Genetic deficiency of the transcription factor NFAT1 confers protection against fibrogenic responses independent of immune influx

Abstract

Idiopathic pulmonary fibrosis (IPF) is marked by unremitting matrix deposition and architectural distortion. Multiple profibrotic pathways contribute to the persistent activation of mesenchymal cells (MCs) in fibrosis, highlighting the need to identify and target common signaling pathways. The transcription factor nuclear factor of activated T cells 1 (NFAT1) lies downstream of second messenger calcium signaling and has been recently shown to regulate key profibrotic mediator autotaxin (ATX) in lung MCs. Herein, we investigate the role of NFAT1 in regulating fibroproliferative responses during the development of lung fibrosis. Nfat1^−/−-deficient mice subjected to bleomycin injury demonstrated improved survival and protection from lung fibrosis and collagen deposition as compared with bleomycin-injured wild-type (WT) mice. Chimera mice, generated by reconstituting bone marrow cells from WT or Nfat1^−/− mice into irradiated WT mice (WT→WT and Nfat1^−/−→WT), demonstrated no difference in bleomycin-induced fibrosis, suggesting immune influx-independent fibroprotection in Nfat1^−/− mice. Examination of lung tissue and flow sorted lineage^neg/platelet-derived growth factor receptor alpha (PDGFRα)^pos MCs demonstrated decreased MC numbers, proliferation [↓ cyclin D1 and 5-ethynyl-2′-deoxyuridine (EdU) incorporation], myofibroblast differentiation [↓ α-smooth muscle actin (α-SMA)], and survival (↓ Birc5) in Nfat1^−/− mice. Nfat1 deficiency abrogated ATX expression in response to bleomycin in vivo and MCs derived from Nfat1^−/− mice demonstrated decreased ATX expression and migration in vitro. Human IPF MCs demonstrated constitutive NFAT1 activation, and regulation of ATX in these cells by NFAT1 was confirmed using pharmacological and genetic inhibition. Our findings identify NFAT1 as a critical mediator of profibrotic processes, contributing to dysregulated lung remodeling and suggest its targeting in MCs as a potential therapeutic strategy in IPF.

NEW & NOTEWORTHY Idiopathic pulmonary fibrosis (IPF) is a fatal disease with hallmarks of fibroblastic foci and exuberant matrix deposition, unknown etiology, and ineffective therapies. Several profibrotic/proinflammatory pathways are implicated in accelerating tissue remodeling toward a honeycombed end-stage disease. NFAT1 is a transcriptional factor activated in IPF tissues. Nfat1-deficient mice subjected to chronic injury are protected against fibrosis independent of immune influxes, with suppression of profibrotic mesenchymal phenotypes including proliferation, differentiation, resistance to apoptosis, and autotaxin-related migration.

INTRODUCTION

Progressive fibrosis, unamenable to therapeutic approaches, is an important cause of subacute and chronic respiratory failure with organ transplantation as the only final viable option. Fibrosis develops in the native lungs in the context of diverse infectious (1–3), environmental (4–6), and autoimmune insults (7, 8) or can be of indeterminate etiology (9) as in idiopathic pulmonary fibrosis (IPF). A myriad of profibrotic signals have been identified and linked to maladaptive persistent mesenchymal cell (MC) activation, which drives the inexorable loss of organ function in these fibrotic lung diseases. Therapeutic targeting has been limited by the redundancy of these signaling pathways, underscoring the need to identify targetable common downstream mediators of diverse upstream signals. Calcium (Ca²⁺) is one such key second messenger of many known profibrotic ligands in mesenchymal cells, and aberrant calcium homeostasis has been extensively documented in fibrosis and specifically IPF (10–12).

A crucial downstream signaling pathway after Ca²⁺ influx is the calmodulin-calcineurin pathway, with the final activation of the nuclear factor of activated T cells (NFAT) (13). Increased intracellular Ca²⁺ concentration increases the calmodulin binding to calcineurin, causing nuclear translocation of NFAT and transcription of putative target genes (14). NFAT family members, although studied most extensively in immune cells, have only been recently implicated in mesenchymal activation. We have identified NFAT1 (NFATC2/NFATP) as a transcriptional promoter of autotaxin (ATX) (15) in mesenchymal cells. ATX, a secreted lysophospholipase D, is strongly implicated in cellular migration and in the pathogenesis of fibrotic diseases including IPF (16) via generation of lipid mediator lysophosphatidic acid (LPA). Our investigations delineated an NFAT1/ATX/LPA1 signaling loop that regulates mesenchymal activation in an autocrine manner (15). Others have demonstrated that NFAT1 mediates lung mesenchymal proliferation downstream of hypoxia inducible factor-2 (HIF-2) (17) and Wnt5a/Frizzled receptor signaling (18), and a recent study using transcriptional profiling found NFAT signaling pathway among the top enriched pathways in aged lung and IPF (19). Although these studies suggest that calcium-dependent NFAT1 activation could be an important converging point for mesenchymal activation, the role of NFAT1 in the development of lung fibrosis via its impact on the nonimmune cell compartment remains to be elucidated.

Here, using the bleomycin model of lung fibrosis in Nfat1^−/− (20) and Nfat1-deficient bone marrow chimera mice, we elucidate the potent profibrotic role of NFAT1, which is independent of its function as a transcriptional promoter of inflammatory responses. Our in vivo murine model studies combined with in vitro investigations in human IPF mesenchymal cells shed novel light on the unique features of NFAT1 activation in these cells.

