The influenza A virus promotes fungal growth of Aspergillus fumigatus via direct interaction in vitro.

Seasonal influenza A virus (IAV) infections still pose a major burden for public health worldwide. Severe disease progression or even death is often related to superinfections of the virus and a secondary bacterial pathogen. However, fungi, especially Aspergillus fumigatus, are also frequently diagnosed during IAV infection. Although, clinical studies have reported the severity of influenza-associated pulmonary aspergillosis, the molecular mechanisms underlying this type of disease are poorly understood. Here, a new in vitro model is introduced that allows the investigation of complex pathogen-host and pathogen-pathogen interactions during coinfection of lung epithelial cells with IAV and A. fumigatus. Our data reveal a reduced IAV load and IAV-induced cytokine and chemokine expression in the presence of A. fumigatus. At the same time, IAV infection promotes the growth of A. fumigatus. Even in the absence of the human host cell, purified IAV particles are able to induce hyphal growth, due to a direct interaction of the virus particles with the fungal surface. Thus, our study gives first insights into the complex interplay between IAV, A. fumigatus and the host cell as well as the two pathogens alone.

D,L-Lysine-Acetylsalicylate + Glycine (LASAG) Reduces SARS-CoV-2 Replication and Shows an Additive Effect with Remdesivir.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing the coronavirus disease-19 (COVID-19) is still challenging healthcare systems and societies worldwide. While vaccines are available, therapeutic strategies are developing and need to be adapted to each patient. Many clinical approaches focus on the repurposing of approved therapeutics against other diseases. However, the efficacy of these compounds on viral infection or even harmful secondary effects in the context of SARS-CoV-2 infection are sparsely investigated. Similarly, adverse effects of commonly used therapeutics against lifestyle diseases have not been studied in detail. Using mono cell culture systems and a more complex chip model, we investigated the effects of the acetylsalicylic acid (ASA) salt D,L-lysine-acetylsalicylate + glycine (LASAG) on SARS-CoV-2 infection in vitro. ASA is commonly known as Aspirin® and is one of the most frequently used medications worldwide. Our data indicate an inhibitory effect of LASAG on SARS-CoV-2 replication and SARS-CoV-2-induced expression of pro-inflammatory cytokines and coagulation factors. Remarkably, our data point to an additive effect of the combination of LASAG and the antiviral acting drug remdesivir on SARS-CoV-2 replication in vitro.

The Inflammatory Profile of Obesity and the Role on Pulmonary Bacterial and Viral Infections.

Obesity is a globally increasing health problem, entailing diverse comorbidities such as infectious diseases. An obese weight status has marked effects on lung function that can be attributed to mechanical dysfunctions. Moreover, the alterations of adipocyte-derived signal mediators strongly influence the regulation of inflammation, resulting in chronic low-grade inflammation. Our review summarizes the known effects regarding pulmonary bacterial and viral infections. For this, we discuss model systems that allow mechanistic investigation of the interplay between obesity and lung infections. Overall, obesity gives rise to a higher susceptibility to infectious pathogens, but the pathogenetic process is not clearly defined. Whereas, viral infections often show a more severe course in obese patients, the same patients seem to have a survival benefit during bacterial infections. In particular, we summarize the main mechanical impairments in the pulmonary tract caused by obesity. Moreover, we outline the main secretory changes within the expanded adipose tissue mass, resulting in chronic low-grade inflammation. Finally, we connect these altered host factors to the influence of obesity on the development of lung infection by summarizing observations from clinical and experimental data.

Heater-Cooler Devices and Risk of Contamination during Cardiac Surgery.

