Olaf Utermöhlen

Host microbiota constantly control maturation and function of microglia in the CNS

As the tissue macrophages of the CNS, microglia are critically involved in diseases of the CNS. However, it remains unknown what controls their maturation and activation under homeostatic conditions. We observed substantial contributions of the host microbiota to microglia homeostasis, as germ-free (GF) mice displayed global defects in microglia with altered cell proportions and an immature phenotype, leading to impaired innate immune responses. Temporal eradication of host microbiota severely changed microglia properties. Limited microbiota complexity also resulted in defective microglia. In contrast, recolonization with a complex microbiota partially restored microglia features. We determined that short-chain fatty acids (SCFA), microbiota-derived bacterial fermentation products, regulated microglia homeostasis. Accordingly, mice deficient for the SCFA receptor FFAR2 mirrored microglia defects found under GF conditions. These findings suggest that host bacteria vitally regulate microglia maturation and function, whereas microglia impairment can be rectified to some extent by complex microbiota.

ISG15, an Interferon-Stimulated Ubiquitin-Like Protein, Is Not Essential for STAT1 Signaling and Responses against Vesicular Stomatitis and Lymphocytic Choriomeningitis Virus

ABSTRACT ISG15 is an interferon-induced ubiquitin-like modifier which can be conjugated to distinct, but largely unknown, proteins. ISG15 has been implicated in a variety of biological activities, which encompass antiviral defense, immune responses, and pregnancy. Mice lacking UBP43 (USP18), the ISG15-deconjugating enzyme, develop a severe phenotype with brain injuries and lethal hypersensitivity to poly(I:C). It has been reported that an augmented conjugation of ISG15 in the absence of UBP43 induces prolonged STAT1 phosphorylation and that the ISG15 conjugation plays an important role in the regulation of JAK/STAT and interferon signaling (O. A. Malakhova, M. Yan, M. P. Malakhov, Y. Yuan, K. J. Ritchie, K. I. Kim, L. F. Peterson, K. Shuai, and D. E. Zhang, Genes Dev. 17:455-460, 2003). Here, we report that ISG15−/− mice are viable and fertile and display no obvious abnormalities. Lack of ISG15 did not affect the development and composition of the main cellular compartments of the immune system. The interferon-induced antiviral state and immune responses directed against vesicular stomatitis virus and lymphocytic choriomeningitis virus were not significantly altered in the absence of ISG15. Furthermore, interferon- or endotoxin-induced STAT1 tyrosine-phosphorylation, as well as expression of typical STAT1 target genes, remained unaffected by the lack of ISG15. Thus, ISG15 is dispensable for STAT1 and interferon signaling.

Subinhibitory Concentrations of Linezolid Reduce Staphylococcus aureus Virulence Factor Expression

ABSTRACT The influence of the antibiotic linezolid on the secretion of exotoxins by Staphylococcus aureus was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis combined with matrix-assisted laser desorption ionization-time of flight mass spectrometry and Western blot analysis. S. aureus suspensions were treated with grading subinhibitory concentrations of linezolid (12.5, 25, 50, and 90% of MIC) at different stages of bacterial growth (i.e., an optical density at 540 nm [OD540] of 0.05 or 0.8). When added to S. aureus cultures at an OD540 of 0.05, linezolid reduced in a dose-dependent manner the secretion of specific virulence factors, including staphylococcal enterotoxin A (SEA) and SEB, bifunctional autolysin, autolysin, protein A, and alpha- and beta-hemolysins. In contrast, other presumably nontoxic exoproteins remained unchanged or even accumulated in supernatants in the presence of linezolid at a 90% MIC. Similarily, when added at OD540 of 0.8, that is, after quorum sensing, linezolid reduced the release of virulence factors, whereas the relative abundance of nontoxic exoproteins such as triacylglycerol lipase, glycerol ester hydrolase, DnaK, or translation elongation factor EF-Tu was found to be increased. Consistently, linezolid reduced in a dose-dependent manner the tumor necrosis factor-inducing activity secreted by S. aureus into the culture supernatants. The results of our study suggest that the expression of virulence factors in S. aureus is especially sensitive to the inhibition of protein synthesis by linezolid, which should be an advantage in the treatment of infections with toxin-producing S. aureus.

