Chronic lung inflammation by Pseudomonas aeruginosa is an important driver of pathogenesis in patients with cystic fibrosis (CF), chronic obstructive pulmonary disease, and non-CF bronchiectasis. Therapies directed at the host inflammatory response hold great promise since it is the major process leading to destruction of lung tissue during infection. The objective of this project is to develop an effective therapy against P. aeruginosa-induced chronic lung inflammation, by targeting bacterial pro-inflammatory mediators expressed in vivo. Nanobodies or variable antigen-binding domains (VHHs) are our method of choice due to the exquisite specificity, small size and high stability, which makes them ideally suited for inhalation delivery. Anti-inflammatory VHHs will be developed against the following targets: 1) pseudolysin, a known pro-inflammatory virulence factor expressed in vivo by P. aeruginosa, 2) novel pro-inflammatory factors that are relevant for the in vivo chronic infection process. These novel mediators will be identified by culturing P. aeruginosa under “in vivo-like” conditions (i.e. synthetic sputum medium and differentiated CF bronchial epithelial cells). Subsequently, VHHs combinations targeting multiple pro-inflammatory targets will be designed and their ability to reduce inflammation in vitro and in vivo will be validated.