Defensa de Tesis Doctoral:

Papel de los receptores TLR4 y de la microbiota intestinal en los procesos inflamatorios y neuroinflamatorios causados por el consumo crónico de alcohol, y posibles terapias con curcuminoides.

Doctorando: Carlos M. Cuesta Díaz

Directoras: Consuelo Guerri, Maria Pascual y Juan Ureña

Abstract:

El alcohol es una de las drogas más aceptadas y consumidas a nivel mundial, y su ingesta crónica causa alteraciones en el sistema nervioso central, desde desmielinización hasta muerte neural. Se ha demostrado que gran parte de estos efectos estarían mediados por la activación del sistema inmune innato, principalmente a través del receptor TLR4, que desemboca en un estado inflamatorio. Para ahondar en estos mecanismos, en esta tesis se pretende analizar si el consumo crónico de etanol puede causar modificaciones en los miARNs de la corteza cerebral, regulando la expresión de genes proinflamatorios asociados a la ruta de señalización del TLR4 y si el uso de la curcumina como terapia es capaz de disminuir estos efectos. Además, analizaremos si el consumo de alcohol a largo plazo es capaz de alterar la microbiota intestinal, y la implicación del TLR4 en estos procesos. Nuestros resultados demuestran que el etanol es capaz de alterar la expresión de diferentes microARNs que regulan la expresión de genes proinflamatorios y asociados a la vía del TLR4. La ausencia del receptor causa un perfil de expresión distinto tanto en los controles como en los animales alcohólicos crónicos.

Puesto que en los últimos años nos hemos interesado en la búsqueda de una posible terapia para paliar la neuroinflamación producida por el etanol, hemos usado el BDMC, un curcuminoide con capacidad antiinflamatoria y antioxidante, para revertir los daños causados por el abuso del alcohol. Para su administración proponemos su conjugación con un polipéptido que elimina los tradicionales problemas asociados al uso de estos compuestos naturales como son su vida media corta y su baja biodisponibilidad, sin que hayamos registrado toxicidad. El uso de este polímero en animales con un consumo crónico de etanol produce una disminución de la inflamación, tanto a nivel proteico como de expresión génica.

Considerando que otra de las regiones del organismo más afectadas por el alcohol es el tracto gastrointestinal, y que la población bacteriana puede ser modificada en determinadas enfermedades o por el uso de sustancias nocivas, produciendo una disbiosis, hemos comparado el efecto del alcohol sobre la población bacteriana y la participación del receptor TLR4 en estos efectos. Mediante análisis de expresión génica, confirmamos que el tejido intestinal ausente del receptor TLR4 es menos susceptible de sufrir cambios debidos al consumo de alcohol. Sin embargo, no hemos encontrado en estos animales la expresión aumentada de genes proinflamatorios característica de los tratamientos alcohólicos. Además, los ratones carentes de este receptor presentan una microbiota intestinal única y diferente a la encontrada en animales de genotipo normal, con sus propios cambios en el equilibrio bacteriano en respuesta a la ingesta de alcohol. Esta microbiota presenta menor cantidad de especies Gram – y mayor resistencia al desequilibrio ocasionado por el alcohol, que su contraparte en los ratones WT.

En general, los resultados científicos que aparecen en esta tesis resaltan el papel del TLR4 en las alteraciones causadas por el consumo crónico de alcohol. Mostramos como cambios causados por el etanol a niveles tan distintos como la regulación mediante miARNs o la microbiota intestinal dependen en gran medida de la respuesta inicial que causa el alcohol en el TLR4. Dado que es un receptor necesario para el correcto funcionamiento de muchos órganos, planteamos un posible tratamiento mediante la inhibición de esta cascada inflamatoria.

