Impact of age on the host response to sepsis in a murine model of fecal-induced peritonitis.

INTRODUCTION: Despite older adults being more vulnerable to sepsis, most preclinical research on sepsis has been conducted using young animals. This results in decreased scientific validity since age is an independent predictor of poor outcome. In this study, we explored the impact of aging on the host response to sepsis using the fecal-induced peritonitis (FIP) model developed by the National Preclinical Sepsis Platform (NPSP). METHODS: C57BL/6 mice (3 or 12 months old) were injected intraperitoneally with rat fecal slurry (0.75 mg/g) or a control vehicle. To investigate the early stage of sepsis, mice were culled at 4 h, 8 h, or 12 h to investigate disease severity, immunothrombosis biomarkers, and organ injury. Mice received buprenorphine at 4 h post-FIP. A separate cohort of FIP mice were studied for 72 h (with buprenorphine given at 4 h, 12 h, and then every 12 h post-FIP and antibiotics/fluids starting at 12 h post-FIP). Organs were harvested, plasma levels of Interleukin (IL)-6, IL-10, monocyte chemoattract protein (MCP-1)/CCL2, thrombin-antithrombin (TAT) complexes, cell-free DNA (CFDNA), and ADAMTS13 activity were quantified, and bacterial loads were measured. RESULTS: In the 12 h time course study, aged FIP mice demonstrated increased inflammation and injury to the lungs compared to young FIP mice. In the 72 h study, aged FIP mice exhibited a higher mortality rate (89%) compared to young FIP mice (42%) (p < 0.001). Aged FIP non-survivors also exhibited a trend towards elevated IL-6, TAT, CFDNA, CCL2, and decreased IL-10, and impaired bacterial clearance compared to young FIP non-survivors. CONCLUSION: To our knowledge, this is the first study to investigate the impact of age on survival using the FIP model of sepsis. Our model includes clinically-relevant supportive therapies and inclusion of both sexes. The higher mortality rate in aged mice may reflect increased inflammation and worsened organ injury in the early stage of sepsis. We also observed trends in impaired bacterial clearance, increase in IL-6, TAT, CFDNA, CCL2, and decreased IL-10 and ADAMTS13 activity in aged septic non-survivors compared to young septic non-survivors. Our aging model may help to increase the scientific validity of preclinical research and may be useful for identifying mechanisms of age-related susceptibility to sepsis as well as age-specific treatment strategies.

Heat shock protein 27 in the pathogenesis of COVID-19 and non-COVID acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is a common cause of hypoxemic respiratory failure in intensive care units that has increased dramatically as a result of the COVID-19 pandemic. In both COVID-19 and non-COVID ARDS, the pathogenesis of lung injury involves local (pulmonary) and systemic inflammation, leading to impaired gas exchange, requirement for mechanical ventilation, and a high risk of mortality. Heat shock protein 27 (HSP27) is a chaperone protein expressed in times of cell stress with roles in modulation of systemic inflammation via the NF-κB pathway. Given its important role as a modulator of inflammation, we sought to investigate the role of HSP27 and its associated auto-antibodies in ARDS caused by both SARS-CoV-2 and non-COVID etiologies. A total of 68 patients admitted to the intensive care unit with ARDS requiring mechanical ventilation were enrolled in a prospective, observational study that included 22 non-COVID-19 and 46 COVID-19 patients. Blood plasma levels of HSP27, anti-HSP27 auto-antibody (AAB), and cytokine profiles were measured on days 1 and 3 of ICU admission along with clinical outcome measures. Patients with COVID-19 ARDS displayed significantly higher levels of HSP27 in plasma, and a higher ratio of HSP27:AAB on both day 1 and day 3 of ICU admission. In patients with COVID-19, higher levels of circulating HSP27 and HSP27:AAB ratio were associated with a more severe systemic inflammatory response and adverse clinical outcomes including more severe hypoxemic respiratory failure. These findings implicate HSP27 as a marker of advanced pathogenesis of disease contributing to the dysregulated systemic inflammation and worse clinical outcomes in COVID-19 ARDS, and therefore may represent a potential therapeutic target.

