Human Respiratory Mucosa Research Articles

Bioengineering Human Upper Respiratory Mucosa: A Systematic Review of the State of the Art of Cell Culture Techniques.

The upper respiratory mucosa plays a crucial role in both the physical integrity and immunological function of the respiratory tract. However, in certain situations such as infections, trauma, or surgery, it might sustain damage. Tissue engineering, a field of regenerative medicine, has found applications in various medical fields including but not limited to plastic surgery, ophthalmology, and urology. However, its application to the respiratory system remains somewhat difficult due to the complex morphology and histology of the upper respiratory tract. To date, a culture protocol for producing a handleable, well-differentiated nasal mucosa has yet to be developed. The objective of this review is to describe the current state of research pertaining to cell culture techniques used for producing autologous healthy human upper respiratory cells and mucosal tissues, as well as describe its clinical applications. A search of the relevant literature was carried out with no time restriction across Embase, Cochrane, PubMed, and Medline Ovid databases. Keywords related to "respiratory mucosa" and "culture techniques of the human airway" were the focus of the search strategy for this review. The risk of bias in retained studies was assessed using the Joanna Briggs Institute's (JBI) critical appraisal tools for qualitative research. A narrative synthesis of our results was then conducted. A total of 33 studies were included in this review, and thirteen of these focused solely on developing a cell culture protocol without further use. The rest of the studies used their own developed protocol for various applications such as cystic fibrosis, pharmacological, and viral research. One study was able to develop a promising model for nasal mucosa that could be employed as a replacement in nasotracheal reconstructive surgery. This systematic review extensively explored the current state of research regarding cell culture techniques for producing tissue-engineered nasal mucosa. Bioengineering the nasal mucosa holds great potential for clinical use. However, further research on mechanical properties is essential, as the comparison of engineered tissues is currently focused on morphology rather than comprehensive mechanical assessments.

A novel approach to investigate severe asthma and COPD: the 3d ex vivo respiratory mucosa model

Purpose: This article illustrates the replication of asthma and COPD conditions in a laboratory setting and the potential applications of this methodology. Introduction: Biologic drugs have been shown to enhance the treatment of severe asthma and COPD. Monoclonal antibodies against specific targets have dramatically changed the management of these conditions. Although the inflammatory pathways of asthma and COPD have already been clearly outlined, alternative mechanisms of action remain mostly unexplored. They could provide additional insights into these diseases and their clinical management. Aims: In vivo or in vitro models have thus been developed to test alternative hypotheses. This study describes sophisticated ex vivo models that mimic the response of human respiratory mucosa to disease triggers, aiming to narrow the gap between laboratory studies and clinical practice. Results: These models successfully replicate crucial aspects of these diseases, such as inflammatory cell presence, cytokine production, and changes in tissue structure, offering a dynamic platform for investigating disease processes and evaluating potential treatments, such as monoclonal antibodies. The proposed models have the potential to enhance personalized medicine approaches and patient-specific treatments, helping to advance the understanding and management of respiratory diseases.

Characterization of a Human Respiratory Mucosa Model to Study Odorant Metabolism.

Nasal xenobiotic metabolizing enzymes (XMEs) are important for the sense of smell because they influence odorant availability and quality. Since the major part of the human nasal cavity is lined by a respiratory mucosa, we hypothesized that this tissue contributed to nasal odorant metabolism through XME activity. Thus, we built human respiratory tissue models and characterized the XME profiles using single-cell RNA sequencing. We focused on the XMEs dicarbonyl and l-xylulose reductase, aldehyde dehydrogenase (ALDH) 1A1, and ALDH3A1, which play a role in food odorant metabolism. We demonstrated protein abundance and localization in the tissue models and showed the metabolic activity of the corresponding enzyme families by exposing the models to the odorants 3,4-hexandione and benzaldehyde. Using gas chromatography coupled with mass spectrometry, we observed, for example, a significantly higher formation of the corresponding metabolites 4-hydroxy-3-hexanone (39.03 ± 1.5%, p = 0.0022), benzyl alcohol (10.05 ± 0.88%, p = 0.0008), and benzoic acid (8.49 ± 0.57%, p = 0.0004) in odorant-treated tissue models compared to untreated controls (0 ± 0, 0.12 ± 0.12, and 0.18 ± 0.18%, respectively). This is the first study that reveals the XME profile of tissue-engineered human respiratory mucosa models and demonstrates their suitability to study nasal odorant metabolism.

