Immunomodulatory effect of MSC on B cells is independent of secreted extracellular vesicles

Altres ajuts: this work was supported in part by Fundació La Marató de TV3 (201516-10, 201502-30). MM-T is sponsored by the PERIS (SLT002/16/00234) from the Generalitat de Catalunya; FB is a researcher from Fundació Institut de Recerca en Ciències de la Salut Germans Trias i Pujol, supported by the Health Department of the Catalan Government (Direcció General de Recerca i Innovació, Department Salut, Generalitat de Catalunya) and MF is funded by the Catalan Health Department (Generalitat de Catalunya) contract PERIS (SLT002/16/00069).Mesenchymal stem or stromal cells (MSC) have proven immunomodulatory properties toward B cell activation and induce regulatory B cells (Breg), through a dual mechanism of action that relies both on cell contact and secreted factors. One of them are MSC-derived extracellular vesicles (EVs), membrane nanovesicles that mediate cell communication and typically reflect the phenotype of the cell of origin. MSC-EVs could resemble MSC functions, and are being contemplated as an improved alternative to the MSC-based immunomodulatory therapy. In the present work, we focused on the factors secreted by MSC and aimed to elucidate the putative role of MSC-EVs in the immunomodulation of B cells. EVs and soluble protein-enriched fractions (PF) were isolated from MSC-conditioned medium (CM) using size-exclusion chromatography (SEC) and their capacity to modulate B cell activation, induction of Breg and B cell proliferation was compared to that of the whole MSCs. Co-culture with MSC or unfractionated CM induced naïve and CD24hiCD38hi, IL-10 producing (Breg) phenotypes on B cells while not affecting proliferation. MSC-PF had a comparable effect to MSCs, inducing a naïve phenotype, and even though they did not induce the shift toward a CD24hiCD38hi population, MSC-PF fostered IL-10 production by B cells. Conversely, MSC-EVs failed to promote naïve B cells and to reduce memory B cells. MSC-EVs induced CD24hiCD38hi B cells to a similar extent of that of MSC, but not bona fide Bregs since they did not produce IL-10. Our results show that B cell modulation by MSC is partially mediated by soluble factors other than EVs

Influence of intestinal microbiota on the response to an experimental infection with Salmonella enterica LT2 in rats

PósterVerrucomicrobiaThe intestinal microbiota is a highly complex community of microorganisms that have a symbiotic relation with the host. The intestinal immune system has evolved together with it to protect the host while permitting the presence and benefits of the resident bacteria. The immune response can differ from individual to individual, and even microbiotaitself is conditionedby the intestinal environment. Therefore, microbiota should be taken into account in studies with animals since it is a notable variable

Size-Exclusion Chromatography-based isolation minimally alters Extracellular Vesicles' characteristics compared to precipitating agents

Extracellular vesicles (EVs) have become an attractive field among the scientific community. Yet, a major challenge is to define a consensus method for EVs isolation. Ultracentrifugation has been the most widely used methodology but rapid methods, including Size Exclusion Chromatography (SEC) and/or precipitating agents such as Polyethylene glycol (PEG) or PRotein Organic Solvent PRecipitation (PROSPR) have emerged. To evaluate the impact of these different methods on the resulting EV preparations, plasma EVs were isolated using SEC, PEG and PROSPR, and their total protein content, NTA and Cryo-electron microscopy profiles, and EV-markers were compared. Also, their effect on recipient cells was tested. Low protein content and Cryo-EM analysis showed that SEC removed most of the overabundant soluble plasma proteins, which were not removed using PEG and partially by PROSPR. Moreover, only SEC allowed the detection of the EV-markers CD9, CD63 and CD81, LGALS3BP and CD5L, suggesting a putative interference of the precipitating agents in the structure/composition of the EVs. Furthermore, PEG and PROSPR-based EV isolation resulted in reduced cell viability in vitro. These results stress that appropriate EV-isolation method should be considered depending on the forthcoming application of the purified EVs

Proteomic Characterization of Urinary Extracellular Vesicles from Kidney-Transplanted Patients Treated with Calcineurin Inhibitors

