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 nonresolving. 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 2-D coculture, direct 3-D coculture, indirect transwell, and in 3-D hydrogels. AM influence on fibroblast function was assessed by gene expression, cytokine/chemokine secretion, and hydrogel contractility. Normal AMs cultured in direct contact with fibroblasts downregulated extracellular matrix (ECM) gene expression whereas IPF AMs had little to no effect. Fibroblast contractility was assessed by encapsulating cocultures in 3-D 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 with normal AMs. TLR ligand stimulation did not alter fibroblast contraction, but stimulation with exogenous TNFα and TGFβ 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 with direct coculture 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.

Keywords: fibroblasts; fibrosis; lung; monocyte/macrophage.

Graphical abstract

Figure 1.

IPF fibroblasts display heightened gene expression and contractility compared with normal pulmonary fibroblasts. Gene expression changes were compared between normal and IPF human fibroblasts for collagen 1, collagen 3, fibronectin, and fibroblast activation marker αSMA (A). Hydrogel contraction was monitored over the course of 7 days for both IPF and normal fibroblasts (B; * significance between IPF and normal on the same day, % significance between normal sample and the previous day, and & significance between IPF sample and the previous day). Regression curves were fit to each sample’s 7-day contraction and decay constants for each curve were plotted for comparison of contraction rate between IPF and normal samples (C). The regression curve plateau values were plotted for both IPF and normal contraction (D). Statistical significance was determined through t test comparisons for gene expression changes, two-way ANOVA for contraction studies with Tukey’s multiple comparisons test, and t tests for comparisons of decay constants and plateau values. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. IPF, idiopathic pulmonary fibrosis.

Figure 2.

Fibroblast-macrophage coculture influences gene expression and contractility of fibroblasts. Normal fibroblasts were cultured alone or directly in contact with normal or IPF AMs and assessed for changes in gene expression for ECM components collagen 1 and 3 and fibronectin and fibroblast activation marker αSMA (A). IPF fibroblasts were cultured independently or in direct contact with normal (blue) or IPF (red) AMs and assessed for changes in gene expression (B). Normal fibroblasts were encapsulated independently (gray) or in conjunction with normal (blue) or IPF (red) AMs and monitored for contractile changes over the course of 7 days (C). Contractility of IPF fibroblasts was then measured independently (gray) or in hydrogel coculture with normal AMs (blue) or IPF AMs (red; D). Best fit curves for exponential decay were fit to all contraction studies and resulting decay constants were plotted for normal (E) and IPF (F) coculture contraction studies. Plateau values for resulting decay curves were also plotted for normal (G) and IPF (H) 7-day contractions. For gene expression changes, statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons when normally distributed (A) and using a nonparametric one-way ANOVA with Kruskal-Wallis test for multiple comparisons when nonnormally distributed (B). Contraction assays were assessed using two-way ANOVAs. Decay constants and plateau values were compared using ordinary one-way ANOVA and uncorrected Fisher’s LSD test for multiple comparisons. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). AMs, alveolar macrophages; ECM, extracellular membrane; IPF, idiopathic pulmonary fibrosis; LSD, least significant difference.

Figure 3.

Alveolar macrophages alter gene expression and protein production in response to TLR stimulation. Bronchoalveolar lavage samples from normal and samples of patients with IPF were stained and quantified for relative proportion of cell populations (A). Normal (B) and IPF (C) human AMs were treated with various TLR ligands including LPS, PAM3Cys, or poly I:C and assessed for gene expression changes compared with nonstimulated controls. Protein production of TNFα (D), MCP1 (E), IL-10 (F), and IL-1β (G) were assessed using ELISA. Significant changes in gene expression were assessed using nonparametric one-way ANOVA with Kruskal-Wallis test for multiple comparisons. Protein production was assessed using a nonparametric one-way ANOVA for either normal AMs under treatment or IPF AM treatments respectively and nonparametric t tests for treatment specific comparison of IPF to normal AMs. The * symbols indicate significance when compared with nonstimulated control from same disease or nondiseased condition (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). The & symbols indicate significance when comparing IPF vs. normal nondiseased samples under same treatment condition (&P < 0.05, &&P < 0.01, &&&P < 0.001, &&&&P < 0.0001). AMs, alveolar macrophages; IPF, idiopathic pulmonary fibrosis; TLR, Toll-like receptor.

Figure 4.

TLR ligands do not affect fibroblast contractility, but TLR4 activation may contribute to CCL2/MCP-1 production from IPF fibroblasts. The functional contractility of normal (A) or IPF (B) fibroblasts was tracked over the course of 1 wk under various TLR ligand treatments (LPS = purple, PAM3Cys = fuchsia, poly I:C = pink). CCL2 protein production due to these treatments was assess using ELISA at days 4 and 7 for both normal (C) and IPF (D) fibroblasts. Two-way ANOVAs were utilized for significance assessment with Tukey’s test for multiple comparisons. Changes in gene expression for normal (E) and IPF (F) fibroblasts were assessed after 24 h of TLR ligand exposure on 2-D plates. Significance was determined using two-way ANOVAs (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). IPF, idiopathic pulmonary fibrosis; TLR, Toll-like receptor.

Figure 5.

Fibroblast respond to exogenous cytokine treatment by altering contraction phenotype and gene expression. Normal (A) or IPF (B) human fibroblast cultures encapsulated within collagen hydrogels were subjected to various cytokine treatments and tracked for changes in contractility over the course of 1 wk. Protein production of CCL2 was measured using ELISA on days 4 and 7 for normal (C) and IPF (D) treatment conditions. Gene expression changes after 24 h of exogenous TNFα treatment on 2-D cultures was investigated for normal (E) and IPF (F) fibroblasts. Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparisons test (A and B), Tukey’s multiple comparison test (C and D), or t tests (E and F; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). IPF, idiopathic pulmonary fibrosis.

Figure 6.

Effects of AMs on fibroblast phenotypes in nondirect coculture. Normal fibroblasts (A) or IPF fibroblasts (B) were cultured in a transwell system with either normal (blue) or IPF (red) AMs and assessed for gene expression changes. Changes in normal fibroblast (C) and IPF fibroblast (D) contraction was assessed when cultured with conditioned media from normal AMs (blue) or IPF AMs (red). Protein production for cytokines CCL2/MCP-1 (G and H) and TGFβ (I and J) was assessed at days 4 and 7 from the contraction assays using ELISA. Dotted lines indicate baseline levels of CCL2/MCP-1 protein (E and F) and TGFβ protein (G and H) in AM-conditioned media. Significant changes in gene expression were determined using one-way ANOVA with Tukey’s multiple comparison tests when data tested with a normal distribution (A), and with nonparametric one-way ANOVA with Kruskal–Wallis test for multiple comparisons when the data was nonnormal (F). All significance in contractility was determined using two-way ANOVA with Sidak’s multiple comparisons test. Statistical significance of protein production was done using two-way ANOVAs Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. AMs, alveolar macrophages; IPF, idiopathic pulmonary fibrosis. % indicates significance between fibroblasts alone and fibroblast + IPF AM conditioned media; # indicates significance between fibroblasts + normal AM conditioned media and fibroblasts + IPF AM conditioned media.

Figure 7.

Summary of fibroblast and alveolar macrophage coculture responses. Pulmonary fibroblasts and alveolar macrophages derived from normal or IPF human patient samples were cocultured in either direct or indirect contact. Changes in gene expression, contraction and CCL2 are summarized for the various coculture settings. IPF, idiopathic pulmonary fibrosis. [Image created with BioRender.com and published with permission.]