabolic level.

Circulating metabolites in progression to islet autoimmunity and type 1 diabetes.

AIMS/HYPOTHESIS: Metabolic dysregulation may precede the onset of type 1 diabetes. However, these metabolic disturbances and their specific role in disease initiation remain poorly understood. In this study, we examined whether children who progress to type 1 diabetes have a circulatory polar metabolite profile distinct from that of children who later progress to islet autoimmunity but not type 1 diabetes and a matched control group. METHODS: We analysed polar metabolites from 415 longitudinal plasma samples in a prospective cohort of children in three study groups: those who progressed to type 1 diabetes; those who seroconverted to one islet autoantibody but not to type 1 diabetes; and an antibody-negative control group. Metabolites were measured using two-dimensional GC high-speed time of flight MS. RESULTS: In early infancy, progression to type 1 diabetes was associated with downregulated amino acids, sugar derivatives and fatty acids, including catabolites of microbial origin, compared with the control group. Methionine remained persistently upregulated in those progressing to type 1 diabetes compared with the control group and those who seroconverted to one islet autoantibody. The appearance of islet autoantibodies was associated with decreased glutamic and aspartic acids. CONCLUSIONS/INTERPRETATION: Our findings suggest that children who progress to type 1 diabetes have a unique metabolic profile, which is, however, altered with the appearance of islet autoantibodies. Our findings may assist with early prediction of the disease.

Alternative oxidase confers nutritional limitation on Drosophila development.

The mitochondrial alternative oxidase, AOX, present in most eukaryotes apart from vertebrates and insects, catalyzes the direct oxidation of ubiquinol by oxygen, by-passing the terminal proton-motive steps of the respiratory chain. Its physiological role is not fully understood, but it is proposed to buffer stresses in the respiratory chain similar to those encountered in mitochondrial diseases in humans. Previously, we found that the ubiquitous expression of AOX from Ciona intestinalis in Drosophila perturbs the development of flies cultured under low-nutrient conditions (media containing only glucose and yeast). Here we tested the effects of a wide range of nutritional supplements on Drosophila development, to gain insight into the physiological mechanism underlying this developmental failure. On low-nutrient medium, larvae contained decreased amounts of triglycerides, lactate, and pyruvate, irrespective of AOX expression. Complex food supplements, including treacle (molasses), restored normal development to AOX-expressing flies, but many individual additives did not. Inhibition of AOX by treacle extract was excluded as a mechanism, since the supplement did not alter the enzymatic activity of AOX in vitro. Furthermore, antibiotics did not influence the organismal phenotype, indicating that commensal microbes were not involved. Fractionation of treacle identified a water-soluble fraction with low solubility in ethanol, rich in lactate and tricarboxylic acid cycle intermediates, which contained the critical activity. We propose that the partial activation of AOX during metamorphosis impairs the efficient use of stored metabolites, resulting in developmental failure.

Serum Serine Peptidase Inhibitor Kazal-Type 1, Trypsinogens 1 to 3, and Complex of Trypsin 2 and α1-Antitrypsin in the Diagnosis of Severe Acute Pancreatitis.

OBJECTIVES: We explored prediction of severe acute pancreatitis (AP) and development of organ dysfunction (OD). METHODS: Serum concentrations of serine peptidase inhibitor Kazal type 1 (SPINK1), trypsinogen 1, trypsinogen 2, and trypsinogen 3, complex between trypsin 2 and α1-antitrypsin, serum C-reactive protein, creatinine, and pancreatic amylase were measured in 239 AP patients with disease onset within 72 hours. RESULTS: SPINK1 distinguished most accurately patients who later developed severe AP. The area under the receiver operating characteristic curve for SPINK1 was 0.742, followed by trypsinogen 2 (0.726), complex between trypsin 2 and α1-antitrypsin (0.657), creatinine (0.656), trypsinogen 1 (0.652), trypsinogen 3 (0.557), and C-reactive protein (0.499). With a cutoff of 166 µg/L, SPINK1 had a specificity of 93%, a sensitivity of 48%, and diagnostic odds ratio of 11.52. In multivariate logistic regression analysis, only SPINK1 was an independent predictor of severe AP among patients presenting without OD on admission (P < 0.001). CONCLUSIONS: Plasma levels of the biomarkers and creatinine correlated with the severity of AP and development of OD. In patients presenting without OD at admission, SPINK1 was an independent marker for later development of severe AP.

Dynamics of Plasma Lipidome in Progression to Islet Autoimmunity and Type 1 Diabetes - Type 1 Diabetes Prediction and Prevention Study (DIPP).

