Nonantimicrobial Actions of Macrolides: Overview and Perspectives for Future Development.

Macrolides are among the most widely prescribed broad spectrum antibacterials, particularly for respiratory infections. It is now recognized that these drugs, in particular azithromycin, also exert time-dependent immunomodulatory actions that contribute to their therapeutic benefit in both infectious and other chronic inflammatory diseases. Their increased chronic use in airway inflammation and, more recently, of azithromycin in COVID-19, however, has led to a rise in bacterial resistance. An additional crucial aspect of chronic airway inflammation, such as chronic obstructive pulmonary disease, as well as other inflammatory disorders, is the loss of epithelial barrier protection against pathogens and pollutants. In recent years, azithromycin has been shown with time to enhance the barrier properties of airway epithelial cells, an action that makes an important contribution to its therapeutic efficacy. In this article, we review the background and evidence for various immunomodulatory and time-dependent actions of macrolides on inflammatory processes and on the epithelium and highlight novel nonantibacterial macrolides that are being studied for immunomodulatory and barrier-strengthening properties to circumvent the risk of bacterial resistance that occurs with macrolide antibacterials. We also briefly review the clinical effects of macrolides in respiratory and other inflammatory diseases associated with epithelial injury and propose that the beneficial epithelial effects of nonantibacterial azithromycin derivatives in chronic inflammation, even given prophylactically, are likely to gain increasing attention in the future. SIGNIFICANCE STATEMENT: Based on its immunomodulatory properties and ability to enhance the protective role of the lung epithelium against pathogens, azithromycin has proven superior to other macrolides in treating chronic respiratory inflammation. A nonantibiotic azithromycin derivative is likely to offer prophylactic benefits against inflammation and epithelial damage of differing causes while preserving the use of macrolides as antibiotics.

Azithromycin induces epidermal differentiation and multivesicular bodies in airway epithelia.

BACKGROUND: Azithromycin (Azm) is a macrolide recognized for its disease-modifying effects and reduction in exacerbation of chronic airway diseases. It is not clear whether the beneficial effects of Azm are due to its anti-microbial activity or other pharmacological actions. We have shown that Azm affects the integrity of the bronchial epithelial barrier measured by increased transepithelial electrical resistance. To better understand these effects of Azm on bronchial epithelia we have investigated global changes in gene expression. METHODS: VA10 bronchial epithelial cells were treated with Azm and cultivated in air-liquid interface conditions for up to 22 days. RNA was isolated at days 4, 10 and 22 and analyzed using high-throughput RNA sequencing. qPCR and immunostaining were used to confirm key findings from bioinformatic analyses. Detailed assessment of cellular changes was done using microscopy, followed by characterization of the lipidomic profiles of the multivesicular bodies present. RESULTS: Bioinformatic analysis revealed that after 10 days of treatment genes encoding effectors of sterol and cholesterol metabolism were prominent. Interestingly, expression of genes associated with epidermal barrier differentiation, KRT1, CRNN, SPINK5 and DSG1, increased significantly at day 22. Together with immunostaining, these results suggest an epidermal differentiation pattern. We also found that Azm induced the formation of multivesicular and lamellar bodies in two different airway epithelial cell lines. Lipidomic analysis revealed that Azm was entrapped in multivesicular bodies linked to different types of lipids, most notably palmitate and stearate. Furthermore, targeted analysis of lipid species showed accumulation of phosphatidylcholines, as well as ceramide derivatives. CONCLUSIONS: Taken together, we demonstrate how Azm might confer its barrier enhancing effects, via activation of epidermal characteristics and changes to intracellular lipid dynamics. These effects of Azm could explain the unexpected clinical benefit observed during Azm-treatment of patients with various lung diseases affecting barrier function.

Conditions associated with the cystic fibrosis defect promote chronic Pseudomonas aeruginosa infection.

