Key Cellular Players Research Articles

Inhalable SARS-CoV-2 Mimetic Particles Induce Pleiotropic Antigen Presentation.

Coronavirus disease 2019 (Covid-19) has caused over 5.5 million deaths worldwide, and viral mutants continue to ravage communities with limited access to injectable vaccines or high rates of vaccine hesitancy. Inhalable vaccines have the potential to address these distribution and compliance issues as they are less likely to require cold storage, avoid the use of needles, and can elicit localized immune responses with only a single dose. Alveolar macrophages represent attractive targets for inhalable vaccines as they are abundant within the lung mucosa (up to 95% of all immune cells) and are important mediators of mucosal immunity, and evidence suggests that they may be key cellular players in early Covid-19 pathogenesis. Here, we report inhalable coronavirus mimetic particles (CoMiP) designed to rapidly bind to, and be internalized by, alveolar macrophages to deliver nucleic acid-encoded viral antigens. Inspired by the SARS-CoV-2 virion structure, CoMiP carriers package nucleic acid cargo within an endosomolytic peptide envelope that is wrapped in a macrophage-targeting glycosaminoglycan coating. Through this design, CoMiP mimic several important features of the SARS-CoV-2 virion, particularly surface topography and macromolecular chemistry. As a result, CoMiP effect pleiotropic transfection of macrophages and lung epithelial cells in vitro with multiple antigen-encoding plasmids. In vivo immunization yields increased mucosal IgA levels within the respiratory tract of CoMiP vaccinated mice.

Engineering DNA Nanostructures to Manipulate Immune Receptor Signaling and Immune Cell Fates.

Immune cells sense, communicate, and logically integrate a multitude of environmental signals to make important cell-fate decisions and fulfill their effector functions. These processes are initiated and regulated by a diverse array of immune receptors and via their dynamic spatiotemporal organization upon ligand binding. Given the widespread relevance of the immune system to health and disease, there have been significant efforts toward understanding the biophysical principles governing immune receptor signaling and activation, as well as the development of biomaterials which exploit these principles for therapeutic immune engineering. Here, how advances in the field of DNA nanotechnology constitute a growing toolbox for further pursuit of these endeavors is discussed. Key cellular players involved in the induction of immunity against pathogens or diseased cells are first summarized. How the ability to design DNA nanostructures with custom shapes, dynamics, and with site-specific incorporation of diverse guests can be leveraged to manipulate the signaling pathways that regulate these processes is then presented. It is followed by highlighting emerging applications of DNA nanotechnology at the crossroads of immune engineering, such as in vitro reconstitution platforms, vaccines, and adjuvant delivery systems. Finally, outstanding questions that remain for further advancing immune-modulatory DNA nanodevices are outlined.

Targeting Neuroimmune Interactions in Diabetic Neuropathy with Nanomedicine.

Significance: Diabetes is a major source of neuropathy and neuropathic pain that is set to continue growing in prevalence. Diabetic peripheral neuropathy (DPN) and pain associated with diabetes are not adequately managed by current treatment regimens. Perhaps the greatest difficulty in treating DPN is the complex pathophysiology, which involves aspects of metabolic disruption and neurotrophic deficits, along with neuroimmune interactions. There is, therefore, an urgent need to pursue novel therapeutic options targeting the key cellular and molecular players. Recent Advances: To that end, cellular targeting becomes an increasingly compelling drug delivery option as our knowledge of neuroimmune interactions continues to mount. These nanomedicine-based approaches afford a potentially unparalleled specificity and longevity of drug targeting, using novel or established compounds, all while minimizing off-target effects. Critical Issues: The DPN therapeutics directly targeted at the nervous system make up the bulk of currently available treatment options. However, there are significant opportunities based on the targeting of non-neuronal cells and neuroimmune interactions in DPN. Future Directions: Nanomedicine-based agents represent an exciting opportunity for the treatment of DPN with the goals of improving the efficacy and safety profile of analgesia, as well as restoring peripheral neuroregenerative capacity. Antioxid. Redox Signal. 36, 122-143.

Fibrotic Idiopathic Interstitial Lung Disease: The Molecular and Cellular Key Players.

