S. aureus biofilm properties correlate with immune B cell subset frequencies and severity of chronic rhinosinusitis.

Staphylococcus aureus mucosal biofilms are associated with recalcitrant chronic rhinosinusitis (CRS). However, S. aureus colonisation of sinus mucosa is frequent in the absence of mucosal inflammation. This questions the relevance of S. aureus biofilms in CRS etiopathogenesis. This study aimed to investigate whether strain-level variation in in vitro-grown S. aureus biofilm properties relates to CRS disease severity, in vitro toxicity, and immune B cell responses in sinonasal tissue from CRS patients and non-CRS controls. S. aureus clinical isolates, tissue samples, and matched clinical datasets were collected from CRS patients with nasal polyps (CRSwNP), CRS without nasal polyps (CRSsNP), and controls. B cell responses in tissue samples were characterised by FACS. S. aureus biofilms were established in vitro, followed by measuring their properties of metabolic activity, biomass, colony-forming units, and exoprotein production. S. aureus virulence was evaluated using whole-genome sequencing, mass spectrometry and application of S. aureus biofilm exoproteins to air-liquid interface cultures of primary human nasal epithelial cells (HNEC-ALI). In vitro S. aureus biofilm properties were correlated with increased CRS severity scores, infiltration of antibody-secreting cells and loss of regulatory B cells in tissue samples. Biofilm exoproteins from S. aureus with high biofilm metabolic activity had enriched virulence genes and proteins, and negatively affected the barrier function of HNEC-ALI cultures. These findings support the notion of strain-level variation in S. aureus biofilms to be critical in the pathophysiology of CRS.

Colloidal silver against macrophage infections and biofilms of atypical mycobacteria.

Skin and soft tissue infection (SSTI) caused by atypical mycobacteria such as Mycobacterium abscessus and Mycobacterium avium intracellulare complex (MAIC) have increased in recent years. Current therapeutic options are limited, and hence new and better therapies are urgently required. Colloidal Silver (CS) has been identified for its widespread antibacterial properties and silver-impregnated dressings have been used for SSTIs caused by various pathogens. The efficacy of Green Synthesized Colloidal Silver (GSCS) was investigated for bacterial growth inhibition (BGI) using a microdilution method and minimum biofilm eradication concentration (MBEC) using resazurin assay and confocal scanning laser microscopy (CSLM) of M. abscessus (n = 5) and MAIC (n = 5). The antibacterial effect of GSCS against M. abscessus infected macrophages was also evaluated. The in vitro cytotoxicity of GSCS on a human keratinocyte cell line (HaCaT) and neonatal foreskin fibroblasts was analyzed by the crystal violet proliferation assay. Average BGI and MBEC of GSCS varied between 0.7 and 22 ppm for M. abscessus and MAIC. The concentration of 3 ppm reduced M. abscessus-infection in macrophages significantly. GSCS was not cytotoxic to HaCaT and neonatal foreskin fibroblast cells at concentrations < 3 ppm up to 2 h exposure time. GSCS therefore, has the potential for topical application against atypical mycobacterial SSTI.

Proteomic analysis of nasal mucus samples of healthy patients and patients with chronic rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis (CRS) has a complex and multifactorial pathogenesis with a heterogeneous inflammatory profile. Proteomic analysis of nasal mucus may enable further understanding of protein abundances and biologic processes present in CRS and its endotypes compared with in healthy patients. OBJECTIVE: Our aim was to determine differences in the nasal mucus proteome of healthy patients and patients with CRS. METHODS: Nasal mucus was obtained from healthy patients, patients with CRS without nasal polyps (CRSsNP), and patients with CRS with nasal polyps (CRSwNP) before surgery. Gel electrophoresis was performed to fractionate the complex protein extracts before mass spectrometry analysis. Gene set enrichment analysis was performed on differentially expressed proteins. RESULTS: A total of 33 patients were included in this study (12 healthy, 10 with CRSsNP, and 11 with CRSwNP). In all, 1142 proteins were identified in mucus samples from healthy patients, 761 in mucus samples from patients with CRSsNP, and 998 in mucus samples from patients with CRSwNP. Dysfunction in immunologic pathways, reduced cellular signaling, and increased cellular metabolism with associated tissue remodeling pathways were present in patients with CRS compared with in healthy patients. CONCLUSION: Significant downregulation of mucosal immunity and antioxidant pathways with increased tissue modeling processes may account for the clinical manifestations of CRS. Ultimately, the differing proteome and biologic processes provide further insight into CRS pathogenesis and its endotypes.

