Combating Pseudomonas aeruginosa lung infections using synthetic host defense peptides

Pseudomonas aeruginosa is a Gram negative bacterium found frequently in the environment. It can infect immunocompromised patients and is a major cause of nosocomial infections. Of particular concern are its roles in lung infections as a causative agent for pneumonia and in respiratory infections in patients with cystic fibrosis and chronic obstructive pulmonary disease. Treatment of P. aeruginosa lung infections is difficult due to its formation of biofilms and the development of multi-drug resistant P. aeruginosa strains. Synthetic derivatives of host defense peptides (HDPs) called innate defense regulators (IDRs) are alternatives to antibiotics that modulate the immune response rather than directly targeting the bacteria, thus limiting the development of antibiotic resistance. IDRs have shown success against many bacteria but had not previously been tested in P. aeruginosa lung infection models. In this work, IDR-1002 reduced the production of inflammatory cytokines by macrophages in response to P. aeruginosa lipopolysaccharide. IDR-1002 also limited the toxicity caused by live P. aeruginosa to macrophages and bronchial epithelial cells. Importantly, IDR-1002 did not show any toxic effects in vitro, unlike the HDP LL-37. In an acute in vivo P. aeruginosa lung infection model, IDR-1002 significantly decreased the bacterial burden as well as the concentrations of MCP-1, KC, and IL-6 in the lungs. In another in vivo P. aeruginosa lung infection model using alginate to mimic a chronic infection, IDR-1002 decreased the infiltration of cells to the infection site and significantly decreased IL-6 levels in the lungs. To improve drug delivery, peptide IDR-1018, which has a strong aggregation propensity, was tested with various formulations, and its combination with a hyperbranched polyglycerol reduced the production of inflammatory cytokines in vitro and trended towards reducing cytokines in vivo in the acute P. aeruginosa lung infection model. Finally, RNA-Seq and downstream bioinformatics were performed on both lung and blood samples from the acute P. aeruginosa lung infection model, providing insights into the impact of P. aeruginosa during infection and the protective mechanisms of IDR-1002 via its anti-inflammatory effects. These data suggest the strong potential of IDR-1002 for the treatment of P. aeruginosa lung infections.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat

IDR-1002 mice demonstrated improved lung appearance in the alginate model.

<p>Mice were given alginate mixed with <i>P</i>. <i>aeruginosa</i> LESB58 (7 x 106 CFU/mouse) IN at 0 h, then IDR-1002 (12 mg/kg) or control IN at 18 h, then euthanized at 42 h. Vehicle control infected mice (A) demonstrated lesions (black arrows), whereas in the IDR-1002 mice (B) the lesions were reduced or eliminated. H&E staining in the control mice (C) showed damage, while in IDR-1002 mice (D) it was reduced. This was also seen with H&E staining in control mice (E) or IDR-1002 mice (F) at 20x.</p

Synthetic host defense peptide IDR-1002 reduces inflammation in <i>Pseudomonas aeruginosa</i> lung infection

<div><p><i>Pseudomonas aeruginosa</i> is a frequent cause of lung infections, particularly in chronic infections in cystic fibrosis patients. However, treatment is challenging due to <i>P</i>. <i>aeruginosa</i> evasion of the host immune system and the rise of antibiotic resistant strains. Host defense peptides (HDPs) and synthetic derivatives called innate defense regulators (IDRs) have shown promise in several infection models as an alternative to antibiotic treatment. Here we tested peptide IDR-1002 against <i>P</i>. <i>aeruginosa</i> in vitro and in vivo. Treatment of bronchial epithelial cells and macrophages with IDR-1002 or in combination with live <i>P</i>. <i>aeruginosa</i> or its LPS led to the reduction of agonist-induced cytokines and chemokines and limited cell killing by live <i>P</i>. <i>aeruginosa</i>. In an in vivo model using <i>P</i>. <i>aeruginosa</i> combined with alginate to mimic a chronic model, IDR-1002 did not reduce the bacterial burden in the lungs, but IDR-1002 mice showed a significant decrease in IL-6 in the lungs and in gross pathology of infection, while histology revealed that IDR-1002 treated mice had reduced alveolar macrophage infiltration around the site of infection and reduced inflammation. Overall, these results indicate that IDR-1002 has promise for combating <i>P</i>. <i>aeruginosa</i> lung infections and their resulting inflammation.</p></div

HBE cells showed increases in IL-6 and IL-8 in response to peptide.

