Coordinated ciliary beating requires Odf2-mediated polarization of basal bodies via basal feet.

Coordinated beating of cilia in the trachea generates a directional flow of mucus required to clear the airways. Each cilium originates from a barrel-shaped basal body, from the side of which protrudes a structure known as the basal foot. We generated mice in which exons 6 and 7 of Odf2, encoding a basal body and centrosome-associated protein Odf2/cenexin, are disrupted. Although Odf2(ΔEx6,7/ΔEx6,7) mice form cilia, ciliary beating is uncoordinated, and the mice display a coughing/sneezing phenotype. Whereas residual expression of the C-terminal region of Odf2 in these mice is sufficient for ciliogenesis, the resulting basal bodies lack basal feet. Loss of basal feet in ciliated epithelia disrupted the polarized organization of apical microtubule lattice without affecting planar cell polarity. The requirement for Odf2 in basal foot formation, therefore, reveals a crucial role of this structure in the polarized alignment of basal bodies and coordinated ciliary beating.

CFAP53 regulates mammalian cilia-type motility patterns through differential localization and recruitment of axonemal dynein components.

Motile cilia can beat with distinct patterns, but how motility variations are regulated remain obscure. Here, we have studied the role of the coiled-coil protein CFAP53 in the motility of different cilia-types in the mouse. While node (9+0) cilia of Cfap53 mutants were immotile, tracheal and ependymal (9+2) cilia retained motility, albeit with an altered beat pattern. In node cilia, CFAP53 mainly localized at the base (centriolar satellites), whereas it was also present along the entire axoneme in tracheal cilia. CFAP53 associated tightly with microtubules and interacted with axonemal dyneins and TTC25, a dynein docking complex component. TTC25 and outer dynein arms (ODAs) were lost from node cilia, but were largely maintained in tracheal cilia of Cfap53-/- mice. Thus, CFAP53 at the base of node cilia facilitates axonemal transport of TTC25 and dyneins, while axonemal CFAP53 in 9+2 cilia stabilizes dynein binding to microtubules. Our study establishes how differential localization and function of CFAP53 contributes to the unique motion patterns of two important mammalian cilia-types.

Histopathology and ultrastructural findings of fatal COVID-19 infections in Washington State: a case series.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of an ongoing pandemic, with increasing deaths worldwide. To date, documentation of the histopathological features in fatal cases of the disease caused by SARS-CoV-2 (COVID-19) has been scarce due to sparse autopsy performance and incomplete organ sampling. We aimed to provide a clinicopathological report of severe COVID-19 cases by documenting histopathological changes and evidence of SARS-CoV-2 tissue tropism. METHODS: In this case series, patients with a positive antemortem or post-mortem SARS-CoV-2 result were considered eligible for enrolment. Post-mortem examinations were done on 14 people who died with COVID-19 at the King County Medical Examiner's Office (Seattle, WA, USA) and Snohomish County Medical Examiner's Office (Everett, WA, USA) in negative-pressure isolation suites during February and March, 2020. Clinical and laboratory data were reviewed. Tissue examination was done by light microscopy, immunohistochemistry, electron microscopy, and quantitative RT-PCR. FINDINGS: The median age of our cohort was 73·5 years (range 42-84; IQR 67·5-77·25). All patients had clinically significant comorbidities, the most common being hypertension, chronic kidney disease, obstructive sleep apnoea, and metabolic disease including diabetes and obesity. The major pulmonary finding was diffuse alveolar damage in the acute or organising phases, with five patients showing focal pulmonary microthrombi. Coronavirus-like particles were detected in the respiratory system, kidney, and gastrointestinal tract. Lymphocytic myocarditis was observed in one patient with viral RNA detected in the tissue. INTERPRETATION: The primary pathology observed in our cohort was diffuse alveolar damage, with virus located in the pneumocytes and tracheal epithelium. Microthrombi, where observed, were scarce and endotheliitis was not identified. Although other non-pulmonary organs showed susceptibility to infection, their contribution to the pathogenesis of SARS-CoV-2 infection requires further examination. FUNDING: None.