Riboflavin kinase couples TNF receptor 1 to NADPH oxidase

Reactive oxygen species (ROS) produced by NADPH oxidase function as defence and signalling molecules related to innate immunity and various cellular responses. The activation of NADPH oxidase in response to plasma membrane receptor activation depends on the phosphorylation of cytoplasmic oxidase subunits, their translocation to membranes and the assembly of all NADPH oxidase components. Tumour necrosis factor (TNF) is a prominent stimulus of ROS production, but the molecular mechanisms by which TNF activates NADPH oxidase are poorly understood. Here we identify riboflavin kinase (RFK, formerly known as flavokinase) as a previously unrecognized TNF-receptor-1 (TNFR1)-binding protein that physically and functionally couples TNFR1 to NADPH oxidase. In mouse and human cells, RFK binds to both the TNFR1-death domain and to p22phox, the common subunit of NADPH oxidase isoforms. RFK-mediated bridging of TNFR1 and p22phox is a prerequisite for TNF-induced but not for Toll-like-receptor-induced ROS production. Exogenous flavin mononucleotide or FAD was able to substitute fully for TNF stimulation of NADPH oxidase in RFK-deficient cells. RFK is rate-limiting in the synthesis of FAD, an essential prosthetic group of NADPH oxidase. The results suggest that TNF, through the activation of RFK, enhances the incorporation of FAD in NADPH oxidase enzymes, a critical step for the assembly and activation of NADPH oxidase.

DNA damage in germ cells induces an innate immune response that triggers systemic stress resistance

DNA damage responses have been well characterized with regard to their cell-autonomous checkpoint functions leading to cell cycle arrest, senescence and apoptosis. In contrast, systemic responses to tissue-specific genome instability remain poorly understood. In adult Caenorhabditis elegans worms germ cells undergo mitotic and meiotic cell divisions, whereas somatic tissues are entirely post-mitotic. Consequently, DNA damage checkpoints function specifically in the germ line, whereas somatic tissues in adult C. elegans are highly radio-resistant. Some DNA repair systems such as global-genome nucleotide excision repair (GG-NER) remove lesions specifically in germ cells. Here we investigated how genome instability in germ cells affects somatic tissues in C. elegans. We show that exogenous and endogenous DNA damage in germ cells evokes elevated resistance to heat and oxidative stress. The somatic stress resistance is mediated by the ERK MAP kinase MPK-1 in germ cells that triggers the induction of putative secreted peptides associated with innate immunity. The innate immune response leads to activation of the ubiquitin–proteasome system (UPS) in somatic tissues, which confers enhanced proteostasis and systemic stress resistance. We propose that elevated systemic stress resistance promotes endurance of somatic tissues to allow delay of progeny production when germ cells are genomically compromised.

Indoleamine 2,3-dioxygenase-expressing dendritic cells form suppurative granulomas following Listeria monocytogenes infection

Control of pathogens by formation of abscesses and granulomas is a major strategy of the innate immune system, especially when effector mechanisms of adaptive immunity are insufficient. We show in human listeriosis that DCs expressing indoleamine 2,3-dioxygenase (IDO), together with macrophages, are major cellular components of suppurative granulomas in vivo. Induction of IDO by DCs is a cell-autonomous response to Listeria monocytogenes infection and was also observed in other granulomatous infections with intracellular bacteria, such as Bartonella henselae. Reporting on our use of the clinically applied anti-TNF-alpha antibody infliximab, we further demonstrate in vitro that IDO induction is TNF-alpha dependent. Repression of IDO therefore might result in exacerbation of granulomatous diseases observed during anti-TNF-alpha therapy. These findings place IDO(+) DCs not only at the intersection of innate and adaptive immunity but also at the forefront of bacterial containment in granulomatous infections.

