Biofilm exopolysaccharides alter sensory-neuron-mediated sickness during lung infection.

Infections of the lung cause observable sickness thought to be secondary to inflammation. Signs of sickness are crucial to alert others via behavioral-immune responses to limit contact with contagious individuals. Gram-negative bacteria produce exopolysaccharide (EPS) that provides microbial protection; however, the impact of EPS on sickness remains uncertain. Using genome-engineered Pseudomonas aeruginosa (P. aeruginosa) strains, we compared EPS-producers versus non-producers and a virulent Escherichia coli (E. coli) lung infection model in male and female mice. EPS-negative P. aeruginosa and virulent E. coli infection caused severe sickness, behavioral alterations, inflammation, and hypothermia mediated by TLR4 detection of the exposed lipopolysaccharide (LPS) in lung TRPV1+ sensory neurons. However, inflammation did not account for sickness. Stimulation of lung nociceptors induced acute stress responses in the paraventricular hypothalamic nuclei by activating corticotropin-releasing hormone neurons responsible for sickness behavior and hypothermia. Thus, EPS-producing biofilm pathogens evade initiating a lung-brain sensory neuronal response that results in sickness.

Dipeptidase-1 Is an Adhesion Receptor for Neutrophil Recruitment in Lungs and Liver.

A hallmark feature of inflammation is the orchestrated recruitment of neutrophils from the bloodstream into inflamed tissue. Although selectins and integrins mediate recruitment in many tissues, they have a minimal role in the lungs and liver. Exploiting an unbiased in vivo functional screen, we identified a lung and liver homing peptide that functionally abrogates neutrophil recruitment to these organs. Using biochemical, genetic, and confocal intravital imaging approaches, we identified dipeptidase-1 (DPEP1) as the target and established its role as a physical adhesion receptor for neutrophil sequestration independent of its enzymatic activity. Importantly, genetic ablation or functional peptide blocking of DPEP1 significantly reduced neutrophil recruitment to the lungs and liver and provided improved survival in models of endotoxemia. Our data establish DPEP1 as a major adhesion receptor on the lung and liver endothelium and identify a therapeutic target for neutrophil-driven inflammatory diseases of the lungs.

Neutrophils are itching to specialize.

Neutrophils are heterogeneous, but the mechanisms underlying their ability to polarize remain unclear. In this issue of Immunity, Gour et al. demonstrate that the GPCR Mrgpra1 and the neuropeptide NPFF, molecules involved in pain and itch, direct neutrophil polarization that impacts host defense and pneumonia susceptibility.

Aged polymorphonuclear leukocytes cause fibrotic interstitial lung disease in the absence of regulation by B cells.

Pulmonary immunity requires tight regulation, as interstitial inflammation can compromise gas exchange and lead to respiratory failure. Here we found a greater number of aged CD11bhiL-selectinloCXCR4+ polymorphonuclear leukocytes (PMNs) in lung vasculature than in the peripheral circulation. Using pulmonary intravital microscopy, we observed lung PMNs physically interacting with B cells via ß2 integrins; this initiated neutrophil apoptosis, which led to macrophage-mediated clearance. Genetic deletion of B cells led to the accumulation of aged PMNs in the lungs without systemic inflammation, which caused pathological fibrotic interstitial lung disease that was attenuated by the adoptive transfer of B cells or depletion of PMNs. Thus, the lungs are an intermediary niche in the PMN lifecycle wherein aged PMNs are regulated by B cells, which restrains their potential to cause pulmonary pathology.

Neonatal imprinting of alveolar macrophages via neutrophil-derived 12-HETE.