<p>Peptide was added to the cells, then supernatants were collected after 24 h for ELISAs. IDR-1002 significantly increased IL-6 (A) and IL-8 (B), and LL-37 significantly increased IL-6 (C) and IL-8 (D). Data represent mean ± SEM from four independent experiments and were analyzed using two-way ANOVA and Dunnett’s multiple comparisons test. **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001 compared to control.</p

<i>P</i>. <i>aeruginosa</i> LESB58 was present two days post-infection when delivered with alginate.

<p>Mice were given alginate or alginate mixed with <i>P</i>. <i>aeruginosa</i> LESB58 (7 x 10⁶ CFU/mouse) IN at 0 h, then euthanized and samples processed at 42 h. (A) CFU counts from the lung homogenate. (B) Health scores over the course of the experiment. (C) Percentage of original weight for mice over the course of the experiment. ELISAs were performed for MCP-1 in lung homogenate (D) and serum (E); KC in lung homogenate (F) and serum (G); IL-6 in lung homogenate (H) and serum (I); and TNF-α in lung homogenate (J). Data represent mean ± SEM for n = 4 or 16 mice per condition from the combination of two experiments and were analyzed using unpaired t-test with Welch’s correction for 6A and 6D-6J, and with a repeated measures two-way ANOVA with Sidak’s multiple comparison for 6B and 6C. **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001.</p

IDR-1002 mice showed decreased inflammation and alveolar macrophages in assessment of histopathology in the alginate model.

<p>IDR-1002 mice showed decreased inflammation and alveolar macrophages in assessment of histopathology in the alginate model.</p

IL-6 and IL-8 production in HBE cells in response to live <i>P</i>. <i>aeruginosa</i> PA103.

<p>PA103 (MOI 4) was added at 0 h, then IDR-1002 (50 μM) or LL-37 (3 μM) was added at -1 h (A, B) or 0 h (C, D). Samples were collected at 6 h and used for ELISAs for IL-6 (A, C) or IL-8 (B, D). Data represent mean ± SEM from four independent experiments and were analyzed using two-way ANOVA and Dunnett’s multiple comparisons test. *: p ≤ 0.05, **: p ≤ 0.01, ****: p ≤ 0.0001 compared to PA103.</p

IDR-1002 limited cytotoxicity of <i>P</i>. <i>aeruginosa</i> in HBE and RAW cells, whereas LL-37 increased it.

<p>(A, B) Live <i>P</i>. <i>aeruginosa</i> PA103 (MOI 4) was added to HBE cells at 0 h, IDR-1002 (50 μM) or LL-37 (3 μM) was added at -1 h (A) or 0 h (B), then samples were collected at 6 h. (C, D) Live <i>P</i>. <i>aeruginosa</i> PA103 (MOI 1) was added to RAW cells at 0 h, IDR-1002 (50 μM) or LL-37 (12.5 μM) was added at -1 h (C) or 0 h (D), then samples were collected at 4 h. For both RAW and HBE cells, Triton X-100 was added at 0 h and samples were compared to a control (0% toxicity) and Triton X-100 (100% toxicity) for each time point. Data represent mean ± SEM from four independent experiments and were analyzed using two-way ANOVA and Tukey’s multiple comparisons test. *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001 when compared to PA103. †††: p ≤ 0.001, ††††: p ≤ 0.0001 when samples compared as indicated in the figure.</p

IDR-1002 and LL-37 reduced LPS-induced cytokines and chemokine MCP-1 in RAW cells.

<p>The listed concentrations of IDR-1002 or LL-37 were added with or without <i>P</i>. <i>aeruginosa</i> LPS (10 ng/ml), then supernatants were collected after 24 h. Without LPS, IDR-1002 and LL-37 did not induce any of the cytokines or chemokine. IDR-1002 decreased LPS-induced IL-6 (A), TNF-α (B), and MCP-1 (C). LL-37 also decreased LPS-induced IL-6 (D), TNF-α (E), and MCP-1 (F). Data represent mean ± SEM from four independent experiments expressed as fold-change relative to LPS and were analyzed using two-way ANOVA and Dunnett’s multiple comparisons test. Only the significance for samples given LPS is displayed. *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001 compared to LPS.</p