Pathogens in exacerbations of chronic rhinosinusitis differ in sensitivity to silver nanoparticles

Introduction: The significance of the microbiome in chronic rhinosinusitis (CRS) is not clear. Antimicrobials are recommended in acute exacerbations of the disease (AECRS). Increasing rates of antibiotic resistance stimulate research on alternative therapeutic options including silver nanoparticles (AgNPs), sometimes referred to as “colloidal silver”. However, there are concerns regarding the safety of silver administration and the emergence of silver resistance. In this cross-sectional observational study, we assessed the sensitivity of sinonasal pathogens to AgNPs and compared it with the toxicity of AgNPs for nasal epithelial cells. Method: Negatively charged AgNPs (13±5 nm) were obtained with the use of tannic acid. Minimal inhibitory concentrations (MIC) of the AgNPs were determined for pathogens isolated from patients with AECRS. Cytotoxicity was tested on human nasal epithelial cells line in vitro. Results: 48 clinical isolates and 4 reference strains were included in the study (Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Acinetobacter baumanii, Serratia marcescens, Enterobacter cloacae). The MIC values differed between isolates, even within the same species. All of the strains were sensitive to AgNPs in concentrations nontoxic to human cells during 24 hours exposition. However, 48h exposition to AgNPs increased toxicity to human cells, narrowing their therapeutic window and enabling 19% of pathogens to resist the AgNPs biocidal action. Conclusions: AgNPs can potentially be used in intranasal drugs to treat most episodes of AECRS. Sensitivity testing may be necessary before application. Results of sensitivity testing for reference strains cannot be extrapolated to other strains of the same species.

Elovanoids Counteract Inflammatory Signaling, Autophagy, Endoplasmic Reticulum Stress, and Senescence Gene Programming in Human Nasal Epithelial Cells Exposed to Allergens

To contribute to further understanding the cellular and molecular complexities of inflammatory-immune responses in allergic disorders, we have tested the pro-homeostatic elovanoids (ELV) in human nasal epithelial cells (HNEpC) in culture challenged by several allergens. ELV are novel bioactive lipid mediators synthesized from the omega-3 very-long-chain polyunsaturated fatty acids (VLC-PUFA,n-3). We ask if: (a) several critical signaling events that sustain the integrity of the human nasal epithelium and other organ barriers are perturbed by house dust mites (HDM) and other allergens, and (b) if ELV would participate in beneficially modulating these events. HDM is a prevalent indoor allergen that frequently causes allergic respiratory diseases, including allergic rhinitis and allergic asthma, in HDM-sensitized individuals. Our study used HNEpC as an in vitro model to study the effects of ELV in counteracting HDM sensitization resulting in inflammation, endoplasmic reticulum (ER) stress, autophagy, and senescence. HNEpC were challenged with the following allergy inducers: LPS, poly(I:C), or Dermatophagoides farinae plus Dermatophagoides pteronyssinus extract (HDM) (30 µg/mL), with either phosphate-buffered saline (PBS) (vehicle) or ELVN-34 (500 nM). Results show that ELVN-34 promotes cell viability and reduces cytotoxicity upon HDM sensitization of HNEpC. This lipid mediator remarkably reduces the abundance of pro-inflammatory cytokines and chemokines IL-1β, IL-8, VEGF, IL-6, CXCL1, CCL2, and cell adhesion molecule ICAM1 and restores the levels of the pleiotropic anti-inflammatory IL-10. ELVN-34 also lessens the expression of senescence gene programming as well as of gene transcription engaged in pro-inflammatory responses. Our data also uncovered that HDM triggered the expression of key genes that drive autophagy, unfolded protein response (UPR), and matrix metalloproteinases (MMP). ELVN-34 has been shown to counteract these effects effectively. Together, our data reveal a novel, pro-homeostatic, cell-protective lipid-signaling mechanism in HNEpC as potential therapeutic targets for allergies.

Nasal epithelium: new insights and differences of the cytokine profile between normal subjects and subjects with allergic rhinitis

Background: The role of the nasal epithelium in the induction of a proper cytokine response in normal subjects and subjects with allergic rhinitis is still not completely elucidated. Methodology: We aimed to compare nasal epithelial immune responses in allergic rhinitis patients of different ages compared to healthy volunteers. Primary nasal epithelial cells from 47 subjects (33 normal and 17 with allergic rhinitis) were collected and cultured. Their unstimulated supernatants were analysed for 21 cytokines and chemokines. Statistical analysis was performed with the R statistical software and the RStudio interface. Results: Differences of the spontaneous release of epithelial cytokines and chemokines were noticed between the two study groups. The levels of GMCSF, MIP1A, MIP1B, IL28A, TNFA, CCL5 were significantly lower in the allergic rhinitis group compared to healthy volunteers’ group, independent of age. Most differences were noticed in the younger allergic rhinitis group (0-12 years old). Conclusions: Despite the cross-sectional nature of the study and the limited number of subjects, allergic rhinitis appears to be associated with dysfunction of cytokine and chemokine spontaneous release from nasal epithelial cells which may represent an abnormal innate immunity maturation pattern.

