**4. Discussion**

In this study, oral OmeGo significantly reduced lung and splenic eosinophilia compared to cod liver oil (vehicle control) in a house dust mite (HDM) mouse model of asthma. These results are consistent with previous work that assessed OmeGo's modulation of eosinophil function, including in vivo studies with OmeGo dosed via intraperitoneal injection [12] and in vitro work in human eosinophils [13].

This study further characterised OmeGo's modulation of inflammatory mediators relevant to asthma pathophysiology. Beyond eosinophil modulation, oral OmeGo significantly reduced BAL (bronchoalveolar lavage) neutrophil levels and total lung collagen content. Airway remodelling is a typical feature in persistent asthma, contributing to airflow limitation. Collagen deposition occurs early in the natural history of asthma [17,18] and is correlated with disease severity [19]. Allergen exposure causes an increase in airway remodelling markers in patients with asthma [20], with IL-13 an underlying driver of the process. Thus, OmeGo's significant impact on IL-13 provides biologic plausibility to this initial result in assessing OmeGo's potential to reduce lung remodelling [21].

IL-4 is an important driver in the initiation of lung inflammation in asthma, including Th2 cell proliferation and IgE synthesis [22,23], and high-dose OmeGo significantly reduced IL-4 by 17% (*p* < 0.05); however, the numeric reduction in serum IgE did not quite achieve significance.

The house dust mite is a common air-borne allergen, with up to 85% of asthma patients being allergic to HDM [24]. The HDM model of induced asthma is, therefore, commonly used to mimic the inflammatory and allergic milieu found in many asthma patients, namely eosinophilia, raised IgE levels and other inflammatory mediators associated with Type 2 inflammation as well as neutrophilia [24,25]. Consistent with the published literature, HDM sensitisation in our study resulted in a leukocytosis driven by eosinophils and neutrophils. We saw no impact on lymphocyte numbers and only a limited impact on macrophage numbers, and previous work indicates that longer duration HDM models

are more likely to induce a significant expansion of macrophage numbers [26] and drive greater fold increases overall in leukocyte recruitment [27,28].

A 5-week HDM mouse model of induced asthma demonstrated a 500-fold increase in BAL eosinophils, equating to 1.5 million eosinophils/mL on flow cytometry [27]. In contrast, a short duration, higher HDM dose (total of 300 μg) in vivo study resulted in a BAL eosinophilia of around 350,000 cells/mL in the saline control group, which was almost totally resolved with intraperitoneal dexamethasone and reduced by approximately 50% with CRTH2 antagonist treatment [29]. We utilized a standard short-exposure HDM model protocol with a total HDM challenge dose of 50 μg; this resulted in an eosinophil count of 97,400 cells/mL in the negative control group (saline). This lower level of induced leukocytosis may explain the smaller magnitude of cell count reductions with CRTH2 antagonism (fevipiprant) in our study, such as a 20% decrease in eosinophilia, compared to previous ex vivo studies with CRTH2 antagonists. This methodological difference may also have influenced the magnitude of effect observed with OmeGo.

The CRTH2 receptor is a well recognised activator of eosinophil-driven inflammation [30,31], hence our choice of the CRTH2 antagonist (fevipiprant) as a positive control. The effects of fevipiprant and OmeGo were generally similar with regard to lung and systemic inflammation, with significant reductions in both eosinophil and neutrophil counts as well as total lung collagen. CRTH2 antagonists are known to inhibit experimental allergen challenge responses in humans [32,33], and the similarity of the effects observed in this in vivo animal model indicates potential for OmeGo to attenuate allergic responses in humans.

Vehicle control, consisting of 0.5 mL cod liver oil, containing DHA (docosahexanoic acid) and EPA (eicosapentaenoic acid) omega-3, did not show an effect on any of the endpoints compared to negative control. This contrasting effect between OmeGo and cod liver oil is consistent with our previous work on the modulation of eosinophil function with OmeGo. This effect appears to be driven by a bioactive fraction not present in highly processed supplements [12,13]. The variable outcomes seen with omega-3 supplementation compared to the consumption of fresh fish [10] suggests that minimal processing helps to retain the full bioactivity of the individual PUFAs to provide health benefits associated with eating fresh fish.

Our study has a number of limitations. By using a shorter-duration HDM exposure model, we did not elicit an elevation in lymphocytes and only a moderate increase in macrophage count, which, therefore, limits the insights on the potential of the interventions to modulate the activity of these immune cells. In addition, analyzing other mediators of inflammation would have been valuable, but there was insufficient serum to analyse changes in IL-5, a cytokine involved in eosinophil activation and recruitment. The elevation of IL-17 was not modulated by OmeGo, and other analyses for inflammatory mediators such as the impact on IL-1β and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the BAL could have provided useful insights into OmeGo's impact on lung barrier function and provided mechanistic explanations for the modulation of lung collagen deposition. Investigation of primary inflammatory pathway mediators, such as nuclear factor kappa B (NFκB), GATA-3, and peroxisome proliferator-activated receptor gamma (PPARγ), would have been valuable to assess the means by which OmeGo reduced lung inflammation. Additionally, further immunohistochemistry work, including assessments of lung damage and collagen deposition, could provide more insights regarding pharmacological effects. Nevertheless, the study builds on previous work and elucidates further the anti-inflammatory action of the whole fish oil, OmeGo.
