MCT4 mediates hypoxia-induced extracellular lactate release from IPF fibroblasts

Abstract

Lactate is increased in the lungs of patients with idiopathic pulmonary fibrosis (IPF) and in mice with experimental lung fibrosis, and lactate has been linked to the pathogenesis of lung fibrosis. IPF fibroblasts in hypoxic conditions generate increased lactate due to an imbalance of lactate dehydrogenase isoforms that induce pyruvate conversion to lactate. Monocarboxylate transporter 4 (MCT4) functions as a key lactate export protein, but its role has not been studied in lung fibrosis. We hypothesized that MCT4 would have a critical role in the ability of IPF fibroblasts to generate extracellular lactate and found that MCT4 was significantly upregulated in IPF fibroblasts under hypoxic conditions. In contrast, the lactate importer protein, monocarboxylate transporter 1 (MCT1) did not significantly change in control or IPF fibroblasts. Pharmacologic inhibition and silencing of MCT4 reduced extracellular lactate generation by IPF fibroblasts. Supporting its role in lung fibrogenesis, MCT4 was increased in bleomycin-injured mice and in lungs from IPF patients. Importantly, normal fibroblasts incubated with conditioned media from IPF fibroblasts exposed to hypoxic conditions had increased α-smooth muscle actin expression that was attenuated by inhibition of MCT4 in the donor IPF fibroblasts or by inhibition of the lactate receptor GPR-81 in the recipient normal fibroblasts. Together, these findings implicate MCT4 in the ability of IPF fibroblasts to increase extracellular lactate and highlight the role of lactate signaling via G-protein coupled receptor-81 in normal fibroblast differentiation. We propose a novel paradigm in which lactate export, driven by increased MCT4 expression, promotes fibrosis in oxygen-deficient microenvironments.

Introduction

Hypoxia, defined as a state in which there is insufficient oxygen available to meet the metabolic demands of a cell or tissue, is associated with wound repair environments, the interior of tumors, and other pathological processes including pulmonary fibrosis.^1–3 Idiopathic Pulmonary Fibrosis (IPF) is an age-related heterogeneous disease process with genetic and environmental risk factors but no specific definable cause. The persistent matrix deposition and remodeling in IPF disrupts parenchymal architecture and function, eventually leading to progressive systemic hypoxia, and ultimately, death from respiratory failure with a median survival of 3–5 years after diagnosis.^4,5 With lung fibrosis, expansion of the interstitial space by cells and extracellular matrix (ECM) presents an increased barrier to oxygen diffusion,⁶ such that fibroblasts in the expanded interstitium are subjected to below-normal oxygen tensions. In the similar settings of wound repair and tumor stromal microenvironments, estimates suggest that fibroblasts are exposed to between 0.5% and 2% oxygen.^7,8

Extracellular lactate levels rise under hypoxic conditions, driven by the balance of lactate dehydrogenase (LDH) enzyme isoforms LDHA and LDHB, which catalyze the conversion of pyruvate to lactate and vice versa.⁹ We previously reported that fibroblasts from IPF patients have an increased LDHA/LDHB ratio leading to a lactate-rich extracellular environment when the IPF cells are cultured under conditions of hypoxia.¹⁰ While increased lactate has been shown in fibrotic lung tissues from patients with IPF and from mice with experimentally induced lung fibrosis, and lactate has been shown to contribute to lung fibrogenesis, the mechanisms by which lactate impacts fibroblast phenotype to promote fibrosis remain poorly defined. In one report, lactate-mediated activation of latent TGF-β was suggested as a pro-fibrotic mechanism.¹¹ We recently reported that in fibroblasts under conditions of hypoxia (but not in normoxia), exogenous lactate could promote differentiation via the cognate lactate receptor GPR-81, which is strongly expressed in the interstitum of IPF lung tissue.¹⁰ Another recent study showed the induction of GPR-81 by hypoxia-induced HIF-1α activation in fibroblasts and implicated GPR-81 in hypoxia-mediated IPF fibroblast fibrogenicity.¹² These studies suggest that the mechanisms behind hypoxia-induced increases in lactate export and GPR-81-dependent cellular differentiation represent potential targets for intervention in fibrotic lung diseases such as IPF.

Lactate homeostasis is regulated by its generation (relative abundance of LDH isoforms and intracellular pyruvate), oxygen availability and utilization to drive mitochondrial respiration, and the expression/activity of transporters which shuttle lactate between the cytoplasm and the extracellular space, thereby affecting the intracellular pyruvate/lactate balance. While the cited recent studies establish a role for the direct effects of lactate on fibroblast phenotype in lung fibrosis, there is little known about the mechanisms that drive increased extracellular lactate in the fibrotic microenvironment or the mechanisms regulating lactate transport to the extracellular space.

