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. 2021 Nov 17:12:645548.
doi: 10.3389/fimmu.2021.645548. eCollection 2021.

Collagen 1a1 Expression by Airway Macrophages Increases In Fibrotic ILDs and Is Associated With FVC Decline and Increased Mortality

Eliza Tsitoura  1 , Athina Trachalaki  1   2 , Eirini Vasarmidi  1 , Semeli Mastrodemou  1 , George A Margaritopoulos  2 , Maria Kokosi  2 , Dionysios Fanidis  3 , Apostolos Galaris  3 , Vassilis Aidinis  3 , Elizabeth Renzoni  2 , Nikos Tzanakis  1 , Athol U Wells  2 , Katerina M Antoniou  1
Affiliations

Collagen 1a1 Expression by Airway Macrophages Increases In Fibrotic ILDs and Is Associated With FVC Decline and Increased Mortality

Eliza Tsitoura et al. Front Immunol. .

Abstract

Within the Interstitial Lung Diseases (ILD), patients with idiopathic pulmonary fibrosis (IPF) and a subset of those with non-IPF fibrotic ILD have a distinct clinical phenotype of progression despite management. This group of patients has been collectively termed the progressive fibrotic phenotype (PFP). Their early recognition may facilitate access to antifibrotic therapies to prevent or slow progression. Macrophages/monocytes within the lung orchestrate the progression and maintenance of fibrosis. A novel role for monocyte-derived macrophages during tissue damage and wound healing is the expression of collagens. We examined Collagen 1a1 expression in airway macrophages from ILD patients at diagnosis. COL1A1 mRNA levels from BAL cells were elevated in IPF and Non-IPF patients. The presence of a UIP pattern and a subsequent progressive phenotype were significantly associated with the higher BAL COL1A1 levels. In Non-IPF patients, higher COL1A1 levels were associated with a more than twofold increase in mortality. The intracellular localisation of COL1A1 in airway macrophages was demonstrated by confocal microscopy in CD45 and CD163 co-staining assays. Additionally, airway macrophages co-expressed COL1A1 with the profibrotic SPP1 gene product osteopontin. The levels of SPP1 mRNA and OPN in the BAL were significantly higher in IPF and Non-IPF patients relative to healthy. Our results suggest that profibrotic airway macrophages are increased in the BAL of patients with IPF and other ILDs and co-express COL1A1 and OPN. Importantly, COL1A1 expression by pro-fibrotic airway macrophages could be a marker of disease progression and poor survival in ILDs.

Keywords: IPF; NSIP; PF-ILD; RA-ILD; SPP1; airway macrophages; collagen 1A1; osteopontin.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
COL1A1 mRNA expression is upregulated in BAL cells from fibrotic ILDs and is higher in ILD patients with UIP versus non-UIP patterns of interstitial pneumonia. COL1A1 relative mRNA levels in: (A) IPF and N-IPF relative to healthy subjects, (One way Anova test and pairwise t tests), (B) COL1A1 expression in whole BAL cells in the ILD diagnostic subgroups relative to heathy. One-way Anova, and pairwise Welch’s T tests, p = 0.024), and H vs RA p = 0.031 (or Welch’s T test p = 0.035). (C) Definitive UIP versus all other non-UIP patterns in IPF, N-IPF and all ILD patients included in the study. (*p < 0.05, **p < 0.01).
Figure 2
Figure 2
COL1A1 is expressed by cells of myeloid origin and AMs. COL1A1 expression in fresh BAL cultures from a Non-IPF patient with CTD-ILD, stained with rabbit anti-human-COL1A1 and ToPro-633 nuclear stain and (A) mouse isotype control, (B) mouse anti-CD45 and (C) mouse anti-CD163 antibodies. Arrows indicate CD45 and CD163 positive cells expressing COL1A1.
Figure 3
Figure 3
Increased expression of COL1A1 in AMs from fibrotic ILDs. (A) Typical images of BAL cytospins stained with anti-human COL1A1 and ToPro-633 nuclear stain from (i) healthy, (ii) IPF, and (iii) a representative N-IPF (RA-ILD) sample. Arrows indicate prominent COL1A1 expression in BAL cells. (B) Violin plots of mean log COL1A1 expression/cell per group. (Brown-Forsythe Anova, with pairwise comparisons, ****p < 0.0001).
Figure 4
Figure 4
SPP1 and OPN expression in BAL cells. (A) SSP1 relative mRNA levels in IPF and N-IPF relative to healthy (Brown-Forsythe Anova, with individual comparisons), (B) Violin plots of mean OPN expression/cell per disease group (Kruskal-Wallis test and individual comparisons) and (C) Typical Images of BAL cytospins stained with anti-human OPN antibody and ToPro-633 nuclear stain in (i) healthy, (ii) IPF and (iii) N-IPF samples (*p < 0.05, **P < 0.01, ****p < 0.0001).
Figure 5
Figure 5
COL1A1 and OPN are coexpressed in AMs from fibrotic ILDs. Fresh BAL cultures were stained with rabbit anti-human-COL1A1and anti-human OPN in healthy (A, D), IPF (B, E, F), and RA-ILD (C, G, H). Panels (A.i, B.i, C.i) show COL1A1 stained cells, panels (A.ii, B.ii, C.ii) show OPN stained cells and panels (A.iii, B.iii,C.iii) are the corresponding merged images. Panels (D–H) show merged COL1A1 and OPN images with corresponding single stainings as smaller insets below.
Figure 6
Figure 6
COL1A1 expression in relation to progression and mortality. Spearman correlation coefficient between relative mRNA expression of COL1A1 and FVC decline in 12 months in: (A) all ILDs combined (IPF and other ILDs), (B) IPF and (C) ILDs other than IPF. Comparison of COL1A1 relative mRNA expression in progressors (≥10% FVC decline at 12 months) and non progressors (<10% FVC decline at 12 months in (D) all ILDs, (E) IPF and (F) ILDs other than IPF unpaired T-test or Mann-Whitney tests. Kaplan Mayer Survival curves of patients with high (above the ROC curve-defined cut-off) and low expression of COL1A1 mRNA expression in: (G) all ILDs combined, (H) IPF and (I) ILDs other than IPF. (*p < 0.05). HR, Hazard ratio).
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