The effect of Dnaaf5 gene dosage on primary ciliary dyskinesia phenotypes.

DNAAF5 is a dynein motor assembly factor associated with the autosomal heterogenic recessive condition of motile cilia, primary ciliary dyskinesia (PCD). The effects of allele heterozygosity on motile cilia function are unknown. We used CRISPR-Cas9 genome editing in mice to recreate a human missense variant identified in patients with mild PCD and a second, frameshift-null deletion in Dnaaf5. Litters with Dnaaf5 heteroallelic variants showed distinct missense and null gene dosage effects. Homozygosity for the null Dnaaf5 alleles was embryonic lethal. Compound heterozygous animals with the missense and null alleles showed severe disease manifesting as hydrocephalus and early lethality. However, animals homozygous for the missense mutation had improved survival, with partially preserved cilia function and motor assembly observed by ultrastructure analysis. Notably, the same variant alleles exhibited divergent cilia function across different multiciliated tissues. Proteomic analysis of isolated airway cilia from mutant mice revealed reduction in some axonemal regulatory and structural proteins not previously reported in DNAAF5 variants. Transcriptional analysis of mouse and human mutant cells showed increased expression of genes coding for axonemal proteins. These findings suggest allele-specific and tissue-specific molecular requirements for cilia motor assembly that may affect disease phenotypes and clinical trajectory in motile ciliopathies.

The effect of Dnaaf5 gene dosage on primary ciliary dyskinesia phenotypes.

DNAAF5 is a dynein motor assembly factor associated with the autosomal heterogenic recessive condition of motile cilia, primary ciliary dyskinesia (PCD). The effects of allele heterozygosity on motile cilia function are unknown. We used CRISPR-Cas9 genome editing in mice to recreate a human missense variant identified in patients with mild PCD and a second, frameshift null deletion in Dnaaf5 . Litters with Dnaaf5 heteroallelic variants showed distinct missense and null gene dosage effects. Homozygosity for the null Dnaaf5 alleles was embryonic lethal. Compound heterozygous animals with the missense and null alleles showed severe disease manifesting as hydrocephalus and early lethality. However, animals homozygous for the missense mutation had improved survival, with partial preserved cilia function and motor assembly observed by ultrastructure analysis. Notably, the same variant alleles exhibited divergent cilia function across different multiciliated tissues. Proteomic analysis of isolated airway cilia from mutant mice revealed reduction in some axonemal regulatory and structural proteins not previously reported in DNAAF5 variants. While transcriptional analysis of mouse and human mutant cells showed increased expression of genes coding for axonemal proteins. Together, these findings suggest allele-specific and tissue-specific molecular requirements for cilia motor assembly that may affect disease phenotypes and clinical trajectory in motile ciliopathies. Brief Summary: A mouse model of human DNAAF5 primary ciliary dyskinesia variants reveals gene dosage effects of mutant alleles and tissue-specific molecular requirements for cilia motor assembly.

The Translational Landscape of SARS-CoV-2-infected Cells Reveals Suppression of Innate Immune Genes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes a number of strategies to modulate viral and host mRNA translation. Here, we used ribosome profiling in SARS-CoV-2-infected model cell lines and primary airway cells grown at an air-liquid interface to gain a deeper understanding of the translationally regulated events in response to virus replication. We found that SARS-CoV-2 mRNAs dominate the cellular mRNA pool but are not more efficiently translated than cellular mRNAs. SARS-CoV-2 utilized a highly efficient ribosomal frameshifting strategy despite notable accumulation of ribosomes within the slippery sequence on the frameshifting element. In a highly permissive cell line model, although SARS-CoV-2 infection induced the transcriptional upregulation of numerous chemokine, cytokine, and interferon-stimulated genes, many of these mRNAs were not translated efficiently. The impact of SARS-CoV-2 on host mRNA translation was more subtle in primary cells, with marked transcriptional and translational upregulation of inflammatory and innate immune responses and downregulation of processes involved in ciliated cell function. Together, these data reveal the key role of mRNA translation in SARS-CoV-2 replication and highlight unique mechanisms for therapeutic development. IMPORTANCE SARS-CoV-2 utilizes a number of strategies to modulate host responses to ensure efficient propagation. Here, we used ribosome profiling in SARS-CoV-2-infected cells to gain a deeper understanding of the translationally regulated events in infected cells. We found that although viral mRNAs are abundantly expressed, they are not more efficiently translated than cellular mRNAs. SARS-CoV-2 utilized a highly efficient ribosomal frameshifting strategy and alternative translation initiation sites that help increase the coding potential of its RNAs. In permissive cells, SARS-CoV-2 infection induced the translational repression of numerous innate immune mediators. Though the impact of SARS-CoV-2 on host mRNA translation was more subtle in primary airway cell cultures, we noted marked transcriptional and translational upregulation of inflammatory and innate immune responses and downregulation of processes involved in ciliated cell function. Together, these data provide new insight into how SARS-CoV-2 modulates innate host responses and highlight unique mechanisms for therapeutic intervention.

