Role of phagocyte extracellular traps during Mycobacterium tuberculosis infections and tuberculosis disease processes.

Mycobacterium tuberculosis (M.tb) infections remain one of the most significant causes of mortality worldwide. The current situation shows an emergence of new antibiotic-resistant strains making it difficult to control the tuberculosis (TB) disease. A large part of its success as a pathogen is due to its ability to persist for years or even decades without causing evident clinical manifestations. M.tb is highly successful in evading the host-defense by manipulating host-signalling pathways. Although macrophages are generally viewed as the key cell type involved in harboring M.tb, growing evidence shows that neutrophils also play a fundamental role. Both cells are known to act in multiple ways when encountering an invading pathogen, including phagocytosis, release of cytokines and chemokines, and oxidative burst. In addition, the formation of neutrophil extracellular traps (NETs) and macrophage extracellular traps (METs) has been described to contribute to M.tb infections. NETs/METs are extracellular DNA fibers with associated granule components, which are released upon activation of the cells by the pathogen or by pro-inflammatory mediators. On one hand, they can lead to a protective immune response by entrapment and killing of pathogens. However, on the other hand, they can also play a severe pathological role by inducing tissue damage. Extracellular traps (ETs) produced in the pulmonary alveoli can expand easily and expose tissue-damaging factors with detrimental effects. Since host-directed therapies offer a complementary strategy in TB, the knowledge of NET/MET formation is important for understanding potential protective versus detrimental pathways during innate immune signaling. In this review, we summarize the progress made in understanding the role of NETs/METs in the pathogenesis of TB

Characterization of young and aged ferrets as animal models for SARS-CoV-2 infection with focus on neutrophil extracellular traps

Neutrophil extracellular traps (NETs) are net-like structures released by activated neutrophils upon infection [e.g., severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)] as part of the innate immune response that have protective effects by pathogen entrapment and immobilization or result in detrimental consequences for the host due to the massive release of NETs and their impaired degradation by nucleases like DNase-1. Higher amounts of NETs are associated with coronavirus disease 2019 (COVID-19) severity and are a risk factor for severe disease outcome. The objective of our study was to investigate NET formation in young versus aged ferrets to evaluate their value as translational model for SARS-CoV-2-infection and to correlate different NET markers and virological parameters. In each of the two groups (young and aged), nine female ferrets were intratracheally infected with 1 mL of 106 TCID50/mL SARS-CoV-2 (BavPat1/2020) and euthanized at 4, 7, or 21 days post-infection. Three animals per group served as negative controls. Significantly more infectious virus and viral RNA was found in the upper respiratory tract of aged ferrets. Interestingly, cell-free DNA and DNase-1 activity was generally higher in bronchoalveolar lavage fluid (BALF) but significantly lower in serum of aged compared to young ferrets. In accordance with these data, immunofluorescence microscopy revealed significantly more NETs in lungs of aged compared to young infected ferrets. The association of SARS-CoV-2-antigen in the respiratory mucosa and NET markers in the nasal conchae, but the absence of virus antigen in the lungs, confirms the nasal epithelium as the major location for virus replication as described for young ferrets. Furthermore, a strong positive correlation was found between virus shedding and cell-free DNA or the level of DNAse-1 activity in aged ferrets. Despite the increased NET formation in infected lungs of aged ferrets, the animals did not show a strong NET phenotype and correlation among tested NET markers. Therefore, ferrets are of limited use to study SARS-CoV-2 pathogenesis associated with NET formation. Nevertheless, the mild to moderate clinical signs, virus shedding pattern, and the lung pathology of aged ferrets confirm those animals as a relevant model to study age-dependent COVID-19 pathogenesis

Companion Animals in Zoonoses Research – Ethical Considerations

Non-human animals are commonly classified according to their "role", such as "livestock", "wild" or "companion" animals. But what if those classifications overlap? This article presents a report of the retreat week "ZooCan – Zoonoses of companion animals as case study for animal ethics" at the University of Veterinary Medicine Hannover, Germany, in November 2022. The workshop included participants from different European countries with interdisciplinary backgrounds (animal law, bioethics, epidemiology, philosophy, biology and veterinary medicine). We address ethically relevant issues that emerge when companion animals are used as research animals, particularly in zoonoses research. The outcomes of the multi-disciplinary approach are used to i) define criteria to classify "companion" and "research" animals, ii) provide guidance to overcome the challenges with classificational overlaps, iii) give insights into cutting-edge zoonoses research with an example of SARS-CoV-2 in cats, and iv) discuss animal ethics approaches with regard to classifications

Immunogenicity of PE18, PE31, and PPE26 proteins from Mycobacterium tuberculosis in humans and mice.

