The increasing number of severe infections with multi-drug-resistant pathogens worldwide highlights the need for alternative treatment options. Given the pivotal role of phagocytes and especially alveolar macrophages in pulmonary immunity, we introduce a new, cell-based treatment strategy to target bacterial airway infections. Here we show that the mass production of therapeutic phagocytes from induced pluripotent stem cells (iPSC) in industry-compatible, stirred-tank bioreactors is feasible. Bioreactor-derived iPSC-macrophages (iPSC-Mac) represent a highly pure population of CD45+CD11b+CD14+CD163+ cells, and share important phenotypic, functional and transcriptional hallmarks with professional phagocytes, however with a distinct transcriptome signature similar to primitive macrophages. Most importantly, bioreactor-derived iPSC-Mac rescue mice from Pseudomonas aeruginosa-mediated acute infections of the lower respiratory tract within 4-8 h post intra-pulmonary transplantation and reduce bacterial load. Generation of specific immune-cells from iPSC-sources in scalable stirred-tank bioreactors can extend the field of immunotherapy towards bacterial infections, and may allow for further innovative cell-based treatment strategies.
Hereditary pulmonary alveolar proteinosis (hPAP) is a rare disorder of pulmonary surfactant accumulation and hypoxemic respiratory failure caused by mutations in CSF2RA (encoding the granulocyte/macrophage colony-stimulating factor [GM-CSF] receptor α-chain [CD116]), which results in reduced GM-CSF-dependent pulmonary surfactant clearance by alveolar macrophages. While no pharmacologic therapy currently exists for hPAP, it was recently demonstrated that endotracheal instillation of wild-type or gene-corrected mononuclear phagocytes (pulmonary macrophage transplantation [PMT]) results in a significant and durable therapeutic efficacy in a validated murine model of hPAP. To facilitate the translation of PMT therapy to human hPAP patients, a self-inactivating (SIN) lentiviral vector was generated expressing a codon-optimized human CSF2RA-cDNA driven from an EF1α short promoter (Lv.EFS.CSF2RA), and a series of nonclinical efficacy and safety studies were performed in cultured macrophage cell lines and primary human cells. Studies in cytokine-dependent Ba/F3 cells demonstrated efficient transduction, vector-derived CD116 expression proportional to vector copy number, and GM-CSF-dependent cell survival and proliferation. Using a novel cell line constructed to express a normal GM-CSF receptor β subunit and a dysfunctional α subunit (due to a function-altering CSF2RA mutation) that reflects the macrophage disease phenotype of hPAP patients, it was demonstrated that Lv.EFS.CSF2RA transduction restored GM-CSF receptor function. Further, Lv.EFS.CSF2RA transduction of healthy primary CD34 cells did not adversely affect cell proliferation or affect the cell differentiation program. Results demonstrate Lv.EFS.CSF2RA reconstituted GM-CSF receptor α expression, restoring GM-CSF signaling in hPAP macrophages, and had no adverse effects in the intended target cells, thus supporting testing of PMT therapy of hPAP in humans.
Primary or secondary immunodeficiencies are characterized by disruption of the cellular and humoral immunity. Respiratory infections are a major cause of morbidity and mortality among immunodeficient or immunocompromised patients with Staphylococcus aureus being a common offending organism. We here propose an adoptive macrophage transfer approach aiming to enhance impaired pulmonary immunity against S. aureus. Our studies, using human induced pluripotent stem cells (iPSC)-derived macrophages (iMφ) demonstrate efficient antimicrobial potential against Methicillin-sensitive and Methicillin-resistant clinical isolates of S. aureus. Using an S. aureus airway infection model in immunodeficient mice, we demonstrate that the adoptive transfer of iMφ is able to reduce the bacterial load more than 10-fold within 20 hours. This effect was associated with reduced granulocyte infiltration and less damage in lung tissue of transplanted animals. Whole transcriptome analysis of iMφ compared to monocyte-derived macrophages indicates a more profound upregulation of inflammatory genes early after infection and faster normalization 24 hours post-infection. Our data demonstrate high therapeutic efficacy of iMφ-based immunotherapy against S. aureus infections and offers an alternative treatment stratgey for immunodeficient or immunocompromised patients.
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