Lung fibrosis and tissue remodeling are features of chronic diseases such as severe asthma, idiopathic pulmonary fibrosis, and systemic sclerosis. However, fibrosis-targeted therapies are currently limited. We demonstrate in mouse models of allergen- and bleomycin-driven airway inflammation that neutralization of the TNF family cytokine TL1A through Ab blocking or genetic deletion of its receptor DR3 restricted increases in peribronchial smooth muscle mass and accumulation of lung collagen, primary features of remodeling. TL1A was found as a soluble molecule in the airways and expressed on the surface of alveolar macrophages, dendritic cells, innate lymphoid type 2 cells, and subpopulations of lung structural cells. DR3 was found on CD4 T cells, innate lymphoid type 2 cells, macrophages, fibroblasts, and some epithelial cells. Suggesting in part a direct activity on lung structural cells, administration of recombinant TL1A into the naive mouse airways drove remodeling in the absence of other inflammatory stimuli, innate lymphoid cells, and adaptive immunity. Correspondingly, human lung fibroblasts and bronchial epithelial cells were found to express DR3 and responded to TL1A by proliferating and/or producing fibrotic molecules such as collagen and periostin. Reagents that disrupt the interaction of TL1A with DR3 then have the potential to prevent deregulated tissue cell activity in lung diseases that involve fibrosis and remodeling.
Severe asthma, idiopathic pulmonary fibrosis (IPF), and systemic sclerosis (SSc; scleroderma with pulmonary fibrosis) are examples of chronic inflammatory disorders characterized by a severe fibrotic and remodeling component of the lungs (1–3). This includes production of extracellular matrix proteins, such as collagen, by lung epithelial cells and fibroblasts, along with proteins such as periostin that can act with collagen to enhance fibrotic activity. Additional consequences are hyperplasia or hypertrophy of smooth muscle cells and the excessive accumulation of myofibroblasts that express α–smooth muscle actin and have smooth muscle cell characteristics (1, 4). An increased understanding of the molecules that can drive lung fibrosis and remodeling is essential for elucidating new targets for therapies that may halt or reverse these diseases.
One molecule of interest is the cytokine TL1A, an inducible TNF family protein, that can be made by dendritic cells (DCs), macrophages, T cells, and endothelial cells and that acts through the cell surface receptor DR3 (5). TL1A plays a strong role in orchestrating inflammation in the intestine through activities on T cells and innate lymphoid cells (6–8). Moreover, TL1A has been associated with acute lung inflammation in that it has been shown to stimulate cytokine production from Th2 cells and innate lymphoid type 2 cells (ILC2) in mouse models with OVA and papain (9–11). Although DR3 has been thought to be primarily expressed on T lymphocytes and innate lymphoid cells (12), some reports have found DR3 on human kidney tubular epithelial cells during renal allograft rejection (13), cells in the mesothelial layer of the peritoneal cavity after bacterial infection (14), and mouse intestinal myofibroblasts (15). Additionally, in the latter study, a reduced number of myofibroblasts was associated with the ability of blocking TL1A to limit fibrosis in the colon driven by dextran sodium sulfate or adoptive transfer of CD4 T cells (15). This directly implies that TL1A might be strongly relevant for stimulating tissue cells in other organs where remodeling takes place. In this paper, we show that TL1A is central to fibrosis and remodeling of the lung using mouse models driven by allergen or bleomycin exposure and that TL1A exhibits remodeling-relevant activities in lung fibroblasts and bronchial epithelial cells that express DR3.
Materials and Methods
Six- to eight-week-old female DR3-deficient mice and wild-type (WT) littermates, derived by Taconic Biosciences (no. TF3529), were bred in-house on the C57BL/6 × 129 background. WT C57BL/6 mice were purchased from The Jackson Laboratory (no. 000664). RAG2-deficient mice and RAG2-γc–deficient mice on the BALB/c × 129 background were purchased from The Jackson Laboratory (no. 014593). All experiments complied with the regulations of the La Jolla Institute for Immunology Animal Care Committee.
Experimental lung remodeling protocols
Mice were challenged with bleomycin (0.2 U/mouse; Sigma), given intratracheally once as in prior studies (16, 17). Analyses were performed after 7 d. For neutralization, 100 μg of Fc fusion protein of DR3 (DR3.Fc) or control IgG was given i.v. 1 d prior to injection of bleomycin and every other day until the end of the experiment.
