induction-ocular-surface-inflammation-collection-involved

Induction of Ocular Surface Inflammation and Collection of Involved Tissues

Ocular surface diseases include a range of disorders that disturb the functions and structures of the cornea, conjunctiva, and the associated ocular ...

Friedrich-Alexander-University Erlangen-Nürnberg and Universitätsklinikum Erlangen

Inappropriate clearance of apoptotic remnants is considered to be the primary cause of systemic autoimmune diseases, like systemic lupus erythematosus. Here we demonstrate that apoptotic cells release distinct types of subcellular membranous particles (scMP) derived from the endoplasmic reticulum (ER) or the plasma membrane. Both types of scMP exhibit desialylated glycotopes resulting from surface exposure of immature ER-derived glycoproteins or from surface-borne sialidase activity, respectively. Sialidase activity is activated by caspase-dependent mechanisms during apoptosis. Cleavage of sialidase Neu1 by caspase 3 was shown to be directly involved in apoptosis-related increase of surface sialidase activity. ER-derived blebs possess immature mannosidic glycoepitopes and are prioritized by macrophages during clearance. Plasma membrane-derived blebs contain nuclear chromatin (DNA and histones) but not components of the nuclear envelope. Existence of two immunologically distinct types of apoptotic blebs may provide new insights into clearance-related diseases.

Macrophages discriminate glycosylation patterns of apoptotic cell-derived microparticles.

In addition to the redundancy of the receptors for the Fc portion of immunoglobulins, glycans result in potential ligands for a plethora of lectin receptors found in immune effector cells. Here we analysed the exposure of glycans containing fucosyl residues and the fucosylated tri-mannose N-type core by complexed native IgG in longitudinal serum samples of well-characterized patients with systemic lupus erythematosus. Consecutive serum samples of a cohort of 15 patients with systemic lupus erythematosus during periods of increased disease activity and remission were analysed. All patients fulfilled the 1982 American College of Rheumatology classification criteria. Sera of 15 sex- and age-matched normal healthy blood donors served as controls. The levels and type of glycosylation of complexed random IgG was measured with lectin enzyme-immunosorbent assays. After specifically gathering IgG complexes from sera, biotinylated lectins Aleuria aurantia lectin and Lens culinaris agglutinin were employed to detect IgG-associated fucosyl residues and the fucosylated tri-mannose N-glycan core, respectively. In sandwich-ELISAs, IgG-associated IgM, IgA, C1q, C3c and C-reactive protein (CRP) were detected as candidates for IgG immune complex constituents. We studied associations of the glycan of complexed IgG and disease activity according to the physician's global assessment of disease activity and the systemic lupus erythematosus disease activity index 2000 documented at the moment of blood taking. Our results showed significantly higher levels of Aleuria aurantia lectin and Lens culinaris agglutinin binding sites exposed on IgG complexes of patients with systemic lupus erythematosus than on those of normal healthy blood donors. Disease activity in systemic lupus erythematosus correlated with higher exposure of Aleuria aurantia lectin-reactive fucosyl residues by immobilized IgG complexes. Top levels of Aleuria aurantia lectin-reactivity were found in samples taken during the highest activity of systemic lupus erythematosus. Our results show that native circulating IgG complexes from active systemic lupus erythematosus patients expose fucosyl residues and their glycan core is accessible to soluble lectins. Two putative mechanisms may contribute to the increased exposure of these glycans: (1) the canonical N-glycosylation site of the IgG-CH2 domain; (2) an IgG binding non-IgG molecule, like complement or C-reactive protein. In both cases the complexed IgG may be alternatively targeted to lectin receptors of effector cells, e.g. dendritic cells. 

Altered glycosylation of complexed native IgG molecules is associated with disease activity of systemic lupus erythematosus.

Glycosylation is well-known to modulate the functional capabilities of immunoglobulin G (IgG)-mediated cellular and humoral responses. Indeed, highly sialylated and desialylated IgG is endowed with anti- and pro-inflammatory activities, respectively, whereas fully deglycosylated IgG is a rather lame duck, with no effector function besides toxin neutralization. Recently, several studies revealed the impact of different glycosylation patterns on the Fc part and Fab fragment of IgG in several autoimmune diseases, including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Here, we provide a synoptic update summarizing the most important aspects of antibody glycosylation, and the current progress in this field. We also discuss the therapeutic options generated by the modification of the glycosylation of IgG in a potential treatment for chronic inflammatory diseases. 