The translational landscape of SARS-CoV-2 and infected cells.

SARS-CoV-2 utilizes a number of strategies to modulate viral and host mRNA translation. Here, we used ribosome profiling in SARS-CoV-2 infected model cell lines and primary airway cells grown at the air-liquid interface to gain a deeper understanding of the translationally regulated events in response to virus replication. We find that SARS-CoV-2 mRNAs dominate the cellular mRNA pool but are not more efficiently translated than cellular mRNAs. SARS-CoV-2 utilized a highly efficient ribosomal frameshifting strategy in comparison to HIV-1, suggesting utilization of distinct structural elements. In the highly permissive cell models, although SARS-CoV-2 infection induced the transcriptional upregulation of numerous chemokines, cytokines and interferon stimulated genes, many of these mRNAs were not translated efficiently. Impact of SARS-CoV-2 on host mRNA translation was more subtle in primary cells, with marked transcriptional and translational upregulation of inflammatory and innate immune responses and downregulation of processes involved in ciliated cell function. Together, these data reveal the key role of mRNA translation in SARS-CoV-2 replication and highlight unique mechanisms for therapeutic development.

Chemokine Receptor 2-targeted Molecular Imaging in Pulmonary Fibrosis. A Clinical Trial.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a progressive inflammatory lung disease without effective molecular markers of disease activity or treatment responses. Monocyte and interstitial macrophages that express the C-C motif CCR2 (chemokine receptor 2) are active in IPF and central to fibrosis.Objectives: To phenotype patients with IPF for potential targeted therapy, we developed 64Cu-DOTA-ECL1i, a radiotracer to noninvasively track CCR2+ monocytes and macrophages using positron emission tomography (PET).Methods: CCR2+ cells were investigated in mice with bleomycin- or radiation-induced fibrosis and in human subjects with IPF. The CCR2+ cell populations were localized relative to fibrotic regions in lung tissue and characterized using immunolocalization, single-cell mass cytometry, and Ccr2 RNA in situ hybridization and then correlated with parallel quantitation of lung uptake by 64Cu-DOTA-ECL1i PET.Measurements and Main Results: Mouse models established that increased 64Cu-DOTA-ECL1i PET uptake in the lung correlates with CCR2+ cell infiltration associated with fibrosis (n = 72). As therapeutic models, the inhibition of fibrosis by IL-1ß blockade (n = 19) or antifibrotic pirfenidone (n = 18) reduced CCR2+ macrophage accumulation and uptake of the radiotracer in mouse lungs. In lung tissues from patients with IPF, CCR2+ cells concentrated in perifibrotic regions and correlated with radiotracer localization (n = 21). Human imaging revealed little lung uptake in healthy volunteers (n = 7), whereas subjects with IPF (n = 4) exhibited intensive signals in fibrotic zones.Conclusions: These findings support a role for imaging CCR2+ cells within the fibrogenic niche in IPF to provide a molecular target for personalized therapy and monitoring.Clinical trial registered with www.clinicaltrials.gov (NCT03492762).

Quantification of image texture in X-ray phase-contrast-enhanced projection images of in vivo mouse lungs observed at varied inflation pressures.

