Loss of Twist1 in the Mesenchymal Compartment Promotes Increased Fibrosis in Experimental Lung Injury by Enhanced Expression of CXCL12

Abstract

Idiopathic pulmonary fibrosis (IPF) is a disease characterized by the accumulation of apoptosis-resistant fibroblasts in the lung. We have previously shown that high expression of the transcription factor Twist1 may explain this prosurvival phenotype in vitro. However, this observation has never been tested in vivo. We found that loss of Twist1 in COL1A2⁺ cells led to increased fibrosis characterized by very significant accumulation of T cells and bone marrow-derived matrix-producing cells. We found that Twist1-null cells expressed high levels of the T cell chemoattractant CXCL12. In vitro, we found that the loss of Twist1 in IPF lung fibroblasts increased expression of CXCL12 downstream of increased expression of the noncanonical NF-κB transcription factor RelB. Finally, blockade of CXCL12 with AMD3100 attenuated the exaggerated fibrosis observed in Twist1-null mice. Transcriptomic analysis of 134 IPF patients revealed that low expression of Twist1 was characterized by enrichment of T cell pathways. In conclusion, loss of Twist1 in collagen-producing cells led to increased bleomycin-induced pulmonary fibrosis, which is mediated by increased expression of CXCL12. Twist1 expression is associated with dysregulation of T cells in IPF patients. Twist1 may shape the IPF phenotype and regulate inflammation in fibrotic lung injury.

FIGURE 1.

Loss of Twist1 in COL1A2⁺ cells leads to increased bleomycin-induced pulmonary fibrosis. (A) Schematic representation of the triple transgenic COL1A2 Cre-ER(T) Twist1 fl/fl ROSA26-tdTomato mouse (Twist1 FL). (B) Gating strategy for tdTomato⁺ cells in the lung. (C) Negative control fluorescent images of spleen showing rare tdTomato⁺ cells (left) and dot plots of splenocytes showing absence of tdTomato⁺ cells (right). (D) Fluorescent images of lungs from bleomycin-injured animals showing tdTomato⁺ (red) cells and staining α-SMA (left, green) or surfactant protein C (SFTPC; right, green). Original magnification, ×200. Arrows indicate α-SMA⁺tdTomato⁺ airway or vascular smooth muscle cells. Arrowheads indicate tdTomato⁻ endothelial cells overlying vascular smooth muscle. Nuclei are counterstained with DAPI. (E) Immunofluorescent images of CD45 expression (green). Yellow arrowheads identify CD45⁺tdTomato⁺ cells and the white arrow identifies a CD45⁺tdTomato⁻ cell. Original magnification, ×400. (F) At 14 d after injury, tdTomato⁺ cells from Twist1 WT or Twist1 FL injured with bleomycin were flow sorted and processed immediately for quantitative RT-PCR of Twist1 (*p < 0.0001, n = 3). (G) H&E staining of lungs at 14 d after bleomycin injury in Twist1 WT or Twist1 FL animals (yellow inset scale bar, 200 μm; original magnification, ×100). Masson trichrome images from bleomycin-injured are magnified (green inset scale bars, 50 μm; original magnification. ×400). (H) Left lungs were processed for detection of acid-soluble collagen. Bleomycin-induced deposition of collagen was increased in Twist1 FL animals compared with WT controls (*p = 0.03 saline plus Twist1 FL versus bleomycin plus Twist1 WT, and **p < 0.003, bleomycin plus Twist1 WT versus bleomycin plus Twist1 FL, by ANOVA, n = 10–14 per group). Quantitative RT-PCR of flow-sorted cells from bleomycin-injured Twist1 WT or FL animals for (I) COL1A1 (*p < 0.0001, n = 3, by t test), (J) FN1 (*p = 0.0001, n = 3, by t test), and (K) Acta2 (α-SMA, *p = 0.033, n = 3, by t test). (L and M) Flow cytometry was performed to quantify the number of CD45⁺ and tdTomato⁺ cells. Total tdTomato⁺ cells were significantly higher in the bleomycin-injured Twist1 FL mice than in their WT counterparts (*p < 0.04, n = 8–9). No significant difference was observed between tdTomato⁺CD45⁻ cells in (N). (O) Significantly more CD45⁺tdTomato⁺ cells were detected in the Twist1 FL animals than in the WT (*p = 0.002, n = 8–9).

