1. Influence of a Ketogenic diet on T cell functions
Nutrition has a strong influence on acute and chronic inflammatory processes. Recent research indicates that particularly carbohydrate metabolism has an important impact: Dror et al. were able to show that both the carbohydrates absorbed, and the consecutive insulin secretion provoke an inflammatory reaction leading to the release of IL-1ß in peritoneal macrophages (Nature immune, 2016). The restriction of carbohydrate intake, on the other hand, has anti-inflammatory effects: Glucose restriction leads to an increased ß oxidation of fatty acids and to the production of ketone bodies, such as ß-hydroxybutyrate (bHB), for energy generation. Ketone bodies significantly reduce the acute and chronic inflammation of the innate immune system by inhibiting the inflammasome and can prevent autoimmunity. Their influence on the adaptive, especially the T-cell induced immune response, has not yet been clarified.
We investigate the effects of a) ketone bodies in vitro and b) a ketogenic diet of healthy subjects in vivo on homeostasis/effector functions of the adaptive immune system. In particular, differentiation, cytotoxicity and cytokine expression of human CD4+ and CD8+ T cells are investigated under different conditions.
For in vitro experiments, PBMCs (peripheral blood mononuclear cell) of healthy subjects are incubated in a glucose-reduced medium supplemented with ketone bodies under stimulation conditions. The respective T-cell populations are subsequently separated and analyzed using qPCR, flow cytometry and functional assays. Our results to date show that bHB supports functions of the adaptive immune system: After bHB supplementation followed by specific T cell stimulation, a forced production of cytokines and differentiation-specific transcription factors as well as a clear induction of cytotoxic markers could be found. In addition, supplementation of bHB leads to a significantly enhanced differentiation of CD4+ T cells to regulatory T cells (Tregs).
In vivo effects are investigated in the context of a three-week ketogenic diet of volunteers under control of blood ketone levels. Blood samples were withdrawn before start of the diet and 3 weeks later with subsequent T-cell analysis. Our results also here show an improvement in the T-cell-specific immune response.
The project addresses a highly topical and medically highly relevant question. Our results indicate that supportive nutritional therapy concepts may be indicated for diseases associated with inflammation in the future.
Initiated further project on the topic:
1. In a mouse model of chronic inflammation, the influence of a ketogenic diet on adaptive immunity, survival and regenerative processes will be investigated.
2. Interaction between malignant brain tumors and the immune system
Glioblastoma multiforme (GBM) is one of the most aggressive tumor entities and the most common brain tumor in adults. The use of external beam radiation in combination with concomitant and adjuvant chemotherapy has improved median survival to currently 15 months. However, no curative treatment exists so far.
GBM closely interact with the immune system: On the one hand, they produce inflammatory mediators themselves and thus create a pro-tumourigenic tumour environment; on the other hand, they induce a suppression of the adaptive immune system and thus prevent a tumour-specific defence.
Our projects focus on exactly these interactions.
a) Modulation of low-Grade inflammation in the tumor and its microenvironment
Glioblastomas lead to an unspecific, low-grade inflammatory reaction in the immediate vicinity of the tumor, which is initially triggered mainly by infiltration of immune cells and hypoxia in the tumor area. This inflammatory environment promotes tumor cell proliferation, invasion and angiogenesis, and stimulates GBM cells to actively secrete proinflammatory mediators themselves. This potentiated inflammatory reaction consecutively leads to an increased recruitment of further immune cells into the tumor microenvironment. A reduction of this self-maintaining inflammatory reaction could significantly inhibit tumor progression. Central targeting hubs to reduce tumor progression could be the inhibition of a) cytokine secretion and b) central cytokine receptors of immune and glioblastoma cells.
This project focuses on evaluating the tumor-inhibitory potential of a) anti-inflammatory miRNAs and b) cytokine receptor antagonists such as Anakinra, an antagonist of the pro-inflammatory IL1-Receptor.
b) Influence of a ketogenic diet on immune functions in malignant gliomas
Malignant brain tumors produce and maintain an undirected, pro-tumorigenic inflammation and induce a suppression of adaptive immunity by secretion of immunomodulating mediators. This inhibits the activation of specific effector T cells and a targeted defense against the malignant process. We investigate the influence of ketones on both non-specific inflammation and tumor-specific CD8 response in malignant gliomas. We analyze this interaction in specifically established in vitro models using primary cell cultures.
Further studies are in preparation:
- In a clinical study enrolling patients with malignant brain tumors, the influence of a ketogenic diet on tumor progression, immune functions and quality of life scores will be investigated.