Lactate shuttling between the cytoplasm and the extracellular space is largely dependent on monocarboxylate transporters (MCTs).^13,14 MCT1, with high L-lactate affinity, is suited for inward lactate transport into cells, whereas MCT4, with low L-lactate affinity, is specialized for lactate export from glycolytic cells.^13–15 No published studies have examined the role of MCT proteins in the pathogenesis of lung fibrosis. One available preprint manuscript¹⁶ reports that inhibition of MCT1 or MCT4 reduces TGF-β stimulated myofibroblast differentiation in vitro and decreases bleomycin-induced lung fibrosis in vivo. This study was undertaken to investigate the mechanisms of lactate shuttling in fibroblasts from normal and IPF patients under oxygen-deficient conditions. Our findings suggest that increased MCT4 expression in IPF fibroblasts facilitates lactate export from the cytoplasm into the extracellular space in oxygen-deficient conditions, thereby creating a lactate-rich microenvironment that promotes myofibroblast differentiation in normal lung fibroblasts.

Materials and methods

For detailed methods for IHC, immunofluorescence, cell culture, chemical inhibitors, siRNAs, Western blot analysis, in vivo murine experiments and general product information, see Materials and Methods in the online supplement.

Human subjects and the isolation of primary lung fibroblasts

Deidentified primary lung fibroblasts were provided after isolation and subculture using protocols as previously reported.¹⁰ Briefly, written informed consent was obtained from all patients, or their surrogates, and protocols were approved by the Institutional Review Board (IRB) at the Ohio State University (protocols 2017H0309 and 2021H0180). Lungs were removed at the time of transplantation or death from non-IPF and IPF patients. All identifiers other than the diagnosis (“IPF” or “normal”) were removed from the specimens provided. Multiple control or IPF cell lines (n = 4–6) were derived from non-IPF or IPF patients. Each distinct cell line from a different donor was considered a biological replicate, and experiments with different cell lines were frequently conducted at different points in time. If a distinct cell line was used more than once, the data were included as technical replicates. In all figures, each data point represents a distinct cell line from an individual donor.

Hypoxic chamber and lactate assay

Using a modular hypoxic chamber (CellXpert, C170i, Eppendorf, Enfield, CT, United States) flushed with a gas mixture containing 1% O₂, 5% CO₂ and 94% N₂, control and IPF fibroblasts were cultured in the serum reduced DMEM under the normoxic and hypoxic conditions without interruption for 3–5 days in the presence or absence of inhibitors as previously described.¹⁰ Extracellular lactate levels in cell culture media were measured using a Lactate Assay kit (Millipore Sigma, MAK064) according to the manufacturer’s protocol.

Medium transfer assay

IPF fibroblasts (n = 3) cultured in medium with reduced serum (5%) were incubated under hypoxic conditions and treated with a HIF-1α inhibitor on days 0 and 3. On day 5, the medium from HIF-1α inhibitor-treated IPF fibroblasts was transferred to control fibroblasts (n = 3), which were then further incubated for 5 days before measuring α-SMA expression. For the medium transfer assay involving MCT4 or GPR-81 inhibition, IPF fibroblasts were treated with 10 μM of MCT4 inhibitor on day 0 and 3. On day 5, the medium was transferred to control fibroblasts, which were then treated with or without a GPR-81 inhibitor on days 0 and 3. Control fibroblasts were then collected on day 5, and α-SMA expression was measured.

Statistics

Data are expressed as the means ± SD. N equals the number of unique and independent biological samples. The significance of data was determined using the student t test (two tailed), and 2 sample equal variance was used when comparing 2 groups. One way ANOVA with multiple comparisons post-test was used for experiments involving more than 2 conditions (analyzed with GraphPad Prism version 10). Significance level was set at P < .05. To adjust for baseline heterogeneity among primary lung fibroblasts, for each experiment, the results from each independent cell line have been normalized such that the baseline for that cell line is equal to 1 and the data therefore show the relative change for that cell line. Then, they have been pooled to allow statistical analysis.

