Low Dose Clodronate Improves Exercise Tolerance, Right Ventricular Function, and Alters Resident Lung Macrophage Phenotypes

Introduction: Pulmonary hypertension (PH) is a fatal disease that is significantly more prevalent in adults with Sickle Cell Disease (SCD) compared to the general population. The prevalence of PH is rare (3 in 1 million) but increases to ~10% within the SCD population. Macrophages (Mf) have been determined to be a key cell type in PH and SCD PH disease progression. Clodronate liposomes have been broadly used to study the function of Mf in immune cell-driven diseases. Although it is well-known that clodronate also induces apoptosis of monocytes and dendritic cells and that the simultaneous death of millions of immune cells leads to inflammation, most studies detecting a clodronate-dependent phenotype have been connected to Mfs, indicating that targeting Mfs alone may be a potential therapeutic intervention. It has been shown that low doses of clodronate targets a subset of Mfs that are CD169⁺. Methods: We determined if a low dose (6.5 mg/kg) administration of clodronate is effective at improving exercise tolerance and cardiopulmonary function in our novel SCD-PH. Berkley-ss mice were exposed to 10 weeks of moderate hypoxia (8,000 ft) which we have previously shown to induce SCD-PH. Three groups were used for analysis - wildtype mice (C57/Bl6; WT Hx) at hypoxia, Berk-ss mice at hypoxia (Berk Hx), and Berk-ss mice at hypoxia with weekly intravenously administration of low doses of clodronate (Berk Hx Tx). After 10 weeks of hypoxic exposure, mice were evaluated for exercise tolerance via critical speed, vascular and pulmonary hemodynamics were measured, and tissue was harvested and processed for multi-omic analysis. Results: After 10 weeks, low dose clodronate administration improved exercise tolerance (determined by critical speed analysis) as well as the right ventricle (RV) functional properties of contractility, ejection fraction, cardiac output, and ventricular-to-vascular coupling ratio. A multi-omics approach was utilized to evaluate whole RV and lung tissue, as well as the remaining resident Mfs from the lung for phenotypic changes. Proteomic analysis of the RV showed ~500 significantly altered proteins between Berk Hx and Berk Hx Tx. The top 100 altered proteins were analyzed via network pathway analysis and showed an increase in phosphorylation and protein modification while the decreased proteins associated with catabolic processes. Metabolomics from the RV showed an increase in alpha linolenic acid and linolenic acid metabolism in Berk Hx Tx when compared to Berk Hx. Surprisingly, Berk Hx Tx had a decrease in mitochondria electron transport chain. We next determined differences in the whole lung omics landscape. 665 proteins were significantly altered between Berk Hx and Berk Hx Tx. Using the top 100 altered proteins, network analysis was performed to show a significantly decrease in macromolecular catabolic processes and an increase in protein targeting to the membrane. Metabolomic analysis showed a decrease in glutamate metabolism. Lastly, since the macrophage population is critical for PH disease progression, the remaining lung macrophages were characterized for phenotypic differences with low dose clodronate treatment. Surprisingly, principal component analysis showed Berk Hx Tx lung macrophages clustered with WT Hx. Both groups were significantly decreased in proteins correlated to mRNA pathways. When Berk Hx were compared directly to Berk Hx Tx, mRNA processes and chromatin alterations were decreased in Berk Hx Tx. Interestingly, innate immune response pathways were increased in the lung macrophage population from Berk Hx Tx. When metabolomics were analyzed, the urea cycle was significantly increased in Berk Hx Tx compared to Berk Hx. Together, these data show that low dose of clodronate in a SCD-PH mouse model alter phenotypes at both the whole organ and cellular (Mf) levels to slow progression of PH and increase exercise tolerance.

George:Agios: Consultancy.