Filling the innate myeloid niche in a 3D human tissue-engineered lung model to study age-dependent immune response to respiratory syncytial virus

Abstract The immune response to respiratory syncytial virus (RSV) is vastly influenced by age. RSV can lead to severe disease in neonates and the elderly. To study human age-dependent innate immune responses, we utilized a 3D Human Tissue-Engineered Lung Model (3D-HTLM) consisting of multiple layers that mimic alveoli, including vascular endothelium, stromal cells, and small airway epithelial cells at air-liquid interface. Our goal is to determine if blood monocytes (Mo) added to the 3D-HTLM will fill the myeloid niche and acquire characteristics of tissue resident cells, including alveolar macrophages (AM), and respond to RSV infection. Based on CD1c and CD14 expression, freshly enriched Mo included small populations of cDC2s, Mo-DCs, and non-classical CD14− Mo, all of which lacked CD206 and CD169 markers typical of innate lung resident cells. The 3D-HTLM was harvested 4 days after Mo addition and 1 day after RSV infection (MOI 10). Independent of RSV, all CD45+ Mo, cDC2s, Mo-DCs, and CD14− Mo had significantly increased expression of CD206, CD169, and HLA-DR, including a minor population resembling AM, identified as CD1c−CD14loHLA-DR+CD169+CD206+CD71+. After seeding the 3D-HTLM, cDC2s had increased expression of activation markers CD86, CD83, and CD40. Mo populations showed few phenotypic changes after RSV infection of the 3D-HTLM, yet cDC2s and Mo-DCs had increased production of IL-12 and TNFα, respectively. These data suggest Mo populations adapt to the 3D-HTLM environment and respond to RSV, but transcriptional profiles will better indicate whether the AM niche is being filled. The 3D-HTLM seeded with myeloid cells from neonates or the elderly will be a significant tool to study age-dependent innate immune responses to RSV.