Resident-tissue macrophages (RTMs) arise from embryonic precursors1,2, yet the developmental signals that shape their longevity remain largely unknown. Here we demonstrate in mice genetically deficient in 12-lipoxygenase and 15-lipoxygenase (Alox15-/- mice) that neonatal neutrophil-derived 12-HETE is required for self-renewal and maintenance of alveolar macrophages (AMs) during lung development. Although the seeding and differentiation of AM progenitors remained intact, the absence of 12-HETE led to a significant reduction in AMs in adult lungs and enhanced senescence owing to increased prostaglandin E2 production. A compromised AM compartment resulted in increased susceptibility to acute lung injury induced by lipopolysaccharide and to pulmonary infections with influenza A virus or SARS-CoV-2. Our results highlight the complexity of prenatal RTM programming and reveal their dependency on in trans eicosanoid production by neutrophils for lifelong self-renewal.

Virus-associated fungal infections and lost immune resistance.

Invasive fungal infections are an increasing threat to human health. Of recent concern is the emergence of influenza- or SARS-CoV-2-virus-associated invasive fungal infections. Understanding acquired susceptibilities to fungi requires consideration of the collective and newly explored roles of adaptive, innate, and natural immunity. Neutrophils are known to provide host resistance, but new concepts are emerging that implicate innate antibodies, the actions of specialized B1 B cell subsets, and B cell-neutrophil crosstalk in mediating antifungal host resistance. Based on emerging evidence, we propose that virus infections impact on neutrophil and innate B cell resistance against fungi, leading to invasive infections. These concepts provide novel approaches to developing candidate therapeutics with the aim of restoring natural and humoral immunity and boosting neutrophil resistance against fungi.

Galectin-3 enhances neutrophil motility and extravasation into the airways during Aspergillus fumigatus infection.

Aspergillus fumigatus is an opportunistic mold that infects patients who are immunocompromised or have chronic lung disease, causing significant morbidity and mortality in these populations. While the factors governing the host response to A. fumigatus remain poorly defined, neutrophil recruitment to the site of infection is critical to clear the fungus. Galectin-3 is a mammalian ß-galactose-binding lectin with both antimicrobial and immunomodulatory activities, however the role of galectin-3 in the defense against molds has not been studied. Here we show that galectin-3 expression is markedly up-regulated in mice and humans with pulmonary aspergillosis. Galectin-3 deficient mice displayed increased fungal burden and higher mortality during pulmonary infection. In contrast to previous reports with pathogenic yeast, galectin-3 exhibited no antifungal activity against A. fumigatus in vitro. Galectin-3 deficient mice exhibited fewer neutrophils in their airways during infection, despite normal numbers of total lung neutrophils. Intravital imaging studies confirmed that galectin-3 was required for normal neutrophil migration to the airspaces during fungal infection. Adoptive transfer experiments demonstrated that stromal rather than neutrophil-intrinsic galectin-3 was necessary for normal neutrophil entry into the airspaces. Live cell imaging studies revealed that extracellular galectin-3 directly increases neutrophil motility. Taken together, these data demonstrate that extracellular galectin-3 facilitates recruitment of neutrophils to the site of A. fumigatus infection, and reveals a novel role for galectin-3 in host defense against fungal infections.

ARDS metabolic fingerprints: characterization, benchmarking, and potential mechanistic interpretation.

In this study, we aimed to identify acute respiratory distress syndrome (ARDS) metabolic fingerprints in selected patient cohorts and compare the metabolic profiles of direct versus indirect ARDS and hypoinflammatory versus hyperinflammatory ARDS. We hypothesized that the biological and inflammatory processes in ARDS would manifest as unique metabolomic fingerprints that set ARDS apart from other intensive care unit (ICU) conditions and could help examine ARDS subphenotypes and clinical subgroups. Patients with ARDS (n = 108) and ICU ventilated controls (n = 27) were included. Samples were randomly divided into 2/3 training and 1/3 test sets. Samples were analyzed using 1H nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry. Twelve proteins/cytokines were also measured. Orthogonal partial least squares discriminant analysis (OPLS-DA) was used to select the most differentiating ARDS metabolites and protein/cytokines. Predictive performance of OPLS-DA models was measured in the test set. Temporal changes of metabolites were examined as patients progressed through ARDS until clinical recovery. Metabolic profiles of direct versus indirect ARDS subgroups and hypoinflammatory versus hyperinflammatory ARDS subgroups were compared. Serum metabolomics and proteins/cytokines had similar area under receiver operator curves when distinguishing ARDS from ICU controls. Pathway analysis of ARDS differentiating metabolites identified a dominant involvement of serine-glycine metabolism. In longitudinal tracking, the identified pathway metabolites generally exhibited correction by 7-14 days, coinciding with clinical improvement. ARDS subphenotypes and clinical subgroups were metabolically distinct. However, our identified metabolic fingerprints are not ARDS diagnostic biomarkers, and further research is required to ascertain generalizability. In conclusion, patients with ARDS are metabolically different from ICU controls. ARDS subphenotypes and clinical subgroups are metabolically distinct.