Interstitial lung diseases (ILDs) that are known as diffuse parenchymal lung diseases (DPLDs) lead to the damage of alveolar epithelium and lung parenchyma, culminating in inflammation and widespread fibrosis. ILDs that account for more than 200 different pathologies can be divided into two groups: ILDs that have a known cause and those where the cause is unknown, classified as idiopathic interstitial pneumonia (IIP). IIPs include idiopathic pulmonary fibrosis (IPF), non-specific interstitial pneumonia (NSIP), cryptogenic organizing pneumonia (COP) known also as bronchiolitis obliterans organizing pneumonia (BOOP), acute interstitial pneumonia (AIP), desquamative interstitial pneumonia (DIP), respiratory bronchiolitis-associated interstitial lung disease (RB-ILD), and lymphocytic interstitial pneumonia (LIP). In this review, our aim is to describe the pathogenic mechanisms that lead to the onset and progression of the different IIPs, starting from IPF as the most studied, in order to find both the common and standalone molecular and cellular key players among them. Finally, a deeper molecular and cellular characterization of different interstitial lung diseases without a known cause would contribute to giving a more accurate diagnosis to the patients, which would translate to a more effective treatment decision.

Myeloid-IL4Rα is an indispensable link in IL-33-ILCs-IL-13-IL4Rα axis of eosinophil recruitment in murine lungs

Increased eosinophil recruitment is a hallmark feature of eosinophilic disorders. Here, we delineated the key molecular and cellular players involved in physiological eosinophilic recruitment during normal postnatal lung development in mice. Physiological eosinophilic recruitment was consistently present in 7-, 10-, and 15-day-old neonatal mice, but not in 42-day-old mice. This feature was completely abolished in interleukin 33 (IL-33)-, interleukin 2 receptor gamma chain (IL2rγ)-, and interleukin 4 receptor alpha (IL4Rα)-knockout mice, but not in recombination activating gene 1 (Rag1)-knockout mice demonstrating an indispensable role for IL-33, innate lymphoid cells (ILCs), and IL4Rα in eosinophil recruitment. Interestingly, myeloid-specific IL4Rα-deficient (mye-IL4Rα−/−) mice had significantly reduced eosinophilia in the airspaces that was associated with reduced levels of IL-4 and IL-5 in the bronchoalveolar lavage fluid (BALF). Further, we tested the effect of myeloid-specific IL4Rα deficiency on IL-13-induced eosinophil recruitment into adult lung airspaces. Eosinophil recruitment into the airspaces was elevated in IL-13-treated WT mice but not in IL-13-treated mye-IL4Rα−/− mice. Consistent with the degree of eosinophilia, the BALF levels of eosinophil recruitment-associated cytokines were significantly elevated in IL-13-treated WT but not in IL-13-treated mye-IL4Rα−/− mice. These data establish that myeloid-IL4Rα is an indispensable component of the IL-33-ILCsIL-13-IL4Rα axis of eosinophil recruitment.

The CD8 T Cell-Epstein-Barr Virus-B Cell Trialogue: A Central Issue in Multiple Sclerosis Pathogenesis.

The cause and the pathogenic mechanisms leading to multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system (CNS), are still under scrutiny. During the last decade, awareness has increased that multiple genetic and environmental factors act in concert to modulate MS risk. Likewise, the landscape of cells of the adaptive immune system that are believed to play a role in MS immunopathogenesis has expanded by including not only CD4 T helper cells but also cytotoxic CD8 T cells and B cells. Once the key cellular players are identified, the main challenge is to define precisely how they act and interact to induce neuroinflammation and the neurodegenerative cascade in MS. CD8 T cells have been implicated in MS pathogenesis since the 80’s when it was shown that CD8 T cells predominate in MS brain lesions. Interest in the role of CD8 T cells in MS was revived in 2000 and the years thereafter by studies showing that CNS-recruited CD8 T cells are clonally expanded and have a memory effector phenotype indicating in situ antigen-driven reactivation. The association of certain MHC class I alleles with MS genetic risk implicates CD8 T cells in disease pathogenesis. Moreover, experimental studies have highlighted the detrimental effects of CD8 T cell activation on neural cells. While the antigens responsible for T cell recruitment and activation in the CNS remain elusive, the high efficacy of B-cell depleting drugs in MS and a growing number of studies implicate B cells and Epstein-Barr virus (EBV), a B-lymphotropic herpesvirus that is strongly associated with MS, in the activation of pathogenic T cells. This article reviews the results of human studies that have contributed to elucidate the role of CD8 T cells in MS immunopathogenesis, and discusses them in light of current understanding of autoreactivity, B-cell and EBV involvement in MS, and mechanism of action of different MS treatments. Based on the available evidences, an immunopathological model of MS is proposed that entails a persistent EBV infection of CNS-infiltrating B cells as the target of a dysregulated cytotoxic CD8 T cell response causing CNS tissue damage.