FIGURE 2.

Loss of Twist1 in COL1A2⁺ cells is associated with enhanced accumulation of T cells following bleomycin injury. (A–E) BAL was processed for flow cytometry for markers of neutrophils, macrophages, and T and B cells. For these experiments, n = 5 per uninjured condition and n = 11–12 for injured conditions. BAL was collected at 14 d after injury. (A) Dot plots of uninjured and bleomycin-injured animals for neutrophils, macrophages, T cells, and B cells. Quantification of (B) Ly6G, (C) CD68 (*p = 0.006, uninjured Twist1 WT versus uninjured Twist1 FL and **p < 0.025 by ANOVA, uninjured plus Twist1 FL versus bleomycin plus Twist1 FL), (D) CD3 (*p = 0.0021, bleomycin plus Twist1 WT versus bleomycin plus Twist1 FL), and (E) B220 (*p = 0.03, uninjured plus Twist1 WT versus uninjured plus Twist1 FL) is shown.

FIGURE 3.

Subphenotyping of T cells from bleomycin-injured Twist1 FL and WT mice. Flow cytometry of single-cell suspensions of bleomycin-injured Twist1 WT or Twist1 FL mouse lungs to describe the T cell subphenotypes following stimulation and intracellular cytokine staining is as described in Materials and Methods. (A) Dot plots for CD4⁺ IFN-γ, IL-4, and IL-17 from bleomycin-injured Twist1 WT and Twist1 FL animals. (B) Dot plot for CD4⁺Foxp3⁺ cells. (C) Dot plots for CD8⁺ IFN-γ, IL-4, and IL-17. Percentage of cells (left y-axis) and the absolute numbers of cells (right y-axis) are reported, n = 8 per condition, and data were analyzed by an unpaired t test. (D) CD4 (*p = 0.003). (E) CD4 plus IFN-γ (*p = 0.04, **p = 0.02). (F) CD4 plus IL-4. (G) CD4 plus IL-17. (H) CD4 plus Foxp3 (*p < 0.05). (I) CD8 (*p = 0.04). (J) CD8 plus IFN-γ (*p = 0.04, **p = 0.03). (K) CD8 plus IL-4 (*p < 0.05, **p = 0.05). (L) CD8 plus IL-17 (*p < 0.03, **p = 0.02).

FIGURE 4.

Loss of Twist1 in COL1A2⁺ cells and in human lung fibroblasts leads to increased expression of CXCL12. (A) BAL from Twist1 WT and Twist1 FL animals at 14 d following bleomycin injury was analyzed by ELISA for CXCL12 as described in Materials and Methods (*p < 0.002, n = 5–8, Twist1 WT plus uninjured versus Twist1 WT plus bleomycin, **p < 0.004, Twist1 WT plus bleomycin versus Twist1 FL plus bleomycin, n = 4–5 per group). (B) Quantitative RT-PCR for CXCL12 was performed on tdTomato⁺ cells flow sorted from Twist1 WT or Twist1 FL animals following bleomycin injury (*p < 0.0002, by t test, n = 3). Normal human lung fibroblasts were cultured in the presence of Twist1 siRNA (siTwist1) or nontargeting controls (siControl) and processed for quantitative RT-PCR for (C) Twist1 (*p < 0.0001, by t test, n = 3) and for (D) CXCL12 (*p < 0.022, by t test, n = 3). (E) IPF-derived lung fibroblasts were also treated with siTwist1 or siControl, and quantitative PCR was performed for CXCL12 (*p < 0.0001, by t test, n = 3). (F) Normal and IPF lung fibroblasts were incubated with siTwist1 or siControl and subjected to immunoblotting for Twist1, CXCL12, collagen I, and α-SMA. All experiments reflect fibroblasts from three independent normal and three IPF lungs. Data were analyzed by two-way ANOVA followed by a Newman–Keuls post hoc test. Band intensity was quantified for (G) Twist1 (*p < 0.0001, IPF siTwist1 versus siControl, n = 3), (H) CXCL12 (*p < 0.03, normal siTwist1 versus siControl, n = 3 and **p < 0.0001, IPF siTwist1 versus siControl, n = 3), (I) collagen I (*p < 0.02, normal siTwist1 versus siControl, n = 3), and (J) α-SMA. (K) Quantitative RT-PCR for CXCL12 was performed on IPF lung fibroblasts in the presence of siControl or siTwist1 with and without hypoxia (*p < 0.03, IPF siTwist1 versus siControl under normoxic conditions, **p < 0.0003, IPF normoxia and hypoxia, and ***p < 0.0001, siControl versus siTwist1 under hypoxic conditions, n = 3). (L) Immunoblot for Twist1 and CXCL12 with siControl or siTwist1 in the presence or absence of hypoxia. (M) ImageJ quantification of the blots in (L) for CXCl12 (*p < 0.0002, IPF siControl versus siTwist1 in normoxia and **p < 0.0001, siControl versus siTwist1 under hypoxic conditions, n = 3).