Mice were sensitized intranasally (i.n.) on day 0, 7, and 14 with 200 and 100 μg house dust mite extract protein (HDM; GREER Laboratories, Lenoir, NC), followed by chronic i.n. challenges of 50 μg of HDM administered twice a week for the following 4 wk as previously described (16). Analyses were performed 24 h after the last challenge. Mouse DR3.Fc (made in-house by Kyowa Kirin, La Jolla, CA) or isotype control IgG were administered i.p. after the initial sensitization period starting at day 14 and were given every 3 d until the end of the experiment (100 μg/injection/mouse).
Activity of recombinant protein.
Mice were given 10 μg of recombinant mouse TL1A (produced in-house by Kyowa Kirin) or PBS intratracheally on days 1 and 2 and sacrificed for analyses 1 d later on day 3.
Lung sections were stained as in previous studies (16, 17). For collagen, Masson trichrome blue was used and scored with defined areas of interest created throughout the entire lung sections, including alveolar and peribronchial regions, to quantify blue color using Image-Pro Plus. Total lung collagen was also measured using a hydroxyproline kit (MAK008; Sigma) or a Sircol Assay (S1000; Biocolor). For smooth muscle mass, sections were stained with Ab to α–smooth muscle actin (clone 1A4, ab7817, 1:200; Abcam, Cambridge, MA) and Rhodamine Red-X AffiniPure Donkey Anti-Rabbit (711295152, 1:500; Jackson ImmunoResearch Laboratories, West Grove, PA). Peribronchial smooth muscle mass was evaluated using Image-Pro Plus from multiple random bronchi throughout the lungs. A similar protocol was used with an Ab to periostin (clone ab14041; Abcam) or with H&E. All images, unless indicated, were captured with the Zeiss Scanner ×20 objective. Soluble TL1A in bronchoalveolar lavage (BAL) fluid was assessed by Western blot using mDR3-Fc and anti-IgG/HRP (109-035-003; Jackson ImmunoResearch Laboratories).
BAL and lung cells were treated with RBC lysing buffer (Sigma). Lungs were dissociated using a Lung Dissociation Kit (Miltenyi Biotec) and Gentle MACS (Miltenyi Biotec). LIVE/DEAD cells were stained with Fixable Aqua Dead Cell Staining Kit (Thermo Fisher Scientific), and after Fc block with the 2.4G2 mAb (eBioscience), cells were stained with the following Abs: DR3-PE (4C12; BioLegend), TL1A-PerCPef710 (Tandys1a; eBioscience), CD45-V500 (30-F11; BioLegend), SiglecF-Brilliant Violet (BV) 605 (E50-2440; BD Biosciences), CD11b-allophycocyanin-Cy7 (M1/70; BD Biosciences), CD11c-PE-Cy7 (HL3; BD Biosciences), Ly6G-Alexa Fluor 700 (1A8; BioLegend), MHC class II–allophycocyanin (M5/114.15.2; BioLegend), CD4-BV570 (RM4-5; BioLegend), CD8a-PEeFluor610 (53-6.7; eBioscience), B220-BV785 (RA3-6B2; BioLegend), CD127-BV785 (A7R34; BioLegend), ST2-BV605 (DIH9; BioLegend), CD90.2-PECy7 (30-H12; BioLegend), Epcam-PECy7 (G8.8; BioLegend), and CD31-BV785 (390; BioLegend). For lineage markers for ILC2 staining, the following Abs were used: CD3-FITC (145-2C11; eBioscience), CD4-FITC (GK1.5; eBioscience), CD8-FITC (5H-10-1; BioLegend), CD19-FITC (1D3; BioLegend), NK1.1-FITC (PK136; BioLegend), CD11b-FITC (M1/70; BioLegend), CD11c-FITC (HL3; BD Biosciences), and Gr1-FITC (RB6-8C5; BioLegend). After fixation and permeabilization using with Foxp3/Transcription Factor Staining Buffer Set (eBioscience), cells were stained intracellularly with vimentin-allophycocyanin (280618; R&D Systems). Flow cytometry analysis was performed on a Fortessa (BD Biosciences), and data were analyzed using FlowJo Software (version 10; FlowJo, Ashland, OR). Live CD45+ lung immune cells were separated into CD4+ T cells (CD3+, CD4+), B cells (CD3−B220+CD11c−), alveolar macrophages (CD11b−CD11c+SiglecF+), DCs (CD11c+MHC class II+), and ILC2 (Lin−CD127+CD90.2+ST2+). Live CD45− lung structural cells were separated by staining with Epcam, CD31, CD90.2, and vimentin.