RATIONALE: Progress has been made in understanding how the cystic fibrosis (CF) basic defect produces lung infection susceptibility. However, it remains unclear why CF exclusively leads to chronic infections that are noninvasive and highly resistant to eradication. Although biofilm formation has been suggested as a mechanism, recent work raises questions about the role of biofilms in CF. OBJECTIVES: To learn how airway conditions attributed to CF transmembrane regulator dysfunction could lead to chronic infection, and to determine if biofilm-inhibiting genetic adaptations that are common in CF isolates affect the capacity of Pseudomonas aeruginosa to develop chronic infection phenotypes. METHODS: We studied P. aeruginosa isolates grown in agar and mucus gels containing sputum from patients with CF and measured their susceptibility to killing by antibiotics and host defenses. We also measured the invasive virulence of P. aeruginosa grown in sputum gels using airway epithelial cells and a murine infection model. MEASUREMENTS AND MAIN RESULTS: We found that conditions likely to result from increased mucus density, hyperinflammation, and defective bacterial killing could all cause P. aeruginosa to grow in bacterial aggregates. Aggregated growth markedly increased the resistance of bacteria to killing by host defenses and antibiotics, and reduced their invasiveness. In addition, we found that biofilm-inhibiting mutations do not impede aggregate formation in gel growth environments. CONCLUSIONS: Our findings suggest that conditions associated with several CF pathogenesis hypotheses could cause the noninvasive and resistant infection phenotype, independently of the bacterial functions needed for biofilm formation.

Cystic fibrosis in Iceland and the high prevalence of the N1303K variant.

BACKGROUND: Cystic fibrosis (CF) is most common in populations of Northern European ancestry where the F508del variant predominates. In 2020, Iceland became a member of the European Cystic Fibrosis Society Patient Registry, and we launched an epidemiological study of CF in Iceland. The study aimed to determine the prevalence and the genetic variants present in the country. Furthermore, we aimed to describe the previous and the current situation regarding lung function, infections, complications, treatment, and follow-up to understand the strengths and weaknesses of CF care in Iceland. METHODS: This retrospective study included all individuals in Iceland with a confirmed CF diagnosis between 1955 and 2021. We conducted a medical records search for CF diagnosis codes and found 30 people with CF who were included in the study. Two hundred sixteen clinical variables were registered. A descriptive analysis of these was performed. RESULTS: The prevalence of CF in Iceland is 0.372:10,000 inhabitants. The F508del is the most common CF transmembrane conductance regulator (CFTR) variant (46.4%), closely followed by N1303K (44.6%). Staphylococcus aureus was the most common airway pathogen, followed by Pseudomonas aeruginosa. Nasal polyps and CF-related diabetes were the most common complications. Modern CF medications, including the recent CFTR modulators, are available. CONCLUSION: Even though Iceland has a relatively low prevalence of CF, it holds the highest known prevalence of the N1303K variant in Europe. Access to necessary treatment is satisfactory, but improvements are advisable for some aspects of the routine assessments by best practice guidelines.

Drug delivery characteristics of the progenitor bronchial epithelial cell line VA10.

PURPOSE: To determine the integrity and permeability properties of the immortalized human VA10 bronchial epithelial cell line for its suitability as an in vitro drug permeation model. METHODS: Cells were grown under liquid-covered culture (LCC) or air-liquid interface (ALI) culture, characterized using electron microscopy and immunostaining. Integrity was measured using transepithelial electrical resistance (TER) and permeability of fluorescein sodium (Flu-Na). General permeability was established with dextrans and model drugs and P-glycoprotein (P-gp) function determined with bidirectional flux of rhodamine-123. RESULTS: ALI culture resulted in 2-3 cell layers with differentiation towards ciliated cells but LCC showed undifferentiated morphology. VA10 cells formed TJ, with higher TER in LCC than ALI (∼2500 vs. ∼1200 Ω*cm(2)) and Flu-Na permeability ∼1-2 × 10(-7) cm/s. ALI cultured cells expressed P-gp and distinguished between compounds depending on lipophilicity and size, consistent with previous data from Calu-3 and 16HBE14o-cell lines. CONCLUSIONS: ALI cultured cell layers capture the in vivo-like phenotype of bronchial epithelium and form functional cell barrier capable of discriminating between compounds depending on physiochemical properties. The VA10 cell line is an important alternative to previously published cell lines and a relevant model to study airway drug delivery in vitro.

Azithromycin maintains airway epithelial integrity during Pseudomonas aeruginosa infection.

