Endothelial eNAMPT amplifies pre-clinical acute lung injury: efficacy of an eNAMPT-neutralising monoclonal antibody.

RATIONALE: The severe acute respiratory syndrome coronavirus 2/coronavirus disease 2019 pandemic has highlighted the serious unmet need for effective therapies that reduce acute respiratory distress syndrome (ARDS) mortality. We explored whether extracellular nicotinamide phosphoribosyltransferase (eNAMPT), a ligand for Toll-like receptor (TLR)4 and a master regulator of innate immunity and inflammation, is a potential ARDS therapeutic target. METHODS: Wild-type C57BL/6J or endothelial cell (EC)-cNAMPT -/- knockout mice (targeted EC NAMPT deletion) were exposed to either a lipopolysaccharide (LPS)-induced ("one-hit") or a combined LPS/ventilator ("two-hit")-induced acute inflammatory lung injury model. A NAMPT-specific monoclonal antibody (mAb) imaging probe (99mTc-ProNamptor) was used to detect NAMPT expression in lung tissues. Either an eNAMPT-neutralising goat polyclonal antibody (pAb) or a humanised monoclonal antibody (ALT-100 mAb) were used in vitro and in vivo. RESULTS: Immunohistochemical, biochemical and imaging studies validated time-dependent increases in NAMPT lung tissue expression in both pre-clinical ARDS models. Intravenous delivery of either eNAMPT-neutralising pAb or mAb significantly attenuated inflammatory lung injury (haematoxylin and eosin staining, bronchoalveolar lavage (BAL) protein, BAL polymorphonuclear cells, plasma interleukin-6) in both pre-clinical models. In vitro human lung EC studies demonstrated eNAMPT-neutralising antibodies (pAb, mAb) to strongly abrogate eNAMPT-induced TLR4 pathway activation and EC barrier disruption. In vivo studies in wild-type and EC-cNAMPT -/- mice confirmed a highly significant contribution of EC-derived NAMPT to the severity of inflammatory lung injury in both pre-clinical ARDS models. CONCLUSIONS: These findings highlight both the role of EC-derived eNAMPT and the potential for biologic targeting of the eNAMPT/TLR4 inflammatory pathway. In combination with predictive eNAMPT biomarker and NAMPT genotyping assays, this offers the opportunity to identify high-risk ARDS subjects for delivery of personalised medicine.

Mice with a Deletion of Rsph1 Exhibit a Low Level of Mucociliary Clearance and Develop a Primary Ciliary Dyskinesia Phenotype.

Primary ciliary dyskinesia (PCD) is a genetically and phenotypically heterogeneous disease caused by mutations in over 40 different genes. Individuals with PCD caused by mutations in RSPH1 (radial spoke head 1 homolog) have been reported to have a milder phenotype than other individuals with PCD, as evidenced by a lower incidence of neonatal respiratory distress, higher nasal nitric oxide concentrations, and better lung function. To better understand genotype-phenotype relationships in PCD, we have characterized a mutant mouse model with a deletion of Rsph1. Approximately 50% of cilia from Rsph1-/- cells appeared normal by transmission EM, whereas the remaining cilia revealed a range of defects, primarily transpositions or a missing central pair. Ciliary beat frequency in Rsph1-/- cells was significantly lower than in control cells (20.2 ± 0.8 vs. 25.0 ± 0.9 Hz), and the cilia exhibited an aberrant rotational waveform. Young Rsph1-/- animals demonstrated a low rate of mucociliary clearance in the nasopharynx that was reduced to zero by about 1 month of age. Rsph1-/- animals accumulated mucus in the nasal cavity but had a lower bacterial burden than animals with a deletion of dynein axonemal intermediate chain 1 (Dnaic1-/-). Thus, Rsph1-/- mice display a PCD phenotype similar to but less severe than that observed in Dnaic1-/- mice, similar to what has been observed in humans. The results suggest that some individuals with PCD may not have a complete loss of mucociliary clearance and further suggest that early diagnosis and intervention may be important to maintain this low amount of clearance.

Loss of ß Epithelial Sodium Channel Function in Meibomian Glands Produces Pseudohypoaldosteronism 1-Like Ocular Disease in Mice.

