Porcine respiratory disease complex induces pulmonary fibrosis related to the aberrant sphingolipid metabolism

Abstract

Porcine respiratory disease complex (PRDC) is a common syndrome in the modern swine industry worldwide, and its pathogenesis remains unclear to date. Our study aimed to investigate PRDC‐induced pulmonary fibrosis and sphingolipid metabolism, and their relationship. Mouse and cell line (A549 and 3D4/21) models exposed to bleomycin and/or transforming growth factor‐β1 (TGF‐β1) were developed. Histopathological and immunohistochemical staining, colorimetry, lipidomics analysis and pharmacologic intervention assays were used to analyse lung fibrosis and sphingolipid profiles. PRDC was validated by the presence of alveolar epithelial cell (AEC) injury and hyperplasia, inflammatory infiltrates, asymmetric macrophage polarization and mast cell phenotypic changes, TGF‐β1 and fibroblast growth factor 2 (FGF‐2) overproduction, extensive collagen deposition, foci of fibroblast/myofibroblast with stress fibres (α‐SMA, γ‐SMA and γ2 actin), cell interaction with increasing frequency, proliferation, apoptosis and autophagy dysregulation, and mucin 6 release—all of which are characteristics of pulmonary fibrosis. Based on the sphingolipidomics and pharmacologic interventions data—the dysregulated sphingolipids, including sphingomyelin (SM), ceramide (Cer), sphingosine‐1‐phosphate (S1P) and cerebroside (Cb), possibly due to serine palmitoyltransferase (SPT; SPTLC1), ceramide synthase (CerS; CerS2, CerS4), sphingomyelin synthase (SMS; SMS1), neutral sphingomyelinase (NSMase), acid sphingomyelinase (ASMase; SMPDL3B) and sphingosine kinase (SphK; SphK1, SphK2), were found to be closely related to pulmonary fibrosis. Furthermore, d18:1 24:1 SM and 18:1 S1P may be conserved biomarkers and tiamulin fumarate (TF) changes have anti‐fibrotic activity. Overall, PRDC induces pulmonary fibrosis, related to the aberrant sphingolipid metabolism, where conserved sphingolipid biomarkers and anti‐fibrotic candidates have been found.

1. INTRODUCTION

Porcine respiratory disease complex (PRDC), one of the most significant problems affecting the swine industry worldwide, is commonly caused by the complex interactions and synergy of viral agents and bacterial pathogens, environmental stressors, production and management types, and animal individual characteristics. ¹ Clinically, PRDC is characterized by slow growth, decreased feed efficiency, lethargy, anorexia, fever, cough, dyspnoea and mortality. ² The histopathology is complex and nearly all pulmonary reaction patterns are overlapped. ³ At present, the mechanism underlying the pathogenesis of PRDC has not been characterized. Generally, once these infectious and non‐infectious risk factors come into play, the following three stages may happen in pigs: (1) acute inflammatory stage with airway epithelial cell damage; inflammatory cell recruitment, polarization and interaction; and pro‐inflammatory mediator release; ⁴ (2) sub‐acute stage with stress fibre formation and fibrogenic mediator secretion; abnormal re‐epithelialization, fibroblast proliferation, migration and differentiation to myofibroblast, and fibroblastic foci (FF) formation; ⁵ and (3) final stage characterized by exaggerated extracellular matrix accumulation, fibrosis and progressive respiratory failure. ³

The persistent co‐infection or superinfection, the acute, chronic and repetitive lung injury, and the final stage (pulmonary fibrosis) are most often associated with modifications in pulmonary lipid metabolism. Sphingolipids are crucial molecules in the lung, where the most prominent bioactive sphingolipids are SM, Cer, sphingosine (Sph) and S1P. These sphingolipids can be converted into each other. ⁶ , ⁷ The balance between SM, Cer and S1P in the lung is tightly regulated and is crucial for maintaining pulmonary function. Sphingolipid metabolism is dysregulated in many pulmonary syndromes, and the imbalances in sphingolipids have different effects on different cell types within the lung owing to differences in their metabolic requirements, functions and responses to signalling molecules. ⁸ However, information about the role of sphingolipid mediators and signalling pathways in PRDC is scarce.

The current study was designed to investigate the lung fibrosis and sphingolipid profile related to PRDC. Our results show for the first time that PRDC induced pulmonary fibrosis is related to sphingolipid metabolism and that TF demonstrates anti‐fibrotic activity.