BACKGROUND: Heater-cooler devices (HCD) have been implicated in a cardiosurgical contamination scenario causing prosthetic valve endocarditis. AIM: We characterized contamination of new HCDs and assessed the risk of intraoperative microorganism transmission from the HCD to the operating field. METHODS: We initially acquired four new FlexTherm and then four new Maquet HCU40 HCDs and assessed occurrence and speed of microbial contamination (including mycobacteria) assessing swab and water samples from the device. In parallel, we collected repeated samples from different sites in the operating room either by swab sticks or by exposing different sample plates to room air. We also reviewed microbiological results from the hospital and compared them to cardiosurgical wound infections and endocarditis cases. Finally, we simulated cardiosurgical conditions and assessed the devices' ability to expel air to the operative field. RESULTS: All new HCDs were clean before first use. Despite authority-mandated decontamination procedures, microbial growth (Fusarium solani, Sphingomonas paucimobilis, Pseudomonas aeruginosa, Mycobacterium chelonae, and gordonae) was identified in all HCDs over time and could not be permanently eliminated. Four of these mircoorganisms were also found in tap water. However, none of the HCD-organisms were found inside the laminar airflow operating area. Importantly, except for P. aeruginosa, none of the HCD organisms were found in patients with surgical wound infections or endocarditis. HCD-expelled air did not rise more than 40 cm above ground. CONCLUSION: HCDs cannot be expected to remain sterile despite extensive decontamination procedures. However, airborne transmission of microorganisms directly from the HCD to the operating field appears unlikely.

Staphylococcus aureus-Derived α-Hemolysin Evokes Generation of Specialized Pro-resolving Mediators Promoting Inflammation Resolution.

Underlying mechanisms of how infectious inflammation is resolved by the host are incompletely understood. One hallmark of inflammation resolution is the activation of specialized pro-resolving mediators (SPMs) that enhance bacterial clearance and promote tissue repair. Here, we reveal α-hemolysin (Hla) from Staphylococcus aureus as a potent elicitor of SPM biosynthesis in human M2-like macrophages and in the mouse peritoneum through selective activation of host 15-lipoxygenase-1 (15-LOX-1). S. aureus-induced SPM formation in M2 is abolished upon Hla depletion or 15-LOX-1 knockdown. Isolated Hla elicits SPM formation in M2 that is reverted by inhibition of the Hla receptor ADAM10. Lipid mediators derived from Hla-treated M2 accelerate planarian tissue regeneration. Hla but not zymosan provokes substantial SPM formation in the mouse peritoneum, devoid of leukocyte infiltration and pro-inflammatory cytokine secretion. Besides harming the host, Hla may also exert beneficial functions by stimulating SPM production to promote the resolution of infectious inflammation.

Co-infection with Staphylococcus aureus after primary influenza virus infection leads to damage of the endothelium in a human alveolus-on-a-chip model.

Pneumonia is one of the most common infectious diseases worldwide. The influenza virus can cause severe epidemics, which results in significant morbidity and mortality. Beyond the virulence of the virus itself, epidemiological data suggest that bacterial co-infections are the major cause of increased mortality. In this context, Staphylococcus aureus represents a frequent causative bacterial pathogen. Currently available models have several limitations in the analysis of the pathogenesis of infections, e.g. some bacterial toxins strongly act in a species-specific manner. Human 2D mono-cell culture models often fail to maintain the differentiation of alveolus-specific functions. A detailed investigation of the underlying pathogenesis mechanisms requires a physiological interaction of alveolus-specific cell types. The aim of the present work was to establish a human in vitro alveolus model system composed of vascular and epithelial cell structures with cocultured macrophages resembling the human alveolus architecture and functions. We demonstrate that high barrier integrity maintained for up to 14 d in our model containing functional tissue-resident macrophages. We show that flow conditions and the presence of macrophages increased the barrier function. The infection of epithelial cells induced a high inflammatory response that spread to the endothelium. Although the integrity of the epithelium was not compromised by a single infection or co-infection, we demonstrated significant endothelial cell damage associated with loss of barrier function. We established a novel immune-responsive model that reflects the complex crosstalk between pathogens and host. The in vitro model allows for the monitoring of spatiotemporal spreading of the pathogens and the characterization of morphological and functional alterations attributed to infection. The alveolus-on-a-chip represents a promising platform for mechanistic studies of host-pathogen interactions and the identification of molecular and cellular targets of novel treatment strategies in pneumonia.