To date, there are very limited noninvasive, regional assays of in vivo lung microstructure near the alveolar level. It has been suggested that x-ray phase-contrast enhanced imaging reveals information about the air volume of the lung; however, the image texture information in these images remains underutilized. Projection images of in vivo mouse lungs were acquired via a tabletop, propagation-based, X-ray phase-contrast imaging system. Anesthetized mice were mechanically ventilated in an upright position. Consistent with previously published studies, a distinct image texture was observed uniquely within lung regions. Lung regions were automatically identified using supervised machine learning applied to summary measures of the image texture data. It was found that an unsupervised clustering within predefined lung regions colocates with expected differences in anatomy along the cranial-caudal axis in upright mice. It was also found that specifically selected inflation pressures-here, a purposeful surrogate of distinct states of mechanical expansion-can be predicted from the lung image texture alone, that the prediction model itself varies from apex to base and that prediction is accurate regardless of overlap with nonpulmonary structures such as the ribs, mediastinum, and heart. Cross-validation analysis indicated low inter-animal variation in the image texture classifications. Together, these results suggest that the image texture observed in a single X-ray phase-contrast-enhanced projection image could be used across a range of pressure states to study regional variations in regional lung function.

Development of Fully Degradable Phosphonium-Functionalized Amphiphilic Diblock Copolymers for Nucleic Acids Delivery.

To expand the range of functional polymer materials to include fully hydrolytically degradable systems that bear bioinspired phosphorus-containing linkages both along the backbone and as cationic side chain moieties for packaging and delivery of nucleic acids, phosphonium-functionalized polyphosphoester- block-poly(l-lactide) copolymers of various compositions were synthesized, fully characterized, and their self-assembly into nanoparticles were studied. First, an alkyne-functionalized polyphosphoester- block-poly(l-lactide) copolymer was synthesized via a one pot sequential ring opening polymerization of an alkyne-functionalized phospholane monomer, followed by the addition of l-lactide to grow the second block. Second, the alkynyl side groups of the polyphosphoester block were functionalized via photoinitiated thiol-yne radical addition of a phosphonium-functionalized free thiol. The polymers of varying phosphonium substitution degrees were self-assembled in aqueous buffers to afford formation of well-defined core-shell assemblies with an average size ranging between 30 and 50 nm, as determined by dynamic light scattering. Intracellular delivery of the nanoparticles and their effects on cell viability and capability at enhancing transfection efficiency of nucleic acids (e.g., siRNA) were investigated. Cell viability assays demonstrated limited toxicity of the assembly to RAW 264.7 mouse macrophages, except at high polymer concentrations, where the polymer of high degree of phosphonium functionalization induced relatively higher cytotoxicity. Transfection efficiency was strongly affected by the phosphonium-to-phosphate (P+/P-) ratios of the polymers and siRNA, respectively. The AllStars Hs Cell Death siRNA complexed to the various copolymers at a P+/P- ratio of 10:1 induced comparable cell death to Lipofectamine. These fully degradable nanoparticles might provide biocompatible nanocarriers for therapeutic nucleic acid delivery.

Establishment of the early cilia preassembly protein complex during motile ciliogenesis.

Motile cilia are characterized by dynein motor units, which preassemble in the cytoplasm before trafficking into the cilia. Proteins required for dynein preassembly were discovered by finding human mutations that result in absent ciliary motors, but little is known about their expression, function, or interactions. By monitoring ciliogenesis in primary airway epithelial cells and MCIDAS-regulated induced pluripotent stem cells, we uncovered two phases of expression of preassembly proteins. An early phase, composed of HEATR2, SPAG1, and DNAAF2, preceded other preassembly proteins and was independent of MCIDAS regulation. The early preassembly proteins colocalized within perinuclear foci that also contained dynein arm proteins. These proteins also interacted based on immunoprecipitation and Förster resonance energy transfer (FRET) studies. FRET analysis of HEAT domain deletions and human mutations showed that HEATR2 interacted with itself and SPAG1 at multiple HEAT domains, while DNAAF2 interacted with SPAG1. Human mutations in HEATR2 did not affect this interaction, but triggered the formation of p62/Sequestosome-1-positive aggregates containing the early preassembly proteins, suggesting that degradation of an early preassembly complex is responsible for disease and pointing to key regions required for HEATR2 scaffold stability. We speculate that HEATR2 is an early scaffold for the initiation of dynein complex assembly in motile cilia.

