The MET-FLAM Faculty

Eva STURM

Scientific Interests:

Janus kinases are crucial components of cytokine signaling pathways which are involved in the regulation of many cellular functions. Cytokine-induced persistent or dysregulated Janus kinase signaling, however, is maladaptive and contributes to many inflammatory diseases. We observed that targeting the JAK1/2 pathway with the JAK inhibitor Baricitinib represents a promising therapeutic strategy for eosinophilic inflammation as observed in severe eosinophilic asthma [11]. In a follow-up project we investigate the therapeutic potential of JAK inhibitors in IL-23 / Th17-driven inflammation which is often associated with steroid resistance.

Apolipoproteins (APO) such as Apo-A1, the predominant apolipoprotein constituent of high-density lipoproteins (HDLs), promote multifunctional roles in immunity and inflammation [3]. Our group could show that ApoA-IV is decreased in serum from allergic patients and inhibits eosinophil function in vitro and in vivo via activation of the circadian nuclear receptor NR1D1 [12]. In our ongoing research we now focus on therapeutic and diagnostic potential of the molecular circadian clock in eosinophilic disorders [13].

Proposed Dissertation Topic:

Background: Macrophage migration inhibitory factor (MIF) is elevated in asthma patients and positively correlates with blood eosinophils, but is negatively associated with lung function indices. MIF functions as a nuclease and participates in the induction of parthanatos, a type of poly (ADP-ribose) polymerase-induced cell death. MIF also acts as a regulator of the NLRP3 inflammasome and has enzymatic activities, such as tautomerization of phenylpyruvate and D-dopachrome. Importantly, MIF acts as a regulator of glucose homeostasis and autophagy. Thus, tautomerase inhibitors might be useful in the treatment of MIF-related diseases, such as sepsis, SARS-CoV-2, and inflammatory bowel disease. Moreover, MIF levels in lungs and blood are significantly higher in asthmatic patients compared to healthy controls and correlate with increased blood eosinophils and FeNO, but are negatively correlated with lung function indices.

Hypothesis and objectives: We aim to elucidate the therapeutic potential of novel inhibitors targeting the enzymatic activities of MIF in patients with severe eosinophilic asthma and respective mouse models. We plan to investigate how MIF and MIF inhibitors influence (i) effector function, (ii) glucose homeostasis and (iii) autophagy in peripheral blood and induced sputum leukocytes from controls and asthmatic patients. (iv) We will elucidate MIF-associated signaling cascades and how they can be modulated with respective inhibitors, and (v) prove the in vivo relevance of these approaches in established murine models of eosinophilic lung inflammation.

Methods and approaches: The PhD candidate will be trained in isolating leukocyte subsets from peripheral blood from healthy donors and patients (asthma and acute respiratory distress syndrome) by negative magnetic selection and cell sorting. The expression of CD74 and NLRP3 will be quantified by flow cytometry, qPCR, western blotting, and fluorescence microscopy. MIF levels and other cytokines in serum, BALF and tissue homogenates will be determined by ELISA, lipid mediators will be measured by mass spectrometry. Functional responses of leukocytes will be investigated in flow cytometric assays of shape change, integrin up-regulation, respiratory burst, and chemotaxis. Autophagic activity and energetic metabolism will be determined using SCENITH and Seahorse analyses (1st and 2nd year). Signaling pathways will be explored by live-cell Ca²⁺ imaging, flow cytometry, and western blotting. For silencing and overexpression studies, bone-marrow-derived mouse eosinophils and monocyte-derived macrophages will be used. Moreover, the student will be trained in established experimental mouse models for LPS-induced acute lung injury and allergen-induced airway inflammation. Lung tissue will be analyzed by immunohistochemistry and in situ hybridization. Lung function testing will be performed by using the flexiVent platform (3rd and 4th year). MIF levels and other cytokines in serum, bronchoalveolar lavage fluid, sputum and tissue homogenates will be determined by ELISA, lipid mediators will be measured by mass spectrometry.

Pitfalls and alternative approaches: Our laboratory has many years of experience with in vitro and in vivo models of lung inflammation. Therefore, we do not expect any technical pitfalls here. However, in the unexpected case, that we do not observe any effects of the respective MIF inhibitors in the planned experiments, we will alternatively (i) monitor other inflammatory cells such as monocytes, macrophages, neutrophils and lymphocytes in blood and sputum of patients as well was in BALF and lung tissue of mice. As MIF also seems to influence epithelial barrier function and tissue remodeling, (ii) we will explore the impact of MIF and MIF inhibitors on energy metabolism, cytokine release and epithelial barrier function in primary bronchial epithelial cells (PBECs) from healthy controls and patients with severe eosinophilic asthma. (iii) Further, organotypic precision-cut lung slices (PCLS) are a well-established model for allergic pulmonary inflammation. Therefore, the beneficial effects of MIF inhibitors on cytokine release, markers of collagen formation and degradation, as well as fibronectin will be studied in activated human and mouse PCLS. (iv) Finally, we will take advantage of publicly available transcriptomic data sets and investigate the expression of MIF-related pathways in samples from healthy and asthmatic individuals.

Involved Faculty members: Eva Sturm (PI), Julia Kargl (multi-color flow cytometry and cell sorting), Ákos Heinemann (leukocyte functional assays), Stefano Angiari (cell signaling assays and Seahorse analysis) and Martin Stradner (SCENITH: autophagy and energetic metabolism).

