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Trabajo presentado en la 13ª Reunión de la Red Española de Bacterias Lácticas (RedBAL), celebrada en Madrid (España) del 17 al 18 de junio de 2019, El uso de probióticos o compuestos bacterianos se ha propuesto como un mecanismo para modular la respuesta inmune del hospedador y remodelar el microbioma humano. Sin embargo, actualmente existe una falta de información sobre las vías moleculares inducidas por las bacterias probióticas, o sobre la interacción entre los probióticos y el sistema inmunológico. Lactobacillus es un género del filo Firmicutes que incluye cepas probióticas y representativas de la microbiota gastrointestinal humana. Estudios independientes han demostrado las propiedades antiinflamatorias de diferentes cepas de Lactobacillus, aunque estamos lejos de comprender la interacción molecular subyacente. En este trabajo, demostramos que una administración diaria de 5e10 células viables de Lactobacillus acidophilus DSM 20079T (DSM20079) a lechones sanos produjo un aumento plasmático de la interleucina antinflamatoria IL10, disminuyendo, a su vez, la interleucina IL12 y la ratio proinflamatorio IL12 + TNF α / IL10. Además, las células mononucleares de sangre preriféricas (PBMCs) extraídas de estos lechones se volvieron menos reactivas contra el lipopolisacárido de Escherichia coli (LPS) y la fitohemaglutinina de Phaseolus vulgaris (PHA), cuando se ven afectadas por la cepa DSM20079. Se identificó la fracción proteica extracelular de DSM20079 como la responsable de esta modulación. El efecto tolerogénico ejercido por las proteínas extracelulares de esta cepa sugiere su uso potencial como coadyuvante para aplicaciones terapéuticas dirigidas a enfermedades inflamatorias crónicas., Este trabajo fue respaldado por el >Programa Estatal de Investigación, Desarrollo e Inovación Orientada a los Retos de la Sociedad> (AGL2016-78311- R); la Asociación Española Contra el Cáncer (>Obtención de péptidos bioactivos contra el Cáncer Colo-Rectal a partir de secuencias genéticas de microbiomas intestinales>, PS-2016).

Trabajo presentado en el 7th International Human Microbiome Congress, IHMC, celebrado en County Kerry (Irlanda) del 28 al 26 de junio de 2018, The human gastrointestinal tract (GIT) is a very complex ecosystem involving a continuous interaction between nutrients, host cells and microorganisms. Recently, we have discovered a new family of immunomodulatory peptides that are encrypted within the sequence of a precise extracellular protein, which is secreted by the lactic acid bacterium Lactobacillus plantarum (Patent WO 2013034795 A1) (1). The genetic information about commensal/probiotic bacteria and gut microbiomes that is present in public repositories is huge, with only a list of 10 million unique bacterial proteins available mainly from the METAHIT and HMP projects (http://gigadb.org/dataset/100064). Our aim is to explore these 10 million unique proteins to obtain information on encrypted peptides which might have immunomodulatory bioactivity on host cells. With this in mind we have developed the MAHMI (Mechanism of Action of the Human Microbiome) database, a unique resource that provides comprehensive information about the sequence of potential immunomodulatory and antiproliferative peptides encrypted in the proteins produced by the human gut microbiota. Fifteen peptides were chosen by its potential immunomodulatory bioactivity. Selected peptides were assayed in vitro at a final concentration of 10 µg/mL using monocyte derived cells (Mo-DCs). Our bioinformatics/in vitro methodology allowed selection of new inmunomodulatory peptides encrypted in the human intestinal bacteria metaproteome. HM14 signaled through an unknown receptor, and imprinted anti-inflammatory traits in immune cells as evidenced by RNASeq and flow cytometry. The mechanisms used by this kind of peptides may be useful for the treatment of inflammatory diseases

