The MET-FLAM Faculty

Ákos HEINEMANN

Scientific Interests:

Another prostaglandin, PGE₂, has also been in the focus of research. We could delineate its protective and anti-inflammatory effects in the lung as a bundle of mechanisms in different cell types mediated by the G protein-coupled receptor, EP₄ [9]. Activation of EP₄ receptors attenuates eosinophil recruitment and activation [10], ILC2 activity [11], and cytokine secretion [12], but strengthens the endothelial barrier [12, 13]. Deposition of collagen, proliferation of myofibroblasts and recruitment of fibrocytes is likewise reversed by blockade of PGE₂ metabolism [14]. We observed corresponding beneficial effects of EP₄ activation in mouse models of allergic airway inflammation, acute lung injury and lung fibrosis [10, 12, 14].

Short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, are produced in high amounts in the gastrointestinal tract by commensal bacteria and can be absorbed into the bloodstream. We observed that SCFAs were able to attenuate human eosinophils at several functional levels, including (i) adhesion to the endothelium, (ii) migration, and (iii) survival. These effects were independent from GPR41 and GPR43 but were mediated by histone deacetylase inhibition. In vivo treatment with butyrate ameliorated allergen-induced airway eosinophilia, reduced type-2 cytokine levels in bronchial fluid, and improved airway hyper­responsiveness in mice [15].

Further, butyrate enhanced barrier function of endothelial cells. In contrast, the classical intermediate Krebs cycle metabolite, succinate [16], reduced endothelial barrier function in HUVECs and prompted hallmarks of angiogenesis such as proliferation, migration and spheroid sprouting in fetoplacental arterial endothelial cells (FpECAs), where it also induced endothelial tube formation. We also measured significantly higher succinate levels in placental tissue lysates from women with gestational diabetes mellitus (GDM) and found that the succinate receptor SUCNR1 was upregulated in GDM tissue lysates as well as in isolated diabetic fetoplacental arterial FpECAs. A positive correlation of SUCNR1 and vascular endothelial growth factor (VEGF) protein levels in tissue lysates indicated a potential link between the succinate-SUCNR1 axis and placental angiogenesis [17].

Proposed Dissertation Topic:

Background: In the course of inflammation, metabolic rewiring of immune cells alters the levels of diverse metabolites. This is particularly the case in acute inflammatory responses, as observed in sepsis and acute lung injury. Succinate, a classically known intermediate metabolite in Krebs cycle, accumulates in macrophages stimulated with lipopolysaccharide (LPS) [18]. We have recently demonstrated that succinate triggers angiogenesis in human umbilical vein endothelial cells via its cognate receptor GPR91 / SUCNR1. However, whether other endothelial responses are also regulated via the succinate – SUCNR1 axis is not yet clear, and the role of succinate in lung disease is completely unknown. A recent study has shown that lactate from bone-marrow-derived inflammatory neutrophils facilitates their mobilization through a GPR81-dependent disruption of the endothelial barrier [19].

Hypothesis and objectives: We hypothesize that the succinate – SUCNR1 axis regulates important endothelial parameters, such as barrier function and adhesion properties and therefore might play a crucial role in the crosstalk between circulating immune cells and endothelial cells in acute inflammatory conditions such as acute lung injury. In detail, the student will investigate whether (i) circulating human immune cells (e. g. monocytes and neutrophils) accumulate and release succinate in response to inflammatory signals such as LPS along with other related metabolites; (ii) succinate affects the barrier function and the adhesiveness of lung endothelial cells to immune cells in a SUCNR1-dependent manner; (iii) targeting SUCNR1 might reduce immune cells infiltration and outcome in mouse models of acute lung injury.

Methods and approaches: The recruited PhD candidate will be trained in and perform isolation of different immune cell populations from donors’ blood, in vitro culture of immune cells and endothelial cells, mass-spectrometry-based metabolomics to quantify succinate and related metabolites in the cells and in their conditioned media (1st year); impedance measurements to investigate the integrity of endothelial barrier, video microscopy to study the adhesion of leukocytes to endothelial cells, multi-color flow cytometry and ELISAs to quantify the expression of adhesion molecules, Western blot, qPCR and siRNA techniques to quantify and target SUCNR1 in endothelial cells (1st and 2nd year); live-cell calcium imaging, phosphoproteomics, and cAMP measurement amongst others to investigate the signaling machinery downstream of SUCNR1 in endothelial cells and to identify candidate molecules to inhibit these responses (3rd year). Finally, the student will use suitable mouse models (e. g. intranasal LPS, bleomycin) to validate his/her findings in vivo (3rd and 4th year).