International Collaborations: Balázs Radnai (Pécs, HU), Hannah Durrington (Manchester, UK), Robert Gurke (Frankfurt, DE).

Facilities: Our team currently consists of two PhD candidates and two technicians. Our laboratory is located on the newly opened basic research campus of the university, just opposite the university hospital. Our division provides the required laboratory and office space, secretarial assistance, and basic laboratory facilities, such as cell culture, flow cytometry, immunofluorescence microscopy and more. Specific equipment for mouse lung function testing and endothelial functional assays (interaction of endothelial cells and immune cells; endothelial barrier function) are also available in our lab. We are constantly isolating fresh human peripheral blood leukocytes from blood donors. Seahorse technology, metabolomics and mass spectrometry are also available on the campus through collaboration partners.

Preparatory Findings:

Novel tautomerase inhibitors attenuate eosinophil and neutrophil migration.
Purified human eosinophils from healthy subjects or polymorphonuclear neutrophils were pretreated with the tautomerase inhibitors ISO-1, KRP6, TE-11 and TE-25 (20 µM) for 30 min at 37 °C and allowed to migrate towards MIF (3 nM) for 60 min at 37 °C in a micro Boyden chamber. Migrated cells were enumerated by flow cytometry. Data are expressed as % of the MIF vehicle response; * P < 0.05, ** P < 0.01, *** P < 0.001; Student’s t test.

1. Peinhaupt M, Sturm EM, Heinemann A: Prostaglandins and Their Receptors in Eosinophil Function and As Therapeutic Targets. Front Med (Lausanne), 2017; 4:104.
   
2. Knuplez E, Sturm EM, Marsche G: Emerging Role of Phospholipase-Derived Cleavage Products in Regulating Eosinophil Activity: Focus on Lysophospholipids, Polyunsaturated Fatty Acids and Eicosanoids. Int J Mol Sci, 2021; 22(9):​4356.
   
3. Sturm EM, Knuplez E, Marsche G: Role of Short Chain Fatty Acids and Apolipoproteins in the Regulation of Eosinophilia-Associated Diseases. Int J Mol Sci, 2021; 22(9):​4377.
   
4. Theiler A, Bärnthaler T, Platzer W, Richtig G, Peinhaupt M, Rittchen S, Kargl J, Ulven T, Marsh LM, Marsche G, Schuligoi R, Sturm EM, Heinemann A: Butyrate ameliorates allergic airway inflammation by limiting eosinophil trafficking and survival. J Allergy Clin Immunol, 2019; 144(3):​764–776.
   
5. Frei RB, Luschnig P, Parzmair GP, Peinhaupt M, Schranz S, Fauland A, Wheelock CE, Heinemann A, Sturm EM: Cannabinoid receptor 2 augments eosinophil responsiveness and aggravates allergen-induced pulmonary inflammation in mice. Allergy, 2016; 71(7):​944–956.
   
6. Kienzl M, Hasenoehrl C, Valadez-Cosmes P, Maitz K, Sarsembayeva A, Sturm E, Heinemann A, Kargl J, Schicho R: IL-33 reduces tumor growth in models of colorectal cancer with the help of eosinophils. Oncoimmunology, 2020; 9(1):​1776059.
   
7. Luschnig-Schratl P, Sturm EM, Konya V, Philipose S, Marsche G, Fröhlich E, Samberger C, Lang-Loidolt D, Gattenlöhner S, Lippe IT, Peskar BA, Schuligoi R, Heinemann A: EP4 receptor stimulation down-regulates human eosinophil function. Cell Mol Life Sci, 2011; 68(21):​3573–87.
   
8. Radnai B, Sturm EM, Stancic A, Jandl K, Labocha S, Ferreirós N, Grill M, Hasenoehrl C, Gorkiewicz G, Marsche G, Heinemann Á, Högenauer C, Schicho R: Eosinophils Contribute to Intestinal Inflammation via Chemoattractant Receptor-homologous Molecule Expressed on Th2 Cells, CRTH2, in Experimental Crohn’s Disease. J Crohns Colitis, 2016; 10(9):​1087–1095.
   
9. Sturm EM, Schratl P, Schuligoi R, Konya V, Sturm GJ, Lippe IT, Peskar BA, Heinemann A: Prostaglandin E₂ inhibits eosinophil trafficking through E-prostanoid 2 receptors. J Immunol, 2008; 181(10):​7273–7283.
   
10. Durchschein F, Eherer A, Grill M, Sturm EM, Pommer V, Langner C, Högenauer C, Schicho R: Involvement of EP2 and EP4 Receptors in Eosinophilic Esophagitis: A Pilot Study. Dig Dis Sci, 2019; 64(10):​2806–2814.
    
11. Luschnig P, Kienzl M, Roula D, Pilic J, Atallah R, Heinemann A, Sturm EM: The JAK1/2 inhibitor baricitinib suppresses eosinophil effector function and restricts allergen-induced airway eosinophilia. Biochem Pharmacol, 2021; 192:​114690.
    
12. Roula D, Theiler A, Luschnig P, Sturm GJ, Tomazic PV, Marsche G, Heinemann A, Sturm EM: Apolipoprotein A-IV acts as an endogenous anti-inflammatory protein and is reduced in treatment-naïve allergic patients and allergen-challenged mice. Allergy, 2020; 75(2):​392–402.
    
13. Krakowiak K, Durrington HJ: The Role of the Body Clock in Asthma and COPD: Implication for Treatment. Pulm Ther, 2018; 4(1):​29–43.