Review: Obesity and COVID-19: A Detrimental Intersection.

Obesity has been recognized as an independent risk factor for critical illness and major severity in subjects with coronavirus disease 2019 (COVID-19). The role of fat distribution, particularly visceral fat (often linked to metabolic abnormalities), is still unclear. The adipose tissue represents a direct source of cytokines responsible for the pathological modifications occurring within adipose tissue in obese subjects. Adipokines are a crucial connection between metabolism and immune system: their dysregulation in obesity contributes to chronic low-grade systemic inflammation and metabolic comorbidities. Therefore the increased amount of visceral fat can lead to a proinflammatory phenotypic shift. This review analyzes the interrelation between obesity and COVID-19 severity, as well as the cellular key players and molecular mechanisms implicated in adipose inflammation, investigating if adipose tissue can constitute a reservoir for viral spread, and contribute to immune activation and cytokines storm. Targeting the underlying molecular mechanisms might have therapeutic potential in the management of obesity-related complications in COVID-19 patients.

Self-Nucleic Acid Sensing: A Novel Crucial Pathway Involved in Obesity-Mediated Metaflammation and Metabolic Syndrome.

Obesity and overweight are a global health problem affecting almost one third of the world population. There are multiple complications associated with obesity including metabolic syndrome that commonly lead to development of type II diabetes and non-alcoholic fatty liver disease. The development of metabolic syndrome and severe complications associated with obesity is attributed to the chronic low-grade inflammation that occurs in metabolic tissues such as the liver and the white adipose tissue. In recent years, nucleic acids (mostly DNA), which accumulate systemically in obese individuals, were shown to aberrantly activate innate immune responses and thus to contribute to metabolic tissue inflammation. This minireview will focus on (i) the main sources and forms of nucleic acids that accumulate during obesity, (ii) the sensing pathways required for their detection, and (iii) the key cellular players involved in this process. Fully elucidating the role of nucleic acids in the induction of inflammation induced by obesity would promote the identification of new and long-awaited therapeutic approaches to limit obesity-mediated complications.

Inhalable SARS-CoV-2 Mimetic Particles Induce Pleiotropic Antigen Presentation

Covid-19 has caused over 4.5 million deaths worldwide and continues to ravage communities with limited access to injectable vaccines, or high rates of vaccine hesitancy. Inhalable vaccines have the potential to address these distribution and compliance issues as they are less likely to require cold storage, avoid the use of needles, and can elicit potent localized immune responses with only a single dose. Alveolar macrophages represent attractive targets for inhalable vaccines as they are abundant within lung mucosa (90-95% of all immune cells), are important mediators of mucosal immunity, and evidence suggests may be key cellular players in early Covid-19 pathogenesis. Here, we report inhalable coronavirus mimetic particles (CoMiP) designed to rapidly bind to, and be internalized by, alveolar macrophages to deliver nucleic acid encoded viral antigens. Inspired by the SARS-CoV-2 virion structure, CoMiPs package nucleic acid cargo within an endosomolytic peptide envelope that is wrapped in a macrophage-targeting glycosaminoglycan coating. Through this design, CoMiP carriers mimic several important features of the SARS-CoV-2 virion, particularly surface topography and macromolecular chemistry. As a result, CoMiPs effect pleiotropic transfection of macrophage and lung epithelial cells in vitro with multiple antigen-encoding plasmids. In vivo immunization yields increased mucosal IgA levels within the respiratory tract of CoMIP vaccinated mice.Funding: Funding for this work was provided by the COVID Research Seed Fund from the Huck Institutes of the Life Sciences and Materials Research Institute at the Pennsylvania State University.Declaration of Interests: None to declare. Ethics Approval Statement: Animal care committee is PSU IACUC, under protocol number 001552.

