The MET-FLAM Faculty

Michael KHALIL

Scientific Interests:

Research findings are being evaluated and interpreted in strong association with imaging (conventional and non-conventional MRI at 3T) findings of the central nervous system and detailed clinical follow-up data. This combined approach shall ultimately serve to define patient subgroups and aid in the development of more effective treatment regimens and drugs.

Current research focuses on investigating the clinical significance of cerebrospinal fluid and blood based biomarkers using ultra-high sensitive detection techniques in neuroimmunological and neurodegenerative disorders.

Proposed Dissertation Topic:

Background: In the chronic immune mediated disease multiple sclerosis (MS), irreversible accumulation of disability can occur at any stage of the disease and may be driven by two main mechanisms, that are relapse-associated worsening (RAW) and progression independent of relapse activity (PIRA). Especially PIRA, which has recently been described as a common feature in MS, seems to be the main driving factor for disease progression in MS without formal diagnosis of a secondary progressive disease course. It is now clear that PIRA can occur even in very early stages of the disease, e. g. in patients after a first demyelinating attack of the central nervous system but also frequently in established MS. Recently, the clinical and neuroimaging predictors of PIRA at the time of the first demyelinating event have been investigated. Patients with PIRA had more brain lesions and had a steeper yearly increase in physical disability. However, the immunological mechanisms underlying PIRA are still unclear.

Hypothesis and objectives: We hypothesize that different phenotypic and metabolic profiles in circulating immune cells may be related to PIRA and may allow to identify patients at risk for early disease worsening. Early identification of immunological mechanisms underlying disease progression is of great importance for treatment decisions to ensure better long-term outcomes and discover novel therapeutic targets. The PhD candidate will investigate whether (i) the inflammatory profile and metabolic features of circulating immune cells allow to identify patients with PIRA; (ii) the inflammatory profile and metabolic features of circulating immune cells correlate with soluble markers of inflammation and neurodegeneration in cerebrospinal fluid and blood; (iii) immunological and clinical characteristics explain MS-related signs of tissue damage as evidenced by magnetic resonance imaging (MRI).

Methods and approaches: The PhD candidate will be trained in isolation of immune cell subsets from human blood (1st year). To potentially link disease worsening to immunological events in MS, the student will perform comprehensive immuno­phenotyping of the immune cells by flow cytometry, looking at (among others): (i) activation markers (e. g., CD71, CD25, CD69); (ii) cytokine production (e. g., GM-CSF, IL-17A, IFN-γ); (iii) markers of cell senescence (e. g., CD28) (1st to 3rd year). The student will also determine the overall metabolic profile of T cells, B cells, and monocytes by Seahorse assay, mass spectrometry, immuno­fluorescence, flow cytometry, qPCR, and western blot (2nd and 3rd year). Examples of readouts are mitochondrial mass and activity, glucose uptake and glycolytic capacity, expression of metabolite transporters and receptors (e. g. GLUT1, SUCNR1, MCT-4), expression and / or activity of metabolic enzymes and sensors (e. g., LDHA, PKM, MTOR, CPT1), accumulation and secretion of metabolic intermediates (e. g. succinate, lactate, fumarate). The immunologic, metabolic and cellular profiles will then be correlated with clinical and MRI data and soluble markers of inflammation and neuro­degeneration in the cerebrospinal fluid and blood (3rd and 4th year).

Pitfalls and alternative approaches: All laboratory tests included in this study have already been established in the participating institutions. Samples will be processed according to international consensus protocols to ensure pre-analytical stability. Comprehensive clinical and MRI data, including post-processing, will be collected according to established protocols. Data documentation will be performed using the database already established at our university. Ethical approval has been obtained to conduct this study. Regular team meetings will be scheduled to ensure early and continuous risk assessment and implementation of effective mitigation strategies.

Involved Faculty members: Michael Khalil (PI), Stefano Angiari (immune cell phenotyping, metabolic profile), Martin Stradner (functional immune cell assays), Vanessa Stadlbauer-Kööllner (neutrophil assays), and Julia Kargl and Eva Sturm (flow cytometry).

International Collaborations: Jens Kuhle (Basel, CH), Charlotte Teunissen (Amsterdam, NL), Manuel Comabella (Barcelona, ES).

Facilities: Our team currently consists of two medical doctors, three PhD candidates and one technicians. Our laboratory is located at 2nd floor of the Department of Neurology at the Medical University of Graz. Our laboratory is equipped with up to date fully automated ultra-high sensitive analytical platforms, such as the Simoa HD-X AnalyzerTM from Quanterix. Neuro­degenerative markers in the CSF are further being analyzed by the automated Lumipulse^® G600II from Fujirebio. CSF and Serum protein analysis are performed on the Optilite^® analyzer from The Binding Site. Our department provides the required laboratory and office space, basic laboratory facilities, such as immuno­fluorescence microscopy, isolating fresh human peripheral blood leukocytes from blood donors, DNA extraction and more. Flow cytometry analysis are mainly performed at the Division of Immunology and the Division of Rheumatology and Immunology. Seahorse technology, metabolomics and mass spectrometry are also available on the campus through collaboration partners.