Ultra- and micro-structural changes of respiratory tracts in SARS-CoV-2 infected Syrian hamsters.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is causing a global crisis. It is still unresolved. Although many therapies and vaccines are being studied, they are still in their infancy. As this pandemic continues, rapid and accurate research for the development of therapies and vaccines is needed. Therefore, it is necessary to understand characteristics of diseases caused by SARS-CoV-2 through animal models. Syrian hamsters are known to be susceptible to SARS-CoV-2. They were intranasally inoculated with SARS-CoV-2. At 2, 4, 8, 12, and 16 days post-infection (dpi), these hamsters were euthanized, and tissues were collected for ultrastructural and microstructural examinations. Microscopic lesions were prominent in the upper and lower respiratory tracts from 2 and 4 dpi groups, respectively. The respiratory epithelium in the trachea, bronchiole, and alveolar showed pathological changes. Inflammatory cells including neutrophils, lymphocytes, macrophages, and eosinophils were infiltrated in/around tracheal lamina propria, pulmonary vessels, alveoli, and bronchiole. In pulmonary lesions, alveolar wall was thickened with infiltrated inflammatory cells, mainly neutrophils and macrophages. In the trachea, epithelial damages started from 2 dpi and recovered from 8 dpi, consistent with microscopic results, High levels of SARS-CoV-2 nucleoprotein were detected at 2 dpi and 4 dpi. In the lung, lesions were most severe at 8 dpi. Meanwhile, high levels of SARS-CoV-2 were detected at 4 dpi. Electron microscopic examinations revealed cellular changes in the trachea epithelium and alveolar epithelium such as vacuolation, sparse micro-organelle, and poor cellular margin. In the trachea epithelium, the number of cytoplasmic organelles was diminished, and small vesicles were prominent from 2 dpi. Some of these electron-lucent vesicles were filled with virion particles. From 8 dpi, the trachea epithelium started to recover. Because of shrunken nucleus and swollen cytoplasm, the N/C ratio of type 2 pneumocyte decreased at 8 and 12 dpi. From 8 dpi, lamellar bodies on type 2 pneumocyte cytoplasm were increasingly observed. Their number then decreased from 16 dpi. However, there was no significant change in type 1 pneumocyte. Viral vesicles were only observed in the cytoplasm of type 2 pneumocyte. In conclusion, ultra- and micro-structural changes presented in this study may provide useful information for SARS-CoV-2 studies in various fields.

Scanning ion conductance microscopy for visualizing the three-dimensional surface topography of cells and tissues.

Scanning ion conductance microscopy (SICM), which belongs to the family of scanning probe microscopy, regulates the tip-sample distance by monitoring the ion current through the use of an electrolyte-filled nanopipette as the probing tip. Thus, SICM enables "contact-free" imaging of cell surface topography in liquid conditions. In this paper, we applied hopping mode SICM for obtaining topographical images of convoluted tissue samples such as trachea and kidney in phosphate buffered saline. Some of the SICM images were compared with the images obtained by scanning electron microscopy (SEM) after drying the same samples. We showed that the imaging quality of hopping mode SICM was excellent enough for investigating the three-dimensional surface structure of the soft tissue samples. Thus, SICM is expected to be used for imaging a wide variety of cells and tissues - either fixed or alive- at high resolution under physiologically relevant liquid conditions.

Comparative Morphological Features of Syrinx in Male Domestic Fowl Gallus gallus domesticus and Male Domestic Pigeon Columba livia domestica: A Histochemical, Ultrastructural, Scanning Electron Microscopic and Morphometrical Study.