FIGURE 5.

Twist1-mediated regulation of CXCL12 expression is downstream of IKKα. (A) Immunoblotting (IB) of CXCL12 and IKKα (encoded by Chuk) from IPF fibroblasts incubated with siTwist1, siChuk, or both. Data were analyzed by two-way ANOVA followed by a Newman–Keuls post hoc test unless otherwise indicated. (B) Band densitometry for IKKα (Chuk) (*p < 0.017, siControl1 plus siControl2 versus siControl1 plus siChuk and **p < 0.03, siTwist1 plus siControl2 versus siTwist1 plus siChuk, n = 3) and (C) CXCL12 (*p < 0.029, siControl1 plus siControl2 versus siTwist1 plus siControl2 and **p = 0.0002, siTwist1 plus siControl2 versus siTwist1 plus siChuk, n = 3). (D) IB for MAP3K1, phospho-IKKα/β, total IKKα, and CXCL12 following silencing of Twist1, MAP3K1, or both. (E–H) Band densitometry was performed for (D). (E) MAP3K1 (*p < 0.0001, siControl1 plus siControl2 versus siControl1 plus siMAP3K1 and **p < 0.0001, siTwist1 plus siControl2 versus siTwist1 plus siMAP3K1, n = 3). (F) Phospho-IKKα/β (*p < 0.031, siControl1 plus siControl2 versus siControl1 plus siMAP3K1, n = 3). (G) Total IKKα. (H) CXCL12 (*p < 0.0001, siControl1 plus siControl2 versus siControl1 plus siMAP3K1 and **p = 0.003, siControl1 plus siMAP3K1 versus siTwist1 plus siMAP3K1).

FIGURE 6.

