Intestinal colonization regulates systemic anti-commensal immune sensitivity and hyperreactivity

Healthy host-microbial mutualism with our intestinal microbiota relies to a large degree on compartmentalization and careful regulation of adaptive mucosal and systemic anti-microbial immune responses. However, commensal intestinal bacteria are never exclusively or permanently restricted to the intestinal lumen and regularly reach the systemic circulation. This results in various degrees of commensal bacteremia that needs to be appropriately dealt with by the systemic immune system. While most intestinal commensal bacteria, except for pathobionts or opportunistic pathogen, have evolved to be non-pathogenic, this does not mean that they are non-immunogenic. Mucosal immune adaptation is carefully controlled and regulated to avoid an inflammatory response, but the systemic immune system usually responds differently and more vigorously to systemic bacteremia. Here we show that germ-free mice have increased systemic immune sensitivity and display anti-commensal hyperreactivity in response to the addition of a single defined T helper cell epitope to the outer membrane porin C (OmpC) of a commensal Escherichia coli strain demonstrated by increased E. coli-specific T cell-dependent IgG responses following systemic priming. This increased systemic immune sensitivity was not observed in mice colonized with a defined microbiota at birth indicating that intestinal commensal colonization also regulates systemic, and not only mucosal, anti-commensal responses. The observed increased immunogenicity of the E. coli strain with the modified OmpC protein was not due to a loss of function and associated metabolic changes as a control E. coli strain without OmpC did not display increased immunogenicity

Interaction of CD44 and hyaluronan is the dominant mechanism for neutrophil sequestration in inflamed liver sinusoids

Adhesion molecules known to be important for neutrophil recruitment in many other organs are not involved in recruitment of neutrophils into the sinusoids of the liver. The prevailing view is that neutrophils become physically trapped in inflamed liver sinusoids. In this study, we used a biopanning approach to identify hyaluronan (HA) as disproportionately expressed in the liver versus other organs under both basal and inflammatory conditions. Spinning disk intravital microscopy revealed that constitutive HA expression was restricted to liver sinusoids. Blocking CD44–HA interactions reduced neutrophil adhesion in the sinusoids of endotoxemic mice, with no effect on rolling or adhesion in postsinusoidal venules. Neutrophil but not endothelial CD44 was required for adhesion in sinusoids, yet neutrophil CD44 avidity for HA did not increase significantly in endotoxemia. Instead, activation of CD44–HA engagement via qualitative modification of HA was demonstrated by a dramatic induction of serum-derived HA-associated protein in sinusoids in response to lipopolysaccharide (LPS). LPS-induced hepatic injury was significantly reduced by blocking CD44–HA interactions. Administration of anti-CD44 antibody 4 hours after LPS rapidly detached adherent neutrophils in sinusoids and improved sinusoidal perfusion in endotoxemic mice, revealing CD44 as a potential therapeutic target in systemic inflammatory responses involving the liver

Multidrug-resistant E. coli encoding high genetic diversity in carbohydrate metabolism genes displace commensal E. coli from the intestinal tract

Extra-intestinal pathogenic Escherichia coli (ExPEC) can cause a variety of infections outside of the intestine and are a major causative agent of urinary tract infections. Treatment of these infections is increasingly frustrated by antimicrobial resistance (AMR) diminishing the number of effective therapies available to clinicians. Incidence of multidrug resistance (MDR) is not uniform across the phylogenetic spectrum of E. coli. Instead, AMR is concentrated in select lineages, such as ST131, which are MDR pandemic clones that have spread AMR globally. Using a gnotobiotic mouse model, we demonstrate that an MDR E. coli ST131 is capable of out-competing and displacing non-MDR E. coli from the gut in vivo. This is achieved in the absence of antibiotic treatment mediating a selective advantage. In mice colonised with non-MDR E. coli strains, challenge with MDR E. coli either by oral gavage or co-housing with MDR E. coli colonised mice results in displacement and dominant intestinal colonisation by MDR E. coli ST131. To investigate the genetic basis of this superior gut colonisation ability by MDR E. coli, we assayed the metabolic capabilities of our strains using a Biolog phenotypic microarray revealing altered carbon metabolism. Functional pangenomic analysis of 19,571 E. coli genomes revealed that carriage of AMR genes is associated with increased diversity in carbohydrate metabolism genes. The data presented here demonstrate that independent of antibiotic selective pressures, MDR E. coli display a competitive advantage to colonise the mammalian gut and points to a vital role of metabolism in the evolution and success of MDR lineages of E. coli via carriage and spread

Molecular mechanisms of neutrophil recruitment in the liver microcirculation

Bibliography: p. 245-291, 317-321Some pages are in colour.Includes copy of Animal Protocol Approval and copyright permissions. Original copies with original Partial Copyright Licence

