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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.

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.

Background Lung inflammation is associated with many respiratory conditions. Consequently, anti-inflammatory medications, like glucocorticoids, have become mainstay intrapulmonary therapeutics. However, their effectiveness for treating inflammation occurring in the alveolar regions of the lung is limited by suboptimal delivery. To improve the pulmonary distribution of glucocorticoids, such as budesonide to distal regions of the lung, exogenous surfactant has been proposed as an ideal delivery vehicle for such therapies. It was therefore hypothesized that fortifying an exogenous surfactant (BLES) with budesonide would enhance efficacy for treating pulmonary inflammation in vivo. Methods An intratracheal instillation of heat-killed bacteria was used to elicit an inflammatory response in the lungs of male and female rats. Thirty minutes after this initial instillation, either budesonide or BLES combined with budesonide was administered intratracheally. To evaluate the efficacy of surfactant delivery, various markers of inflammation were measured in the bronchoalveolar lavage and lung tissue. Results Although budesonide exhibited anti-inflammatory effects when administered alone, delivery with BLES enhanced those effects by lowering the lavage neutrophil counts and myeloperoxidase activity in lung tissue. Combining budesonide with BLES was also shown to reduce several other pro-inflammatory mediators. These results were shown across both sexes, with no observed sex differences. Conclusion Based on these findings, it was concluded that exogenous surfactant can enhance the delivery and efficacy of budesonide in vivo.

Background Inflammation associated with diseases like Acute Respiratory Distress Syndrome (ARDS) and Bacterial Pneumonia, often occurs in the deeper, alveolar, areas of the lung. In these circumstances the complex branching structure of the lung, its large surface area, and associated areas of airway collapse provide substantial hurdles for adequate delivery of anti‐inflammatory drugs to remote regions of inflammation. To address this, our lab has utilized a bovine derived exogenous surfactant (BLES) as a pulmonary vehicle to facilitate the transport of a glucocorticoid (budesonide). Budesonide is a strong anti‐inflammatory drug currently used in the lung to treat asthma, while BLES can open collapsed airways and spread to distal sites within the lung. Hypothesis Combining budesonide with a bovine derived exogenous surfactant will enhance its delivery and efficacy for treating pulmonary inflammation. Methods Our hypothesis was tested using both in vitro and in vivo methodology. For in vitro studies the wet bridge transfer system was utilized to assess spreading and efficacy of budesonide alone or in combination with BLES across an air‐liquid interface. In this system, macrophages were seeded to a remote site and stimulated with heat‐killed bacteria (HKB). Treatments were then administered to a delivery site and IL‐6 concentrations were measured at the remote site. An in vivo model of pulmonary inflammation was created by instilling either saline (control) or HKB into the lungs of male and female rats. This first instillation was followed 30 minutes later by a second instillation of either saline, budesonide or BLES/budesonide. Rats were then monitored for six hours before being euthanized. A bronchoalveolar lavage (BAL) was performed, followed by cell counts and differentials. Results The in vitro data showed that administering BLES or budesonide alone had no effect on IL‐6 concentrations at the remote site, across the air‐liquid interface. However, the administration of BLES/budesonide significantly reduced IL‐6 content at the remote site. Data collected from the in vivo experiment indicates that instillation of HKB significantly increased the number of inflammatory cells and neutrophils in the BAL compared to the control. Budesonide alone was able to show a reduction in the number of neutrophils in the BAL. However, BLES/budesonide showed significant reductions in both the number of inflammatory cells and neutrophils in the BAL compared to budesonide and HKB groups. Discussion The in vitro data indicates that BLES/budesonide is more effective at reaching and eliciting an anti‐inflammatory effect at a distal site than budesonide alone. Moreover, administering budesonide with BLES in vivo resulted in significant improvements in drug delivery and efficacy. Further measurements of pulmonary inflammation will include myeloperoxidase assays as well as quantifying pro‐inflammatory cytokine mRNA and protein through qPCR and ELISA assays respectively. This novel strategy of utilizing a spreading agent to delivery budeso...