METHODS

Study Approval

Frozen tissues from patients with IPF were obtained from the Lung Tissue Research Consortium, sponsored by the National Institutes of Health-National Heart, Lung, and Blood Institute. Mesenchymal cells derived from lung tissue biopsies of patients with IPF were procured from the University of Michigan Pulmonary Biorepository. All experiments were performed according to protocols approved by the University of Michigan Institutional Review Board and all participants signed informed consent. All animal experiments were approved by the Institutional Animal Care and Use Committee.

Human Primary Cell Culture

Mesenchymal cells from passages between 3 and 5 were seeded onto culture dishes until 70% confluence in DMEM medium containing 10% fetal bovine serum, along with penicillin and fungizone. Subsequently, cells were placed in serum-free media for 24 h to attain quiescence before treatment. Nuclear fractions were isolated as per the manufacturer’s protocol (Cat. No. 78833, Thermo Scientific., Waltham, MA).

Animals and Animal Models of Fibrosis

Specific pathogen-free female and male C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, ME). Nfat1^−/− C57BL/6 mice were a generous donation by Dr. Anjana Rao (21, 22), La Jolla Institute for Allergy and Immunology, San Diego, CA.

Bleomycin Injury Model

Six- to 8-wk-old wild-type (WT) or B6. Nfat1^−/− mice were anesthetized with isoflurane, and an oropharyngeal administration of bleomycin (50 µL of 0.025 units; Sigma, St. Louis, MO) was given to induce lung injury as previously described (23).

Bone Marrow Transplant Model

As shown in the schematic in Fig. 3A, recipient wild-type (WT) mice were treated with 13 Gy total body irradiation (split dose) using a ¹³⁷Cs irradiator, followed by tail vein injection of 5 × 10⁶ whole bone marrow cells from healthy wild-type or B6. Nfat1^−/− mice. Chimeras (WT→WT or Nfat1^−/−→WT) were provided acid water from weeks 3–5. Five weeks after bone marrow transplant (BMT), when total numbers of hematopoietic cells were fully reconstituted in the lungs and spleen (24), the mice were given oropharyngeal instillations of bleomycin (0.025 units; Sigma, St. Louis, MO). Mice were euthanized 3 wk after bleomycin injury.

Figure 3.

Nfat1 deficiency in radioresistant hematopoietic cells does not confer fibroprotection with bleomycin injury. A: schematic strategy shows chimera generation and subsequent instillation of bleomycin. Lethal irradiation of WT mice was followed by reconstitution of 5 × 10⁶ bone marrow cells from WT or Nfat1^−/− mice to generate chimeras (WT→WT and Nfat1^−/−→WT). Five weeks after bone marrow transplant, the chimera mice were oropharyngeally instilled with bleomycin or saline. Three weeks later, lungs were harvested. B: representative formalin-fixed paraffin-embedded sections with Masson’s trichrome staining (blue) demonstrated parenchymal and airway fibrosis indicating collagen deposition. n = 5 mice/group. Scale bar: 100 μm. C: quantitative analysis of collagen deposition. Hydroxyproline content in acid-digested lung homogenates was measured in triplicates and repeated twice. Values: means ± SD. Statistics: one-way ANOVA, post hoc Bonferroni. **P < 0.01, ****P < 0.0001. NFAT1, nuclear factor of activated T cells isoform 1; WT, wild-type. [Image created with a licensed version of BioRender.com.]

5-Ethynyl-2′-Deoxyuridine Infusion

Two weeks after bleomycin injury of wild-type or B6. Nfat1^−/− mice, 0.1 mL of intraperitoneal injections of 5-ethynyl-2′-deoxyuridine (EdU) in PBS (50 mg/kg body wt) were administered. Mice were harvested 24 h later and analyzed for EdU.

Survival Analysis

To evaluate the role of NFAT1 on the survival rates in bleomycin-injured mice, wild-type or B6. Nfat1^−/− mice were monitored from the date of bleomycin instillation to the end of the 3-wk study period in three cohorts.

Histopathological Evaluation and Immunohistochemistry

Three weeks after bleomycin injury, the heart and lungs were removed en bloc, fixed in 10% neutral buffered-formalin, and embedded in paraffin. Tissue sections of 5 µm thickness were stained with hematoxylin and eosin (H&E) and Masson’s trichrome stain (NovaUltra Masson’s Trichrome Stain Kit; IHC WORLD, Woodstock, MD) to determine the presence of fibrosis and the extent of collagen deposition in the lesions.

Immunofluorescent Staining

Quiescent mesenchymal cells were fixed with 4% paraformaldehyde for 10 min, followed by permeabilization with 0.1% Triton X-100 for 20 min. Cells were rinsed with PBS, blocked, and incubated with NFAT1 antibody (1:100, 24 h; R&D Systems). Cells were rinsed and probed with TRITC-conjugated secondary antibody, and nucleus was counterstained with DAPI. Histology sections were incubated with primary antibodies specific to α-smooth muscle actin (α-SMA) (1:100) for 1 h and then incubated with rhodamine-conjugated secondary antibodies. The sections were counterstained with DAPI. Images were visualized with Nikon Ti microscope and captured with NIS-Elements v.2.0 (Nikon, Melville, NY).

EdU Infusion and Staining

EdU-infused lungs were subjected to an assay for EdU detection using the Click-iT EdU AlexaFluor-647 Kit (Invitrogen C10340). α-SMA-specific antibody conjugated with Cy3 (C6198, Sigma, St. Louis, MO, 1:200) was coimmunostained to trace the architecture. Images were captured with a Nikon Eclipse 50i microscope.