A Novel Preparation Technique for Human Nasal Respiratory Mucosa to Disclose Its Glycosylation Pattern for Bioadhesive Drug Delivery

To shed some light on glycotargeting as a potential strategy for nasal drug delivery, a reliable preparation method for human nasal mucosa samples and a tool to investigate the carbohydrate building blocks of the glycocalyx of the respiratory epithelium are required. Applying a simple experimental setup in a 96-well plate format together with a panel of six fluorescein-labeled lectins with different carbohydrate specificities allowed for the detection and quantification of accessible carbohydrates in the mucosa. As confirmed by binding experiments at 4 °C, both quantitatively by fluorimetry and qualitatively by microscopy, the binding of wheat germ agglutinin exceeded that of the others by 150% on average, indicating a high content of N-acetyl-D-glucosamine and sialic acid. Providing energy by raising the temperature to 37 °C revealed uptake of the carbohydrate-bound lectin into the cell. Moreover, repeated washing steps during the assay gave a slight hint as to the influence of mucus renewal on bioadhesive drug delivery. All in all, the experimental setup reported here for the first time is not only a suitable approach to estimating the basics and potential of nasal lectin-mediated drug delivery but also meets the needs for answering a broad variety of scientific questions involving the use of ex vivo tissue samples.

Type 2 inflammation drives an airway basal stem cell program through insulin receptor substrate signaling

Chronic rhinosinusitis with nasal polyposis (CRSwNP) is a type 2 (T2) inflammatory disease associated with an increased number of airway basal cells (BCs). Recent studies have identified transcriptionally distinct BCs, but the molecular pathways that support or inhibit human BC proliferation and differentiation are largely unknown. We sought to determine the role of T2 cytokines in regulating airway BCs. Single-cell and bulk RNA sequencing of sinus and lung airway epithelial cells was analyzed. Human sinus BCs were stimulated with IL-4 and IL-13 in the presence and absence of inhibitors of IL-4R signaling. Confocal analysis of human sinus tissue and murine airway was performed. Murine BC subsets were sorted for RNA sequencing and functional assays. Fate labeling was performed in a murine model of tracheal injury and regeneration. Two subsets of BCs were found in human and murine respiratory mucosa distinguished by the expression of basal cell adhesion molecule (BCAM). BCAM expression identifies airway stem cells among P63+KRT5+NGFR+ BCs. In the sinonasal mucosa, BCAMhi BCs expressing TSLP, IL33, CCL26, and the canonical BC transcription factor TP63 are increased in patients with CRSwNP. In cultured BCs, IL-4/IL-13 increases the expression of BCAM and TP63 through an insulin receptor substrate-dependent signaling pathway that is increased in CRSwNP. These findings establish BCAM as a marker of airway stem cells among the BC pool and demonstrate that airway epithelial remodeling in T2 inflammation extends beyond goblet cell metaplasia to the support of a BC stem state poised to perpetuate inflammation.

Streamlined copper defenses make Bordetella pertussis reliant on custom-made operon

Copper is both essential and toxic to living beings, which tightly controls its intracellular concentration. At the host–pathogen interface, copper is used by phagocytic cells to kill invading microorganisms. We investigated copper homeostasis in Bordetella pertussis, which lives in the human respiratory mucosa and has no environmental reservoir. B. pertussis has considerably streamlined copper homeostasis mechanisms relative to other Gram-negative bacteria. Its single remaining defense line consists of a metallochaperone diverted for copper passivation, CopZ, and two peroxide detoxification enzymes, PrxGrx and GorB, which together fight stresses encountered in phagocytic cells. Those proteins are encoded by an original, composite operon assembled in an environmental ancestor, which is under sensitive control by copper. This system appears to contribute to persistent infection in the nasal cavity of B. pertussis-infected mice. Combining responses to co-occurring stresses in a tailored operon reveals a strategy adopted by a host-restricted pathogen to optimize survival at minimal energy expenditure.