Association between mucosal barrier disruption by Pseudomonas aeruginosa exoproteins and asthma in patients with chronic rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis (CRS) is a common chronic respiratory condition, frequently associated with asthma and affecting the majority of cystic fibrosis (CF) patients. Pseudomonas aeruginosa infections and biofilms have been implicated in recalcitrant CRS. One of the mechanisms of action for bacteria in CRS and CF is mucosal barrier disruption by secreted products that contribute to the inflammation. However, the role of biofilm and planktonic forms of P. aeruginosa in this process is not known. The aim is to determine the effect of P. aeruginosa exoproteins isolated from CF and non-CF CRS patients on the mucosal barrier. METHODS: Exoproteins from 40 P. aeruginosa isolates were collected in planktonic and biofilm forms and applied to air-liquid interface (ALI) cultures of primary human nasal epithelial cells (HNECs). Mucosal barrier integrity was evaluated by transepithelial electrical resistance (TEER), passage of FITC-dextrans and immunofluorescence of tight junction proteins. Cytotoxicity assays were performed to measure cell viability, and IL-6 ELISA was carried out to evaluate pro-inflammatory effects. RESULTS: Planktonic exoproteins from 20/40 (50%) clinical isolates had a significant detrimental effect on the barrier and significantly increased IL-6 production. Barrier disruption was characterized by a reduced TEER, increased permeability of FITC-dextrans and discontinuous immunolocalization of tight junction proteins and was significantly more prevalent in isolates harvested from patients with comorbid asthma (P < .05). CONCLUSION: Exoproteins from planktonic P. aeruginosa clinical isolates from asthmatic CRS patients have detrimental effects on the mucosal barrier and induce IL-6 production potentially contributing to the mucosal inflammation in CRS patients.

The international sinonasal microbiome study: A multicentre, multinational characterization of sinonasal bacterial ecology.

The sinonasal microbiome remains poorly defined, with our current knowledge based on a few cohort studies whose findings are inconsistent. Furthermore, the variability of the sinus microbiome across geographical divides remains unexplored. We characterize the sinonasal microbiome and its geographical variations in both health and disease using 16S rRNA gene sequencing of 410 individuals from across the world. Although the sinus microbial ecology is highly variable between individuals, we identify a core microbiome comprised of Corynebacterium, Staphylococcus, Streptococcus, Haemophilus and Moraxella species in both healthy and chronic rhinosinusitis (CRS) cohorts. Corynebacterium (mean relative abundance = 44.02%) and Staphylococcus (mean relative abundance = 27.34%) appear particularly dominant in the majority of patients sampled. Amongst patients suffering from CRS with nasal polyps, a statistically significant reduction in relative abundance of Corynebacterium (40.29% vs 50.43%; P = .02) was identified. Despite some measured differences in microbiome composition and diversity between some of the participating centres in our cohort, these differences would not alter the general pattern of core organisms described. Nevertheless, atypical or unusual organisms reported in short-read amplicon sequencing studies and that are not part of the core microbiome should be interpreted with caution. The delineation of the sinonasal microbiome and standardized methodology described within our study will enable further characterization and translational application of the sinus microbiota.

Sirtuin-1 Controls Poly (I:C)-Dependent Matrix Metalloproteinase 9 Activation in Primary Human Nasal Epithelial Cells.

