Pro-regenerative biomaterials recruit immunoregulatory dendritic cells after traumatic injury.

During wound healing and surgical implantation, the body establishes a delicate balance between immune activation to fight off infection and clear debris and immune tolerance to control reactivity against self-tissue. Nonetheless, how such a balance is achieved is not well understood. Here we describe that pro-regenerative biomaterials for muscle injury treatment promote the proliferation of a BATF3-dependent CD103+XCR1+CD206+CD301b+ dendritic cell population associated with cross-presentation and self-tolerance. Upregulation of E-cadherin, the ligand for CD103, and XCL-1 in injured tissue suggests a mechanism for cell recruitment to trauma. Muscle injury recruited natural killer cells that produced Xcl1 when stimulated with fragmented extracellular matrix. Without cross-presenting cells, T-cell activation increases, pro-regenerative macrophage polarization decreases and there are alterations in myogenesis, adipogenesis, fibrosis and increased muscle calcification. These results, previously observed in cancer progression, suggest a fundamental mechanism of immune regulation in trauma and material implantation with implications for both short- and long-term injury recovery.

Development of a High-Color Flow Cytometry Panel for Immunologic Analysis of Tissue Injury and Reconstruction in a Rat Model.

The rat model is an important resource in biomedical research due to its similarities to the human immune system and its use for functional studies. However, because of the preponderance of mouse models in foundational and mechanistic immunological studies, there is a relative lack of diverse, commercially available flow cytometry antibodies for immunological profiling in the rat model. Available antibodies are often conjugated to common fluorophores with similar peak emission wavelengths, making them hard to distinguish on conventional flow cytometers and restricting more comprehensive immune analysis. This can become a limitation when designing immunological studies in rat injury models to investigate the immune response to tissue injury. In addition, this lack of available antibodies limits the number of studies that can be done on the immune populations in lymphoid organs in other research areas. To address this critical unmet need, we designed a spectral flow cytometry panel for rat models. Spectral cytometry distinguishes between different fluorophores by capturing their full emission spectra instead of their peak emission wavelengths. This flow cytometry panel includes 24 distinct immune cell markers to analyze the innate and adaptive immune response. Importantly, this panel identifies different immune phenotypes, including tolerogenic, Type 1, and Type 2 immune responses. We show that this panel can identify unique immune populations and phenotypes in a rat muscle trauma model. We further validated that the panel can identify distinct adaptive and innate immune populations and their unique phenotypes in lymphoid organs. This panel expands the scope of previous rat panels providing a tool for scientists to examine the immune system in homeostasis and injury while pairing mechanistic immunological studies with functional studies.

Neutrophils in asthma: the good, the bad and the bacteria.

Airway inflammation plays a key role in asthma pathogenesis but is heterogeneous in nature. There has been significant scientific discovery with regard to type 2-driven, eosinophil-dominated asthma, with effective therapies ranging from inhaled corticosteroids to novel biologics. However, studies suggest that approximately 1 in 5 adults with asthma have an increased proportion of neutrophils in their airways. These patients tend to be older, have potentially pathogenic airway bacteria and do not respond well to classical therapies. Currently, there are no specific therapeutic options for these patients, such as neutrophil-targeting biologics.Neutrophils comprise 70% of the total circulatory white cells and play a critical defence role during inflammatory and infective challenges. This makes them a problematic target for therapeutics. Furthermore, neutrophil functions change with age, with reduced microbial killing, increased reactive oxygen species release and reduced production of extracellular traps with advancing age. Therefore, different therapeutic strategies may be required for different age groups of patients.The pathogenesis of neutrophil-dominated airway inflammation in adults with asthma may reflect a counterproductive response to the defective neutrophil microbial killing seen with age, resulting in bystander damage to host airway cells and subsequent mucus hypersecretion and airway remodelling. However, in children with asthma, neutrophils are less associated with adverse features of disease, and it is possible that in children, neutrophils are less pathogenic.In this review, we explore the mechanisms of neutrophil recruitment, changes in cellular function across the life course and the implications this may have for asthma management now and in the future. We also describe the prevalence of neutrophilic asthma globally, with a focus on First Nations people of Australia, New Zealand and North America.

New insights in neutrophilic asthma.