2. MATERIALS AND METHODS

2.1. Reagents

Bleomycin sulphate (BMS) was purchased from MedChemExpress (Shanghai, China). Standard lipids, including 1‐myristoyl‐2‐hydroxy‐sn‐glycero‐3‐phosphate (14:0 LPA) and 1‐lauroyl‐2‐hydroxy‐sn‐glycero‐3‐phosphocholine (12:0 LPC), were purchased from Avanti Polar lipids (Birmingham, AL, USA). TGF‐β1 was purchased from R&D Systems (Minneapolis, Minnesota, USA). TF (98 ≥ %) was a gift from ECO‐BIOK Animal Health (Deqing, Zhejiang, China).

2.2. Mouse model

Six‐to eight‐week‐old female C57BL/6 mice (Laboratory Animal Research Center, Peking University, Beijing, China) were maintained under standard conditions with free access to food and water. The mice were intratracheally instilled with BMS (50 mg/kg, 50 μL/mouse) in phosphate‐buffered saline (PBS) or PBS as a control on Day 0. Subsequently, the mice were euthanized on Day 21, and bronchoalveolar lavage fluid (BALF), plasma, lung and spleen tissues were harvested for subsequent experiments. All tissue specimens were split in half, one for snap freezing and stored at −80°C and the other for fixation in 4% paraformaldehyde. All animal experiments in this study were approved by the Ethical Committee on Laboratory Animals Care and Use of the Institute of Animal Science, China Academy of Agricultural Sciences, Beijing.

2.3. PRDC subjects

During a study period from January 2013 to December 2019, pigs with respiratory signs were diagnosed and screened at Veterinary Teaching & Collaboration Hospitals according to Figures S1 and 1. PRDC pigs were enrolled in this study. The pigs' clinical data were obtained through medical record reviews with the veterinarians and/or owners. Pathogen identifications were done as described previously. ⁹ Healthy, age‐ and strain‐matched control pigs free of respiratory signs were obtained from commercial farms.

FIGURE 1.

Porcine respiratory disease complex (PRDC) induces pulmonary fibrosis. (A) PRDC induces pulmonary fibrosis with cell survival and death paradox. The right lung lower lobes from the healthy control (HC) and PRDC pigs were stained with haematoxylin and eosin (HE), Masson's trichrome (MT) and immunohistochemical staining (IHC) of markers of proliferation (Ki67), apoptosis (CC3) and autophagy (LC3B). The staining was analysed by light microscopy, and 12 high power fields (HPFs) were examined at a magnification of ×400, followed by quantitative analysis of histology score, Ashcroft score and Cells/HPF. Lung structure distortion and alveolar architecture obliteration (arrow a1), hyperplastic epithelial cells (arrow a2), inflammatory infiltrates (arrow a3), foamy macrophage accumulation within alveolar spaces and foamy change within alveolar septa (arrow a4), myofibroblast core (arrow a5) and the active fibrotic front (arrow a6), honeycomb zone (arrow a7), fibroblastic foci (FF) (arrow a8), collagen fibres (blue, arrow a9) and Masson body (arrow a10) were present in PRDC lung parenchyma. The Ki67‐positive cells were present in FF (arrow a1), alveolar epithelium (arrow a2) and inflammatory infiltrates (arrow a3). The CC3‐positive cells were shown in alveolar epithelium (arrow a2). The LC3B‐positive cells were present in alveolar epithelium (arrow a2). (B) Stress fibres formation and fibrogenic mediators secretion contribute to pulmonary fibrosis induced by PRDC. The right lung lower lobes from HC and PRDC pigs were stained with IHC of stress fibres (α‐SMA, γ‐SMA and γ2 Actin) and fibrogenic mediator (TGF‐β1 and FGF‐2). The staining was analysed as described above. The positive cells were predominantly distributed within FF (arrow a1), alveolar epithelium (arrow a2) and inflammatory infiltrates (arrow a3). (C) Macrophage/mast cell polarization and myofibroblast–mast cells interaction contribute to pulmonary fibrosis induced by PRDC. The right lung lower lobes from HC and PRDC pigs were stained with IHC of M2 macrophage (CD68 and CD163) and mast cells (Tryptase and Chymase). The staining was analysed as described above. The positive cells (macrophages and mast cells) were predominantly distributed within airspaces and alveolar septa (black arrow). Dual‐stained Chymase (brown) and α‐SMA (pink)‐positive cells were mainly distributed within FF and pulmonary vessel walls (black arrow). (D) Mucin 6 is related to pulmonary fibrosis induced by PRDC. The right lung lower lobes from HC and PRDC pigs were stained with IHC of Mucin 6. The staining was analysed as described above. The Mucin 6‐positive cells were present in hyperplastic alveolar epithelium (arrow a2) and inflammatory infiltrates (arrow a3). (E) Hydroxyproline is positively related to pulmonary fibrosis induced by PRDC. Hydroxyproline in the lungs from HC and PRDC pigs was measured by the colorimetric assay. (F) Average daily weight gain (ADWG) is negatively related to pulmonary fibrosis induced by PRDC. ADWG of the HC and PRDC pigs was from 10 weeks of age. Representative photomicrographs from 10 pigs were shown. Nuclei were stained with haematoxylin (blue). Scale bar = 50 μm. All data were presented as mean ± SD. *p < .05, **p < .01.