Hydroxyproline Assay

Hydroxyproline concentrations were analyzed as previously described (15, 25). In brief, lung explants were acid-digested using phosphate-buffered saline:12 N HCl (1:1) at 120°C for 24 h. A known aliquot of this sample was sequentially reacted against citrate/acetate buffer, chloramine T solution, and Ehrlich’s reagent followed by incubation at 65°C for 30 min. The absorbance of each sample was measured at 550 nm. Standard curves for the experiment were generated using known concentrations of hydroxyproline (Sigma-Aldrich, St. Louis, MO).

Real-Time PCR

Total RNA was isolated using the RNeasy mini kit (Qiagen, Hilden, Germany), and cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Real-time PCR was conducted using an Applied Biosystems StepOne machine with commercially validated primer sets. Real-time PCR was performed using TaqMan Gene Expression Assays (Applied Biosystems, Waltham, MA) for all genes measured, whereas mouse Nfatc1 and Nfatc2 were measured using QuantiTect Primer Assays (Qiagen, Hilden, Germany), in TaqMan Gene Expression Master Mix (4369016, Applied Biosystems, Waltham, MA). The relative transcript level for each target gene was expressed as 2^−ΔΔCt relative quantitation compared to the endogenous control.

Flow Cytometric Analysis

• 1)To characterize the immune cell types in Nfat1^−/− and wild-type mice at day 7 after bleomycin injury, multichannel flow cytometric analysis was used to quantify inflammatory cell infiltration. Single-cell suspensions enriched for lung leukocytes were obtained from perfused and collagenase A-digested lungs and immunostained for 30 minutes with specific conjugated antibodies (BD Biosciences, San Jose, CA) or isotype-matched control at recommended concentrations. Antibodies used to identify specific immune populations are presented in Supplemental Table S1.
• 2)Lineage negative (CD45⁻/CD31⁻EpCAM⁻) populations were gated for platelet-derived growth factor receptor alpha positive (PDGFRα⁺) mesenchymal cells in Nfat1^−/− and wild-type mice at day 14 after bleomycin injury. The following antibodies were used: BD Pharmingen PerCP rat anti-mouse CD45 (557235, BD Biosciences), EpCAM PerCP-eFluor 710 (46-5791-82, eBioscience), CD31(PECAM-1) PerCP-eFluor 710 (46-0311-82, eBioscience), and PDGFRα PE-Cyanine7 (25-1401-82, eBioscience).
• 3)To determine the intracellular autotaxin expression in mesenchymal cells derived from the lungs of patients with IPF, control cells and cells treated with VIVIT peptide (NFAT Inhibitor-1, HY-P1026, MedChemExpress; Monmouth Junction, NJ) were washed and stained for intracellular autotaxin using the ectonucleotide pyrophosphatase (ENPP2) antibody (PAS-12478, Invitrogen) and detected by an AF488-labeled secondary Ab (A11034, Invitrogen). All washed and stained cells were analyzed by FACS analysis on a BD LSRFortessa (Becton Dickinson, Franklin Lakes, NJ) and FlowJo software ver.10.7.1 was used to calculate specific populations using established gating strategies.

Murine Primary Cell Isolation and Culture

Lungs from 4- to 6-wk-old mice were harvested, digested with collagenase A (Roche, Basle, Switzerland), mechanically dissociated using an 18 G needle, and grown in DMEM supplemented with 10% fetal bovine serum and 100 U/mL penicillin/streptomycin as previously described (25). Cells were passaged and expanded at least once before use at passage 2.

siRNA-Mediated Silencing

Dharmacon SMARTpool ON-TARGETplus human siRNAs specific to NFATC2 (D-00360601-04-05-06) or nontargeting pool (D-001810-10) were used. In brief, mesenchymal cells were seeded at 50% confluence and transfected with 100 nM siRNA per well using Oligofectamine (Invitrogen) suspended in opti-MEM medium. The next day, medium was changed to serum-free DMEM for 48–72 h before protein harvesting and immunoblot analysis.

Immunoblot Analysis

Bleomycin-injured lungs were homogenized in PBS and centrifuged at 10,000 rpm for 10 min at 4°C. Supernatants were stored in −80°C until analyzed by immunoblotting. Primary cultured mesenchymal cells from the whole lungs of adult Nfat1^−/− and wild-type mice were cultured to 60% confluence at passage 2 and the mesenchymal cells were then serum-starved overnight and harvested for immunoblot analysis as described previously (22). The following primary antibodies were used in this study, anti-NFAT1 (MAB6499, R&D Systems, Minneapolis, MN), anti-autotaxin (10005375, Cayman Chemical, Ann Arbor, MI; 1:100), anti-cyclin D1 (sc-718, Santa Cruz Biotechnology, Dallas, TX; 1:1,000), and anti-GAPDH (MAB374, Millipore, Burlington, MA; 1:5,000). Corresponding secondary antibodies of horseradish peroxidase (HRP)-conjugated anti-mouse (A8924, Sigma-Aldrich, St. Louis, MO; 1:20,000) or anti-rabbit (A0545, Sigma-Aldrich, St. Louis, MO; 1:10,000) were used, respectively. Densitometry was performed using NIH ImageJ software (ver.1.50i). The ratio of the band density for the target protein and the corresponding GAPDH was determined.

ELISA

Conditioned media from normal and IPF MCs were clarified by centrifugation at 10,000 rpm for 10 min at 4°C. The samples were then analyzed using Human ENPP-2/Autotaxin Quantikine ELISA Kit (Cat. No. DENP20; R&D Systems, Minneapolis, MN). Murine lungs were homogenized in 1 mL phosphate-buffered saline, followed by centrifugation at 10,000 rpm for 10 min at 4°C. Samples were analyzed using Autotaxin sandwich ELISA (Cat. No. K600, Echelon Biosciences, Salt Lake City, UT).