Stimulation of structural cells
Normal human lung fibroblasts (NHLF; Lonza, Walkersville, MD) and human bronchial epithelial cells (BEAS-2B cell line; American Type Culture Collection), were stimulated with 100 ng/ml of recombinant human TL1A, a dose predetermined to produce a maximal response (made in-house by Kyowa Kirin), or varying doses of human recombinant TGF-β (R&D Systems, Minneapolis, MN) for 72 h in EpiLife media (Life Technologies/Thermo Fisher Scientific, Carlsbad, CA). DR3 was visualized using anti-human DR3 (clone JD3; BioLegend, San Diego, CA). Periostin and collagen protein were visualized with anti-human periostin (clone ABT292; MilliporeSigma) or anti-human collagen (clone MAB4165; R&D Systems). Proliferation was studied by uptake of 0.5 μCi [3H]-TdR (PerkinElmer, Waltham, MA) added 16 h before the end of the culture. PCR was performed as previously described (16, 17).
Patient human lung fibroblasts
Fibroblasts were obtained from normal, SSc with pulmonary fibrosis, or IPF patients undergoing lung transplantation at the University of Pittsburgh Medical Center and from donors whose lungs were not used for transplantation as previously described (18). Samples were analyzed for expression of mRNA for DR3 by PCR.
Quantitative real-time PCR
Total RNA was isolated from lungs or in vitro stimulated cells using TRIzol (Invitrogen). RNeasy Fibrous Tissue Mini Kit (74704; QIAGEN, Valencia, CA) was used to further purify RNA from lung samples. Single-strand cDNA was prepared by reverse transcribing 5 μg of total RNA using Transcriptor First Strand cDNA kit (Roche Diagnostics, Indianapolis, IN). Samples were amplified in IQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA) using the following primer pairs: human collagen, forward, 5′-CCT CAA GGG CTC CAA C-3′, reverse, 5′-GGT TTT GTA TTC AAT CAC TGT CTT GC-3′; murine collagen, forward, 5′-GAG CCC TCG CTT CCG TAC TC-3′, reverse, 5′-TGT TCC CTA CTC AGC CGT CTG-3′; human periostin, forward, 5′-GAC TCA AGA TGA TTC CCT TT-3′, reverse, 5′-GGT GCA AAG TAA GTG AAG G-3′; murine periostin, forward, 5′-CCC TCC AGC AAA TTC TGG GCA CCA-3′, reverse, 5′-GAG ACT CAC GTT TTC TTC CCG CAG A-3′; human α–smooth muscle actin, forward, 5′-CTG GCA TCG TGC TGG ACT CT-3′, reverse, 5′-GATCTCGGCCAGCCAGATC-3′; murine α-smooth muscle actin, forward, 5′-TCT CTA TGC TAA CAA CGT CCT GTCA-3′, reverse, 5′-CCA CCG ATC CAG ACA GAG TAC TT-3′; and human DR3, forward, 5′-AAG GCG AAG AAG CAC GAA CGA ATG-3′, reverse, 5′-ACT CCG GCC GAG AAG TTG AGA AAT-3′. All samples were run in triplicate, and the mean values were used for quantification.
One-way ANOVA or nonparametric Mann–Whitney U test was used where indicated. A p value <0.05 was considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.001).
