Riboflavin, also known as vitamin B₂, is converted by riboflavin kinase (RFK) into flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are essential cofactors of dehydrogenases, reductases, and oxidases including the phagocytic NADPH oxidase 2 (Nox2). Riboflavin deficiency is common in young adults and elderly individuals, who are at the coincidental risk for listeriosis. To address the impact of acute riboflavin deficiency on host defense against Listeria monocytogenes (L.m.), we generated conditional RFK knockout (KO) strains of mice. Phagocyte‐specific RFK KO impaired the capability of phagocytes to control intracellular L.m., which corresponded to a greater susceptibility of mice to in vivo challenge with L.m. The oxidative burst of RFK‐deficient phagocytes in response to L.m. infection was significantly reduced. Mechanistically, TNF‐induced priming of Nox2, which is needed for oxidative burst, was defective in RFK‐deficient phagocytes. Lack 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. Vice versa, ROS production by riboflavin‐deprived macrophages was rapidly restored by riboflavin supplementation. Our results suggest that acute riboflavin deficiency immediately impairs priming of Nox2, which is of crucial relevance for an effective phagocytic immune response in vivo.

Background: The shiga toxin-producing E. coli (STEC) O104:H4 caused a major outbreak in Germany in spring 2011. STEC are usually susceptible to common antibiotics. However, antibiotic treatment of STEC-infected patients is not recommended because STEC may enhance production and release of shiga toxins (STX) in response to antibiotics, which eventually enhances the frequency and severity of clinical symptoms, including haemolytic uraemic syndrome (HUS) and fatalities. Results: We characterized the response to antibiotics of STEC O104:H4 isolates from two HUS patients during the German STEC outbreak in spring 2011 in comparison to the common STEC O157:H7. Liquid cultures of STEC O157:H7 and O104:H4 were incubated with graded dilutions of the antibiotics ciprofloxacin, meropenem, fosfomycin, gentamicin, rifampicin, and chloramphenicol. At defined times of antibiotic treatment, transcriptional activation of the STX2 gene, contents of STX and STX-activity in the culture supernatants were quantified. Unlike the common serotype O157:H7, STEC O104:H4 does not release STX in response to therapeutic concentrations of ciprofloxacin, meropenem, fosfomycin, and chloramphenicol. Conclusions: In future outbreaks, the response of the respective epidemiologic STEC strain to antibiotics should be rapidly characterized in order to identify antibiotics that do not enhance the release of STX. This will eventually allow clinical studies tackling the question whether antibiotic treatment impacts on the eradication of STEC, clinical course of disease, and frequency of carriers.

Severe defects in human IFNγ immunity predispose individuals to both Bacillus Calmette–Guérin disease and tuberculosis, whereas milder defects predispose only to tuberculosis¹. Here we report two adults with recurrent pulmonary tuberculosis who are homozygous for a private loss-of-function TNF variant. Neither has any other clinical phenotype and both mount normal clinical and biological inflammatory responses. Their leukocytes, including monocytes and monocyte-derived macrophages (MDMs) do not produce TNF, even after stimulation with IFNγ. Blood leukocyte subset development is normal in these patients. However, an impairment in the respiratory burst was observed in granulocyte–macrophage colony-stimulating factor (GM-CSF)-matured MDMs and alveolar macrophage-like (AML) cells² from both patients with TNF deficiency, TNF- or TNFR1-deficient induced pluripotent stem (iPS)-cell-derived GM-CSF-matured macrophages, and healthy control MDMs and AML cells differentiated with TNF blockers in vitro, and in lung macrophages treated with TNF blockers ex vivo. The stimulation of TNF-deficient iPS-cell-derived macrophages with TNF rescued the respiratory burst. These findings contrast with those for patients with inherited complete deficiency of the respiratory burst across all phagocytes, who are prone to multiple infections, including both Bacillus Calmette–Guérin disease and tuberculosis³. Human TNF is required for respiratory-burst-dependent immunity to Mycobacterium tuberculosis in macrophages but is surprisingly redundant otherwise, including for inflammation and immunity to weakly virulent mycobacteria and many other infectious agents.

Loss of skeletal muscle mass is one of the most widespread and deleterious processes in aging humans. However, the mechanistic metabolic principles remain poorly understood. In the framework of a multi‐organ investigation of age‐associated changes of ceramide species, a unique and distinctive change pattern of C_16:0 and C_18:0 ceramide species was detected in aged skeletal muscle. Consistently, the expression of CerS1 and CerS5 mRNA, encoding the ceramide synthases (CerS) with substrate preference for C_16:0 and C_18:0 acyl chains, respectively, was down‐regulated in skeletal muscle of aged mice. Similarly, an age‐dependent decline of both CerS1 and CerS5 mRNA expression was observed in skeletal muscle biopsies of humans. Moreover, CerS1 and CerS5 mRNA expression was also reduced in muscle biopsies from patients in advanced stage of chronic heart failure (CHF) suffering from muscle wasting and frailty. The possible impact of CerS1 and CerS5 on muscle function was addressed by reversed genetic analysis using CerS1^Δ/Δ and CerS5^Δ/Δ knockout mice. Skeletal muscle from mice deficient of either CerS1 or CerS5 showed reduced caliber sizes of both slow (type 1) and fast (type 2) muscle fibers, fiber grouping, and fiber switch to type 1 fibers. Moreover, CerS1‐ and CerS5‐deficient mice exhibited reduced twitch and tetanus forces of musculus extensor digitorum longus. The findings of this study link CerS1 and CerS5 to histopathological changes and functional impairment of skeletal muscle in mice that might also play a functional role for the aging skeletal muscle and for age‐related muscle wasting disorders in humans.

Ceramide synthase 5 is one of six enzymes that catalyze the production of ceramides from sphingosine or sphinganine. Ceramides are important components of cell membranes and act as signaling molecules. Previously it has been shown that ceramide synthase 6 and 2 influence colitis in several animal models with sometimes opposite effects. Here, we investigated the disease course of dextran sodium sulfate-induced acute colitis and azoxymethane/dextran sodium sulfate-induced colitis-associated colon cancer in mice with global ceramide synthase 5 knockout (CerS5-ko) or with ceramide synthase 5 knockout restricted to the colon epithelium (CerS5fl/fl VilCre). We monitored disease development and analyzed colon barrier function as well as the immune cell status in these mice. CerS5-ko mice but not CerS5fl/fl-VilCre mice were more susceptible to acute and chronic inflammation. However, the cell barrier function of colon epithelial cells was not disturbed by downregulation of ceramide synthase 5. Instead, untreated CerS5-ko mice displayed reduced numbers of CD3⁺ immune cells in the spleen, colon, and blood, especially of intraepithelial CD8⁺ T-cells, which was not obvious in CerS5fl/fl Vil Cre mice. Reduced T-cell number in colon tissue of CerS5-ko mice was accompanied by a reduced expression of IL-1β, IFNγ, and IL-4. In vitro investigations revealed that knockdown of ceramide synthase 5 in T-cells impaired T-cell activation. In summary, we show that CerS5-ko mice were more susceptible to dextran sodium sulfate-induced colitis and azoxymethane/dextran sodium sulfate-induced colitis-associated colon cancer. A reduced number of T-cells in the colon epithelium that was already the case in untreated CerS5-ko mice might have contributed to this effect.