Human subjects with pseudohypoaldosteronism-1 because of loss-of-function mutations in epithelial sodium channel (ENaC) subunits exhibit meibomian gland (MG) dysfunction. A conditional ßENaC MG knockout (KO) mouse model was generated to elucidate the pathogenesis of absent ENaC function in the MG and associated ocular surface disease. ßENaC MG KO mice exhibited a striking age-dependent, female-predominant MG dysfunction phenotype, with white toothpaste-like secretions observed obstructing MG orifices at 7 weeks of age. There were compensatory increases in tear production but higher tear sodium and indexes of mucin concentration in ßENaC MG KO mice. Histologically, MG acinar atrophy was observed with ductal enlargement and ductal epithelial hyperstratification. Inflammatory cell infiltration was observed in both MG and conjunctiva of ßENaC MG KO mice. In older ßENaC MG KO mice (5 to 11 months), significant ocular surface pathologies were noted, including corneal opacification, ulceration, neovascularization, and ectasia. Inflammation in MG and conjunctiva was confirmed by increased cytokine gene and protein expression and positive Ly-6B.2 immunostaining. Cell proliferation assays revealed lower proliferation rates of MG cells derived from ßENaC MG KO than control mice, suggesting that ßENaC plays a role in cell renewal of mouse MG. Loss of ßENaC function resulted in MG disease and severe ocular surface damage that phenocopied aspects of human pseudohypoaldosteronism-1 MG disease and was sex dependent.

Role of Spdef in the Regulation of Muc5b Expression in the Airways of Naive and Mucoobstructed Mice.

Understanding how expression of airway secretory mucins MUC5B and MUC5AC is regulated in health and disease is important to elucidating the pathogenesis of mucoobstructive respiratory diseases. The transcription factor SPDEF (sterile α-motif pointed domain epithelial specific transcription factor) is a key regulator of MUC5AC, but its role in regulating MUC5B in health and in mucoobstructive lung diseases is unknown. Characterization of Spdef-deficient mice upper and lower airways demonstrated region-specific, Spdef-dependent regulation of basal Muc5b expression. Neonatal Spdef-deficient mice exhibited reductions in BAL Muc5ac and Muc5b. Adult Spdef-deficient mice partially phenocopied Muc5b-deficient mice as they exhibited reduced Muc5b in nasopharyngeal and airway epithelia but not in olfactory Bowman glands, 75% incidence of nasopharyngeal hair/mucus plugs, and mild bacterial otitis media, without defective mucociliary clearance in the nasopharynx. In contrast, tracheal mucociliary clearance was reduced in Spdef-deficient mice in the absence of lung disease. To evaluate the role of Spdef in the development and persistence of Muc5b-predominant mucoobstructive lung disease, Spdef-deficient mice were crossed with Scnn1b-transgenic (Scnn1b-Tg) mice, which exhibit airway surface dehydration-induced airway mucus obstruction and inflammation. Spdef-deficient Scnn1b-Tg mice exhibited reduced Muc5ac, but not Muc5b, expression and BAL content. Airway mucus obstruction was not decreased in Spdef-deficient Scnn1b-Tg mice, consistent with Muc5b-dominant Scnn1b disease, but increased airway neutrophilia was observed compared with Spdef-sufficient Scnn1b-Tg mice. Collectively, these results indicate that Spdef regulates baseline Muc5b expression in respiratory epithelia but does not contribute to Muc5b regulation in a mouse model of Muc5b-predominant mucus obstruction caused by airway dehydration.

Lung disease phenotypes caused by overexpression of combinations of α-, ß-, and γ-subunits of the epithelial sodium channel in mouse airways.

