Background and Aims: Pulmonary hypertension (PH) is accompanied by detrimental pulmonary vascular and right ventricular myocardial remodeling. One strategy to overcome this problem is the targeted delivery of Interleukin-9 using the human recombinant F8 antibody as vehicle in terms of fusion proteins (F8-IL9). Preliminary data of our group could demonstrate beneficial effects of F8-IL9 treatment in a preclinical PH mouse model. Nevertheless, the local mode of IL-9 action has not been elucidated yet. Thus, our current study was aimed to address this question by focusing on mast cells (MCs) and regulatory T cells (Tregs) as two main cells types being regulated by IL-9.

Methods: The mouse model of Monocrotaline (MCT) induced PH (PH, n=12) was chosen to evaluate treatment effects of F8-IL9 formats (n=18) compared to sham-treated animals (sham, n=10), a dual endothelin receptor antagonist (dual ERA, n=12) or IL9 based immunocytokines with irrelevant antibody specificity (KSF-IL9, n=18). Besides validation of treatment effects on the hemodynamic (right heart catheterization), echocardiographic and histological levels, the study was focused on the detection of MCs and Tregs by immunohistochemistry in lung and cardiac tissue as well as using FACS analysis in the circulation. In addition, pre-selected potential down-stream cytokines, e.g., Interleukin-10 (IL-10), Interleukin-35 (IL-35) or CXCL12 were detected in the circulation via ELISA.

Results:

There was a significantly elevated systolic right ventricular pressure (RVP_sys) as well as an impairment of a variety of echocardiographic parameters of right ventricular form and function, e.g., right ventricular basal diameter (RV_basal) or tricuspid annular plane systolic excursion (TAPSE), in untreated PH compared to the sham group (p<0.05). Although these effects could be reversed for single parameters, e.g., RVP_sys, by dual ERA treatment (p<0.05), only the group treated with F8-IL9 but not the group treated with KSF-IL9 showed consistent beneficial effects for the majority of hemodynamic and echocardiographic parameters (p<0.05). On the histological level, only F8-IL9 treatment significantly reduced lung tissue damage (p<0.05). For MCs, no relevant quantitative differences in both, lung and right ventricular tissue, could be detected between the groups (p=n.s.). With respect to Tregs, we observed a significant increase in lung but not cardiac tissue compared to sham animals in all experimental groups except of the dual ERA group (p<0.05). Most strikingly, there was an enormous increase especially in the IL9 treated animals (p<0.05). FACS analysis of circulating of MCs and Tregs revealed not differences for MCs between the groups (p=n.s.). In contrast, there was a significant decrease of circulating Tregs in PH compared to sham animals (p=0.014), which was reversed in the IL9 treated groups (p<0.05). There was a significant increase in IL-10 serum levels when comparing the targeted IL-9 (F8-IL9) to the sham treated (p=0.044) or the dual ERA group (p=0.017).        

Conclusions: In conclusion, the beneficial treatment effects of targeted IL-9 delivery in a preclinical model of PH could be convincingly validated in a large number of animals. IL-9 mediated recruitment of Tregs into lung tissue might play a crucial role for the induction of anti-inflammatory and antiproliferative mechanisms potentially contributing to a novel disease-modifying concept for the treatment of PH.