Bronchoalveolar lavage (BAL) cells in idiopathic pulmonary fibrosis express a complex pro-inflammatory, pro-repair, angiogenic activation pattern, likely associated with macrophage iron accumulation

Abstract

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease of unknown cause characterized by alveolar epithelial damage, patchy interstitial fibrosis and diffuse microvascular abnormalities. In IPF, alveolar clustering of iron-laden alveolar macrophages-a common sign of microhemorrhage, has been associated with vascular abnormalities and worsening of pulmonary hypertension. As iron-dependent ROS generation has been shown to induce unrestrained macrophage activation in disease models of vascular damage, we explored alveolar macrophage activation phenotype in IPF patients (n = 16) and healthy controls (CTR, n = 7) by RNA sequencing of bronchoalveolar lavage (BAL) cells. The frequencies of macrophages in BAL cells were 86+4% and 83.4+8% in IPF and CTR groups, respectively (p-value = 0.41). In IPF patients, BAL cells showed increased iron-dependent ROS generation (p-value<0.05 vs CTR). Gene expression analysis showed overrepresentation of Gene Ontology processes/functions and KEGG pathways enriched in upregulated M1-type inflammatory (p-value<0.01), M2-type anti-inflammatory/tissue remodeling (p-value<0.0001), and MTPP-type chronic inflammatory/angiogenic (p-value<0.0001) chemokine and cytokine genes. The ex vivo finding was confirmed by the induction of iron-dependent ROS generation and chemokine/cytokine overexpression of Ccl4, Cxcl10 (M1), Il1rn (M2), Cxcl2, and Cxcl7 (MTPP) in MH-S murine immortalized alveolar macrophages exposed to ferric ammonium citrate in culture (p-value<0.05 vs CTR). The data show alveolar macrophage expression of a pro-inflammatory, tissue remodeling and angiogenic complex activation pattern, suggesting that iron accumulation may play a role in macrophage activation.

Fig 1. Intracellular iron and ROS levels in BAL cells.

Alveolar macrophage intracellular iron was assessed using the Prussian Blue Iron Staining and the Golde score was calculated based on the staining intensity 14 IPF patients and 7 controls. Reactive oxygen species (ROS) were measured in 11 IPF patients and 7 controls, using CM-H2DCFDA fluorimetry. Shown are Golde scores and chelatable iron-dependent ROS levels per 10⁵ BAL cells in healthy controls and IPF affected individuals.

Fig 2. Principal Component Analysis of healthy controls and IPF patients.

The PCA plot was computed using the differentially expressed gene list (FDR<0.05, log2FC>0.6 filter).

Fig 3. RNA-seq differential gene expression validation by qRT-PCR.

Relative expression of selected chemokines in IPF (N = 7, closed circles) vs. Healthy control (N = 4, open circles) BAL cells represented by normalized ΔCt value. ΔCt value was normalized to the GNB2L1 housekeeping gene expression by subtracting GNB2L1 raw Ct from the selected gene raw Ct and by further adding the absolute GNB2L1 minimum value (the same for all samples) to the gene ΔCt. Lower cycle values correspond to higher expression level. Mann-Whitney test (*) and (**) denote statistical significance (P < 0.05, P < 0.01, respectively) vs. control.

Fig 4. Quantification of BAL fluid chemokine levels by protein immunochemistry.

Chemokines were measured in un-concentrated BAL fluid by protein immunochemistry on IPF affected individuals (closed circles) and Healthy Controls (open circles). CXCL-5 was quantified using the Chemokine Human 5-Plex Panel II for the Luminex 100/200 platform. PPBP/CXCL-7, CXCL-10, CCL13 and CCL18 were measured using enzyme-linked immunosorbent assay (ELISA) kits. Mann-Whitney test (*) and (**) denote statistical significance (P < 0.05, P < 0.01, respectively) vs. control.

Fig 5. FAC-treated murine alveolar macrophage cells, MH-S.

Murine alveolar macrophage cells (MH-S, ATCC CRL-2019) derived from a BALB/C strain mouse were incubated with 100 μM of Ferric ammonium citrate (FAC) for 1, 3, and 5 days. A. Shown are iron and ROS levels in FAC-treated cells. B. Shown are relative mRNA expression levels of selected chemokines/cytokines in FAC-treated mouse alveolar macrophages examined by qRT-PCR. Relative expression levels of the cytokines were normalized to the GNB2L1 housekeeping gene and calculated using the 2^ΔΔCt method. Mann-Whitney test (*) and (**) denote statistical significance (P < 0.05, P < 0.01, respectively) vs. control.