Nanoparticle-Directed Targeting of Clustered PSGL-1 Mitigates Neutrophil-Derived Thrombosis in Antiphospholipid Antibody Syndrome

Multiorgan thromboses and subsequent organ failure are among the more devastating consequences of antiphospholipid antibody syndrome (APS). APS poses a relatively unique risk to patients by virtue of its propensity to cause thrombi of both the arterial and venous vascular systems. While recent studies have explored mechanisms of thrombosis in singular vascular beds, there remains a dearth of targetable effectors of arteriovenous thrombosis in APS. Here, we examine the potential of neutrophils as key players of both arterial and venous thrombosis in APS. From this and previous studies, we have also identified a critical transcriptional regulator in neutrophils whose loss mimics arterial and venous thrombosis susceptibility seen in APS. This factor, Krüppel-like factor 2 (KLF2), is down regulated in APS patients as well as in cells treated with antiphospholipid antibody (APLA) ex vivo. Loss of KLF2 in neutrophils contributes to classic neutrophil activation phenotypes including NETosis, increased tissue factor release, and heightened chemotaxis; remarkably, blocking each of these functions greatly abrogates the thrombotic phenotype. Utilizing the myeloid KLF2 knockout (K2KO) system as a model for aberrant neutrophil activation seen in APS, we have identified clustering of P-selectin glycoprotein ligand 1 (PSGL-1) secondary to actin rearrangement to be operative in neutrophil-mediated thrombosis in arterial and venous systems. While neutralizing antibodies against PSGL-1 have been explored as therapeutic options in APS, there remains a challenge in establishing local specificity to activated immune cells in order to increase efficacy. To circumvent this, we have designed a nanoparticle (NP) based approach that utilizes NPs decorated with anti-PSGL-1 immunoglobulins to better target clustered PSGL-1 on activated neutrophils. PSGL-1 NPs are more effective at inhibiting neutrophil rolling and adhesion in in vitro assays while, incubating activated neutrophils in PSGL-1 NPs prior to infusion attenuates their ability to contribute to both arterial and venous thrombosis. Further, injection of PSGL-1 NPs directly into K2KO mice reduces their extensive thrombotic phenotype. Importantly, this effect occurs at a substantially lower dose of PSGL-1 NPs than that which would be needed of PSGL-1 antibody alone. Finally, PSGL-1 NPs greatly reduce thrombotic burden in mice injected with APLA, diminishing clotting in both arteries and veins. Together, this work identifies key molecular and cellular players in APLA-induced arteriovenous thrombosis. Furthermore, it provides early pre-clinical evidence at specifically targeting clustered PSGL-1 on neutrophils as a viable option in combatting thrombosis in this particularly vulnerable patient population. Disclosures No relevant conflicts of interest to declare.

Updated perspectives on vascular cell specification and pluripotent stem cell-derived vascular organoids for studying vasculopathies.

Vasculopathy is a pathological process occurring in the blood vessel wall, which could affect the haemostasis and physiological functions of all the vital tissues/organs and is one of the main underlying causes for a variety of human diseases including cardiovascular diseases. Current pharmacological interventions aiming to either delay or stop progression of vasculopathies are suboptimal, thus searching novel, targeted, risk-reducing therapeutic agents, or vascular grafts with full regenerative potential for patients with vascular abnormalities are urgently needed. Since first reported, pluripotent stem cells (PSCs), particularly human-induced PSCs, have open new avenue in all research disciplines including cardiovascular regenerative medicine and disease remodelling. Assisting with recent technological breakthroughs in tissue engineering, in vitro construction of tissue organoid made a tremendous stride in the past decade. In this review, we provide an update of the main signal pathways involved in vascular cell differentiation from human PSCs and an extensive overview of PSC-derived tissue organoids, highlighting the most recent discoveries in the field of blood vessel organoids as well as vascularization of other complex tissue organoids, with the aim of discussing the key cellular and molecular players in generating vascular organoids.

Neuro-innate immune interactions in gut mucosal immunity.

The gastrointestinal (GI) tract performs a set of vital physiological functions related to food and water consumption. To help regulate these complex physiological processes, the GI tract is innervated by extensive neural networks. The GI tract also serves as the largest immune organ aimed to protect hosts from harmful microbes and toxins ingested with food. It emerges that the enteric nervous and immune systems are highly integrated to optimize digestion while reinforcing immune protection. In this review, we will discuss key cellular players involved in the neuro-immune interactions within the GI mucosa with the focus on the recently uncovered neural pathways that regulate mucosal immunity in a context relevant to GI health and disease.