The Lung Microvasculature Is a Functional Immune Niche.

Lung capillaries, best known for vital gas exchange, also contribute to neutrophil margination, a phenomenon resulting in large numbers of pulmonary vascular neutrophils. Importantly, the functional relevance of neutrophil margination is unknown. Recent advances in microscopy have altered our understanding of why neutrophils marginate. Specifically, data show that lung capillaries provide a unique anatomical site for neutrophils to capture bloodstream pathogens, which contrasts the conventional monophagocytic-dominated vascular host defense of the spleen and liver. Moreover, lung capillaries provide an efficient site for direct cell-cell communication required for the induction of apoptosis in aged neutrophils. These new ideas transform our views of the pulmonary circulation as a site for immediate neutrophil-mediated host defense and regulation of their life cycle.

Proceedings from the 8th Annual University of Calgary Leaders in Medicine Research Symposium.

On November 14, 2016, the Leaders in Medicine (LIM) program at the Cumming School of Medicine, University of Calgary hosted its 8th Annual Research Symposium. Professor Stephen Sawcer, Professor of Neurological Genetics at the University of Cambridge and an Honorary Consultant Neurologist at Addenbrooke's Hospital, was the keynote speaker and presented a lecture entitled, "Multiple sclerosis genetics - prospects and pitfalls". This was not only a cutting edge address on genetics but also a thoughtful overview on Dr. Sawcer's career and career choices. We were extremely grateful for the opportunity to have Dr. Sawcer participate in our annual symposium.

The Need for an Executive Leadership Curriculum in Scientist-Clinician Training Programs.

The health of Canadians depends on effective leadership among health care providers to facilitate the translation of new health discoveries into clinical practice. Clinician-scientists play an important role in bridging the gap between research and clinical practice, and require effective leadership skills to advance clinical practice successfully. To accelerate the leadership development in clinician scientist trainees, with the aim of developing strong leaders in administration and health advocacy, the Leaders in Medicine (LIM) training program at the University of Calgary created an Executive Leadership Coaching Program involving three phases: 1) an evidence-based evaluation tool, the Core Values IndexTM (CVI), that was used to identify the key drivers behind how individuals can be most effective in making their contribution; 2) small group workshops to debrief the results of the CVI assessment; and 3) one-on-one executive coaching sessions to facilitate the discovery, development and deployment of individual leadership capabilities. Coaching in leadership strategies enables clinician-scientist trainees to lead, influence, manage and deliver science-based improvements into the practice of medicine. We strongly recommend that other Canadian scientist-clinician training programs consider opportunities like the ones we offer to our LIM trainees. This training has important implications for the delivery of healthcare in Canada.

NETosis: how vital is it?

In this review, we examine the evidence that neutrophil extracellular traps (NETs) play a critical role in innate immunity. We summarize how NETs are formed in response to various stimuli and provide evidence that NETosis is not universally a cell death pathway. Here we describe at least 2 different mechanisms by which NETs are formed, including a suicide lytic NETosis and a live cell or vital NETosis. We also evaluate the evidence for NETs in catching and killing pathogens. Finally, we examine how infections are related to the development of autoimmune and vasculitic diseases through unintended but detrimental bystander damage resulting from NET release.