Use of immunosuppressive drugs is still unavoidable in kidney-transplanted patients. Since their discovery, calcineurin inhibitors (CNI) have been considered the first-line immunosuppressive agents, in spite of their known nephrotoxicity. Chronic CNI toxicity (CNIT) may lead to kidney fibrosis, a threatening scenario for graft survival. However, there is still controversy regarding CNIT diagnosis, monitoring and therapeutic management, and their specific effects at the molecular level are not fully known. Aiming to better characterize CNIT patients, in the present study, we collected urine from kidney-transplanted patients treated with CNI who (i) had a normal kidney function, (ii) suffered CNIT, or (iii) presented interstitial fibrosis and tubular atrophy (IFTA). Urinary extracellular vesicles (uEV) were enriched and the proteome was analyzed to get insight into changes happening during CNI. Members of the uroplakin and plakin families were significantly upregulated in the CNIT group, suggesting an important role in CNIT processes. Although biomarkers cannot be asserted from this single pilot study, our results evidence the potential of uEV as a source of non-invasive protein biomarkers for a better detection and monitoring of this renal alteration in kidney-transplanted patients

Mesenchymal stromal cells induced regulatory B cells are enriched in extracellular matrix genes and IL-10 independent modulators

Regulatory B cells (Breg) are essential players in tolerance and immune homeostasis. However, lack of specific Breg markers limit their potential in clinical settings. Mesenchymal stromal cells (MSC) modulate B cell responses and are described to induce Breg in vitro. The aim of this work was to characterize MSC induced Breg (iBreg) and identify specific Breg biomarkers by RNAseq. After 7-day coculture with adipose tissue-derived MSC, B cells were enriched in transitional B cell populations, with increased expression and secretion of IL-10 and no TNFα. In addition, iBreg showed potential to modulate T cell proliferation at 2 to 1 cell ratios and their phenotype remained stable for 72h. RNAseq analysis of sorted IL-10 positive and negative iBreg populations identified over 1500 differentially expressed genes (DEG) among both populations. Analysis of biological processes of DEG highlighted an enrichment of immune regulation and extracellular matrix genes in IL-10 - iBreg populations, while IL-10 + iBreg DEG were mostly associated with cell activation. This was supported by T cells modulation assays performed in the presence of anti-IL-10 neutralizing antibodies showing the non-essential role of IL-10 in the immunomodulatory capacity of iBregs on T cells. However, based on RNAseq results we explored the role of TGF-β and found out that it plays a major role on iBreg induction and iBreg immunomodulatory properties. Therefore, we report that MSC induce B cell populations characterized by the generation of extracellular matrix and immune modulation independently of IL-10

Inflammatory conditions dictate the effect of mesenchymal stem or stromal cells on B cell function

The immunomodulatory capacity of mesenchymal stem or stromal cells (MSC) makes them a promising tool for treatment of immune disease and organ transplantation. The effects of MSC on B cells are characterized by an abrogation of plasmablast formation and induction of regulatory B cells (Bregs). It is, however, unknown how MSC interact with B cells under inflammatory conditions. In this study, adipose tissue-derived MSC were pretreated with 50 ng/ml IFN-γ for 96 h (MSC-IFN-γ) to simulate inflammatory conditions. Mature B cells were obtained from spleens by CD43- selection. B cells were co-cultured with MSC and stimulated with anti-IgM, anti-CD40, and IL-2; and after 7 days, B cell proliferation, phenotype, Immunoglobulin-G (IgG), and IL-10 production were analyzed. MSC did not inhibit B cell proliferation but increased the percentage of CD38high CD24high B cells (Bregs) and IL-10 production, while MSC-IFN-γ significantly reduced B cell proliferation and inhibited IgG production by B cells in a more potent fashion but did not induce Bregs or IL-10 production. Both MSC and MSC-IFN-γ required proximity to target cells and being metabolically active to exert their effects. Indoleamine 2,3 dioxygenase expression was highly induced in MSC-IFN-γ and was responsible of the anti-proliferative and Breg reduction since addition of tryptophan (TRP) restored MSC properties. Immunological conditions dictate the effect of MSC on B cell function. Under immunological quiescent conditions, MSC stimulate Breg induction; whereas, under inflammatory conditions, MSC inhibit B cell proliferation and maturation through depletion of TRP. This knowledge is useful for customizing MSC therapy for specific purposes by appropriate pretreatment of MSC

