Predictors of COVID-19 in an outpatient fever clinic.

BACKGROUND: The objective of this study was to identify clinical risk factors for COVID-19 in a German outpatient fever clinic that allow distinction of SARS-CoV-2 infected patients from other patients with flu-like symptoms. METHODS: This is a retrospective, single-centre cohort study. Patients were included visiting the fever clinic from 4th of April 2020 to 15th of May 2020. Symptoms, comorbidities, and socio-demographic factors were recorded in a standardized fashion. Multivariate logistic regression was used to identify risk factors of COVID-19, on the bases of those a model discrimination was assessed using area under the receiver operation curves (AUROC). RESULTS: The final analysis included 930 patients, of which 74 (8%) had COVID-19. Anosmia (OR 10.71; CI 6.07-18.9) and ageusia (OR 9.3; CI 5.36-16.12) were strongly associated with COVID-19. High-risk exposure (OR 12.20; CI 6.80-21.90), especially in the same household (OR 4.14; CI 1.28-13.33), was also correlated; the more household members, especially with flu-like symptoms, the higher the risk of COVID-19. Working in an essential workplace was also associated with COVID-19 (OR 2.35; CI 1.40-3.96), whereas smoking was inversely correlated (OR 0.19; CI 0.08-0.44). A model that considered risk factors like anosmia, ageusia, concomitant of symptomatic household members and smoking well discriminated COVID-19 patients from other patients with flu-like symptoms (AUROC 0.84). CONCLUSIONS: We report a set of four readily available clinical parameters that allow the identification of high-risk individuals of COVID-19. Our study will not replace molecular testing but will help guide containment efforts while waiting for test results.

DJ-1 (Park7) affects the gut microbiome, metabolites and the development of innate lymphoid cells (ILCs).

The proper communication between gut and brain is pivotal for the maintenance of health and, dysregulation of the gut-brain axis can lead to several clinical disorders. In Parkinson's disease (PD) 85% of all patients experienced constipation many years before showing any signs of motor phenotypes. For differential diagnosis and preventive treatment, there is an urgent need for the identification of biomarkers indicating early disease stages long before the disease phenotype manifests. DJ-1 is a chaperone protein involved in the protection against PD and genetic mutations in this protein have been shown to cause familial PD. However, how the deficiency of DJ-1 influences the risk of PD remains incompletely understood. In the present study, we provide evidence that DJ-1 is implicated in shaping the gut microbiome including; their metabolite production, inflammation and innate immune cells (ILCs) development. We revealed that deficiency of DJ-1 leads to a significant increase in two specific genera/species, namely Alistipes and Rikenella. In DJ-1 knock-out (DJ-1-/-) mice the production of fecal calprotectin and MCP-1 inflammatory proteins were elevated. Fecal and serum metabolic profile showed that malonate which influences the immune system was significantly more abundant in DJ-1-/- mice. DJ-1 appeared also to be involved in ILCs development. Further, inflammatory genes related to PD were augmented in the midbrain of DJ-1-/- mice. Our data suggest that metabolites and inflammation produced in the gut could be used as biomarkers for PD detection. Perhaps, these metabolites and inflammatory mediators could be involved in triggering inflammation resulting in PD pathology.

Oral intake of lipopolysaccharide regulates toll-like receptor 4-dependent granulopoiesis.

IMPACT STATEMENT: In our present study, we investigated the impact of LPS on neutrophil homeostasis and found that oral intake is sufficient to induce hematopoietic stem and progenitor cell fate decisions towards the neutrophil lineage independent of G-CSF. In addition, TLR4 has been identified as the indispensable sensor for oral LPS-modulated steady-state granulopoiesis. We provide evidence that the gastrointestinal microbiome is critical for neutrophil homeostasis, which has implications for patients being treated with chemotherapy or antimicrobial therapy, since both are significantly influencing the composition of the intestinal microbiome.