INTRODUCTION: The large family of PE and PPE proteins accounts for as much as 10% of the genome of Mycobacterium tuberculosis. In this study, we explored the immunogenicity of three proteins from this family, PE18, PE31, and PPE26, in humans and mice. METHODS: The investigation involved analyzing the immunoreactivity of the selected proteins using sera from TB patients, IGRA-positive household contacts, and IGRA-negative BCG vaccinated healthy donors from the TB endemic country Mozambique. Antigen-recall responses were examined in PBMC from these groups, including the evaluation of cellular responses in healthy unexposed individuals. Moreover, systemic priming and intranasal boosting with each protein, combined with the Quil-A adjuvant, were conducted in mice. RESULTS: We found that all three proteins are immunoreactive with sera from TB patients, IGRA-positive household contacts, and IGRA-negative BCG vaccinated healthy controls. Likewise, antigen-recall responses were induced in PBMC from all groups, and the proteins stimulated proliferation of peripheral blood mononuclear cells from healthy unexposed individuals. In mice, all three antigens induced IgG antibody responses in sera and predominantly IgG, rather than IgA, responses in bronchoalveolar lavage. Additionally, CD4+ and CD8+ effector memory T cell responses were observed in the spleen, with PE18 demonstrating the ability to induce tissue-resident memory T cells in the lungs. DISCUSSION: Having demonstrated immunogenicity in both humans and mice, the protective capacity of these antigens was evaluated by challenging immunized mice with low-dose aerosol of Mycobacterium tuberculosis H37Rv. The in vitro Mycobacterial Growth Inhibition Assay (MGIA) and assessment of viable bacteria in the lung did not demonstrate any ability of the vaccination protocol to reduce bacterial growth. We therefore concluded that these three specific PE/PPE proteins, while immunogenic in both humans and mice, were unable to confer protective immunity under these conditions

Immunogenicity of PE18, PE31, and PPE26 proteins from Mycobacterium tuberculosis in humans and mice

IntroductionThe large family of PE and PPE proteins accounts for as much as 10% of the genome of Mycobacterium tuberculosis. In this study, we explored the immunogenicity of three proteins from this family, PE18, PE31, and PPE26, in humans and mice.MethodsThe investigation involved analyzing the immunoreactivity of the selected proteins using sera from TB patients, IGRA-positive household contacts, and IGRA-negative BCG vaccinated healthy donors from the TB endemic country Mozambique. Antigen-recall responses were examined in PBMC from these groups, including the evaluation of cellular responses in healthy unexposed individuals. Moreover, systemic priming and intranasal boosting with each protein, combined with the Quil-A adjuvant, were conducted in mice.ResultsWe found that all three proteins are immunoreactive with sera from TB patients, IGRA-positive household contacts, and IGRA-negative BCG vaccinated healthy controls. Likewise, antigen-recall responses were induced in PBMC from all groups, and the proteins stimulated proliferation of peripheral blood mononuclear cells from healthy unexposed individuals. In mice, all three antigens induced IgG antibody responses in sera and predominantly IgG, rather than IgA, responses in bronchoalveolar lavage. Additionally, CD4+ and CD8+ effector memory T cell responses were observed in the spleen, with PE18 demonstrating the ability to induce tissue-resident memory T cells in the lungs.DiscussionHaving demonstrated immunogenicity in both humans and mice, the protective capacity of these antigens was evaluated by challenging immunized mice with low-dose aerosol of Mycobacterium tuberculosis H37Rv. The in vitro Mycobacterial Growth Inhibition Assay (MGIA) and assessment of viable bacteria in the lung did not demonstrate any ability of the vaccination protocol to reduce bacterial growth. We therefore concluded that these three specific PE/PPE proteins, while immunogenic in both humans and mice, were unable to confer protective immunity under these conditions

Case study on the pathophysiology of Fabry disease: abnormalities of cellular membranes can be reversed by substrate reduction in vitro

It is still not entirely clear how α-galactosidase A (GAA) deficiency translates into clinical symptoms of Fabry disease (FD). The present communication investigates the effects of the mutation N215S in FD on the trafficking and processing of lysosomal GAA and their potential association with alterations in the membrane lipid composition. Abnormalities in lipid rafts (LRs) were observed in fibroblasts isolated from a male patient with FD bearing the mutation N215S. Interestingly, LR analysis revealed that the distribution of cholesterol and flotillin-2 are distinctly altered in the Fabry fibroblasts when compared with that of the wild-type cells. Furthermore, increased levels of glycolipid globotriaosylceramide 3 (Gb3) and sphingomyelin (SM) were observed in non-raft membrane fractions of Fabry cells. Substrate reduction with N-butyldeoxynojirimycin (NB-DNJ) in vitro was capable of reversing these abnormalities in this patient. These data led to the hypothesis that alterations of LRs may contribute to the pathophysiology of Morbus Fabry. Furthermore, it may be suggested that substrate reduction therapy with NB-DNJ might be a promising approach for the treatment of GAA deficiency at least for the selected patients