Results

Hypoxia reduces intracellular pyruvate and elevates extracellular lactate in IPF fibroblasts

LDHA converts pyruvate to lactate, while LDHB reverses this reaction. Our previous findings showed that enhanced LDHA and reduced LDHB expression in IPF fibroblasts strongly skews the kinetics of this reaction toward lactate production under hypoxic conditions.¹⁰ Lactate export from the cells leads to extracellular lactate accumulation and acidification, suggesting that available pyruvate is preferentially converted to lactate instead of entering the TCA cycle under these conditions.^17–19 To test this, we examined intracellular pyruvate levels in both control and IPF fibroblasts under normoxic and hypoxic conditions. Under hypoxia, IPF fibroblasts displayed decreased intracellular pyruvate with concomitantly increased extracellular lactate levels compared to the normoxic conditions (Figure 1A, left and right). In contrast, control fibroblasts showed no significant changes in either intracellular pyruvate or extracellular lactate levels between normoxic and hypoxic conditions (Figure 1B, left and right). These results are consistent with enhanced intracellular reduction of pyruvate to lactate and subsequent extracellular export from IPF fibroblasts exposed to hypoxia.

Figure 1.

(A) IPF Fibroblasts (n = 5) were cultured in normoxic or hypoxic (1% oxygen) conditions for 5 days. Intracellular pyruvate (left) and extracellular lactate levels (right) were measured. Results are expressed as the % change for each individual cell line using the normoxia baseline as 100%.

(B) Intracellular pyruvate (left) and extracellular lactate (right) levels in control fibroblasts (n = 25) were measured under the same conditions as described above. Each dot represents a separate control or IPF fibroblast cell line.

Hypoxia induces MCT4 expression in IPF fibroblasts

Increased extracellular lactate reflects a shift in the homeostatic balance of lactate generation coupled with the balance of lactate export and import under hypoxic conditions.^20–22 Having previously shown that hypoxia promotes a shift in lactate generation mediated by differential regulation of LDHA and LDHB,¹⁰ we assessed the effects of hypoxia on the expression of MCT4, the primary protein responsible for lactate export from the cytosol to the extracellular space, and MCT1, which primarily imports lactate into the cytoplasm.^13,15 MCT4 levels were statistically unchanged in control fibroblasts exposed to hypoxia (Figure 2A). In contrast, while baseline expression levels were heterogenous, MCT4 levels were significantly increased in IPF fibroblasts exposed to hypoxia (Figure 2B). Decreased MCT1 levels were observed in control fibroblasts under hypoxia, although the decline was not statistically significant (Figure 2C). There was a slight increase in MCT1 in IPF fibroblasts that was also not statistically significant (Figure 2D). These findings indicate that MCT4 upregulation represents a fundamental response of IPF fibroblasts in oxygen-deficient conditions and suggests that MCT4 may be critical to lactate export from these cells.

Figure 2.

(A) Control fibroblasts (n = 6) were cultured in normoxic (N) and hypoxic (H, 1% oxygen) conditions for 5 days, and MCT4 protein levels were measured by Western blot. (B) MCT4 protein in IPF fibroblasts (n = 6) under normoxic and hypoxic conditions. (C) MCT1 expression in control and (D) IPF fibroblasts (n = 6, each). for each cohort, we have shown 3 representative Western blots along with the protein/GAPDH densitometry ratio for each of the 6 different cell lines. We also show the pooled densitometry in which the protein/GAPDH ratio for each cell line was normalized to its own baseline to allow for comparative analysis in the setting of baseline heterogeneity. * P < .05.

To further assess the role of MCT4 in the elaboration of extracellular lactate in IPF fibroblasts, we next treated cells with either the MCT4 inhibitor AZD0095 (MCT4-i) or the MCT1 inhibitor AZD3965 (MCT1-i) and assessed the impact on hypoxia-induced extracellular lactate and intracellular pyruvate levels. Under conditions of hypoxia, MCT4 inhibition significantly reduced extracellular lactate levels in media from IPF fibroblasts (Figure 3A). Notably, this MCT4 inhibitor did not alter LDHA or LDHB expression in normal or IPF fibroblasts, indicating that the decrease in extracellular lactate was not due to alterations of the LDHA/LDHB ratio that drives lactate homeostasis (Figure S1). MCT4 inhibition did not significantly affect intracellular pyruvate levels in IPF fibroblasts under hypoxic conditions, although a trend for increased pyruvate was observed (Figure 3B). MCT1 inhibition had no effect on extracellular lactate levels (Figure 3C). Consistent with the lack of change in intracellular pyruvate, extracellular lactate, MCT1 or MCT4 in control fibroblasts under hypoxic conditions, neither the MCT4 nor the MCT1 inhibitor significantly impacted extracellular lactate or intracellular pyruvate levels when control fibroblasts were exposed to hypoxic conditions (Figure 3D-F). Together, these findings suggest that MCT4 induction in IPF fibroblasts under hypoxic conditions is required for increased extracellular lactate.