The Effect of IL-35 on the Expression of Nasal Epithelial-Derived Proinflammatory Cytokines

Background. Airway epithelium plays an important role during the development of allergic rhinitis (AR), which is characterized by production of thymic stromal lymphopoietin (TSLP), interleukin 33 (IL-33), and interleukin 25 (IL-25). IL-35, mainly expressed by Treg cells, have negative regulation in Th2, Th17, and ILC2 inflammation. However, the effect of IL-35 on human nasal epithelial cells (HNECs) especially the secretion of nasal epithelial-derived proinflammatory cytokines as well as the possible mechanism is still unclear. Methods. HNECs were cultured and stimulated by various stimulators. The expression of IL-33, IL-25, TSLP, eotaxin-1, eotaxin-2, and eotaxin-3 from supernatant was measured using real-time reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). AR mice were developed to verify the effect of IL-35 on nasal epithelial cells in vivo. Results. After Poly I:C stimulation, IL-35 inhibited the production of IL-25, and TSLP from HNECs increased significantly compared with baseline levels ( P < 0.05 ). After Dermatophagoides pteronyssinus or Aspergillus fumigatus stimulation, IL-35 inhibited the production of IL-25, IL-33, and TSLP from HNECs increased significantly compared with baseline levels ( P < 0.05 ). After Dermatophagoides pteronyssinus, IL-35 inhibited the production of eotaxin-1, eotaxin-2, and eotaxin-3 released from HNECs increased significantly compared with baseline levels ( P < 0.05 ). Similarly, IL-35-treated AR mice presented with decreased expression of IL-33, IL-25, TSLP, eotaxin-1, eotaxin-2, and eotaxin-3 in nasal lavage fluid. Conclusion. IL-35 suppressed both type 2 inflammation-inducing cytokines and eosinophil chemotactic factor from HNECs, suggesting the important role of IL-35 during the development of AR.

Major Complex Trait for Early De Novo Programming ‘CoV-MAC-TED’ Detected in Human Nasal Epithelial Cells Infected by Two SARS-CoV-2 Variants Is Promising to Help in Designing Therapeutic Strategies

Background: Early metabolic reorganization was only recently recognized as an essentially integrated part of immunology. In this context, unbalanced ROS/RNS levels connected to increased aerobic fermentation, which is linked to alpha-tubulin-based cell restructuring and control of cell cycle progression, were identified as a major complex trait for early de novo programming (‘CoV-MAC-TED’) during SARS-CoV-2 infection. This trait was highlighted as a critical target for developing early anti-viral/anti-SARS-CoV-2 strategies. To obtain this result, analyses had been performed on transcriptome data from diverse experimental cell systems. A call was released for wide data collection of the defined set of genes for transcriptome analyses, named ‘ReprogVirus’, which should be based on strictly standardized protocols and data entry from diverse virus types and variants into the ‘ReprogVirus Platform’. This platform is currently under development. However, so far, an in vitro cell system from primary target cells for virus attacks that could ideally serve for standardizing the data collection of early SARS-CoV-2 infection responses has not been defined. Results: Here, we demonstrate transcriptome-level profiles of the most critical ‘ReprogVirus’ gene sets for identifying ‘CoV-MAC-TED’ in cultured human nasal epithelial cells infected by two SARS-CoV-2 variants differing in disease severity. Our results (a) validate ‘Cov-MAC-TED’ as a crucial trait for early SARS-CoV-2 reprogramming for the tested virus variants and (b) demonstrate its relevance in cultured human nasal epithelial cells. Conclusion: In vitro-cultured human nasal epithelial cells proved to be appropriate for standardized transcriptome data collection in the ‘ReprogVirus Platform’. Thus, this cell system is highly promising to advance integrative data analyses with the help of artificial intelligence methodologies for designing anti-SARS-CoV-2 strategies.

Hypertonic saline and aprotinin based blockage of SARS-CoV-2 specific furin site cleavage by inhibition of nasal protease activity

SARS-CoV-2 enters into the human body mainly through the nasal epithelial cells. Cell entry of SARS-CoV-2 needs to be pre-activated by S1/S2 boundary furin motif cleavage by furin and/or relevant proteases. It is important to locally block SARS-CoV-2 S1/S2 site cleavage caused by furin and other relevant protease activity in the nasal cavity. We tested hypertonic saline and aprotinin-based blockage of SARS-CoV-2 specific furin site cleavage by furin, trypsin and nasal swab samples containing nasal proteases. Our results show that saline and aprotinin block SARS-Cov-2 specific furin site cleavage and that a saline and aprotinin combination could significantly reduce SARS-Cov-2 wild-type and P681R mutant furin site cleavage by inhibition of nasal protease activity.