Cell Migration Assay

Transwell dishes (8 µm pore size; Fisher Scientific, Hampton, NH) were coated with 1 mg/mL Matrigel (Corning, Corning, NY) as previously described (26). MCs were subcultured into the upper chamber using serum-free DMEM, while the bottom chamber was filled with serum-free DMEM containing 10 µM lysophosphatidic acid (LPA). After 18 h, all cells on the upper surface of the filter were mechanically removed and cells that migrated through the matrigel to the underside of the filter were quantified using a standard 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; Sigma-Aldrich, St. Louis, MO) assay.

Statistical Analysis

The Student’s two-tailed t test was used to determine P values when comparing two groups. When comparing three or more groups, one-way analysis of variance was performed with a post hoc Bonferroni test to determine which groups showed significant differences unless otherwise specified. A P value of less than 0.05 was considered significant using GraphPad Prism (ver.8.0.0) for Windows 64-bit. Survival analyses were performed in STATA (ver.12.0, College Station, TX) using the log-rank test (27, 28), and results were presented as Kaplan–Meier curves (29).

RESULTS

NFAT1 Activation in IPF and Protective Effect of Nfat1 Deficiency in Murine Model of Bleomycin-Induced Lung Fibrosis

To determine the potential relevance of NFAT1 in IPF, we first assessed NFAT1 expression in human IPF lung tissues and mesenchymal cells (MCs) derived from lungs of patients with IPF. Higher mRNA expression of NFAT1 (NFATC2) was noted in IPF lung homogenates (n = 12) compared with pathologically “normal” lung tissues (n = 3) by real-time PCR (Fig. 1A; P = 0.0099). Comparison of control and IPF MCs demonstrated higher total and nuclear NFAT1 protein expression in IPF cells by Western blot analysis (Fig. 1, B and C). Increased nuclear localization of NFAT1 was further confirmed by immunofluorescence staining (Fig. 1D). Next, to ascertain the pathogenic role of NFAT1 signaling in the development of lung fibrosis, we investigated the effect of Nfat1 deficiency on fibrogenesis in the bleomycin lung injury model. Lung tissue from Nfat1^−/− mice (21, 30) did not demonstrate differences in the expression of other NFAT family members (Nfat2, Nfat3, Nfat4, Nfat5) as analyzed by real-time PCR (Supplemental Fig. S1). A difference in survival was noted between WT and Nfat1^−/− mice exposed to oropharyngeal instillations of bleomycin (0.025 U/mouse) with no deaths noted in the Nfat1^−/− mice (Fig. 1E, P = 0.002). To determine the extent of lung fibrosis in bleomycin-injured mice due to genetic depletion of Nfat1, bleomycin-injured adult Nfat1^−/− and wild-type (WT) mice were euthanized at day 21 after injury, and the collagen expression was quantitatively evaluated by hydroxyproline assay. Bleomycin-treated Nfat1^−/− mice demonstrated significantly lower collagen expression than bleomycin-treated WT mice with no significant difference noted in hydroxyproline content between saline- and Bleomycin-treated Nfat1^−/− mice (Fig. 1F). On histopathological evaluation, Nfat1^−/− mice demonstrated a significant reduction in the extent of scarring and focal accumulation of activated mesenchymal cells as revealed by H&E staining (Fig. 1G) and α-SMA labeling (Fig. 1H). Masson’s trichrome staining confirmed decreased fibrotic injury and collagen expression in bleomycin-treated Nfat1^−/− as compared with WT mice (Fig. 1I).

Figure 1.

Upregulation of NFAT1 in IPF tissues and murine Nfat1 deficiency promotes survival and attenuates collagen deposition due to bleomycin injury. A: RNA isolated from pathologically “normal” and IPF lung biopsies were analyzed by real-time PCR for NFATC2. Values: means ± SE. Statistics: unpaired t test, **P < 0.01. B: protein lysates from quiescent normal and IPF lung mesenchymal cells were subjected to immunoblotting and probed against NFAT1 and GAPDH (loading control). Densitometry shows higher NFAT1 expression in IPF MCs. Values: means ± SE. Statistics: unpaired t test, *P < 0.05. C: nuclear fractions from quiescent normal and IPF mesenchymal cells were immunoblotted and probed against NFAT1 and SAM68 (loading control for nuclear fraction). D: immunofluorescent staining of quiescent normal and IPF mesenchymal cells against NFAT1. Nuclear counterstain was with DAPI. Original magnification: ×40. Scale bar: 100 µm. E–I: C57BL6/J wild-type (WT) and Nfat1^−/− mice were subjected to bleomycin injury (0.025 U) and euthanized 3 wk later. E: Kaplan–Meier survival curve for bleomycin-injured WT and Nfat1^−/− mice. WT: 35; Nfat1^−/−: 36. Statistics: log-rank test. F: quantitative analysis of collagen deposition. Hydroxyproline content in acid-digested lung homogenates was measured in triplicates and repeated twice. WT saline: 6; WT bleo: 18; Nfat1^−/− saline: 5; Nfat1^−/− bleo: 19. Values: means ± SD. Statistics: one-way ANOVA; post hoc Bonferroni, ****P < 0.0001. G–I: histopathology examination of formalin-fixed paraffin-embedded lung tissues. G: hematoxylin and eosin staining showed bleomycin-injured extensive lung remodeling in wild-type but not in injured Nfat1^−/− mice. H: fluorescent labeling of mesenchymal differentiation marker, α-smooth muscle actin (α-SMA), to demonstrate presence or absence of fibroblastic foci (FF) around the vessels (V) and airways (AW). Nuclei were counterstained with DAPI. I: formalin-fixed paraffin-embedded representative sections with Masson’s trichrome staining (blue) demonstrated extent of parenchymal and airway fibrosis indicating collagen deposition. n = 5 mice/group. Scale bar: 100 μm. IPF, idiopathic pulmonary fibrosis; MC, mesenchymal cell; NFAT1, nuclear factor of activated T cells 1; SAM68, SRC associated in mitosis of 68 kDa.