Wheat Consumption Aggravates Colitis in Mice via Amylase Trypsin Inhibitor-mediated Dysbiosis.

BACKGROUND & AIMS: Wheat has become the world's major staple and its consumption correlates with prevalence of noncommunicable disorders such as inflammatory bowel diseases. Amylase trypsin inhibitors (ATIs), a component of wheat, activate the intestine's innate immune response via toll-like receptor 4 (TLR4). We investigated the effects of wheat and ATIs on severity of colitis and fecal microbiota in mice. METHODS: C57BL/6 wild-type and Tlr4-/- mice were fed wheat- or ATI-containing diets or a wheat-free (control) diet and then given dextran sodium sulfate to induce colitis; we also studied Il10-/- mice, which develop spontaneous colitis. Changes in fecal bacteria were assessed by taxa-specific quantitative polymerase chain reaction and 16S ribosomal RNA metagenomic sequencing. Feces were collected from mice on wheat-containing, ATI-containing, control diets and transplanted to intestines of mice with and without colitis on control or on ATI-containing diets. Intestinal tissues were collected and analyzed by histology, immunohistochemistry, and flow cytometry. Bacteria with reported immunomodulatory effects were incubated with ATIs and analyzed in radial diffusion assays. RESULTS: The wheat- or ATI-containing diets equally increased inflammation in intestinal tissues of C57BL/6 mice with colitis, compared with mice on control diets. The ATI-containing diet promoted expansion of taxa associated with development of colitis comparable to the wheat-containing diet. ATIs inhibited proliferation of specific human commensal bacteria in radial diffusion assays. Transplantation of microbiota from feces of mice fed the wheat- or ATI-containing diets to intestines of mice on control diets increased the severity of colitis in these mice. The ATI-containing diet did not increase the severity of colitis in Tlr4-/- mice. CONCLUSIONS: Consumption of wheat or wheat ATIs increases intestinal inflammation in mice with colitis, via TLR4, and alters their fecal microbiota. Wheat-based, ATI-containing diets therefore activate TLR4 signaling and promote intestinal dysbiosis.

Gut Commensal-Induced IκBζ Expression in Dendritic Cells Influences the Th17 Response.

Intestinal commensal bacteria can have a large impact on the state of health and disease of the host. Regulation of Th17 cell development by gut commensals is known to contribute to their dichotomous role in promoting gut homeostasis and host defense, or development of autoimmune diseases. Yet, the underlying mechanisms remain to be fully elucidated. One candidate factor contributing to Th17 differentiation, and the expression of which could be influenced by commensals is the atypical nuclear IκB protein IκBζ. IκBζ acts as a transcriptional regulator of the expression of Th17-related secondary response genes in many cell types including dendritic cells (DCs). Insights into the regulation of IκBζ in DCs could shed light on how these immune sentinel cells at the interface between commensals, innate and adaptive immune system drive an immune-tolerogenic or inflammatory Th17 cell response. In this study, the influence of two gut commensals of low (Bacteroides vulgatus) or high (Escherichia coli) immunogenicity on IκBζ expression in DCs and its downstream effects was analyzed. We observed that the amount of IκBζ expression and secretion of Th17-inducing cytokines correlated with the immunogenicity of these commensals. However, under immune-balanced conditions, E. coli also strongly induced an IκBζ-dependent secretion of anti-inflammatory IL-10, facilitating a counter-regulative Treg response as assessed in in vitro CD4+ T cell polarization assays. Yet, in an in vivo mouse model of T cell-induced colitis, prone to inflammatory and autoimmune conditions, administration of E. coli promoted an expansion of rather pro-inflammatory T helper cell subsets whereas administration of B. vulgatus resulted in the induction of protective T helper cell subsets. These findings might contribute to the development of new therapeutic strategies for the treatment of autoimmune diseases using commensals or commensal-derived components.

Weak Agonistic LPS Restores Intestinal Immune Homeostasis.

