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Metabolomics, lipidomics, and the study of cellular metabolism are gaining increasing interest particularly in the field of immunology, since the activation and effector functions of immune cells are profoundly controlled by changes in cellular metabolic asset. Among the different techniques that can be used for the evaluation of cellular metabolism, the Seahorse Extracellular Flux Analyzer allows the real time measurement of both glycolytic and mitochondrial respiration pathways in cells of interest, through the assessment of extracellular acidification and oxygen consumption rate. Metabolomics, on the other hand, is the high-throughput analysis of metabolites, i.e., the substrates, intermediates, and products of cellular metabolism, starting from biofluids, cells or tissues. The metabolome does not include lipids as their properties are different from water-soluble metabolites and are classified under the lipidome. Lipidomics analysis allows the identification and quantification of lipid species. Metabolomics and lipidomics are currently performed with mass-spectrometry coupled with liquid or gas chromatography (LC-MS or GC-MS) and/or nuclear-magnetic resonance (NMR). Here we describe the protocol for the evaluation of metabolic rate, metabolomics, and lipidomics in T cells, examining the detailed experimental approaches.

Introduction: Cryopyrin associated periodic syndromes (CAPS) are rare autoinflammatory disorders associated with dominantly gain-offunction mutations in the NLRP3 gene that result in overactivation of the inflammasome, increased secretion of interleukin (IL)-1beta and IL-18, and systemic inflammation. It has been reported that oligomeric particles of the adaptor ASC (apoptosis-associated Speck-like protein with a caspase-recruitment domain) are released together with IL-1beta and active caspase-1 subunits after activation of the inflammosome complex and that patients with CAPS show an increased serum concentration of ASC+ particles. Objectives: The diagnosis of CAPS is a critical factor due to both the lack of specific laboratory results and the sharing of similar clinical manifestations with other autoinflammatory diseases, our aim is to develop a simple assay to evaluate the levels of ASC particles in the serum of CAPS patients to provide novel biomarkers facilitating early disease diagnosis and able to monitor treatment responses. Methods: We developed an ELISA for the quantification of ASC particles in serum and plasma of normal and pathological subjects. We analysed samples from CAPS patients and from patients with autoimmune disorders (Multiple Sclerosis (MS), Type 1 Diabetes (T1D) and juvenile idiopathic arthritis), to confirm that ASC presence in the serum is not due to other chronic inflammatory processes characterizing autoimmunity. In addition, we also evaluated the concentration of ASC in the sera of TNF receptor–associated periodic syndrome (TRAPS) patients to reinforce the concept of specificity of this biomarker in CAPS patients and not in individuals suffering from others inflammatory disorders. Results: We observed that untreated CAPS patients are characterized by the presence of a significant higher amount of ASC particles when compared with healthy controls (HS) and with patients suffering from MS and T1D. This tendency was also evident in patients with arthritis and TRAPS even if the difference was not statistically significant due to the small number of samples. In addition there is a tendency through a reduction of ASC levels in CAPS patients after pharmacological treatment, which require future investigations. Conclusion: These data suggest that ELISA quantitation of ASC protein could represent a novel and additional strategy for the diagnosis and monitoring of CAPS.

Background: Regulatory T cells (Tregs) play a critical role in maintaining immune homeostasis. We investigated two main types of Tregs, the CD4+FOXP3+ and IL-10+ Tr1, in pediatric subjects with inflammatory bowel disease (IBD) both at diagnosis and after the clinical remission. Methods: Peripheral blood Tregs were analyzed in 16 children with Crohn's disease (CD), 19 with ulcerative colitis (UC), and 14 healthy controls (HC). Two cocktails of fluoresceinated antibodies were used to discriminate between CD4+FOXP3+ and Tr1. Results: We observed in both CD and UC groups a higher frequency of Tr1 at diagnosis compared to controls, which decreased at follow-up compared to diagnosis, in particular in UC. Similarly, in UC patients the percentage of CD4+FOXP3+ Tregs markedly decreased at follow-up compared to the same patients at diagnosis and compared to HC. The expression of CTLA-4 in CD4+FOXP3+ Tregs increased in both groups at clinical remission. Conclusion: This study shows that IBD children present at diagnosis an increased frequency of circulating Tregs, probably as a compensative reaction to tissue inflammation. During the clinical remission, the Treg frequency diminishes, and concomitantly, their activation status increases. Notwithstanding, the high Treg density at diagnosis is not sufficient to counteract the inflammation in the childhood IBD.