The Japanese Experience with Basic Fibroblast Growth Factor in Cutaneous Wound Management and Scar Prevention: A Systematic Review of Clinical and Biological Aspects.

IntroductionBasic fibroblast growth factor (bFGF) plays several key roles in wound healing. Over the last 2 decades, clinical and basic research on bFGF has been actively conducted in Japan with reports on its potent efficacy in accelerating the healing of chronic ulcers and burn wounds by stimulating key cellular players in the skin. However, its efficacy remains unrecognized internationally. Thus, this study reviews current knowledge about the therapeutic value of bFGF in wound management and scar prevention accumulated in Japan over the last 2 decades.MethodsWe review the Japanese literature that demonstrates the anti-scarring effects of bFGF and exhaustively assess how these effects are exerted. Using the search terms “bFGF OR growth factors AND wound healing in Japan” and “bFGF AND scar prevention in Japan,” we conducted a search of the PubMed database for publications on the role of bFGF in wound and scar management in Japan. All eligible papers published between 1988 and December 2019 were retrieved and reviewed.ResultsOur search yielded 208 articles; 82 were related to the application of bFGF for dermal wound healing in Japan. Of these, 27 fulfilled all inclusion criteria; 11 were laboratory studies, 7 were case reports, 4 were clinical studies, and 5 were randomized controlled trials.ConclusionFurther research, with recognition of the therapeutic value of bFGF in wound and scar management and its clinical applications, is needed to provide additional clinical advantages while improving wound healing and reducing the risk of post-surgical scar formation.

4097 Transcriptome and molecular analysis of erythropoietin-induced hypertension

OBJECTIVES/GOALS: High blood-pressure (BP) is a common adverse effect of erythropoietin (EPO) therapy among patients with chronic kidney disease on hemodialysis, and even among otherwise healthy individuals who receive EPO. In human genetics, EPO is associated with not only red blood cell traits, but hypertension (HTN) as well. Currently, there is no vascular gene expression data available in the setting of EPO-induced HTN that may explain precise role of key cellular players in its hypertensive etiology. Our aim is to characterize vascular transcriptome to identify key cellular players in EPO-induced HTN. METHODS/STUDY POPULATION: 10-12 week C57BL/6 male and female mice were randomly divided into two groups 1. Vehicle (0.9% saline-VEH), 2. EPO, (N = 4). VEH and EPO were intraperitoneal administered (EPO 75U/30g, 3 times/week) for 20 days. Blood-pressure was measured non-invasively via tail-cuff plethysmography. We characterized in-vivo transcriptome response of mouse descending aorta to EPO-HTN and vehicle control group by high-throughput RNA-sequencing. RESULTS/ANTICIPATED RESULTS: Systolic blood pressure (SBP) was significantly higher in EPO treatment, compared to vehicle (males and females combined SBP VEH 116.29 ± 6.21, EPO 129.57 ± 4.59, mean ± s.d., adjusted P = 0.0012). Comparison of in-vivo transcriptional differences between vehicle and EPO-treated reveal statistically significant changes in cellular pathways consistent with hypertension such as upregulation of RAS signaling pathway and oxidative stress. In-vitro mouse aortic smooth muscle cells, EPO markedly increased phosphorylated-ERK activity, suggesting increased RAS activity. DISCUSSION/SIGNIFICANCE OF IMPACT: This study highlights the importance of previously unknown vascular key players and advances our understanding of the transcriptional events associated with EPO-induced hypertension.

Glial Cell-Axonal Growth Cone Interactions in Neurodevelopment and Regeneration

The developing nervous system is a complex yet organized system of neurons, glial support cells, and extracellular matrix that arranges into an elegant, highly structured network. The extracellular and intracellular events that guide axons to their target locations have been well characterized in many regions of the developing nervous system. However, despite extensive work, we have a poor understanding of how axonal growth cones interact with surrounding glial cells to regulate network assembly. Glia-to-growth cone communication is either direct through cellular contacts or indirect through modulation of the local microenvironment via the secretion of factors or signaling molecules. Microglia, oligodendrocytes, astrocytes, Schwann cells, neural progenitor cells, and olfactory ensheathing cells have all been demonstrated to directly impact axon growth and guidance. Expanding our understanding of how different glial cell types directly interact with growing axons throughout neurodevelopment will inform basic and clinical neuroscientists. For example, identifying the key cellular players beyond the axonal growth cone itself may provide translational clues to develop therapeutic interventions to modulate neuron growth during development or regeneration following injury. This review will provide an overview of the current knowledge about glial involvement in development of the nervous system, specifically focusing on how glia directly interact with growing and maturing axons to influence neuronal connectivity. This focus will be applied to the clinically-relevant field of regeneration following spinal cord injury, highlighting how a better understanding of the roles of glia in neurodevelopment can inform strategies to improve axon regeneration after injury.

