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.

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.

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.

Post-mortem molecular investigations of SARS-CoV-2 in an unexpected death of a recent kidney transplant recipient.

Solid organ transplant recipients are vulnerable to severe infection during induction therapy. We report a case of a 67-year-old male who died unexpectedly 10 days after receiving a kidney transplant on February 10, 2020. There was no clear cause of death, but COVID-19 was considered retrospectively, as the death occurred shortly after the first confirmed case of COVID-19 in Canada. We confirmed the presence of SARS-CoV-2 components in the renal allograft and native lung tissue using immunohistochemistry for SARS-CoV-2 spike protein and RNA scope in situ hybridization for SARS-CoV-2 RNA. Results were reaffirmed with the Food and Drug Administration Emergency Use Authorization approved Bio-Rad SARS-CoV-2 digital droplet PCR for the kidney specimen. Our case highlights the importance of patient autopsies in an unfolding global pandemic and demonstrates the utility of molecular assays to diagnose SARS-CoV-2 post-mortem. SARS-CoV-2 infection during induction therapy may portend a fatal clinical outcome. We also suggest COVID-19 may be transmittable via renal transplant.

Identification of hypercoagulability with thrombelastography in patients with hip fracture receiving thromboprophylaxis.

Background: Venous thromboembolism (VTE) is the second most common complication after hip fracture surgery. We used thrombelastography (TEG), a whole-blood, point-of-care test that can provide an overview of the clotting process, to determine the duration of hypercoagulability after hip fracture surgery. Methods: In this prospective study, consecutive patients aged 51 years or more with hip fractures (trochanteric region or neck) amenable to surgical treatment who presented to the emergency department were eligible for enrolment. Thrombelastography, including calculation of the coagulation index (CI) (combination of 4 TEG parameters for an overall assessment of coagulation) was performed daily from admission until 5 days postoperatively, and at 2 and 6 weeks postoperatively. All patients received 28 days of thromboprophylaxis. We used single-sample t tests to compare mean maximal amplitude (MA) values (a measure of clot strength) to the hypercoagulable threshold of greater than 65 mm, a predictor of in-hospital VTE. Results: Of the 35 patients enrolled, 11 (31%) were hypercoagulable on admission based on an MA value greater than 65 mm, and 29 (83%) were hypercoagulable based on a CI value greater than 3.0; the corresponding values at 6 weeks were 23 (66%) and 34 (97%). All patients had an MA value greater than 65 mm at 2 weeks. Patients demonstrated normal coagulation on admission (mean MA value 62.2 mm [standard deviation (SD) 6.3 mm], p = 0.01) but became significantly hypercoagulable at 2 weeks (mean 71.6 mm [SD 2.6 mm], p < 0.001). There was a trend toward persistent hypercoagulability at 6 weeks (mean MA value 66.2 mm [SD 3.8 mm], p = 0.06). Conclusion: More than 50% of patients remained hypercoagulable 6 weeks after fracture despite thromboprophylaxis. Thrombelastography MA thresholds or a change in MA over time may help predict VTE risk; however, further study is needed.

Contexte: La thromboembolie veineuse (TEV) est la deuxième complication la plus courante après une chirurgie pour fracture de la hanche. Nous avons eu recours à la thromboélastographie, un test de sang total effectué au point d'intervention et donnant une idée du processus de coagulation, pour évaluer la durée de l'hypercoagulabilité à la suite d'une chirurgie pour fracture de la hanche. Méthodes: Cette étude prospective a été menée auprès de patients consécutifs admissibles de 51 ans et plus qui se sont présentés à l'urgence pour une fracture de la hanche (région trochantérienne ou col du fémur) pouvant faire l'objet d'un traitement chirurgical. Une thromboélastographie (TEG), qui comprenait le calcul de l'indice de coagulation (IC) [combinaison de 4 paramètres du TEG permettant une évaluation globale de la coagulation], a été réalisée chaque jour, de l'admission au cinquième jour postopératoire, de même qu'à 2 et à 6 semaines postopératoires. Tous les patients ont suivi une thromboprophylaxie de 28 jours. Nous avons réalisé des tests t pour échantillon unique afin de comparer l'amplitude maximale (AM) moyenne (une mesure de la résistance d'un caillot) au seuil d'hypercoagulabilité de plus de 65 mm, un prédicteur de TEV à l'hôpital. Résultats: Des 35 patients recrutés, 11 (31 %) présentaient une hypercoagulabilité à l'admission selon une AM supérieure à 65 mm, et 29 (83 %) présentaient une hypercoagulabilité selon un IC supérieur à 3,0; les valeurs correspondantes à 6 semaines étaient de 23 (66 %) et de 34 (97 %), respectivement. Tous les patients avaient une AM de plus de 65 mm à 2 semaines. Dans l'ensemble, les patients avaient une coagulation normale à l'admission (AM moyenne 62,2 mm [écart type (E.T.) 6,3 mm], p = 0,01), mais présentaient une hypercoagulabilité importante à 2 semaines (moyenne 71,6 mm [E.T. 2,6 mm], p < 0,001). L'hypercoagulabilité avait tendance à persister à 6 semaines (AM moyenne 66,2 mm [E.T. 3,8 mm], p = 0,06). Conclusion: Malgré la thromboprophylaxie, plus de 50 % des patients présentaient toujours une hypercoagulabilité 6 semaines après leur fracture. Les seuils d'AM à la thromboélastographie et les changements de l'AM au fil du temps pourraient aider à prédire le risque de TEV, mais d'autres études sur le sujet sont nécessaires.