Highlights from the 6th Annual University of Calgary Leaders in Medicine Research Symposium and the Keynote Address by Dr. Danuta Skowronski.

The Leaders in Medicine (LIM) Program at the University of Calgary hosted its 6th Annual Research Symposium on November 14, 2014, showcasing the quality and breadth of work performed by students at the Cumming School of Medicine. Participation at this year's event was our most successful to date, with a total of six oral and 77 poster presentations during the afternoon symposium. For a detailed description of the work presented at the symposium, please see the Proceedings from the 6th Annual University of Calgary Leaders in Medicine Research Symposium published in this issue of Clinical and Investigative Medicine.

Proceedings from the 6th Annual University of Calgary Leaders in Medicine Research Symposium.

On November 14, 2014, the Leaders in Medicine (LIM) program at the Cumming School of Medicine, University of Calgary hosted its 6th Annual Research Symposium. Dr. Danuta Skowronski, Epidemiology Lead for Influenza and Emerging Respiratory Pathogens at the British Columbia Centre for Disease Control (BCCDC), was the keynote speaker and presented a lecture entitled "Rapid response research during emerging public health crises: influenza and reflections from the five year anniversary of the 2009 pandemic". The LIM symposium provides a forum for both LIM and non-LIM medical students to present their research work, either as an oral or poster presentation. There were a total of six oral presentations and 77 posters presented. The oral presentations included: Swathi Damaraju, "The role of cell communication and 3D Cell-Matrix environment in a stem cell-based tissue engineering strategy for bone repair"; Menglin Yang, "The proteolytic activity of Nepenthes pitcher fluid as a therapeutic for the treatment of celiac disease"; Amelia Kellar, "Monitoring pediatric inflammatory bowel disease - a retrospective analysis of transabdominal ultrasound"; Monica M. Faria-Crowder, "The design and application of a molecular profiling strategy to identify polymicrobial acute sepsis infections"; Waleed Rahmani, "Hair follicle dermal stem cells regenerate the dermal sheath, repopulate the dermal papilla and modulate hair type"; and, Laura Palmer, "A novel role for amyloid beta protein during hypoxia/ischemia". The article on the University of Calgary Leaders in Medicine Program, "A Prescription that Addresses the Decline of Basic Science Education in Medical School," in a previous issue of CIM (2014 37(5):E292) provides more details on the program. Briefly, the LIM Research Symposium has the following objectives: (1) to showcase the impressive variety of projects undertaken by students in the LIM Program as well as University of Calgary medical students; (2) to encourage medical student participation in research and special projects; and, (3) to inform students and faculty about the diversity of opportunities available for research and special projects during medical school and beyond.The following abstracts were submitted for publication.

Endotyping in ARDS: one step forward in precision medicine.

BACKGROUND: The Berlin definition of acute respiratory distress syndrome (ARDS) includes only clinical characteristics. Understanding unique patient pathobiology may allow personalized treatment. We aimed to define and describe ARDS phenotypes/endotypes combining clinical and pathophysiologic parameters from a Canadian ARDS cohort. METHODS: A cohort of adult ARDS patients from multiple sites in Calgary, Canada, had plasma cytokine levels and clinical parameters measured in the first 24 h of ICU admission. We used a latent class model (LCM) to group the patients into several ARDS subgroups and identified the features differentiating those subgroups. We then discuss the subgroup effect on 30 day mortality. RESULTS: The LCM suggested three subgroups (n1 = 64, n2 = 86, and n3 = 30), and 23 out of 69 features made these subgroups distinct. The top five discriminating features were IL-8, IL-6, IL-10, TNF-a, and serum lactate. Mortality distinctively varied between subgroups. Individual clinical characteristics within the subgroup associated with mortality included mean PaO2/FiO2 ratio, pneumonia, platelet count, and bicarbonate negatively associated with mortality, while lactate, creatinine, shock, chronic kidney disease, vasopressor/ionotropic use, low GCS at admission, and sepsis were positively associated. IL-8 and Apache II were individual markers strongly associated with mortality (Area Under the Curve = 0.84). PERSPECTIVE: ARDS subgrouping using biomarkers and clinical characteristics is useful for categorizing a heterogeneous condition into several homogenous patient groups. This study found three ARDS subgroups using LCM; each subgroup has a different level of mortality. This model may also apply to developing further trial design, prognostication, and treatment selection.

