Dysregulated alveolar epithelial cell progenitor function and identity in Hermansky-Pudlak syndrome pulmonary fibrosis.

Hermansky-Pudlak syndrome (HPS) is a genetic disorder of endosomal protein trafficking associated with pulmonary fibrosis in specific subtypes, including HPS-1 and HPS-2. Single mutant HPS1 and HPS2 mice display increased fibrotic sensitivity while double mutant HPS1/2 mice exhibit spontaneous fibrosis with aging, which has been attributed to HPS mutations in alveolar epithelial type II (AT2) cells. We utilized HPS mouse models and human lung tissue to investigate mechanisms of AT2 cell dysfunction driving fibrotic remodeling in HPS. Starting at 8 weeks of age, HPS mice exhibited progressive loss of AT2 cell numbers. HPS AT2 cell was impaired ex vivo and in vivo. Incorporating AT2 cell lineage tracing in HPS mice, we observed aberrant differentiation with increased AT2-derived alveolar epithelial type I cells. Transcriptomic analysis of HPS AT2 cells revealed elevated expression of genes associated with aberrant differentiation and p53 activation. Lineage tracing and modeling studies demonstrated that HPS AT2 cells were primed to persist in a Krt8+ reprogrammed transitional state, mediated by p53 activity. Intrinsic AT2 progenitor cell dysfunction and p53 pathway dysregulation are novel mechanisms of disease in HPS-related pulmonary fibrosis, with the potential for early targeted intervention before the onset of fibrotic lung disease.

Excess neuropeptides in lung signal through endothelial cells to impair gas exchange.

Although increased neuropeptides are often detected in lungs that exhibit respiratory distress, whether they contribute to the condition is unknown. Here, we show in a mouse model of neuroendocrine cell hyperplasia of infancy, a pediatric disease with increased pulmonary neuroendocrine cells (PNECs), excess PNEC-derived neuropeptides are responsible for pulmonary manifestations including hypoxemia. In mouse postnatal lung, prolonged signaling from elevated neuropeptides such as calcitonin gene-related peptide (CGRP) activate receptors enriched on endothelial cells, leading to reduced cellular junction gene expression, increased endothelium permeability, excess lung fluid, and hypoxemia. Excess fluid and hypoxemia were effectively attenuated by either prevention of PNEC formation, inactivation of CGRP gene, endothelium-specific inactivation of CGRP receptor gene, or treatment with CGRP receptor antagonist. Neuropeptides were increased in human lung diseases with excess fluid such as acute respiratory distress syndrome. Our findings suggest that restricting neuropeptide function may limit fluid and improve gas exchange in these conditions.

Inhaled Antibiotics for Mycobacterial Lung Disease.

Mycobacterial lung diseases are an increasing global health concern. Tuberculosis and nontuberculous mycobacteria differ in disease severity, epidemiology, and treatment strategies, but there are also a number of similarities. Pathophysiology and disease progression appear to be relatively similar between these two clinical diagnoses, and as a result these difficult to treat pulmonary infections often require similarly extensive treatment durations of multiple systemic drugs. In an effort to improve treatment outcomes for all mycobacterial lung diseases, a significant body of research has investigated the use of inhaled antibiotics. This review discusses previous research into inhaled development programs, as well as ongoing research of inhaled therapies for both nontuberculous mycobacterial lung disease, and tuberculosis. Due to the similarities between the causative agents, this review will also discuss the potential cross-fertilization of development programs between these similar-yet-different diseases. Finally, we will discuss some of the perceived difficulties in developing a clinically utilized inhaled antibiotic for mycobacterial diseases, and potential arguments in favor of the approach.

Clofazimine inhalation suspension for the aerosol treatment of pulmonary nontuberculous mycobacterial infections.

BACKGROUND: Nontuberculous mycobacteria are recognized as a concern for cystic fibrosis (CF) patients due to increasing disease prevalence and the potential for detrimental effects on pulmonary function and mortality. Current standard of care involves prolonged systemic antibiotics, which often leads to severe side effects and poor treatment outcomes. In this study, we investigated the tolerability and efficacy of a novel inhaled therapeutic in various mouse models of NTM disease. METHODS: We developed clofazimine inhalation suspension (CIS), a novel formulation of clofazimine developed for inhaled administration. To determine the efficacy, minimum inhibitory concentrations were evaluated in vitro, and tolerability of CIS was determined in naïve mouse models over various durations. After establishing tolerability, CIS efficacy was tested in in vivo infection models of both Mycobacterium avium and M. abscessus. Lung and plasma clofazimine levels after chronic treatments were evaluated. RESULTS: Clofazimine inhalation suspension demonstrated antimycobacterial activity in vitro, with MIC values between 0.125 and 2 µg/ml for M. avium complex and M. abscessus. Administration into naïve mice showed that CIS was well tolerated at doses up to 28 mg/kg over 28 consecutive treatments. In vivo, CIS was shown to significantly improve bacterial elimination from the lungs of both acute and chronic NTM-infected mouse models compared to negative controls and oral clofazimine administration. Clofazimine concentrations in lung tissue were approximately four times higher than the concentrations achieved by oral dosing. CONCLUSION: Clofazimine inhalation suspension is a well tolerated and effective novel therapeutic candidate for the treatment of NTM infections in mouse models.

The Antibacterial and Anti-inflammatory Activity of Chicken Cathelicidin-2 combined with Exogenous Surfactant for the Treatment of Cystic Fibrosis-Associated Pathogens.