Defensa de Tesis Doctoral:

Multi-omic data integration study of immune system alterations in the development of minimal hepatic encephalopathy in patients with liver cirrhosis

Doctorando:

Teresa Rubio Martínez-Abarca

Directores:

Resumen:

A high proportion of patients with liver cirrhosis develop minimal hepatic encephalopathy (MHE), a neuropsychiatric syndrome that produces attention deficits, cognitive impairment, and motor incoordination. MHE reduces both the quality of life and the life span of patients and results in high economic costs for healthcare systems given that it has now become a major socioeconomic problem. Although the etiology of MHE is not completely understood, it has been demonstrated that the appearance of mild cognitive and coordination impairments in patients with MHE is due to the synergistic action of both hyperammonemia and peripheral inflammation arising from liver failure. The main objective of this work was to understand the immunological alterations associated with the peripheral inflammation that trigger MHE in patients with cirrhosis. These changes can be monitored through the signaling cascades of different immune system cell types. In this work, in a preliminary study, changes in gene expression (transcriptomics), plasma metabolites (metabolomics), and a panel of extracellular cytokines were analyzed in blood samples from patients with cirrhosis with and without MHE.

Transcriptomics analysis supported the hypothesis that alternations in the Th1/Th2 and Th17 lymphocyte cell populations are the major drivers of MHE. Cluster analysis of serum molecules highlighted 6 groups of chemically similar compounds, suggesting that functional modules operate during the induction of MHE. We also developed a multi-omic integration analysis pipeline to detect covariation between intra- and extracellular components that could contribute to the induction of cognitive impairment. Results of this integrative analysis suggested a relationship between cytokines CCL20, CX3CL1, CXCL13, IL-15, IL-22, and IL-6 and altered chemotaxis, as well as a link between long-chain unsaturated phospholipids and increased fatty acid transport and prostaglandin production.

A shift in peripheral inflammation in patients with MHE, mainly orchestrated by CD4⁺ T cells, had been proposed in previous studies as a critical factor that triggers cognitive impairment. Thus, the second part of this thesis focused on understanding the pathways and mechanisms by which alterations in CD4⁺ lymphocytes may contribute to peripheral inflammation in MHE. Thus, the expression levels of genes, transcription factors, and miRNAs were analyzed in this lymphocyte subtype by high throughput sequencing (RNA-seq and miRNA-seq). Separate analysis of each dataset showed mRNA and miRNA expression differences and altered biological pathways in CD4⁺ lymphocytes when compared to patients with cirrhosis with and without MHE. We found alterations in 167 mRNAs and 20 pathways in patients with MHE, including toll-like receptors, IL-17 signaling, histidine, and tryptophan metabolism pathways. In addition, 13 miRNAs and 7 transcription factors presented alterations in patients with MHE. We used public databases to determine the target genes of these regulatory molecules and found key genes involved in the immunological shift triggering MHE. For instance, increased miR-494-39, miR-656-3p, and miR-130b-3p expression may modulate TNFAIP3 (A20) and ZFP36 (TTP) to increase levels of pro-inflammatory cytokines such as IL-17 and TNFα.

The last part of this thesis comprised a case study of the T-cell receptor (TCR) repertoire profiles of control patients and patients with cirrhosis with and without MHE obtained from the bulk RNA-seq dataset previously generated from isolated CD4⁺ T cells. Given that RNA-seq experiments contain the TCR genes in a fraction of the data, an opportunity for receptor repertoire analysis without the need to generate additional data was created, thereby reducing the sample number required and the associated economic cost. After read alignment to the VDJ genes was performed with the MiXCR tool, we successfully recovered 498–1,114 distinct TCR beta chains per patient. In addition, profiling of the CD4⁺ TCR repertoire could be used to help us understand the immune status of patients with MHE. Results showed fewer public clones (clonal convergence), higher diversity (clonal expansion), and elevated sequence architecture similarity within repertoires, independently of the immune status of the 3 groups of patients. Additionally, we detected significant overrepresentation of celiac disease and inflammatory bowel disease related TCRs in MHE patient repertoires. To the best of our knowledge, this is one of the few studies to have shown a step-by-step pipeline for the analysis of immune repertoires using whole transcriptome RNA-seq reads as source data.

In conclusion, our work identified the molecular mechanisms of the changes in the immune system associated with the onset of MHE in patients with cirrhosis. Nonetheless, future work with a large sample cohort will be required to validate these results in terms of biomarker determination and the development of new, more effective treatments for MHE.