2.4. Pharmacologic interventions

On Day 14, mice from the BMS model were randomly allocated to the positive control (BMS) (n = 6) negative control and medication group (TF) (n = 6). The medication group was administered intraperitoneally once daily for 7 days with TF (50 mg/kg/200 μL). The BMS group was administered intraperitoneally once daily for 7 days with an equivalent volume of the vehicle. Clinical signs were recorded daily, and animals were weighed daily. On Day 21, the mice were necropsied, and the samples were collected as described above.

Twenty PRDC pigs between 6 and 8 weeks of age were screened from the indicated farm and randomly allocated to control and medication groups (n = 10) in Longyan, Xinyang and Beijing, respectively. Healthy, age‐and strain‐matched control pigs (HC) were obtained from the same farm. Pigs of the medication group received a feed containing 100 p.p.m. TF for 21 days. Clinical signs were recorded daily, and animals were weighed on a weekly basis. On Day 21 post‐medication, the pigs were necropsied, and all organs were examined with emphasis on lungs. Meanwhile, tissues were collected, fixed and pulmonary fibrosis was evaluated as described above.

2.5. Histopathology

Paraffin‐embedded tissue sections were stained with haematoxylin and eosin (HE), Masson's trichrome (MT), Toluidine blue (TB) and 12 high power fields (HPFs, 100×, 400× and/or 1000×) were scored. ¹⁰ , ¹¹ , ¹²

2.6. Hydroxyproline assay

Ten milligrams of upper and lower lobes were hydrolysed in 1 mL of 6 M HCl at 120°C overnight. The resulting hydrolysate was assayed by a colorimetric method as described previously. ¹³

2.7. Enzyme‐linked immunosorbent assay (ELISA)

TGF‐β1 in the lung homogenate and BALF supernatant was measured using a commercial ELISA kit (eBioscience, San Diego, CA) according to the manufacturers' instructions.

2.8. Immunohistochemical staining (IHC)

Paraffin‐embedded and/or frozen tissue sections were stained with primary antibodies as previously described. ¹² Twelve HPFs were examined at a magnification of ×100, ×400 or ×1000. Positive cells were counted (Cells/HPF). Alternatively, mean fluorescent intensity (MFI) was quantified using NIH ImageJ v1.46.

2.9. Lipidomics analysis

Human lung epithelial cell line A549 and porcine monomyeloid cell line 3D4/21 were maintained and cultured routinely. A549 and 3D4/21 cells were treated with BMS (100 μM), TGF‐β1 (10 ng/mL) or solvent control (DMEM). At 24 h post‐treatment, the cell pellets (10⁵) were resuspended with 100 μL ddH₂O and mixed thoroughly. The mixtures were added to 900 μL methanol with internal standards (14:0 LPA and 12:0 LPC). The following steps were the same as the below description.

The frozen tissues were homogenized, and 50 μL homogenate per sample was added to 950 μL methanol with internal standards. After vortexing, the mixture was centrifuged (10,000 g, 10 min, 4°C). The supernatants were analysed using an LC system (I‐class Acquity ultra performance liquid chromatography; Waters, Milford, MA, USA) with an autosampler and an API QTrap® 4500 mass spectrometer (Applied Biosystems/MDS SCIEX, Forster City, CA, USA; LC‐MS/MS) with the software Analyst 3.0, followed by Tables S1–S3.

2.10. Statistical analysis

All data were presented as mean ± SD. GraphPad Prism v6.02 (La Jolla, CA, USA) was used to perform Student's t‐test or one‐way analysis of variance (one‐way ANOVA) and post hoc multiple comparisons between the groups. *p < .05, **p < .01.