On the in vivo origin of human nasal mesenchymal stem cell cultures.

ObjectivesMesenchymal stem cells (MSCs), classically expanded in culture from bone marrow, are of broad interest to the regenerative medicine community. Human nasal turbinate mesenchymal‐like stem cell cultures have also been described, defined by an in vitro phenotype similar to bone marrow MSCs. Nonetheless, the identity in vivo of the cells that give rise to nasal MSC‐like cultures remains unclear, and these cells are often suggested to be related to olfactory lineages. Here, we sought to define the in vivo phenotype of human nasal MSC‐like cells.MethodsHuman turbinate tissue samples were used for RNA and immunohistochemical analysis. We also analyzed a recently published single cell RNA‐sequencing dataset from adult human olfactory and respiratory mucosa samples from our lab, to focus on cell populations expressing MSC markers. Immunochemistry was performed to stain turbinate sections and nasal MSC cultures for selected markers.ResultsWhile there is no single MSC‐specific gene, we identified a human nasal mucosal cell population in vivo that uniquely expressed transcripts characteristic of typical MSC cultures, including ENG (CD105), NES, and CD34, and lacked expression of other transcripts associated with surface epithelia. The expression of transcription factors such as SOX17, EBF1, and FOXP1 suggests cells in the MSC‐like cluster maintain an ability to direct cell fate, consistent with the behavior of nasal MSC‐like cells in vitro. SOX17 was found to be uniformly expressed by nasal MSC cultures, consistent with the in vivo data. Immunohistochemistry of human nasal tissue samples indicated that ENG, CD34, and SOX17 expression localized selectively to cells surrounding blood vessels in the lamina propria.ConclusionOur findings provide evidence that the in vivo origin of nasal MSC‐like cultures is likely a vascular or pericyte population, rather than cells related to the olfactory neuronal lineage.Level of evidenceNA.

The influence of interferon-lambda on restricting Middle East Respiratory Syndrome Coronavirus replication in the respiratory epithelium

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) causes severe respiratory in human with high mortality and it has been a challenge to determine optimum treatment for MERS-CoV-induced respiratory infection. Here, we observed the distribution of MERS-CoV receptors using human respiratory mucosa and also evaluated the contribution of interferon-lambdas (IFN-λs) in response to MERS-CoV infection using in vitro normal human nasal epithelial (NHNE) and bronchial epithelial (NHBE) cells. We found that the gene and protein expression of DPPIV, MERS-CoV receptor, were more dominantly located in nasal and bronchial epithelium although human nasal mucosa exhibited relatively lower DPPIV expression than lung parenchymal tissues. The quantitative mRNA level of the MERS-CoV envelope (upE) gene was significantly induced in MERS-CoV-infected cultured NHNE and NHBE cells until 3 days after infection. The induction of IFNs was identified in NHNE and NHBE cells after MERS-CoV infection and IFN-λs were predominantly increased in MERS-CoV-infected respiratory epithelial cells. Inoculation of IFN-λs to NHNE and NHBE cells suppressed MERS-CoV replication and in particular, IFN-λ4 showed a strong therapeutic effect in reducing MERS-CoV infection with higher induction of IFN-stimulated genes. Thus, IFN-λ has a decisive function in the respiratory epithelium that greatly limits MERS-CoV replication, and may be a key cytokine for better therapeutic outcomes against MERS-CoV infection in respiratory tract.

Effect of oxidized cellulose on human respiratory mucosa and submucosa and its implications for endoscopic skull-base approaches.

Regenerated oxidized cellulose (ROC) sheets have gained popularity as an adjunct to a vascularized nasoseptal flap for closure of dural defects after endoscopic endonasal skull-base approaches (EESBS). However, evidence supporting its impact on the healing process is uncertain. This study was performed to evaluate the impact of ROC on the nasal mucosa and assess its effects on tissue pH, structure, and cell viability. In 5 patients, a 1-cm2 piece of ROC gauze was placed on the surface of the middle turbinate before it was resected as part of a standard EESBS. Mucosa treated with ROC was separated from untreated mucosa and a histologic examination of structural changes in the respiratory epithelium was performed. To assess the effect of ROC on pH, increasing amounts of ROC were added to culture medium. Nasal fibroblasts viability was assessed in the presence of ROC before and after the pH was neutralized. Compared with unexposed controls, treated mucosa exhibited a higher incidence of cell necrosis and epithelial cell detachment. When added to Dulbecco's modified Eagle medium, ROC caused a dose-dependent decrease in pH of the medium. Only 1 ± 0.8% of cultured fibroblasts exposed to the ROC-induced acidic medium were alive, whereas 98.25 ± 0.5% of the cells were viable when the pH was neutralized (p < 0.001). ROC applied in vivo to nasal mucosa induced epithelial necrosis likely by diminishing the medium pH, because pH neutralization prevents its effect. The ultimate effect of this material on the healing process is yet to be determined.