ClpC affects the intracellular survival capacity of Staphylococcus aureus in non-professional phagocytic cells.

Invasion and persistence of bacteria within host cells requires that they adapt to life in an intracellular environment. This adaptation induces bacterial stress through events such as phagocytosis and enhanced nutrient-restriction. During stress, bacteria synthesize a family of proteins known as heat shock proteins (HSPs) to facilitate adaptation and survival. Previously, we determined the Staphylococcus aureus HSP ClpC temporally alters bacterial metabolism and persistence. This led us to hypothesize that ClpC might alter intracellular survival. Inactivation of clpC in S. aureus strain DSM20231 significantly enhanced long-term intracellular survival in human epithelial (HaCaT) and endothelial (EA.hy926) cell lines, without markedly affecting adhesion or invasion. This phenotype was similar across a genetically diverse collection of S. aureus isolates, and was influenced by the toxin/antitoxin encoding locus mazEF. Importantly, MazEF alters mRNA synthesis and/or stability of S. aureus virulence determinants, indicating ClpC may act through the mRNA modulatory activity of MazEF. Transcriptional analyses of total RNAs isolated from intracellular DSM20231 and isogenic clpC mutant cells identified alterations in transcription of α-toxin (hla), protein A (spa), and RNAIII, consistent with the hypothesis that ClpC negatively affects the intracellular survival of S. aureus in non-professional phagocytic cells, via modulation of MazEF and Agr.

Mitogen-activated protein kinases (MAPKs) regulate IL-6 over-production during concomitant influenza virus and Staphylococcus aureus infection.

Bacterial super-infections are a major complication of influenza virus (IV) infections and often lead to severe pneumonia. One hallmark of IV-associated Staphylococcus aureus (S. aureus) infection is rapid progression to a serious disease outcome. Changes in immune and inflammatory host responses increase morbidity and complicate efficient therapy. A key player during inflammation is the multifunctional cytokine IL-6. Although increased IL-6 levels have been observed after severe disease upon IV and/or bacterial super-infection, the underlying molecular mechanisms still remain to be elucidated. In the present study, we focused on cellular signalling pathways regulating IL-6 production upon IV/S. aureus super-infection. Additionally, infection with viable bacteria was mimicked by lipoteichoic acid stimulation in this model. Analyses of cellular signalling mechanisms revealed synergistically increased activation of the MAPK p38 as well as enhanced phosphorylation of the MAPKs ERK1/2 and JNK in the presence of super-infecting bacteria. Interestingly, inhibition of MAPK activity indicated a strong dependence of IL-6 expression on p38 and ERK1/2, while the MAPK JNK seems not to be involved. Thus, our results provide new molecular insights into the regulation of IL-6, a marker of severe disease, which might contribute to the lethal synergism of IV and S. aureus.

Per2 induction limits lymphoid-biased haematopoietic stem cells and lymphopoiesis in the context of DNA damage and ageing.

Ageing-associated impairments in haemato-lymphopoiesis are associated with DNA damage accumulation and reduced maintenance of lymphoid-biased (Ly-biased) compared with myeloid-biased (My-biased) haematopoietic stem cells (HSCs). Here, in vivo RNAi screening identifies period circadian clock 2 (Per2) as a critical factor limiting the maintenance of HSCs in response to DNA damage and ageing. Under these conditions, Per2 is activated predominantly in Ly-biased HSCs and stimulates DNA damage signalling and p53-dependent apoptosis in haematopoietic cells. Per2 deletion ameliorates replication stress and DNA damage responses in haematopoietic cells, thereby improving the maintenance of Ly-biased HSCs, lymphopoiesis, and immune function in ageing mice without increasing the accumulation of DNA damage. Per2-deficient mice retain Batf/p53-dependent induction of differentiation of HSCs in response to DNA damage and exhibit an elongated lifespan. Together, these results identify Per2 as a negative regulator of Ly-biased HSCs and immune functions in response to DNA damage and ageing.