El campo de investigación de esta tesis es el envejecimiento del sistema inmunológico y sus efectos sobre el estado de salud y la calidad de vida de las personas de edad avanzada. Se sabe que el envejecimiento humano se caracteriza por cambios en el sistema inmune con un profundo impacto en el compartimiento de las células T, así como en la mayoría de células del sistema inmune innato e immunomediadores. Este deterioro progresivo conduce a una mayor incidencia o reactivación de las enfermedades infecciosas, así como al desarrollo de enfermedades autoinmunes y el cáncer. Esta respuesta inmune defectuosa también se manifiesta en una disminución de la capacidad de inducir memoria inmunológica a vacunas e infecciones, lo cual tiene un impacto clínico profundo. La senescencia del sistema inmune se puede producir no sólo por el envejecimiento fisiológico, sino también por diferentes enfermedades crónicas e infecciosas, como artritis reumatoide, aterosclerosis, deterioro cognitivo, infecciones por CMV o VIH... Intervenir para frenar el deterioro de la respuesta del sistema inmune podría mejorar la calidad de vida de los ancianos y de los jóvenes con patologías caracterizadas por inmunosenescencia. En este sentido, estrategias tales como el aumento de la actividad física y la ingesta de probióticos inmunomoduladores han sido evaluadas en nuestro laboratorio. De los numerosos resultados obtenidos a lo largo de estos años realizando la tesis doctoral podemos destacar: - La expresión de la molécula NKG2D en los linfocitos T CD4+ se encuentra significativamente elevada en los individuos ancianos respecto a los adultos jóvenes. Esta población celular presenta el fenotipo y las funciones típicas de las células altamente diferenciadas que han sufrido un gran número de divisiones. - La citocina IL-15 posee un efecto muy notorio sobre las células T CD4+, con gran capacidad de activar células CD28null, mucho más que sobre la población CD28+. La IL-15 aumenta la proliferación y la frecuencia de la población CD4+CD28null frente a la población CD4+CD28+. Además, la activación en las células CD4+CD28null está claramente inducida por la IL-15, como se puede comprobar por la expresión de diversos marcadores de activación y por el aumento de sus propiedades citotóxicas. También encontramos un aumento de la respuesta antígeno-específica tras el tratamiento con IL-15. - Existe una relación entre el envejecimiento, la seropositividad a CMV y los niveles de anticuerpos frente a CMV, que están claramente asociados con la frecuencia de las células T CD4+ específicas frente a CMV en ancianos. Además, tanto la seropositividad a CMV como el título de anticuerpos se relacionan con el grado de diferenciación de las células T CD4+ y con el perfil de riesgo inmunológico en ancianos. La capacidad in vivo en ancianos de responder a nuevos antígenos también se encuentra influenciada por la infección por CMV. - El envejecimiento del sistema inmune se relaciona con el declive funcional en ancianos. Existen diferencias significativas en la distribución y en el estado de diferenciación de las distintas poblaciones celulares, en la respuesta celular in vitro y a su capacidad de responder in vivo a la inmunización. Además, los ancianos con una peor capacidad funcional tienen los títulos de anticuerpos frente a CMV y la respuesta T específica frente al virus más altos que los individuos con una mejor capacidad funcional. - Los pacientes con insuficiencia cardiaca crónica (ICC) muestran un envejecimiento de su respuesta inmune adaptativa que se correlaciona con el estadio de la patología. Las concentraciones séricas de IL-6, una de las citocinas pro-inflamatorias más importantes, se relaciona con el desarrollo del proceso de inmunosenescencia en los pacientes con ICC. - Existe una asociación clara entre altos niveles de actividad física mantenidos a lo largo de toda la vida y el envejecimiento del sistema inmune, contrariamente a lo que cabría esperar. Los altos niveles de actividad física mantenidos a lo largo de muchos años se correlacionan con el estado de la respuesta inmune adaptativa. Esto puede afectar a la eficacia de la respuesta inmune en atletas, a su estado de salud y a su rendimiento deportivo, de gran importancia para estos individuos. - El consumo de Lactobacillus delbrueckii subsp. bulgaricus 8481 se relaciona con una mejora en el estado del sistema inmune en ancianos. El consumo de este probiótico podría favorecer el mantenimiento de una adecuada respuesta inmune, ralentizando el envejecimiento de las subpoblaciones de linfocitos T y aumentando el número de células inmaduras potencialmente respondedoras frente a nuevos antígenos.

This work was supported by Red de Investigación Renal (REDinREN), Spanish grant FIS PI080566 from Instituto ‘‘Carlos III’’ (European FEDER founds).

Background Exercise induces a series of changes on the immune system depending on their intensity and duration. 1 2 In fact, transient states of immunosuppression are induced after intense physical activity 3 and yet beneficial exercise anti-inflammatory effects have been described over many diseases and longevity. 1 Methods To study the impact of intense exercise for long periods of life on adaptive immunity at different ages we compared phenotypical and functional features of T lymphocytes of young (n=27) and elderly athletes (n=15) with young (n=30) and elderly non-athletes (n=30). We characterized leukocyte and lymphocyte subpopulations by flow cytometry and measured the T cell proliferation and activation response (CD69) against anti-CD3. We also studied the percentage of recent thymic emigrants (RTEs) by quantification of TREC by real-time PCR. Specific antibody titers against CMV were determined by ELISA. Results Leucopenia was found in both groups of athletes, mainly explained by low levels of neutrophils and lymphocytes. Exercise induced higher frequencies of NK, B lymphocytes and CD8+ T cells, whereas CD4+ T lymphocytes showed significant lower levels in the elderly athletes. Moreover, young athletes showed significant differences in all parameters that define the immune risk profile (IRP), with characteristics of an aging immune system, but we did not find differences between elderly groups. Less differentiated subsets of T lymphocytes were more frequents in non-athletes, with the exception of CD8+ T lymphocytes in young individuals. The analysis of TREC content in the elderly groups showed no significant difference in either the CD4+ or CD8+ T lymphocytes. In the young non-athletes group we observe an increase in the content of TREC in CD8+ T cells, but not in CD4+ T cells. Moreover, functional response of CD4+ and CD8+ T lymphocytes was significantly impaired only in young but no in elderly athletes. Conclusions Intensive training for long periods throughout life induces important phenotypical and functional changes on the adaptive immune response. These changes are most striking in young individuals and they damped with physiologic immune aging.