Matrix metalloproteinase (MMP)-9 is thought to be involved in the etiopathogenesis of chronic rhinosinusitis (CRS) with nasal polyps and cleaves collagen IV, causing hyperpermeability of the basement membrane within mucosal tissue. It is known that MMP-9 expression is negatively affected by sirtuin (SIRT)-1 in human monocytotic cells, retinal endothelial cells, and epithelial carcinoma cells. However, it is unknown which factors affect MMP-9 expression and activity in human nasal epithelial cells (HNECs). To examine factors affecting MMP-9 expression and activity in HNECs, HNECs were stimulated with Toll-like receptor (TLR) agonists, followed by quantitative PCR, immunofluorescence, and zymography to examine MMP-9 expression and activity. MMP-9 expression was evaluated in sinonasal tissue of control subjects without CRS, and patients with CRS without nasal polyps and those with CRS with nasal polyps, in relation to the expression of SIRT1 using a tissue microarray. The effect of SIRT1 stimulation/inhibition on MMP-9 expression in HNECs was also tested. TLR3 agonists increased MMP-9 mRNA expression (473 fold, P = 0.0198) and activity (20.4-fold, P < 0.05). SIRT1 activation or inhibition reciprocally affected MMP-9 expression in the presence of TLR3 agonists. MMP-9 and SIRT1 expression within the epithelial layer of sinonasal tissue was inversely correlated only in patients with CRS but not in control subjects. TLR3 agonists increased MMP-9 expression and activity in HNECs, and the effect was abolished in the presence of SIRT1 activation. SIRT1 and MMP-9 expression was inversely correlated in CRS tissue, supporting SIRT1 as a possible therapeutic target for nasal polyp formation.

Th17 Cytokines Disrupt the Airway Mucosal Barrier in Chronic Rhinosinusitis.

Cytokine mediated changes in paracellular permeability contribute to a multitude of pathological conditions including chronic rhinosinusitis (CRS). The purpose of this study was to investigate the effect of interferons and of Th1, Th2, and Th17 cytokines on respiratory epithelium barrier function. Cytokines and interferons were applied to the basolateral side of air-liquid interface (ALI) cultures of primary human nasal epithelial cells (HNECs) from CRS with nasal polyp patients. Transepithelial electrical resistance (TEER) and permeability of FITC-conjugated dextrans were measured over time. Additionally, the expression of the tight junction protein Zona Occludens-1 (ZO-1) was examined via immunofluorescence. Data was analysed using ANOVA, followed by Tukey HSD post hoc test. Our results showed that application of interferons and of Th1 or Th2 cytokines did not affect the mucosal barrier function. In contrast, the Th17 cytokines IL-17, IL-22, and IL-26 showed a significant disruption of the epithelial barrier, evidenced by a loss of TEER, increased paracellular permeability of FITC-dextrans, and discontinuous ZO-1 immunolocalisation. These results indicate that Th17 cytokines may contribute to the development of CRSwNP by promoting a leaky mucosal barrier.

Venom present in sea anemone (Heteractis magnifica) induces apoptosis in non-small-cell lung cancer A549 cells through activation of mitochondria-mediated pathway.

Lung cancer is a major cause of cancer deaths throughout the world and the complexity of apoptosis resistance in lung cancer is apparent. Venom from Heteractis magnifica caused dose-dependent decreases in survival of the human non-small-cell lung cancer cell line, as determined by the MTT and Crystal Violet assays. The H. magnifica venom induced cell cycle arrest and induced apoptosis of A549 cells, as confirmed by annexin V/propidium iodide staining. The venom-induced apoptosis in A549 cells was characterized by cleavage of caspase-3 and a reduction in the mitochondrial membrane potential. Interestingly, crude extracts from H. magnifica had less effect on the survival of non-cancer cell lines. In the non-cancer cells, the mechanism via which cell death occurred was through necrosis not apoptosis. These findings are important for future work using H. magnifica venom for pharmaceutical development to treat human lung cancer.

Development of novel iron(III) crosslinked bioinks comprising carboxymethyl cellulose, xanthan gum, and hyaluronic acid for soft tissue engineering applications.

The advent of three-dimensional (3D) bioprinting offers a feasible approach to construct complex structures for soft tissue regeneration. Carboxymethyl cellulose (CMC) has been emerging as a very promising biomaterial for 3D bioprinting. However, due to the inability to maintain the post-printed stability, CMC needs to be physically blended and/or chemically crosslinked with other polymers. In this context, this study presents the combination of CMC with xanthan gum (XG) and hyaluronic acid (HA) to formulate a multicomponent bioink, leveraging the printability of CMC and XG, as well as the cellular support properties of HA. The ionic crosslinking of printed constructs with iron(III) via the metal-ion coordination between ferric cations and carboxylate groups of the three polymers was introduced to induce improved mechanical strength and long-term stability. Moreover, immortalized human epidermal keratinocytes (HaCaT) and human foreskin fibroblasts (HFF) encapsulated within iron-crosslinked printed hydrogels exhibited excellent cell viability (more than 95%) and preserved morphology. Overall, the presented study highlights that the combination of these three biopolymers and the ionic crosslinking with ferric ions is a valuable strategy to be considered for the development of new and advanced hydrogel-based bioinks for soft tissue engineering applications.