Background: Oxidative stress is a driver of multiple sclerosis (MS) pathology. We evaluated the effect of coenzyme Q10 (CoQ10) on laboratory markers of oxidative stress and inflammation, and on MS clinical severity. Methods: We included 60 relapsing–remitting patients with MS treated with interferon beta1a 44μg (IFN-β1a) with CoQ10 for 3 months, and with IFN-β1a 44μg alone for 3 more months (in an open-label crossover design). At baseline and at the 3 and 6-month visits, we measured markers of scavenging activity, oxidative damage and inflammation in the peripheral blood, and collected data on disease severity. Results: After 3 months, CoQ10 supplementation was associated with improved scavenging activity (as mediated by uric acid), reduced intracellular reactive oxygen species production, reduced oxidative DNA damage, and a shift towards a more anti-inflammatory milieu in the peripheral blood [with higher interleukin (IL)-4 and IL-13, and lower eotaxin, granulocyte-macrophage colony-stimulating factor (GM-CSF), hepatocyte growth factor (HGF), interferon (IFN)-γ, IL-1α, IL-2R, IL-9, IL-17F, macrophage inflammatory proteins (MIP)-1α, regulated on activation-normal T cell expressed and secreted (RANTES), tumor necrosis factor (TNF)-α, and vascular endothelial growth factor (VEGF). Also, CoQ10 supplementation was associated with lower Expanded Disability Status Scale, fatigue severity scale, Beck’s depression inventory, and the visual analogue scale for pain. Conclusions: CoQ10 supplementation improved scavenging activity, reduced oxidative damage, and induced a shift towards a more anti-inflammatory milieu, in the peripheral blood of relapsing–remitting MS patients treated with 44μg IFN-β1a 44μg. A possible clinical effect was noted but deserves to be confirmed over longer follow ups.

Background: Dominantly gain-of-function mutations in the NLRP3 gene lead to Cryopyrin associated periodic syndromes (CAPS) characterized by constitutive activation of the inflammasome, increased secretion of interleukin (IL)-1beta and IL-18, and systemic inflammation. IL-1beta and active caspase-1 subunits are released in the serum together with the oligomeric particles of the adaptor ASC (apoptosis-associated Speck-like protein with a caspase-recruitment domain) after activation of the inflammosome complex and, as a consequence, patients with CAPS show an increased serum concentration of ASC+ particles. Objectives: Patients suffering from CAPS are characterized by clinical manifestation similar to other autoinflammatory diseases. This phenomenon together with the lack of specific laboratory tests makes difficult the diagnosis of CAPS. In this context the development of a test for the evaluation of serum ASC levels could provide novel biomarkers facilitating early disease diagnosis and able to monitor treatment responses. Methods: We analysed, with a novel ELISA assay, the levels of ASC particles in serum and plasma of normal subjects, CAPS patients and patients with autoimmune disorders (Multiple Sclerosis (MS), Type 1 Diabetes (T1D) and juvenile idiopathic arthritis), to confirm that ASC presence in the serum is not due to other chronic inflammatory processes characterizing autoimmunity. To evaluate the specificity of this biomarker in CAPS patients and not in individuals suffering from others inflammatory disorders, we also analysed sera from TNF receptor–associated periodic syndrome (TRAPS) patients. Results: ASC particles were higher in untreated CAPS patients with respect to healthy controls and patients suffering from MS and T1D. This tendency was also evident in patients with arthritis and TRAPS even if the difference was not statistically significant due to the small number of samples. In addition after pharmacological treatment there is a tendency to be confirmed through a reduction of ASC levels in CAPS patients. Conclusion: These data suggest that ELISA quantitation of ASC protein could represent a novel and additional strategy for the diagnosis and monitoring of CAPS. Disclosure of Interests:: Fortunata Carbone: None declared, Teresa Micillo: None declared, Luca Cantarini: None declared, Maria Alessio: None declared, Alma Nunzia Olivieri: None declared, Maria Francesca Gicchino: None declared, Antonella Insalaco: None declared, Maria Cristina Maggio: None declared, Orso Maria Lucherini: None declared, Roberto Scarpioni: None declared, Matteo Piga: None declared, Maria Maddalena Angioni: None declared, Laura Obici: None declared, Antonella Simpatico: None declared, Pietro Leccese: None declared, Rita Consolini: None declared, Raffaele Manna: None declared, Paolo Sfriso: None declared, Sara Bindoli: None declared, Paola Galozzi: None declared, Ida Orlando: None declared, Sabrina Chiesa: None declared, Marco Gattorno Grant/research support from: MG has received unrestricted grants from Sobi and Novartis, Giuseppe Matarese Grant/research support from: Matarese reports research grants from Merck-Serono, Biogen Idec, Novartis and IBSA.