Inflammatory conditions dictate the effect of Mesenchymal stem or Stromal cells on B cell function

The immunomodulatory capacity of mesenchymal stem or stromal cells (MSC) makes them a promising tool for treatment of immune disease and organ transplantation. The effects of MSC on B cells are characterized by an abrogation of plasmablast formation and induction of regulatory B cells (Bregs). It is, however, unknown how MSC interact with B cells under inflammatory conditions. In this study, adipose tissue-derived MSC were pretreated with 50 ng/ml IFN-γ for 96 h (MSC-IFN-γ) to simulate inflammatory conditions. Mature B cells were obtained from spleens by CD43− selection. B cells were co-cultured with MSC and stimulated with anti-IgM, anti-CD40, and IL-2; and after 7 days, B cell proliferation, phenotype, Immunoglobulin-G (IgG), and IL-10 production were analyzed. MSC did not inhibit B cell proliferation but increased the percentage of CD38high CD24high B cells (Bregs) and IL-10 production, while MSC-IFN-γ significantly reduced B cell proliferation and inhibited IgG production by B cells in a more potent fashion but did not induce Bregs or IL-10 production. Both MSC and MSC-IFN-γ required proximity to target cells and being metabolically active to exert their effects. Indoleamine 2,3 dioxygenase expression was highly induced in MSC-IFN-γ and was responsible of the anti-proliferative and Breg reduction since addition of tryptophan (TRP) restored MSC properties. Immunological conditions dictate the effect of MSC on B cell function. Under immunological quiescent conditions, MSC stimulate Breg induction; whereas, under inflammatory conditions, MSC inhibit B cell proliferation and maturation through depletion of TRP. This knowledge is useful for customizing MSC therapy for specific purposes by appropriate pretreatment of MSC

Study of urine extracellular vesicles-derived protein biomarkers for the non-invasive monitoring of kidney-transplanted patients