PET-based Imaging of Chemokine Receptor 2 in Experimental and Disease-related Lung Inflammation.

Purpose To characterize a chemokine receptor type 2 (CCR2)-binding peptide adapted for use as a positron emission tomography (PET) radiotracer for noninvasive detection of lung inflammation in a mouse model of lung injury and in human tissues from subjects with lung disease. Materials and Methods The study was approved by institutional animal and human studies committees. Informed consent was obtained from patients. A 7-amino acid CCR2 binding peptide (extracellular loop 1 inverso [ECL1i]) was conjugated to tetraazacyclododecane tetraacetic acid (DOTA) and labeled with copper 64 (64Cu) or fluorescent dye. Lung inflammation was induced with intratracheal administration of lipopolysaccharide (LPS) in wild-type (n = 19) and CCR2-deficient (n = 4) mice, and these mice were compared with wild-type mice given control saline (n = 5) by using PET performed after intravenous injection of 64Cu-DOTA-ECL1i. Lung immune cells and those binding fluorescently labeled ECL1i in vivo were detected with flow cytometry. Lung inflammation in tissue from subjects with nondiseased lungs donated for lung transplantation (n = 11) and those with chronic obstructive pulmonary disease (COPD) who were undergoing lung transplantation (n = 16) was evaluated for CCR2 with immunostaining and autoradiography (n = 6, COPD) with 64Cu-DOTA-ECL1i. Groups were compared with analysis of variance, the Mann-Whitney U test, or the t test. Results Signal on PET images obtained in mouse lungs after injury with LPS was significantly greater than that in the saline control group (mean = 4.43% of injected dose [ID] per gram of tissue vs 0.99% of injected dose per gram of tissue; P < .001). PET signal was significantly diminished with blocking studies using nonradiolabeled ECL1i in excess (mean = 0.63% ID per gram of tissue; P < .001) and in CCR2-deficient mice (mean = 0.39% ID per gram of tissue; P < .001). The ECL1i signal was associated with an elevated level of mouse lung monocytes. COPD lung tissue displayed significantly elevated CCR2 levels compared with nondiseased tissue (median = 12.8% vs 1.2% cells per sample; P = .002), which was detected with 64Cu-DOTA-ECL1i by using autoradiography. Conclusion 64Cu-DOTA-ECL1i is a promising tool for PET-based detection of CCR2-directed inflammation in an animal model and in human tissues as a step toward clinical translation. © RSNA, 2017 Online supplemental material is available for this article.

In vivo fate tracking of degradable nanoparticles for lung gene transfer using PET and Cerenkov imaging.

Nanoparticles (NPs) play expanding roles in biomedical applications including imaging and therapy, however, their long-term fate and clearance profiles have yet to be fully characterized in vivo. NP delivery via the airway is particularly challenging, as the clearance may be inefficient and lung immune responses complex. Thus, specific material design is required for cargo delivery and quantitative, noninvasive methods are needed to characterize NP pharmacokinetics. Here, biocompatible poly(acrylamidoethylamine)-b-poly(dl-lactide) block copolymer-based degradable, cationic, shell-cross-linked knedel-like NPs (Dg-cSCKs) were employed to transfect plasmid DNA. Radioactive and optical beacons were attached to monitor biodistribution and imaging. The preferential release of cargo in acidic conditions provided enhanced transfection efficiency compared to non-degradable counterparts. In vivo gene transfer to the lung was correlated with NP pharmacokinetics by radiolabeling Dg-cSCKs and performing quantitative biodistribution with parallel positron emission tomography and Cerenkov imaging. Quantitation of imaging over 14 days corresponded with the pharmacokinetics of NP movement from the lung to gastrointestinal and renal routes, consistent with predicted degradation and excretion. This ability to noninvasively and accurately track NP fate highlights the advantage of incorporating multifunctionality into particle design.

