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Influence of protein prenylation and its modulation on the immune system

Final Report Summary - PREN-IBD (Influence of protein prenylation and its modulation on the immune system)

Focusing on new research avenues in the field of gut homeostasis and Inflammatory Bowel Disease (IBD), prenylation appears to be an attractive target. Post-translational attachment of isoprenoids, namely prenylation, is required for optimal function of a wide spectrum of proteins, such as small GTPases. Upon this lipidation, prenylated proteins modify significantly their physicochemical properties and therefore their subcellular location and overall function. Recent observations support the link between prenylation and regulation of the mucosal immune system in inflammation and cancer. In line with this hypothesis, we aimed to characterize the impact of abrogation of geranylgeranylation for maintenance of gut homeostasis, and development of inflammation and colorectal cancer. Intestinal mucosa is a complex system in which innate and immune cells work together in a tightly regulated manner in order to avoid the activation of local immune responses, representing the declutching event for chronic inflammatory disorders. Therefore, we have focused on the role of geranylgeranylation within two cell types representing innate and immune players within intestine, such as T cells and in intestinal epithelial cells (IECs). In both cases, we have demonstrated the indispensable role of geranylgeranylation for maintenance of intestinal homeostasis.
In order to characterize the impact of prenylation and geranylgeranylation within intestinal mucosa for gut homeostasis, we have proposed four research objectives. First, we intend to generate engineered modified mice which serve us as main scientific tool for our studies: conditional knock-out mice of GGTase-Iβ in T cells and IECs. Second, we intended to characterize the physiological consequence of abrogation of geranylgeranylation in T cells and IECs by means of the use of generated mice. In a pathological approach, we aimed to assess the impact of GGTase-Iβ deficient T cells and IECs for the development of relevant intestinal disorders, such as inflammatory Bowel Disease and Colorectal Cancer, by performing experimental colitis and tumor models in mentioned mouse strains. Finally, in a translational approach we intended to describe GGTase-Iβ expression profile and its potential regulation due to inflammation or tumourigenesis, both in murine as well as in human gut samples.

In order to generate genetically modified mice with cell-specific blockade of geranylgeranylation, the Lox/Cre recombination system has been used. Mice carrying a LoxP-flanked GGTase-Iβ gene (exon 7) were kindly provided by Prof. Martin O. Bergo (University of Gothenburg, Sweden); they were crossbred with mice with dependency of Cre-recombinase expression on one specific cell type protein promoter, such as Villin for IECs or CD4 for lymphocytes. A conditional system was also generated with CreERT2 mice (Tamoxifen administration is required for ERT2-dependent Cre-recombinase expression), in both cases. Thus, generated Pggt-IβΔIEC and Pggt-IβTcell have represented our main experimental tool in order to assess the role of prenylation within intestinal mucosa in the murine system. But we have even generated additional mouse strains, such as Pggt-IβΔIEC-Casp8ΔIEC, Pggt-IβΔIEC-RIP3-/- or Pggt-IβΔIEC-ATG7ΔIEC. We have performed a deep description and mechanistic study for characterizing the intestinal phenotype of these mouse strains. In the case of Pggt-IβΔIEC mice, we have focused on intestinal integrity and permeability, and its alteration due to cytoskeleton rearrangement. On the other hand, T cells trafficking and gut homing of GGTase-Iβ deficient T cells was the key event studied in the case of Pggt-IβTcell mice. In the context of colitis, Pggt-IβTcell mice were submitted to several experimental models, such as TNBS, acute DSS, chronic DSS and adoptive lymphocyte transfer colitis. Since deletion of GGTase-Iβ in IECs causes the death of the animal, heterozygous Pggt-Iβflx-wt-VillinCre mice were investigated under experimental colitis models, mainly acute and chronic DSS. Regarding colorectal cancer, we have induced colitis-associated colorectal cancer by means of the use of the AOM-DSS model, both in Pggt-IβTcell and Pggt-Iβflx-wt-VillinCre mice. Lastly, we have analyzed GGTase-Iβ expression in murine and human gut in both cell compartments: IECs and lamina propria cells. Furthermore, we have compared the expression and downstream signaling of GGTase-Iβ in inflamed/uninflamed and tumor/normal tissues from murine experimental colitis, CAC and sporadic tumors as well as from IBD and tumor versus control patients.