The epithelial Na+ channel (ENaC) regulates airway surface hydration. In mouse airways, ENaC is composed of three subunits, α, ß, and γ, which are differentially expressed (α > ß > γ). Airway-targeted overexpression of the ß subunit results in Na+ hyperabsorption, causing airway surface dehydration, hyperconcentrated mucus with delayed clearance, lung inflammation, and perinatal mortality. Notably, mice overexpressing the α- or γ-subunit do not exhibit airway Na+ hyperabsorption or lung pathology. To test whether overexpression of multiple ENaC subunits produced Na+ transport and disease severity exceeding that of ßENaC-Tg mice, we generated double (αß, αγ, ßγ) and triple (αßγ) transgenic mice and characterized their lung phenotypes. Double αγENaC-Tg mice were indistinguishable from WT littermates. In contrast, double ßγENaC-Tg mice exhibited airway Na+ absorption greater than that of ßENaC-Tg mice, which was paralleled by worse survival, decreased mucociliary clearance, and more severe lung pathology. Double αßENaC-Tg mice exhibited Na+ transport rates comparable to those of ßENaC-Tg littermates. However, αßENaC-Tg mice had poorer survival and developed severe parenchymal consolidation. In situ hybridization (RNAscope) analysis revealed both alveolar and airway αENaC-Tg overexpression. Triple αßγENaC-Tg mice were born in Mendelian proportions but died within the first day of life, and the small sample size prevented analyses of cause(s) of death. Cumulatively, these results indicate that overexpression of ßENaC is rate limiting for generation of pathological airway surface dehydration. Notably, airway co-overexpression of ß- and γENaC had additive effects on Na+ transport and disease severity, suggesting dose dependency of these two variables.

Neonatal Pulmonary Macrophage Depletion Coupled to Defective Mucus Clearance Increases Susceptibility to Pneumonia and Alters Pulmonary Immune Responses.

Resident immune cells (e.g., macrophages [MΦs]) and airway mucus clearance both contribute to a healthy lung environment. To investigate interactions between pulmonary MΦ function and defective mucus clearance, a genetic model of lysozyme M (LysM) promoter-mediated MΦ depletion was generated, characterized, and crossed with the sodium channel ß subunit transgenic (Scnn1b-Tg) mouse model of defective mucus clearance. Diphtheria toxin A-mediated depletion of LysM(+) pulmonary MΦs in wild-type mice with normal mucus clearance resulted in lethal pneumonia in 24% of neonates. The pneumonias were dominated by Pasteurella pneumotropica and accompanied by emaciation, neutrophilic inflammation, and elevated Th1 cytokines. The incidence of emaciation and pneumonia reached 51% when LysM(+) MΦ depletion was superimposed on the airway mucus clearance defect of Scnn1b-Tg mice. In LysM(+) MΦ-depleted Scnn1b-Tg mice, pneumonias were associated with a broader spectrum of bacterial species and a significant reduction in airway mucus plugging. Bacterial burden (CFUs) was comparable between Scnn1b-Tg and nonpneumonic LysM(+) MΦ-depleted Scnn1b-Tg mice. However, the nonpneumonic LysM(+) MΦ-depleted Scnn1b-Tg mice exhibited increased airway inflammation, the presence of neutrophilic infiltration, and increased levels of inflammatory cytokines in bronchoalveolar lavage fluid compared with Scnn1b-Tg mice. Collectively, these data identify key MΦ-mucus clearance interactions with respect to both infectious and inflammatory components of muco-obstructive lung disease.

ZMYND10 is mutated in primary ciliary dyskinesia and interacts with LRRC6.

Defects of motile cilia cause primary ciliary dyskinesia (PCD), characterized by recurrent respiratory infections and male infertility. Using whole-exome resequencing and high-throughput mutation analysis, we identified recessive biallelic mutations in ZMYND10 in 14 families and mutations in the recently identified LRRC6 in 13 families. We show that ZMYND10 and LRRC6 interact and that certain ZMYND10 and LRRC6 mutations abrogate the interaction between the LRRC6 CS domain and the ZMYND10 C-terminal domain. Additionally, ZMYND10 and LRRC6 colocalize with the centriole markers SAS6 and PCM1. Mutations in ZMYND10 result in the absence of the axonemal protein components DNAH5 and DNALI1 from respiratory cilia. Animal models support the association between ZMYND10 and human PCD, given that zmynd10 knockdown in zebrafish caused ciliary paralysis leading to cystic kidneys and otolith defects and that knockdown in Xenopus interfered with ciliogenesis. Our findings suggest that a cytoplasmic protein complex containing ZMYND10 and LRRC6 is necessary for motile ciliary function.

A new tool improves diagnostic test performance for transmission em evaluation of axonemal dynein arms.