Preparatory Findings:

Figure 1. A metabolic modulator limits proliferation of T cells from multiple sclerosis (MS) patients.
Peripheral blood mononuclear cells (PBMCs) from MS patients were stained with fluorescent Cell-Trace Violet (CTV) and restimulated in vitro with anti-CD3 / anti-CD28 antibodies, in the presence of vehicle (CTRL) or a metabolic modulator (Mod; undisclosed). After 5 days of culture, cells were stained with anti-CD3, anti-CD4, and anti-CD8 antibodies and analyzed by flow cytometry. As the cells divide, CTV is diluted in daughter cells. Thus, the lowest the CTV fluorescence intensity, the higher the cells proliferation. Mod significantly increased CTV mean fluorescence intensity (MFI) in both CD4⁺ and CD8⁺ T cells, suggesting less proliferative capacity of cells treated with Mod. ** p < 0.01 and *** p < 0.001 by Wilcoxon signed-rank test; n = 13.

Figure 2. A metabolic modulator limits pro-inflammatory cytokine production by MS T cells.
PBMCs from MS patients were restimulated in vitro with anti-CD3 / anti-CD28 antibodies, in the presence of vehicle (CTRL) or a metabolic modulator (Mod; undisclosed). After 24 hours of culture, cells were restimulated with PMA / ionomycin / brefeldin A for 5%bvsp;hours. Cells were then collected and stained with anti-CD3, anti-CD4, and anti-CD8 antibodies plus antibodies against several pro-inflammatory cytokines relevant for MS pathogenesis, and analyzed by flow cytometry. Our results indicate that Mod reduces the percentage of CD4⁺ and CD8⁺ T cells producing granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFN-γ), interleukin-17A (IL-17A), and tumour necrosis factor-alpha (TNF-α). * p < 0.05, ** p < 0.01, and *** p < 0.001 by Wilcoxon signed-rank test; n = 13.

1. Buchmann A, Pirpamer L, Pinter D, Voortman M, Helmlinger B, Pichler A, Maceski AM, Benkert P, Bachmaier G, Ropele S, Reindl M, Leppert D, Kuhle J, Enzinger C, Khalil M: High serum neurofilament light chain levels correlate with brain atrophy and physical disability in multiple sclerosis. Eur J Neurol, 2023; 30(5):​1389–1139.
   
2. Meier S, Willemse EAJ, Schaedelin S, Oechtering J, Lorscheider J, Melie-Garcia L, Cagol A, Barakovic M, Galbusera R, Subramaniam S, Barro C, Abdelhak A, Thebault S, Achtnichts L, Lalive P, Müller S, Pot C, Salmen A, Disanto G, Zecca C, D’Souza M, Orleth A, Khalil M, Buchmann A, Du Pasquier R, Yaldizli Ö, Derfuss T, Berger K, Hermesdorf M, Wiendl H, Piehl F, Battaglini M, Fischer U, Kappos L, Gobbi C, Granziera C, Bridel C, Leppert D, Maleska Maceski A, Benkert P, Kuhle J: Serum Glial Fibrillary Acidic Protein Compared With Neurofilament Light Chain as a Biomarker for Disease Progression in Multiple Sclerosis. JAMA Neurol, 2023; 80(3):​287–297.
   
3. Cagol A, Schaedelin S, Barakovic M, Benkert P, Todea RA, Rahmanzadeh R, Galbusera R, Lu PJ, Weigel M, Melie-Garcia L, Ruberte E, Siebenborn N, Battaglini M, Radue EW, Yaldizli Ö, Oechtering J, Sinnecker T, Lorscheider J, Fischer-Barnicol B, Müller S, Achtnichts L, Vehoff J, Disanto G, Findling O, Chan A, Salmen A, Pot C, Bridel C, Zecca C, Derfuss T, Lieb JM, Remonda L, Wagner F, Vargas MI, Du Pasquier R, Lalive PH, Pravatà E, Weber J, Cattin PC, Gobbi C, Leppert D, Kappos L, Kuhle J, Granziera C: Association of Brain Atrophy With Disease Progression Independent of Relapse Activity in Patients With Relapsing Multiple Sclerosis. JAMA Neurol, 2022; 79(7):​682–692.
   
4. Weiss B, Pichler A, Damulina A, Buchmann A, Hochmeister S, Seifert-Held T, Enzinger C, Archelos JJ, Khalil M: Evidence for an Intrathecal Immunoglobulin Synthesis by Kappa Free Light Chains in Neurological Patients with an Isolated Band in Isoelectric Focusing. Biomedicines, 2022; 10(9):​2202.
   
5. Voortman MM, Damulina A, Pirpamer L, Pinter D, Pichler A, Enzinger C, Ropele S, Bachmaier G, Archelos JJ, Marsche G, Khalil M: Decreased Cerebrospinal Fluid Antioxidative Capacity Is Related to Disease Severity and Progression in Early Multiple Sclerosis. Biomolecules, 2021; 11(9):1264.
   