- The influence of a ketogenic diet on tumor progression and immune functions will be investigated in a mouse model with syngeneic brain tumors.
c) Photodynamic therapy of malignant gliomas as a therapeutic tool for the induction of long lasting antitumoral immune responses
Interstitial photodynamic therapy represents a new stereotactic, minimally invasive procedure for the treatment of glioblastoma currently being under clinical and experimental evaluation. The procedure enables accurate irradiation of a glioblastoma via stereotactically implanted laser probes after prior photosensitization of the tumor. In a pilot study conducted by the Neurosurgical Department of the LMU, photodynamic therapy was applied as an initial treatment for non-resectable glioblastomas. Surprisingly, 40% of the treated patients experienced long-term survival (>60 months). As local tumor destruction by laser irradiation cannot sufficiently explain long-lasting anti-tumor effects of photodynamic treatment, we assumed the induction of anti-tumor immunological effects as the underlying molecular mechanism of PDT. Therefore, we developed a novel in vitro PDT model to investigate the effect of PDT on cells of the adaptive immune system using co-cultures of glioblastoma cells and PBMCs (peripheral blood mononuclear cells). Initial analysis of the expression of CD8+ cells showed a significant increase in cytolytic proteins perforin (PRF) and Interferon-γ (IFNγ) after PDT. In agreement, we were able to demonstrate an improvement in the specific lysis capacity of these cells in functional assays. These effects were observed in allogeneic models with a glioblastoma cell line and immune cells of healthy subjects as well as in autologous approaches with isolated GBM primary cultures and PBMCs of the same patients.
These results show that activation of the adaptive immune response contributes to long-term survival after iPDT in glioblastoma patients. This is currently being further investigated, especially with regard to the development of memory cells and a possible use of the model as a preoperative screening method for the individual therapeutic success of iPDT.
4. Control of T-cell functions by miRNAs during sepsis.
Sepsis is a life-threatening disease leading to death in a high percentage of patients despite modern treatment strategies. The basic problem of the clinical syndrome is a complex systemic immune reaction triggered by the invasion of pathogens. While this reaction generally is beneficial, it is out of balance in sepsis: An uncontrolled, excessive activation of immune cells leads to damage to the body's own tissues and even organ failure. Despite intensive research efforts, the pathophysiology of sepsis is poorly understood. It currently is clear, however, that is a heterogeneous dynamic syndrome caused by imbalances in the "inflammatory networks". T-lymphocytes occupy a central position in this network. The influence of miRNAs on the activation and differentiation of T lymphocytes in sepsis has not yet been elucidated and is the subject of our research project. Using array analyses and in-vitro experiments, we have already been able to identify specific miRNAs differentially expressed in sepsis, their target genes and their biological functions. We have also established an immunological "fingerprint" that can be used to determine a patient's current immune status. Our work aims to contribute to the future use of miRNAs in the diagnosis and therapy of sepsis.
5. Regulation of endothelial barrier function in acute inflammatory processes by miRNAs
The endothelial barrier is a fine-tuned system based on the fast interaction of cell junction molecules with the cytoskeleton and the extracellular matrix. Its function is to regulate the directed transport of fluids, gases, electrolytes, proteins and circulating cells from the microvascular system towards tissues.
A disrupted endothelial vascular barrier can lead to severe volume shifts and the formation of massive edemas in acute inflammatory processes. Particularly in sepsis, an inflammatory clinical syndrome associated with high mortality, pathological permeability of the endothelial barrier with consecutively disturbed microcirculation occurs in the course of the disease. This usually causes hemodynamic instability, which is difficult to treat, up to complete organ failure.
Molecular mechanisms of endothelial permeability in acute inflammation are still elusive. In this project we will experimentally investigate the role of mircoRNAs (miRNAs), as central control elements of cells, in the regulation of the endothelial vascular barrier.
Goal of this project is to investigate the regulatory role of miRNAs on the expression of cell junction molecules and on the involved upstream signaling pathways. We have already identified miRNA 125a-5p as an important regulator of key molecules of the endothelial vascular barrier. In a next step the impact of specific miRNAs on endothelial permeability in a translational in-vitro model of sepsis is going to be tested..
6. Analysis of MDSCs as cells of endogenous immunosuppression after cardiopulmonary bypass
Myeloid-Derived Suppressor Cells (MDSCs) are a heterogeneous group of immature myeloid cells with strong immunosuppressive properties. They can be detected in chronic inflammatory diseases as well as in peripheral blood of tumor patients. In particular, the competence of the adaptive immune system is significantly impaired by even a small number of MDSCs. So far, there is only limited data on the molecular mechanisms leading to the occurrence of MDSCs and on the clinical relevance of MDSC-specific immune paralysis.
Particularly in the perioperative and intensive care context, the occurrence of MDSCs and their effects on immune competence have not been investigated so far. However, this could be of great relevance since immunoanalytical conditions, especially after major interventions, often lead to intensive care complications such as susceptibility to infections or wound healing disorders. This may particularly apply to patients who have undergone major cardiosurgical procedures using the heart-lung machine (HLM).
In this project we thus have investigated whether MDSCs occur after HLM and whether they may be involved in postoperative immune paralysis. We could show that MDSCs can be detected during cardiac surgery using the heart-lung machine and that postoperative paralysis of the adaptive immune system is significantly supported by MDSCs. Currently, we are characterizing the underlying molecular mechanisms and are investigating possible therapeutic strategies.