Generated by gram-negative bacteria, lipopolysaccharides (LPSs) are one of the most abundant and potent immunomodulatory substances present in the intestinal lumen. Interaction of agonistic LPS with the host myeloid-differentiation-2/Toll-like receptor 4 (MD-2/TLR4) receptor complex results in nuclear factor κB (NF-κB) activation, followed by the robust induction of pro-inflammatory immune responses. Here we have isolated LPS from a common gut commensal, Bacteroides vulgatus mpk (BVMPK), which provides only weak agonistic activity. This weak agonistic activity leads to the amelioration of inflammatory immune responses in a mouse model for experimental colitis, and it was in sharp contrast to strong agonists and antagonists. In this context, the administration of BVMPK LPS into mice with severe intestinal inflammation re-established intestinal immune homeostasis within only 2 weeks, resulting in the clearance of all symptoms of inflammation. These inflammation-reducing properties of weak agonistic LPS are grounded in the induction of a special type of endotoxin tolerance via the MD-2/TLR4 receptor complex axis in intestinal lamina propria CD11c+ cells. Thus, weak agonistic LPS represents a promising agent to treat diseases involving pathological overactivation of the intestinal immune system, e.g., in inflammatory bowel diseases.

Flagellin hypervariable region determines symbiotic properties of commensal Escherichia coli strains.

Escherichia coli represents a classical intestinal gram-negative commensal. Despite this commensalism, different E. coli strains can mediate disparate immunogenic properties in a given host. Symbiotic E. coli strains such as E. coli Nissle 1917 (EcN) are attributed beneficial properties, e.g., promotion of intestinal homeostasis. Therefore, we aimed to identify molecular features derived from symbiotic bacteria that might help to develop innovative therapeutic alternatives for the treatment of intestinal immune disorders. This study was performed using the dextran sodium sulphate (DSS)-induced colitis mouse model, which is routinely used to evaluate potential therapeutics for the treatment of Inflammatory Bowel Diseases (IBDs). We focused on the analysis of flagellin structures of different E. coli strains. EcN flagellin was found to harbor a substantially longer hypervariable region (HVR) compared to other commensal E. coli strains, and this longer HVR mediated symbiotic properties through stronger activation of Toll-like receptor (TLR)5, thereby resulting in interleukin (IL)-22-mediated protection of mice against DSS-induced colitis. Furthermore, using bone-marrow-chimeric mice (BMCM), CD11c+ cells of the colonic lamina propria (LP) were identified as the main mediators of these flagellin-induced symbiotic effects. We propose flagellin from symbiotic E. coli strains as a potential therapeutic to restore intestinal immune homeostasis, e.g., for the treatment of IBD patients.

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses.

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal host-microbe interactions. It is well established that different commensals exhibit a different potential to stimulate the host intestinal immune system. Such investigations involve vertebrate animals, especially rodents. Since increasing ethical concerns are linked with experiments involving vertebrates, there is a high demand for invertebrate replacements models. Here, we provide a Galleria mellonella oral administration model using commensal non-pathogenic bacteria and the possible assessment of the immunogenic potential of commensals on the G. mellonella immune system. We demonstrate that G. mellonella is a useful alternative invertebrate replacement model that allows the analysis of commensals with different immunogenic potential such as Bacteroides vulgatus and Escherichia coli. Interestingly, the bacteria exhibited no killing effect on the larvae, which is similar to mammals. The immune responses of G. mellonella were comparable with vertebrate innate immune responses and involve recognition of the bacteria and production of antimicrobial molecules. We propose that G. mellonella was able to restore previous microbiota balance, which is well known from healthy mammalian individuals. Although providing comparable innate immune responses in both G. mellonella and vertebrates, G. mellonella does not harbor an adaptive immune system. Since the investigated components of the innate immune system are evolutionary conserved, the model allows a prescreening and first analysis of bacterial immunogenic properties.

Bacterial Immunogenicity Is Critical for the Induction of Regulatory B Cells in Suppressing Inflammatory Immune Responses.