Tight junctions (TJs) play a key role in maintaining bronchial epithelial integrity, including apical-basolateral polarity and paracellular trafficking. Patients with chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) often suffer from chronic infections by the opportunistic Gram-negative bacterium Pseudomonas aeruginosa, which produces multiple virulence factors, including rhamnolipids. The macrolide antibiotic azithromycin (azm) has been shown to improve lung function in patients with CF without reducing the bacterial count within the lung. However, the mechanism of this effect is still debated. It has previously been shown that azm increased transepithelial electrical resistance (TER) in a bronchial epithelial cell line. In this study we used an air-liquid interface model of human airway epithelia and measured TER, changes in TJ expression and architecture after exposure to live P. aeruginosa PAO1, and PAO1-Deltarhl which is a PAO1 mutant lacking rhlA and rhlB, which encode key enzymes for rhamnolipid production. In addition, the cells were challenged with bacterial culture medium conditioned by these strains, purified rhamnolipids, or synthetic 3O-C(12)-HSL. Virulence factors secreted by P. aeruginosa reduced TER and caused TJ rearrangement in the bronchial epithelium, exposing the epithelium to further bacterial infiltration. Pretreatment of the bronchial epithelium with azm attenuated this effect and facilitated epithelial recovery. These data suggest that azm protects the bronchial epithelium during P. aeruginosa infection independent of antimicrobial activity, and could explain in part the beneficial results seen in clinical trials of patients with CF.

Airway branching morphogenesis in three dimensional culture.

BACKGROUND: Lungs develop from the fetal digestive tract where epithelium invades the vascular rich stroma in a process called branching morphogenesis. In organogenesis, endothelial cells have been shown to be important for morphogenesis and the maintenance of organ structure. The aim of this study was to recapitulate human lung morphogenesis in vitro by establishing a three dimensional (3D) co-culture model where lung epithelial cells were cultured in endothelial-rich stroma. METHODS: We used a human bronchial epithelial cell line (VA10) recently developed in our laboratory. This cell line cell line maintains a predominant basal cell phenotype, expressing p63 and other basal markers such as cytokeratin-5 and -14. Here, we cultured VA10 with human umbilical vein endothelial cells (HUVECs), to mimic the close interaction between these cell types during lung development. Morphogenesis and differentiation was monitored by phase contrast microscopy, immunostainings and confocal imaging. RESULTS: We found that in co-culture with endothelial cells, the VA10 cells generated bronchioalveolar like structures, suggesting that lung epithelial branching is facilitated by the presence of endothelial cells. The VA10 derived epithelial structures display various complex patterns of branching and show partial alveolar type-II differentiation with pro-Surfactant-C expression. The epithelial origin of the branching VA10 colonies was confirmed by immunostaining. These bronchioalveolar-like structures were polarized with respect to integrin expression at the cell-matrix interface. The endothelial-induced branching was mediated by soluble factors. Furthermore, fibroblast growth factor receptor-2 (FGFR-2) and sprouty-2 were expressed at the growing tips of the branching structures and the branching was inhibited by the FGFR-small molecule inhibitor SU5402. DISCUSSION: In this study we show that a human lung epithelial cell line can be induced by endothelial cells to form branching bronchioalveolar-like structures in 3-D culture. This novel model of human airway morphogenesis can be used to study critical events in human lung development and suggests a supportive role for the endothelium in promoting branching of airway epithelium.

Incidence, Etiology, and Outcomes of Community-Acquired Pneumonia: A Population-Based Study.

BACKGROUND: The microbial etiology of community-acquired pneumonia (CAP) is often unclear in clinical practice, and previous studies have produced variable results. Population-based studies examining etiology and incidence are lacking. This study examined the incidence and etiology of CAP requiring hospitalization in a population-based cohort as well as risk factors and outcomes for specific etiologies. METHODS: Consecutive admissions due to CAP in Reykjavik, Iceland were studied. Etiologic testing was performed with cultures, urine-antigen detection, and polymerase chain reaction analysis of airway samples. Outcomes were length of stay, intensive care unit admission, assisted ventilation, and mortality. RESULTS: The inclusion rate was 95%. The incidence of CAP requiring hospitalization was 20.6 cases per 10000 adults/year. A potential pathogen was detected in 52% (164 of 310) of admissions and in 74% (43 of 58) with complete sample sets. Streptococcuspneumoniae was the most common pathogen (61 of 310, 20%; incidence: 4.1/10000). Viruses were identified in 15% (47 of 310; incidence: 3.1/10000), Mycoplasmapneumoniae were identified in 12% (36 of 310; incidence: 2.4/10000), and multiple pathogens were identified in 10% (30 of 310; incidence: 2.0/10000). Recent antimicrobial therapy was associated with increased detection of M pneumoniae (P < .001), whereas a lack of recent antimicrobial therapy was associated with increased detection of S pneumoniae (P = .02). Symptoms and outcomes were similar irrespective of microbial etiology. CONCLUSIONS: Pneumococci, M pneumoniae, and viruses are the most common pathogens associated with CAP requiring hospital admission, and they all have a similar incidence that increases with age. Symptoms do not correlate with specific agents, and outcomes are similar irrespective of pathogens identified.