Prognostic Value of the Pretreatment Neutrophil-to-Lymphocyte Ratio in Different Phenotypes of Locally Advanced Breast Cancer During Neoadjuvant Systemic Treatment

PurposeNeutrophils are among the key cellular players in the inflammatory milieu produced in patients with breast cancer (BC), and strong evidence exists in terms of the prognostic value of assessing the neutrophil-to-lymphocyte ratio (NLR) in patients with BC. In this study we sought to determine whether the baseline NLR correlates with pathological complete response (pCR), disease-free survival (DFS), and overall survival (OS) in patients with locally advanced BC in the neoadjuvant chemotherapy (NAC) setting. MethodsWe analyzed the pretreatment NLR from the first blood count of patients treated from 2007 to 2015 in terms of pCR, DFS, and OS in patients with locally advanced BC. Patients received standard medical care based on national guidelines. ResultsA total of 1519 patients were included in the study. Median age was 49 years (22-88). The cutoff point for NLR was 2.0. NLR was not associated with pCR or DFS. However, patients with high NLR had worse OS in the presence of triple-negative BC (105.9 months; 95% confidence interval [CI], 100.2-111.5] vs. 98.7 months; 95% CI, 91.1-106.3; P = .029), Her2 overexpression (114.0 months; 95% CI, 110.5-118.0 vs. 100.8 months; 95% CI 95.7-105.9; P = .019), and residual disease after NAC for both phenotypes. Multivariate analysis showed that NLR was independently associated with OS (hazard ratio, 1.4; 95% CI, 1.02-1.95; P = .037). ConclusionsPretreatment NLR in patients with locally advanced BC correlates with OS as an independent prognostic factor. This influence depends on phenotype and residual disease. Routine assessment of this parameter could be an easy and affordable tool for defining prognosis.

The Nose-To-Brain Transport of Polymeric Nanoparticles Is Mediated by Immune Sentinels and Not by Olfactory Sensory Neurons.

The nose-to-brain (N-to-B) transport mechanism of nanoparticles through the olfactory epithelium (OE) is not fully understood. Most research utilized nasal epithelial cell models completely deprived of olfactory cells. Aiming to shed light into key cellular pathways, in this work, for the first time, the interaction of polymeric nanoparticles in a 17-483 nm size range and with neutral and negatively and positively charged surfaces with primary olfactory sensory neurons, cortical neurons, and microglia isolated from olfactory bulb (OB), OE, and cortex of newborn rats is investigated. After demonstrating the good cell compatibility of the different nanoparticles, the nanoparticle uptake by confocal laser scanning fluorescence microscopy is monitored. Our findings reveal that neither olfactory nor forebrain neurons internalize nanoparticles. Conversely, it is demonstrated that olfactory and cortical microglia phagocytose the nanoparticles independently of their features. Overall, our findings represent the first unambiguous evidence of the possible involvement of microglia in N-to-B nanoparticle transport and the unlikely involvement of neurons. Furthermore, this approach emerges as a completely new experimental tool to screen the biocompatibility, uptake, and transport of nanomaterials by key cellular players of the N-to-B pathway in nanosafety and nanotoxicology and nanomedicine.

Eosinophils and Macrophages within the Th2-Induced Granuloma: Balancing Killing and Healing in a Tight Space.