3. RESULTS

3.1. Clinical signs and gross lesions of PRDC

Clinically, affected pigs show signs of lethargy, cough and dyspnoea as well as weight loss (10%). Grossly, there is a predominance of swelling or atrophy, consolidation, hyperaemia or haemorrhage, and fibrous exudation (85%), with the chronic case being the most prevalent (70%; Figure S1).

3.2. PRDC induces pulmonary fibrosis

PRDC lungs presented diffused structural alterations, pneumocyte hyperplasia, interstitial cellular infiltration, including foamy macrophage accumulation and foamy change within alveolar septa, a more heterogeneous pattern with areas of dense fibrosis and extensive deposition of collagen fibres (Figure 1A). These data indicate that PRDC induces pulmonary fibrosis. Compared with HC, Ki67 nuclei‐positive cells, preferentially localized in hyperplastic epithelial cells, fibroblasts and inflammatory infiltrate cells, increased in PRDC lungs (Figure 1A). The cleaved caspase‐3 (CC3)‐positive cells were primarily detected in alveolar epithelium and largely absent within FF (Figure 1A). Microtubule associated protein 1 light chain 3β (LC3B) immunoreactivity was observed in alveolar epithelium of HC lungs, and the number of LC3B‐positive cells within hyperplastic alveolar epithelium and FF was significantly decreased in PRDC lungs (Figure 1A).

The stress fibres (α‐SMA, γ‐SMA and γ2 actin) were strongly detected in fibroblast/myofibroblast foci and/or hyperplastic AECs of PRDC lungs (Figure 1B). Meanwhile, the fibrogenic mediators (TGF‐β1 and FGF‐2) were overexpressed in fibroblast/myofibroblast foci and inflammatory infiltrate cells (Figure 1B).

CD68 was highly expressed in airspaces and alveolar septa neighbouring severe fibrotic lesions of PRDC lungs (Figure 1C). CD163, a M2 macrophage marker, was only expressed in fibrotic areas of PRDC lung, suggesting that PRDC‐induced pulmonary fibrosis might be associated with M2 macrophages. Compared with HC, the increased numbers of mast cells (MCs) were found within PRDC lungs, and most of them appeared in an activated state (Figures S2 and 1C). MCs were dominated by Chymase expression, and Chymase‐positive MCs were diffusely distributed within fibrotic areas, while a few Tryptase‐positive MCs were found in this area. The colocalization of MCs and myofibroblasts was found within PRDC lung (Figure 1C).

The hyperplastic alveolar epithelium and inflammatory infiltrate cells within alveolar spaces and septa were enriched in Mucin 6 (Figure 1D), associated with pulmonary alveoli obstruction and hydroxyproline content (Figure 1E). In addition, average daily weight gain (ADWG) showed a negative correlation with fibrosis (Figure 1F).

In conclusion, PRDC induces pulmonary fibrosis, and may be a novel surrogate to study pulmonary fibrosis.

3.3. BMS induces mouse pulmonary fibrosis

The BMS‐treated lungs showed the characteristics of pulmonary fibrosis (Figure 2A), with the increase of α‐SMA, TGF‐β1 and hydroxyproline (Figure 2B–D).

FIGURE 2.

Bleomycin sulphate (BMS) induces pulmonary fibrosis. On Day 21, the lungs from phosphate‐buffered saline (PBS) and BMS‐treated mice were paraformaldehyde‐fixed and stained with haematoxylin and eosin (HE), Masson's trichrome (MT) (A), immunohistochemical staining (IHC) (α‐SMA, TGF‐β1) (B). The staining was analysed as described above. The characteristics of pulmonary fibrosis were indicated by the arrow a1, a2, a3, a4, a8, a9 and a10, respectively. α‐SMA‐ and TGF‐β1‐positive staining within fibroblastic foci (FF), alveolar epithelium and inflammatory infiltrates were indicated by the arrow a11, a12 and a13, respectively. Nuclei were stained with haematoxylin (blue). The number of α‐SMA⁺ and TGF‐β1⁺ cells per high power fields (HPF) was counted. Hydroxyproline (C) in the lungs, TGF‐β1 (D) in the BALF and lungs were measured by the colorimetric assay, respectively. The representative micrographs from three independent experiments were presented. Scale bar = 50 μm. All data were presented as mean ± SD. *p < .05, **p < .01.