Differentiation potential of a basal epithelial cell line established from human bronchial explant.

Due to the cellular complexity of the airway epithelium, it is important to carefully define bronchial cell lines that capture the phenotypic traits of a particular cell type. We describe the characterization of a human bronchial epithelial cell line, VA10. It was established by transfection of primary bronchial epithelial cells with retroviral constructs containing the E6 and E7 oncogenes from HPV16. The cell line has been cultured for over 2 yr, a total of 60 passages. Although prolonged culture resulted in increased chromosomal instability, no major phenotypic drift in marker expression was observed. The cells expressed cytokeratins 5, 13, 14, and 17 suggesting a basal-like phenotype. This was further supported by the expression of alpha6beta4 integrins and the basal cell-associated transcription factor p63. The VA10 cell line generated high transepithelial electrical resistance in suspended and air-liquid interface culture, indicating functionally active tight junction (TJ) complexes. Immunocytochemistry showed the typical reticular structures of occludin and TJ-associated F-actin. VA10 produced pseudostratified layer in air-liquid interface culture with expression of p63 restricted to the basal layer. Furthermore, VA10 produced round colonies when cultured in laminin-rich reconstituted basement membrane, and immunostaining of claudin-1 and the basolateral marker beta4 integrin revealed colonies that generated polarization as expected in vivo. These data indicate that VA10 epithelia have the potential to model the bronchial epithelium in vivo and may be useful to study epithelial regeneration and repair and the effect of chemicals and potential drug candidates on TJ molecules in airway epithelia.

Mortality in healthcare-associated pneumonia in a low resistance setting: a prospective observational study.

BACKGROUND: The classification of pneumonia as community-acquired pneumonia (CAP) or healthcare-associated pneumonia (HCAP) has implications for selection of initial antimicrobial therapy. HCAP has been associated with an increased prevalence of multidrug-resistant (MDR) pathogens and with high mortality leading to recommendations for broad empiric therapy. METHODS: We performed a prospective, population-based study on consecutive adults (≥ 18 years) admitted for pneumonia over 1 calendar year. Patients were classified by pneumonia type and severity. Microbial etiologic testing was performed on all patients. Treatment, length of stay, and mortality rates were compared. RESULTS: A total of 373 admissions were included, 94% of all eligible patients. They were classified as CAP (n = 236, 63%) or HCAP (n = 137, 37%). Chronic underlying disease was more commonly found among patients with HCAP compared with CAP (74% vs 51%, p < 0.001). Mycoplasma pneumoniae was more common among CAP patients (p < 0.01), while gram-negative bacteria were more often found among HCAP patients (p = 0.02). No MDR pathogens were detected, and rates of Staphylococcus aureus were similar in the two groups. HCAP patients were not more likely to receive ineffective initial antimicrobial therapy. HCAP patients had worse prognostic scores on admission and higher in-house mortality than CAP patients (10% vs 1%, respectively, p < 0.01). CONCLUSIONS: Even in a low resistance setting, patients with HCAP have increased mortality compared with patients with CAP. This is most likely explained by a higher prevalence of co-morbidities. Our data do not support broad-spectrum empiric antibiotic therapy for HCAP.

Curcumin, bisdemethoxycurcumin and dimethoxycurcumin complexed with cyclodextrins have structure specific effect on the paracellular integrity of lung epithelia in vitro.