Many studies have been carried out to investigate the morphological structure of the syrinx in many bird species. However, the cellular organization of the syrinx in the fowls and pigeons is still unclear. The current study revealed that in fowl and pigeon, the syrinx is formed of three main parts including tympanum (cranial) part, intermediate syringeal part, and bronchosyringeal (caudal) part, in addition to pessulus and tympaniform membranes. A great variation in the structural characteristics of syrinx of fowl and pigeon was recorded. In fowl, the tympaniform membranes showed a characteristic distribution of elastic and collagen fibers which increase the elasticity of tympaniform membranes. Moreover, the bony pessulus helps the medial tympaniform membranes to be stiffer, vibrate more strongly so that louder sound will be generated. In pigeon, the lateral tympaniform membrane is of greater thickness so that the oscillation of this membrane is reduced and the amplitude is lower. Moreover, the pessulus is smaller in size and is formed mainly of connective tissue core (devoid of cartilaginous or bony plates), resulting in the failure of stretching and vibrating of the medial tympaniform membranes, that leads to the generation of deeper sound. Electron microscopic examination of the syringes of fowls and pigeons revealed numerous immune cells including dendritic cells, plasma cells, mast cells, and lymphocytes distributed within syringeal mucosa and invading the syringeal epithelium. Telocytes were first recorded in the syrinx of fowls and pigeons in this study. They presented two long telopodes that made up frequent close contacts with other neighboring telocytes, immune cells, and blood capillaries.

Early studies of airway submucosal glands.

This historical article provides a comprehensive review of early research on the structure and function of airway submucosal glands. The literature before 1950 or so, is virtually unknown, but in addition to being of historical interest it contains much of relevance to current research. Airway glands were first mentioned in 1602. The first description of their general form, size, and distribution was in 1712. Gland morphology was determined in 1827 by injecting mercury into their openings. Wax was later used. Detailed comparative information for all regions of the tracheobronchial tree was provided by Frankenhauser in 1879 (Untersuchungen uber den bau der Tracheo-Bronchial-Schleimhaut). Histological studies began in 1870, and by the end of the 19th century, all the major histological features had been described. The first physiological studies on airway mucous secretion were published in 1892. Kokin, in 1896 (Archiv für die gesamte Physiologie des Menschen und der Tiere 63: 622-630), was the first to measure secretion from individual glands. It was not, however, until 1933 that gland secretion was quantified. This early literature raises important questions as to the role of the collecting duct epithelium in modifying primary secretions. It also provides perhaps the most accurate measure of basal gland secretion in vivo.

PLGA-PTMC-Cultured Bone Mesenchymal Stem Cell Scaffold Enhances Cartilage Regeneration in Tissue-Engineered Tracheal Transplantation.

The treatment of long-segment tracheal defect requires the transplantation of effective tracheal substitute, and the tissue-engineered trachea (TET) has been proposed as an ideal tracheal substitute. The major cause of the failure of segmental tracheal defect reconstruction by TET is airway collapse caused by the chondromalacia of TET cartilage. The key to maintain the TET structure is the regeneration of chondrocytes in cartilage, which can secrete plenty of cartilage matrices. To address the problem of the chondromalacia of TET cartilage, this study proposed an improved strategy. We designed a new cell sheet scaffold using the poly(lactic-co-glycolic acid) (PLGA) and poly(trimethylene carbonate) (PTMC) to make a porous membrane for seeding cells, and used the PLGA-PTMC cell-scaffold to pack the decellularized allogeneic trachea to construct a new type of TET. The TET was then implanted in the subcutaneous tissue for vascularization for 2 weeks. Orthotopic transplantation was then performed after implantation. The efficiency of the TET we designed was analyzed by histological examination and biomechanical analyses 4 weeks after surgery. Four weeks after surgery, both the number of chondrocytes and the amount of cartilage matrix were significantly higher than those contained in the traditional stem-cell-based TET. Besides, the coefficient of stiffness of TET was significantly larger than the traditional TET. This study provided a promising approach for the long-term functional reconstruction of long-segment tracheal defect, and the TET we designed had potential application prospects in the field of TET reconstruction.

