Alveolar macrophages drive lung fibroblast function in cocultures of IPF and normal patient samples

Abstract

Idiopathic pulmonary fibrosis (IPF) is characterized by increased collagen accumulation that is progressive and non-resolving. Although fibrosis progression may be regulated by fibroblasts and alveolar macrophage (AM) interactions, this cellular interplay has not been fully elucidated. To study AM-fibroblast interactions, cells were isolated from IPF and normal human lung tissue and cultured independently or together in direct 2D co-culture, direct 3D co-culture, indirect transwell, and in 3D hydrogels. AM influence on fibroblast function was assessed by gene expression, cytokine/chemokine secretion, and gel/matrix contractility. Normal AMs cultured in direct contact with fibroblasts down-regulated ECM gene expression while IPF AMs showed little to no effect. Fibroblast contractility was assessed by encapsulating co-cultures in 3D collagen hydrogels and monitoring gel diameter over time. Both normal and IPF AMs reduced baseline contractility of normal fibroblasts but had little to no effect on IPF fibroblasts. When stimulated with Toll-like receptor (TLR) agonists, IPF AMs increased production of pro-inflammatory cytokines TNFα and IL-1β, compared to normal AMs. TLR ligand stimulation did not alter fibroblast contraction, but stimulation with exogenous TNFα and TGF-β did alter contraction. To determine if the observed changes required cell-to-cell contact, AM-conditioned media and transwell systems were utilized. Transwell culture showed decreased ECM gene expression changes compared to direct co-culture and conditioned media from AMs did not alter fibroblast contraction regardless of disease state. Taken together, these data indicate that normal fibroblasts are more responsive to AM crosstalk, and that AM influence on fibroblast behavior depends on cell proximity.

Association between mosaic loss of chromosome Y and pulmonary fibrosis susceptibility and severity

“…Significantly reduced efferocytosis in alveolar macrophages and impaired mitochondrial homeostasis in alveolar macrophages observed in IPF patients link alveolar macrophages to the pathogenesis of IPF. Alveolar macrophage can also impact normal and IPF fibroblasts to differing extents through cell-cell communication (51). Pro-inflammatory cytokine expressed classically activated (M1) and anti-inflammatory cytokine expressed alternatively activated (M2) phenotypes exert polarised effects on the responses to the injury (50).…”

In vitro co-culture studies and the crucial role of fibroblast-immune cell crosstalk in IPF pathogenesis

“…Further, Novak et al, isolated IPF and control-derived primary alveolar macrophages (AMs) and lung fibroblasts. Various combinations of CM experiments, direct co-cultures in tissue culture plates, transwell and 3D collagen hydrogel cocultures were then set up to assess the interaction between different combinations of IPF-and control-derived lung fibroblasts and AMs [42]. After experiments, it was found that while coculturing control-derived AMs with control-derived fibroblasts led to a reduction in fibroblast-α-SMA expression, direct cocultures of control-derived AMs with IPF-derived fibroblasts resulted in increased fibronectin, collagen I and III as well as α-SMA gene expression pointing to potential myofibroblast differentiation [42].…”

Pulmonary Matrix Derived Hydrogels from Patients with Idiopathic Pulmonary Fibrosis Induce a Proinflammatory State in Lung FibroblastsIn Vitro

“…To better understand the role of immune cells in IPF pathogenesis, a few recent studies have focused on generating innovative in vitro systems in which fibroblasts are co-cultured with macrophages in 3D collagen matrices (Ullm et al, 2020;Novak et al, 2023). For example, Novak et al demonstrated that direct co-cultures of fibroblasts and macrophages in 3D collagen matrices result in altered crosstalk due to the proximity of the cells, suggesting that collagen matrices are a suitable platform for in vitro crosstalk studies (Novak et al, 2023). Additionally, Ullm et al found that macrophage and fibroblast co-culture systems using 3D collagen matrices result in a significant secretion of interleukin-10 (IL-10) from macrophages, eliciting fibroblast to myofibroblast differentiation (Ullm et al, 2020).…”

“…To better understand the role of immune cells in IPF pathogenesis, a few recent studies have focused on generating innovative in vitro systems in which fibroblasts are co-cultured with macrophages in 3D collagen matrices(Ullm et al ., 2020; Novak et al ., 2023). For example, Novak et al .…”

“…For example, Novak et al . demonstrated that direct co-cultures of fibroblasts and macrophages in 3D collagen matrices result in altered crosstalk due to the proximity of the cells, suggesting that collagen matrices are a suitable platform for in vitro crosstalk studies(Novak et al ., 2023). Additionally, Ullm et al .…”