Clinical Significance and Pathogenesis of Staphylococcal Small Colony Variants in Persistent Infections.

Small colony variants (SCVs) were first described more than 100 years ago for Staphylococcus aureus and various coagulase-negative staphylococci. Two decades ago, an association between chronic staphylococcal infections and the presence of SCVs was observed. Since then, many clinical studies and observations have been published which tie recurrent, persistent staphylococcal infections, including device-associated infections, bone and tissue infections, and airway infections of cystic fibrosis patients, to this special phenotype. By their intracellular lifestyle, SCVs exhibit so-called phenotypic (or functional) resistance beyond the classical resistance mechanisms, and they can often be retrieved from therapy-refractory courses of infection. In this review, the various clinical infections where SCVs can be expected and isolated, diagnostic procedures for optimized species confirmation, and the pathogenesis of SCVs, including defined underlying molecular mechanisms and the phenotype switch phenomenon, are presented. Moreover, relevant animal models and suggested treatment regimens, as well as the requirements for future research areas, are highlighted.

Dietary restriction improves repopulation but impairs lymphoid differentiation capacity of hematopoietic stem cells in early aging.

Dietary restriction (DR) improves health, delays tissue aging, and elongates survival in flies and worms. However, studies on laboratory mice and nonhuman primates revealed ambiguous effects of DR on lifespan despite improvements in health parameters. In this study, we analyzed consequences of adult-onset DR (24 h to 1 yr) on hematopoietic stem cell (HSC) function. DR ameliorated HSC aging phenotypes, such as the increase in number of HSCs and the skewing toward myeloid-biased HSCs during aging. Furthermore, DR increased HSC quiescence and improved the maintenance of the repopulation capacity of HSCs during aging. In contrast to these beneficial effects, DR strongly impaired HSC differentiation into lymphoid lineages and particularly inhibited the proliferation of lymphoid progenitors, resulting in decreased production of peripheral B lymphocytes and impaired immune function. The study shows that DR-dependent suppression of growth factors and interleukins mediates these divergent effects caused by DR. Supplementation of insulin-like growth factor 1 partially reverted the DR-induced quiescence of HSCs, whereas IL-6/IL-7 substitutions rescued the impairment of B lymphopoiesis exposed to DR. Together, these findings delineate positive and negative effects of long-term DR on HSC functionality involving distinct stress and growth signaling pathways.

MRI visualization of Staphyloccocus aureus-induced infective endocarditis in mice.

Infective endocarditis (IE) is a severe and often fatal disease, lacking a fast and reliable diagnostic procedure. The purpose of this study was to establish a mouse model of Staphylococcus aureus-induced IE and to develop a MRI technology to characterize and diagnose IE. To establish the mouse model of hematogenous IE, aortic valve damage was induced by placing a permanent catheter into right carotid artery. 24 h after surgery, mice were injected intravenously with either iron particle-labeled or unlabeled S. aureus (strain 6850). To distinguish the effect of IE from mere tissue injury or recruited macrophages, subgroups of mice received sham surgery prior to infection (n = 17), received surgery without infection (n = 8), or obtained additionally injection of free iron particles to label macrophages (n = 17). Cardiac MRI was performed 48 h after surgery using a self-gated ultra-short echo time (UTE) sequence (TR/TE, 5/0.31 ms; in-plane/slice, 0.125/1 mm; duration, 12∶08 min) to obtain high-resolution, artifact-free cinematographic images of the valves. After MRI, valves were either homogenized and plated on blood agar plates for determination of bacterial titers, or sectioned and stained for histology. In the animal model, both severity of the disease and mortality increased with bacterial numbers. Infection with 105 S. aureus bacteria reliably caused endocarditis with vegetations on the valves. Cinematographic UTE MRI visualised the aortic valve over the cardiac cycle and allowed for detection of bacterial vegetations, while mere tissue trauma or labeled macrophages were not detected. Iron labeling of S. aureus was not required for detection. MRI results were consistent with histology and microbial assessment. These data showed that S. aureus-induced IE in mice can be detected by MRI. The established mouse model allows for investigation of the pathophysiology of IE, testing of novel drugs and may serve for the development of a clinical diagnostic strategy.