Concerted Action of the FasL/Fas and Perforin/Granzyme A and B Pathways Is Mandatory for the Development of Early Viral Hepatitis but Not for Recovery from Viral Infection

ABSTRACT Cytotoxic T lymphocytes (CTL) play a major role in the recovery from primary viral infections and the accompanying tissue injuries. However, it is unclear to what extent the two main cytolytic pathways, perforin-granzyme A and B exocytosis and Fas ligand (FasL)-Fas interaction, contribute to these processes. Here we have employed mouse strains with either spontaneous mutations or targeted gene defects in one or more components of either of the two cytolytic pathways to analyze the molecular basis of viral clearance and induction of hepatitis during lymphocytic choriomeningitis virus infection. Our results reveal that viral clearance is solely dependent on perforin but that virus-induced liver damage only occurs when both the FasL/Fas and the perforin pathways, including granzymes A and B, are simultaneously activated. The finding that development of hepatitis but not viral clearance is dependent on the concomitant activation of FasL-Fas and perforin-granzymes may be helpful in designing novel strategies to prevent hepatic failures during viral infections.

Reexamination of the Role of Ubiquitin-Like Modifier ISG15 in the Phenotype of UBP43-Deficient Mice

ABSTRACT UBP43/USP18 was described as a specific protease that removes conjugated ubiquitin-like modifier ISG15 from target proteins. The severe phenotype of UBP43−/− mice characterized by premature death, brain cell injury, and deregulated STAT1 signaling was ascribed to an enhanced conjugation of ISG15. In contrast, no phenotypic changes were detected in ISG15−/− mice. To verify the role of ISG15 in the phenotype of UBP43−/− mice, we employed mice deficient for both ISG15 and UBP43. Here, we show that the phenotype of UBP43−/− mice was not rescued by the absence of ISG15, as evident from unchanged mortality, neurological symptoms, and occurrence of hydrocephalus. Also, the reported hypersensitivity of UBP43−/− mice to an interferon inducer, poly(I · C), was ISG15 independent. Furthermore, no evidence for a role of ISG15 in the modulation of STAT1 signaling or in the resistance against lymphocytic choriomeningitis virus and vesicular stomatitis virus was found. Presented results clearly demonstrate that the phenotypic alterations of UBP43−/− mice are not caused by the lack of ISG15 deconjugation and must be due to another, non-ISG15-mediated molecular mechanism.

Severe Impairment in Early Host Defense Against Listeria monocytogenes in Mice Deficient in Acid Sphingomyelinase

The phagolysosomal compartment is crucial for the defense against infection with intracellular pathogens. Within this compartment, the TNF- and IFN-γ-responsive acid sphingomyelinase (ASMase) generates the signaling molecule ceramide, resulting in the activation of proteases like cathepsin D. To investigate the possible role of ASMase as a mediator of the antibacterial effects of TNF and IFN-γ, ASMase−/− mice were infected with Listeria monocytogenes. ASMase−/− mice showed a dramatically increased susceptibility to L. monocytogenes (LD50 ∼100 CFU) when compared with syngeneic wild-type mice (LD50 ∼10,000 CFU). In L. monocytogenes-challenged ASMase−/− mice, IFN-γ serum levels as well as IL-1β and IL-6 secretion by macrophages were similar to those observed in wild-type C57BL/6 mice. Although macrophages and granulocytes from ASMase−/− mice showed intact production of reactive nitrogen intermediates and oxidative burst, ASMase−/− macrophages proved completely incapable of restricting the growth of L. monocytogenes in vitro. The results of this study suggest that ASMase is crucially required for the intracellular control of L. monocytogenes in macrophages and granulocytes by nonoxidative mechanisms.

Fusogenicity of membranes: The impact of acid sphingomyelinase on innate immune responses

Acid sphingomyelinase (ASMase) has been implemented in cellular signaling mainly because its reaction product, ceramide, has been assumed to be a mediator within signaling pathways. Our studies of three independent infection systems show that ASMase is required for phago-lysosomal fusion in macrophages infected with Listeria monocytogenes, for exocytosis of secretory lysosomes by lymphocytic choriomeningitis virus-specific cytotoxic T cells, and for generation of multinucleated giant cells in granuloma of mice infected with Mycobacterium avium. Because of its neutral lipid nature, ceramide is confined to the membranes of phagosomes and lysosomes or the extracellular leaflet of the plasma membrane. In light of the biochemical and biophysical properties of ceramide, we provide a model suggesting that ASMase regulates select vesicular fusion processes by modifying the steric conformation of cellular membranes.