Hydrocortisone metabolism by Staphylococcus: Effects on anti-inflammatory and antimicrobial action.

KEY POINTS: Hydrocortisone 21-hemisuccinate (HCHS) influenced the growth and metabolism of Staphylococcus aureus S. aureus metabolic activity was high and antibiotic susceptibility low at 1.4 mg/mL HCHS S. aureus metabolized HCHS to cortisol and reduced poly(I:C)-induced IL-6 secretion.

Increased antibiotic resistance of Pseudomonas aeruginosa isolates from chronic rhinosinusitis patients grown in anaerobic conditions.

Introduction: In chronic rhinosinusitis (CRS), the congestion and blockage of the nose can cause anaerobic conditions within the sinus cavities which may promote the expression of virulence and antibiotic resistance genes in invading pathogens. Pseudomonas aeruginosa is a facultative anaerobic bacteria and causes severe recalcitrant CRS. In this study, we aimed to evaluate the antimicrobial resistance of P. aeruginosa isolates of CRS patients in planktonic and biofilm form grown in aerobic and anaerobic conditions. Methods: P. aeruginosa clinical isolates of CRS patients (n = 25) were grown in planktonic and biofilm form in aerobic and anaerobic conditions. Minimum inhibitory concentrations (MIC) of planktonic forms and minimum biofilm eradication concentrations (MBEC) were determined. Additionally, metabolic activity by fluorescein diacetate assay, biofilm biomass by crystal violet assay and eDNA concentration were assessed in both conditions. Results: P. aeruginosa planktonic cells grown in anaerobic condition exhibited increased gentamicin resistance (p < .01), whereas P. aeruginosa biofilms grown in anaerobic condition displayed significantly increased MBEC values for gentamicin (p < .0001) and levofloxacin (p < .001). The metabolic activity of anaerobic biofilms was significantly higher compared with aerobic biofilms (p < .0001). However, the biofilm biomass of isolates grown in aerobic conditions was higher than anaerobic conditions (p < .5). Conclusion: P. aeruginosa isolates from CRS patients grown in anaerobic conditions showed significantly increased resistance to antibiotics with an increased metabolic activity but decreased biofilm biomass. Level of Evidence: NA.

Proteomic characterisation of perhexiline treatment on THP-1 M1 macrophage differentiation.

Background: Dysregulated inflammation is important in the pathogenesis of many diseases including cancer, allergy, and autoimmunity. Macrophage activation and polarisation are commonly involved in the initiation, maintenance and resolution of inflammation. Perhexiline (PHX), an antianginal drug, has been suggested to modulate macrophage function, but the molecular effects of PHX on macrophages are unknown. In this study we investigated the effect of PHX treatment on macrophage activation and polarization and reveal the underlying proteomic changes induced. Methods: We used an established protocol to differentiate human THP-1 monocytes into M1 or M2 macrophages involving three distinct, sequential stages (priming, rest, and differentiation). We examined the effect of PHX treatment at each stage on the polarization into either M1 or M2 macrophages using flow cytometry, quantitative polymerase chain reaction (qPCR) and enzyme linked immunosorbent assay (ELISA). Quantitative changes in the proteome were investigated using data independent acquisition mass spectrometry (DIA MS). Results: PHX treatment promoted M1 macrophage polarization, including increased STAT1 and CCL2 expression and IL-1ß secretion. This effect occurred when PHX was added at the differentiation stage of the M1 cultures. Proteomic profiling of PHX treated M1 cultures identified changes in metabolic (fatty acid metabolism, cholesterol homeostasis and oxidative phosphorylation) and immune signalling (Receptor Tyrosine Kinase, Rho GTPase and interferon) pathways. Conclusion: This is the first study to report on the action of PHX on THP-1 macrophage polarization and the associated changes in the proteome of these cells.

Chronic Rhinosinusitis, S. aureus Biofilm and Secreted Products, Inflammatory Responses, and Disease Severity.