Platelets and neutrophil extracellular traps collaborate to promote intravascular coagulation during sepsis in mice.

Neutrophil extracellular traps (NETs; webs of DNA coated in antimicrobial proteins) are released into the vasculature during sepsis where they contribute to host defense, but also cause tissue damage and organ dysfunction. Various components of NETs have also been implicated as activators of coagulation. Using multicolor confocal intravital microscopy in mouse models of sepsis, we observed profound platelet aggregation, thrombin activation, and fibrin clot formation within (and downstream of) NETs in vivo. NETs were critical for the development of sepsis-induced intravascular coagulation regardless of the inciting bacterial stimulus (gram-negative, gram-positive, or bacterial products). Removal of NETs via DNase infusion, or in peptidylarginine deiminase-4-deficient mice (which have impaired NET production), resulted in significantly lower quantities of intravascular thrombin activity, reduced platelet aggregation, and improved microvascular perfusion. NET-induced intravascular coagulation was dependent on a collaborative interaction between histone H4 in NETs, platelets, and the release of inorganic polyphosphate. Real-time perfusion imaging revealed markedly improved microvascular perfusion in response to the blockade of NET-induced coagulation, which correlated with reduced markers of systemic intravascular coagulation and end-organ damage in septic mice. Together, these data demonstrate, for the first time in an in vivo model of infection, a dynamic NET-platelet-thrombin axis that promotes intravascular coagulation and microvascular dysfunction in sepsis.

Chemokines: sirens of neutrophil recruitment-but is it just one song?

Neutrophil trafficking to inflamed tissues requires the integration of multiple chemoattractant guidance signals. In this issue of Immunity, Chou et al. (2010) demonstrate that collaborative "cascades" of chemoattractant mediators control neutrophil recruitment to arthritic joints in mice.

Neutrophils in critical illness.

During critical illness, dramatic alterations in neutrophil biology are observed including abnormalities of granulopoeisis and lifespan, cell trafficking and antimicrobial effector functions. As a result, neutrophils transition from powerful antimicrobial protectors into dangerous mediators of tissue injury and organ dysfunction. In this article, the role of neutrophils in the pathogenesis of critical illness (sepsis, trauma, burns and others) will be explored, including pathological changes to neutrophil function during critical illness and the utility of monitoring aspects of the neutrophil phenotype as biomarkers for diagnosis and prognostication. Lastly, we review findings from clinical trials of therapies that target the harmful effects of neutrophils, providing a bench-to-bedside perspective on neutrophils in critical illness.

Innate Immune Cell Trafficking and Function During Sterile Inflammation of the Liver.

The sterile inflammatory response (inflammation in the absence of infection) to tissue injury and cell death is required for normal wound healing. However, dysregulated sterile inflammation leads to various acute and chronic inflammatory diseases, including those of the liver and gastrointestinal tract. It is therefore important to increase our understanding of the mechanisms that control physiological versus pathological sterile inflammation. We have begun to clarify the cellular and molecular mechanisms that coordinate the innate immune response to tissue damage and cell death in the liver. In this review, we summarize the mechanisms that alert the immune system to the presence of tissue damage and highlight recent advances in our understanding of innate immune cell trafficking to sites of hepatic sterile inflammation. We explore the functions of various innate immune cells in the coordination of tissue repair, including previously underappreciated roles of peritoneal macrophages and platelets. We propose that dysregulation of immune cell trafficking or function at sites of tissue injury contributes to the misdirection of sterile inflammation to promote chronic inflammatory disease.

Imaging the dynamic platelet-neutrophil response in sterile liver injury and repair in mice.

UNLABELLED: Although platelets have been extensively studied in hemostasis and inflammation, their role is not well understood in sterile liver injury and repair. Using a thermally induced focal liver injury and repair model and multichannel spinning disk confocal microscopy allowed visualization of the dynamic behavior of platelets and neutrophils in this insult. Platelets instantaneously adhered to molecularly altered sinusoidal endothelium adjacent to the afflicted area, paving approximately 200 µm abutting the injury. Platelets remained adherent for at least 4 hours, but dissipated by 8 hours. The early recruitment occurred by GPIIbIIIa (CD41) and the later recruitment was dependent upon both GPIIbIIIa and GPIb (CD42B). Platelets did not occlude the vessels, but rather paved the altered endothelium. Endothelin-induced vasoconstriction by hepatic stellate cells, and not platelet accumulation or coagulation, was responsible for temporarily restricted perfusion around the injury. Neutrophils crawled into the injury from significant distances through the sinusoids. The crawling neutrophils required the platelet-paved endothelium given that very little neutrophil recruitment was noted in thrombocytopenic or CD41-deficient mice. As platelets slowly dissipated, neutrophil recruitment was also halted. Previous work suggested that platelets binding to immobilized neutrophils induced neutrophil extracellular trap (NET) formation in response to infection as well as during thrombosis and other forms of sterile injury. In this model of neutrophils crawling on immobilized platelets, very few NETs were observed and no additional injury was noted. In fact, GPIIbIIIa-deficient mice had delayed repair. CONCLUSION: In a liver model of sterile injury and repair, platelets play a critical role in forming a substratum and pave the way for neutrophils to enter the injured site for subsequent repair.

Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood.

It has been known for many years that neutrophils and platelets participate in the pathogenesis of severe sepsis, but the inter-relationship between these players is completely unknown. We report several cellular events that led to enhanced trapping of bacteria in blood vessels: platelet TLR4 detected TLR4 ligands in blood and induced platelet binding to adherent neutrophils. This led to robust neutrophil activation and formation of neutrophil extracellular traps (NETs). Plasma from severely septic humans also induced TLR4-dependent platelet-neutrophil interactions, leading to the production of NETs. The NETs retained their integrity under flow conditions and ensnared bacteria within the vasculature. The entire event occurred primarily in the liver sinusoids and pulmonary capillaries, where NETs have the greatest capacity for bacterial trapping. We propose that platelet TLR4 is a threshold switch for this new bacterial trapping mechanism in severe sepsis.

Pathogenesis and therapeutic opportunities of gut microbiome dysbiosis in critical illness.

For 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.

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.

Kupffer cells and activation of endothelial TLR4 coordinate neutrophil adhesion within liver sinusoids during endotoxemia.

A key pathological feature of the systemic inflammatory response of sepsis/endotoxemia is the accumulation of neutrophils within the microvasculature of organs such as the liver, where they cause tissue damage and vascular dysfunction. There is emerging evidence that the vascular endothelium is critical to the orchestration of inflammatory responses to blood-borne microbes and microbial products in sepsis/endotoxemia. In this study, we aimed to understand the role of endothelium, and specifically endothelial TLR4 activation, in the regulation of neutrophil recruitment to the liver during endotoxemia. Intravital microscopy of bone marrow chimeric mice revealed that TLR4 expression by non-bone marrow-derived cells was required for neutrophil recruitment to the liver during endotoxemia. Furthermore, LPS-induced neutrophil adhesion in liver sinusoids was equivalent between wild-type mice and transgenic mice that express TLR4 only on endothelium (tlr4(-/-)Tie2(tlr4)), revealing that activation of endothelial TLR4 alone was sufficient to initiate neutrophil adhesion. Neutrophil arrest within sinusoids of endotoxemic mice requires adhesive interactions between neutrophil CD44 and endothelial hyaluronan. Intravital immunofluorescence imaging demonstrated that stimulation of endothelial TLR4 alone was sufficient to induce the deposition of serum-derived hyaluronan-associated protein (SHAP) within sinusoids, which was required for CD44/hyaluronan-dependent neutrophil adhesion. In addition to endothelial TLR4 activation, Kupffer cells contribute to neutrophil recruitment via a distinct CD44/HA/SHAP-independent mechanism. This study sheds new light on the control of innate immune activation within the liver vasculature during endotoxemia, revealing a key role for endothelial cells as sentinels in the detection of intravascular infections and coordination of neutrophil recruitment to the liver.

The role of CD14 in neutrophil recruitment within the liver microcirculation during endotoxemia.

During Gram-negative sepsis and endotoxemia, CD14 is essential for the recognition of LPS by the TLR4 complex and subsequent generation of systemic inflammation. However, CD14-independent responses to LPS have been reported in vitro and in vivo in selected tissues including the skin. As the liver is a key target organ for neutrophil sequestration and inflammatory pathology during sepsis and endotoxemia, we investigated the role of CD14 in the recruitment of neutrophils into the liver in a mouse model of endotoxemia. Using dynamic in vivo imaging of the liver, we observed that neutrophil recruitment within the sinusoids and post-sinusoidal venules occurred equivalently between LPS-treated wild-type and CD14-knockout mice. Neutrophil recruitment within the liver was completely independent of CD14 regardless of whether it was expressed on cells of hematopoietic or nonhematopoietic origin or in serum as soluble CD14. Whereas CD14 expression was essential for activation of circulating neutrophils and for the development of LPS-induced systemic inflammation (pulmonary neutrophil sequestration, leukopenia, and increased serum proinflammatory cytokine levels), deficiency of CD14 did not limit the adhesion strength of neutrophils in vitro. Furthermore, wild-type and CD14-knockout mice displayed identical deposition of serum-derived hyaluronan-associated protein within liver sinusoids in response to LPS, indicating that the sinusoid-specific CD44/hyaluronan/serum-derived hyaluronan-associated protein-dependent pathway of neutrophil adhesion is activated independently of CD14. Therefore, the liver microcirculation possesses a unique CD14-independent mechanism of LPS detection and activation of neutrophil recruitment.

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.

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.

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.

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.