Ultrastructure of human tracheal smooth muscle from subjects with asthma and nonasthmatic subjects. Standardized methods for comparison.

A characteristic feature of asthma is exaggerated airway narrowing, termed airway hyper-responsiveness (AHR) due to contraction of airway smooth muscle (ASM). Although smooth muscle (SM)-specific asthma susceptibility genes have been identified, it is not known whether asthmatic ASM is phenotypically different from nonasthmatic ASM in terms of subcellular structure or mechanical function. The present study is the first to systematically quantify, using electron microscopy, the ultrastructure of tracheal SM from subjects with asthma and nonasthmatic subjects. Methodological details concerning tissue sample preparation, ultrastructural quantification, and normalization of isometric force by appropriate morphometric parameters are described. We reasoned that genetic and/or acquired differences in the ultrastructure of asthmatic ASM could be associated with functional changes. We recently reported that asthmatic ASM is better able to maintain and recover active force generation after length oscillations simulating deep inspirations. The present study was designed to seek structural evidence to account for this observation. Contrary to our hypotheses, no significant qualitative or quantitative differences were found in the subcellular structure of asthmatic versus nonasthmatic tracheal SM. Specifically, there were no differences in average SM cell cross-sectional area; fraction of the cell area occupied by nonfilamentous area; amounts of mitochondria, dense bodies, and dense plaques; myosin and actin filament densities; basal lamina thickness; and the number of microtubules. These results indicate that functional differences in ASM do not necessarily translate into observable structural changes.

The first tissue-engineered airway transplantation: 5-year follow-up results.

BACKGROUND: In 2008, the first transplantation of a tissue-engineered trachea in a human being was done to replace an end-staged left main bronchus with malacia in a 30-year-old woman. We report 5 year follow-up results. METHODS: The patient was followed up approximately every 3 months with multidetector CT scan and bronchoscopic assessment. We obtained mucosal biopsy samples every 6 months for histological, immunohistochemical, and electron microscopy assessment. We also assessed quality of life, respiratory function, cough reflex test, and production and specificity of recipient antibodies against donor human leucocyte antigen. FINDINGS: By 12 months after transplantation, a progressive cicatricial stenosis had developed in the native trachea close to the tissue-engineered trachea anastomosis, which needed repeated endoluminal stenting. However, the tissue-engineered trachea itself remained open over its entire length, well vascularised, completely re-cellularised with respiratory epithelium, and had normal ciliary function and mucus clearance. Lung function and cough reflex were normal. No stem-cell-related teratoma formed and no anti-donor antibodies developed. Aside from intermittent bronchoscopic interventions, the patient had a normal social and working life. INTERPRETATION: These clinical results provide evidence that a tissue-engineering strategy including decellularisation of a human trachea, autologous epithelial and stem-cell culture and differentiation, and cell-scaffold seeding with a bioreactor is safe and promising. FUNDING: European Commission, Knut and Alice Wallenberg Foundation, Swedish Research Council, ALF Medicine.

GBF1 (Gartenzwerg)-dependent secretion is required for Drosophila tubulogenesis.

Here we report on the generation and in vivo analysis of a series of loss-of-function mutants for the Drosophila ArfGEF, Gartenzwerg. The Drosophila gene gartenzwerg (garz) encodes the orthologue of mammalian GBF1. garz is expressed ubiquitously in embryos with substantially higher abundance in cells forming diverse tubular structures such as salivary glands, trachea, proventriculus or hindgut. In the absence of functional Garz protein, the integrity of the Golgi complex is impaired. As a result, both vesicle transport of cargo proteins and directed apical membrane delivery are severely disrupted. Dysfunction of the Arf1-COPI machinery caused by a loss of Garz leads to perturbations in establishing a polarized epithelial architecture of tubular organs. Furthermore, insufficient apical transport of proteins and other membrane components causes incomplete luminal diameter expansion and deficiencies in extracellular matrix assembly. The fact that homologues of Garz are present in every annotated metazoan genome indicates that secretion processes mediated by the GBF-type ArfGEFs play a universal role in animal development.