Soluble CD163 masks fibronectin-binding protein A-mediated inflammatory activation of Staphylococcus aureus infected monocytes.

Binding to fibronectin (FN) is a crucial pathogenic factor of Staphylococcus aureus mediated by fibronectin-binding protein A (FnBP-A) and extracellular adherence protein (Eap). Recently, we have shown that binding of soluble CD163 (sCD163) to FN linked to these molecules exhibits anti-microbial effects by enhancing phagocytosis and killing activity of S. aureus-infected monocytes. However, it remained unclear whether sCD163 also influences the monocytic activation status. Using genetically modified staphylococcal strains we now identified FnBP-A, but not Eap, as activator of the inflammatory response of monocytes to infection. FnBP-A-mediated inflammatory activation was masked by sCD163 binding to S. aureus promoting efficient pathogen elimination. Thus, sCD163 protects monocytes from overwhelming activation upon staphylococcal infection by dampening the secretion of pro-inflammatory cytokines TNFα, IL-1ß, IL-6 and IL-8 and DAMP molecule MRP8/14. Moreover, sCD163 limited expression of pro-apoptotic transcription factor NR4A1 induced during S. aureus infection and inhibited induction of chemokine CXCL2promoting survival of staphylococci in vivo. sCD163-mediated effects were not due to general immunosuppression since MAP kinase activation and ROS production were unaltered during infection of monocytes with sCD163-bound bacteria. Thus, sCD163 promotes a specific defence of the immune system against FnBP-A-mediated inflammatory activation enabling successful pathogen elimination, tissue recovery and resolution of inflammation.

Bacteria tracking by in vivo magnetic resonance imaging.

BACKGROUND: Different non-invasive real-time imaging techniques have been developed over the last decades to study bacterial pathogenic mechanisms in mouse models by following infections over a time course. In vivo investigations of bacterial infections previously relied mostly on bioluminescence imaging (BLI), which is able to localize metabolically active bacteria, but provides no data on the status of the involved organs in the infected host organism. In this study we established an in vivo imaging platform by magnetic resonance imaging (MRI) for tracking bacteria in mouse models of infection to study infection biology of clinically relevant bacteria. RESULTS: We have developed a method to label Gram-positive and Gram-negative bacteria with iron oxide nano particles and detected and pursued these with MRI. The key step for successful labeling was to manipulate the bacterial surface charge by producing electro-competent cells enabling charge interactions between the iron particles and the cell wall. Different particle sizes and coatings were tested for their ability to attach to the cell wall and possible labeling mechanisms were elaborated by comparing Gram-positive and -negative bacterial characteristics. With 5-nm citrate-coated particles an iron load of 0.015 ± 0.002 pg Fe/bacterial cell was achieved for Staphylococcus aureus. In both a subcutaneous and a systemic infection model induced by iron-labeled S. aureus bacteria, high resolution MR images allowed for bacterial tracking and provided information on the morphology of organs and the inflammatory response. CONCLUSION: Labeled with iron oxide particles, in vivo detection of small S. aureus colonies in infection models is feasible by MRI and provides a versatile tool to follow bacterial infections in vivo. The established cell labeling strategy can easily be transferred to other bacterial species and thus provides a conceptual advance in the field of molecular MRI.