Figure 3.

(A) IPF Fibroblasts (n = 5) were treated with 10 μM of MCT4 inhibitor (MCT4-i),²³ AZD0095, and cultured in hypoxic (H, 1% oxygen) conditions for 3 days prior to measurement of extracellular lactate levels. Shown is the % change in the extracellular lactate levels in the presence of absence of MCT4-i expressed as % of lactate normalized to hypoxia without inhibitor in the same cell line (expressed as 100%). each dot represents a separate cell line. (B) Intracellular pyruvate levels in IPF fibroblasts (n = 5) in the presence or absence of the MCT4 inhibitor (MCT4-i), AZD0095 under the same conditions as described above. (C) Extracellular lactate levels in IPF fibroblasts (n = 5) in the presence or absence of the MCT1 inhibitor (MCT1-i), AZD3965 under the same conditions as described above. (D) Extracellular lactate levels in control fibroblasts (n = 5) in the presence or absence of MCT4-ie,) Intracellular pyruvate levels in IPF fibroblasts (n = 5) in the presence or absence of the MCT4 inhibitor (MCT4-i), AZD0095 under the same conditions as described above. (F) Extracellular lactate levels in control fibroblasts (n = 5) in the presence or absence of MCT inhibitor (MCT1-i), AZD3965 as described above. NT: No treatment.

HIF-1α regulates MCT4 upregulation

HIF-1α is strongly expressed in the interstitium of fibrotic lung tissue,^24–27 and exposure to hypoxic conditions increases HIF-1α expression in normal lung fibroblasts.^10,28 Prior studies suggest that HIF-1α transcriptionally activates MCT4.²⁹ To examine the role of HIF-1α in MCT4-dependent lactate export in IPF fibroblasts, we treated the cells with the selective HIF-1α inhibitor, PX47822 (HIF1α-i) and measured MCT4 expression and extracellular lactate levels. First, we confirmed that HIF-1α expression was increased in IPF fibroblasts exposed to hypoxia (Figure 4A). Consistent with a key role for HIF-1α in its induction, MCT4 expression was markedly decreased in IPF fibroblasts treated with PX47822 under hypoxic conditions (Figure 4B, upper & lower). Likewise, HIF-1α siRNA diminished MCT4 and reduced extracellular lactate levels (Figure 4C and D; Figures S2 and S3A), supporting HIF-1α regulation of MCT4 in lactate export from IPF fibroblasts in a hypoxic environment.

Figure 4.

(A) IPF Fibroblasts were cultured under normoxic (N) or hypoxic (H) conditions for 5 days, and HIF-1α expression was measured. Blot is representative of 3 distinct IPF cell lines. (B) IPF fibroblasts were cultured in the presence or absence of the HIF-1α inhibitor, PX478 (HIF1α-i; 10 μM) in the hypoxic chamber for 3 days. Shown is a representative image of MCT4 protein levels in IPF fibroblasts (n = 3). below, MCT4/GAPDH densitometry was determined. (C) IPF fibroblasts were treated with 50 nM of HIF-1α or control siRNA and incubated for 3 days under hypoxic conditions. HIF-1α, MCT4 and β-actin levels were then measured. Shown is a representative image of HIF-1α, MCT4 and β-actin levels in IPF fibroblasts (n = 4). See Figures S2 and S3A for HIF-1α and MCT4 densitometry in cells transfected with the siRNA targeting HIF-1α. D) IPF fibroblasts (n = 4) treated with 50 nM of HIF-1α siRNA were cultured under hypoxic conditions for 3 days and extracellular lactate levels were measured. (E) IPF fibroblasts treated with MCT4 siRNA or control siRNA were cultured for 3 days under hypoxic conditions. Shown are representative images of MCT4 and β-actin expression in IPF fibroblasts (n = 5). See Figure S3B for MCT4 densitometry in cells transfected with the siRNA targeting MCT4. (F) Extracellular lactate (% of baseline) in IPF fibroblasts (n = 5) transfected with control siRNA or siRNA targeting MCT4 cultured under hypoxic conditions. In (D) and (F), 100% represents the baseline lactate level for each IPF fibroblast cell line transfected with control siRNA. Each dot represents a distinct IPF fibroblast line.