Distribution of ciliated epithelial cells in the trachea of common marmosets (Callithrix jacchus).

BACKGROUND: The distribution of ciliated cells in the tracheal epithelium of common marmosets was evaluated. METHODS: Light and scanning electron microscopy of tracheal epithelium was performed. RESULTS: Ciliated cells were concentrated in cartilage-free areas and virtually absent in cartilage-supported epithelial regions. CONCLUSIONS: Heterogeneous distribution of ciliated cells in the trachea has to be considered when using animal models for translational respiratory research approaches.

Tracheole investment does not vary with body size among bumblebee (Bombus impatiens) sisters.

Body size is a key organism trait with critical implications for the physiology, life history, and ecology of organisms. Modern insects vary in body mass by over 6 orders of magnitude, but are small by comparison to many other metazoan taxa. The small size of modern insects may reflect limitations imposed by their open respiratory systems which rely, in part, on diffusion. Diffusion rates decline with distance such that, absent compensation, the capacity for larger insects to deliver oxygen to their tissues may be compromised. To compensate, larger grasshoppers, beetles, and bumblebees devote proportionally more of their body volume to the respiratory system, as demonstrated by hypermetric scaling of tracheal volume with body mass(>1). Among bumblebee sisters, total respiratory volume scaled with mass(2.6), but it is unclear at what level or levels of the tracheal system (main tracheal trunks, air sacs, tracheoles) bumblebees express this extreme hypermetry. Here we use transmission electron microscopy to examine the morphology of tracheoles in bumblebee flight muscle among sister bumblebees varying nearly four-fold in body mass. Neither tracheole density nor tracheole diameter changed with body mass. The total cross-sectional area of tracheoles was also invariant with body mass. Together, these results reveal that bumblebees do not compensate for size-related limitations on oxygen delivery by increasing investment at the level of the tracheoles.

Synergism of ochratoxin B and calcium-channel antagonist verapamil caused mitochondrial dysfunction.

We examined the mechanism by which the ochratoxin B induced interaction with calcium-channel antagonist verapamil and mitochondrial dysfunction of the rat trachea in vitro experiment. The tracheas were cut into 2-3 mm wide rings and suspended in a tissue bath. Isometric tension was continuously measured with an isometric force transducer connected to a computer-based data acquisition system. Verapamil (1 × 10(-6) M) produced a concentration-dependent contraction response in rat's tracheal rings pre-contracted by acetylcholine. Incubation of rat's tracheal rings with the ochratoxin B significantly potentiated the contraction responses of verapamil. Verapamil and OTB accelerate the overloading of Ca(2+) in tracheal smooth muscle contributes the tissue toxicity as shown in electron microscopy and mitochondrial enzymes, through a mechanism that could involve perturbations of Ca(2+) homeostasis. These results proved that ochratoxin B is a potential vasoconstrictor mycotoxin with the presence of calcium-channel antagonist. In conclusion, disturbance of Ca(2+) homeostasis caused by OTA and plays a significant role in produces toxicity through mitochondrial enzyme inhibition.

Correlative ultrastructural investigations of airway epithelium following experimental exposure to defined air pollutants and lifestyle exposure to tobacco smoke.