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.

Development and characterization of a fecal-induced peritonitis model of murine sepsis: results from a multi-laboratory study and iterative modification of experimental conditions.

BACKGROUND: Preclinical sepsis models have been criticized for their inability to recapitulate human sepsis and suffer from methodological shortcomings that limit external validity and reproducibility. The National Preclinical Sepsis Platform (NPSP) is a consortium of basic science researchers, veterinarians, and stakeholders in Canada undertaking standardized multi-laboratory sepsis research to increase the efficacy and efficiency of bench-to-bedside translation. In this study, we aimed to develop and characterize a 72-h fecal-induced peritonitis (FIP) model of murine sepsis conducted in two independent laboratories. The experimental protocol was optimized by sequentially modifying dose of fecal slurry and timing of antibiotics in an iterative fashion, and then repeating the experimental series at site 1 and site 2. RESULTS: Escalating doses of fecal slurry (0.5-2.5 mg/g) resulted in increased disease severity, as assessed by the modified Murine Sepsis Score (MSS). However, the MSS was poorly associated with progression to death during the experiments, and mice were found dead without elevated MSS scores. Administration of early antibiotics within 4 h of inoculation rescued the animals from sepsis compared with late administration of antibiotics after 12 h, as evidenced by 100% survival and reduced bacterial load in peritoneum and blood in the early antibiotic group. Site 1 and site 2 had statistically significant differences in mortality (60% vs 88%; p < 0.05) for the same dose of fecal slurry (0.75 mg/g) and marked differences in body temperature between groups. CONCLUSIONS: We demonstrate a systematic approach to optimizing a 72-h FIP model of murine sepsis for use in multi-laboratory studies. Alterations to experimental conditions, such as dose of fecal slurry and timing of antibiotics, have clear impact on outcomes. Differences in mortality between sites despite rigorous standardization warrants further investigations to better understand inter-laboratory variation and methodological design in preclinical studies.

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.

Vascular traffic control of neutrophil recruitment to the liver by microbiota-endothelium crosstalk.

During bloodstream infections, neutrophils home to the liver as part of an intravascular immune response to eradicate blood-borne pathogens, but the mechanisms regulating this crucial response are unknown. Using in vivo imaging of neutrophil trafficking in germ-free and gnotobiotic mice, we demonstrate that the intestinal microbiota guides neutrophil homing to the liver in response to infection mediated by the microbial metabolite D-lactate. Commensal-derived D-lactate augments neutrophil adhesion in the liver independent of granulopoiesis in bone marrow or neutrophil maturation and activation in blood. Instead, gut-to-liver D-lactate signaling primes liver endothelial cells to upregulate adhesion molecule expression in response to infection and promote neutrophil adherence. Targeted correction of microbiota D-lactate production in a model of antibiotic-induced dysbiosis restores neutrophil homing to the liver and reduces bacteremia in a model of Staphylococcus aureus infection. These findings reveal long-distance traffic control of neutrophil recruitment to the liver by microbiota-endothelium crosstalk.

Dysbiosis of a microbiota-immune metasystem in critical illness is associated with nosocomial infections.