IL13 activates autophagy to regulate secretion in airway epithelial cells.

Cytokine modulation of autophagy is increasingly recognized in disease pathogenesis, and current concepts suggest that type 1 cytokines activate autophagy, whereas type 2 cytokines are inhibitory. However, this paradigm derives primarily from studies of immune cells and is poorly characterized in tissue cells, including sentinel epithelial cells that regulate the immune response. In particular, the type 2 cytokine IL13 (interleukin 13) drives the formation of airway goblet cells that secrete excess mucus as a characteristic feature of airway disease, but whether this process is influenced by autophagy was undefined. Here we use a mouse model of airway disease in which IL33 (interleukin 33) stimulation leads to IL13-dependent formation of airway goblet cells as tracked by levels of mucin MUC5AC (mucin 5AC, oligomeric mucus/gel forming), and we show that these cells manifest a block in mucus secretion in autophagy gene Atg16l1-deficient mice compared to wild-type control mice. Similarly, primary-culture human tracheal epithelial cells treated with IL13 to stimulate mucus formation also exhibit a block in MUC5AC secretion in cells depleted of autophagy gene ATG5 (autophagy-related 5) or ATG14 (autophagy-related 14) compared to nondepleted control cells. Our findings indicate that autophagy is essential for airway mucus secretion in a type 2, IL13-dependent immune disease process and thereby provide a novel therapeutic strategy for attenuating airway obstruction in hypersecretory inflammatory diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis lung disease. Taken together, these observations suggest that the regulation of autophagy by Th2 cytokines is cell-context dependent.

Visualization of pulmonary clearance mechanisms via noninvasive optical imaging validated by near-infrared flow cytometry.

In vivo optical imaging with near-infrared (NIR) probes is an established method of diagnostics in preclinical and clinical studies. However, the specificities of these probes are difficult to validate ex vivo due to the lack of NIR flow cytometry. To address this limitation, we modified a flow cytometer to include an additional NIR channel using a 752 nm laser line. The flow cytometry system was tested using NIR microspheres and cell lines labeled with a combination of visible range and NIR fluorescent dyes. The approach was verified in vivo in mice evaluated for immune response in lungs after intratracheal delivery of the NIR contrast agent. Flow cytometry of cells obtained from the lung bronchoalveolar lavage demonstrated that the NIR dye was taken up by pulmonary macrophages as early as 4-h post-injection. This combination of optical imaging with NIR flow cytometry extends the capability of imaging and enables complementation of in vivo imaging with cell-specific studies.

Synthesis, characterization, and in vivo efficacy of shell cross-linked nanoparticle formulations carrying silver antimicrobials as aerosolized therapeutics.

The use of nebulizable, nanoparticle-based antimicrobial delivery systems can improve efficacy and reduce toxicity for treatment of multi-drug-resistant bacteria in the chronically infected lungs of cystic fibrosis patients. Nanoparticle vehicles are particularly useful for applying broad-spectrum silver-based antimicrobials, for instance, to improve the residence time of small-molecule silver carbene complexes (SCCs) within the lung. Therefore, we have synthesized multifunctional, shell cross-linked knedel-like polymeric nanoparticles (SCK NPs) and capitalized on the ability to independently load the shell and core with silver-based antimicrobial agents. We formulated three silver-loaded variants of SCK NPs: shell-loaded with silver cations, core-loaded with SCC10, and combined loading of shell silver cations and core SCC10. All three formulations provided a sustained delivery of silver over the course of at least 2-4 days. The two SCK NP formulations with SCC10 loaded in the core each exhibited excellent antimicrobial activity and efficacy in vivo in a mouse model of Pseudomonas aeruginosa pneumonia. SCK NPs with shell silver cation-load only, while efficacious in vitro, failed to demonstrate efficacy in vivo. However, a single dose of core SCC10-loaded SCK NPs (0.74 ± 0.16 mg Ag) provided a 28% survival advantage over sham treatment, and administration of two doses (0.88 mg Ag) improved survival to 60%. In contrast, a total of 14.5 mg of Ag(+) delivered over 5 doses at 12 h intervals was necessary to achieve a 60% survival advantage with a free-drug (SCC1) formulation. Thus, SCK NPs show promise for clinical impact by greatly reducing antimicrobial dosage and dosing frequency, which could minimize toxicity and improve patient adherence.