B cells fulfill multifaceted functions that influence immune responses during health and disease. In autoimmune diseases, such as inflammatory bowel disease, multiple sclerosis and rheumatoid arthritis, depletion of functional B cells results in an aggravation of disease in humans and respective mouse models. This could be due to a lack of a pivotal B cell subpopulation: regulatory B cells (Bregs). Although Bregs represent only a small proportion of all immune cells, they exhibit critical properties in regulating immune responses, thus contributing to the maintenance of immune homeostasis in healthy individuals. In this study, we report that the induction of Bregs is differentially triggered by the immunogenicity of the host microbiota. In comparative experiments with low immunogenic Bacteroides vulgatus and strong immunogenic Escherichia coli, we found that the induction and longevity of Bregs depend on strong Toll-like receptor activation mediated by antigens of strong immunogenic commensals. The potent B cell stimulation via E. coli led to a pronounced expression of suppressive molecules on the B cell surface and an increased production of anti-inflammatory cytokines like interleukin-10. These bacteria-primed Bregs were capable of efficiently inhibiting the maturation and function of dendritic cells (DCs), preventing the proliferation and polarization of T helper (Th)1 and Th17 cells while simultaneously promoting Th2 cell differentiation in vitro. In addition, Bregs facilitated the development of regulatory T cells (Tregs) resulting in a possible feedback cooperation to establish immune homeostasis. Moreover, the colonization of germfree wild type mice with E. coli but not B. vulgatus significantly reduced intestinal inflammatory processes in dextran sulfate sodium (DSS)-induced colitis associated with an increase induction of immune suppressive Bregs. The quantity of Bregs directly correlated with the severity of inflammation. These findings may provide new insights and therapeutic approaches for B cell-controlled treatments of microbiota-driven autoimmune disease.

Galleria mellonella: A Novel Invertebrate Model to Distinguish Intestinal Symbionts From Pathobionts.

Insects and mammals share evolutionary conserved innate immune responses to maintain intestinal homeostasis. We investigated whether the larvae of the greater wax moth Galleria mellonella may be used as an experimental organism to distinguish between symbiotic Bacteroides vulgatus and pathobiotic Escherichia coli, which are mammalian intestinal commensals. Oral application of the symbiont or pathobiont to G. mellonella resulted in clearly distinguishable innate immune responses that could be verified by analyzing similar innate immune components in mice in vivo and in vitro. The differential innate immune responses were initiated by the recognition of bacterial components via pattern recognition receptors. The pathobiont detection resulted in increased expression of reactive oxygen and nitrogen species related genes as well as antimicrobial peptide gene expression. In contrast, the treatment/application with symbiotic bacteria led to weakened immune responses in both mammalian and insect models. As symbionts and pathobionts play a crucial role in development of inflammatory bowel diseases, we hence suggest G. mellonella as a future replacement organism in inflammatory bowel disease research.

Genome Sequence of Galleria mellonella (Greater Wax Moth).

The larvae of the greater wax moth, Galleria mellonella, are pests of active beehives. In infection biology, these larvae are playing a more and more attractive role as an invertebrate host model. Here, we report on the first genome sequence of Galleria mellonella.

Outer membrane vesicles blebbing contributes to B. vulgatus mpk-mediated immune response silencing.