To confirm the direct role of MCT4 in regulating lactate export in hypoxic IPF fibroblasts, MCT4 was then silenced in IPF fibroblasts under hypoxic conditions and extracellular lactate was measured. Treating IPF fibroblasts with MCT4 siRNA reduced MCT4 expression (Figure 4E and Figure S3B), and extracellular lactate levels were also significantly decreased under hypoxic conditions when MCT4 was knocked down (Figure 4F). These results confirm that HIF-1α regulates MCT4, and that MCT4 regulates extracellular lactate levels in IPF fibroblasts under hypoxia.

TGF-β is a pro-fibrotic cytokine previously implicated in a mechanism by which lactic acid contributes to lung fibrosis.¹¹ To test the role of TGF-β in hypoxia-mediated regulation of MCT4, control and IPF fibroblasts were treated with or without TGF-β under normoxic or hypoxic conditions. TGF-β did not significantly impact MCT4 expression in either control and IPF fibroblasts (Figure S4A and B), suggesting that TGF-β is not required for hypoxia-mediated induction of MCT4 expression or lactate expression.

Enhanced MCT4 expression in fibrotic lung tissues from IPF patients

Previous studies have shown elevated lactate levels in human lungs from patients with IPF,¹¹ and in lungs from mice with experimentally induced fibrosis.^30,31 To assess the potential role of MCT4 as a critical mediator of increased lactate in IPF lungs, we first examined the expression of MCT1 and MCT4 in lung tissues from IPF and non-IPF patients using immunohistochemistry. MCT1 expression was almost absent or very low in both control and IPF lung tissues (Figure 5A, right panel). In contrast, MCT4 expression was robust in IPF lung tissues, while it remained low or absent in non-fibrotic lung tissues (Figure 5A, left panel). To extend these findings and specifically examine MCT4 expression in mesenchymal cells, normal and IPF tissue was further assessed by immunofluorescence staining for MCT4 (red), PDGFRα (green), and αSMA (white). Qualitatively and quantitatively, we observed substantially greater MCT4 expression in the IPF tissues (Figure 5B). To quantify MCT4 expression in fibroblasts, the Corrected Total Cell Fluorescence (CTCF) for MCT4 and PDGFRα was measured by 3 investigators who were blinded to the origin of the tissues from non-IPF and IPF patients (n = 3 each). MCT4 and PDGFRα were significantly increased in IPF patient lung tissues (Figure 5C; Figures S5 and S6). Next, blinded investigators measured colocalization by individually counting DAPI + cells and determining if they had no stain, red-stain (MCT4), or green-stain (PDGFRα) (Figure 5D). This objective quantification clearly showed that the IPF tissue had significantly increased “double-positive” (MCT4+/PDGFRα+) cells (48.5% of total counted cells in IPF lungs vs 29.9% of total counted cells in control lungs), strongly supporting increased MCT4 expression in fibroblasts within lung tissues from patients with IPF (Figure 5D; Figures S5 and S6).

Figure 5.

(A) Immunohistochemistry images of MCT4 (n = 3) and MCT1 (n = 2) in normal and IPF lung tissues. Each image shows lung tissue from a distinct patient, with low power images shown in the upper right of each. Magnified regions are shown to highlight MCT4 expression in the IPF specimens (bottom row). bars indicate the size of tissues (10X: 275 μm). arrows in the magnified images indicate some of the brown-stained MCT4 positive cells. NC: Negative control IHC image of IPF lung tissue. (B) MCT4, PDGFRα and αSMA expression in control and IPF fibroblasts analyzed by immunofluorescence. C) CTCF analysis for PDGFRα (left) and MCT4 (right) was performed as described at the materials and methods in the supplemental figure legend section. Scale: (20X: 50 μm). (D) For the analysis of double positive (MCT4 + and PDGFRα+) cell population, 2–4 blinded investigators counted cells in images taken from 3 normal and 3 IPF lung samples. Cells were defined by DAPI + nuclear staining. Each DAPI + cell was characterized as “negative” (no staining for MCT4 or PDGFRα), MCT4+, PDGFRα+ or “double positive”. Double + cells expressing both MCT4 and PDGFRα increased from 29.9% in control tissue to 48.5% in IPF tissue (P = .016) while the double-negative cells decreased from 55.1% to 34.6% (P = .007). Total cells counted (average +/− S.D.) were 656.3 ± 201.2 in control images and 628 ± 123.1 in IPF images.

