The therapeutic effect of mesenchymal stem cells on pulmonary myeloid cells following neonatal hyperoxic lung injury in mice

Exposure to high levels of oxygen (hyperoxia) after birth leads to lung injury. Our aims were to investigate the modulation of myeloid cell sub-populations and the reduction of fibrosis in the lungs following administration of human mesenchymal stem cells (hMSC) to neonatal mice exposed to hyperoxia.

Newborn mice were exposed to 90% O₂ (hyperoxia) or 21% O₂ (normoxia) from postnatal days 0–4. A sub-group of hyperoxia mice were injected intratracheally with 2.5X10⁵ hMSCs. Using flow cytometry we assessed pulmonary immune cells at postnatal days 0, 4, 7 and 14. The following markers were chosen to identify these cells: CD45⁺ (leukocytes), Ly6C⁺Ly6G⁺ (granulocytes), CD11b⁺CD11c⁺ (macrophages); macrophage polarisation was assessed by F4/80 and CD206 expression. hMSCs expressing enhanced green fluorescent protein (eGFP) and firefly luciferase (fluc) were administered via the trachea at day 4. Lung macrophages in all groups were profiled using next generation sequencing (NGS) to assess alterations in macrophage phenotype. Pulmonary collagen deposition and morphometry were assessed at days 14 and 56 respectively.

At day 4, hyperoxia increased the number of pulmonary Ly6C⁺Ly6G⁺ granulocytes and F4/80^lowCD206^low macrophages but decreased F4/80^highCD206^high macrophages. At days 7 and 14, hyperoxia increased numbers of CD45⁺ leukocytes, CD11b⁺CD11c⁺ alveolar macrophages and F4/80^lowCD206^low macrophages but decreased F4/80^highCD206^high macrophages. hMSCs administration ameliorated these effects of hyperoxia, notably reducing numbers of CD11b⁺CD11c⁺ and F4/80^lowCD206^low macrophages; in contrast, F4/80^highCD206^high macrophages were increased. Genes characteristic of anti-inflammatory ‘M2’ macrophages (Arg1, Stat6, Retnla, Mrc1, Il27ra, Chil3, and Il12b) were up-regulated, and pro-inflammatory ‘M1’ macrophages (Cd86, Stat1, Socs3, Slamf1, Tnf, Fcgr1, Il12b, Il6, Il1b, and Il27ra) were downregulated in isolated lung macrophages from hyperoxia-exposed mice administered hMSCs, compared to mice without hMSCs. Hydroxyproline assay at day 14 showed that the 2-fold increase in lung collagen following hyperoxia was reduced to control levels in mice administered hMSCs. By day 56 (early adulthood), hMSC administration had attenuated structural changes in hyperoxia-exposed lungs.

Our findings suggest that hMSCs reduce neonatal lung injury caused by hyperoxia by modulation of macrophage phenotype. Not only did our cell-based therapy using hMSC induce structural repair, it limited the progression of pulmonary fibrosis.