The Gram negative intestinal symbiont Bacteroides vulgatus mpk is able to prevent from induction of colonic inflammation in Rag1-/- mice and promotes immune balance in Il2-/- mice. These inflammation-silencing effects are associated with B. vulgatus mpk-mediated induction of semi-mature dendritic cells, especially in the colonic lamina propria (cLP). However the beneficial interaction of bacteria with host immune cells is limited due to the existence of a large mucus layer covering the intestinal epithelium. How can intestinal bacteria overcome this physical barrier and contact the host immune system? One mechanism is the production of outer membrane vesicles (OMVs) via ubiquitous blebbing of the outer membrane. These proteoliposomes have the ability to traverse the mucus layer. Hence, OMVs play an important role in immunomodulation and the maintenance of a balanced gut microbiota. Here we demonstrate that the stimulation of bone marrow derived dendritic cells (BMDCs) with isolated OMVs originated from B. vulgatus mpk leads to the induction of a tolerant semi-mature phenotype. Thereby, microbe- associated molecular patterns (MAMPs) delivered by OMVs are crucial for the interaction and the resulting maturation of immune cells. Additional to the binding to host TLR4, a yet unknown ligand to TLR2 is indispensable for the conversion of immature BMDCs into a semi-mature state. Thus, crossing the epithelial mucus layer and directly contact host cells, OMV mediate cross-tolerance via the transport of various Toll-like receptor antigens. These features make OMVs to a key attribute of B. vulgatus mpk for a vigorous acellular prevention and treatment of systemic diseases.

Proteome and phosphoproteome analysis of commensally induced dendritic cell maturation states.

Dendritic cells (DCs) can shape the immune system towards an inflammatory or tolerant state depending on the bacterial antigens and the environment they encounter. In this study we provide a proteomic catalogue of differentially expressed proteins between distinct DC maturation states, brought about by bacteria that differ in their endotoxicity. To achieve this, we have performed proteomics and phosphoproteomics on murine DC cultures. Symbiont and pathobiont bacteria were used to direct dendritic cells into a semi-mature and fully-mature state, respectively. The comparison of semi-mature and fully-mature DCs revealed differential expression in 103 proteins and differential phosphorylation in 118 phosphosites, including major regulatory factors of central immune processes. Our analyses predict that these differences are mediated by upstream elements such as SOCS1, IRF3, ABCA1, TLR4, and PTGER4. Our analyses indicate that the symbiont bacterial strain affects DC proteome in a distinct way, by downregulating inflammatory proteins and activating anti-inflammatory upstream regulators. Biological significance In this study we have investigated the responses of immune cells to distinct bacterial stimuli. We have used the symbiont bacterial strain B. vulgatus and the pathobiont E. coli strain to stimulate cultured primary dendritic cells and performed a shotgun proteome analysis to investigate the protein expression and phosphorylation level differences on a genome level. We have observed expression and phosphorylation level differences in key immune regulators, transcription factors and signal transducers. Moreover, our subsequent bioinformatics analysis indicated regulation at several signaling pathways such as PPAR signaling, LXR/RXR activation and glucocorticoid signaling pathways, which are not studied in detail in an inflammation and DC maturation context. Our phosphoproteome analysis showed differential phosphorylation in 118 phosphosites including those belonging to epigenetic regulators, transcription factors and major cell cycle regulators. We anticipate that our study will facilitate further investigation of immune cell proteomes under different inflammatory and non-inflammatory conditions.

Phosphotransferase systems in Enterococcus faecalis OG1RF enhance anti-stress capacity in vitro and in vivo.

Phosphotransferase systems are common and essential in bacteria, which are in charge of sugar transportation and phosphorylation. However, phosphotransferase systems were found in recent years to be associated with environmental stress factors. This study investigated the role of the mannose/fructose/sorbose phosphotransferase systems in Enterococcus faecalis OG1RF in adaption to harsh environments by construction of pts mutants. More than one mannose/fructose/sorbose phosphotransferase system was found in E. faecalis OG1RF, and the elimination of pts gene at different loci generated different after-effects corresponding to different ambiences. An in vitro study showed that the presence of intact phosphotransferase systems in E. faecalis OG1RF promoted resistance to hydrogen peroxide and acid and enhanced susceptibility to pediocin. In vivo study demonstrated that the presence of intact phosphotransferase systems induced more hazardous substances like superoxide dismutase (SOD) in Caenorhabditis elegans and enhanced bacterial infection and survival in macrophages J774A.1 and BMM. In addition, phosphotransferase systems regulated transcription of antioxidant and catabolite genes such as katA, gor, lysR, hypR, rex, hprK and tpx to different extents (-6.3- to 3.5-fold). It is therefore suggested that pts genes are regulatory factors promoting adaption of E. faecalis OG1RF to stressful conditions, thereby enhancing the possibility of bacterial survival and infectivity.