Sweet but dangerous - the role of immunoglobulin G glycosylation in autoimmunity and inflammation.

Neutrophils, the most abundant leukocytes in the human body, are considered to be the first line of defense in the fight against microorganisms. In this fight neutrophils employ weaponry such as reactive oxygen species produced via the NADPH oxidase complex 2 together with the release of intracellular granules containing antimicrobial agents. The discovery that activated neutrophils release decondensed chromatin as DNase-sensitive neutrophil extracellular traps (NETs) lead to a renewed interest in these leukocytes and the function of NETs in vivo. In this review, we will focus on desirable as well as detrimental features of NETs by the example of gout and pancreatitis. In our models we observed that neutrophils drive the initiation of inflammation and are required for the resolution of inflammation. 

Neutrophils and neutrophil extracellular traps orchestrate initiation and resolution of inflammation.

The critical size for strong interaction of hydrophobic particles with phospholipid bilayers has been predicted to be 10 nm. Because of the wide spreading of nonpolar nanoparticles (NPs) in the environment, we aimed to reveal the ability of living organisms to entrap NPs via formation of neutrophil extracellular traps (NETs). Upon interaction with various cell types and tissues, 10- to 40-nm-sized NPs induce fast (<20 min) damage of plasma membranes and instability of the lysosomal compartment, leading to the immediate formation of NETs. In contrast, particles sized 100-1,000 nm behaved rather inertly. Resulting NET formation (NETosis) was accompanied by an inflammatory reaction intrinsically endowed with its own resolution, demonstrated in lungs and air pouches of mice. Persistence of small NPs in joints caused unremitting arthritis and bone remodeling. Small NPs coinjected with antigen exerted adjuvant-like activity. This report demonstrates a cellular mechanism that explains how small NPs activate the NETosis pathway and drive their entrapping and resolution of the initial inflammatory response.

Nanoparticles size-dependently initiate self-limiting NETosis-driven inflammation.

A novel mechanism is revealed by which clinical isolates of adherent-invasive  (AIEC) penetrate into the epithelial cell layer, replicate, and establish biofilms in Crohn's disease. AIEC uses the FimH fimbrial adhesin to bind to oligomannose glycans on the surface of host cells. Oligomannose glycans exposed on early apoptotic cells are the preferred binding targets of AIEC, so apoptotic cells serve as potential entry points for bacteria into the epithelial cell layer. Thereafter, the bacteria propagate laterally in the epithelial intercellular spaces. We demonstrate oligomannosylation at two distinct sites of a glycoprotein receptor for AIEC, carcinoembryonic antigen related cell adhesion molecule 6 (CEACAM6 or CD66c), on human intestinal epithelia. After bacterial binding, FimH interacts with CEACAM6, which then clusters. The presence of the highest-affinity epitope for FimH, oligomannose-5, on CEACAM6 is demonstrated using LC-MS/MS. As mannose-dependent infections are abundant, this mechanism might also be used by other adherent-invasive pathogens.

Oligomannose-Rich Membranes of Dying Intestinal Epithelial Cells Promote Host Colonization by Adherent-Invasive .

Following strong activation signals, several types of immune cells reportedly release chromatin and granular proteins into the extracellular space, forming DNA traps. This process is especially prominent in neutrophils but also occurs in other innate immune cells such as macrophages, eosinophils, basophils and mast cells. Initial reports demonstrated that extracellular traps belong to the bactericidal and anti-fungal armamentarium of leukocytes, but subsequent studies also linked trap formation to a variety of human diseases. These pathological roles of extracellular DNA traps are now the focus of intensive biomedical research. The type of pathology associated with the release of extracellular DNA traps is mainly determined by the site of trap formation and the way in which these traps are further processed. Targeting the formation of aberrant extracellular DNA traps or promoting their efficient clearance are attractive goals for future therapeutic interventions, but the manifold actions of extracellular DNA traps complicate these approaches.

Extracellular DNA traps in inflammation, injury and healing.

The presence of gallstones (cholelithiasis) is a highly prevalent and severe disease and one of the leading causes of hospital admissions worldwide. Due to its substantial health impact, we investigated the biological mechanisms that lead to the formation and growth of gallstones. We show that gallstone assembly essentially requires neutrophil extracellular traps (NETs). We found consistent evidence for the presence of NETs in human and murine gallstones and describe an immune-mediated process requiring activation of the innate immune system for the formation and growth of gallstones. Targeting NET formation via inhibition of peptidyl arginine deiminase type 4 or abrogation of reactive oxygen species (ROS) production, as well as damping of neutrophils by metoprolol, effectively inhibit gallstone formation in vivo. Our results show that after the physicochemical process of crystal formation, NETs foster their assembly into larger aggregates and finally gallstones. These insights provide a feasible therapeutic concept to prevent cholelithiasis in patients at risk.