El rebuig crònic en el trasplantament renal és la causa principal de fallada de l'empelt. Donat que la biòpsia renal (l'actual mètode diagnòstic considerat com a el 'gold standard') és invasiva, costosa, implica una alta subjectivitat i no és representativa de tot el ronyó, hi ha una necessitat clara de desenvolupar testos diagnòstics no invasius i rendibles. Per cobrir aquesta necessitat, en aquesta tesis ens vam proposar com a objectiu trobar biomarcadors proteics que es puguin utilitzar com a un mètode diagnòstic no invasiu d'alteracions renals en pacients trasplantats de ronyó. L'orina s'ha investigat extensament buscant biomarcadors de diverses alteracions renals en el context del trasplantament perquè proporciona una visió holística de tot el sistema urinari. A més, es pot mostrejar de manera no invasiva i repetidament. No obstant, els complexos constituents de l'orina poden dificultar la detecció de proteïnes menys abundants mitjançant tècniques de proteòmica. En aquest sentit, l'anàlisi de vesícules extracel·lulars (VE) presents en l'orina profereix una solució atractiva. Degut al fet que la composició de VE varia segons l'estat fisiològic i l'origen de la cèl·lula productora, les VE urinàries (uVE) són una font ideal de biomarcadors urinaris. La introducció de la tesis conté una breu història del ronyó i del trasplantament de ronyó que, malgrat que poden ser interesants pel lector, no és necessari per poder comprendre els resultats. La introducció 'formal' se centra en l'epidemiologia del trasplantament renal, els tipus de rebuig, els mètodes diagnòstics actuals i defineix els problemes a solucionar. Els resultats i discussió del projecte s'han dividit en tres parts basats en l'estructura d'articles publicats, amb algunes modificacions per acomodar-ho a l'estructura d'una tesi doctoral. L'estudi exposat en aquesta tesi segueix un sistema comú en la recerca de biomarcadors proteics, que consisteix en una fase de descobriment, una fase de verificació i una fase de validació. En cada fase, vam analitzar el proteoma de les uVE de pacients trasplantats de ronyó utilitzant espectrometria de masses (EM) o tècniques basades en anticossos. Els pacients van ser classificats en quatre grups segons l'alteració renal que patien, diagnosticada a partir de biòpsia renal i paràmetres clínics (sigles segons terminologia anglesa): funció renal normal (NKF), fibrosi intersticial i atròfia tubular (IFTA), toxicitat per inhibidors de la calcineurina (CNIT) i rebuig cel·lular agut (ACR). La primera part es focalitza en la CNIT, els mecanismes de la qual estan poc caracteritzats, així com també el seu diagnosis i tractament. Els resultats van mostrar que hi havia una major regulació de proteïnes relacionades amb processos epitelials, de uroplaquines i plaquines en les uVE de CNIT, el que suggereix la participació d'aquestes proteïnes en la CNIT específicament però no en el desenvolupament de lesions fibròtiques. A la segona part es descriuen altres proteïnes diferencialment expressades en mostres patològiques en comparació amb el grup NKF trobades en la fase de descobriment. Vam procedir amb una segona fase (fase de verificació) mitjançant proteòmica dirigida en una cohort de pacients més gran. Entre altres troballes, la vitronectina es va trobar més expressada en pacients amb un alt grau de fibrosi renal (definida segons paràmetres histològics de Banff), assenyalant el potencial d'aquesta proteïna com a biomarcadors de fibrosi renal. Finalment, a la tercera part, vam dur a terme un estudi pilot per demostrar la detecció de vitronectina segons el grau de fibrosi renal utilitzant un assaig d'ELISA. Amb aquest mètode, molt més rendible que l'EM, aconseguim apropar les troballes del projecte a la pràctica clínica.El rechazo crónico en el trasplante renal es la causa principal de fallo del injerto. Dado que la biopsia renal (el actual método diagnóstico considerado como el ""gold standard"") es invasiva, costosa, implica una alta subjetividad y no es representativa de todo el riñón, hay una necesidad clara de desarrollar macetas diagnósticos no invasivos y rentables. Para cubrir esta necesidad, en esta tesis nos propusimos como objetivo encontrar biomarcadores proteicos que se puedan utilizar como un método diagnóstico no invasivo de alteraciones renales en pacientes trasplantados de riñón. La orina se ha investigado extensamente buscando biomarcadores de diversas alteraciones renales en el contexto del trasplante porque proporciona una visión holística de todo el sistema urinario. Además, se puede muestrear de manera no invasiva y repetidamente. Sin embargo, los complejos constituyentes de la orina pueden dificultar la detección de proteínas menos abundantes mediante técnicas de proteómica. En este sentido, el análisis de vesículas extracelulares (VE) presentes en la orina profiere una solución atractiva. Debido a que la composición de VE varía según el estado fisiológico y el origen de la célula productora, las VE urinarias (UVE) son una fuente ideal de biomarcadores urinarios. La introducción de la tesis contiene una breve historia del riñón y del trasplante de riñón que, a pesar de que pueden ser interesantes para el lector, no es necesario para poder comprender los resultados. La introducción ""formal"" se centra en la epidemiología del trasplante renal, los tipos de rechazo, los métodos diagnósticos actuales y define los problemas a solucionar. Los resultados y discusión del proyecto se han dividido en tres partes basados ​​en la estructura de artículos publicados, con algunas modificaciones para acomodarlo a la estructura de una tesis doctoral. El estudio expuesto en esta tesis sigue un sistema común en la búsqueda de biomarcadores proteicos, que