6. Koini M, Pirpamer L, Hofer E, Buchmann A, Pinter D, Ropele S, Enzinger C, Benkert P, Leppert D, Kuhle J, Schmidt R, Khalil M: Factors influencing serum neurofilament light chain levels in normal aging. Aging (Albany NY), 2021; 13(24):​25729–25738.
   
7. Kappos L, Wolinsky JS, Giovannoni G, Arnold DL, Wang Q, Bernasconi C, Model F, Koendgen H, Manfrini M, Belachew S, Hauser SL: Contribution of Relapse-Independent Progression vs Relapse-Associated Worsening to Overall Confirmed Disability Accumulation in Typical Relapsing Multiple Sclerosis in a Pooled Analysis of 2 Randomized Clinical Trials. JAMA Neurol, 2020; 77(9):​1132–1140.
   
8. Khalil M, Pirpamer L, Hofer E, Voortman MM, Barro C, Leppert D, Benkert P, Ropele S, Enzinger C, Fazekas F, Schmidt R, Kuhle J: Serum neurofilament light levels in normal aging and their association with morphologic brain changes. Nat Commun, 2020; 11(1):812.
   
9. Angiari S, Runtsch MC, Sutton CE, Palsson-McDermott EM, Kelly B, Rana N, Kane H, Papadopoulou G, Pearce EL, Mills KHG, O'Neill LAJ: Pharmacological Activation of Pyruvate Kinase M2 Inhibits CD4⁺ T Cell Pathogenicity and Suppresses Autoimmunity. Cell Metab, 2020; 31(2):​391–405.e8.
   
10. Pinter D, Gattringer T, Enzinger C, Seifert-Held T, Kneihsl M, Fandler S, Pichler A, Barro C, Eppinger S, Pirpamer L, Bachmaier G, Ropele S, Wardlaw JM, Kuhle J, Khalil M, Fazekas F: Longitudinal MRI dynamics of recent small subcortical infarcts and possible predictors. J Cereb Blood Flow Metab, 2019; 39(9):​1669–1677.
    
11. Bridel C, van Wieringen WN, Zetterberg H, Tijms BM, Teunissen CE; and the NFL Group; Alvarez-Cermeño JC, Andreasson U, Axelsson M, Bäckström DC, Bartos A, Bjerke M, Blennow K, Boxer A, Brundin L, Burman J, Christensen T, Fialová L, Forsgren L, Frederiksen JL, Gisslén M, Gray E, Gunnarsson M, Hall S, Hansson O, Herbert MK, Jakobsson J, Jessen-Krut J, Janelidze S, Johannsson G, Jonsson M, Kappos L, Khademi M, Khalil M, Kuhle J, Landén M, Leinonen V, Logroscino G, Lu CH, Lycke J, Magdalinou NK, Malaspina A, Mattsson N, Meeter LH, Mehta SR, Modvig S, Olsson T, Paterson RW, Pérez-Santiago J, Piehl F, Pijnenburg YAL, Pyykkö OT, Ragnarsson O, Rojas JC, Romme Christensen J, Sandberg L, Scherling CS, Schott JM, Sellebjerg FT, Simone IL, Skillbäck T, Stilund M, Sundström P, Svenningsson A, Tortelli R, Tortorella C, Trentini A, Troiano M, Turner MR, van Swieten JC, Vågberg M, Verbeek MM, Villar LM, Visser PJ, Wallin A, Weiss A, Wikkelsø C, Wild EJ: Diagnostic Value of Cerebrospinal Fluid Neurofilament Light Protein in Neurology: A Systematic Review and Meta-analysis. JAMA Neurol, 2019; 76(9):​1035–1048.
    
12. Khalil M, Teunissen CE, Otto M, Piehl F, Sormani MP, Gattringer T, Barro C, Kappos L, Comabella M, Fazekas F, Petzold A, Blennow K, Zetterberg H, Kuhle J: Neurofilaments as biomarkers in neurological disorders. Nat Rev Neurol, 2018; 14(10):​577–589.
    
13. Khalil M, Renner A, Langkammer C, Enzinger C, Ropele S, Stojakovic T, Scharnagl H, Bachmaier G, Pichler A, Archelos JJ, Fuchs S, Seifert-Held T, Fazekas F: Cerebrospinal fluid lipocalin 2 in patients with clinically isolated syndromes and early multiple sclerosis. Mult Scler, 2016; 22(12):​1560–1568.
    
14. Khalil M, Langkammer C, Pichler A, Pinter D, Gattringer T, Bachmaier G, Ropele S, Fuchs S, Enzinger C, Fazekas F: Dynamics of brain iron levels in multiple sclerosis: A longitudinal 3T MRI study. Neurology, 2015; 84(24):​2396 2402.
    
15. O'Neill LA, Kishton RJ, Rathmell J: A guide to immunometabolism for immunologists. Nat Rev Immunol, 2016; 16(9):​553–565.
    
16. Pearce EL, Pearce EJ: Metabolic pathways in immune cell activation and quiescence. Immunity, 2013; 38(4):𔁧633–643.