Fibroprotection due to Genetic Deficiency of Nfat1 Is Independent of Immune Cell Influx

To investigate whether fibroprotection in Nfat1^−/− mice may be due to potential participation of recruited inflammatory leukocyte subsets, collagenase-digested lungs of bleomycin-injured Nfat1^−/− and wild-type (WT) mice were analyzed by FACS analysis to enable the identification of innate immune cells and myeloid populations as shown in the gating strategy in Fig. 2A. Total CD45⁺ immune cells were twofold lower in bleomycin-treated Nfat1^−/− compared with WT murine lungs (Fig. 2B), but no changes were noted in the recruited CD3⁺/CD4⁺ T cell populations (Fig. 2B). Total and interstitial macrophages were approximately twofold lower in injured lungs from Nfat1^−/− mice (Fig. 2C), with no changes noted in total alveolar macrophages (Fig. 2C) or in the tissue-resident and monocyte-derived alveolar macrophages (Supplemental Fig. S2). Bleomycin injury induces an influx of CD11b⁺ and CD11b⁻ dendritic cells, but only the latter subset was lower in injured lungs from the Nfat1^−/− mice(Fig. 2D). No significant differences were observed in the bleomycin-mediated influx of monocytes (Fig. 2E) or B cells (Fig. 2F) in the WT mice, but lower total and inflammatory monocytes were noted in injured lungs from Nfat1^−/− mice. No differences were noted in the populations of neutrophils and eosinophils due to bleomycin injury in the WT and Nfat1^−/− mice (data not shown).

Figure 2.

Fibroprotection in Nfat1-deficient mice is independent of immune influx. A: contour plots of gating strategy used to identify major immune cell populations (R1–R7) in saline-treated murine lungs. Immune populations include T and B cells; total, alveolar, and interstitial macrophages (Macs); total, inflammatory, and resident monocytes (Mono); and CD11b^+/− dendritic cells (DCs). The flow panels showing distribution of cells are representative of 3 mice. B–F: immune profiling of wild-type (WT) and Nfat1^−/− mice 7 days after bleomycin injury. FACS analyses were performed on single-cell suspensions of collagenase-digested lungs. Values: means ± SE. Statistics: one-way ANOVA, post hoc Bonferroni test compared with wild-type bleomycin-injured group. *P < 0.05, **P < 0.01, ***P < 0.001. NFAT1, nuclear factor of activated T cells isoform 1; ns, not significant.

Nfat1 Deficiency in Radioresistant Hematopoietic Cells Does Not Confer Fibroprotection

To further delineate the contribution of NFAT1 loss in the hematopoietic compartment to the protection against fibrosis noted in Nfat1^−/− mice, we created chimera mice by reconstituting bone marrow cells from healthy WT or Nfat1^−/− mice into irradiated WT mice (WT→WT and Nfat1^−/−→WT). Bleomycin treatment was used in these mice as shown in Fig. 3A. At the end of the 3-wk study period, lung tissue sections were examined by histopathology analyses for collagen deposition. Masson’s trichrome staining of the tissue sections revealed that the extent of injury in the lung architecture and collagen deposition was comparable in both WT→WT and Nfat1^−/−→WT chimera mice (Fig. 3B). Furthermore, quantitative analyses of collagen deposition demonstrated no significant difference in hydroxyproline concentrations between Nfat1^−/−→WT chimera and WT→WT chimera mice, with both showing significant increase as compared with saline treatment (Fig. 3C).

Nfat1 Deficiency Attenuates Mesenchymal Proliferation and Differentiation

Our data using bone marrow chimera mice suggested a role for NFAT1 in directly regulating fibroproliferative responses. To investigate this further, we first assessed the impact of NFAT1 deficiency on mesenchymal cell expansion in response to bleomycin. Lineage^neg/PDGFRα^pos (Lin⁻/PDGFRα⁺) mesenchymal cells were measured by flow cytometry in single-cell suspensions of lungs isolated from saline- or bleomycin-treated WT and Nfat1^−/− mice (Fig. 4A). A significant increase in Lin⁻/PDGFRα⁺ cells was noted in response to bleomycin in WT but not in Nfat1^−/− mice; bleomycin-treated Nfat1^−/− demonstrated significantly lower mesenchymal cell numbers as compared with WT mice (Fig. 4B, P < 0.05). Lung homogenates derived from bleomycin-injured lungs from wild-type and Nfat1^−/− mice were then assessed by Western blotting for the expression of the cell cycle progression marker, cyclin D1 (Fig. 4C). Bleomycin-induced upregulation of cyclin D1 was significantly diminished due to Nfat1^−/− deficiency as demonstrated by densitometry analyses (P < 0.05). In vivo proliferation was further investigated by using EdU incorporation (31). Nfat1^−/− and wild-type mice were injected with EdU (50 mg/kg body wt) at day 14 after bleomycin injury and euthanized 24 h later. Subsequently, formalin-fixed paraffin-embedded tissues were subjected to dual immunostaining for EdU and α-SMA (Fig. 4D). A significant decrease in the extent of fibrosis and cell cycle activity marked by EdU positive cells was noted in the bleomycin-injured lungs from Nfat1^−/− mice compared with wild-type controls. Confocal microscopy images confirmed nuclear staining of EdU in cells positive for α-SMA-positive cells in WT bleomycin-treated lungs (Fig. 4E). We next investigated the extent of mesenchymal differentiation in these lungs by measuring the Lin⁻/PDGFRα⁺ MC subset for Acta2 (α-SMA) mRNA expression by quantitative real-time PCR analysis. Lin⁻/PDGFRα⁺ MCs derived from WT mice with bleomycin injury expressed fourfold higher levels of Acta2 mRNA compared with saline (Fig. 4F, P < 0.001), and this upregulation was repressed in Nfat1^−/− mice with bleomycin injury (Fig. 4F, P < 0.0001). Lung fibrosis has been associated with resistance to apoptosis in mesenchymal cells and NFAT1 is a known transcriptional activator of the apoptosis inhibitor, survivin (32). Hence, we investigated the differences in apoptosis by analyzing the Lin⁻/PDGFRα⁺ MCs for the mRNA expression of survivin (Birc5) by quantitative real-time PCR. Lin⁻/PDGFRα⁺ MCs derived from bleomycin-injured WT mice expressed ∼2.5-fold higher levels of Birc5 mRNA compared with mice treated with saline (Fig. 4F, P < 0.05), and this upregulation was repressed in Nfat1^−/− mice with bleomycin injury (Fig. 4F, P < 0.0001).