PURPOSE OF REVIEW: Recent advances in both murine models and clinical research of neutrophilic asthma are improving our understanding on the etiology and pathophysiology of this enigmatic endotype of asthma. We here aim at providing an overview of our current and latest insights on the pathophysiology and treatment of neutrophilic asthma. RECENT FINDINGS: Activation of the NLRP3 inflammasome pathway with increased IL-1ß has been demonstrated in various studies involving patients with asthma. It has been suggested that type 3 innate lymphoid cells are implicated in the inflammatory cascade leading to neutrophilic inflammation. The role of neutrophil extracellular traps is only at the start of being understood and might be an attractive novel therapeutic target. A diverse panel of nonallergic stimuli, such as cigarette smoke, intensive exercise, cold air or saturated fatty acids, have been linked with neutrophilic airway inflammation. Azithromycin treatment could reduce asthma exacerbations and quality of life in patients with persistent asthma. SUMMARY: Research of the last few years has accelerated our insights in mechanisms underlying neutrophilic asthma. This is in stark contrast with the lack of efficacy of different therapies targeting neutrophil chemotaxis and/or signalling cascade, such as IL-17A or CXCR2. Macrolide therapy might be a useful add-on therapy for patients with persistent asthma.

Intersection of Immunity, Metabolism, and Muscle Regeneration in an Autoimmune-Prone MRL Mouse Model.

Due to the limited capacity of mammals to regenerate complex tissues, researchers have worked to understand the mechanisms of tissue regeneration in organisms that maintain that capacity. One example is the MRL/MpJ mouse strain with unique regenerative capacity in ear pinnae that is absent from other strains, such as the common C57BL/6 strain. The MRL/MpJ mouse has also been associated with an autoimmune phenotype even in the absence of the mutant Fas gene described in its parent strain MRL/lpr. Due to these findings, the differences between the responses of MRL/MpJ versus C57BL/6 strain are evaluated in volumetric muscle injury and subsequent material implantation. One salient feature of the MRL/MpJ response to injury is robust adipogenesis within the muscle. This is associated with a decrease in M2-like polarization in response to biologically derived extracellular matrix scaffolds. In pro-fibrotic materials, such as polyethylene, there are fewer foreign body giant cells in the MRL/MpJ mice. As there are reports of both positive and negative influences of adipose tissue and adipogenesis on wound healing, this model can provide an important lens to investigate the interplay between stem cells, adipose tissue, and immune responses in trauma and material implantation.

Conserved and tissue-specific immune responses to biologic scaffold implantation.

Upon implantation into a patient, any biomaterial induces a cascade of immune responses that influences the outcome of that device. This cascade depends upon several factors, including the composition of the material itself and the location in which the material is implanted. There is still significant uncertainty around the role of different tissue microenvironments in the immune response to biomaterials and how that may alter downstream scaffold remodeling and integration. In this study, we present a study evaluating the immune response to decellularized extracellular matrix materials within the intraperitoneal cavity, the subcutaneous space, and in a traumatic skeletal muscle injury microenvironment. All different locations induced robust cellular recruitment, specifically of macrophages and eosinophils. The latter was most prominent in the subcutaneous space. Intraperitoneal implants uniquely recruited B cells that may alter downstream reactivity as adaptive immunity has been strongly implicated in the outcome of scaffold remodeling. These data suggest that the location of tissue implants should be taken together with the composition of the material itself when designing devices for downline therapeutics. STATEMENT OF SIGNIFICANCE: Different tissue locations have unique immune microenvironments, which can influence the immune response to biomaterial implants. By considering the specific immune profiles of the target tissue, researchers can develop implant materials that promote better integration, reduce complications, and improve the overall outcome of the implantation process.

Conserved and tissue-specific immune responses to biologic scaffold implantation.

Upon implantation into a patient, any biomaterial induces a cascade of immune responses that influences the outcome of that device. This cascade depends upon several factors, including the composition of the material itself and the location in which the material is implanted. There is still significant uncertainty around the role of different tissue microenvironments in the immune response to biomaterials and how that may alter downstream scaffold remodeling and integration. In this study, we present a study evaluating the immune response to decellularized extracellular matrix materials within the intraperitoneal cavity, the subcutaneous space, and in a traumatic skeletal muscle injury microenvironment. All different locations induced robust cellular recruitment, specifically of macrophages and eosinophils. The latter was most prominent in the subcutaneous space. Intraperitoneal implants uniquely recruited B cells that may alter downstream reactivity as adaptive immunity has been strongly implicated in the outcome of scaffold remodeling. These data suggest that the location of tissue implants should be taken together with the composition of the material itself when designing devices for downline therapeutics.