CONTEXT: Investigations of cell/molecular level effects of in vivo exposure of airway mucosa of experimental animals to common irritant gases have demonstrated structural and physiological changes reflective of breaches in epithelial barrier function, presence of inflammatory cell infiltrate and compromised ciliary function. These experimental animal studies provided useful perspectives of plausible, but more subtle pathologic outcomes having relevance to lifestyle exposure to gaseous environmental irritants including tobacco smoke. METHODS: Freeze-fracture technology was applied to ultrastructural examination of large airway epithelium, with appropriate controls, from guinea pigs exposed to ozone and of nasal mucosa of human subjects exposed to ozone or sulfur dioxide, and nasal mucosa of active smokers. RESULTS: We documented substantive membrane structural changes to tight junctional complexes and cilia as well as an infiltrate of neutrophils into the surface mucosal layer in exposed animals. These patterns also were evident but not as pervasive among human subjects acutely exposed experimentally to irritant gases and those chronically exposed by their lifestyle to tobacco smoke. DISCUSSION: Our intent was to characterize respiratory tract mucosal membrane disorganization associated with high level acute irritant exposures in an experimental animal model and to evaluate evidence of similar but perhaps more subtle pathologic change associated with lower level experimental or lifestyle exposures. Our studies demonstrate continuity, albeit subtle, of pathologic change from high dosage experimental animal exposure to low dosage human exposures. CONCLUSIONS: This study represents the first report of ultrastructural airway epithelial membrane anomalies associated with lifestyle exposure to tobacco smoke irritants.

Comparative study on the pathogenesis of the generated 9a5b Newcastle disease virus mutant isolate between chickens and waterfowl.

Lentogenic Newcastle disease viruses (NDVs), circulating among waterfowl, have the potential to become highly pathogenic by replication in chickens. The pathological studies that compare NDV infections in chickens and waterfowl are rare. The virulent 9a5b mutant NDV isolate was generated by passaging the lentogenic Goose/Alaska/415/91 NDV isolate in chickens. The pathogenesis of the virulent 9a5b mutant isolate is unknown in both chickens and waterfowl. In this study, the virulent 9a5b mutant NDV isolate was inoculated intranasally in 32-day-old specific pathogen-free white Leghorn chickens and Japanese commercial ducks. Unlike ducks, which remained clinically normal throughout the study, chickens had depression, gasping, oral discharges, and greenish-white soft feces. Gross and histologic lesion patterns as well as viral replication supported the differing clinical outcome. Ducks had slight inflammation mainly in respiratory and digestive tracts, whereas slight nonpurulent encephalitis, necrotizing pancreatitis, tubulointerstitial nephritis, and mild inflammation in respiratory and digestive tracts were detected in chickens. In agreement, interferon-beta (IFN-ß)-immunopositive signals were more intense in lung tissue of ducks than that of chickens, and NDV replications were detected intensively in chicken tissues. These results suggest that the 9a5b mutant NDV isolate is more virulent in chickens, and slight histological lesions were induced in ducks even with virulent NDVs.

Decellularized tracheal scaffold as a promising 3D scaffold for tissue engineering applications.

Tissue engineering is a science that uses the combination of scaffolds, cells, and active biomolecules to make tissue in order to restore or maintain its function and improve the damaged tissue or even an organ in the laboratory. The purpose of this research was to study the characteristics and biocompatibility of decellularized sheep tracheal scaffolds and also to investigate the differentiation of Adipose-derived stem cells (AD-MSCs) into tracheal cells. After the decellularization of sheep tracheas through the detergent-enzyme method, histological evaluations, measurement of biochemical factors, measurement of DNA amount, and photographing the ultrastructure of the samples by scanning electron microscopy (SEM), they were also evaluated mechanically. Further, In order to check the viability and adhesion of stem cells to the decellularized scaffolds, adipose mesenchymal stem cells were cultured on the scaffolds, and the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay was performed. The expression analysis of the intended genes for the differentiation of mesenchymal stem cells into tracheal cells was evaluated by the real-time PCR method. These results show that the prepared scaffolds are an ideal model for engineering applications, have high biocompatibility, and that the tracheal scaffold provides a suitable environment for the differentiation of ADMSCs. This review provides a basis for future research on tracheal decellularization scaffolds, serves as a suitable model for organ regeneration, and paves the way for their use in clinical medicine.

Telocyte morphologies and potential roles in diseases.