Neutrophil Extracellular Traps Initiate Gallstone Formation.

Neutrophils respond to various stimuli by decondensing and releasing nuclear chromatin characterized by citrullinated histones as neutrophil extracellular traps (NETs). This achieves pathogen immobilization or initiation of thrombosis, yet the molecular mechanisms of NET formation remain elusive. Peptidyl arginine deiminase-4 (PAD4) achieves protein citrullination and has been intricately linked to NET formation. Here we show that citrullination represents a major regulator of proteolysis in the course of NET formation. Elevated cytosolic calcium levels trigger both peptidylarginine deiminase-4 (PAD4) and calpain activity in neutrophils resulting in nuclear decondensation typical of NETs. Interestingly, PAD4 relies on proteolysis by calpain to achieve efficient nuclear lamina breakdown and chromatin decondensation. Pharmacological or genetic inhibition of PAD4 and calpain strongly inhibit chromatin decondensation of human and murine neutrophils in response to calcium ionophores as well as the proteolysis of nuclear proteins like lamin B1 and high mobility group box protein 1 (HMGB1). Taken together, the concerted action of PAD4 and calpain induces nuclear decondensation in the course of calcium-mediated NET formation.

Citrullination Licenses Calpain to Decondense Nuclei in Neutrophil Extracellular Trap Formation.

SARS-CoV-2 infection poses a major threat to the lungs and multiple other organs, occasionally causing death. Until effective vaccines are developed to curb the pandemic, it is paramount to define the mechanisms and develop protective therapies to prevent organ dysfunction in patients with COVID-19. Individuals that develop severe manifestations have signs of dysregulated innate and adaptive immune responses. Emerging evidence implicates neutrophils and the disbalance between neutrophil extracellular trap (NET) formation and degradation plays a central role in the pathophysiology of inflammation, coagulopathy, organ damage, and immunothrombosis that characterize severe cases of COVID-19. Here, we discuss the evidence supporting a role for NETs in COVID-19 manifestations and present putative mechanisms, by which NETs promote tissue injury and immunothrombosis. We present therapeutic strategies, which have been successful in the treatment of immunο-inflammatory disorders and which target dysregulated NET formation or degradation, as potential approaches that may benefit patients with severe COVID-19.

Patients with COVID-19: in the dark-NETs of neutrophils.

The subgingival biofilm attached to tooth surfaces triggers and maintains periodontitis. Previously, late-onset periodontitis has been considered a consequence of dysbiosis and a resultant polymicrobial disruption of host homeostasis. However, a multitude of studies did not show "healthy" oral microbiota pattern, but a high diversity depending on culture, diets, regional differences, age, social state etc. These findings relativise the aetiological role of the dysbiosis in periodontitis. Furthermore, many late-onset periodontitis traits cannot be explained by dysbiosis; e.g. age-relatedness, attenuation by anti-ageing therapy, neutrophil hyper-responsiveness, and microbiota shifting by dysregulated immunity, yet point to the crucial role of dysregulated immunity and neutrophils in particular. Furthermore, patients with neutropenia and neutrophil defects inevitably develop early-onset periodontitis. Intra-gingivally injecting lipopolysaccharide (LPS) alone causes an exaggerated neutrophil response sufficient to precipitate experimental periodontitis. Vice versa to the surplus of LPS, the increased neutrophil responsiveness characteristic for late-onset periodontitis can effectuate gingiva damage likewise. The exaggerated neutrophil extracellular trap (NET) response in late-onset periodontitis is blameable for damage of gingival barrier, its penetration by bacteria and pathogen-associated molecular patterns (PAMPs) as well as stimulation of Th17 cells, resulting in further neutrophil activation. This identifies the dysregulated immunity as the main contributor to periodontal disease.

Neutrophils Orchestrate the Periodontal Pocket.

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition).