The phytochemical curcumin may improve translocation of the cystic fibrosis transmembrane regulatory (CFTR) protein in lung epithelium and therefore be helpful in the treatment of cystic fibrosis (CF) symptoms. However, previous studies often use commercial curcumin that is a combination of curcumin, demethoxycurcumin and bisdemethoxycurcumin which could affect the investigated cells differently. In the present study, we investigated the potential difference between curcumin, bisdemethoxycurcumin and dimethoxycurcumin on the epithelial tight junction complex, in the bronchial epithelial cell line VA10, by measuring transepithelial electrical resistance (TER), immunofluorescence and western blotting of tight junction proteins. The curcuminoids were complexed with hydroxypropyl-γ-cyclodextrin for increased solubility and stability. Curcumin (10 µg/ml) increased the TER significantly after 24 h of treatment while four times higher concentration of bisdemethoxycurcumin was required to obtain similar increase in TER as curcumin. Interestingly, dimethoxycurcumin did not increase TER. Curcumin clearly affected the F-actin structures both apically and basolaterally. These results begin to define possible effects of curcuminoids on healthy bronchial epithelia and shows that difference in the phenyl moiety structure of the curcuminoids influences the paracellular epithelial integrity.

Challenges in evaluation of chitosan and trimethylated chitosan (TMC) as mucosal permeation enhancers: From synthesis to in vitro application.

The polysaccharide chitosan and the water soluble chitosan derivative N,N,N-trimethyl chitosan (TMC) have been widely investigated as permeation enhancers of mucosal surfaces with numerous papers published over the last two decades. Although both chitosan and TMC increase permeation of markers through mucosal membranes, such as the intestinal and airway epithelium as well as in in vivo models, these investigations have not led to their use in marketed drug formulations. In this review, the reported extent of the permeation enhancement and cell viability after chitosan or TMC treatment in intestinal and airway models is critically evaluated and concluded that the apparent discrepancies can be explained by differences in polymer structure, experimental conditions and in vitro models. Additionally, aspects regarding the synthesis of TMC and its structural characterization are described, focusing on new synthetic strategies implemented to reduce O-methylation. Finally recommendations are provided on how studies can be conducted to improve understanding of the structure­activity relationship and elucidate possible mechanism of action.

N-alkylation of highly quaternized chitosan derivatives affects the paracellular permeation enhancement in bronchial epithelia in vitro.

This study describes the structure-activity relationship for carefully characterized N-alkyl-N-quaternary chitosan derivatives as permeation enhancers for drugs that are mainly absorbed through the paracellular pathway, such as macromolecular drugs and hydrophilic drugs, in a well defined bronchial epithelial cell line. The O-methyl free derivatives used in the study were fully trimethylated (100%) N,N,N-trimethyl chitosan (TMC) and N-propyl-(QuatPropyl), N-butyl-(QuatButyl) and N-hexyl (QuatHexyl)-N,N-dimethyl chitosan, with 85-91% degree of quaternization. The fully trimethylated TMC, from 0.25mg/ml, decreased transepithelial electrical resistance (TER) in a reversible manner and enhanced the permeation of the macromolecule FITC-dextran 4kDa (FD4) 2-5 fold. TMC did not cause any alterations in the tight junction (TJ) protein claudin-4 or in F-actin architecture. QuatHexyl was the most effective polymer to produce enhanced permeation and decreased TER from 0.016mg/ml. Nevertheless, this enhanced permeation was accompanied by reduced viability and dissociation of F-actin and claudin-4 proteins. The structure-activity relationship suggests that more lipophilic derivatives show more permeation enhancement, TJ disassembly, and less viability in the order of hexyl≈butyl>propyl>methyl and demonstrates that the permeation effect is not only mediated by permanent positive charge but also by the extent of N-alkylation. These results are relevant to elucidate the structural factors contributing to the permeation enhancement of chitosan derivatives and for potential use in pulmonary applications.

deltaNp63 has a role in maintaining epithelial integrity in airway epithelium.