Abstract Diagnosis of primary ciliary dyskinesia (PCD) by identification of dynein arm loss in transmission electron microscopy (TEM) images can be confounded by high background noise due to random electron-dense material within the ciliary matrix, leading to diagnostic uncertainty even for experienced morphologists. The authors developed a novel image analysis tool to average the axonemal peripheral microtubular doublets, thereby increasing microtubular signal and reducing random background noise. In a randomized, double-blinded study that compared two experienced morphologists and three different diagnostic approaches, they found that use of this tool led to improvement in diagnostic TEM test performance.

Prenatal Exposure to Environmentally Relevant Low Dosage Dibutyl Phthalate Reduces Placental Efficiency in CD-1 Mice.

Introduction: Dibutyl phthalate (DBP), a phthalate congener, is widely utilized in consumer products and medication coatings. Women of reproductive age have a significant burden of DBP exposure through consumer products, occupational exposure, and medication. Prenatal DBP exposure is associated with adverse pregnancy/fetal outcomes and cardiovascular diseases in the offspring. However, the role of fetal sex and the general mechanisms underlying DBP exposure-associated adverse pregnancy outcomes are unclear. We hypothesize that prenatal DBP exposure at an environmentally relevant low dosage adversely affects fetal-placental development and function during pregnancy in a fetal sex-specific manner. Methods: Adult female CD-1 mice (8-10wks) were orally treated with vehicle (control) or with environmentally relevant low DBP dosages at 0.1 µg/kg/day (refer as DBP0.1) daily from 30 days before pregnancy through gestational day (GD) 18.5. Dam body mass composition was measured non-invasively using the echo-magnetic resonance imaging system. Lipid disposition in fetal labyrinth and maternal decidual area of placentas was examined using Oil Red O staining. Results: DBP0.1 exposure did not significantly affect the body weight and adiposity of non-pregnant adult female mice nor the maternal weight gain pattern and adiposity during pregnancy in adult female mice. DBP0.1 exposure does not affect fetal weight but significantly increased the placental weight at GD18.5 (indicative of decreased placental efficiency) in a fetal sex-specific manner. We further observed that DBP0.1 significantly decreased lipid disposition in fetal labyrinth of female, but not male placentas, while it did not affect lipid disposition in maternal decidual. Conclusions: Prenatal exposure to environmentally relevant low-dosage DBP adversely impacts the fetal-placental efficiency and lipid disposition in a fetal sex-specific manner.

High prevalence of respiratory ciliary dysfunction in congenital heart disease patients with heterotaxy.

BACKGROUND: Patients with congenital heart disease (CHD) and heterotaxy show high postsurgical morbidity/mortality, with some developing respiratory complications. Although this finding is often attributed to the CHD, airway clearance and left-right patterning both require motile cilia function. Thus, airway ciliary dysfunction (CD) similar to that of primary ciliary dyskinesia (PCD) may contribute to increased respiratory complications in heterotaxy patients. METHODS AND RESULTS: We assessed 43 CHD patients with heterotaxy for airway CD. Videomicrocopy was used to examine ciliary motion in nasal tissue, and nasal nitric oxide (nNO) was measured; nNO level is typically low with PCD. Eighteen patients exhibited CD characterized by abnormal ciliary motion and nNO levels below or near the PCD cutoff values. Patients with CD aged >6 years show increased respiratory symptoms similar to those seen in PCD. Sequencing of all 14 known PCD genes in 13 heterotaxy patients with CD, 12 without CD, 10 PCD disease controls, and 13 healthy controls yielded 0.769, 0.417, 1.0, and 0.077 novel variants per patient, respectively. One heterotaxy patient with CD had the PCD causing DNAI1 founder mutation. Another with hyperkinetic ciliary beat had 2 mutations in DNAH11, the only PCD gene known to cause hyperkinetic beat. Among PCD patients, 2 had known PCD causing CCDC39 and CCDC40 mutations. CONCLUSIONS: Our studies show that CHD patients with heterotaxy have substantial risk for CD and increased respiratory disease. Heterotaxy patients with CD were enriched for mutations in PCD genes. Future studies are needed to assess the potential benefit of prescreening and prophylactically treating heterotaxy patients for CD.