Ectopic adipogenesis in response to injury and material implantation in an autoimmune mouse model.

Due to the limited capacity of mammals to regenerate complex tissues, researchers have worked to understand the mechanisms of tissue regeneration in organisms that maintain that capacity. One example is the MRL/MpJ mouse strain with unique regenerative capacity in ear pinnae that is absent from other strains, such as the common C57BL/6 strain. The MRL/MpJ mouse has also been associated with an autoimmune phenotype even in the absence of the mutant Fas gene described in its parent strain MRL/lpr. Due to these findings, we evaluated the differences between the responses of MRL/MpJ versus C57BL/6 strain in traumatic muscle injury and subsequent material implantation. One salient feature of the MRL/MpJ response to injury was a robust adipogenesis within the muscle. This was associated with a decrease in M2-like polarization in response to biologically derived extracellular matrix scaffolds. In pro-fibrotic materials, such as polyethylene, there were fewer foreign body giant cells in the MRL/MpJ mice. As there are reports of both positive and negative influences of adipose tissue and adipogenesis on wound healing, this model could provide an important lens to investigate the interplay between stem cells, adipose tissue, and immune responses in trauma and materials implantation.

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues.

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Mapping a Pandemic: SARS-CoV-2 Seropositivity in the United States.

Asymptomatic SARS-CoV-2 infection and delayed implementation of diagnostics have led to poorly defined viral prevalence rates. To address this, we analyzed seropositivity in US adults who have not previously been diagnosed with COVID-19. Individuals with characteristics that reflect the US population (n = 11,382) and who had not previously been diagnosed with COVID-19 were selected by quota sampling from 241,424 volunteers (ClinicalTrials.gov NCT04334954). Enrolled participants provided medical, geographic, demographic, and socioeconomic information and 9,028 blood samples. The majority (88.7%) of samples were collected between May 10th and July 31st, 2020. Samples were analyzed via ELISA for anti-Spike and anti-RBD antibodies. Estimation of seroprevalence was performed by using a weighted analysis to reflect the US population. We detected an undiagnosed seropositivity rate of 4.6% (95% CI: 2.6 - 6.5%). There was distinct regional variability, with heightened seropositivity in locations of early outbreaks. Subgroup analysis demonstrated that the highest estimated undiagnosed seropositivity within groups was detected in younger participants (ages 18-45, 5.9%), females (5.5%), Black/African American (14.2%), Hispanic (6.1%), and Urban residents (5.3%), and lower undiagnosed seropositivity in those with chronic diseases. During the first wave of infection over the spring/summer of 2020 an estimate of 4.6% of adults had a prior undiagnosed SARS-CoV-2 infection. These data indicate that there were 4.8 (95% CI: 2.8-6.8) undiagnosed cases for every diagnosed case of COVID-19 during this same time period in the United States, and an estimated 16.8 million undiagnosed cases by mid-July 2020.

Undiagnosed SARS-CoV-2 seropositivity during the first 6 months of the COVID-19 pandemic in the United States.

Asymptomatic SARS-CoV-2 infection and delayed implementation of diagnostics have led to poorly defined viral prevalence rates in the United States and elsewhere. To address this, we analyzed seropositivity in 9089 adults in the United States who had not been diagnosed previously with COVID-19. Individuals with characteristics that reflected the U.S. population (n = 27,716) were selected by quota sampling from 462,949 volunteers. Enrolled participants (n = 11,382) provided medical, geographic, demographic, and socioeconomic information and dried blood samples. Survey questions coincident with the Behavioral Risk Factor Surveillance System survey, a large probability-based national survey, were used to adjust for selection bias. Most blood samples (88.7%) were collected between 10 May and 31 July 2020 and were processed using ELISA to measure seropositivity (IgG and IgM antibodies against SARS-CoV-2 spike protein and the spike protein receptor binding domain). The overall weighted undiagnosed seropositivity estimate was 4.6% (95% CI, 2.6 to 6.5%), with race, age, sex, ethnicity, and urban/rural subgroup estimates ranging from 1.1% to 14.2%. The highest seropositivity estimates were in African American participants; younger, female, and Hispanic participants; and residents of urban centers. These data indicate that there were 4.8 undiagnosed SARS-CoV-2 infections for every diagnosed case of COVID-19, and an estimated 16.8 million infections were undiagnosed by mid-July 2020 in the United States.