Type 1 diabetes (T1D) is one of the most prevalent autoimmune diseases among children in Western countries. Earlier metabolomics studies suggest that T1D is preceded by dysregulation of lipid metabolism. Here we used a lipidomics approach to analyze molecular lipids in a prospective series of 428 plasma samples from 40 children who progressed to T1D (PT1D), 40 children who developed at least a single islet autoantibody but did not progress to T1D during the follow-up (P1Ab) and 40 matched controls (CTR). Sphingomyelins were found to be persistently downregulated in PT1D when compared to the P1Ab and CTR groups. Triacylglycerols and phosphatidylcholines were mainly downregulated in PT1D as compared to P1Ab at the age of 3 months. Our study suggests that distinct lipidomic signatures characterize children who progressed to islet autoimmunity or overt T1D, which may be helpful in the identification of at-risk children before the initiation of autoimmunity.

Genetic Variants in the Manganese Superoxide Dismutase 2 Gene and in the Catalase Gene are not Associated With Alcoholic Chronic Pancreatitis.

AIMS: Oxidative stress may contribute to the development of chronic pancreatitis (CP). The enzymes manganese superoxide dismutase 2 (MnSOD, SOD2) and catalase (CAT) counteract free radical activity within the mitochondria and the cytosol. Moreover, CAT activity contributes to the transformation of ethanol to acetaldehyde, a toxic intermediate product of ethanol metabolism, which has been associated with pancreatic damage. Common functional polymorphisms have been described in the MnSOD gene [rs4880, NM_000636.3:c.47 T > C, alanine (ALA) to valine (Val)] and in the CAT promoter region [rs1001179, NG_013339.1:g.4760 C > T]. We investigated whether these polymorphisms are associated with alcoholic CP. METHODS: We genotyped 470 patients with alcoholic CP for these MnSOD and CAT polymorphisms. We also analysed these variants in 357 healthy control subjects, and in an additional control group of 113 individuals with non-alcoholic CP. We used the age at onset of CP as marker of disease severity and investigated whether different genotypes are associated with different ages at onset. In patients with alcoholic CP, we investigated whether an interaction exists between smoking behaviour and genotypes by comparing genotype distributions in smokers and non-smokers. RESULTS: We did not observe significant differences of genotype frequencies between patient groups and controls. In patient groups, we did not find significant differences in the ages at onset between different genotypes. We did not observe an interaction between these polymorphisms. We did not find an association of these variants with smoking behaviour. CONCLUSIONS: The investigated MnSOD and CAT polymorphisms do not predispose to the development of alcoholic CP. SHORT SUMMARY: Patients with alcoholic pancreatitis and controls were genotyped for polymorphisms in oxidative stress genes. There were no significant differences of genotype frequencies between patients and controls, and no association with the age at onset of disease was observed. The polymorphisms are not associated with the development of alcoholic pancreatitis.

Tumour-associated macrophages activate migration and STAT3 in pancreatic ductal adenocarcinoma cells in co-cultures.

OBJECTIVES: Tumour-associated macrophages participate in tumour development and progression. The aim of this study was to assess the interactions of pancreatic cancer cells and pro-inflammatory M1 and anti-inflammatory M2 macrophages, specifically their effect on pancreatic cancer cell migration and the changes in STAT-signalling. METHODS: Monocytes were isolated from healthy subjects and differentiated into macrophages with M-CSF. The macrophages were polarized towards M1 by IL-12 and towards M2 by IL-10. We studied also the effect of pan-JAK/STAT-inhibitor P6. Macrophage polarization and STAT and NFkB-activation in both MiaPaCa-2 and macrophages were assessed by flow cytometry. We recorded the effect of co-culture on migration rate of pancreatic cancer cells MiaPaCa-2. RESULTS: Macrophages increased the migration rate of pancreatic cancer cells. Co-culture activated STAT1, STAT3, STAT5, AKT, and NFkB in macrophages and STAT3 in MiaPaCa-2 cells. IL-12 polarized macrophages towards M1 and decreased the migration rate of pancreatic cancer cells in co-cultures as well as P6. IL-10 skewed macrophage polarization towards M2 and induced increase of pancreatic cancer cells in co-cultures. CONCLUSION: Co-culture with macrophages increased pancreatic cancer cell migration and activated STAT3. It is possible to activate and deactivate migration of pancreatic cancer cells trough macrophage polarization.

RNA Polymerase III Subunit POLR3G Regulates Specific Subsets of PolyA+ and SmallRNA Transcriptomes and Splicing in Human Pluripotent Stem Cells.