Figure 4.

Nfat1 deficiency ameliorates mesenchymal proliferation and differentiation. Wildtype (WT) or Nfat1^−/− mice instilled with bleomycin were harvested at day 14 after injury. A: scatter plot showing distribution of lineage^neg/PDGFRα^pos mesenchymal cells (Lin⁻/PDGFRα⁺ MCs). Single-cell suspensions of collagenase-digested lungs were sorted by flow cytometry for lineage^neg/PDGFRα^pos mesenchymal cells (Lin⁻/PDGFRα⁺ MCs). B: data showing diminished number of MCs in injured Nfat1^−/− lungs. C: lung homogenates of WT and Nfat1^−/− mice were analyzed for cyclin D1 by western blotting. Representative immunoblot and corresponding densitometry analyses are shown. D: proliferating cells were labeled with 5-ethynyl-2′-deoxyuridine (EdU; 50 mg/kg body wt ip) 24 h before harvest. Representative tissues sections were dual labeled with α-SMA (green) and EdU (red). Nuclei were counterstained with DAPI. Scale bar: 100 μm. Original magnification: ×200. Yellow arrowheads highlight colocalization of EdU in labeled mesenchymal cells. E: laser scanning confocal microscopy images of the injured WT and Nfat1^−/− lungs. F: Lin⁻/PDGFRα⁺ MCs were analyzed for Acta2 and Birc5 by real-time PCR. B, C, and F: values: means ± SE. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Statistics: one-way ANOVA; post hoc Bonferroni (B and F); unpaired t test (C). α-SMA, α-smooth muscle actin; MC, mesenchymal cell; NFAT1, nuclear factor of activated T cells isoform 1; PDGFRα, platelet-derived growth factor receptor alpha.

NFAT1 Regulates Autotaxin in Vivo and in IPF MCs

Among potential mechanisms by which NFAT1 can regulate mesenchymal cell activation and fibrosis is through its transcriptional activation of fibrotic mediator ATX (15). To investigate the in vivo relevance of this pathway, autotaxin expression was compared between bleomycin-injured murine lungs from WT and Nfat1^−/− mice. Autotaxin protein expression, quantitated by ELISA in whole lung homogenates, demonstrated an approximately threefold induction in wild-type mice after bleomycin injury. This induction was significantly attenuated in Nfat1^−/− mice with bleomycin injury (Fig. 5A, P < 0.01). Western blot analyses showed lower autotaxin protein expression in lung homogenate from bleomycin-treated Nfat1^−/− mice as compared with bleomycin-treated WT mice (Fig. 5A; P < 0.01). Autotaxin mRNA expression was approximately fivefold higher in bleomycin-injured lungs from WT mice (Fig. 5A, P < 0.01) and this induction was suppressed in bleomycin-treated Nfat1^−/− lungs (Fig. 5A, P < 0.05). MC-derived autotaxin uniquely drives their migration capacity via autocrine synthesis of the profibrotic lysophosphatidic acid (LPA) (15), which led us to hypothesize that the migration capacity of MCs from lungs of Nfat1^−/− will be lower than those of WT mice. Primary cultured lung mesenchymal cells derived from Nfat1^−/− and WT mice were grown in matrigel-coated transwell chambers for a period of 18 h in the presence or absence of LPA. The migrated mesenchymal cells were harvested and subjected to a quantitative MTT assay. We observed that LPA-induced migration rate in wild-type cells was 50% higher than those derived from bleomycin-injured lungs from Nfat1^−/− mice (Fig. 5B; P < 0.0001). Our findings in Fig. 1, A–D indicated high NFAT1 expression in IPF lungs and MCs, therefore we investigated the constitutive autotaxin secretion by these cells using ELISA. We observed approximately fourfold higher autotaxin levels in the conditioned media of IPF MCs compared with that of normal MCs (Fig. 5C; P < 0.05). We next examined the role of NFAT1 in regulating autotaxin expression using pharmacological and genetic approaches. IPF MCs were treated with a pharmacological inhibitor against NFAT, NFAT Inhibitor-1 (VIVIT peptide; 30 µM × 48 h), and the cells were examined for intracellular autotaxin expression by FACS analysis. We observed that NFAT inhibition caused a shift to the left in the histogram, thus suggesting a significant decrease in autotaxin expression (Fig. 5C; P < 0.05). We then subjected IPF MCs to RNAi-specific NFATC2 silencing and analyzed autotaxin mRNA and protein expression by real-time PCR and immunoblotting analysis, respectively. NFATC2 silencing leads to ∼50% decrease in autotaxin mRNA levels (Fig. 5D; P < 0.05) and protein expression (Fig. 5E; P < 0.05).

