Immunomodulatory effect of gut microbiota-derived bioactive peptides on human immune system from healthy controls and patients with inflammatory bowel disease

Bioactive peptides secreted by probiotic Bifidobacterium longum (peptide B7) and opportunistic pathogen Bacteroides fragilis (peptide B12) modulate the intestinal cytokine milieu in health. Here, we characterized their capacity to modulate both the mucosal cytokine production and the phenotype of circulating antigen presenting cells (APCs) in active inflammatory bowel disease (IBD). The IBD mucosa produced higher levels of pro-inflammatory cytokines referred to healthy controls (HCs). Peptides B7 and B12, however, did not ameliorate the mucosal cytokine milieu in IBD. Human circulating APCs (B-cells, monocytes, plasmacytoid dendritic cells (pDCs), and conventional dendritic cells (cDCs)) were characterized by flow cytometry in presence/absence of the peptides. Circulating B-cells, monocytes, and cDCs from IBD patients were more activated than those from HCs. Peptide B7, but not B12, decreased CCR2 expression on all APC subsets from HC, but not IBD patients. Moreover, both peptides tend to further increase their pro-inflammatory profile in IBD. In summary, IBD patients display mucosal and circulating APC pro-inflammatory properties. Peptide B7 immunomodulatory capacity elicited over circulating APCs from HC, but not IBD patients, suggests the presence of disrupted modulatory mechanisms for this peptide in IBD. Future studies should address the effect of bacteria-derived immunomodulatory peptides in non-inflamed (quiescent) IBD patientsThis work was supported by the Spanish Ministry of Economy (SAF2014-56642-JIN), the Spanish Ministry of Health (PIE13/00041), GETECCU (Grupo Español de Trabajo en Enfermedad Crohn y Colitis Ulcerosa), and the Community of Madrid (Consejería de Educación, Juventud y Deporte, Programa de Garantía Juvenil 2015 and 2016)

Urinary exosomes reveal protein signatures in hypertensive patients with albuminuria

Albuminuria is an indicator of cardiovascular risk and renal damage in hypertensive individuals. Chronic renin-angiotensin system (RAS) suppression facilitates blood pressure control and prevents development of new-onset-albuminuria. A significant number of patients, however, develop albuminuria despite chronic RAS blockade, and the physiopathological mechanisms are underexplored. Urinary exosomes reflect pathological changes taking place in the kidney. The objective of this work was to examine exosomal protein alterations in hypertensive patients with albuminuria in the presence of chronic RAS suppression, to find novel clues underlying its development. Patients were followed-up for three years and were classified as: a) patients with persistent normoalbuminuria; b) patients developing de novo albuminuria; and c) patients with maintained albuminuria. Exosomal protein alterations between groups were identified by isobaric tag quantitation (iTRAQ). Confirmation was approached by target analysis (SRM). In total, 487 proteins were identified with high confidence. Specifically, 48 proteins showed an altered pattern in response to hypertension and/or albuminuria. Out of them, 21 proteins interact together in three main functional clusters: glycosaminoglycan degradation, coagulation and complement system, and oxidative stress. The identified proteins constitute potential targets for drug development and may help to define therapeutic strategies to evade albuminuria progression in hypertensive patients chronically treated.Instituto de Salud Carlos III, fondos FEDER/FSE (PI11/01401, PI13/01873, PI14/01841, IF08/3667-1, PI11-02239, PI 14/0917, PI11/02432, PI13/01746, PI14/01650, PI16/01334, PT13/0001/0013, CP09/00229, CP15/00129, CPII15/00027), Fundacion SENEFRO, Fundacion Conchita Rabago de Jimenez Diaz, and Redes Tematicas de Investigacion Cooperativa (fondos FEDER/FSE, RD12/0021/0001, RD12/0042/0071). These results are lined up with the Spanish initiative on the Human Proteome Project (SpHPP).S

Differential effects of Anti-TNFα and Anti-α4β7 drugs on circulating dendritic cells migratory capacity in inflammatory bowel disease