Increased MCT4 in mice with bleomycin-induced lung fibrosis

Prior studies have shown that HIF-1α is strongly expressed in human and murine fibrotic lungs, and that hypoxia and lactate are associated with pathogenesis of IPF.^26,32 Our in vitro experiments link hypoxia and HIF-1α with MCT4 induction and lactate generation in IPF fibroblasts, so we next examined MCT1 and MCT4 levels in lung tissues from mice with bleomycin-induced lung fibrosis. As expected in this established murine model of fibrosis, Col1a1 expression was increased in bleomycin-treated mice compared to saline-treated controls (Figure 6A). MCT4 was also significantly increased in the lungs of bleomycin-injured mice (Figure 6A and B, left) while MCT1 levels were unchanged (Figure 6A and B, right). These findings were confirmed by immunofluorescence showing that MCT4 expression is clearly increased in bleomycin-injured mouse lungs compared to saline controls (Figure 6C-E; Figure S7-S12). Like the human samples, MCT4 was strongly expressed in the PDGFRα positive cells. Given the link between increased lactic acid and lung fibrosis, these findings demonstrate a novel role for MCT4 in lung fibroblasts in the development, maintenance and progression of lung fibrosis. Moreover, these data suggest a potential mechanism by which lung injury promotes tissue hypoxia within local lung microenvironments, leading to the upregulation of MCT4 and increased elaboration of lactate by the reparative fibroblasts.

Figure 6.

(A) C57/BL6 Mice (n = 4) were instilled with bleomycin via oropharyngeal aspiration (3.0 U/Kg) or saline (n = 3) at day 0, and whole lungs were harvested on day 21. MCT4, MCT1, Col1a1 and GAPDH expression levels were measured. (B) Densitometric analysis of MCT4/GAPDH (left), and MCT1/GAPDH (right). (C) immunofluorescence images of MCT4 expression in saline or bleomycin-injured mouse lungs (day 21). DAPI ⁺ nuclear staining, MCT4+, PDGFRα+, α-SMA and merged images are shown. (D) CTCF analysis for PDGFRα (left) and MCT4 (right) was performed as described at the materials and methods in the supplemental figure legend section. Scale: (20X: 50 μm). (E) For the analysis of double positive (MCT4 + and PDGFRα+) cell population, 2 blinded investigators counted cells in images taken from 3 saline or 3 bleomycin-injured mouse lung samples. Each point represents a mean (%) of total cell counting from each investigator. Cells were defined by DAPI + nuclear staining. Each DAPI + cell was characterized as “negative” (no staining for MCT4 or PDGFRα), MCT4+, PDGFRα+ or “double positive”. Figures S7-S12 show all images that were analyzed.

Lactate-rich extracellular medium from IPF fibroblasts induces normal fibroblast differentiation through GPR-81

Our previous study showed that lactate induces myofibroblast differentiation in control fibroblasts under hypoxic conditions via the lactate receptor GPR-81.¹⁰ Since IPF fibroblasts produce enhanced extracellular lactate in a HIF-1α and MCT-4-dependent manner, we next examined whether the treatment of IPF fibroblasts with the HIF-1α inhibitor would impair the differentiation of normal fibroblasts exposed to the conditioned medium from IPF fibroblasts. For this assay, culture medium from IPF fibroblasts incubated under hypoxic conditions with or without a HIF-1α inhibitor was transferred to the control fibroblasts, and α-SMA expression was measured. When control fibroblasts were cultured with the medium from HIF-1α inhibitor-treated IPF fibroblasts, αSMA expression was clearly reduced compared to that from non-treated IPF cells (Figure 7A, left and right). To further demonstrate that MCT4-dependent lactate secretion by IPF fibroblasts induces normal fibroblast differentiation via GPR-81, IPF cells under hypoxic conditions were treated with or without a MCT4 inhibitor and the medium was then transferred to recipient control fibroblasts that were pretreated with or without a GPR-81 inhibitor (Figure 7B). When control fibroblasts were cultured with conditioned medium from IPF fibroblasts, αSMA expression was significantly increased (Figure 7C, lane 2) compared to control cells that were not exposed to any conditioned medium (lane 1). However, αSMA expression in the recipient control fibroblasts was markedly reduced when the control fibroblasts were cultured with medium from IPF cells that had received the MCT4 inhibitor (lane 3). Similarly, αSMA levels remained low when control fibroblasts were pre-treated with an inhibitor that blocks the GPR-81 lactate receptor prior to incubation with medium from IPF fibroblasts (lane 4). Consistently, low αSMA expression was observed in GPR-81 inhibitor-treated control cells that were exposed to medium from MCT4 inhibitor-treated IPF fibroblasts (lane 5).

Figure 7.