Critically ill patients in intensive care units experience profound alterations of their gut microbiota that have been linked to a high risk of hospital-acquired (nosocomial) infections and adverse outcomes through unclear mechanisms. Abundant mouse and limited human data suggest that the gut microbiota can contribute to maintenance of systemic immune homeostasis, and that intestinal dysbiosis may lead to defects in immune defense against infections. Here we use integrated systems-level analyses of fecal microbiota dynamics in rectal swabs and single-cell profiling of systemic immune and inflammatory responses in a prospective longitudinal cohort study of critically ill patients to show that the gut microbiota and systemic immunity function as an integrated metasystem, where intestinal dysbiosis is coupled to impaired host defense and increased frequency of nosocomial infections. Longitudinal microbiota analysis by 16s rRNA gene sequencing of rectal swabs and single-cell profiling of blood using mass cytometry revealed that microbiota and immune dynamics during acute critical illness were highly interconnected and dominated by Enterobacteriaceae enrichment, dysregulated myeloid cell responses and amplified systemic inflammation, with a lesser impact on adaptive mechanisms of host defense. Intestinal Enterobacteriaceae enrichment was coupled with impaired innate antimicrobial effector responses, including hypofunctional and immature neutrophils and was associated with an increased risk of infections by various bacterial and fungal pathogens. Collectively, our findings suggest that dysbiosis of an interconnected metasystem between the gut microbiota and systemic immune response may drive impaired host defense and susceptibility to nosocomial infections in critical illness.

Sex-based analysis of treatment responses in animal models of sepsis: a preclinical systematic review protocol.

BACKGROUND: The importance of investigating sex- and gender-dependent differences has been recently emphasized by major funding agencies. Notably, the influence of biological sex on clinical outcomes in sepsis is unclear, and observational studies suffer from the effect of confounding factors. The controlled experimental environment afforded by preclinical studies allows for clarification and mechanistic evaluation of sex-dependent differences. We propose a systematic review to assess the impact of biological sex on baseline responses to disease induction as well as treatment responses in animal models of sepsis. Given the lack of guidance surrounding sex-based analyses in preclinical systematic reviews, careful consideration of various factors is needed to understand how best to conduct analyses and communicate findings. METHODS: MEDLINE and Embase will be searched (2011-present) to identify preclinical studies of sepsis in which any intervention was administered and sex-stratified data reported. The primary outcome will be mortality. Secondary outcomes will include organ dysfunction, bacterial load, and IL-6 levels. Study selection will be conducted independently and in duplicate by two reviewers. Data extraction will be conducted by one reviewer and audited by a second independent reviewer. Data extracted from included studies will be pooled, and meta-analysis will be conducted using random effects modeling. Primary analyses will be stratified by animal age and will assess the impact of sex at the following time points: pre-intervention, in response to treatment, and post-intervention. Risk of bias will be assessed using the SYRCLE's risk-of-bias tool. Illustrative examples of potential methods to analyze sex-based differences are provided in this protocol. DISCUSSION: Our systematic review will summarize the current state of knowledge on sex-dependent differences in sepsis. This will identify current knowledge gaps that future studies can address. Finally, this review will provide a framework for sex-based analysis in future preclinical systematic reviews. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42022367726.

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

PURPOSE OF REVIEW: This 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 FINDINGS: The 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. SUMMARY: Although 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.

A B1a-natural IgG-neutrophil axis is impaired in viral- and steroid-associated aspergillosis.

The lung naturally resists Aspergillus fumigatus (Af) in healthy individuals, but multiple conditions can disrupt this resistance, leading to lethal invasive infections. Core processes of natural resistance and its breakdown are undefined. We investigated three distinct conditions predisposing to lethal aspergillosis-severe SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection, influenza A viral pneumonia, and systemic corticosteroid use-in human patients and murine models. We found a conserved and essential coupling of innate B1a lymphocytes, Af-binding natural immunoglobulin G antibodies, and lung neutrophils. Failure of this axis concealed Af from neutrophils, allowing rapid fungal invasion and disease. Reconstituting the axis with immunoglobulin therapy reestablished resistance, thus representing a realistic pathway to repurpose currently available therapies. Together, we report a vital host resistance pathway that is responsible for protecting against life-threatening aspergillosis in the context of distinct susceptibilities.

On the same page? A qualitative study protocol on collaboration in a multi-laboratory preclinical study.