Figure 5.

NFAT1-related downregulation of autotaxin in vivo and in IPF MCs. A: lung homogenates of wild-type (WT) and Nfat1^−/− mice at day 21 after bleomycin injury were analyzed for autotaxin (ATX) by ELISA and Western blotting. Representative immunoblot from the same gel and corresponding densitometry analyses is shown. RNA isolated from lung homogenates was analyzed for ATX by real-time PCR. B: primary mesenchymal cells derived from WT and Nfat1^−/− mice were seeded on matrigel-coated upper transwell chambers and cultured ± LPA for 18 h. Migrated cells in the lower chamber were qualitatively analyzed by cytology and quantitatively by MTT assay. C: conditioned media from quiescent normal and IPF MCs were analyzed for secreted Autotaxin by ELISA. D: IPF MCs were treated ± NFAT1 inhibitor (NFAT1i; 30 µM × 48 h) and analyzed for autotaxin by FACS analysis. Representative histogram and analyses are shown. E and F: mesenchymal cells derived from the lungs of patients with IPF (IPF MCs) were transfected with scrambled or NFATC2 siRNA. RNA lysates were analyzed by real-time PCR for NFATC2 and ATX as shown in E. Protein lysates were analyzed by Western blot for NFAT1 and autotaxin. Representative immunoblots and corresponding densitometry are shown in F. Values: means ± SE. *P < 0.05, **P < 0.01, ****P < 0.0001. Statistics: unpaired t test (A, C–F); one-way ANOVA; post hoc Bonferroni (A and B). IPF, idiopathic pulmonary fibrosis; LPA, lysophosphatidic acid; NFAT1, nuclear factor of activated T cells isoform 1.

DISCUSSION

Calcium signaling is downstream of multiple pathways of profibrotic activation in mesenchymal cells and can regulate gene signature via target NFAT family of transcription factors (13). However, although NFAT targets are widely recognized in immune cells, their transcriptional regulation of cellular processes such as cell cycle, proliferation, differentiation, and migration in mesenchymal cells and their pathogenic role in lung fibrosis have not been fully elucidated. In the current report, as shown in the graphical abstract, we demonstrate that 1) Nfat1 deficiency protects against bleomycin-induced lung fibrosis and improves survival rates, 2) the fibro-protected Nfat1-deficient mice present immune flux comparable with wild-type controls, and mice with hematopoietic cell-specific genetic deletion of Nfat1 are not protected against bleomycin-induced fibrosis, 3) NFAT1 is required for mesenchymal proliferation, resistance to apoptosis, and differentiation during fibrogenesis, and 4) NFAT1 regulates key profibrotic mediator autotaxin in the context of IPF. The findings reported here suggest a paradigm shift in that targeting Nfat1 modulates fibrosis independent of immune responses and identifies NFAT1 as a common downstream pathway through which diverse profibrotic signals can regulate mesenchymal activation.

Nfat1 deficiency protected against fibroproliferative responses in the lung in response to instillation of bleomycin, a commonly used subacute experimental fibrosis model of the lung, with findings suggesting the impact of lack of NFAT1 on mesenchymal proliferation, migration, resistance to apoptosis, and differentiation. Along with improved survival and lower quantitative expression of collagen, Nfat1^−/− mice exposed to bleomycin demonstrated a lower number of Lin⁻/PDGFRα⁺ mesenchymal cells as well as downregulation of cyclin D1 as compared with control mice, a finding consistent with known effect of NFAT1 on cell cycle progression (26, 33) and fibroblast proliferation (34, 35). Furthermore, although control mice demonstrated significant EdU activity in areas of α-SMA expression marking activated mesenchymal cells, both decreased activated mesenchymal infiltration and EdU staining were noted in Nfat1^−/− mice. That mesenchymal cell activation and differentiation are abrogated in the absence of Nfat1 was further confirmed by decreased α-SMA expression in sorted PDGFRα⁺ cells from Nfat1^−/− mice. Survivin is an apoptosis inhibitor upregulated in IPF lungs and associated with mesenchymal resistance to apoptosis (36, 37). BIRC5 was identified as a candidate effector gene for the NFATC2-dependent enhancer loci, as it is a gene member within the topologically associating domains (32). Our data indicate decreased resistance to apoptosis in sorted PDGFRα⁺ cells from injured Nfat1^−/− mice. Although our future studies will further dissect the role of NFAT1 in mesenchymal resistance to apoptosis, our current findings represent the first in vivo demonstration of the necessary role of NFAT1 in regulating fibroproliferative responses in the lung.