Pitfalls and alternative approaches: From a technical stand, isolation of immune cells from donors’ blood is well established in the applicant’s laboratory and ethical approval for this procedure is in place. Culture and handling of human endothelial cells from different vascular beds is also well established. All methods and techniques planned for this project are performed regularly either in the applicant’s laboratory or by collaborators within the frame of the university or beyond. While planning the proposed project, avoidance of artificial overexpression cellular systems is intended. Using primary human cells with the receptor at its native expression levels will be the closest we can get to the in vivo biosystem. However, we are aware that specific GPCRs can promiscuously couple to and activate multiple G proteins and noting the low expression levels of GPCRs, the study of their downstream signaling at endogenous levels might be challenging. If this is the case, we will transfect the cells to stably express SUCNR1. Furthermore, the possibility that SUCNR1 activation only potentiates the effects of other factors regulating endothelial adhesion without exerting effects by its own cannot be excluded, Therefore, we might need to use succinate in combination with other established substrates / ligands to induce cellular response such as TNF-α if we fail to see a response to succinate alone.

Involved Faculty members: Ákos Heinemann (PI), Eva Sturm (mouse models and lung function testing), Susanne Sattler (myocardial infarction model), Dagmar Kratky (metabolic phenotyping), Stefano Angiari (SUCNR1 signaling).

International Collaborations: Marc Rothenberg (Cincinnati, OH), Evi Kostenis (Uni Bonn, DE), Thomas Vorup-Jensen (Uni Aarhus, DK), James Pease (Imperial College, London, UK), Jenny Mjösberg (Karolinska, SE).

Facilities: Our team currently consists of two PhD candidates, two postdocs 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:

Figure 1: SUCNR1 is expressed in endothelial cells (HUVECs).
Immunofluorescence staining of SUCNR1 (green) in HUVECs. DAPI (blue) was used for counterstaining. Image isrepresentative for 3 independent experiments.

Figure 2: Succinate reduces the barrier function of HUVECs.
(A) Resistance of HUVECs monolayer in response to succinate or vehicle recorded with ECIS-Z-Theta device. (B) Immunofluorescence staining of VE-cadherin (green) and F-actin (red) in HUVECs treated with succinate or vehicle. DAPI (blue) was used for counterstaining. For A, two-way ANOVA for repeated measurements followed by Tukey’s post-hoc test was used. *** P < 0,0001 (n = 3). Data are shown as mean ± SEM. For B, images are representative of 4 independent experiments. [Figure adapted from the PhD thesis of Reham Atallah]

1. Schuligoi R, Sturm E, Luschnig P, Konya V, Philipose S, Sedej M, Waldhoer M, Peskar BA, Heinemann A: CRTH2 and D-type prostanoid receptor antagonists as novel therapeutic agents for inflammatory diseases. Pharmacology, 2010; 85(6):​372–382.
   
2. Rittchen S, Jandl K, Lanz I, Reiter B, Ferreirós N, Kratz D, Lindenmann J, Brcic L, Bärnthaler T, Atallah R, Olschewski H, Sturm EM, Heinemann A: Monocytes and Macrophages Serve as Potent Prostaglandin D₂ Sources during Acute, Non-Allergic Pulmonary Inflammation. Int J Mol Sci, 2021; 22(21):11697.
   
3. Jandl K, Heinemann A: The therapeutic potential of CRTH2/DP2 beyond allergy and asthma. Prostaglandins Other Lipid Mediat, 2017; 133:​42–48.
   
4. Mathiesen JM, Christopoulos A, Ulven T, Royer JF, Campillo M, Heinemann A, Pardo L, Kostenis E: On the mechanism of interaction of potent surmountable and insurmountable antagonists with the prostaglandin D2 receptor CRTH2. Mol Pharmacol, 2006; 69(4):​1441–1453.
   
5. Royer JF, Schratl P, Lorenz S, Kostenis E, Ulven T, Schuligoi R, Peskar BA, Heinemann A: A novel antagonist of CRTH2 blocks eosinophil release from bone marrow, chemotaxis and respiratory burst. Allergy, 2007; 62(12):​1401–1409.
   
6. Sedej M, Schröder R, Bell K, Platzer W, Vukoja A, Kostenis E, Heinemann A, Waldhoer M: D-type prostanoid receptor enhances the signaling of chemoattractant receptor-homologous molecule expressed on T_H2 cells. J Allergy Clin Immunol, 2012; 129(2):​492-500.e1-9.
   