Inflammatory bowel disease (IBD) is an idiopathic and chronic disorder that includes ulcerative colitis (UC) and Crohn’s disease (CD). Both diseases show an uncontrolled intestinal immune response that generates tissue inflammation. Dendritic cells (DCs) are antigen-presenting cells that play a key role in tolerance maintenance in the gastrointestinal mucosa. Although it has been reported that DC recruitment by the intestinal mucosa is more prominent in IBD patients, the specific mechanisms governing this migration are currently unknown. In this study, the expression of several homing markers and the migratory profile of circulating DC subsets towards intestinal chemo-attractants were evaluated and the effect of biological drugs with different mechanisms of action, such as anti-TNFα or anti-integrin α4β7 (vedolizumab), on this mechanism in healthy controls (HCs) and IBD patients was also assessed. Our results revealed that type 2 conventional DCs (cDC2) express differential homing marker profiles in UC and CD patients compared to HCs. Indeed, integrin β7 was differentially modulated by vedolizumab in CD and UC. Additionally, although CCL2 displayed a chemo-attractant effect over cDC2, while biological therapies did not modulate the expression of the homing markers, we paradoxically found that anti-TNF-treated cDC2 increased their migratory capacity towards CCL2 in HCs and IBD. Our results therefore suggest a key role for cDC2 migration towards the intestinal mucosa in IBD, something that could be explored in order to develop novel diagnostic biomarkers or to unravel new immunomodulatory targets in IBD.This study has been funded through the Instituto de Salud Carlos III (Sara Borrell fellowships, CD17/00014; CD21/00014), Asociación Española de Gastroenterología (Beca del Grupo Joven), Programa Estratégico Instituto de Biología y Genética Molecular (IBGM Junta de Castilla y León. Ref. CCVC8485), Plan Nacional (PID2019-104218RB-I00) from the Spanish Government, Janssen and MSD

Lunasin Peptide is a Modulator of the Immune Response in the Human Gastrointestinal Tract

[Introduction]: Lunasin is a soybean bioactive peptide with a variety of beneficial properties against chronic disorders. However, its effect in human primary intestinal cells remains unknown. Hence, this study aims to characterize its ex vivo biological activity in the human intestinal mucosa. [Methods and Results]: Human intestinal biopsies, obtained from healthy controls, are ex vivo conditioned with lunasin both in the presence/absence of lipopolysaccharide (LPS). Peptide maintains its stability during biopsy culture by HPLC-MS/MS analysis. Lunasin is bioactive in the human mucosa, as it induces IL-1β, TNF-α, IL-17A, CCL2, and PGE2/COX-2 gene expression together with an increased expression of tolerogenic IL-10 and TGFβ, while it also downregulates the expression of iNOS and subunit p65 from NF-κB. Indeed, lunasin also abrogates the LPS-induced pro-inflammatory response, downregulating IL-17A, IFNγ, and IL-8 expression, and inducing IL-10 and TGFβ expression. These results are also mirrored in the cell-free culture supernatants at the protein level by Multiplex. Moreover, lunasin further induces a regulatory phenotype and function on human intestinal conventional dendritic cell and macrophage subsets as assessed by flow cytometry. [Conclusions]: We hereby have characterized lunasin as an immunomodulatory peptide with potential capacity to prevent immune and inflammatory-mediated disorders in the human gastrointestinal tract.This work was supported by the Spanish Ministry of Economy (SAF2014-56642-JIN), the Spanish Ministry of Health (PIE13/00041), the Spanish Ministry of Science, Innovation and Universities (AGL2015-66886-R, PID2019-104218RB-I00), and the Community of Madrid (Consejería de Educación, Juventud y Deporte, Programa de Garantía Juvenil 2015 and 2016). S.F.T. is currently funded by the Instituto de Salud Carlos III (Sara Borrell fellowship CD17/00014). L.O.M. is funded by the Community of Madrid (BMD-5800). D.B. is funded by the Spanish Ministry of Science (RYC-2017-21606)

Potential role of new molecular plasma signatures on cardiovascular risk stratification in asymptomatic individuals

The evaluation of cardiovascular (CV) risk is based on equations derived from epidemiological data in individuals beyond the limits of middle age such as the Framingham and SCORE risk assessments. Lifetime Risk calculator (QRisk®), estimates CV risk throughout a subjects' lifetime, allowing those. A more aggressive and earlier intervention to be identified and offered protection from the consequences of CV and renal disease. The search for molecular profiles in young people that allow a correct stratification of CV risk would be of great interest to adopt preventive therapeutic measures in individuals at high CV risk. To improve the selection of subjects susceptible to intervention with aged between 30-50 years, we have employed a multiple proteomic strategy to search for new markers of early CV disease or reported CV events and to evaluate their relationship with Lifetime Risk. Blood samples from 71 patients were classified into 3 groups according to their CV risk (healthy, with CV risk factors and with a previously reported CV event subjects) and they were analyzed using a high through quantitative proteomics approach. This strategy allowed three different proteomic signatures to be defined, two of which were related to CV stratification and the third one involved markers of organ damage.This work was supported by grants from the Instituto de Salud Carlos III (PI070537, IF08/3667-1, PI11-02239, PI 14/01917, PI11/01401, PI11/02432, PI13/01873, PI13/01746, PI13/01581, PI14/01650, PI14/01841), PT13/0001/0013, PIE13/00051, PIE13/00045, CP09/00229, CP15/00129, IDC Salud (3371/002), the MutuaMadrileña Foundation, the SENEFRO Foundation and FONDOS FEDER (RD06/0014/1015, RD12/0042/0071). Sociedad Española de cardiología para la Investigación Básica 2017. Grant PRB3 (IPT17/0019 - ISCIII-SGEFI / ERDF. These results are in line with the Spanish initiative on the Human Proteome Project.S