A significant finding of our study is the demonstration that these protective effects of lack of NFAT1 on fibrosis are not mediated via well-characterized immune regulatory functions of NFAT. Hematopoietic cell-specific Nfat1-deficient chimera mice demonstrated collagen deposition that was not statistically different from wild-type chimera, suggesting that the protection from fibrosis noted in Nfat1^−/− mice is dependent on the lung-resident cells and not on the hematopoietic compartment. Consistent with these findings, Fonseca et al. (38) have reported that Nfat1-deficient mice had lower bronchoconstriction in response to methacholine stimulation, which was independent of the enhanced lung-allergic inflammation observed in these mice. Prior studies have described the proinflammatory role of NFAT1 in LPS-induced acute lung injury through the miR155/IRF2BP2/NFAT1 signaling axis (39) and IL-33-induced nuclear translocation of NFAT1 in innate lymphoid cells during asthma (40). NFAT1 is a key mediator of T cell activation, differentiation, and development (41, 42). However, Nfat1-deficient mice were paradoxically shown to have increased lymphocytic proliferation in allergen-sensitized mice (34). Immune influx has been well characterized in the bleomycin injury model (43–45), and CD3⁺ (46) and CD4⁺ (47, 48) T cells are implicated in profibrotic responses. Characterization of immune response components in WT and Nfat1^−/− mice in context of bleomycin injury in our study demonstrated intriguing findings. No differences were noted between Nfat1^−/− and WT injured mice in T or B cells, suggesting that loss-of-function does not affect immune influx in the lung and the fibroprotection is independent of lymphocytic recruitment. We did find a decrease in total/interstitial macrophages, CD11c⁺CD11b⁻ dendritic cells, macrophages, and inflammatory monocytes in Nfat1^−/− mice. Whether those differences reflect the effect of loss of NFAT1 in the immune cells or resident somatic cells remains to be elucidated. Lung-resident cells play an important role in the regulation of the immune milieu of an injured lung (49) with an increasing understanding of the paracrine actions of activated mesenchymal cells, through which they can promote immune cell recruitment (15). Future studies will focus on elucidating the role of NFAT1 in modulating mesenchymal immune cell interaction.

Although NFAT downstream targets in mesenchymal cells remain to be fully elucidated, we provide further evidence that a specific mechanism by which NFAT1 can regulate mesenchymal cell function is via regulation of ATX, a secreted enzyme that has been demonstrated to play a vital role in tissue fibrosis (50). Enzymatic activity of ATX leads to the generation of the majority of extracellular LPA and regulates cellular processes via the ATX/LPA/LPA1 signaling axis. NFAT1 is a known transcriptional enhancer of ATX expression with two NFAT1-binding sites described in the ATX promoter region (51). We have previously demonstrated that NFAT1 regulates ATX in lung mesenchymal cells and delineated an autocrine activation loop where ATX generates LPA via LPA1 ligation and its known second messenger calcium, which leads to NFAT1 nuclear translocation and further ATX expression. Stable increased ATX expression is well recognized in human mesenchymal cells derived from fibrotic tissues, including IPF lungs, and it has been shown that local ATX increases in the bleomycin lung model. We found that Nfat1^−/− mice subjected to bleomycin demonstrated lower ATX expression in lung homogenates and lung mesenchymal cells from Nfat1^−/− mice demonstrated diminished migratory capacity. Furthermore, we confirm increased ATX secretion by IPF MCs and demonstrate the regulation of ATX by NFAT1 in IPF mesenchymal cells, underscoring the translational relevance of this pathway.

The in vivo demonstration of the significance of NFAT signaling in the lung-resident compartment in regulating fibrosis highlights the future need to further delineate the regulation of NFAT1 activation in mesenchymal cells in homeostatic and fibrotic conditions. We have previously demonstrated that unstimulated lung mesenchymal cells in vitro demonstrate both cytoplasmic and nuclear NFAT1 expression with increased nuclear translocation noted in response to profibrotic stimuli like LPA (15). This is unlike immune and even epithelial cells, where nuclear NFAT1 expression is absent in unstimulated cells. A previous study reported similar findings in skin fibroblasts and demonstrated that they differed significantly from keratinocytes studied in the same experimental conditions (52). In the present study, we demonstrate increased nuclear NFAT1 expression in IPF mesenchymal cells, consistent with their activated status. Immunofluorescence staining again confirmed the presence of both cytoplasmic and nuclear NFAT1 in control and IPF cells with increased nuclear concentration in IPF. NFAT1 activation in skin fibroblasts demonstrated muted response to calcineurin inhibitors cyclosporine, which is highly effective at inhibiting NFAT1 activation and nuclear translocation in epithelial cells and immune cells (51). We observed comparable results in lung mesenchymal cells with variable response to cyclosporine (data not shown), further underscoring the need to delineate cell-specific differences in NFAT signaling. Identifying a robust NFAT inhibitor in mesenchymal cells can potentially allow for therapeutic targeting of fibrosis and will be the topic of future investigations.

In summary, we provide the first in vivo evidence for the role of NFAT1 in promoting lung fibrosis independent of its immunomodulatory role and suggest a future potential benefit of further investigating and targeting this final common pathway downstream of calcium dysregulation in mesenchymal cells.

DATA AVAILABILITY

All data generated during this study are included in this article.

SUPPLEMENTAL DATA

Supplemental Table S1 and Supplemental Figs. S1 and S2: https://doi.org/10.6084/m9.figshare.24328492.

GRANTS

This work was supported by NIH-NHLBI-R01 (HL118017, HL162171, HL094622), the Taubman Institute, the Cystic Fibrosis Foundation (LAMA16XX0), and the Campbell Gift Fund awarded to V.N.L.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

R.V., N.M.W., and V.N.L. conceived and designed research; R.V., N.M.W., A.P.M., R.R.B., F.K., F.F., K.M., Y.A., C.A.W., S.K.H., and P.C. performed experiments; R.V., N.M.W., A.P.M., R.R.B., F.K., F.F., M.P.C., D.S.W., S.K.H., and P.C. analyzed data; R.V., N.M.W., R.R.B., B.B.M., and V.N.L. interpreted results of experiments; R.V., N.M.W., and R.R.B. prepared figures; R.V., N.M.W., and V.N.L. drafted manuscript; R.V., N.M.W., and V.N.L. edited and revised manuscript; V.N.L. approved final version of manuscript.