POLR3G is expressed at high levels in human pluripotent stem cells (hPSCs) and is required for maintenance of stem cell state through mechanisms not known in detail. To explore how POLR3G regulates stem cell state, we carried out deep-sequencing analysis of polyA+ and smallRNA transcriptomes present in hPSCs and regulated in POLR3G-dependent manner. Our data reveal that POLR3G regulates a specific subset of the hPSC transcriptome, including multiple transcript types, such as protein-coding genes, long intervening non-coding RNAs, microRNAs and small nucleolar RNAs, and affects RNA splicing. The primary function of POLR3G is in the maintenance rather than repression of transcription. The majority of POLR3G polyA+ transcriptome is regulated during differentiation, and the key pluripotency factors bind to the promoters of at least 30% of the POLR3G-regulated transcripts. Among the direct targets of POLR3G, POLG is potentially important in sustaining stem cell status in a POLR3G-dependent manner.

Perspectives on Systems Modeling of Human Peripheral Blood Mononuclear Cells.

Human peripheral blood mononuclear cells (PBMCs) are the key drivers of the immune responses. These cells undergo activation, proliferation and differentiation into various subsets. During these processes they initiate metabolic reprogramming, which is coordinated by specific gene and protein activities. PBMCs as a model system have been widely used to study metabolic and autoimmune diseases. Herein we review various omics and systems-based approaches such as transcriptomics, epigenomics, proteomics, and metabolomics as applied to PBMCs, particularly T helper subsets, that unveiled disease markers and the underlying mechanisms. We also discuss and emphasize several aspects of T cell metabolic modeling in healthy and disease states using genome-scale metabolic models.

Systemic Inflammatory Response and Elevated Tumour Markers Predict Worse Survival in Resectable Pancreatic Ductal Adenocarcinoma.

BACKGROUND: Estimation of the prognosis of resectable pancreatic ductal adenocarcinoma (PDAC) currently relies on tumour-related factors such as resection margins and on lymph-node ratio (LNR) both inconveniently available only postoperatively. Our aim was to assess the accuracy of preoperative laboratory data in predicting PDAC prognosis. METHODS: Collection of laboratory and clinical data was retrospective from 265 consecutive patients undergoing surgery for PDAC at Helsinki University Hospital. Cancer-specific survival assessment utilized Kaplan-Meier analysis, and independent associations between factors were by the Cox regression model. RESULTS: During follow-up, 76% of the patients died of PDAC, with a median survival time of 19.6 months. In univariate analysis, CRP, albumin, CEA, and CA19-9 were significantly associated with postoperative cancer-specific survival. In multivariate analysis, taking into account age, gender, LNR, resection margins, tumour status, and adjuvant chemotherapy, the preoperative biomarkers independently associated with adverse prognosis were hypoalbuminemia (< 36 g/L, hazard ratio (HR) 1.56, 95% confidence interval (CI) 1.10-2.19, p = 0.011), elevated CRP (> 5 mg/L, HR 1.44, 95% CI 1.03-2.02, p = 0.036), CEA (> 5 µg/L, HR 1.60, 95% CI 1.07-2.53, p = 0.047), and CA19-9 (≥555 kU/L, HR 1.91, 95% CI 1.18-3.08, p = 0.008). CONCLUSION: For patients with resectable PDAC, preoperative CRP, along with albumin and tumour markers, is useful for predicting prognosis.

Increasing the Inflammatory Competence of Macrophages with IL-6 or with Combination of IL-4 and LPS Restrains the Invasiveness of Pancreatic Cancer Cells.

Recent studies suggest that pro-inflammatory type M1 macrophages inhibit tumor progression and that anti-inflammatory M2 macrophages enhance it. The aim of this study was to examine the interaction of type M1 and M2 macrophages with pancreatic cancer cells. We studied the migration rate of fluorescein stained pancreatic cancer cells on Matrigel cultured alone or with Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) differentiated macrophages or with Macrophage Colony Stimulating Factor (M-CSF) differentiated macrophages, skewing the phenotype towards pro- and anti-inflammatory direction, respectively. Macrophage differentiation was assessed with flow cytometry and the cytokine secretion in cell cultures with cytokine array. Both GM-CSF and M-CSF differentiated macrophages increased the migration rate of primary pancreatic adenocarcinoma cell line (MiaPaCa-2) and metastatic cell line (HPAF-II). Stimulation with IL6 or IL4+LPS reversed the macrophages' increasing effect on the migration rate of MiaPaCa-2 completely and partly of HPAF-II. Co-culture with MiaPaCa-2 reduced the inflammatory cytokine secretion of GM-CSF differentiated macrophages. Co-culture of macrophages with pancreatic cancer cells seem to change the inflammatory cytokine profile of GM-CSF differentiated macrophages and this might explain why also GM-CSF differentiated macrophages promoted the invasion. Adding IL6 or IL4+LPS to the cell culture with MiaPaCa-2 and GM-CSF or M-CSF differentiated macrophages increased the secretion of inflammatory cytokines and this could contribute to the reversion of the macrophage induced increase of cancer cell migration rate.