Proteoglycans as Immunomodulators of the Innate Immune Response to Lung Infection

Abstract

Proteoglycans (PGs) are complex, multifaceted molecules that participate in diverse interactions vital for physiological and pathological processes. As structural components, they provide a scaffold for cells and structural organization that helps define tissue architecture. Through interactions with water, PGs enable molecular and cellular movement through tissues. Through selective ionic interactions with growth factors, chemokines, cytokines, and proteases, PGs facilitate the ability of these soluble ligands to regulate intracellular signaling events and to influence the inflammatory response. In addition, recent findings now demonstrate that PGs can activate danger-associated molecular patterns (DAMPs) and other signaling pathways to influence production of many of these soluble ligands, indicating a more direct role for PGs in influencing the immune response and tissue inflammation. This review will focus on PGs that are selectively expressed during lung inflammation and will examine the novel emerging concept of PGs as immunomodulatory regulators of the innate immune responses in lungs.

Keywords: extracellular matrix; innate immunity; pulmonary inflammation; versican.

Figure 1.

Versican accumulation during embryonic development in healthy adult lungs and in lungs of a mouse with Pseudomonas aeruginosa lung infection. (A) Versican accumulation in fetal lung tissue at E14.5 days. (B) Versican accumulation in the lungs of an 8- to 12-week-old mouse treated with PBS as vehicle control. (C) Versican accumulation from an 8- to 12-week-old mouse infected with live P. aeruginosa for 5 days. Brown = positive staining for versican β-GAG, blue = hematoxylin counterstain. The scale bars for A, B, and C = 100 μm and for C inset = 50 μm. Br = bronchiole, Di = diaphragm, Ri = rib, PV = postcapillary vein, TB = terminal bronchiole, arrows = versican staining in alveolar septa, * = area where positive staining of the alveolar septa and cells in alveolar space makes it difficult to distinguish these two anatomical compartments. (D) The amount of versican-stained lung tissue as a percentage of total lung tissue in control mice (PBS) and those exposed to live P. aeruginosa for up to 5 days. Values are the mean ± SEM (n=3–6) with a = significantly different from PBS, b = significantly different from 4 hr, c = significantly different from 24 hr and p<.00001 using one-way ANOVA with Tukey’s multiple comparison test. Source. Originally published as Figure 8 in Snyder et al. Copyright © 2015 The Histochemical Society. Reuse permitted.

Figure 2.

Development of a provisional matrix with selective changes in expression of HSPGs and CSPGs in lungs of mice exposed to LPS. (A) Adaptation of data extrapolated from the work of Blackwood et al., showing a shift from an ECM in lungs that is composed predominately of HS to that of lungs composed predominately of CS and DS 24 h after exposure to LPS. (B) Adaptation of data extrapolated from the work of Blackwood et al., showing changes to CS-4, CS-6, and DS after exposure to LPS. (C) Changes in the relative amounts of mRNA for the HSPGs, syndecan-1, -2, -4, and perlecan, were determined using mRNA collected from whole lung homogenates and quantitative real-time PCR. (D) Changes in the relative amounts of mRNA for the CSPGs, biglycan, decorin, and versican, were determined using mRNA collected from whole lung homogenates and quantitative real-time PCR. (C, D) Comparisons of mRNA recovered from lungs of mice treated with PBS (open symbols) and 1 μg/g LPS at (closed symbols) were made at 2, 6, and 24 hr. An asterisk (*) shows groups that are significantly different (p≤0.05) when mice treated with PBS and LPS were compared. Abbreviations: HSPGs, heparan sulfate proteoglycans; CSPGs, chondroitin sulfate proteoglycans; LPS, lipopolysaccharide; CS, chondroitin sulfate; DS, dermatan sulfate; HS, heparan sulfate; PCR, polymerase chain reaction. Source. Figures 2A and B are based on data extrapolated from Blackwood et al. Figure 2C was originally published as Figure 1A in Tanino et al. Reprinted with permission of the American Thoracic Society. Copyright © 2017 American Thoracic Society. The American Journal of Respiratory Cell and Molecular Biology is an official journal of the American Thoracic Society. Figure 2D was originally published as Figure 1B in Chang et al. Copyright © 2014 International Society of Matrix Biology. Published by Elsevier B.V. Reuse permitted under https://creativecommons.org/licenses/by-nc-nd/4.0/.

Figure 3.