Degradable cationic shell cross-linked knedel-like nanoparticles: synthesis, degradation, nucleic acid binding, and in vitro evaluation.

In this work, degradable cationic shell cross-linked knedel-like (deg-cSCK) nanoparticles were developed as an alternative platform to replace similar nondegradable cSCK nanoparticles that have been utilized for nucleic acids delivery. An amphiphilic diblock copolymer poly(acrylamidoethylamine)(90)-block-poly(DL-lactide)(40) (PAEA(90)-b-PDLLA(40)) was synthesized, self-assembled in aqueous solution, and shell cross-linked using a hydrolyzable cross-linker to afford deg-cSCKs with an average core diameter of 45 ± 7 nm. These nanoparticles were fluorescently labeled for in vitro tracking. The enzymatic- and hydrolytic-degradability, siRNA binding affinity, cell uptake and cytotoxicity of the deg-cSCKs were evaluated. Esterase-catalyzed hydrolysis of the nanoparticles resulted in the degradation of ca. 24% of the PDLLA core into lactic acid within 5 d, as opposed to only ca. 9% degradation from aqueous solutions of the deg-cSCK nanoparticles in the absence of enzyme. Cellular uptake of deg-cSCKs was efficient, while exhibiting low cytotoxicity with LD50 values of ca. 90 and 30 µg/mL in RAW 264.7 mouse macrophages and MLE 12 cell lines, respectively, ca. 5- to 6-fold lower than the cytotoxicity observed for nondegradable cSCK analogs. Additionally, deg-cSCKs were able to complex siRNA at an N/P ratio as low as 2, and were efficiently able to facilitate cellular uptake of the complexed nucleic acids.

Antisense peptide nucleic acid-functionalized cationic nanocomplex for in vivo mRNA detection.

Acute lung injury (ALI) is a complex syndrome with many aetiologies, resulting in the upregulation of inflammatory mediators in the host, followed by dyspnoea, hypoxemia and pulmonary oedema. A central mediator is inducible nitric oxide synthase (iNOS) that drives the production of NO and continued inflammation. Thus, it is useful to have diagnostic and therapeutic agents for targeting iNOS expression. One general approach is to target the precursor iNOS mRNA with antisense nucleic acids. Peptide nucleic acids (PNAs) have many advantages that make them an ideal platform for development of antisense theranostic agents. Their membrane impermeability, however, limits biological applications. Here, we report the preparation of an iNOS imaging probe through electrostatic complexation between a radiolabelled antisense PNA-YR9 · oligodeoxynucleotide (ODN) hybrid and a cationic shell-cross-linked knedel-like nanoparticle (cSCK). The Y (tyrosine) residue was used for (123)I radiolabelling, whereas the R9 (arginine9) peptide was included to facilitate cell exit of untargeted PNA. Complete binding of the antisense PNA-YR9 · ODN hybrid to the cSCK was achieved at an 8 : 1 cSCK amine to ODN phosphate (N/P) ratio by a gel retardation assay. The antisense PNA-YR9 · ODN · cSCK nanocomplexes efficiently entered RAW264.7 cells, whereas the PNA-YR9 · ODN alone was not taken up. Low concentrations of (123)I-labelled antisense PNA-YR9 · ODN complexed with cSCK showed significantly higher retention of radioactivity when iNOS was induced in lipopolysaccharide+interferon-γ-activated RAW264.7 cells when compared with a mismatched PNA. Moreover, statistically, greater retention of radioactivity from the antisense complex was also observed in vivo in an iNOS-induced mouse lung after intratracheal administration of the nanocomplexes. This study demonstrates the specificity and sensitivity by which the radiolabelled nanocomplexes can detect iNOS mRNA in vitro and in vivo and their potential for early diagnosis of ALI.

Azithromycin attenuates airway inflammation in a mouse model of viral bronchiolitis.