Chronic rhinosinusitis (CRS) is a persistent inflammation of the nasal cavity and paranasal sinuses associated with tissue remodelling, dysfunction of the sinuses' natural defence mechanisms, and induction of different inflammatory clusters. The etiopathogenesis of CRS remains elusive, and both environmental factors, such as bacterial biofilms and the host's general condition, are thought to play a role. Bacterial biofilms have significant clinical relevance due to their potential to cause resistance to antimicrobial therapy and host defenses. Despite substantial medical advances, some CRS patients suffer from recalcitrant disease that is unresponsive to medical and surgical treatments. Those patients often have nasal polyps with tissue eosinophilia, S. aureus-dominant mucosal biofilm, comorbid asthma, and a severely compromised quality of life. This review aims to summarise the contemporary knowledge of inflammatory cells/pathways in CRS, the role of bacterial biofilm, and their impact on the severity of the disease. Here, an emphasis is placed on S. aureus biofilm and its secreted products. A better understanding of these factors might offer important diagnostic and therapeutic perceptions for recalcitrant disease.

Characterization of human nasal organoids from chronic rhinosinusitis patients.

Patient-derived organoids grown in three-dimensional cultures provide an excellent platform for phenotypic high-throughput screening and drug-response research. Organoid technology has been applied to study stem cell biology and various human pathologies. This study investigates the characteristics and cellular morphology of organoids derived from primary human nasal epithelial cells (HNECs) of chronic rhinosinusitis (CRS) patients. Nasal organoids were cultured up to 20 days and morphological, cell composition and functional parameters were measured by immunofluorescence, RT-qPCR, western blot and FACS analysis. The results showed that nasal organoids expressed the stem cell marker leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), and markers for apical junction genes, goblet cells and ciliated cells. Moreover, we were able to regrow and expand the nasal organoids well after freezing and thawing. This study provides an effective and feasible method for development of human nasal organoids, suitable for the phenotypic high-throughput screening and drug response research.

3D bioprinting of a cell-laden antibacterial polysaccharide hydrogel composite.

Bioink with inherent antibacterial activity is of particular interest for tissue engineering application due to the growing number of bacterial infections associated with impaired wound healing or bone implants. However, the development of cell-laden bioink with potent antibacterial activity while supporting tissue regeneration proved to be challenging. Here, we introduced a cell-laden antibacterial bioink based on Methylcellulose/Alginate (MC/Alg) hydrogel for skin tissue engineering via elimination of the risks associated with a bacterial infection. The key feature of the bioink is the use of gallium (Ga+3) in the design of bioink formulation with dual functions. First, Ga+3 stabilized the hydrogel bioink by the formation of ionic crosslinking with Alg chains. Second, the gallium-crosslinked bioink exhibited potent antibacterial activity toward both Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria with a bactericidal rate of 99.99 %. In addition, it was found that the developed bioink supported encapsulated fibroblast cellular functions.

In vitro and in vivo evaluation of probiotic properties of Corynebacterium accolens isolated from the human nasal cavity.

Corynebacterium accolens strains are increasingly recognized as beneficial bacteria that can confer a health benefit on the host. In the current study, the probiotic potential of three C. accolens strains, C779, C781 and C787 derived from a healthy human nasal cavity were investigated. These strains were examined for their adhesion to HNECs, competition with Staphylococcus aureus for adhesion, toxicity, induction of IL-6, antibiotic susceptibility and the presence of antibiotic resistance and virulence genes. Furthermore, the safety and efficacy of strains were evaluated in vivo using Caenorhabditis elegans. The adhesion capacity of C. accolens to HNECs was strain-dependent. Highest adhesion was observed for strain C781. None of the C. accolens strains tested caused cell lysis. All strains were able to outcompete S. aureus for cell adhesion and caused a significant decrease of IL-6 production by HNECs co-exposed to S. aureus when compared to the control groups. All strains were sensitive or showed intermediate sensitivity to 10 different antibiotics. Whole Genome Sequence analysis showed C. accolens C781 and C787 did not possess antibiotic resistance genes whereas strain C779 harboured 5 genes associated with resistance to Aminoglycoside, Chloramphenicol and Erythromycin. In addition, no virulence genes were detected in any of the 3 strains. Moreover, the tested strains had no detrimental effect on worm survival and induced protection from S. aureus-mediated infection. Taken all together, C. accolens strains, C781 and C787 displayed probiotic potential and hold promise for use in clinical applications for combating dysbiosis in chronic rhinosinusitis.