This diverse methodology and complex working plan allowed us for confirming the important role of geranylgeranylation within IECs and T cells for gut homeostasis, as hypothesized. In brief, our results are summarized as follows:
- Lack of GGTase-Iβ expression in Intestinal Epithelial Cells (IECs).
Intestinal homeostasis depends on an adequate level of geranylgeranylation within IECs. Inhibition of prenylation in IECs due to deletion of GGTase-Iβ gene (Pggt-IβΔIEC mice) led to embryonic lethality or to a severe enteric disease, resulting in the death of the animals, in the case of adult mice. Intestinal mucosa appeared extremely damaged, epithelial architecture destroyed and intestinal permeability dramatically increased. Electron microscopy analysis showed a clear modification of epithelial morphology, disposition and integrity. Gene expression as well as proteomic assay clearly demonstrated the dramatic impact of geranylgeranylation in IEC biology, involving IEC integrity, morphology, and architecture in this effect. Regarding the underlying mechanism, our data can rule out intestinal microbiota as well as cell death as key mediators. However, they strongly support cytoskeleton rearrangement as main consequence of abrogation of geranylgeranylation in IECs. GGTase-Iβ deficient IECs experienced a complex cytoskeletal alteration (altered actin-myosin complex) which in turn affect cell shedding leading to instability of epithelium. This fact was associated with a dysfunction of Rho-A pathway after GGTase-I deletion: decreased levels of activated GTP-bound Rho-A, translocation of Rho-A from membrane to cytosol, and altered activation of downstream signaling pathways. Together, impaired Rho-A prenylation is crucial for cytoskeleton arrangement within IECs in order to maintain epithelial integrity in the gut.

- Lack of GGTase-Iβ expression T-cells.

Protein geranylgeranylation is relevant in the case of T cell biology as well. Mice lacking GGTase-Iβ expression in T cells (Pggt-IβTcell mice) developed a peripheral lymphopenia, with decreased numbers of CD4+ and CD8+ T cells in blood, spleen and lymph nodes. However, intrathymic T cell development turned out to be less affected and the number of single positive T cells within the thymus was even increased in the absence of geranylgeranylation. Thymocytes lacking GGTase-Iβ expression reached their mature status and responded to S1P for emigration from thymus; however they were not able to migrate to secondary lymphoid tissues. In contrast, normal numbers of infiltrated GGTase-Iβ deficient T cells could be detected within the intestinal lamina propria and intraepithelial compartment in gut, implicating a possible intestine-directed migration of GGTase-Iβ deficient T cells. Indeed, LPMC as well as single positive thymocytes in Pggt-IβTcell showed increased expression of α4β7 integrins (gut homing marker). Regarding the resulting intestinal phenotype, young Pggt-IβTcell mice (8-10 weeks) show no signs of intestinal inflammation, while a spontaneous development of rectal prolapse and inflammatory alterations of the distal colon could be observed in elder animals, with massive immune cell infiltration. With regard to T cell function, GGTase-Iβ deficient T cells isolated from spleen, mesenteric lymph nodes and lamina propria showed a tendency towards a proinflammatory cytokine profile with increased production of IL-17A. Regarding the impact of GGTase-Iβ function in T cells on the development of colitis, the severity of acute chemical-induced colitis, such as TNBS or DSS colitis was not modified by the abrogation of geranylgeranylation. However, mice lacking GGTase-Iβ expression in T cells, showed an increased severity of intestinal inflammation in chronic DSS colitis, including increased mucosal cell infiltration and a higher degree of tissue damage. Likewise in non-challenged mice, proinflammatory cytokine production in GGTase-Iβ deficient spleen, MLN and lamina propria cells turned out to be also increased in the context of chronic colitis. However, our data implicated a protective role of GGTase-Iβ deficient T cells in the development of colitis-associated tumors in the AOM-DSS model. Strikingly, adoptively transferred GGTase-Iβ deficient naïve T cells are protective in transfer colitis, both in a preventive and curative therapeutic approach. Summing up, T cells lacking GGTase-Iβ expression might be directed to the gut from thymocyte precursors, probably due to increased expression of gut homing markers. Within the gut, these cells could lead to an increased severity in colitis, although they seemed to play a protective anti-tumour response.

Focusing on the GGTase-Iβ profile, the overall expression is reduced in AOM-DSS and APCmin tumors compared to normal tissue, which is accompanied by accumulation of non prenylated proteins (Rap1A). Although, this enzyme is expressed in both IECs and immune cells in the lamina propria in gut, the epithelial expression is more consistent and important than in the case of lamina propria cells, both in murine and human tissues. Inflamed areas of tissues from T-cell dependent and T-cell independent colitis, as well as IBD patients, show a significant reduction of GGTase-Iβ expression within the epithelium.

Covering technical, description, mechanism and translational purposes, we can state that this project represents a fundamental step for the identification and characterization of prenylation and geranylgeranylation as a novel key player for maintenance of intestinal homeostasis. Importantly, this project set the basis for potential future research linking prenylation and its targets to intestinal disorders, such IBD or colorectal cancer. Translationally, this basic research might open new avenues in the search for new therapeutic strategies in pathological conditions such as the latter, which are devoid of satisfactory causative treatment. In a close future, results obtained in this project may be useful in the pharmaceutical industry for the development of novel drugs which improve the quality of life and prognosis of gut-related diseases patients.

Researcher: Rocío López Posadas.
Project Co-ordinator: Markus F. Neurath
Medical Clinic 1. Universitätsklinikum Erlangen