TL1A injection into the lungs promotes fibrosis and remodeling
We previously found that one member of the TNF superfamily, LIGHT (TNFSF14), induced fibrosis and tissue remodeling when administered directly to the mouse lungs (16, 17). To assess if another TNF-like protein might also be relevant for lung remodeling, we injected recombinant TL1A (TNFSF15) into the airways of naive WT mice. Two intratracheal injections of rTL1A, given over 3 d, induced primary remodeling features, namely the accumulation of collagen, based on the extent of Masson trichrome blue stain, around the bronchioles and in the alveolar regions and an increase in smooth muscle mass, based on α–smooth muscle actin staining, around the bronchioles (Fig. 1A). The effect on lung remodeling was generally quicker than has been reported for remodeling in other systems, for example with IL-13 transgenic mice, but is in line with the rapidity with which we found LIGHT could induce a similar lung remodeling response (16, 17) and was likely due to the high dose of the TL1A protein used and that its receptor was readily available on relevant cells to signal for these activities. Because T cells and innate lymphoid cells express DR3, we asked whether these cells were necessary for the remodeling activity of rTL1A and found that the effect was still evident in RAG2-deficient and RAG2γc-deficient mice that lack one or both of these cell populations (Fig. 1B, 1C). Showing specificity, rTL1A failed to induce fibrosis and remodeling in mice lacking DR3 (Fig. 1D). Thus, TL1A exerts profibrotic and remodeling activity in the lungs through DR3, independently of T cells and ILC.
Inhibiting DR3 reduces fibrosis and lung tissue remodeling induced by bleomycin
To determine if the physiological production of TL1A played any role in lung tissue remodeling, we first used intratracheal administration of the antibiotic bleomycin at high dose. We have previously shown that this also rapidly induces upregulation of lung collagen and increases in peribronchial smooth muscle mass over a 1-wk period (17), as opposed to other models in which lower doses of bleomycin generally only drive accumulation of lung collagen after 2–3 wk. In line with our studies of recombinant TL1A, genetic deletion of DR3 significantly attenuated increases in collagen deposition and lung smooth muscle mass (Fig. 2A). Inflammatory lung infiltrates were also affected to an extent in mice lacking DR3 with a trend toward a reduction in specific lung cell populations seen with flow cytometry analyses, albeit not statistically significant (Supplemental Fig. 1A). Showing the reduced remodeling response was not due to a developmental defect in the knockout animals or the background of these mice; TL1A blocking with a DR3.Fc in WT BL/6 mice replicated the effect and inhibited fibrosis and increases in smooth muscle mass (Fig. 2B). The reduction in collagen deposition observed by scoring the extent of trichrome staining was confirmed with assays of soluble (Sircol score) and total collagen (hydroxyproline score) extracted from the whole lungs (Fig. 2C).
TL1A–DR3 neutralization limits allergen-induced lung fibrosis and tissue remodeling
To further implicate TL1A and DR3 as being strong regulators of lung fibrotic activity, we used an allergen-driven model (16) in which mice were sensitized and chronically challenged with HDM given i.n. We have previously shown that this protocol also results in fibrosis in the lungs with significant remodeling of the smooth muscle mass around the bronchioles. In mice lacking DR3, a strong decrease in fibrosis was visualized in lung tissue (trichrome blue stain for collagen deposition), along with reduced remodeling (α–smooth muscle actin staining around the bronchioles) (Fig. 3A). We then tested whether blocking TL1A–DR3 signaling could produce a similar reduction in remodeling features and specifically if the intervention was performed after the initial Th2 response that characterizes this model had developed (16). Treatment of WT mice with DR3.Fc given starting after day 14 also demonstrated less fibrosis and remodeling in the lungs with a significant decrease in collagen deposition and smooth muscle mass (Fig. 3B). Both CD4+ T cells and eosinophils were significantly reduced in the lungs of HDM-challenged DR3-deficient mice, whereas DR3-Fc–treated mice showed a moderate decrease in the CD4+ T cell and DC infiltrates but no difference in eosinophilia (Supplemental Fig. 1B). These results show that endogenous TL1A–DR3 signaling is central to the development of lung fibrosis and tissue remodeling downstream of two diverse stimulants, bleomycin, and HDM allergen.