(A) Left, representative images of α-SMA expression in control fibroblasts (n = 3) incubated in 1% oxygen cultured with conditioned medium from IPF fibroblasts (n = 3) treated with or without 10 μM HIF-1α inhibitor (HIF1α-I) under hypoxic conditions. Right, α-SMA/GAPDH expression in the presence or absence of HIF1α inhibitor under the same condition as described above. (B) Medium transfer assay experiment: IPF fibroblasts under hypoxic conditions were treated with or without an MCT4 inhibitor, and the conditioned media was transferred to recipient normal fibroblasts that had been incubated in the presence or absence of the lactate receptor inhibitor (GPR81-I) under hypoxic conditions. (C) Upper, representative images of α-SMA expression in control fibroblasts (n = 5) treated with/without the GPR81 inhibitor (GPR81-I) and then cultured with medium from IPF fibroblasts (n = 3), treated with or without 10 μM of MCT4-I. Lower, fold change of α-SMA/β actin. Experimental replicates (n = 5). *P < .05, **P < .01. (D) Proposed model for HIF1α-mediated MCT4 regulation and extracellular lactate expression, which induces fibroblast differentiation.

Collectively, these results demonstrate that lactate export from IPF fibroblasts is mediated by upregulation of MCT4 in a hypoxic environment and promotes fibroblast differentiation through the GPR-81 receptor in control fibroblasts. These findings further implicate lactate, which is abundantly produced by IPF fibroblasts,²¹ as a signaling molecule that directly impacts normal lung fibroblast phenotypes and highlights the pathological role of lactate as a mediator of lung fibrosis.

Discussion

Distal airway and alveolar interstitial remodeling associated with lung fibrosis presents a barrier to oxygen diffusion from the alveolar space through the epithelium, interstitium, and endothelium into the blood, leading to systemic hypoxemia. Importantly, evidence suggests that mesenchymal cells within the fibrotic interstitium, like those within wound-repair environments and tumor stroma, exist in a hypoxic environment with partial pressures of oxygen below 5% and as low as 0.5%.^24,33,34 This premise of cellular hypoxia is supported by studies showing increased expression of HIF-1α, a transcription factor which commonly functions as an “oxygen sensor,” in interstitial tissues and within fibroblastic foci of IPF patients.^2,26,35 Consistently, lactate accumulation is observed in IPF lung tissue and in the lungs of mice with bleomycin-induced pulmonary fibrosis, and lactate has been implicated in disease pathogenesis.^11,30 Given the accumulating evidence of metabolic dysregulation as a driving mechanism in lung fibrogenesis,³⁶ there is a compelling need to better understand how lactate generation is regulated and can directly contribute to the development and progression of lung fibrosis.

During wound repair, highly active and proliferating cells require continuous oxygen delivery to meet their metabolic demands.^19,37 With normal oxygen availability, metabolic demands are primarily met from mitochondrial respiration, beginning with the conversion of glucose to pyruvate through glycolysis.^18,37 Most pyruvate is then converted to Acetyl-CoA in the mitochondria, entering the tricarboxylic acid (TCA) cycle and driving oxidative phosphorylation.^19,37 However, in hypoxic conditions in which oxygen availability is not adequate to meet cellular metabolic demands, pyruvate is redirected towards anaerobic glycolysis leading to lactate production.³⁷ Despite the strong associations between hypoxia, lactate accumulation and lung fibrosis, the cellular mechanisms linking a hypoxic microenvironment to lung fibrosis remain unclear. In a previous study, we showed that IPF fibroblasts under hypoxic conditions elaborate increased lactate due to a shift in LDHA/LDHB favoring lactate production, and that under conditions of hypoxia lactate can directly promote fibroblast differentiation via its cognate receptor, GPR-81.¹⁰ In this study, we sought to determine whether hypoxia impacted fibroblast differentiation through regulation of lactate import and export proteins. We found that the lactate export protein MCT4 was upregulated in IPF fibroblasts exposed to hypoxia via HIF-1α and that MCT4 was necessary for the hypoxia-mediated increase in extracellular lactate. Together, these findings suggest that the coordinated increase in LDHA/LDHB combines with HIF-1α-dependent increased expression of MCT4 in IPF fibroblasts to enhance lactate production and export under hypoxic conditions (Figure 7D). This concept is further supported by our demonstration of increased MCT4 expression in IPF tissues and the lungs of mice with bleomycin-induced lung fibrosis. Coupled with the demonstration by our group and others that GPR-81 and LDHA are increased, while LDHB is decreased, in IPF fibroblasts exposed to hypoxia and in IPF lung tissue, these findings lay the foundation for hypoxia-mediated regulation of lactate homeostasis as a critical contributing factor to the pathobiology of lung fibrosis that holds potential as a therapeutic target.

