The family of NADPH oxidase (Nox) isoenzymes, i.e. in humans Nox 1-5 and Duox 1 and 2, generates reactive oxygen species (ROS). Previously, ROS produced by the prototypic phagocyte Nox isoenzyme (Nox2) were regarded as crude antimicrobial compounds. Today, it is widely accepted that Nox-derived ROS exert in different cell types diverse and subtle functions, e.g. signaling and regulation of gene expression. This project aims at revealing specific functions of Nox-derived ROS in antimicrobial immunity.
Identification of the members of the entire family of Nox isoenzymes, i. e. in humans Nox1-5 and Duox1 and 2, led to the notion that ROS generated by defined Nox isoenzymes exert many tissue-specific effects beyond direct anti-microbial activity, e.g. intracellular signaling or activation and inactivation of enzymes. Such subtle effects of Nox-derived ROS largely remain to be elucidated in antimicrobial immune responses.
This project aims at characterizing the regulatory and effector functions of ROS generated by defined isoenzymes of the Nox family during the murine model infections with the gram-positive bacterium Listeria (L.) monocytogenes, the gram-negative bacterium Shigella (S.) flexneri and the RNA virus Lymphocytic Choriomeningitis virus (LCMV). Specifically, the Nox family members Nox1, Nox2, Nox4, Duox1, and Duox2 are in the focus of this project.
A superordinate regulatory mechanism controlling the activity of Nox isoenzymes depends on the activity of riboflavinkinase (RFK). RFK activity, by converting riboflavin (vitamin B2) to flavin-mononucleotide (FMN), is rate-limiting for providing flavin-adenin-dinucleotide (FAD) which is an essential cofactor of each Nox isoenzyme. In resting cells, usually less than half of the cellular pool of Nox isoenzymes is saturated with FAD. Therefore, interfering with RFK activity may provide a master switch for general inhibition of undesired Nox activities during pathophysiological reactions involving overshooting production of ROS.
In the current CMMC funding period, we revealed that riboflavin availability and riboflavinkinase activity impact on the defense against Listeria monocytogenes (Schramm et al.). Specifically, by using cell type-specific RFK knockout (KO) mice, we elucidated that in phagocytes RFK is required to control intracellular L. monocytogenes. The oxidative burst of RFK-deficient phagocytes in response to L. monocytogenes infection was significantly reduced. Mechanistically, TNF priming of Nox2 was defective in RFK-deficient phagocytes secondary to failure of saturation of Nox2 with FAD through RFK-dependent de novo generation of FMN/FAD. Similarly, withdrawal of riboflavin in wild-type macrophages for only 6 h shut down TNF-induced, RFK-mediated de novo FMN/FAD generation, which was accompanied by diminished ROS production and impaired anti-listerial activity.
These data show that TNF priming of Nox2 represents a riboflavin-dependent mechanism that is crucial for optimal ROS production in innate immune responses.
These results may have therapeutic implications for the treatment of select bacterial infections. Specifically, the intracellular life style of L. monocytogenes presents a therapeutic challenge, because none of the antibiotics of choice penetrate the host cells, distribute within the cells, and remain stable in the intracellular environment. Thus, riboflavin supplementation should also be considered next to antibiotics as a complementary therapeutic modality of listeriosis of elderly individuals to ensure optimal ROS production by neutrophils and macrophages, which represents an effective host defense mechanism protecting young and riboflavin-proficient individuals from listeriosis.
Insight into the involvement of ROS in antiviral immune responses is very limited. Due to its superordinate regulatory effects on the activity of Nox isoenzymes, we investigated the impact of RFK activity on the paradigmatic antiviral immune response during acute infection with the Lymphopcytic Choriomeningitis Virus (LCMV).