TL1A and DR3 are expressed in immune cells and lung structural cells
TL1A can be expressed on the membrane of cells and also can be cleaved from the membrane and act as a soluble cytokine, similar to TNF (19–21). Our data with recombinant TL1A (Fig. 1) suggested the soluble molecule could be most relevant, and a recent report found elevated levels of soluble TL1A in sputum from severe asthmatics, a population of patients most likely to display lung fibrosis and airway remodeling (22). In line with this, we detected soluble TL1A in the bronchoalveolar fluid of mice with lung remodeling (Fig. 4A). To understand which cells might produce TL1A, we stained both immune lung infiltrates and structural cells for membrane TL1A (Fig. 4B). TL1A was expressed in the naive lung on a subpopulation of epithelial cells (CD31−Epcam+) and on CD31+Epcam− cells, the latter likely a mixture of endothelial cells and pericytes (see Supplemental Fig. 1C for gating). In mice challenged with bleomycin or HDM, this expression was lost, perhaps reflecting production of the soluble molecule. In addition, membrane TL1A was found after challenge with either bleomycin or HDM on subpopulations of alveolar macrophages, ILC2, and vimentin+CD90− cells that might represent smooth muscle cells. Not surprisingly, based on prior reports, DR3 was found constitutively expressed on CD4 T cells and ILC2 in naive lungs and those receiving bleomycin and HDM (Fig. 4C). DR3 was also expressed on alveolar macrophages in all situations and on DC after HDM challenge. Most interestingly, DR3 was strongly expressed on vimentin+CD90+ (CD31−Epcam−) cells, a phenotype consistent with fibroblasts. We also observed DR3 on a small population of epithelial cells (Epcam+CD31−), but as our recovery of epithelial cells from the lungs was poor (Supplemental Fig. 1C), this might have been an underestimate of the expression of DR3 on these cells. Last, we examined human fibroblasts for expression of DR3 isolated from the lungs of normal individuals or patients with SSc with pulmonary fibrosis and patients with IPF. Significantly, all samples expressed mRNA for DR3 at high levels (Fig. 4D), correlating with the mouse data.
TL1A can act directly on lung fibroblasts
As rTL1A rapidly upregulated collagen and α–smooth muscle actin expression in the lungs in the absence of DR3-expressing T cells and ILC2 (Fig. 1), and these are products of fibroblasts and/or smooth muscle cells; this suggested that TL1A might partly have functioned via a direct action on lung fibroblasts. This was supported by finding DR3 expression on fibroblasts, but not on putative smooth muscle cells (vimentin+CD90−) in the mouse lung. We tested if this was possible using commercially purchased NHLF, which were also found to have significant expression of surface DR3 (Fig. 5A). The division of fibroblasts can lead to their excessive accumulation in the lung tissue where they produce extracellular matrix proteins. Moreover, fibroblasts expressing α–smooth muscle actin (myofibroblasts), in addition to mature smooth muscle cells, can contribute to the smooth muscle mass surrounding the bronchioles in patients with asthma and SSc (23–25). We then asked if recombinant human TL1A could promote proliferation of fibroblasts and differentiation into a myofibroblast. rTL1A induced division of NHLF, leading to an approximate doubling of the basal level of proliferation of these cells (Fig. 5B), but it did not induce α–smooth muscle actin expression in isolation (Fig. 5C). Because TGF-β, a cytokine ubiquitously expressed by many cells in the lungs, is one of the primary factors that can enhance myofibroblast differentiation (26), we tested whether TL1A acted together with TGF-β to promote either division or differentiation of lung fibroblasts. TGF-β alone did not induce proliferation but strongly upregulated α–smooth muscle actin, as previously reported by many groups in the literature (Fig. 5B, 5C). However, TGF-β in a dose dependent fashion significantly enhanced the extent of division induced by TL1A (Fig. 5B). Interestingly, TL1A also moderately augmented the induction of α–smooth muscle actin by TGF-β (Fig. 5C). Last, we asked whether TL1A could promote production of extracellular matrix proteins by lung fibroblasts and found strong induction of collagen, the cardinal feature of lung fibrosis (Fig. 5D). Collectively, these data show that TL1A can directly drive remodeling-relevant activity in lung fibroblasts, and it can work together with TGF-β to enhance the accumulation of myofibroblasts that make collagen, corresponding in part to the requirement of TL1A in contributing to fibrosis and smooth muscle mass observed in the mouse studies.
DR3 activity in lung fibroblasts and epithelial cells promotes periostin expression
Next, we asked whether TL1A could regulate the expression of periostin, a protein that has recently emerged as a potential marker of fibrosis and tissue remodeling associated with steroid-resistant asthma, IPF, and SSc in humans and that is thought to contribute to lung dysfunction (27–30). Periostin is primarily expressed by fibroblasts and epithelial cells and has been described to possess a number of activities, including acting as a scaffold by binding to extracellular matrix proteins like collagen as well as synergizing with TGF-β to enhance collagen deposition or chemokine production that further contributes to tissue remodeling (27, 31, 32). Significantly, we found that periostin expression was upregulated in human lung fibroblasts after stimulation with TL1A in vitro (Fig. 6A). We additionally tested the human bronchial epithelial cell line BEAS-2B and found that DR3 was expressed well in these cells. Moreover, they also responded to TL1A by making periostin (Fig. 6B).