Plasma Molecular Signatures in Hypertensive Patients With Renin-Angiotensin System Suppression: New Predictors of Renal Damage and De Novo Albuminuria Indicators

Albuminuria is a risk factor strongly associated with cardiovascular disease, the first cause of death in the general population. It is well established that renin-angiotensin system suppressors prevent the development of new-onset albuminuria in naïf hypertensive patients and diminish its excretion, but we cannot forget the percentage of hypertensive patients who develop de novo albuminuria. Here, we applied multiple proteomic strategy with the purpose to elucidate specific molecular pathways involved in the pathogenesis and provide predictors and chronic organ damage indicators. Briefly, 1143 patients were followed up for a minimum period of 3 years. One hundred and twenty-nine hypertensive patients chronically renin-angiotensin system suppressed were recruited, classified in 3 different groups depending on their albuminuria levels (normoalbuminuria, de novo albuminuria, and sustained albuminuria), and investigated by multiple proteomic strategies. Our strategy allowed us to perform one of the deepest plasma proteomic analysis to date, which has shown 2 proteomic signatures: (1) with predictive value of de novo albuminuria and (2) sustained albuminuria indicator proteins. These proteins are involved in inflammation, immune as well as in the proteasome activation occurring in situations of endoplasmic reticulum stress. Furthermore, these results open the possibility of a future strategy based on anti-immune therapy to treat hypertension which could help to prevent the development of albuminuria and, hence, the progression of kidney damage.N

Comprehensive Proteomic Profiling of Pressure Ulcers in Patients with Spinal Cord Injury Identifies a Specific Protein Pattern of Pathology

Objective: Severe pressure ulcers (PUs) do not respond to conservative wound therapy and need surgical repair. To better understand the pathogenesis and to advance on new therapeutic options, we focused on the proteomic analysis of PU, which offers substantial opportunities to identify significant changes in protein abundance during the course of PU formation in an unbiased manner. Approach: To better define the protein pattern of this pathology, we performed a proteomic approach in which we compare severe PU tissue from spinal cord injury (SCI) patients with control tissue from the same patients. Results: We found 76 proteins with difference in abundance. Of these, 10 proteins were verified as proteins that define the pathology: antithrombin-III, alpha-1-antitrypsin, kininogen-1, alpha-2-macroglobulin, fibronectin, apolipoprotein A-I, collagen alpha-1 (XII) chain, haptoglobin, apolipoprotein B-100, and complement factor B. Innovation: This is the first study to analyze differential abundance protein of PU tissue from SCI patients using high-throughput protein identification and quantification by tandem mass tags followed by liquid chromatography tandem mass spectrometry. Conclusion: Differential abundance proteins are mainly involved in tissue regeneration. These proteins might be considered as future therapeutic options to enhance the physiological response and permit cellular repair of damaged tissue.This work was supported by grants from the Instituto de Salud Carlos III (PI14/01917, PI18/00995, PT13/0001/0013) for Funding: ISCIII (‘‘PI14/01917, PI18/00995, PT13/0001/0013)’’, co-funded by ERDF/ESF, ‘‘Investing in your future’’. Redes Tema ticas de Investigacion Cooperativa (FONDOS FEDER, RD12/0042/0071). Sociedad Espanola de Cardiologıa para la Investigacion Basica 2017. Grant PRB3 (IPT17/0019—ISCIII-SGEFI/ERDF). These results are aligned with the Spanish initiative on the Human Proteome Project (SpHPP).S