Plasma membrane localization is required for RasA-mediated polarized morphogenesis and virulence of Aspergillus fumigatus.

Ras is a highly conserved GTPase protein that is essential for proper polarized morphogenesis of filamentous fungi. Localization of Ras proteins to the plasma membrane and endomembranes through posttranslational addition of farnesyl and palmitoyl residues is an important mechanism through which cells provide specificity to Ras signal output. Although the Aspergillus fumigatus RasA protein is known to be a major regulator of growth and development, the membrane distribution of RasA during polarized morphogenesis and the role of properly localized Ras signaling in virulence of a pathogenic mold remain unknown. Here we demonstrate that Aspergillus fumigatus RasA localizes primarily to the plasma membrane of actively growing hyphae. We show that treatment with the palmitoylation inhibitor 2-bromopalmitate disrupts normal RasA plasma membrane association and decreases hyphal growth. Targeted mutations of the highly conserved RasA palmitoylation motif also mislocalized RasA from the plasma membrane and led to severe hyphal abnormalities, cell wall structural changes, and reduced virulence in murine invasive aspergillosis. Finally, we provide evidence that proper RasA localization is independent of the Ras palmitoyltransferase homolog, encoded by erfB, but requires the palmitoyltransferase complex subunit, encoded by erfD. Our results demonstrate that plasma membrane-associated RasA is critical for polarized morphogenesis, cell wall stability, and virulence in A. fumigatus.

Human-relevant exposure to di-n-butyl phthalate tampers with the ovarian insulin-like growth factor 1 system and disrupts folliculogenesis in young adult mice.

Phthalates are compounds used in consumer and medical products worldwide. Phthalate exposure in women has been demonstrated by detection of phthalate metabolites in their urine and ovarian follicular fluid. High urinary phthalate burden has been associated with reduced ovarian reserve and oocyte retrieval in women undergoing assisted reproduction. Unfortunately, no mechanistic explanation for these associations is available. In short term in vivo and in vitro animal studies modeling human-relevant exposures to di-n-butyl phthalate (DBP), we have identified ovarian folliculogenesis as a target for phthalate exposures. In the present study, we investigated whether DBP exposure negatively influences insulin-like growth factor 1 (IGF1) signaling in the ovary and disrupts ovarian folliculogenesis. CD-1 female mice were exposed to corn oil (vehicle) or DBP (10 µg/kg/day, 100 µg/kg/day, or 1000 mg/kg/day) for 20-32 days. Ovaries were collected as animals reached the proestrus stage to achieve estrous cycle synchronization. Levels of mRNAs encoding IGF1 and 2 (Igf1 and Igf2), IGF1 receptor (Igf1r), and IGF-binding proteins 1-6 (Ifgbp1-6) were measured in whole ovary homogenates. Ovarian follicle counts and immunostaining for phosphorylated IGF1R protein (pIGF1R) were used to evaluate folliculogenesis and IGF1R activation, respectively. DBP exposure, at a realistic dose that some women may experience (100 µg/kg/day for 20-32 days), reduced ovarian Igf1 and Igf1r mRNA expression and reduced small ovarian follicle numbers and primary follicle pIGF1R positivity in DBP-treated mice. These findings reveal that DBP tampers with the ovarian IGF1 system and provide molecular insight into how phthalates could influence the ovarian reserve in females.

Human relevant exposure to di-n-butyl phthalate tampers with the ovarian insulin-like growth factor 1 system and disrupts folliculogenesis in young adult mice.