A Multi-Modal Toolkit for Studying Neutrophils in Cancer and Beyond

As key effector cells of the innate immune response, neutrophils are rapidly deployed to sites of inflammation where they deliver a payload of potent effector mechanisms that are essential for host defense against pathogens as well as tissue homeostasis. In addition, neutrophils are central contributors to the pathogenesis of a vast spectrum of inflammatory, degenerative, and neoplastic diseases. As our understanding of neutrophils in health and disease continually expands, so too does our appreciation of their complex and dynamic nature in vivo; from development, maturation, and trafficking to cellular heterogeneity and functional plasticity. Therefore, contemporary neutrophil research relies on multiple complementary methodologies to perform integrated analysis of neutrophil phenotypic heterogeneity, organ- and stimulus-specific trafficking mechanisms, as well as tailored effector functions in vivo. This review discusses established and emerging technologies used to study neutrophils, with a focus on in vivo imaging in animal models, as well as next-generation ex vivo model systems to study mechanisms of neutrophil function. Furthermore, we discuss how high-dimensional single-cell analysis technologies are driving a renaissance in neutrophil biology by redefining our understanding of neutrophil development, heterogeneity, and functional plasticity. Finally, we discuss innovative applications and emerging opportunities to integrate these high-dimensional, multi-modal techniques to deepen our understanding of neutrophils in cancer research and beyond

Leveraging the microbiome in the treatment of sepsis: potential pitfalls and new perspectives

Purpose of reviewThis review aims to provide an overview of the current knowledge about microbiota-targeted therapies in sepsis, and calls out - despite recent negative studies - not to halt our efforts of translating these tools into regular medical practice.Recent findingsThe intestinal microbiome has an important role in shaping our immune system, and microbiota-derived metabolites prime innate and adaptive inflammatory responses to infectious pathogens. Microbiota composition is severely disrupted during sepsis, which has been linked to increased risk of mortality and secondary infections. However, efforts of using these microbes as a tool for prognostic or therapeutic purposes have been unsuccessful so far, and recent trials studying the impact of probiotics in critical illness did not improve patient outcomes. Despite these negative results, researchers must continue their attempts of harnessing the microbiome to improve sepsis survival in patients with a high risk of clinical deterioration. Promising research avenues that could potentially benefit sepsis patients include the development of next-generation probiotics, use of the microbiome as a theranostic tool to direct therapy, and addressing the restoration of microbial communities following ICU discharge.SummaryAlthough research focused on microbiome-mediated therapy in critically ill patients has not yielded the results that were anticipated, we should not abandon our efforts to translate promising preclinical findings into clinical practice

Pathogenesis and therapeutic opportunities of gut microbiome dysbiosis in critical illness

ABSTRACTFor many years, it has been hypothesized that pathological changes to the gut microbiome in critical illness is a driver of infections, organ dysfunction, and other adverse outcomes in the intensive care unit (ICU). The advent of contemporary microbiome methodologies and multi-omics tools have allowed researchers to test this hypothesis by dissecting host–microbe interactions in the gut to better define its contribution to critical illness pathogenesis. Observational studies of patients in ICUs have revealed that gut microbial communities are profoundly altered in critical illness, characterized by markedly reduced alpha diversity, loss of commensal taxa, and expansion of potential pathogens. These key features of ICU gut dysbiosis have been associated with adverse outcomes including life-threatening hospital-acquired (nosocomial) infections. Current research strives to define cellular and molecular mechanisms connecting gut dysbiosis with infections and other outcomes, and to identify opportunities for therapeutic modulation of host–microbe interactions. This review synthesizes evidence from studies of critically ill patients that have informed our understanding of intestinal dysbiosis in the ICU, mechanisms linking dysbiosis to infections and other adverse outcomes, as well as clinical trials of microbiota-modifying therapies. Additionally, we discuss novel avenues for precision microbial therapeutics to combat nosocomial infections and other life-threatening complications of critical illness

INTERACTION OF CD44 AND HYALURONAN IS THE DOMINANT MECHANISM FOR NEUTROPHIL ADHESION IN INFLAMED LIVER SINUSOIDS

Background: Previous studies have been unable to identify adhesion molecules that mediate neutrophil recruitment within the liver sinusoids. We hypothesise that involved adhesion molecules may represent novel therapeutic targets for combating pathologic liver inflammation. Methods: Candidate adhesion molecules were identified using a novel in vivo biopanning approach (dual radiolabelled antibody technique) to quantify endothelial expression levels within the liver compared to other organs. Spinning disk intravital microscopy demonstrated the localization of adhesion molecule expression withinthe liver microvasculature. Using knockout mice, bone marrow chimeric mice, and blocking antibodies, candidate adhesion molecules were systematically investigated for a role in neutrophil recruitment in the liver sinusoids of endotoxemic mice using intravital microscopy and in vitro flow chamber assays. Results: Hyaluronan was identified as disproportionately expressed in the liver versus other organs, and hyaluronan expression was restricted to liver sinusoids. Blocking CD44-hyaluronan interactions reduced neutrophil adhesion in the sinusoids of endotoxemic mice, but had no effect on neutrophil rolling or adhesion in post-sinusoidal venules. Neutrophil but not endothelial CD44 was required for adhesionin sinusoids. Surprisingly, neutrophil CD44 avidity for hyaluronan was not increased in endotoxemia. Instead, activation of CD44-hyaluronan engagement was the result of qualitative modification of hyaluronan by a dramatic induction of serum-derived hyaluronan-associated protein (SHAP) in sinusoids in response to lipopolysaccharide. Lipopolysaccharide-induced hepatic injury was significantly reduced by inhibiting CD44-hyaluronan interactions. Therapeutic administration of anti-CD44 antibody to endotoxemic mice rapidly detached adherent neutrophils and improved sinusoidal perfusion. Conclusion: These findings reveal CD44 as a potential therapeutic target in systemic inflammatory responses involving liver