Global deletion of RFK in mice during the first days of infection with LCMV massively impairs expansion and maturation of the cytolytic effector mechanisms of virus-specific CD8+ T cells. Consequently, the control and elimination LCMV is significantly impaired in vivo. Deletion of RFK in dendritic cells, which are necessary for induction of antiviral CD8+ T cells, did not impair the expansion or maturation of LCMV-specific CD8+ T cells. In contrast, deletion of RFK in T cells themselves abolished the LCMV-induced expansion and maturation of CD8+ T cells. In contrast, the virus-induced function of CD4+ T cells from the same mice appears to be much less impaired. This is of special interest, because the floxed RFK allels are deleted from both CD4+ and CD8+ T cells due to expression of Cre recombinase in the CD4/CD8 double positive common thymic precursors of both T cell subpopulations.
The strong effect of RFK deficiency on CD8+ effector cells suggests that inhibiting RFK activity, e.g. by RF analogs, may have potential as a future immunosuppressive approach.
The biomedical relevance of this project is suggestive, because both RFK as well as Nox isoenzymes are suitable targets for small molecule inhibitors (Krause, K.H., Lambeth, D., and Krönke, M. (2012) NOX enzymes as drug targets. Cell Mol Life Sci 69:2279-2282). Therefore, specific inhibitors and/or cell type-specific delivery of such compounds may become future therapeutic approaches in immune-mediated diseases.
Meijles, D.N., Fan, L.M., Ghazaly, M. M., Howlin, B., Krönke, M., Brooks, G., Li, J.M. (2016) p22phox C242T Single-Nucleotide Polymorphism Inhibits Inflammatory Oxidative Damage to Endothelial Cells and Vessels. Circulation, 133:2391-403
Jakobshagen, K., Ward, B., Baschuk, N., Huss, S., Brunn, A., Malecki, M., Fiolka, M., Rappl, G., Corogeanu, D., Karow, U., Schiller, P., Abken, H., Heukamp, L.C., Deckert, M., Krönke, M., Utermöhlen, O. (2015). Endogenous IL-10 alleviates the systemic antiviral cellular immune response and T cell-mediated immunopathology in select organs of acutely LCMV-infected mice. Am J Pathol, 185: 3025-38.
Dannhausen, K., Karlstetter, M., Caramoy, A., Volz, C., Jaegle, H., Liebisch, G., Utermöhlen, O., Langmann, T. (2015). Acid sphingomyelinase (aSMase) deficiency leads to abnormal microglia behavior and disturbed retinal function. Biochem Biophys Res Com, 464(2):434-40.
Erny, D., Hrabě de Angelis, A.L. Jaitin, D., Wieghofer, P., Staszewski, O., David, E., Keren-Shaul, H., Mahlakoiv, T., Jakobshagen, K., Buch, T., Schwierzeck, V. Utermöhlen, O., Chun, E., Garrett, W.S., McCoy, K.D., Diefenbach, A., Staeheli, P., Stecher, B., Amit, O., Prinz, M. (2015). Host microbiota constantly control maturation and function of microglia in the CNS. Nat Neurosci. 18: 965-77.
Ketscher, L., Hannß, R., Morales, D., Basters, A., Guerra, S., Goldmann, T., Hausmann, A., Prinz, M., Naumann, R., Pekosz, A., Utermöhlen, O., Lenschow, D.J., Knobeloch, K-P. (2015). Selective inactivation of USP18 isopeptidase activity in vivo enhances ISG15 conjugation and viral resistance. Proc Natl Acad Sci U S A. 112:1577-82.
Schramm, M., Wiegmann, K., Schramm, S., Utermöhlen, O., and Krönke, M. (2014) Riboflavin (vitamin B2) deficiency impairs phagocytic ROS production and defense against Listeria monocytogenes. Eur. J. Immunol., 44:728-41.
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Inst. for Med. Microbiology, Immunology and Hygiene
CMMC - PI - SRG 03
Executive Board Member
Institute for Medical Microbiology, Immunology and Hygiene
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Institute for Medical Microbiology, Immunology and Hygiene
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Curriculum Vitae (CV)
Michael Schramm (PostDoc)
Birgit Blissenbach (PostDoc)
Marc Herb (doctoral student)
Alexander Gluschko (doctoral student)
Arlette Paillard (doctoral student)
Andrew Berdel (physician)
Pailin Pongratanakul (cand. med.)
Ulrike Karow (technician)
Katja Wiegmann (technician)