consiste en una fase de descubrimiento, una fase de verificación y una fase de validación. En cada fase, analizamos el proteoma de las UVE de pacientes trasplantados de riñón utilizando espectrometría de masas (EM) o técnicas basadas en anticuerpos. Los pacientes fueron clasificados en cuatro grupos según la alteración renal que sufrían, diagnosticada a partir de biopsia renal y parámetros clínicos (siglas según terminología inglesa): función renal normal (NKF), fibrosis intersticial y atrofia tubular (IFTA), toxicidad por inhibidores de la calcineurina (CNIT) y rechazo celular agudo (ACR). La primera parte se focaliza en la CNIT, los mecanismos de la que están poco caracterizados, así como también su diagnosis y tratamiento. Los resultados mostraron que había una mayor regulación de proteínas relacionadas con procesos epiteliales, de uroplaquines y plaquines en las UVE de CNIT, lo que sugiere la participación de estas proteínas en la CNIT específicamente pero no en el desarrollo de lesiones fibróticas. En la segunda parte se describen otras proteínas diferencialmente expresadas en muestras patológicas en comparación con el grupo NKF encontradas en la fase de descubrimiento. Procedimos con una segunda fase (fase de verificación) mediante proteómica dirigida en una cohorte de pacientes mayor. Entre otros hallazgos, la vitronectina se encontró más expresada en pacientes con un alto grado de fibrosis renal (definida según parámetros histológicos de Banff), señalando el potencial de esta proteína como biomarcadores de fibrosis renal. Finalmente, en la tercera parte, llevamos a cabo un estudio piloto para demostrar la detección de vitronectina según el grado de fibrosis renal utilizando un ensayo de ELISA. Con este método, mucho más rentable que la EM, conseguimos acercar los hallazgos del proyecto a la práctica clínica.Chronic rejection in kidney transplantation is the main cause of graft failure. Since renal biopsy -the current gold standard diagnostic method- is invasive, costly, entails a high subjectivity factor and is not representative of the whole kidney, there is a clear need for more precise, non-invasive and cost-effective diagnosis tests. To meet this need, the objective of this thesis was to find protein biomarkers that could be utilized as a non-invasive diagnosis method of renal alterations in kidney-transplanted patients. Urine has been extensively investigated to find biomarkers of different renal alterations in kidney transplantation because it provides a holistic view of the whole urinary system, and it can be collected non-invasively, and repeatedly. However, the complex constituents of urine can hamper the detection of lower abundant proteins by proteomic technologies. In this sense, the analysis of extracellular vesicles (EV) found in urine provides an attractive solution. Since the composition of EV varies upon the origin and physiological state of the producing cell, urinary EV (uEV) are an ideal source of biomarkers for renal alterations. Moreover, high abundance contaminant proteins are usually removed during the isolation process, while the protein cargo of uEV remains protected. The introduction of the thesis contains a brief history of the kidney and kidney transplantation, which although interesting for the reader, it is not strictly relevant for the comprehension of the results. The 'formal' introduction focuses on the epidemiology of kidney transplantation, types of graft rejection, current diagnosis methods and defines the problem to solve. The results and discussion of the project were divided into three parts based on the structure of published articles, with some modifications to accommodate to the structure of a doctoral thesis. The study exposed in this thesis follows a common pipeline for protein biomarker search, consisting of a discovery phase, a verification phase and a validation phase. In each phase, we analysed the uEV proteome from kidney-transplanted patients using mass-spectrometry (MS) or antibody-based techniques. Patients were classified into four groups according to their renal alteration diagnosed by biopsy and clinical parameters: normal kidney function (NKF), interstitial fibrosis and tubular atrophy (IFTA), calcineurin inhibitors toxicity (CNIT) and acute cellular rejection (ACR). The first part is focused on CNIT, a kidney alteration of poorly characterized mechanisms, diagnosis and treatment. Although in kidney biopsy CNIT often shows IFTA lesions, the treatment is consistently different. We scanned the CNIT uEV proteome by MS searching for differences with IFTA group that could reveal potential biomarkers of CNI nephrotoxicity. The results showed upregulation of terms related to epithelial processes and overexpression of uroplakins and plakins in the CNIT group uEV, suggesting the participation of these proteins specifically in CNIT, but not in the development of fibrotic lesions. In the second part, we expose other proteins differentially expressed in pathological samples compared to NKF patients found in the discovery phase. Then, a second phase (verification phase) was set up using targeted MS in a bigger cohort of patients to confirm the results of the candidate proteins selected from the first phase. Among other findings, vitronectin was found to be more expressed in patients with high degree of kidney fibrosis (defined by Banff histological criteria), pointing out the potential of this protein as a biomarker for renal fibrosis. Finally, in the third part, we carried out a pilot study to demonstrate the detection of urinary vitronectin using ELISA. With this method, much more cost-efficient than MS, we can make the findings in this project more translationable to the clinical practice