Loss of Twist1 in human lung fibroblasts leads to increased expression of RelB. (A) IPF fibroblasts were incubated with siTwist1 or siControl. Cells were processed for quantitative RT-PCR for the noncanonical NF-κB transcription factor RelB (*p < 0.003, n = 3, by t test). (B) Immunoblot of IPF fibroblasts following silencing of Twist1, RelB, or both. Data were analyzed by two-way ANOVA followed by a Newman–Keuls post hoc test. (C) ImageJ quantification of RelB (*p < 0.004, siTwist1 plus siControl2 versus siControl1 plus siControl2 and **p = 0.0014, siTwist1 plus siControl2 versus siTwist1 plus siRelB, n = 3) and (D) CXCL12 (*p < 0.0001, siTwist1 plus siControl2 versus siControl1 plus siControl2 and **p < 0.0001, siTwist1 plus siControl2 versus siTwist1 plus siRelB, n = 3). (E) Immunoblot of IPF fibroblasts following silencing of Twist1, the RelB regulator, RelA, or both. (F) ImageJ quantification of RelA (*p = 0.002, siRelA plus siControl2 versus siControl1 plus siControl2 and **p < 0.01, siTwist1 plus siControl2 versus siTwist1 plus siRelA, by two-way ANOVA, n = 3) and (G) RelB (*p < 0.002, siControl1 plus siRelA versus siControl1 plus siControl2 and **p = 0.01, siTwist1 plus siControl2 versus siTwist1 plus siRelA, by two-way ANOVA, n = 3. (H) Immunoblot of A549 cells with siTwist1 or siControl. (I) Locations of potential E-box motifs identified in the CXCL12 (top) and RelB (bottom) gene. The starting nucleotide position relative to the transcription start site was listed for each of the potential E-box motifs identified in the upstream sequences of each gene. Blue stars identify regions of DNA that were amplified by PCR of ChIP products. (J and K) PCR amplification of CXCL12 and RelB upstream sequences using ChIP products from (J) A549 cells and (K) normal, IPF, and MRC5 fibroblasts. For normal and IPF lung fibroblasts, n = 3. ChIP assay of the CXCL12 and RelB upstream sequences were performed using mouse Ab specific for Twist1. Ab specific for DNA polymerase II was used as a positive control and IgG1 purified from rabbit and mouse serum, respectively, were used as negative controls.

FIGURE 7.

Treatment of bleomycin-injured Twist1 FL animals with the CXCR4 blocker AMD3100 decreased the enhanced pulmonary fibrosis associated with the loss of Twist1 and significantly reduced the accumulation of T cells to the lung. Twist1 FL mice were injured with bleomycin or saline control and then treated with or without the CXCR4 blocker AMD3100. Animals were sacrificed on day 14. (A) H&E staining and Masson trichrome staining of lung sections (inset scale bars, 200 μm; original magnification, ×200). (B) The left lungs were processed for collagen determination by the Sircol assay (*p < 0.0001, uninjured versus bleomycin, n = 8–9 and **p = 0.0004 by ANOVA, bleomycin plus vehicle versus bleomycin plus AMD3100, n = 8–9). BAL was processed for flow cytometry for (C) and (D). Dot plots for Ly6G⁺ neutrophils, CD68⁺ macrophages, CD3⁺ T cells, and B220⁺ B cells from bleomycin-injured Twist1 FL mice treated with or without AMD3100. (E) Ly6G (*p < 0.0001, uninjured plus vehicle versus bleomycin plus vehicle, n = 8–9 and **p < 0.04, bleomycin plus vehicle versus bleomycin plus AMD3100, n = 8–9), (F) CD68 (*p < 0.0001, uninjured plus vehicle versus bleomycin plus vehicle, n = 8–9 and **p < 0.002, bleomycin plus vehicle versus bleomycin plus AMD3100, n = 8–9), (G) CD3 (*p < 0.0001, uninjured plus vehicle versus bleomycin plus vehicle, n = 8–9 and **p < 0.0001, bleomycin plus vehicle versus bleomycin plus AMD3100, n = 8–9), and (H) B220 (*p < 0.0001, uninjured plus vehicle versus bleomycin plus vehicle, n = 8–9). All data were analyzed by ANOVA.

FIGURE 8.

Twist1 gene expression is increased in IPF and, among IPF patients, negatively correlates with DLCO in IPF and is associated with a distinct gene expression profile. (A) Twist1 gene expression was measured in IPF patients (n = 134) and control patients (n = 107). Gene expression was 2.3-fold higher in IPF (*p < 0.0001, by unpaired t test). (B) IPF patients were organized into three tertiles based on increasing expression of Twist1 (p = 0.003 for trend by one way ANOVA, n = 44–45 per tertile). (C) Heat map of differentially expressed genes in IPF patients based on Twist1 expression. Each column represents a patient, and each row represents a gene. Between the lowest and the highest tertiles, 387 genes are differentially expressed. (D) FVC (percent predicted) and (E) DLCO (percent predicted) as a function of natural log-transformed Twist1 gene expression.