TL1A regulates periostin production in the lungs
Finally, we then assessed whether TL1A was upstream of periostin production in vivo to provide additional evidence of the relevance of the prior in vitro findings in lung fibroblasts and bronchial epithelial cells and as a further feature linking TL1A activity to lung tissue fibrotic activity. The in vivo administration of rTL1A to the airways of naive mice upregulated periostin protein expression in the bronchial epithelium and in cells in the parenchyma as shown by immunofluorescence (Fig. 7A). Confirming this, an increase in periostin mRNA was also induced in the lungs. Most importantly, when the TL1A–DR3 interaction was inhibited by ligand blocking in the allergen-induced model, we saw an almost complete abrogation of periostin protein expression in both the bronchial epithelium and parenchyma, and correspondingly, mRNA expression in the lungs of these mice was reduced (Fig. 7B). These results further the contention that TL1A–DR3 signaling is a major driver of lung fibrosis and tissue remodeling.
Lung fibrosis and tissue remodeling are common features of many chronic inflammatory disorders, such as asthma, IPF, and SSc, but knowledge of molecules that promote these responses is still limited. In this study, we reveal, to our knowledge, a new role for TL1A–DR3 signaling in contributing to lung fibrosis and remodeling, potentially independent of its activity in enhancing adaptive immunity that has been reported previously in the literature. We elucidate new functional activities of TL1A in lung structural cells, demonstrating significant effects on proliferation, collagen production, or periostin expression, in fibroblasts or myofibroblasts and bronchial epithelial cells, all phenotypes that were strongly reduced when DR3 signaling was abrogated in mouse models of lung fibrosis. Our data highlight an unappreciated role of TL1A/DR3 signaling as a central driver of lung fibrotic activity downstream of several inflammatory insults and suggest that TL1A and DR3 could be targets for therapeutics aimed at reducing fibrosis and tissue remodeling in humans with severe lung disease.
TL1A could potentially contribute to lung tissue deregulation in both indirect and direct manners. It has previously been linked with the induction of acute lung inflammation from experiments showing reduced eosinophilia and production of the Th2 cytokines IL-4, IL-5, IL-13, and IL-9 in the lungs of TL1A-deficient mice in models driven by OVA and papain. DR3 was found to be expressed by Th2 cells and ILC2s and was shown to either enhance the expansion of these cells or their ability to secrete Th2 cytokines (9–11). By contributing to a Th2-dominant environment, TL1A could therefore be instrumental in generating the initial response that creates an environment favorable to fibrosis and tissue remodeling in, for example, asthmatics that progress from moderate to severe. Our results now add to this literature but highlight a potential downstream role of TL1A independent of the Th2/ILC2 response whereby TL1A may provide inflammatory signals to lung structural cells that directly rather than indirectly drive the fibrotic and remodeling response. Indeed, we found that DR3 was expressed in mouse and human lung fibroblasts and bronchial epithelial cells, and TL1A induced several remodeling-related activities in these cells, including their ability to proliferate and/or upregulate collagen and periostin. Importantly, these effects were observed in vitro in the absence of other Th2 factors, and further substantiating this new activity, in vivo injection of rTL1A into the lungs of mice demonstrated that a tissue remodeling phenotype could be induced in the absence of Th2 cells and ILC2. This leads to the contention that if TL1A is expressed and available in the lungs and DR3 is present on both adaptive immune cells and tissue structural cells, it will be a major contributor to the tissue remodeling and fibrosis that is seen in chronic lung disease. We primarily focused on fibroblasts, and in the mouse lung, they constitutively expressed DR3. This was further supported by mRNA data from human lung fibroblasts from normal donors as well as those with SSc and IPF and by surface expression seen in NHLF from a commercial source. However, we found a small subset of epithelial cells that were DR3 positive in the mouse, and the human bronchial epithelial cell line, BEAS-2B, also expressed DR3, suggesting that lung epithelial cells could also be a major target of TL1A activity in addition to fibroblasts. It is possible the mouse data were an underestimate of the true expression of DR3 on epithelial cells as we recovered only small numbers of these cells or that given six or seven distinct bronchial epithelial subsets have recently been described, DR3 expression might be restricted to only a subpopulation of these cells. Future studies are then needed to further understand the extent of DR3 expression on epithelial cells in the lung and the range of potential activities of TL1A on these cells. Additional questions remain about whether extrinsic factors can positively or negatively modulate the availability of DR3 on the various structural cell types.