INTRODUCTION: Medical advancements are slow to reach the patient bedside due to issues with knowledge translation from preclinical studies. Multi-laboratory preclinical studies are a promising strategy for addressing the methodological deficiencies that weaken the translational impact of single laboratory findings. However, multi-laboratory preclinical studies are rare and difficult, requiring strong collaboration to plan and execute a shared protocol. In multiteam systems such as these, collaboration is enhanced when members have cohesive ways of thinking about their goals and how to achieve them-that is, when they have "shared mental models". In this research project, we will examine how members of Canada's first multi-laboratory preclinical study build shared mental models and collaborate in the execution of their study. METHODS: Six independent labs in Canada will conduct a preclinical study using a common protocol. To investigate mental models and collaboration in this multiteam system we will conduct a longitudinal qualitative study involving interviews at four time points, team observation, and document analysis. We will analyze interview transcripts using deductive coding to produce a matrix analysis of mental model content over time and inductive coding to produce a thematic analysis of members' experiences of collaboration over time. We will also triangulate data sources to "tell the story" of teamwork, capturing events and contextual information that explain changes in mental models and collaboration over time. DISCUSSION: This study will be one of the most comprehensive longitudinal analyses of a real-world multiteam system, and the first within a preclinical laboratory setting. The results will contribute to our understanding of collaboration in multiteam systems, an organizational form increasingly used to tackle complex scientific and social problems. The results will also inform the implementation of future multi-laboratory preclinical studies, enhancing the likelihood of effective collaboration and improved 'bench to bedside' translation.

Small intestinal resident eosinophils maintain gut homeostasis following microbial colonization.

The intestine harbors a large population of resident eosinophils, yet the function of intestinal eosinophils has not been explored. Flow cytometry and whole-mount imaging identified eosinophils residing in the lamina propria along the length of the intestine prior to postnatal microbial colonization. Microscopy, transcriptomic analysis, and mass spectrometry of intestinal tissue revealed villus blunting, altered extracellular matrix, decreased epithelial cell turnover, increased gastrointestinal motility, and decreased lipid absorption in eosinophil-deficient mice. Mechanistically, intestinal epithelial cells released IL-33 in a microbiota-dependent manner, which led to eosinophil activation. The colonization of germ-free mice demonstrated that eosinophil activation in response to microbes regulated villous size alterations, macrophage maturation, epithelial barrier integrity, and intestinal transit. Collectively, our findings demonstrate a critical role for eosinophils in facilitating the mutualistic interactions between the host and microbiota and provide a rationale for the functional significance of their early life recruitment in the small intestine.

Long-distance relationships - regulation of systemic host defense against infections by the gut microbiota.

Despite compartmentalization within the lumen of the gastrointestinal tract, the gut microbiota has a far-reaching influence on immune cell development and function throughout the body. This long-distance relationship is crucial for immune homeostasis, including effective host defense against invading pathogens that cause systemic infections. Herein, we review new insights into how commensal microbes that are spatially restricted to the gut lumen can engage in long-distance relationships with innate and adaptive immune cells at systemic sites to fortify host defenses against infections. In addition, we explore the consequences of intestinal dysbiosis on impaired host defense and immune-mediated pathology during infections, including emerging evidence linking dysbiosis with aberrant systemic inflammation and immune-mediated organ damage in sepsis. As such, therapeutic modification of the gut microbiota is an emerging target for interventions to prevent and/or treat systemic infections and sepsis by harnessing the long-distance relationships between gut microbes and systemic immunity.

"Molding" immunity-modulation of mucosal and systemic immunity by the intestinal mycobiome in health and disease.

Fungi are important yet understudied contributors to the microbial communities of the gastrointestinal tract. Starting at birth, the intestinal mycobiome undergoes a period of dynamic maturation under the influence of microbial, host, and extrinsic influences, with profound functional implications for immune development in early life, and regulation of immune homeostasis throughout life. Candida albicans serves as a model organism for understanding the cross-talk between fungal colonization dynamics and immunity, and exemplifies unique mechanisms of fungal-immune interactions, including fungal dimorphism, though our understanding of other intestinal fungi is growing. Given the prominent role of the gut mycobiome in promoting immune homeostasis, emerging evidence points to fungal dysbiosis as an influential contributor to immune dysregulation in a variety of inflammatory and infectious diseases. Here we review current knowledge on the factors that govern host-fungi interactions in the intestinal tract and immunological outcomes in both mucosal and systemic compartments.