Identification of urban particulate matter-induced disruption of human respiratory mucosa integrity using whole transcriptome analysis and organ-on-a chip

BackgroundExposure to air particulate matter (PM) is associated with various diseases in the human respiratory system. To date, most in vitro studies showing cellular responses to PM have been performed in cell culture using a single cell type. There are few studies considering how multicellular networks communicate in a tissue microenvironment when responding to the presence of PM. Here, an in vitro three-dimensional (3D) respiratory mucosa-on-a-chip, composed of human nasal epithelial cells, fibroblasts, and endothelial cells, is used to recapitulate and better understand the effects of urban particulate matter (UPM) on human respiratory mucosa.ResultsWe hypothesized that the first cells to contact with UPM, the nasal epithelial cells, would respond similar to the tissue microenvironment, and the 3D respiratory mucosa model would be a suitable platform to capture these events. First, whole transcriptome analysis revealed that UPM induced gene expression alterations in inflammatory and adhesion-related genes in human nasal epithelial cells. Next, we developed an in vitro 3D respiratory mucosa model composed of human nasal epithelial cells, fibroblasts, and endothelial cells and demonstrated that the model is structurally and functionally compatible with the respiratory mucosa. Finally, we used our model to expose human nasal epithelial cells to UPM, which led to a disruption in the integrity of the respiratory mucosa by decreasing the expression of zonula occludens-1 in both the epithelium and endothelium, while also reducing vascular endothelial cadherin expression in the endothelium.ConclusionsWe demonstrate the potential of the 3D respiratory mucosa model as a valuable tool for the simultaneous evaluation of multicellular responses caused by external stimuli in the human respiratory mucosa. We believe that the evaluation strategy proposed in the study will move us toward a better understanding of the detailed molecular mechanisms associated with pathological changes in the human respiratory system.

IgA tetramerization improves target breadth but not peak potency of functionality of anti-influenza virus broadly neutralizing antibody.

Mucosal immunoglobulins comprise mainly secretory IgA antibodies (SIgAs), which are the major contributor to pathogen-specific immune responses in mucosal tissues. These SIgAs are highly heterogeneous in terms of their quaternary structure. A recent report shows that the polymerization status of SIgA defines their functionality in the human upper respiratory mucosa. Higher order polymerization of SIgA (i.e., tetramers) leads to a marked increase in neutralizing activity against influenza viruses. However, the precise molecular mechanisms underlying the effects of SIgA polymerization remain elusive. Here, we developed a method for generating recombinant tetrameric monoclonal SIgAs. We then compared the anti-viral activities of these tetrameric SIgAs, which possessed variable regions identical to that of a broadly neutralizing anti-influenza antibody F045-092 against influenza A viruses, with that of monomeric IgG or IgA. The tetrameric SIgA showed anti-viral inhibitory activity superior to that of other forms only when the antibody exhibits low-affinity binding to the target. By contrast, SIgA tetramerization did not substantially modify anti-viral activity against targets with high-affinity binding. Taken together, the data suggest that tetramerization of SIgA improved target breadth, but not peak potency of antiviral functions of the broadly neutralizing anti-influenza antibody. This phenomenon presumably represents one of the mechanisms by which SIgAs present in human respiratory mucosa prevent infection by antigen-drifted influenza viruses. Understanding the mechanisms involved in cross neutralization of viruses by SIgAs might facilitate the development of vaccine strategies against viral infection of mucosal tissues.