7. Maric J, Ravindran A, Mazzurana L, Van Acker A, Rao A, Kokkinou E, Ekoff M, Thomas D, Fauland A, Nilsson G, Wheelock CE, Dahlén SE, Ferreirós N, Geisslinger G, Friberg D, Heinemann A, Konya V, Mjösberg J: Cytokine-induced endogenous production of prostaglandin D₂ is essential for human group 2 innate lymphoid cell activation. J Allergy Clin Immunol, 2019; 143(6):​2202–2214.e5.
   
8. Jandl K, Stacher E, Bálint Z, Sturm EM, Maric J, Peinhaupt M, Luschnig P, Aringer I, Fauland A, Konya V, Dahlen SE, Wheelock CE, Kratky D, Olschewski A, Marsche G, Schuligoi R, Heinemann A: Activated prostaglandin D₂ receptors on macrophages enhance neutrophil recruitment into the lung. J Allergy Clin Immunol, 2016; 137(3):​833–843.
   
9. Konya V, Marsche G, Schuligoi R, Heinemann A: E-type prostanoid receptor 4 (EP4) in disease and therapy. Pharmacol Ther, 2013; 138(3):​485–502.
   
10. 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.
    
11. Maric J, Ravindran A, Mazzurana L, Björklund ÅK, Van Acker A, Rao A, Friberg D, Dahlén SE, Heinemann A, Konya V, Mjösberg J: Prostaglandin E₂ suppresses human group 2 innate lymphoid cell function. J Allergy Clin Immunol, 2018; 141(5):​1761–1773.e6.
    
12. Konya V, Maric J, Jandl K, Luschnig P, Aringer I, Lanz I, Platzer W, Theiler A, Bärnthaler T, Frei R, Marsche G, Marsh LM, Olschewski A, Lippe IT, Heinemann A, Schuligoi R: Activation of EP₄ receptors prevents endotoxin-induced neutrophil infiltration into the airways and enhances microvascular barrier function. Br J Pharmacol, 2015; 172(18):​4454–4468.
    
13. Konya V, Üllen A, Kampitsch N, Theiler A, Philipose S, Parzmair GP, Marsche G, Peskar BA, Schuligoi R, Sattler W, Heinemann A: Endothelial E-type prostanoids 4 receptors promote barrier function and inhibit neutrophil trafficking. J Allergy Clin Immunol, 2013; 131(2):​532–540.e1-2.
    
14. Bärnthaler T, Theiler A, Zabini D, Trautmann S, Stacher-Priehse E, Lanz I, Klepetko W, Sinn K, Flick H, Scheidl S, Thomas D, Olschewski H, Kwapiszewska G, Schuligoi R, Heinemann A: Inhibiting eicosanoid degradation exerts antifibrotic effects in a pulmonary fibrosis mouse model and human tissue. J Allergy Clin Immunol, 2020; 145(3):​818–833.e11.
    
15. 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.
    
16. Atallah R, Olschewski A, Heinemann A: Succinate at the Crossroad of Metabolism and Angiogenesis: Roles of SDH, HIF1α and SUCNR1. Biomedicines, 2022; 10(12):​3089.
    
17. Atallah R, Gindlhuber J, Platzer W, Bärnthaler T, Tatzl E, Toller W, Strutz J, Rittchen S, Luschnig P, Birner-Gruenberger R, Wadsack C, Heinemann A: SUCNR1 Is Expressed in Human Placenta and Mediates Angiogenesis: Significance in Gestational Diabetes. Int J Mol Sci, 2021; 22(21):​12048.
    
18. Khatib-Massalha E, Bhattacharya S, Massalha H, Biram A, Golan K, Kollet O, Kumari A, Avemaria F, Petrovich-Kopitman E, Gur-Cohen S, Itkin T, Brandenburger I, Spiegel A, Shulman Z, Gerhart-Hines Z, Itzkovitz S, Gunzer M, Offermanns S, Alon R, Ariel A, Lapidot T: Lactate released by inflammatory bone marrow neutrophils induces their mobilization via endothelial GPR81 signaling. Nat Commun, 2020; 11(1):​3547.
    
19. Mills EL, Kelly B, Logan A, Costa ASH, Varma M, Bryant CE, Tourlomousis P, Däbritz JHM, Gottlieb E, Latorre I, Corr SC, McManus G, Ryan D, Jacobs HT, Szibor M, Xavier RJ, Braun T, Frezza C, Murphy MP, O'Neill LA: Succinate Dehydrogenase Supports Metabolic Repurposing of Mitochondria to Drive Inflammatory Macrophages. Cell, 2016; 167(2):​457–470.e13.