Airway dehydration causes mucus stasis and bacterial overgrowth in cystic fibrosis and chronic bronchitis (CB). Rehydration by hypertonic saline is efficacious but suffers from a short duration of action. We tested whether epithelial sodium channel (ENaC) inhibition would rehydrate normal and dehydrated airways to increase mucociliary clearance (MCC) over a significant time frame. For this, we used a tool compound (Compound A), which displays nanomolar ENaC affinity and retention in the airway surface liquid (ASL). Using normal human bronchial epithelial cultures (HBECs) grown at an air-liquid interface, we evaluated in vitro potency and efficacy using short-circuit current (I(sc)) and ASL height measurements where it inhibited I(sc) and increased ASL height by ∼ 50% (0.052 μM at 6 h), respectively. The in vivo efficacy was investigated in a modified guinea pig tracheal potential difference model, where we observed an effective dose (ED50) of 5 μg/kg (i.t.), and by MCC measures in rats and sheep, where we demonstrated max clearance rates at 100 μg/kg (i.t.) and 75 μg/kg (i.t.), respectively. Acute cigarette smoke-induced ASL height depletion in HBECs was used to mimic the situation in patients with CB, and pretreatment prevented both cigarette smoke-induced ASL dehydration and lessened the decrease in ciliary beat frequency. Furthermore, when added after cigarette smoke exposure, Compound A increased the rate of ASL rehydration. In conclusion, Compound A demonstrated significant effects and a link between increased airway hydration, ciliary function, and MCC. These data support the hypothesis that ENaC inhibition may be efficacious in the restoration of mucus hydration and transport in patients with CB.

Type 2 Innate lymphoid cells (ILC2s) are implicated in helminth infections and asthma where they play a role in the production of Th2-type cytokines. ILC2s express the IL-33 receptor and are a major cell type thought to mediate the effects of this cytokine in vivo. To study the signalling pathways that mediate IL-33 induced cytokine production, a culture system was set up to obtain pure populations of ILC2s from mice. Inhibitors of the p38α/β and ERK1/2 MAPK pathways reduced the production of IL-5, IL-6, IL-9, IL-13 and GM-CSF by ILC2 in response to IL-33, with inhibition of p38 having the greatest effect. MK2 and 3 are kinases activated by p38α; MK2/3 inhibitors or knockout of MK2/3 in mice reduced the production of IL-6 and IL-13 (two cytokines implicated in asthma) but not IL-5, IL-9 or GM-CSF in response to IL-33. MK2/3 inhibition also suppressed IL-6 and IL-13 production by human ILC2s. MK2/3 were required for maximal S6 phosphorylation, suggesting an input from the p38α-MK2/3 pathway to mTOR1 activation in ILC2s. The mTORC1 inhibitor rapamycin also reduced IL-6 and IL-13 production, which would be consistent with a model in which MK2/3 regulate IL-6 and IL-13 via mTORC1 activation in ILC2s.Innate lymphoid cells (ILCs) are a recently identified group of lymphocytes acting within the innate immune system. ILCs lack antigen specific receptors, however in terms of the cytokines they produce they mimic various Th cell subsets. ILCs are found in both lymphoid and non-lymphoid tissues, particularly at the barrier sites including skin, respiratory and gastrointestinal systems 1-4 . Based on their effector function, cell surface markers and the transcription factors required for their development, ILCs can be subdivided into several distinct groups 1,2 . Group 1 includes NK cells and ILC1 cells, which are involved in protective immunity to viral infections and antimicrobial responses. The second group comprises of type 2 innate lymphoid cells (ILC2). ILC2 cells secrete type two cytokines and are implicated in responses to helminth infection, asthma and atopic diseases. The third group includes the lymphoid tissue inducer cells and ILC3. In 2017, a potential new group of ILC with regulatory functions was discovered. ILCreg are found in human and mouse gut and limit the activity of ILC1 and ILC3 by secreting the anti-inflammatory cytokine IL-10 5 .The first evidence for the existence of ILC2 cells was found in 2006 when Fallon et al. showed that during infection with the helminth Nippostrongylus brasilienis a population of non-T and non-B cells secrete IL-4, IL-5 and IL-13 and promote helminth expulsion 6 . In 2010, several labs reported the characterisation of ILC2s as a distinct population of cells, which were initially referred to as either neuocytes, innate helper 2 cells or natural helper cells 7-9 . ILC2s are found in the lymphoid tissues such as spleen and mesenteric lymph nodes, as well as in some non-lymphoid organs including fat associated lymphoid clusters, lungs, skin and liver. In mice, ILC2 are character...

In preclinical research, allergic asthma is investigated in rats sensitised with the antigen ovalbumin (OVA), followed by a challenge with aerosolised OVA to induce an inflammatory reaction of the lower airways. This causes diffuse, nonfocal ventilation defects that lead to heterogeneously distributed signal intensities in hyperpolarised (HP) (3)He MR images, which are difficult to assess directly by diagnostic grading or volumetry. Texture analysis can characterise these changes and does not require segmentation of the lung structures prior to the analysis. The aim of this work was to evaluate a texture analysis approach to quantify changes in lung ventilation in HP (3)He MRI of OVA-challenged rats. OVA-challenged animals were treated with two different compound doses to evaluate the sensitivity of the texture analysis. Four groups were investigated using HP (3)He MRI at 4.7 T: controls, vehicle-treated, and low- and high-dose budesonide-treated rats. In addition, broncho-alveolar lavage was performed and the eosinophil cell count was used as a biological reference marker. First-order texture, geometrical features and features based on second-order statistics using run-length and grey-level co-occurrence matrices were calculated. In addition, wavelet transforms were applied to compute first-order statistics on multiple scales. The texture analysis was able to show significant differences between the control and untreated vehicle groups as well as between the vehicle and treatment groups. This is in agreement with the findings of the eosinophil cell counts, which were used as a marker for the severity of inflammation. However, not all features used in the different texture analysis methods could differentiate between the treatment groups. In conclusion, texture analysis can be used to quantify changes in lung ventilation as measured with HP (3)He MRI after therapeutic intervention with budesonide.

Pulmonary diseases are known to be largely inhomogeneous. To evaluate such inhomogeneities, we are testing an image-based method to measure gas flow in the lung regionally. Dynamic, spin-density-weighted hyperpolarized (3)He MR images performed during slow inhalation of this gas were analyzed to quantify regional inflation rate. This parameter was measured in regions of interest (ROIs) that were defined by a rectangular grid that covered the entire rat lung and grew dynamically with it during its inflation. We used regional inflation rate to quantify elastase-induced emphysema and to differentiate healthy (n = 8) from elastase-treated (n = 9) rat lungs as well as healthy from elastase-treated areas of one rat unilaterally treated with elastase in the left lung. Emphysema was also assessed by gold standard morphological and well-established hyperpolarized (3)He MRI diffusion measurements. Mean values of regional inflation rates were significantly different for healthy and elastase-treated animals and correlated well with the apparent diffusion coefficient of (3)He and morphological measurements. The image-based biomarker inflation rate may be useful for the assessment of regional lung ventilation.