IgA polymerization contributes to efficient virus neutralization on human upper respiratory mucosa after intranasal inactivated influenza vaccine administration

ABSTRACTUnlike the current injectable influenza vaccines, intranasally administered influenza vaccines induce influenza virus-specific IgA antibodies in the local respiratory mucosa as well as IgG antibodies in the systemic circulation. Our previous study showed that after five volunteers underwent intranasal administration with inactivated H3N2 or H5N1 vaccines, their IgA antibodies on the upper respiratory tract were present as monomers, dimers, and multimers (trimers and tetramers). Moreover, the multimers associated with the highest virus neutralizing activity. However, it has remained elusive whether a more practical intranasal vaccination strategy could induce the high-performance IgA multimers in the nasal mucosa. In the present study, volunteers were administered with two doses of the intranasal trivalent whole-virus inactivated influenza vaccine and showed that in nasal wash samples the amount of multimeric IgA correlated positively with virus neutralizing titers, indicating that the multimeric IgA antibodies play an important role in the antiviral activity at the nasal mucosa. Surface plasmon resonance analysis of the binding dynamics of nasal wash derived IgA monomers, dimers, and multimers against recombinant trimeric influenza virus HA showed that sample fractions containing IgA multimers dissociated from HA less well than sample fractions without IgA multimers. Thus, IgA multimers may “stick” to the antigen more tightly than the other structures. In summary, intranasal administration of two doses of multivalent inactivated influenza vaccines induced multimeric IgA. Multimerization of mucosal IgA antibodies conferred higher neutralizing activity against viruses in the nasal mucosa, possibly by increasing their cohesion to virus antigens.

Absorption of Nasal and Bronchial Fluids: Precision Sampling of the Human Respiratory Mucosa and Laboratory Processing of Samples

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the human respiratory tract. NA is a non-invasive technique which absorbs fluid from the inferior turbinate, and causes minimal discomfort. NA has yielded reproducible results with the ability to frequently repeat sampling of the upper airway. By comparison, alternative methods of sampling the respiratory mucosa, such as nasopharyngeal aspiration (NPA) and conventional swabbing, are more invasive and may result in greater data variability. Other methods have limitations, for instance, biopsies and bronchial procedures are invasive, sputum contains many dead and dying cells and requires liquefaction, exhaled breath condensate (EBC) contains water and saliva, and lavage samples are dilute and variable. BA can be performed through the working channel of a bronchoscope in clinic. Sampling is well tolerated and can be conducted at multiple sites in the airway. BA results in MLF samples being less dilute than bronchoalveolar lavage (BAL) samples. This article demonstrates the techniques of NA and BA, as well as the laboratory processing of the resulting samples, which can be tailored to the desired downstream biomarker being measured. These absorption techniques are useful alternatives to the conventional sampling techniques used in clinical respiratory research.

Absorption of Nasal and Bronchial Fluids: Precision Sampling of the Human Respiratory Mucosa and Laboratory Processing of Samples.

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the human respiratory tract. NA is a non-invasive technique which absorbs fluid from the inferior turbinate, and causes minimal discomfort. NA has yielded reproducible results with the ability to frequently repeat sampling of the upper airway. By comparison, alternative methods of sampling the respiratory mucosa, such as nasopharyngeal aspiration (NPA) and conventional swabbing, are more invasive and may result in greater data variability. Other methods have limitations, for instance, biopsies and bronchial procedures are invasive, sputum contains many dead and dying cells and requires liquefaction, exhaled breath condensate (EBC) contains water and saliva, and lavage samples are dilute and variable.BA can be performed through the working channel of a bronchoscope in clinic. Sampling is well tolerated and can be conducted at multiple sites in the airway. BA results in MLF samples being less dilute than bronchoalveolar lavage (BAL) samples.This article demonstrates the techniques of NA and BA, as well as the laboratory processing of the resulting samples, which can be tailored to the desired downstream biomarker being measured. These absorption techniques are useful alternatives to the conventional sampling techniques used in clinical respiratory research.

Anti-Aspergillus Activities of the Respiratory Epithelium in Health and Disease.

