Registration of the extracellular matrix components constituting the fibroblastic focus in idiopathic pulmonary fibrosis

Abstract

The extracellular matrix (ECM) in idiopathic pulmonary fibrosis (IPF) drives fibrosis progression; however, the ECM composition of the fibroblastic focus (the hallmark lesion in IPF) and adjacent regions remains incompletely defined. Herein, we serially sectioned IPF lung specimens constructed into tissue microarrays and immunostained for ECM components reported to be deregulated in IPF. Immunostained sections were imaged, anatomically aligned, and 3D reconstructed. The myofibroblast core of the fibroblastic focus (defined by collagen I, α-smooth muscle actin, and procollagen I immunoreactivity) was associated with collagens III, IV, V, and VI; fibronectin; hyaluronan; and versican immunoreactivity. Hyaluronan immunoreactivity was also present at the fibroblastic focus perimeter and at sites where early lesions appear to be forming. Fibrinogen immunoreactivity was often observed at regions of damaged epithelium lining the airspace and the perimeter of the myofibroblast core but was absent from the myofibroblast core itself. The ECM components of the fibroblastic focus were distributed in a characteristic and reproducible manner in multiple patients. This information can inform the development of high-fidelity model systems to dissect mechanisms by which the IPF ECM drives fibrosis progression.

Keywords: Fibrosis; Pulmonology.

Figure 1. The myofibroblast core of the fibroblastic focus is defined by collagen I and cells expressing α-smooth muscle actin and procollagen I.

(A and B) Twelve IPF specimens were assembled into a tissue microarray, serially sectioned at 5 μm, and immunostained for collagen I (left), α-smooth muscle actin (αSMA; middle), and procollagen I (right). (A) Low-magnification images of 2 IPF specimen serial section core punches. Red arrows show regions of high collagen I deposition traced through the serial stains. Scale bar: 500 μm. (B) High-magnification images of 3 IPF specimens. The myofibroblast core of the fibroblastic focus is outlined by red dotted lines. Scale bar: 100 μm. (C) The myofibroblast core of the fibroblastic focus, characterized by collagen I immunoreactivity, was scored as either positive (score = 1) or negative (score = 0) immunoreactivity for αSMA or procollagen I. Each myofibroblast core was serially immunostained up to 4 times for αSMA and procollagen I, and their averaged score is represented as a single data point on the graph. A χ² test was performed for αSMA and procollagen I association with collagen I; P = 0.9999 for both (n = 29 fibroblastic foci for αSMA, n = 29 fibroblastic foci for procollagen I; n = 12 IPF patients total [1–6 fibroblastic foci per patient]).

Figure 2. Distribution of collagen V and hyaluronan in the fibroblastic focus.

(A and B) Twelve IPF specimens were assembled into a tissue microarray, serially sectioned at 5 μm, and immunostained for collagen I (left), collagen V (middle), and hyaluronan (right). (A) Low-magnification images of 2 IPF specimen serial section core punches. Red arrows show regions of high collagen I deposition traced through the serial stains. Scale bar: 500 μm. (B) High-magnification images of 3 IPF specimens. Each myofibroblast core of the fibroblastic focus is outlined by a red dotted line. Scale bar: 100 μm. (C) The myofibroblast core of the fibroblastic focus, characterized by collagen I immunoreactivity, was scored as either positive (score = 1) or negative (score = 0) immunoreactivity for collagen V or hyaluronan. Each myofibroblast core was serially immunostained up to 4 times for collagen V and hyaluronan, and their averaged score is represented as a single data point on the graph. A χ² test was performed for collagen V and hyaluronan association with collagen I; P = 0.9999 for both (n = 40 fibroblastic foci for collagen V, n = 40 fibroblastic foci for hyaluronan; n = 9 IPF patients total [1–7 fibroblastic foci per patient]).

Figure 3. Hyaluronan is present at the interface between fibrotic and morphologically normal alveoli and absent from morphologically normal alveolar walls.

FFPE serial sections of 6 IPF specimens were stained for collagen I (left) and hyaluronan (middle), with a higher-magnification inset for hyaluronan (right). Shown are specimens with a fibrotic and normal interphase highlighted on the collagen I immunostain (left). High magnification of the hyaluronan stain shows strong reactivity (right, red arrows) along the active fibrotic front (thickened alveolar septa), with gradual attenuation of reactivity as it transitions into normal alveoli (red asterisk). Scale bar: 100 μm.

Figure 4. Distribution of fibronectin and collagen VI in the fibroblastic focus.