Phthalates are compounds used in consumer and medical products worldwide. Phthalate exposure in women has been demonstrated by detection of phthalate metabolites in their urine and ovarian follicular fluid. High urinary phthalate burden has been associated with reduced ovarian reserve and oocyte retrieval in women undergoing assisted reproduction. Unfortunately, no mechanistic explanation for these associations is available. In short term in vivo and in vitro animal studies modeling human relevant exposures to di-n-butyl phthalate (DBP), we have identified ovarian folliculogenesis as a target for phthalate exposures. In the present study, we investigated whether DBP exposure negatively influences insulin-like growth factor 1 (IGF) signaling in the ovary and disrupts ovarian folliculogenesis. CD-1 female mice were exposed to corn oil (vehicle) or DBP (10 or 100 µg/kg/day) for 20-32 days. Ovaries were collected as animals reached the proestrus stage to achieve estrous cycle synchronization. Levels of mRNAs encoding IGF1 and 2 ( Igf1 and Igf2 ), IGF1 receptor ( Igf1r ), and IGF binding proteins 1-6 ( Ifgbp1-6 ) were measured in whole ovary homogenates. Ovarian follicle counts and immunostaining for phosphorylated IGF1R protein (pIGF1R) were used to evaluate folliculogenesis and IGF1R activation, respectively. DBP exposure, at a realistic dose that some women may experience (100 µg/kg/day for 20-32 days), reduced ovarian Igf1 and Igf1r mRNA expression and reduced small ovarian follicle numbers and primary follicle pIGF1R positivity in DBP-treated mice. These findings reveal that DBP tampers with the ovarian IGF1 system and provide molecular insight into how phthalates could influence the ovarian reserve in females.

Localization and activity of the calcineurin catalytic and regulatory subunit complex at the septum is essential for hyphal elongation and proper septation in Aspergillus fumigatus.

Calcineurin, a heterodimer composed of the catalytic (CnaA) and regulatory (CnaB) subunits, plays key roles in growth, virulence and stress responses of fungi. To investigate the contribution of CnaA and CnaB to hyphal growth and septation, ΔcnaB and ΔcnaAΔcnaB strains of Aspergillus fumigatus were constructed. CnaA colocalizes to the contractile actin ring early during septation and remains at the centre of the mature septum. While CnaB's septal localization is CnaA-dependent, CnaA's septal localization is CnaB-independent, but CnaB is required for CnaA's function at the septum. Catalytic null mutations in CnaA caused stunted growth despite septal localization of the calcineurin complex, indicating the requirement of calcineurin activity at the septum. Compared to the ΔcnaA and ΔcnaB strains, the ΔcnaAΔcnaB strain displayed more defective growth and aberrant septation. While three Ca(2+) -binding motifs in CnaB were sufficient for its association with CnaA at the septum, the amino-terminal arginine-rich domains (16-RRRR-19 and 44-RLRKR-48) are dispensable for septal localization, yet required for complete functionality. Mutation of the 51-KLDK-54 motif in CnaB causes its mislocalization from the septum to the nucleus, suggesting it is a nuclear export signal sequence. These findings confirm a cooperative role for the calcineurin complex in regulating hyphal growth and septation.

Mutations of DNAH11 in patients with primary ciliary dyskinesia with normal ciliary ultrastructure.

RATIONALE: Primary ciliary dyskinesia (PCD) is an autosomal recessive, genetically heterogeneous disorder characterised by oto-sino-pulmonary disease and situs abnormalities (Kartagener syndrome) due to abnormal structure and/or function of cilia. Most patients currently recognised to have PCD have ultrastructural defects of cilia; however, some patients have clinical manifestations of PCD and low levels of nasal nitric oxide, but normal ultrastructure, including a few patients with biallelic mutations in dynein axonemal heavy chain 11 (DNAH11). OBJECTIVES: To test further for mutant DNAH11 as a cause of PCD, DNAH11 was sequenced in patients with a PCD clinical phenotype, but no known genetic aetiology. METHODS: 82 exons and intron/exon junctions in DNAH11 were sequenced in 163 unrelated patients with a clinical phenotype of PCD, including those with normal ciliary ultrastructure (n=58), defects in outer and/or inner dynein arms (n=76), radial spoke/central pair defects (n=6), and 23 without definitive ultrastructural results, but who had situs inversus (n=17), or bronchiectasis and/or low nasal nitric oxide (n=6). Additionally, DNAH11 was sequenced in 13 subjects with isolated situs abnormalities to see if mutant DNAH11 could cause situs defects without respiratory disease. RESULTS: Of the 58 unrelated patients with PCD with normal ultrastructure, 13 (22%) had two (biallelic) mutations in DNAH11; and two patients without ultrastructural analysis had biallelic mutations. All mutations were novel and private. None of the patients with dynein arm or radial spoke/central pair defects, or isolated situs abnormalities, had mutations in DNAH11. Of the 35 identified mutant alleles, 24 (69%) were nonsense, insertion/deletion or loss-of-function splice-site mutations. CONCLUSIONS: Mutations in DNAH11 are a common cause of PCD in patients without ciliary ultrastructural defects; thus, genetic analysis can be used to ascertain the diagnosis of PCD in this challenging group of patients.