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jff.2020.103969., This work supports the massive presence of potential immunomodulatory peptides in the human gut metaproteome. These peptides were identified through the MAHMI database as potentially anti-inflammatory, and sixteen of them synthesized for characterize their mechanism of action. From them, peptide HM14 was encrypted in an extracellular protein produced by Bifidobacterium longum, a common member of the human microbiota, and displayed the highest anti-inflammatory capability. Molecular mechanism of action of HM14 pointed to a specific interaction between this immunomodulatory peptide and antigen presenting cells, which resulted in a higher formation of iTreg cells. Moreover, HM14 was effective in decreasing pro-inflammatory parameters in PBMCs isolated from a cohort of Crohns patients. Finally, non-targeted metabolomics confirmed the ability of HM14 to modulate the metabolic activity of PBMCs to fulfil its energy and biosynthetic requirements. Overall, our combined in silico/multiomics approach supports the human gut metaproteome as a source for immunomodulatory peptides., Our work is supported by the Spanish “Programa Estatal de Investigación. Desarrollo e Innovación Orientada a los Retos de la Sociedad” (grants AGL2013-44761-P and AGL2016-78311-R); the Asociación Española Contra el Cancer (“Obtención de péptidos bioactivos contra el Cáncer Colo-Rectal a partir de secuencias genéticas de microbiomas intestinales”, Grant PS-2016), by the Asturias Regional Plan I + D + i for research groups (FYCYT-IDI/2018/000236) and by the Autonomic “Investigadores Emerxentes do Sistema Universitario de Galicia” (Grant EM2014/046). This work was partially supported by the Consellería de Educación. Universidades e Formación Profesional (Xunta de Galicia) under the scope of the strategic funding of ED431C2018/55-GRC Competitive Reference Group. Finally, the authors wish to thank Jaume Morales and Rubén García form Agilent Technologies for technical support., info:eu-repo/semantics/publishedVersion

Lactobacillus genus includes both probiotic and representative strains of the human gut microbiota. Independent studies have reported on the anti-inflammatory properties of different Lactobacillus strains, although we are far from understanding the underlying molecular interplay. In this work we show that a daily administration of Lactobacillus acidophilus DSM20079T (DSM20079) to healthy piglets resulted in plasmatic increases of the anti-inflammatory IL10, whilst IL12 and the pro-inflammatory ratio IL12+TNF/IL10 decreased. The extracellular protein fraction of DSM20079 was identified as the responsible for the crosstalk interaction that elicited these tolerogenic effects. This strain was able to activate innate immune pathways in dendritic cells and to decrease the production of pro-inflammatory cytokines in both CD4+/CD8+ T cell subsets in healthy donors and in Crohns Disease patients. The tolerogenic effect exerted by the extracellular proteins of this strain suggests their potential use as coadjutant for therapeutic applications targeting chronic inflammatory illnesses., Our work is supported by the Spanish “Programa Estatal de Investigación, Desarrollo e Inovación Orientada a los Retos de la Sociedad” (grant AGL2016-78311-R); the Asociación Española Contra el Cancer (“Obtención de péptidos bioactivos contra el Cáncer ColoRectal a partir de secuencias genéticas de microbiomas intestinales”, Grant PS-2016) and by the Asturias Regional Plan I+D+i for research groups (FYCYT-IDI/2018/000236). This study was also supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER006684). SING group thanks CITI (Centro de Investigación, Transferencia e Innovación) from University of Vigo for hosting its IT infrastructure. LJR was supported by the Principado de Asturias, PCTI 2018–2020 (GRUPIN: IDI2018-000237) and FEDER. This work was partially supported by the Consellería de Educación, Universidades e Formación Profesional (Xunta de Galicia) under the scope of the strategic funding of ED431C2018/55-GRC Competitive Reference Group., info:eu-repo/semantics/publishedVersion