Contribution of proteomics to the management of vascular disorders

AbstractVascular disorders, and in particular atherothrombosis, are currently a leading cause of morbidity and mortality in Western societies. Proteomics research into these disorders has helped improving our knowledge of the underlying mechanisms involved in the development of atherothrombosis, as well as providing novel biomarkers to diagnose and for the prognosis of this disease. However, the application of these advances into clinical use has not followed this trend. In this review we explore the potential of Proteomics and Metabolomics for the management of vascular disorders, paying special attention to atherothrombosis and aiming to guide the reader from the experimental design of proteomic analysis through the initial discovery phase to the clinical implementation of biomarkers or therapeutic targets (Fig. 1), providing state-of-the-art proteomic studies to exemplify the concepts addressed

Serum adipokines as non-invasive biomarkers in Crohn’s disease

© The Author(s) 2020.Adipose tissue secretes molecules that can promote activity in Crohn’s disease. We aimed to evaluate the role of serum adipokines as possible biomarkers in Crohn’s disease. Serum samples were obtained from 40 patients with endoscopically active or quiescent Crohn’s disease and 36 healthy controls. Serum leptin, ghrelin, resistin and adiponectin levels were analysed by Multiplex in a Luminex 200 system technology. Receiver Operating Characteristic curves were performed to evaluate the adipokines discriminatory capacity. A logistic regression adjusted by possible confounders (i.e. gender, age, BMI) was performed for those adipokines that showed an area under the curve > 0.7. No differences were found in age, gender or BMI among groups. Distribution for serum resistin was different among the three groups of study, and only this adipokine showed an area under the curve of 0.75 comparing actives patients and healthy control groups. Resistin median concentration was selected as a cut-off for a logistic regression analysis; odds ratio along its 95% confidence interval adjusted by gender, age, and BMI yielded a value of 5.46 (1.34–22.14) comparing actives patients and healthy controls. High concentration of serum resistin is probably associated to activity, being this association independent of gender, age or BMI.This work was supported by Ayudas Atracción de Talento modalidad 2 2017, BMD-5800 (Universidad Autónoma de Madrid and Comunidad de Madrid), the Ramón y Cajal Research Programme from the Spanish Ministry of Science (RYC-2017-21606), and the Sara Borrell fellowship from the Instituto de Salud Carlos III (CD17/00014). This study was possible thanks to Dr. Domingo Barber, scientifc director of Instituto de Medicina Molecular Aplicada (IMMA), Facultad de Medicina, Universidad San Pablo CEU, Madrid, Spain

Profiling of Human Circulating Dendritic Cells and Monocyte Subsets Discriminates between Type and Mucosal Status in Patients with Inflammatory Bowel Disease

[Background]: Intestinal dendritic cells (DC) and macrophages drive disease progression in patients with inflammatory bowel disease (IBD). We aimed to characterize the activation and homing profile of human circulating DC and monocyte subsets in healthy control patients (CP) and IBD patients.[Methods]: Eighteen CP and 64 patients with IBD were categorized by diagnoses of Crohn disease (CD) and ulcerative colitis (UC), either endoscopically active (inflamed) or quiescent. Circulating type 1 conventional DC, type 2 conventional DC, plasmacytoid DC, classical monocytes, nonclassical monocytes, and intermediate monocytes were identified by flow cytometry in each individual and characterized for the expression of 18 markers. Association between DC/monocytes and IBD risk was tested by logistic regression. Discriminant canonical analyses were performed to classify the patients in their own endoscopy category considering all markers on each subset.[Results]: CCRL1, CCR3, and CCR5 expression on circulating type 1 DC; CCRL1 expression on nonclassical monocytes; and CCR9 and β7 expression on classical monocytes allowed us to discriminate among the different study groups. Indeed, the same markers (excluding β7) were also associated with IBD when all DC and monocyte subsets were considered at the same time.[Conclusions]: Monitoring the phenotype of human circulating DC and monocyte subsets may provide novel tools as biomarkers for disease diagnosis (CD/UC) or mucosal status (inflamed/noninflamed) in the absence of an invasive colonoscopy.This work was supported by the Spanish Ministry of Science (SAF2014-56642-JIN; RYC-2017–21606), the Instituto de Salud Carlos III (PIE13/00041), the Sara Borrell fellowship CD17/00014), the Asociación Española de Gastroenterología (Becas Nuevos Investigadores 2016), and the Community of Madrid (Consejería de Educación, Juventud y Deporte, Programa de Garantía Juvenil 2015 and 2016; Universidad Autónoma de Madrid, Ayudas Atracción de Talento modalidad 2 2017 [BMD-5800])