BACKGROUND: Viral bronchiolitis is the leading cause of hospitalization in young infants. It is associated with the development of childhood asthma and contributes to morbidity and mortality in the elderly. Currently no therapies effectively attenuate inflammation during the acute viral infection, or prevent the risk of post-viral asthma. We hypothesized that early treatment of a paramyxoviral bronchiolitis with azithromycin would attenuate acute and chronic airway inflammation. METHODS: Mice were inoculated with parainfluenza type 1, Sendai Virus (SeV), and treated daily with PBS or azithromycin for 7 days post-inoculation. On day 8 and 21 we assessed airway inflammation in lung tissue, and quantified immune cells and inflammatory mediators in bronchoalveolar lavage (BAL). RESULTS: Compared to treatment with PBS, azithromycin significantly attenuated post-viral weight loss. During the peak of acute inflammation (day 8), azithromycin decreased total leukocyte accumulation in the lung tissue and BAL, with the largest fold-reduction in BAL neutrophils. This decreased inflammation was independent of changes in viral load. Azithromycin significantly attenuated the concentration of BAL inflammatory mediators and enhanced resolution of chronic airway inflammation evident by decreased BAL inflammatory mediators on day 21. CONCLUSIONS: In this mouse model of paramyxoviral bronchiolitis, azithromycin attenuated acute and chronic airway inflammation. These findings demonstrate anti-inflammatory effects of azithromycin that are not related to anti-viral activity. Our findings support the rationale for future prospective randomized clinical trials that will evaluate the effects of macrolides on acute viral bronchiolitis and their long-term consequences.

Temporal relationship between primary and motile ciliogenesis in airway epithelial cells.

Cilia are traditionally classified as motile or primary. Motile cilia are restricted to specific populations of well-differentiated epithelial cells, including those in the airway, brain ventricles, and oviducts. Primary cilia are nonmotile, solitary structures that are present in many cell types, and often have sensory functions such as in the retina and renal tubules. Primary cilia were also implicated in the regulation of fundamental processes in development. Rare depictions of primary cilia in embryonic airways led us to hypothesize that primary cilia in airway cells are temporally related to motile ciliogenesis. We identified primary cilia in undifferentiated, cultured airway epithelial cells from mice and humans and in developing lungs. The solitary cilia in the airways express proteins considered unique to primary cilia, including polycystin-1 and polycystin-2. A temporal analysis of airway epithelial cell differentiation showed that cells with primary cilia acquire markers of motile ciliogenesis, suggesting that motile ciliated cells originate from primary ciliated cells. Whereas motile ciliogenesis requires Foxj1, primary ciliogenesis does not, and the expression of Foxj1 was associated with a loss of primary cilia, just before the appearance of motile cilia. Primary cilia were not found in well-differentiated airway epithelial cells. However, after injury, they appear in the luminal layer of epithelium and in basal cells. The transient nature of primary cilia, together with the temporal and spatial patterns of expression in the development and repair of airway epithelium, suggests a critical role of primary cilia in determining outcomes during airway epithelial cell differentiation.

Impact of hydrogel nanoparticle size and functionalization on in vivo behavior for lung imaging and therapeutics.

Polymer chemistry offers the possibility of synthesizing multifunctional nanoparticles which incorporate moieties that enhance diagnostic and therapeutic targeting of cargo delivery to the lung. However, since rules for predicting particle behavior following modification are not well-defined, it is essential that probes for tracking fate in vivo are also included. Accordingly, we designed polyacrylamide-based hydrogel particles of differing sizes, functionalized with a nona-arginine cell-penetrating peptide (Arg(9)), and labeled with imaging components to assess lung retention and cellular uptake after intratracheal administration. Radiolabeled microparticles (1-5 microm diameter) and nanoparticles (20-40 nm diameter) without and with Arg(9) showed diffuse airspace distribution by positron emission tomography imaging. Biodistribution studies revealed that particle clearance and extrapulmonary distribution was, in part, size dependent. Microparticles were rapidly cleared by mucociliary routes but, unexpectedly, also through the circulation. In contrast, nanoparticles had prolonged lung retention enhanced by Arg(9) and were significantly restricted to the lung. For all particle types, uptake was predominant in alveolar macrophages and, to a lesser extent, lung epithelial cells. In general, particles did not induce local inflammatory responses, with the exception of microparticles bearing Arg(9). Whereas microparticles may be advantageous for short-term applications, nanosized particles constitute an efficient high-retention and non-inflammatory vehicle for the delivery of diagnostic imaging agents and therapeutics to lung airspaces and alveolar macrophages that can be enhanced by Arg(9). Importantly, our results show that minor particle modifications may significantly impact in vivo behavior within the complex environments of the lung, underscoring the need for animal modeling.