Hypersegmented airway neutrophils and its association with reduced lung function in adults with obstructive airway disease: an exploratory study.

OBJECTIVES: The significance of neutrophilic inflammation in obstructive airway disease remains controversial. Recent studies have demonstrated presence of an active neutrophil population in systemic circulation, featuring hypersegmented morphology, with high oxidative burst and functional plasticity in inflammatory conditions. The aim of this study was to characterise neutrophil subsets in bronchial lavage (BL) of obstructive airway disease participants (asthma, chronic obstructive pulmonary disease (COPD) and bronchiectasis) and healthy controls on the basis of nuclear morphology and to assess the association between neutrophil subsets and the clinical parameters of the obstructive airway disease participants. DESIGN: A cross-sectional exploratory study. SETTING: John Hunter Hospital and Hunter Medical Research Institute, Australia. PARTICIPANTS: Seventy-eight adults with obstructive airway disease comprised those with stable asthma (n=39), COPD (n=20) and bronchiectasis (n=19) and 20 healthy controls. MATERIALS AND METHODS: Cytospins were prepared and neutrophil subsets were classified based on nuclear morphology into hypersegmented (>4 lobes), normal (2-4 lobes) and banded (1 lobe) neutrophils and enumerated. RESULTS: Neutrophils from each subset were identified in all participants. Numbers of hypersegmented neutrophils were elevated in participants with airway disease compared with healthy controls (p<0.001). Both the number and the proportion of hypersegmented neutrophils were highest in COPD participants (median (Q1-Q3) of 1073.6 (258.8-2742) × 102/mL and 24.5 (14.0-46.5)%, respectively). An increased proportion of hypersegmented neutrophils in airway disease participants was significantly associated with lower forced expiratory volume in 1 s/forced vital capacity per cent (Spearman's r=-0.322, p=0.004). CONCLUSION: Neutrophil heterogeneity is common in BL and is associated with more severe airflow obstruction in adults with airway disease. Further work is required to elucidate the functional consequences of hypersegmented neutrophils in the pathogenesis of disease.

Blood Neutrophils In COPD But Not Asthma Exhibit A Primed Phenotype With Downregulated CD62L Expression.

Purpose: To characterize neutrophils in obstructive airway disease by measuring their surface adhesion molecules and oxidative burst along with characterizing them into different subsets as per their adhesion molecule expression. Patients and methods: Peripheral blood from adults with COPD (n=17), asthma (n=20), and healthy participants (n=19) was examined for expression of CD16, CD62L, CD11b, CD11c, and CD54, and analyzed by flow cytometry. For oxidative burst and CD62L shedding analysis, CD16 and CD62L stained leukocytes were loaded with Dihydrorhodamine-123 (DHR-123) and stimulated with N-Formylmethionine-leucyl-phenylalanine (fMLF). Neutrophil subsets were characterized based on CD16 and CD62L expression. Marker surface expression was recorded on CD16+ neutrophils as median fluorescence intensity (MFI). Results: Neutrophil surface expression of CD62L was significantly reduced in COPD (median (IQR) MFI: 1156 (904, 1365)) compared with asthma (1865 (1157, 2408)) and healthy controls (2079 (1054, 2960)); p=0.028. COPD neutrophils also demonstrated a significant reduction in CD62L expression with and without fMLF stimulation. Asthma participants had a significantly increased proportion and number of CD62Lbright/CD16dim neutrophils (median: 5.4% and 0.14 × 109/L, respectively), in comparison with healthy (3.54% and 0.12 × 109/L, respectively); p<0.017. Conclusion: Reduced CD62L expression suggests blood neutrophils have undergone priming in COPD but not in asthma, which may be the result of systemic inflammation. The increased shedding of CD62L receptor by COPD blood neutrophils suggests a high sensitivity for activation.