Neutrophil swarming: Is a good offense the best defense?

The phenomenon of swarming has long been observed in nature as a strategic event that serves as a good offense toward prey and predators. Imaging studies have uncovered that neutrophils employ this swarm-like tactic within infected and inflamed tissues as part of the innate immune response. Much of our understanding of neutrophil swarming builds from observations during sterile inflammation and various bacterial, fungal, and parasitic infections of the skin. However, the architecture and function of the skin differ significantly from vital organs where highly specialized microenvironments carry out critical functions. Therefore, the detrimental extent this perturbation may have on organ function remains unclear. In this review, we examine organ-specific swarming within the skin, liver, and lungs, with a detailed focus on swarming within microvascular environments. In addition, we examine potential "swarmulants" that initiate both transient and persistent swarms that have been implicated in disease.

Case report: Immune profiling links neutrophil and plasmablast dysregulation to microvascular damage in post-COVID-19 Multisystem Inflammatory Syndrome in Adults (MIS-A).

Despite surviving a SARS-CoV-2 infection, some individuals experience an intense post-infectious Multisystem Inflammatory Syndrome (MIS) of uncertain etiology. Children with this syndrome (MIS-C) can experience a Kawasaki-like disease, but mechanisms in adults (MIS-A) are not clearly defined. Here we utilize a deep phenotyping approach to examine immunologic responses in an individual with MIS-A. Results are contextualized to healthy, convalescent, and acute COVID-19 patients. The findings reveal systemic inflammatory changes involving novel neutrophil and B-cell subsets, autoantibodies, complement, and hypercoagulability that are linked to systemic vascular dysfunction. This deep patient profiling generates new mechanistic insight into this rare clinical entity and provides potential insight into other post-infectious syndromes.

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.

A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response to Staphylococcus aureus.

Neutrophil extracellular traps (NETs) are webs of DNA covered with antimicrobial molecules that constitute a newly described killing mechanism in innate immune defense. Previous publications reported that NETs take up to 3-4 h to form via an oxidant-dependent event that requires lytic death of neutrophils. In this study, we describe neutrophils responding uniquely to Staphylococcus aureus via a novel process of NET formation that did not require neutrophil lysis or even breach of the plasma membrane. The multilobular nucleus rapidly became rounded and condensed. During this process, we observed the separation of the inner and outer nuclear membranes and budding of vesicles, and the separated membranes and vesicles were filled with nuclear DNA. The vesicles were extruded intact into the extracellular space where they ruptured, and the chromatin was released. This entire process occurred via a unique, very rapid (5-60 min), oxidant-independent mechanism. Mitochondrial DNA constituted very little if any of these NETs. They did have a limited amount of proteolytic activity and were able to kill S. aureus. With time, the nuclear envelope ruptured, and DNA filled the cytoplasm presumably for later lytic NET production, but this was distinct from the vesicular release mechanism. Panton-Valentine leukocidin, autolysin, and a lipase were identified in supernatants with NET-inducing activity, but Panton-Valentine leukocidin was the dominant NET inducer. We describe a new mechanism of NET release that is very rapid and contributes to trapping and killing of S. aureus.

Inflammatory platelet death drives sepsis.

Thrombocytopenia is common in severe sepsis and is associated with an increased risk of mortality. A new study shows that platelet pyroptosis initiated during infection promotes a feedforward loop of neutrophil-mediated inflammation that worsens outcomes during sepsis.