Azithromycin attenuates airway inflammation in a noninfectious mouse model of allergic asthma.

BACKGROUND: Definitive conclusions regarding the antiinflammatory effects of macrolide antibiotics for treatment of asthma are difficult to formulate since their beneficial effects may be related to their antimicrobial action. We hypothesized that azithromycin possesses distinct antiinflammatory properties and tested this assumption in a noninfectious mouse model of allergic asthma. METHODS: To induce allergic airway inflammation, 7-week-old BALB/cJ mice underwent intraperitoneal ovalbumin sensitization on days 0 and 7 followed by an intranasal challenge on day 14. Mice were treated with azithromycin or phosphate-buffered saline (PBS) solution on days 13 through 16. On day 17, airway inflammation was assessed by quantifying leukocytes in the airway, expression of multiple inflammatory mediators in the BAL fluid, and mucous cell metaplasia. In a separate set of experiments, azithromycin or PBS solution treatment were initiated after the ovalbumin challenge. Each experiment was repeated 3 times (a total of 9 to 11 mice in each group). RESULTS: Compared to treatment with PBS solution, azithromycin attenuated the ovalbumin-dependent airway inflammation. We observed a decrease in total leukocytes in the lung tissue and BAL fluid. In addition, azithromycin attenuated the expression of cytokines (eg, interleukin [IL]-13 and IL-5) and chemokines (eg, CCL2, CCL3, and CCL4) in the BAL fluid and abrogated the extent of mucous cell metaplasia. Similar antiinflammatory effects were observed when azithromycin treatment was initiated after the ovalbumin challenge. CONCLUSION: In this noninfectious mouse model of allergic asthma, azithromycin attenuated allergic airway inflammation. These findings demonstrate an antiinflammatory effect of azithromycin and suggest azithromycin may have beneficial effects in treating noninfectious airway inflammatory diseases, including asthma.

IL-12 p80-dependent macrophage recruitment primes the host for increased survival following a lethal respiratory viral infection.

A protective immune response to a respiratory viral infection requires a series of coordinated cellular and molecular responses. We have previously demonstrated that increased expression of airway epithelial cell interleukin (IL)-12 p80, a macrophage chemoattractant, is associated with human respiratory viral infection and mediates post-viral mortality in the mouse. To better understand the role of IL-12 p80-dependent macrophage chemotaxis in mediating viral immunity, we generated a transgenic mouse strain utilizing a promoter to drive IL-12 p40 gene expression in the airway epithelium. This transgenic strain secreted biologically active IL-12 p80 in a lung-specific manner, and demonstrated a selective increase in the number of resident, unactivated airway macrophages at baseline. Following infection with a sublethal dose of mouse parainfluenza virus type 1 (Sendai virus), the transgenic mice demonstrated an earlier peak and decline in the number of airway inflammatory cells. The transgenic mice were resistant to a lethal dose of virus and this viral resistance was dependent on the increased number of airway macrophages at baseline as partial depletion prior to infection abrogated this phenotype. The survival advantage in the transgenic mice was independent of viral load but was associated with a more rapid decline in the number of airway inflammatory cells and concentrations of multiple chemokines including the CC chemokine ligand 2 (CCL2)/JE, CCL3/macrophage inflammatory protein (MIP)-1alpha, CCL4/MIP-1beta, and CCL5/RANTES. Collectively, these results suggest that IL-12 p80-driven increases in the number of resident airway macrophages prime the host for a protective immune response that can confer increased survival following a lethal respiratory viral infection.