(A and B) Twelve IPF specimens were assembled into a tissue microarray, serially sectioned at 5 μm, and immunostained for collagen I (left), fibronectin (middle), and collagen VI (right). (A) Low-magnification images of 2 IPF specimen serial section core punches. Red arrows show regions of high collagen I deposition traced through the serial stains. Scale bar: 500 μm. (B) High-magnification images of 3 IPF specimens. The myofibroblast core of the fibroblastic focus is outlined by a red dotted line. Scale bar: 100 μm. (C) The myofibroblast core of the fibroblastic focus, characterized by collagen I immunoreactivity, was scored as either positive (score = 1) or negative (score = 0) immunoreactivity for fibronectin or collagen VI. Each myofibroblast core was serially immunostained up to 4 times for fibronectin and collagen VI, and their averaged score is represented as a single data point on the graph. A χ² test was performed for fibronectin and collagen VI association with collagen I; P = 0.9999 for both (n = 41 fibroblastic foci for fibronectin, n = 41 fibroblastic foci for collagen VI; n = 9 IPF patients total [2–8 fibroblastic foci per patient]).

Figure 5. Distribution of collagen IV and versican in the fibroblastic focus.

(A and B) Twelve IPF specimens were assembled into a tissue microarray, serially sectioned at 5 μm, and immunostained for collagen I (left), collagen IV (middle), and versican (right). (A) Low-magnification images of 2 IPF specimen serial section core punches. Red arrows show regions of high collagen I deposition traced through the serial stains. Scale bar: 500 μm. (B) High-magnification images of 3 IPF specimens. The myofibroblast core of the fibroblastic focus is outlined by a red dotted line. Scale bar: 100 μm. (C) The myofibroblast core of the fibroblastic focus, characterized by collagen I immunoreactivity, was scored as either positive (score = 1) or negative (score = 0) immunoreactivity for collagen IV or versican. Each myofibroblast core was serially immunostained up to 4 times for collagen IV and versican, and their averaged score is represented as a single data point on the graph. A χ² test was performed for collagen IV and versican association with collagen I; P = 0.9999 for both (n = 53 fibroblastic foci for collagen IV, n = 53 fibroblastic foci for versican; n = 9 IPF patients total [1–10 fibroblastic foci per patient]).

Figure 6. Distribution of collagen III and fibrinogen in the fibroblastic focus.

(A and B) Twelve IPF specimens were assembled into a tissue microarray, serially sectioned at 5 μm, and immunostained for collagen I (left), collagen III (middle), and fibrinogen (right). (A) Low-magnification images of 2 IPF specimen serial section core punches. Red arrows show regions of high collagen I deposition traced through the serial stains. Scale bar: 500 μm. (B) High-magnification images of 3 IPF specimens. The myofibroblast core of the fibroblastic focus is outlined by a red dotted line. Scale bar: 100 μm. (C) The myofibroblast core of the fibroblastic focus, characterized by collagen I immunoreactivity, was scored as positive (score = 1), weak (score = 0.5), or negative (score = 0) immunoreactivity for collagen III or fibrinogen. Each myofibroblast core was serially immunostained up to 4 times for collagen III and fibrinogen, and their averaged score is represented as a single data point on the graph. A χ² test was performed for collagen III and fibrinogen association with collagen I; *P < 0.0001 (n = 22 fibroblastic foci for collagen III, n = 24 fibroblastic foci for fibrinogen; n = 7 IPF patients total [1–4 fibroblastic foci per patient]).

Figure 7. Fibrinogen is expressed at the interface between the myofibroblast core and small airways.

FFPE sections of 6 IPF specimens were sectioned and immunostained for collagen I (left) and fibrinogen (middle), with a higher-magnification inset (right). Higher magnification reveals strong fibrinogen immunoreactivity (right, red arrows) along the interface between the myofibroblast core and airspace, where there is evidence of damage to the epithelium (red asterisk). Scale bar: 100 μm.

Figure 8. A model of ECM-mediated fibrosis progression.

(A) The myofibroblast core of the fibroblastic focus contains collagens I, III, IV, V, and VI; fibronectin; and versican, ECM components also found in the normal lung. In addition, unlike in the normal lung, hyaluronan is ubiquitously present. (B) A fibroblastic focus immunostained with collagen I (top) was traced (center), with nuclei shown as circles/ovals. Surrounding the tracing, immunostaining for each component is shown for the same fibroblastic focus. The fibroblastic focus is comprised of a myofibroblast core and an active fibrotic front, defined as a highly cellular and mitotically active region at the myofibroblast core perimeter that extends into thickened alveolar septa adjacent to morphologically normal regions. This morphology is consistent with a mechanism of fibrosis progression in which myofibroblasts, at thickened alveolar septa, invade into normal regions in response to hyaluronan (red arrows). Fibrinogen marks regions of damaged/stressed epithelium where myofibroblasts begin to encroach into the airspaces, mimicking the situation in wound healing (green arrow).