A novel mouse model of Staphylococcus aureus chronic osteomyelitis that closely mimics the human infection: an integrated view of disease pathogenesis.

Osteomyelitis is a serious bone infection typically caused by Staphylococcus aureus. The pathogenesis of osteomyelitis remains poorly understood, mainly for lack of experimental models that closely mimic human disease. We describe a novel murine model of metastatic chronic osteomyelitis initiated after intravenous inoculation of S. aureus microorganisms. The bacteria entered bones through the bloodstream and, after an acute phase with progressive growth (first 2 weeks after infection), they remained at constant numbers for up to 56 days (chronic phase). Clinical signs of illness and systemic inflammation were apparent only during the acute phase. Bone destruction and remodeling processes were readily detectable by magnetic resonance and X-ray imaging 3 weeks after infection, and high levels of bone deformation were observed during the chronic phase. Histological examination of infected bones demonstrated suppurative inflammation with foci of intense bacterial multiplication and necrosis during acute infection and osteoclastic resorption accompanied by new woven bone formation during chronic infection. Transmission electron microscopy revealed S. aureus microorganisms forming microcolonies within the nonmineralized collagen matrix or located intracellularly within neutrophils. In summary, our mouse model of staphylococcal hematogenous osteomyelitis precisely reproduces most features of the human disease. Although the extent of lesions in the chronic phase was subject to variation, this model is ideal for testing and monitoring novel treatment modalities via noninvasive imaging.

Early detection of lung inflammation: exploiting T1-effects of iron oxide particles using UTE MRI.

At high magnetic fields diagnostic proton MRI of the lung is problematic, because of fast T2* relaxation. The application of superparamagnetic contrast agents and the exploitation of the corresponding T2* effect is inefficient with conventional MRI methods, which limits the early detection of lung diseases. However, a simple theoretical treatment shows that in the lung, by the use of ultra-short echo time sequences, T2* effects can be neglected while T(1) shortening effects can be used for signal detection. In our study, we have applied a theoretically and experimentally optimized 3D ultra-short echo time sequence to lung phantoms and to a mouse model of lung inflammation, which was induced by systemic bacterial infection. Following the systemic application of very small superparamagnetic iron oxide nanoparticles, a significant signal increase in the lung of infected animals was detected already at 24 h postinfection, compared to control mice (17%, P < 0.001). Iron accumulation in the lung parenchyma as consequence of the host immune response was histologically confirmed. By conventional T2*- and T(2)-weighted imaging, neither structural changes nor formation of substantial edema were observed.

Soluble CD163 promotes recognition, phagocytosis and killing of Staphylococcus aureus via binding of specific fibronectin peptides.

CD163 is a multi-ligand scavenger receptor exclusively expressed by monocytes and macrophages, which is released after their activation during sepsis (sCD163). The biological relevance of sCD163, however, is not yet clear. We now demonstrate that sCD163 exhibits direct antimicrobial effects by recognizing a specific subfragment ((6) F1(1) F2(2) F2(7) F1) of fibronectin (FN) bound to staphylococcal surface molecules. Moreover, contact with staphylococci promotes sCD163-shedding from monocyte surface via induction of metalloproteinases ADAM10 and ADAM17. sCD163 subsequently binds to Staphylococcus aureus via FN peptides and strongly amplifies phagocytosis as well as killing by monocytes and to a lesser extend by neutrophils. This mechanism exhibits additional paracrine effects because staphylococci additionally opsonized by sCD163 induce higher activation and more efficient killing activity of non-professional phagocytes like endothelial cells. Targeting pathogen-bound FN by sCD163 would be a very sophisticated strategy to attack S. aureus as any attempt of the pathogen to avoid this defence mechanism will automatically bring about loss of adherence to the host protein FN, which is a pivotal patho-mechanism of highly invasive staphylococcal strains. Thus, we report a novel function for sCD163 that is of particular importance for immune defence of the host against S. aureus infections.