Inflammation is a multifactorial defense process involved in numerous pathologies. Its exacerbation is a hallmark of several pulmonary diseases, particularly cystic fibrosis (CF), a genetic disorder caused by mutations in the chloride channel CFTR, most commonly F508del. This mutation leads to chronic pulmonary inflammation, often present from the early stages of the disease, independently of any infection. It is now well established that the lack of the activity of the CFTR channel generates an environment that facilitate the infiltration of neutrophils (PMNs) by transmigration across epithelial and endothelial barriers, thereby worsening the inflammatory response.
Although CFTR modulators have improved patients' quality of life by partially restoring the activity of CFTR-F508del chloride channel, their anti-inflammatory effects remain limited. This suggests the involvement of additional mechanisms in CF-related inflammation, that could be independent of CFTR transport activity. Among these mechanisms, are interactions between epithelial and endothelial cells via inflammatory and angiogenic factors.
Our team focused on PROK1 (also known as EG-VEGF), a circulating factor from the prokineticin family, and its receptors PROKR1 and PROKR2. Although dysregulation of this signaling axis has been observed in CF, its role in inflammation, infection, and the integrity of cellular barriers remains poorly understood.
My PhD project was structured around two main objectives:
(1) Characterizing the inflammatory response of bronchial epithelial cells in relation to CFTR modulators and CFTR restoration.
(2) Exploring the role of PROK1 in inflammation and the permeability of human pulmonary microvascular endothelial cells (HPMECs).
First, we compared the anti-inflammatory effects of CFTR-F508del modulators to wild-type CFTR overexpression, using various bronchial cell lines (HBE, CFBE, CFBE-dF, CFBE-wt). Our results show that modulators exert only a limited effect on the inflammatory response, unlike the overexpression of wild-type CFTR. The limited response to modulators is likely due to insufficient folding and function of the CFTR-F508del protein, as confirmed by in situ protease resistance assays.
Next, we evaluated the response of HPMECs to inflammatory stimuli by assessing their proliferation, migration, invasion, and ability to form vascular-like structures. Our analyses demonstrate that these cells express PROKR1 and PROKR2, secrete PROK1, and display either pro- or anti-inflammatory phenotypes depending on the type of stimulus. Moreover, we demonstrated that the integrity of the endothelial barrier was impaired in the presence of inflammation, infection (LPS), or PROK1, which facilitate neutrophil transmigration. This was confirmed by transmigration assays using HL60 cells differentiated into neutrophil-like cells, which crosse the HPMEC barrier more easily under stressful conditions.
Overall, this work highlights the limited anti-inflammatory effect of CFTR modulators and reveals the involvement of PROK1 in promoting vascular permeability. A better understanding of the functioning of endothelial in pathological setting such as CF, may open new therapeutic avenue through the specific and early targeting of inflammation.

Supervision of the thesis :

Mohamed BENHAROUGA