Phagocytosis, degranulation, and neutrophil extracellular traps (NETs) formation build the armory of neutrophils for the first line of defense against invading pathogens. All these processes are modulated by the microenvironment including tonicity, pH and oxygen levels. Here we investigated the neutrophil infiltration in cardiac tissue autopsy samples of patients with acute myocardial infarction (AMI) and compared these with tissues from patients with sepsis, endocarditis, dermal inflammation, abscesses and diseases with prominent neutrophil infiltration. We observed many neutrophils infiltrating the heart muscle after myocardial infarction. Most of these had viable morphology and only few showed signs of nuclear de-condensation, a hallmark of early NET formation. The abundance of NETs was the lowest in acute myocardial infarction when compared to other examined diseases. Since cardiac oxygen supply is abruptly abrogated in acute myocardial infarction, we hypothesized that the resulting tissue hypoxia increased the longevity of the neutrophils. Indeed, the viable cells showed increased nuclear hypoxia inducible factor-1α (HIF-1α) content, and only neutrophils with low HIF-1α started the process of NET formation (chromatin de-condensation and nuclear swelling). Prolonged neutrophil survival, increased oxidative burst and reduced NETs formation were reproduced under low oxygen tensions and by HIF-1α stabilization . We conclude that nuclear HIF-1α is associated with prolonged neutrophil survival and enhanced oxidative stress in hypoxic areas of AMI.

Hypoxia Promotes Neutrophil Survival After Acute Myocardial Infarction.

The frequent severe COVID-19 course in patients with periodontitis suggests a link of the aetiopathogenesis of both diseases. The formation of intravascular neutrophil extracellular traps (NETs) is crucial to the pathogenesis of severe COVID-19. Periodontitis is characterised by an increased level of circulating NETs, a propensity for increased NET formation, delayed NET clearance and low-grade endotoxemia (LGE). The latter has an enormous impact on innate immunity and susceptibility to infection with SARS-CoV-2. LPS binds the SARS-CoV-2 spike protein and this complex, which is more active than unbound LPS, precipitates massive NET formation. Thus, circulating NET formation is the common denominator in both COVID-19 and periodontitis and other diseases with low-grade endotoxemia like diabetes, obesity and cardiovascular diseases (CVD) also increase the risk to develop severe COVID-19. Here we discuss the role of propensity for increased NET formation, DNase I deficiency and low-grade endotoxaemia in periodontitis as aggravating factors for the severe course of COVID-19 and possible strategies for the diminution of increased levels of circulating periodontitis-derived NETs in COVID-19 with periodontitis comorbidity.

Article	Year
Macrophages discriminate glycosylation patterns of apoptotic cell-derived microparticles. The Journal of biological chemistry\| PubMed ID: 22074924	2012
Altered glycosylation of complexed native IgG molecules is associated with disease activity of systemic lupus erythematosus. Lupus\| PubMed ID: 25389233	2015
Sweet but dangerous - the role of immunoglobulin G glycosylation in autoimmunity and inflammation. Lupus\| PubMed ID: 27252272	2016
Neutrophils and neutrophil extracellular traps orchestrate initiation and resolution of inflammation. Clinical and experimental rheumatology\| PubMed ID: 27586795	2016
Nanoparticles size-dependently initiate self-limiting NETosis-driven inflammation. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 27647892	2016
Oligomannose-Rich Membranes of Dying Intestinal Epithelial Cells Promote Host Colonization by Adherent-Invasive . Frontiers in microbiology\| PubMed ID: 29720971	2018
Extracellular DNA traps in inflammation, injury and healing. Nature reviews. Nephrology\| PubMed ID: 31213698	2019
Neutrophil Extracellular Traps Initiate Gallstone Formation. Immunity\| PubMed ID: 31422870	2019
Citrullination Licenses Calpain to Decondense Nuclei in Neutrophil Extracellular Trap Formation. Frontiers in immunology\| PubMed ID: 31695698	2019
Patients with COVID-19: in the dark-NETs of neutrophils. Cell death and differentiation\| PubMed ID: 34031543	2021
Neutrophils Orchestrate the Periodontal Pocket. Frontiers in immunology\| PubMed ID: 34899756	2021
Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition). European journal of immunology\| PubMed ID: 34910301	2021
Hypoxia Promotes Neutrophil Survival After Acute Myocardial Infarction. Frontiers in immunology\| PubMed ID: 35222361	2022
Periodontitis-Derived Dark-NETs in Severe Covid-19. Frontiers in immunology\| PubMed ID: 35493525	2022

Martin Herrmann

Biography

JoVE Journal Publications

Other Publications