Respiratory epithelia fulfil multiple roles beyond that of gaseous exchange, also acting as primary custodians of lung sterility and inflammatory homeostasis. Inhaled fungal spores pose a continual antigenic, and potentially pathogenic, challenge to lung integrity against which the human respiratory mucosa has developed various tolerance and defence strategies. However, respiratory disease and immune dysfunction frequently render the human lung susceptible to fungal diseases, the most common of which are the aspergilloses, a group of syndromes caused by inhaled spores of Aspergillus fumigatus. Inhaled Aspergillus spores enter into a multiplicity of interactions with respiratory epithelia, the mechanistic bases of which are only just becoming recognized as important drivers of disease, as well as possible therapeutic targets. In this mini-review we examine current understanding of Aspergillus-epithelial interactions and, based upon the very latest developments in the field, we explore two apparently opposing schools of thought which view epithelial uptake of Aspergillus spores as either a curative or disease-exacerbating event.

Evidence for altered levels of IgD in the nasal airway mucosa of patients with chronic rhinosinusitis

IgD is an enigmatic antibody isotype best known when coexpressed with IgM on naive B cells. However, increased soluble IgD (sIgD) levels and increased IgD+IgM- B-cell populations have been described in the human upper respiratory mucosa. We assessed whether levels of sIgD and IgD+ B cell counts are altered in nasal tissue from patients with chronic rhinosinusitis (CRS). We further characterized IgD+ B-cell populations and explored clinical and local inflammatory factors associated with tissue sIgD levels. sIgD levels were measured by means of ELISA in nasal tissues, nasal lavage fluid, sera, and supernatants of dissociated nasal tissues. IgD+ cells were identified by using immunofluorescence and flow cytometry. Inflammatory mediator levels in tissues were assessed by using real-time PCR and multiplex immunoassays. Bacterial cultures from the middle meatus were performed. Underlying medical history and medicine use were obtained from medical records. sIgD levels and numbers of IgD+ cells were significantly increased in uncinate tissue (UT) of patients with chronic rhinosinusitis without nasal polyps (CRSsNP) compared with that of control subjects (4-fold, P<.05). IgD+ cells were densely scattered in the periglandular regions of UT from patients with CRSsNP. We also found that IgD+CD19+CD38bright plasmablast numbers were significantly increased in tissues from patients with CRSsNP compared with control tissues (P<.05). Among numerous factors tested, IL-2 levels were increased in UT from patients with CRSsNP and were positively correlated with tissue IgD levels. Additionally, supernatants of IL-2-stimulated dissociated tissue from patients with CRSsNP had significantly increased sIgD levels compared with those in IL-2-stimulated dissociated control tissue exvivo (P<.05). Tissue from patients with CRS with preoperative antibiotic use or those with pathogenic bacteria showed higher IgD levels compared with tissue from patients without these variables (P<.05). sIgD levels and IgD+CD19+CD38bright plasmablast counts were increased in nasal tissue of patients with CRSsNP. IgD levels were associated with increased IL-2 levels and the presence of pathogenic bacteria. These findings suggest that IgD might contribute to enhancement mucosal immunity or inflammation or respond to bacterial infections in patients with CRS, especially CRSsNP.

Interactions of surface-displayed glycolytic enzymes of Mycoplasma pneumoniae with components of the human extracellular matrix

Mycoplasma pneumoniae is a major cause of community-acquired respiratory infections worldwide. Due to the strongly reduced genome, the number of virulence factors expressed by this cell wall-less pathogen is limited. To further understand the processes during host colonization, we investigated the interactions of the previously confirmed surface-located glycolytic enzymes of M. pneumoniae (pyruvate dehydrogenase A-C [PdhA-C], glyceraldehyde-3-phosphate dehydrogenase [GapA], lactate dehydrogenase [Ldh], phosphoglycerate mutase [Pgm], pyruvate kinase [Pyk] and transketolase [Tkt]) to the human extracellular matrix (ECM) proteins fibrinogen (Fn), fibronectin (Fc), lactoferrin (Lf), laminin (Ln) and vitronectin (Vc), respectively. Concentration-dependent interactions between Fn and Vc and all eight recombinant proteins derived from glycolytic enzymes, between Ln and PdhB-C, GapA, Ldh, Pgm, Pyk and Tkt, between Lf and PdhA-C, GapA and Pyk, and between Fc and PdhC and GapA were demonstrated. In most cases, these associations are significantly influenced by ionic forces and by polyclonal sera against recombinant proteins. In immunoblotting, the complex of human plasminogen, activator (tissue-type or urokinase plasminogen activator) and glycolytic enzyme was not able to degrade Fc, Lf and Ln, respectively. In contrast, degradation of Vc was confirmed in the presence of all eight enzymes tested. Our data suggest that the multifaceted associations of surface-localized glycolytic enzymes play a potential role in the adhesion and invasion processes during infection of human respiratory mucosa by M. pneumoniae.