Involvement of eNAMPT/TLR4 signaling in murine radiation pneumonitis: protection by eNAMPT neutralization.

Therapeutic strategies to prevent or reduce the severity of radiation pneumonitis are a serious unmet need. We evaluated extracellular nicotinamide phosphoribosyltransferase (eNAMPT), a damage-associated molecular pattern protein (DAMP) and Toll-Like Receptor 4 (TLR4) ligand, as a therapeutic target in murine radiation pneumonitis. Radiation-induced murine and human NAMPT expression was assessed in vitro, in tissues (IHC, biochemistry, imaging), and in plasma. Wild type C57Bl6 mice (WT) and Nampt+/- heterozygous mice were exposed to 20Gy whole thoracic lung irradiation (WTLI) with or without weekly IP injection of IgG1 (control) or an eNAMPT-neutralizing polyclonal (pAb) or monoclonal antibody (mAb). BAL protein/cells and H&E staining were used to generate a WTLI severity score. Differentially-expressed genes (DEGs)/pathways were identified by RNA sequencing and bioinformatic analyses. Radiation exposure increases in vitro NAMPT expression in lung epithelium (NAMPT promoter activity) and NAMPT lung tissue expression in WTLI-exposed mice. Nampt+/- mice and eNAMPT pAb/mAb-treated mice exhibited significant histologic attenuation of WTLI-mediated lung injury with reduced levels of BAL protein and cells, and plasma levels of eNAMPT, IL-6,  and IL-1ß. Genomic and biochemical studies from WTLI-exposed lung tissues highlighted dysregulation of NFkB/cytokine and MAP kinase signaling pathways which were rectified by eNAMPT mAb treatment. The eNAMPT/TLR4 pathway is essentially involved in radiation pathobiology with eNAMPT neutralization an effective therapeutic strategy to reduce the severity of radiation pneumonitis.

Mucus concentration-dependent biophysical abnormalities unify submucosal gland and superficial airway dysfunction in cystic fibrosis.

Cystic fibrosis (CF) is characterized by abnormal transepithelial ion transport. However, a description of CF lung disease pathophysiology unifying superficial epithelial and submucosal gland (SMG) dysfunctions has remained elusive. We hypothesized that biophysical abnormalities associated with CF mucus hyperconcentration provide a unifying mechanism. Studies of the anion secretion-inhibited pig airway model of CF revealed elevated SMG mucus concentrations, osmotic pressures, and SMG mucus accumulation. Human airway studies revealed hyperconcentrated CF SMG mucus with raised osmotic pressures and cohesive forces predicted to limit SMG mucus secretion/release. Using proline-rich protein 4 (PRR4) as a biomarker of SMG secretion, CF sputum proteomics analyses revealed markedly lower PRR4 levels compared to healthy and bronchiectasis controls, consistent with a failure of CF SMGs to secrete mucus onto airway surfaces. Raised mucus osmotic/cohesive forces, reflecting mucus hyperconcentration, provide a unifying mechanism that describes disease-initiating mucus accumulation on airway surfaces and in SMGs of the CF lung.

Characterization of exosome-like vesicles released from human tracheobronchial ciliated epithelium: a possible role in innate defense.