Cystic fibrosis (CF) is characterized by recurrent airway infections with antibiotic-resistant bacteria and chronic inflammation. Chicken cathelicin-2 (CATH-2) has been shown to exhibit antimicrobial activity against antibiotic-resistant bacteria and to reduce inflammation. In addition, exogenous pulmonary surfactant has been suggested to enhance pulmonary drug delivery. It was hypothesized that CATH-2 when combined with an exogenous surfactant delivery vehicle, bovine lipid extract surfactant (BLES), would exhibit antimicrobial activity against CF-derived bacteria and downregulate inflammation. Twelve strains of CF-pathogens were exposed to BLES+CATH-2 in vitro and killing curves were obtained to determine bactericidal activity. Secondly, heat-killed bacteria were administered in vivo to elicit a pro-inflammatory response with either a co-administration or delayed administration of BLES+CATH-2 to assess the antimicrobial-independent, anti-inflammatory properties of BLES+CATH-2. CATH-2 alone exhibited potent antimicrobial activity against all clinical strains of antibiotic-resistant bacteria, while BLES+CATH-2 demonstrated a reduction, but significant antimicrobial activity against bacterial isolates. Furthermore, BLES+CATH-2 reduced inflammation in vivo when either co-administered with killed bacteria or after delayed administration. The use of a host-defense peptide combined with an exogenous surfactant compound, BLES+CATH-2, is shown to exhibit antimicrobial activity against antibiotic-resistant CF bacterial isolates and reduce inflammation.

Killing of Pseudomonas aeruginosa by Chicken Cathelicidin-2 Is Immunogenically Silent, Preventing Lung Inflammation In Vivo.

The development of antibiotic resistance by Pseudomonas aeruginosa is a major concern in the treatment of bacterial pneumonia. In the search for novel anti-infective therapies, the chicken-derived peptide cathelicidin-2 (CATH-2) has emerged as a potential candidate, with strong broad-spectrum antimicrobial activity and the ability to limit inflammation by inhibiting Toll-like receptor 2 (TLR2) and TLR4 activation. However, as it is unknown how CATH-2 affects inflammation in vivo, we investigated how CATH-2-mediated killing of P. aeruginosa affects lung inflammation in a murine model. First, murine macrophages were used to determine whether CATH-2-mediated killing of P. aeruginosa reduced proinflammatory cytokine production in vitro Next, a murine lung model was used to analyze how CATH-2-mediated killing of P. aeruginosa affects neutrophil and macrophage recruitment as well as cytokine/chemokine production in the lung. Our results show that CATH-2 kills P. aeruginosa in an immunogenically silent manner both in vitro and in vivo Treatment with CATH-2-killed P. aeruginosa showed reduced neutrophil recruitment to the lung as well as inhibition of cytokine and chemokine production, compared to treatment with heat- or gentamicin-killed bacteria. Together, these results show the potential for CATH-2 as a dual-activity antibiotic in bacterial pneumonia, which can both kill P. aeruginosa and prevent excessive inflammation.

The effect of diet-induced serum hypercholesterolemia on the surfactant system and the development of lung injury.

Acute respiratory distress syndrome (ARDS) is a pulmonary disorder associated with alterations to the pulmonary surfactant system. Recent studies showed that supra-physiological levels of cholesterol in surfactant contribute to impaired function. Since cholesterol is incorporated into surfactant within the alveolar type II cells which derives its cholesterol from serum, it was hypothesized that serum hypercholesterolemia would predispose the host to the development of lung injury due to alterations of cholesterol content in the surfactant system. Wistar rats were randomized to a standard lab diet or a high cholesterol diet for 17-20 days. Animals were then exposed to one of three models of lung injury: i) acid aspiration ii) ventilation induced lung injury, and iii) surfactant depletion. Following physiological monitoring, lungs were lavaged to obtain and analyze the surfactant system. The physiological results showed there was no effect of the high cholesterol diet on the severity of lung injury in any of the three models of injury. There was also no effect of the diet on surfactant cholesterol composition. Rats fed a high cholesterol diet had a significant impairment in surface tension reducing capabilities of isolated surfactant compared to those fed a standard diet exposed to the surfactant depletion injury. In addition, only rats that were exposed to ventilation induced lung injury had elevated levels of surfactant associated cholesterol compared to non-injured rats. It is concluded that serum hypercholesterolemia does not predispose rats to altered surfactant cholesterol composition or to lung injury. Elevated cholesterol within surfactant may be a marker for ventilation induced lung damage.

Antimicrobial and biophysical properties of surfactant supplemented with an antimicrobial peptide for treatment of bacterial pneumonia.

Antibiotic-resistant bacterial infections represent an emerging health concern in clinical settings, and a lack of novel developments in the pharmaceutical pipeline is creating a "perfect storm" for multidrug-resistant bacterial infections. Antimicrobial peptides (AMPs) have been suggested as future therapeutics for these drug-resistant bacteria, since they have potent broad-spectrum activity, with little development of resistance. Due to the unique structure of the lung, bacterial pneumonia has the additional problem of delivering antimicrobials to the site of infection. One potential solution is coadministration of AMPs with exogenous surfactant, allowing for distribution of the peptides to distal airways and opening of collapsed lung regions. The objective of this study was to test various surfactant-AMP mixtures with regard to maintaining pulmonary surfactant biophysical properties and bactericidal functions. We compared the properties of four AMPs (CATH-1, CATH-2, CRAMP, and LL-37) suspended in bovine lipid-extract surfactant (BLES) by assessing surfactant-AMP mixture biophysical and antimicrobial functions. Antimicrobial activity was tested against methillicin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. All AMP/surfactant mixtures exhibited an increase of spreading compared to a BLES control. BLES+CATH-2 mixtures had no significantly different minimum surface tension versus the BLES control. Compared to the other cathelicidins, CATH-2 retained the most bactericidal activity in the presence of BLES. The BLES+CATH-2 mixture appears to be an optimal surfactant-AMP mixture based on in vitro assays. Future directions involve investigating the potential of this mixture in animal models of bacterial pneumonia.