Granuloma formation is a key host immune response generated to confine invading pathogens and limit extensive host damage. It consists of an accumulation of host immune cells around a pathogen. This host response has been extensively studied in the context of inflammatory diseases. However, there is much less known about Th2-type granulomas generated in response to parasitic worms. Based on in vitro data, innate immune cells within the granuloma are thought to immobilize and kill parasites but also act to repair damaged tissue. Understanding this dual function is key. The two billion people and many livestock/wild animals infected with helminths demonstrate that granulomas are not effective at clearing infection. However, the lack of high mortality highlights their importance in ensuring that parasite migration/tissue damage is restricted and wound healing is effective. In this review, we define two key cellular players (macrophages and eosinophils) and their associated molecular players involved in Th2 granuloma function. To date, the underlying mechanisms remain poorly understood, which is in part due to a lack of conclusive studies. Most have been performed in vitro rather than in vivo, using cells that have not been obtained from granulomas. Experiments using genetically modified mouse strains and/or antibody/chemical-mediated cell depletion have also generated conflicting results depending on the model. We discuss the caveats of previous studies and the new tools available that will help fill the gaps in our knowledge and allow a better understanding of the balance between immune killing and healing.

The Possible Role of Neutrophils in the Induction of Osteoclastogenesis.

The ligand of the receptor activator of NF-κB (RANKL) is a key molecule in the formation of osteoclasts, the key cells that cause the disease-associated alveolar bone resorption in periodontitis. We hypothesized that polymorphonuclear leukocytes (PMNs), found as the most prominent cells of inflamed periodontal tissues, could play an important role in providing signals to trigger osteoclastogenesis and thus activating pathological bone resorption in periodontitis. RANKL expression was investigated on circulatory PMNs (cPMNs) and oral PMNs (oPMNs) taken from both controls and periodontitis patients. On average, 2.3% and 2.4% RANKL expression was detected on the cPMNs and oPMNs from periodontitis patients, which did not differ significantly from healthy controls. Since cPMNs may acquire a more osteoclastogenesis-facilitating phenotype while migrating into the inflamed periodontium, we next investigated whether stimulated (with LPS, TNF-α, or IL-6) cPMNs have the capacity to contribute to osteoclastogenesis. Enduring surface expression of RANKL for short-lived cells as cPMNs was achieved by fixating stimulated cPMNs. RANKL expression on stimulated cPMNs, as assessed by flow cytometry and immunohistochemistry, was limited (6.48 ± 0.72%, mean expression ± SEM) after 24 and 48 hours of stimulation with LPS. Likewise, stimulation with TNF-α and IL-6 resulted in limited RANKL expression levels. These limited levels of expression did not induce osteoclastogenesis when cocultured with preosteoclasts for 10 days. We report that, under the aforementioned experimental conditions, neither cPMNs nor oPMNs directly induced osteoclastogenesis. Further elucidation of the key cellular players and immune mediators that stimulate alveolar bone resorption in periodontitis will help to unravel its pathogenesis.

Super-Resolution Microscopy Reveals Local Accumulation of Plasma Membrane Gangliosides at Neisseria meningitidis Invasion Sites.

Neisseria meningitidis (meningococcus) is a Gram-negative bacterium responsible for epidemic meningitis and sepsis worldwide. A critical step in the development of meningitis is the interaction of bacteria with cells forming the blood-cerebrospinal fluid barrier, which requires tight adhesion of the pathogen to highly specialized brain endothelial cells. Two endothelial receptors, CD147 and the β2-adrenergic receptor, have been found to be sequentially recruited by meningococci involving the interaction with type IV pilus. Despite the identification of cellular key players in bacterial adhesion the detailed mechanism of invasion is still poorly understood. Here, we investigated cellular dynamics and mobility of the type IV pilus receptor CD147 upon treatment with pili enriched fractions and specific antibodies directed against two extracellular Ig-like domains in living human brain microvascular endothelial cells. Modulation of CD147 mobility after ligand binding revealed by single-molecule tracking experiments demonstrates receptor activation and indicates plasma membrane rearrangements. Exploiting the binding of Shiga (STxB) and Cholera toxin B (CTxB) subunits to the two native plasma membrane sphingolipids globotriaosylceramide (Gb3) and raft-associated monosialotetrahexosylganglioside GM1, respectively, we investigated their involvement in bacterial invasion by super-resolution microscopy. Structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM) unraveled accumulation and coating of meningococci with GM1 upon cellular uptake. Blocking of CTxB binding sites did not impair bacterial adhesion but dramatically reduced bacterial invasion efficiency. In addition, cell cycle arrest in G1 phase induced by serum starvation led to an overall increase of GM1 molecules in the plasma membrane and consequently also in bacterial invasion efficiency. Our results will help to understand downstream signaling events after initial type IV pilus-host cell interactions and thus have general impact on the development of new therapeutics targeting key molecules involved in infection.