Background: Previous studies evidenced a link between metabolic dysregulation, inflammation, and neurodegeneration in multiple sclerosis (MS). Objectives: To explore whether increased adipocyte mass expressed as body mass index (BMI) and increased serum lipids influence cerebrospinal fluid (CSF) inflammation and disease severity. Methods: In this cross-sectional study, 140 consecutive relapsing-remitting (RR)-MS patients underwent clinical assessment, BMI evaluation, magnetic resonance imaging scan, and blood and CSF collection before any specific drug treatment. The CSF levels of the following cytokines, adipocytokines, and inflammatory factors were measured: interleukin (IL)-6, IL-13, granulocyte macrophage colony-stimulating factor, leptin, ghrelin, osteoprotegerin, osteopontin, plasminogen activator inhibitor-1, resistin, and Annexin A1. Serum levels of triglycerides, total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C) were assessed. Results: A positive correlation emerged between BMI and Expanded Disability Status Scale score. Obese RR-MS patients showed higher clinical disability, increased CSF levels of the proinflammatory molecules IL-6 and leptin, and reduced concentrations of the anti-inflammatory cytokine IL-13. Moreover, both the serum levels of triglycerides and TC/HDL-C ratio showed a positive correlation with IL-6 CSF concentrations. Conclusion: Obesity and altered lipid profile are associated with exacerbated central inflammation and higher clinical disability in RR-MS at the time of diagnosis. Increased adipocytokines and lipids can mediate the negative impact of high adiposity on RR-MS course.

Reliable immunologic biomarkers able to monitor disease course during multiple sclerosis (MS) are still missing. We aimed at identifying possible immunometabolic biomarkers able to predict the clinical outcome in MS patients during treatment with interferon (IFN)-beta-1a. We measured in 45 relapsing-remitting (RR) MS patients, blood circulating levels of several immunometabolic markers, at enrolment, and correlated their levels to disease activity and progression over time. Higher levels of interleukin (IL)-6, soluble-CD40-ligand (sCD40L) and leptin at baseline associated with a higher relapse rate and a greater risk of experiencing at least one relapse in the following year. Higher values of soluble tumor necrosis factor receptor (sTNF-R) and leptin at baseline were predictive of a higher number of lesions in the following one-year of follow up. In conclusion, our data suggest that an immunometabolic profiling measuring IL-6, sCD40L, leptin and sTNF-R at baseline, could represent a useful tool to predict disease course in RRMS patients during treatment with IFN-beta-1a.

The filed that links immunity and metabolism is rapidly expanding. The adipose tissue, by secreting a series of immune regulators called adipokines, represents the common mediator linking metabolic processes and immune system functions. The dysregulation of adipokine secretion, occurring in obese individuals or in conditions of malnutrition or dietary restriction, affects the activity of immune cells resulting in inflammatory autoimmune responses or increased susceptibility to infectious diseases. Alterations of cell metabolism that characterize several autoimmune diseases strongly support the idea that the immune tolerance is also regulated by metabolic pathways. The comprehension of the molecular mechanisms underlying these alterations may lead to the development of novel therapeutic strategies to control immune cell differentiation and function in conditions of autoimmunity.