Telocytes (TCs) are a new type of interstitial cells, a small cellular body with the presence of 2-5 prolongations named as telopode (Tp)-very thin (less than 0.2 µm) and extremely long (10-1,000 µm), a moniliform aspect, and caveolae, containing a nucleus surrounded by a small amount of cytoplasm. The nucleus occupies about 25% of TC body volume and contains clusters of heterochromatin attached to the nuclear envelope. The perinuclear cytoplasm is rich in mitochondria and contains a small Golgi complex, rough and smooth endoplasmic reticulum and cytoskeletal elements. TCs have several immunophenotypes such as CD34, c-kit, and vimentin. TCs were found in many organs of mammals with potential biological functions, even though the exact function remains unclear. Recently, we identified and isolated TCs from the trachea for the first time and confirmed the existence of TC in lung tissues, which could have the potential significance in the pathogenesis of pulmonary diseases. Future efforts are required to clarify pathophysiological functions of TCs in the disease.

Sequential pulses of apical epithelial secretion and endocytosis drive airway maturation in Drosophila.

The development of air-filled respiratory organs is crucial for survival at birth. We used a combination of live imaging and genetic analysis to dissect respiratory organ maturation in the embryonic Drosophila trachea. We found that tracheal tube maturation entails three precise epithelial transitions. Initially, a secretion burst deposits proteins into the lumen. Solid luminal material is then rapidly cleared from the tubes, and shortly thereafter liquid is removed. To elucidate the cellular mechanisms behind these transitions, we identified gas-filling-deficient mutants showing narrow or protein-clogged tubes. These mutations either disrupt endoplasmatic reticulum-to-Golgi vesicle transport or endocytosis. First, Sar1 is required for protein secretion, luminal matrix assembly, and diametric tube expansion. Subsequently, a sharp pulse of Rab5-dependent endocytic activity rapidly internalizes and clears luminal contents. The coordination of luminal matrix secretion and endocytosis may be a general mechanism in tubular organ morphogenesis and maturation.

Inhibitory influence of the hexapeptidic sequence SLIGRL on influenza A virus infection in mice.

Proteinase-activated receptor 2 (PAR(2)) is widely expressed in the respiratory tract and is an integral component of the host antimicrobial defense system. The principal aim of this study was to investigate the influence of a PAR(2)-activating peptide, SLIGRL, on influenza A virus (IAV)-induced pathogenesis in mice. Intranasal inoculation of BALB/c mice with influenza A/PR/8/34 virus caused time-dependent increases in the number of pulmonary leukocytes (recovered from bronchoalveolar lavage fluid), marked airway histopathology characterized by extensive epithelial cell damage, airway hyper-responsiveness to the bronchoconstrictor methacholine, and elevated levels of inflammatory chemokines (keratinocyte-derived chemokine and macrophage inflammatory protein 2) and cytokines (interferon-γ). It is noteworthy that these IAV-induced effects were dose-dependently attenuated in mice treated with a PAR(2)-activating peptide, SLIGRL, at the time of IAV inoculation. However, SLIGRL also inhibited IAV-induced increases in pulmonary leukocytes in PAR(2)-deficient mice, indicating these antiviral actions were not mediated by PAR(2). The potency order obtained for a series of structural analogs of SLIGRL for anti-IAV activity (IGRL > SLIGRL > LSIGRL >2-furoyl-LIGRL) was also inconsistent with a PAR(2)-mediated effect. In further mechanistic studies, SLIGRL inhibited IAV-induced propagation in ex vivo perfused segments of trachea from wild-type or PAR(2)(-/-) mice, but did not inhibit viral attachment or replication in Madin-Darby canine kidney cells and chorioallantoic membrane cells, which are established hosts for IAV. In summary, SLIGRL protected mice from IAV infection independently of PAR(2) and independently of direct inhibition of IAV attachment or replication, potentially through the activation of endogenous antiviral pathways within the mouse respiratory tract.