The exact source of TL1A that might be crucial to remodeling in the lung is not known, but prior publications have observed TL1A to be made by macrophages, DCs, T cells, and endothelial cells (33–36). Our mouse staining data for membrane TL1A further support multiple possible sources in the lungs, including alveolar macrophages, DC, ILC2, epithelial cells, and endothelial cells or pericytes (CD31+Epcam− cells). Another intriguing idea is that TL1A could be a product of the structural cells that are central to tissue remodeling, and it is of interest in future studies to determine if normal lung cells or those from patients with severe lung disease can produce TL1A. This could imply that an autocrine feedback loop might exist whereby fibroblasts or epithelial cells autonomously drive their own deregulated activity during the later stages of chronic disease that may be independent of adaptive immunity, a notion previously discussed as potentially being central to several diseases, including rheumatoid arthritis as well as SSc and IPF. We observed surface TL1A on a subset of epithelial cells in the naive mouse lung and on a subset of vimentin+CD90− cells in bleomycin-challenged mice that might have been smooth muscle, although none on fibroblasts, but given that TL1A can be cleaved from the membrane, these data might also be an underestimation of whether lung structural cells could be a primary source of TL1A. In fact, there is some support for this in the literature in that synovial fibroblasts and chondrocytes and intestinal subepithelial myofibroblasts were found to be capable of making TL1A after stimulation with TNF or IL-1 (37–39).
Our studies suggest that either TL1A or DR3 are potential targets for therapeutics aimed at limiting the development or persistence of tissue remodeling and fibrosis in severe lung disease but raise additional questions that will need to be addressed before translational work can be pursued. It is becoming increasingly apparent that patients with severe asthma, SSc, and IPF are heterogenous in terms of the extent and type of lung inflammatory response that underlies their clinical symptoms. Understanding whether DR3 or TL1A are differentially expressed in the lung tissue of subsets of these patients will be important, including whether the expression of DR3 in individual patients varies between lymphoid cell types and structural cells of the lung. Last, as TL1A can be made as a soluble molecule (19–21), a further question relates to whether soluble TL1A is found in biological fluids of these patients (serum, BAL, and sputum) and if this can be used as a biomarker to indicate progression of disease or simply patients amenable to therapeutic interventions targeting TL1A and DR3. In this regard, a recent study found soluble TL1A was more strongly expressed in the sputum of a subset of severe eosinophilic asthmatics compared with mild asthmatics after challenge with allergen (22), suggesting this could inform future treatments.
In summary, we reveal new activities of TL1A in structural cells of the lungs that are highly relevant for lung remodeling that is seen in severe diseases such as asthma and SSc as well as IPF. Using mouse models, we demonstrate that TL1A–DR3 activity is directly or indirectly involved in promoting several cardinal features of these diseases. Given that TL1A has previously been shown to be a regulator of Th2 cells and ILC2 associated with acute lung inflammatory responses, our results further extend the biology of this molecule and suggest it is likely to be a central mediator of both acute and chronic lung inflammation and a realistic target for therapy of lung fibrosis and tissue dysregulation.
We acknowledge La Jolla Institute Facility cores, the Division of Laboratory Animal Care, Microscopy, and Flow cytometry cores.
This work was supported by funds from Kyowa Kirin Pharmaceutical Research to M.C. and National Institutes of Health, National Institute of Allergy and Infectious Diseases Grant AI070535 to M.C. and D.H.B.
The online version of this article contains supplemental material.
Abbreviations used in this article:
Fc fusion protein of DR3
house dust mite extract protein
innate lymphoid type 2 cell
idiopathic pulmonary fibrosis
normal human lung fibroblast
The authors have no financial conflicts of interest.