The rising incidence of antibiotic-resistant lung infections has instigated a much-needed search for new therapeutic strategies. One proposed strategy is the use of exogenous surfactants to deliver antimicrobial peptides (AMPs), like CATH-2, to infected regions of the lung. CATH-2 can kill bacteria through a diverse range of antibacterial pathways and exogenous surfactant can improve pulmonary drug distribution. Unfortunately, mixing AMPs with commercially available exogenous surfactants has been shown to negatively impact their antimicrobial function. It was hypothesized that the phosphatidylglycerol component of surfactant was inhibiting AMP function and that an exogenous surfactant, with a reduced phosphatidylglycerol composition would increase peptide mediated killing at a distal site. To better understand how surfactant lipids interacted with CATH-2 and affected its function, isothermal titration calorimetry and solid-state nuclear magnetic resonance spectroscopy as well as bacterial killing curves against Pseudomonas aeruginosa were utilized. Additionally, the wet bridge transfer system was used to evaluate surfactant spreading and peptide transport. Phosphatidylglycerol was the only surfactant lipid to significantly inhibit CATH-2 function, showing a stronger electrostatic interaction with the peptide than other lipids. Although diluting the phosphatidylglycerol content in an existing surfactant, through the addition of other lipids, significantly improved peptide function and distal killing, it also reduced surfactant spreading. A synthetic phosphatidylglycerol-free surfactant however, was shown to further improve CATH-2 delivery and function at a remote site. Based on these in vitro experiments synthetic phosphatidylglycerol-free surfactants seem optimal for delivering AMPs to the lung.

BackgroundBacterial pneumonia is a leading cause of death worldwide. Unfortunately, new treatments are faced with several major hurdles. Firstly, the incidence of antibiotic resistance is increasing. Secondly, both acute and chronic lung infections are often accompanied by maladaptive inflammatory responses linked to poor outcomes. Finally, the structure of the lung makes delivery of therapeutics to the sites of infection challenging. As a potential treatment for bacterial pneumonia, the current study combines a host‐defense peptide (CATH‐2), previously shown to kill antibiotic‐resistant bacteria and reduce inflammation, with an exogenous surfactant (BLES), capable of enhancing spreading throughout the lung.Objectives1) Quantify the transport CATH‐2 by BLES in vitro, 2) Assess the antimicrobial and anti‐inflammatory properties of BLES+CATH‐2 subsequent to spreading across a surface and 3) Investigate the immunomodulatory effects of BLES+CATH‐2 in vivo.HypothesisThe mixture of BLES+CATH‐2 will improve transport of CATH‐2 allowing for effective bacterial killing and reductions in inflammation at distal sites in vitro and in vivo.MethodsFluorescently‐labelled CATH‐2 was used to track its movement as it spread across a Wet Bridge Transfer system alone or in combination with BLES. Bacterial killing and anti‐inflammatory properties were assessed by seeding either a lab strain of Pseudomonas aeruginosa or RAW 264.7 macrophages to the distal well of the wet bridge system. The macrophages were stimulated with heat‐killed P. aeruginosa 15 minutes prior to the administration of saline, BLES, CATH‐2 or BLES+CATH‐2 in the proximal well. The fluid in each well was analyzed for cytokine content and bacterial killing. Additionally, a non‐infectious model of bacterial pneumonia was used, where mice were instilled with heat‐killed P. aeruginosa or saline. This first instillation was then followed by either saline, BLES, CATH‐2 or BLES+CATH‐2. All mice were monitored for 4 hours before being euthanized. Bronchoalveolar lavage fluid was collected and analyzed for cell counts, cell differentials, and cytokine concentrations.ResultsFluorescence spectrometry revealed that significantly more CATH‐2 was transferred across the bridge when combined with BLES compared to CATH‐2 by itself. Additionally, only the combination of BLES+CATH‐2 showed significant improvements in bacterial killing and reducing inflammation across the wet bridge. Mice administered heat‐killed bacteria showed significant increases in the number of inflammatory cells, neutrophils and lavage IL‐6, TNF‐α and KC content compared to saline control. Instillation of BLES+CATH‐2 after an instillation of heat‐killed bacteria showed significant reductions across all markers of inflammation compared to saline, BLES or CATH‐2 alone.DiscussionThese results support BLES as an effective vehicle for the transport of CATH‐2 and that the mixture has potent antimicrobial and anti‐inflammatory properties. Our novel approach allowed us to rapidly assess the efficacy and spreading capabilities of BLES+CATH‐2. Additionally, the results support BLES+CATH‐2 as a therapy which can overcome the delivery problem hindering pulmonary therapies and reach distal sites of inflammation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.