Dexamethasone modulates immature neutrophils and interferon programming in severe COVID-19.

Although critical for host defense, innate immune cells are also pathologic drivers of acute respiratory distress syndrome (ARDS). Innate immune dynamics during Coronavirus Disease 2019 (COVID-19) ARDS, compared to ARDS from other respiratory pathogens, is unclear. Moreover, mechanisms underlying the beneficial effects of dexamethasone during severe COVID-19 remain elusive. Using single-cell RNA sequencing and plasma proteomics, we discovered that, compared to bacterial ARDS, COVID-19 was associated with expansion of distinct neutrophil states characterized by interferon (IFN) and prostaglandin signaling. Dexamethasone during severe COVID-19 affected circulating neutrophils, altered IFNactive neutrophils, downregulated interferon-stimulated genes and activated IL-1R2+ neutrophils. Dexamethasone also expanded immunosuppressive immature neutrophils and remodeled cellular interactions by changing neutrophils from information receivers into information providers. Male patients had higher proportions of IFNactive neutrophils and preferential steroid-induced immature neutrophil expansion, potentially affecting outcomes. Our single-cell atlas (see 'Data availability' section) defines COVID-19-enriched neutrophil states and molecular mechanisms of dexamethasone action to develop targeted immunotherapies for severe COVID-19.

A functionally distinct neutrophil landscape in severe COVID-19 reveals opportunities for adjunctive therapies.

Acute respiratory distress syndrome (ARDS) is a life-threatening syndrome, constituted by respiratory failure and diffuse alveolar damage that results from dysregulated local and systemic immune activation, causing pulmonary vascular, parenchymal, and alveolar damage. SARS-CoV-2 infection has become the dominant cause of ARDS worldwide, and emerging evidence implicates neutrophils and their cytotoxic arsenal of effector functions as central drivers of immune-mediated lung injury in COVID-19 ARDS. However, key outstanding questions are whether COVID-19 drives a unique program of neutrophil activation or effector functions that contribute to the severe pathogenesis of this pandemic illness and whether this unique neutrophil response can be targeted to attenuate disease. Using a combination of high-dimensional single-cell analysis and ex vivo functional assays of neutrophils from patients with COVID-19 ARDS, compared with those with non-COVID ARDS (caused by bacterial pneumonia), we identified a functionally distinct landscape of neutrophil activation in COVID-19 ARDS that was intrinsically programmed during SARS-CoV-2 infection. Furthermore, neutrophils in COVID-19 ARDS were functionally primed to produce high amounts of neutrophil extracellular traps. Surprisingly, this unique pathological program of neutrophil priming escaped conventional therapy with dexamethasone, thereby revealing a promising target for adjunctive immunotherapy in severe COVID-19.

Platelet-Mediated NET Release Amplifies Coagulopathy and Drives Lung Pathology During Severe Influenza Infection.

The influenza A virus (IAV) causes a respiratory tract infection with approximately 10% of the population infected by the virus each year. Severe IAV infection is characterized by excessive inflammation and tissue pathology in the lungs. Platelet and neutrophil recruitment to the lung are involved in the pathogenesis of IAV, but the specific mechanisms involved have not been clarified. Using confocal intravital microscopy in a mouse model of IAV infection, we observed profound neutrophil recruitment, platelet aggregation, neutrophil extracellular trap (NET) production and thrombin activation within the lung microvasculature in vivo. Importantly, deficiency or antagonism of the protease-activated receptor 4 (PAR4) reduced platelet aggregation, NET production, and neutrophil recruitment. Critically, inhibition of thrombin or PAR4 protected mice from virus-induced lung tissue damage and edema. Together, these data imply thrombin-stimulated platelets play a critical role in the activation/recruitment of neutrophils, NET release and directly contribute to IAV pathogenesis in the lung.

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.