While MCT1 and MCT4 are the primary proteins responsible for lactate import and export,^22,38–40 respectively, as a passive transporter, MCT1 may also operate bidirectionally and has also been reported to facilitate lactic acid export from cancer cells.^41,42 Notably, we did not observe any statistically significant regulation of the lactate export by MCT1 under hypoxia in normal or IPF fibroblasts, although there was a trend for MCT1 decline in the control cells. We did not observe evidence that MCT1 itself is regulated in IPF fibroblasts or expressed in fibrotic lung tissue. Our studies indicate that MCT4 has a critical role in lactate export from IPF fibroblasts under hypoxic conditions. Our medium transfer assay in the presence of MCT4 inhibitor or GPR-81 inhibitor further demonstrated that lactate produced from IPF fibroblasts via MCT4 alters the cellular phenotype of non-fibrotic fibroblasts through the GPR-81 receptor.

Extracellular lactate has traditionally been considered a metabolic by-product, but recent studies have linked anaerobic glycolysis and lactate to a variety of human diseases, including fibrotic disease of the lungs and skin.^{11,30,43–45} Given the recognition of lactate as a direct contributor in disease pathogenesis, emerging studies have assessed the role of lactate transport proteins in malignancies. For example, high MCT4, but not MCT1, expression correlates with worse overall and progression-free survival in patients with non-small cell lung cancer.⁴⁶ MCT4 has also been identified as a potential diagnostic and prognostic marker in triple-negative breast cancer and may serve as a therapeutic target in pancreatic cancer.⁴⁷ As such, MCT4 targeting by AZD0095 is a feasible strategy being evaluated for translation into clinical studies for cancer.^48,49 Interestingly, the inhibition of MCT4 in cancer cells is thought to lead to the accumulation of cytotoxic levels of intracellular lactate.⁴⁸ We have previously discussed the similarities between fibrotic lung fibroblasts and cancer cells and indicated that one approach to fibrosis would be to enhance the elimination of pathologic and apoptosis-resistant fibroblasts, suggesting an additional and complementary mechanism by which MCT4 inhibition may be effective for fibrotic lung disease.^50,51 The current study shows that MCT4 inhibition by AZD0095 and MCT4 knockdown by siRNA reduce extracellular lactate levels in IPF fibroblasts exposed to hypoxia. Our prior study identified extracellular lactate as a signaling molecule that promotes differentiation of normal lung fibroblasts via the lactate receptor GPR-81.¹⁰ Our medium transfer assay demonstrated that a lactate-rich environment can drive the phenotypic alteration of normal fibroblasts, indicating the plausible role of lactate as a key mediator in initiating and propagating fibrosis through intercellular signaling.

Together, these studies suggest that: (1) tissue hypoxia promotes enhanced lactate production (via increased LDHA/LDHB) and export (via MCT4) by IPF fibroblasts; (2) dysregulation of lactate homeostasis serves to drive fibroblast differentiation via paracrine mechanisms that may function to propagate lung fibrosis; and (3) selective inhibition of MCT4, or combined inhibition of MCT4 and GPR-81, may serve as a promising therapeutic strategy for fibrotic lung disease.

In summary, we found that MCT4 upregulation facilitates increased extracellular lactate in IPF fibroblasts under hypoxic conditions. High MCT4 expression is observed in IPF patient tissues and in the lungs of mice exposed to hypoxia and in mouse lungs injured by bleomycin. Pharmacologic inhibition and molecular knockdown of MCT4 reduces extracellular lactate in the media of IPF fibroblasts exposed to hypoxia. These findings suggest that MCT4 contributes to the aberrant lactate homeostasis observed in IPF fibroblasts and in fibrotic lung diseases, and positions MCT4 and lactate signaling as promising therapeutic targets to mitigate lung fibrosis.

Supplementary Material

Supplementary material

Supplementary material is available at American Journal of Respiratory and Critical Care Medicine online.

Clinical Relevance.

Increased lactate production in the hypoxic microenvironment of fibrotic lung tissue is a hallmark of Idiopathic Pulmonary Fibrosis (IPF). We show that extracellular lactate derived from IPF fibroblasts with increased MCT4 expression induces myofibroblast differentiation in non-fibrotic fibroblasts via activation of the lactate receptor GPR81. These findings identify lactate signaling as a driver of fibroblast activation and suggest MCT4 and GPR81 as potential therapeutic targets in lung fibrosis.

Data availability

The data underlying this article will be shared on reasonable request to the corresponding author.