Versican staining in lung tissue sections following treatment with cockroach antigen (CRA). Depicted are representative images of control (A–G) and CRA-treated mice (H–N) stained for versican. Sections obtained from control and CRA-treated mice are shown at low magnification (Panels A and H, respectively; 50×, scale bar = 500 μm). Higher magnification images (200× B–D and I–K, scale bar = 100 μm; 630× E–F and L–N, scale bar = 20 μm) are shown for comparison, including bronchioles and pulmonary arteries (B, E, I, and L), pulmonary veins (C, F, J, and M), and lung parenchyma (D, G, K, and N). Black arrows denote increased versican staining in the subepithelial space, perivascular space, and parenchyma. Source. Originally published as Figure 2 in Reeves et al. Copyright © 2016 The Histochemical Society. Reuse permitted.

Figure 4.

Sensograms obtained using surface plasmon resonance (SPR) reveal the chemokines bind heparin with different kinetics. The binding of various concentrations of rhCXCL8 (A), rmCXCL1/MIP-2 (B), and rmCXCL2/KC (C) to heparinized CM-4 chips is shown. (A) The concentration of CXCL8 was (upper to lower curves) 1,000, 500, 250, 125, 62.5, and 0 nM. (B) The concentrations of CXCL2/MIP-2 were (upper to lower curves) 1,000, 333, 111, 37, 12.4, and 4.12 nM. (C) The concentrations of CXCL1/KC were (upper to lower curves) 1,000, 333, 111, 37, 12.4, 4.12, 1.37, 0.46, and 0.15 nM. Source. Originally published as Figure 6 in Tanino et al. Copyright © 2010. The American Association of Immunologists, Inc.

Figure 5.

Monocyte binding to pericellular matrices of control (A, D) and poly I:C-treated (B, E, C, F) fibroblasts. (A, B) Hyaluronan is stained green and nuclei are stained blue (4′,6-diamidino-2-phenylindole). These specimens were fixed with acetic acid–ethanol–formalin. (C) This specimen was fixed with formalin and stained for hyaluronan (red) and versican (green). Monocytes are bound in the matrix and along the uropod/tail of a partially rounded fibroblast. The inset in C shows a more-condensed aggregate of closely spaced punctate versican staining within a looser network (arrow). The size (~100 nm) and spacing (~100 nm) is consistent with each green signal representing antibody bound to one versican monomer in a compact aggregate. (D–F) Scanning electron micrographs reveal monocytes that have adhered to matrix associated with fibroblast protrusions or uropods (arrows). The monocytes have extended their own filopodia (arrowheads) into the matrix and are partially guided by the fibroblast protrusions. Source. Originally published as Figure 12 in Evanko et al. Copyright © 2009 The Histochemical Society. Reuse permitted.

Figure 6.

Increased expression of the ECM protein, versican, by alveolar macrophages in lungs of mice exposed to lipopolysaccharide. Immunohistochemistry for versican (red) showing alveolar macrophages that have positive staining for versican (black arrows). In contrast, several alveolar macrophages, identified by colloidal carbon uptake, do not show positive staining for versican (gray arrows). For these studies, the IT instillation of Escherichia coli 0111:B4 LPS (1 μg/g) was performed in mice anesthetized with 3–4% isoflurane. The mice were euthanized 6 hr after exposure to LPS, and the lungs were fixed in neutral buffered formalin and processed into paraffin. Immunohistochemistry was performed with an antibody specific for the β-GAG domain of versican and the tissues were counterstained with hematoxylin. The scale bar is 10 µm. Abbreviations: ECM, extracellular matrix; IT, intratracheal; LPS, lipopolysaccharide. Source. Originally published as Figure 3E in Chang et al. Copyright © 2014 International Society of Matrix Biology. Published by Elsevier B.V. Reuse permitted under https://creativecommons.org/licenses.

Figure 7.

Schematic depicting pathways by which LPS and poly(I:C) regulate the expression of versican, HAS1, and syndecan-4. Engagement of macrophage toll-like receptors TLR4 and TLR3 by LPS and poly(I:C), respectively, results in enhanced versican expression. Subsequent to activation of TLR4 and TLR3, engagement of the TRIF adaptor molecule is known to activate transcription factors IRF3/7 that lead to production of type I interferons (IFNα/β) and recognition by type I interferon receptors (IFNAR1/2). Signaling events downstream of IFNAR activation lead to production of versican; the transcription factor(s) mediating expression of versican in this response are still to be determined. In contrast, expression of both HAS1 and syndecan-4 result from TLR4- and TLR2-mediated engagement of the MyD88 adaptor molecule. In addition, syndecan-4 expression results from TLR4- and TLR3-mediated engagement of TRIF and downstream signaling events which are independent of type I IFNs. Abbreviations: LPS, lipopolysaccharide; TLR, toll-like receptor; IFN, interferon. Source. Originally published as Figure 11 in Chang et al. Copyright © 2017, American Journal of Physiology-Lung Cellular and Molecular Physiology. Reuse permitted.