Airway mucus forms the structural basis of the local innate immune defense mechanism. It is an integrated, active, viscoelastic gel matrix evolved to protect the exposed lung from physical, chemical, and pathological erosion. Exosomes are biologically active vesicles secreted by different cell types including epithelial, hematopoietic, and some tumor cells. They are also present in some biological fluids such as serum, urine, breast milk, and bronchoalveolar lavage fluid. In this study, we demonstrate for the first time that exosome-like vesicles with antiviral properties are present in human tracheobronchial epithelial (HTBE) cell culture secretions. These vesicles have been isolated by differential centrifugation and are characterized further by mass spectrometry, flow cytometry, immunoblotting, electron microscopy, and light-scattering methods. HTBE vesicles exhibited characteristic exosomal size (30-100 nm) and morphology (cup-shaped) with a buoyant density in sucrose of 1.12-1.18 g/ml. Biochemical characterization further revealed typical surface, cytoskeletal, and cytoplasmic proteins characteristic of exosomes, including the multivesicular and late endosomal membrane markers Tsg101 and CD63. The presence of RNA was also observed. The epithelial mucins MUC1, MUC4, and MUC16 also contributed to the vesicles' structure. Notably, alpha-2,6-linked sialic acid was associated with these mucin molecules and subsequent functional analysis showed that these vesicles have a neutralizing effect on human influenza virus, which is known to bind sialic acid. Taken together, these findings suggest that airway epithelial cells release exosome-like vesicles and that these structures may be involved in diverse physiological processes in airway biology, including innate mucosal defense.

Cytoplasmic "ciliary inclusions" in isolation are not sufficient for the diagnosis of primary ciliary dyskinesia.

BACKGROUND: The diagnosis of primary ciliary dyskinesia (PCD) is difficult and requires a combination of clinical features, nasal nitric oxide testing, cilia ultrastructural analysis by electron microscopy (EM), and genetics. A recently described cytoplasmic ultrastructural change termed "ciliary inclusions" was reported to be diagnostic of PCD; however, no supporting evidence of PCD was provided. In this study, we sought to confirm, or refute, the diagnosis of PCD in subjects with "ciliary inclusions" on EM. METHODS: Six subjects from five families with previous lab reports of "ciliary inclusions" on EMs of ciliated cells were identified and evaluated at a Genetic Disorders of Mucociliary Clearance Consortium site. We performed a detailed clinical history, nasal nitric oxide measurement, genetic testing including whole-exome sequencing (WES), and when possible, repeat ciliary EM study. RESULTS: Only one of six subjects had multiple and persistent clinical features congruent with PCD. No subject had situs inversus. Only one of six subjects had a very low nasal nitric oxide level. No "ciliary inclusions" were found in three subjects who had a repeat ciliary EM, and ciliary axonemal ultrastructures were normal. Genetic testing, including WES, was negative for PCD-causing genes, and for pathogenic variants in gene pathways that might cause "ciliary inclusions," such as ciliary biogenesis. CONCLUSION: "Ciliary Inclusions", in isolation, are not sufficient to diagnosis PCD. If seen, additional studies should be done to pursue an accurate diagnosis.

Characterization of Rat Meibomian Gland Ion and Fluid Transport.

PURPOSE: We establish novel primary rat meibomian gland (MG) cell culture systems and explore the ion transport activities of the rat MG. METHODS: Freshly excised rat MG tissues were characterized as follows: (1) mRNA expression of selected epithelial ion channels/transporters were measured by RT-PCR, (2) localization of epithelial sodium channel (ENaC) mRNAs was performed by in situ hybridization, and (3) protein expression and localization of ßENaC, the Na+/K+/Cl- cotransporter (NKCC), and the Na+/K+ ATPase were evaluated by immunofluorescence. Primary isolated rat MG cells were cocultured with 3T3 feeder cells and a Rho-associated kinase (ROCK) inhibitor (Y-27632) for expansion. Passaged rat MG cells were cultured as planar sheets under air-liquid interface (ALI) conditions for gene expression and electrophysiologic studies. Passaged rat MG cells also were cultured in matrigel matrices to form spheroids, which were examined ultrastructurally by transmission electron microscopy (TEM) and functionally using swelling assays. RESULTS: Expression of multiple ion channel/transporter genes was detected in rat MG tissues. ß-ENaC mRNA and protein were localized more to MG peripheral acinar cells than central acinar cells or ductular epithelial cells. Electrophysiologic studies of rat MG cell planar cultures demonstrated functional sodium, chloride, and potassium channels, and cotransporters activities. Transmission electron microscopic analyses of rat MG spheroids revealed highly differentiated MG cells with abundant lysosomal lamellar bodies. Rat MG spheroids culture-based measurements demonstrated active volume regulation by ion channels. CONCLUSIONS: This study demonstrates the presence and function of ion channels and volume transport by rat MG. Two novel primary MG cell culture models that may be useful for MG research were established.