M1T1 group A streptococcal pili promote epithelial colonization but diminish systemic virulence through neutrophil extracellular entrapment

Group A Streptococcus is a leading human pathogen associated with a diverse array of mucosal and systemic infections. Cell wall anchored pili were recently described in several species of pathogenic streptococci, and in the case of GAS, these surface appendages were demonstrated to facilitate epithelial cell adherence. Here we use targeted mutagenesis to evaluate the contribution of pilus expression to virulence of the globally disseminated M1T1 GAS clone, the leading agent of both GAS pharyngitis and severe invasive infections. We confirm that pilus expression promotes GAS adherence to pharyngeal cells, keratinocytes, and skin. However, in contrast to findings reported for group B streptococcal and pneumococcal pili, we observe that pilus expression reduces GAS virulence in murine models of necrotizing fasciitis, pneumonia and sepsis, while decreasing GAS survival in human blood. Further analysis indicated the systemic virulence attenuation associated with pilus expression was not related to differences in phagocytic uptake, complement deposition or cathelicidin antimicrobial peptide sensitivity. Rather, GAS pili were found to induce neutrophil IL-8 production, promote neutrophil transcytosis of endothelial cells, and increase neutrophil release of DNA-based extracellular traps, ultimately promoting GAS entrapment and killing within these structures

Catheter Colonization and Abscess Formation Due to Staphylococcus epidermidis with Normal and Small-Colony-Variant Phenotype Is Mouse Strain Dependent

Coagulase-negative staphylococci (CoNS) form a thick, multilayered biofilm on foreign bodies and are a major cause of nosocomial implant-associated infections. Although foreign body infection models are well-established, limited in vivo data are available for CoNS with small-colony-variant (SCV) phenotype described as causative agents in implant-associated infections. Therefore, we investigated the impact of the Staphylococcus epidermidis phenotype on colonization of implanted PVC catheters and abscess formation in three different mouse strains. Following introduction of a catheter subcutaneously in each flank of 8- to 12-week-old inbred C57BL/6JCrl (B6J), outbred Crl:CD1(ICR) (CD-1), and inbred BALB/cAnNCrl (BALB/c) male mice, doses of S. epidermidis O-47 wild type, its hemB mutant with stable SCV phenotype, or its complemented mutant at concentrations of 106 to 109 colony forming units (CFUs) were gently spread onto each catheter. On day 7, mice were sacrificed and the size of the abscesses as well as bacterial colonization was determined. A total of 11,500 CFUs of the complemented mutant adhered to the catheter in BALB/c followed by 9,960 CFUs and 9,900 CFUs from S. epidermidis wild type in BALB/c and CD-1, respectively. SCV colonization was highest in CD-1 with 9,500 CFUs, whereas SCVs were not detected in B6J. The minimum dose that led to colonization or abscess formation in all mouse strains was 107 or 108 CFUs of the normal phenotype, respectively. A minimum dose of 108 or 109 CFU of the hemB mutant with stable SCV phenotype led to colonization only or abscess formation, respectively. The largest abscesses were detected in BALB/c inoculated with wild type bacteria or SCV (64 mm2 vs. 28 mm2). Our results indicate that colonization and abscess formation by different phenotypes of S. epidermidis in a foreign body infection model is most effective in inbred BALB/c followed by outbred CD-1 and inbred B6J mice

Neutrophil responses to Aspergillosis : new roles for old players

Neutrophils are professional phagocytic cells that play a crucial role in innate immunity. Through an assortment of antifungal effector mechanisms, neutrophils are essential in controlling the early stages of fungal infection. These mechanisms range from the production of reactive oxygen intermediates and release of antimicrobial enzymes to the formation of complex extracellular traps that aid in the elimination of the fungus. Their importance in antifungal immunity is supported by the extreme susceptibility to infection of patients with primary (e.g., chronic granulomatous disease) or acquired (e.g., undergoing immunosuppressive therapy) neutrophil deficiency. More recently, common genetic variants affecting neutrophil antifungal capacity have also been disclosed as major risk factors for aspergillosis in conditions of generalized immune deficiency. The present review revisits the role of neutrophils in the host response against Aspergillus and highlights the consequences of their deficiency in susceptibility to aspergillosis.This work was supported by a Research Grant from the European Society of Clinical Microbiology and Infectious Diseases (ESCMID). Cristina Cunha was supported by the Fundacao para a Ciencia e Tecnologia, Portugal (contract SFRH/BPD/96176/2013)