Background: Inflammation and consecutive pulmonary vascular remodeling are two main key processes in the pathogenesis of pulmonary hypertension (PH). Recently, we investigated a novel treatment strategy applying Interleukin-9 (IL9) capable to induce both, pro- and anti-inflammatory signals, directly to the site of disease in a mouse model of PH. Therefore, IL9 was fused to the F8 antibody specifically recognizing the extra-domain A of Fibronectin, which is not expressed in healthy adult tissue but shows a strong re-occurrence in PH. We observed beneficial effects in terms of disease attenuation not occurring in sham-treated controls.

Purpose: We hypothesize F8IL9 therapy to exert immunomodulatory properties creating an anti-inflammatory milieu. Therefore, we performed pathway-focused gene expression analysis of inflammatory response and autoimmunity in lung tissue.

Methods: The Monocrotaline (MCT) method was used to induce PH in mice (n=44) and the following experimental groups were subjected to RT-PCR based gene expression analysis of inflammatory response and autoimmunity (84 genes) in lung tissue: sham-induced animals without PH (control, n=4), MCT-induced PH without treatment (PH, n=8), with dual endothelin receptor antagonist treatment (dual ERA, n=8), with F8IL9 treatment (n=12, 2 formats with n=6 each) or with KSFIL9 treatment (KSFIL9, n=12, 2 formats with n=6 each, KSF: control antibody with irrelevant antigen specificity).

Results: In the PH group without treatment, 20 genes showed relevant (±2.5-fold) expression dynamics compared to controls, 19 of these genes were up-regulated with Chemokine (C-C-motif) ligand 20 (CCL20; +58.9-fold), Chemokine (C-X-C-motif) ligand 5 (CXCL5; +26.1-fold) and Interleukin-17A (IL17A; +7.5-fold) as the most prominent genes. The only gene showing down-regulation was inducible nitric oxidase synthase 2 (NOS2) (-4.6-fold). F8IL9 treatment was, in general, associated with less increased gene expression levels compared to the PH group without treatment, most strikingly observable for CCL20, CXCL5, C-reactive protein, pentraxin related (CRP) and Kininogen-1 (KNG1), which showed a trend of reversibility towards or even a down-regulation compared to controls. These effects were, in principle, also observable in KSFIL9 treated mice but to a much lesser extent. Moreover, in the dual ERA group, there was a less increased fold change compared to the PH group without treatment. This effect was, however, noticeably less pronounced compared to the F8IL9 treated mice. Figure 1 summarizes relevant gene expression dynamics in the different experimental groups.

Conclusion: F8IL9 treatment led to a relevant attenuation of upregulation of certain genes of inflammatory response and autoimmunity in a preclinical model of PH. Thus, targeted delivery of IL9, known to be effective in PH treatment in this animal model, likely mediates its beneficial effects via diverse anti-inflammatory actions leading to reduced pulmonary vascular remodeling.