There are strong evidences that probiotics influence the immune status of the host, in a strain-specific manner, acting in the gastrointestinal tract. On the hypothesis that certain extracellular proteins and peptides from gut bacteria may mediate part of this immunomodulation and assuming they are able to diffuse through the mucus layer and interact with immune cells we have developed this work. Our study attempts to understand the immunomodulatory mechanisms of (i) Pext, the extracellular protein fraction of Lactobacillus acidophilus DSM20079T, (ii) HM14, a peptide encrypted in an extracellular glycoside hydrolase from Bifidobacterium longum NCIMB 8809 and (iii) Escherichia coli O111:B4 lipopolysaccharide (LPS), a well-known pro-inflammatory molecule, over human peripheral blood mononuclear cells (PBMCs). An untargeted LC-ESI-QTOF-MS metabolomics approach was applied to reveal intracellular changes in treated-PBMCs isolated from healthy donors. Differences in NADH arrest, NAD+ concentration reduction, as well as increases in palmitic acid and methanephrin were observed in HM14 and Pext treated-cells compared to those stimulated with LPS. This would support an anti-inflammatory molecular mechanism of action of such proteinaceous molecules. Moreover, this methodology has confirms the importance of metabolomics approaches to better understanding immune cell responses to gut bacterial-derived molecules., This work was funded by the Autonomic Investigadores Emerxentes do Sistema Universitario de Galicia (Grant EM2014/046) and the Spanish Programa Estatal de Investigación, Desarrollo e Innovación Orientada a los Retos de la Sociedad (Grant AGL2013-44039R). This work has received also financial support from the Xunta de Galicia (Plan de mellora do Centro de Investigacións Agroalimentarias CIA3 do Campus de Ourense).

Probiotics are used to improve human health due to their capability to induce beneficial effects in gastrointestinal diseases and modulate the immune response. However, scarce information is known about the bacterial active compounds and the metabolic pathways of interaction with immune cells. In the present study, we analyzed the immune response elicited by surface-associated proteins, the whole surface extract (WSE) and the genomic DNA of three strains from the main representative probiotic species for human consumption, L. acidophilus DSM20079T, L. rhamnosus GG, B. longum NCIMB 8809, using an in vitro model of human PBMCs from healthy donors. Eighteen cytokines were quantified using Luminex Technology to understand the immune pathways modulate by bacterial extracts. Overall, the bacterial fractions elicited a different immune response in PBMCs, whereas the isolated DNA was able to induce a higher Th17-like response that could lead to a protective effect in the epithelial barrier function., This work was funded by the Spanish “Programa Estatal de Investigación, Desarrollo e Innovación Orientada a los Retos de la Sociedad” (Grant AGL2013-44039R) and the “Fundación Científica Asociación Española contra el Cáncer” (Grant PS-2016).

Trabajo presentado en el IX Workshop de la Sociedad Española de Probióticos y Prebióticos (SEPyP), celebrado en Zaragoza (España) el 15 y 16 de febrero de 2018, [Introduction] Bifidobacteria and lactobacilli include both probiotic strains and representative microorganisms of the human gut microbiota, and their presence has been associated to a proper immune development and maturation. In addition, several independent works report the anti-inflammatory properties of several strains of those groups. However, we are far from understanding the molecular interplay behind/supporting these beneficial interactions. [Objective] To study the effect of different fractions of lactobacilli and bifidobacteria in human peripheral blood mononuclear cells (PBMCs) isolated from healthy donors and patients with Inflammatory Bowel Disease (IBD) at the time of diagnosis. [Methods] The fractions were obtained from strains of Bifidobacterium longum NCIMB 8809, Lactobacillus rhamnosus GG and Lactobacillus acidophilus DSM 20079: DNA, surface-associated proteins, whole cell soluble extract and extracellular proteins. Different concentrations of the fractions were incubated with PBMCs, and key T-cell cytokines, T regulatory cell differentiation and RNASeq of CD4+ cells were performed. [Results] Among the extracts, extracellular proteins from L. acidophilus DSM 20079 increased IL-2 production by PBMCs isolated from healthy donors, and were able to increment the proportion of functional T regulatory cells. Data on the signaling mechanisms involved were obtained by RNASeq of CD4+ Th cell subpopulations. Finally, this fraction decreased drastically the production of the pro-inflammatory mediator TNF-alpha and induced increments of IL-12 in PBMCs isolated from IBD patients at the diagnosis. [Conclusions] Extracellular proteins from strain L. acidophilus DSM 20079 had a positive impact on the immunological status of both healthy donors and IBD patients. This fraction will be on the basis of new clinical studies focused in the immunomodulation of the human host targeting pro-inflammatory effectors and the T-cell regulatory response. This fraction may represent a new therapeutic approach for chronic inflammatory illnesses. [Competing interests] Borja Sánchez and Claudio Hidalgo are members of the scientific committee of Microviable Therapeutics SL.