Microbiome and chronic inflammatory bowel diseases.

It is nowadays generally accepted that the microbiome is a central driver of chronic inflammatory bowel diseases based on observations from human patients as well as inflammatory rodent models. Many studies focussed on different aspects of microbiota and some scientists believe that a primary dis-balance results in a direct microbial induced inflammatory situation. It is also clear that the microbiome is influenced by environmental and genetic factors and is also tightly regulated by host defense molecules such as antimicrobial peptides (defensins et al.). Different lines of investigations showed different complex antimicrobial barrier defects in inflammatory bowel diseases which also influence the composition of the microbiome and generally impact on the microbial-mucosal interface. In this review, we aim to discuss the bigger picture of these different aspects and current views and conclude about therapeutic consequences for future concepts beyond anti-inflammatory treatment.

Non-invasive detection and successful treatment of a Helicobacter pylori infection in a captive rhesus macaque.

Gastritis is a commonly diagnosed condition in non-human primates used in biomedical research. As in humans, Helicobacter pylori infection may cause gastritis. The following report presents a method of non-invasive detection and a successful treatment protocol for this common pathogen.

Symbiotic gut commensal bacteria act as host cathepsin S activity regulators.

Cathepsin S (CTSS) is a lysosomal protease whose activity regulation is important for MHC-II signaling and subsequent activation of CD4+ T cell mediated immune responses. Dysregulation of its enzymatic activity or enhanced secretion into extracellular environments is associated with the induction or progression of several autoimmune diseases. Here we demonstrate that commensal intestinal bacteria influence secretion rates and intracellular activity of host CTSS and that symbiotic bacteria, i.e. Bacteroides vulgatus mpk, may actively regulate this process and help to maintain physiological levels of CTSS activities in order to prevent from induction of pathological inflammation. The symbiont-controlled regulation of CTSS activity is mediated by anticipating reactive oxygen species induction in dendritic cells which, in turn, maintains cystatin C (CysC) monomer binding to CTSS. CysC monomers are potent endogenous CTSS inhibitors. This Bacteroides vulgatus caused and CysC dependent CTSS activity regulation is involved in the generation of tolerant intestinal dendritic cells contributing to prevention of T-cell mediated induction of colonic inflammation. Taken together, we demonstrate that symbionts of the intestinal microbiota regulate host CTSS activity and secretion and might therefore be an attractive approach to deal with CTSS associated autoimmune diseases.

Extensive Mobilome-Driven Genome Diversification in Mouse Gut-Associated Bacteroides vulgatus mpk.

Like many other Bacteroides species, Bacteroides vulgatus strain mpk, a mouse fecal isolate which was shown to promote intestinal homeostasis, utilizes a variety of mobile elements for genome evolution. Based on sequences collected by Pacific Biosciences SMRT sequencing technology, we discuss the challenges of assembling and studying a bacterial genome of high plasticity. Additionally, we conducted comparative genomics comparing this commensal strain with the B. vulgatus type strain ATCC 8482 as well as multiple other Bacteroides and Parabacteroides strains to reveal the most important differences and identify the unique features of B. vulgatus mpk. The genome of B. vulgatus mpk harbors a large and diverse set of mobile element proteins compared with other sequenced Bacteroides strains. We found evidence of a number of different horizontal gene transfer events and a genome landscape that has been extensively altered by different mobilization events. A CRISPR/Cas system could be identified that provides a possible mechanism for preventing the integration of invading external DNA. We propose that the high genome plasticity and the introduced genome instabilities of B. vulgatus mpk arising from the various mobilization events might play an important role not only in its adaptation to the challenging intestinal environment in general, but also in its ability to interact with the gut microbiota.