Effect of in Vivo Oxidized Cellulose on in Vitro Growth of Human Respiratory Mucosa and Sub-mucosa During Endoscopic Skull Base Approaches

Effect of in Vivo Oxidized Cellulose on in Vitro Growth of Human Respiratory Mucosa and Sub-mucosa During Endoscopic Skull Base Approaches

Dual RNA-seq of Nontypeable Haemophilus influenzae and Host Cell Transcriptomes Reveals Novel Insights into Host-Pathogen Cross Talk

ABSTRACTThe ability to adhere and adapt to the human respiratory tract mucosa plays a pivotal role in the pathogenic lifestyle of nontypeable Haemophilus influenzae (NTHi). However, the temporal events associated with a successful colonization have not been fully characterized. In this study, by reconstituting the ciliated human bronchial epithelium in vitro, we monitored the global transcriptional changes in NTHi and infected mucosal epithelium simultaneously for up to 72 h by dual RNA sequencing. The initial stage of colonization was characterized by the binding of NTHi to ciliated cells. Temporal profiling of host mRNA signatures revealed significant dysregulation of the target cell cytoskeleton elicited by bacterial infection, with a profound effect on the intermediate filament network and junctional complexes. In response to environmental stimuli of the host epithelium, NTHi downregulated its central metabolism and increased the expression of transporters, indicating a change in the metabolic regime due to the availability of host substrates. Concurrently, the oxidative environment generated by infected cells instigated bacterial expression of stress-induced defense mechanisms, including the transport of exogenous glutathione and activation of the toxin-antitoxin system. The results of this analysis were validated by those of confocal microscopy, Western blotting, Bio-plex, and real-time quantitative reverse transcription-PCR (qRT-PCR). Notably, as part of our screening for novel signatures of infection, we identified a global profile of noncoding transcripts that are candidate small RNAs (sRNAs) regulated during human host infection in Haemophilus species. Our data, by providing a robust and comprehensive representation of the cross talk between the host and invading pathogen, provides important insights into NTHi pathogenesis and the development of efficacious preventive strategies.

Highly differentiated human airway epithelial cells: a model to study host cell–parasite interactions in pertussis

Background: Bordetella pertussis colonizes the human respiratory mucosa. Most studies on B. pertussis adherence have relied on cultured mammalian cells that lack key features present in differentiated human airway cells or on animal models that are not natural hosts of B. pertussis. The objectives of this work were to evaluate B. pertussis infection in highly differentiated human airway cells in vitro and to show the role of B. pertussis fimbriae in cell adherence. Methods: Primary human airway epithelial (PHAE) cells from human bronchi and a human bronchial epithelial (HBE) cell line were grown in vitro under air–liquid interface conditions. Results: PHAE and HBE cells infected with B. pertussis wild-type strain revealed bacterial adherence to the apical surface of cells, bacteria-induced cytoskeleton changes, and cell detachment. Mutations in the major fimbrial subunits Fim2/3 or in the minor fimbrial adhesin subunit FimD affected B. pertussis adherence to predominantly HBE cells. This cell model recapitulates the morphologic features of the human airway infected by B. pertussis and confirms the role of fimbriae in B. pertussis adherence. Furthermore, HBE cells show that fimbrial subunits, and specifically FimD adhesin, are critical in B. pertussis adherence to airway cells. Conclusions: The relevance of this model to study host–parasite interaction in pertussis lies in the striking physiologic and morphologic similarity between the PHAE and HBE cells and the human airway ciliated and goblet cells in vivo. These cells can proliferate in vitro, differentiate, and express the same genetic profile as human respiratory cells in vivo.