The upper airways are lined with a pseudostratified bronchial epithelium that forms a barrier against unwanted substances in breathing air. The transcription factor p63, which is important for stratification of skin epithelium, has been shown to be expressed in basal cells of the lungs and its ΔN isoform is recognized as a key player in squamous cell lung cancer. However, the role of p63 in formation and maintenance of bronchial epithelia is largely unknown. The objective of the current study was to determine the expression pattern of the ΔN and TA isoforms of p63 and the role of p63 in the development and maintenance of pseudostratified lung epithelium in situ and in culture. We used a human bronchial epithelial cell line with basal cell characteristics (VA10) to model bronchial epithelium in an air-liquid interface culture (ALI) and performed a lentiviral-based silencing of p63 to characterize the functional and phenotypic consequences of p63 loss. We demonstrate that ΔNp63 is the major isoform in the human lung and its expression was exclusively found in the basal cells lining the basement membrane of the bronchial epithelium. Knockdown of p63 affected proliferation and migration of VA10 cells and facilitated cellular senescence. Expression of p63 is critical for epithelial repair as demonstrated by wound healing assays. Importantly, generation of pseudostratified VA10 epithelium in the ALI setup depended on p63 expression and goblet cell differentiation, which can be induced by IL-13 stimulation, was abolished by the p63 knockdown. After knockdown of p63 in primary bronchial epithelial cells they did not proliferate and showed marked senescence. We conclude that these results strongly implicate p63 in the formation and maintenance of differentiated pseudostratified bronchial epithelium.

Regioselective fluorescent labeling of N,N,N-trimethyl chitosan via oxime formation.

Fluorescent labeling of chitosan and its derivatives is widely used for in vitro visualization and is accomplished by random introduction of the fluorophore to the polymer backbone, conceivably altering the bioactivity of the polymer. Here, we report for the first time the regioselective conjugation of a fluorophore to the reducing end of a fully N,N,N-trimethylated chitosan (TMC) by oxime formation. End-labeled conjugation of 5-(2-((aminooxyacetyl)amino)ethylamino)naphthalene-1-sulfonic acid (EDANS-O-NH(2)) fluorophore to TMC to form TMC-oxime-EDANS (f-TMC) was confirmed by (1)H NMR and fluorescence spectroscopy. Average molecular weight calculations of f-TMC with (1)H NMR and fluorescence spectroscopy gave similar results or ∼7.7kDa. f-TMC in human bronchial epithelial cells was both cell membrane bound as well as intracellularly localized. This demonstrates the proof-of-concept for selective oxime formation at the reducing end of a chitosan derivative, which can be used for tracking chitosan in gene and drug delivery purposes and gives rise to further modifications with other functional groups.

Severity of influenza A 2009 (H1N1) pneumonia is underestimated by routine prediction rules. Results from a prospective, population-based study.

BACKGROUND: Characteristics of patients with community-acquired pneumonia (CAP) due to pandemic influenza A 2009 (H1N1) have been inadequately compared to CAP caused by other respiratory pathogens. The performance of prediction rules for CAP during an epidemic with a new infectious agent are unknown. METHODS: Prospective, population-based study from November 2008-November 2009, in centers representing 70% of hospital beds in Iceland. Patients admitted with CAP underwent evaluation and etiologic testing, including polymerase chain reaction (PCR) for influenza. Data on influenza-like illness in the community and overall hospital admissions were collected. Clinical and laboratory data, including pneumonia severity index (PSI) and CURB-65 of patients with CAP due to H1N1 were compared to those caused by other agents. RESULTS: Of 338 consecutive and eligible patients 313 (93%) were enrolled. During the pandemic peak, influenza A 2009 (H1N1) patients constituted 38% of admissions due to CAP. These patients were younger, more dyspnoeic and more frequently reported hemoptysis. They had significantly lower severity scores than other patients with CAP (1.23 vs. 1.61, P= .02 for CURB-65, 2.05 vs. 2.87 for PSI, P<.001) and were more likely to require intensive care admission (41% vs. 5%, P<.001) and receive mechanical ventilation (14% vs. 2%, P= .01). Bacterial co-infection was detected in 23% of influenza A 2009 (H1N1) patients with CAP. CONCLUSIONS: Clinical characteristics of CAP caused by influenza A 2009 (H1N1) differ markedly from CAP caused by other etiologic agents. Commonly used CAP prediction rules often failed to predict admissions to intensive care or need for assisted ventilation in CAP caused by the influenza A 2009 (H1N1) virus, underscoring the importance of clinical acumen under these circumstances.