3.4. Pulmonary fibrosis affects sphingolipid metabolism

In this study, SPT (SPTLC1, a major subunit of SPT), CerS (CerS2 and CerS4), SMS (SMS1), ASMase (SMPDL3B, a paralog of ASM) and SphK (SphK1 and SphK2) were elevated in PRDC pigs, while NSMase decreased (Figure S3). Meanwhile, SMS1 was decreased in the lungs of BMS‐treated mice (Figure S4). These data demonstrated that these sphingolipid metabolizing enzymes were probably relevant to pulmonary fibrosis.

Furthermore, LC‐MS/MS‐based analyses of sphingolipidomics, including 4 subgroups (SM, Cer, S1P and Cb) and 32 molecular species, were carried out (Tables S4–S8 and Figure 3). Among these species in lung and plasma, d18:1 24:1 SM, 18:1 S1P and 18:0 S1P were all significantly elevated. Similar to BMS, all species, including d18:1 24:1 SM, 24:1 Cer and 18:1 S1P in TGF‐β1‐treated A549 cells were all significantly elevated. Compared with A549 cells, only limited sphingolipids in 3D4/21 cells were affected. In all, PRDC affects lung sphingolipid metabolism (Figure 3), and d18:1 24:1 SM, 18:1 S1P may be conserved biomarkers with pulmonary fibrosis.

FIGURE 3.

Porcine respiratory disease complex (PRDC) affects porcine lung sphingolipid metabolism. Sphingolipids (pmol/mg) in the upper lobes of right lungs from the healthy control (HC) and PRDC pigs were analysed with LC‐MS/MS as described in materials and methods. (A) SM, (B) Cer, (C) S1P, (D) Cb. The representative micrographs from 10 pigs were shown. All data were presented as mean ± SD. *p < .05, **p < .01, versus corresponding control.

3.5. Pulmonary fibrosis is positively related to the level of sphingolipid biomarker

TF attenuated pulmonary fibrosis and improved ADWG (Figures 4 and 5). Meanwhile, the level of d18:1 24:1 SM, 24:1 Cer and 18:1 S1P was downregulated (Figures 4E and 5E). Thus, pulmonary fibrosis is positively related to the level of sphingolipid biomarker. The spleen histology score was also positively related to mouse pulmonary fibrosis (Figure 5F), possibly related to other sphingolipids, including Cb (Table S5).

FIGURE 4.

Pulmonary fibrosis induced by porcine respiratory disease complex (PRDC) is positively related to the level of sphingolipid biomarker. The lungs from healthy control (HC), PRDC and PRDC+TF (TF)‐treated pigs were paraformaldehyde‐fixed or frozen‐fixed, stained with haematoxylin and eosin (HE), Masson's trichrome (MT) (A) and IHC (α‐SMA, TGF‐β1, A, B), and analysed by light microscopy and/or by fluorescence microscopy. Twelve high power fields (HPFs) were examined at a magnification of ×100 or ×400. The characteristics of pulmonary fibrosis were described as shown above. Positive cell was indicated by the arrow. Nuclei were stained with haematoxylin or DAPI (blue). Hydroxyproline (C) and TGF‐β1 (D) in the lungs were measured by the colorimetric assay, respectively. The sphingolipid biomarkers (d18:1 24:1 SM, 18:1 S1P) (E) in the lungs were analysed with LC‐MS/MS as described in Materials and Methods. The representative micrographs from three independent experiments were presented. Scale bar = 50 or 20 μm. The histology score, Ashcroft score, Cells/HPF, hydroxyproline (μg/mg), TGF‐β1 (pg/mg) and sphingolipid biomarker (pmol/mg) were analysed using GraphPad Prism v6.02. All data were presented as mean ± SD. *p < .05, **p < .01, versus corresponding control.

FIGURE 5.

Pulmonary fibrosis induced by bleomycin sulphate (BMS) is positively related to the level of sphingolipid biomarker. On Day 21, the tissues from phosphate‐buffered saline (PBS), BMS and BMS + TF (TF)‐treated mice were paraformaldehyde‐fixed or frozen‐fixed, stained with haematoxylin and eosin (HE) (A, F), Masson's trichrome (MT) (A) and IHC (α‐SMA, B), and analysed by light microscopy and/or by fluorescence microscopy. Twelve HPFs were examined at a magnification of ×400 or ×1000. The characteristics of pulmonary fibrosis were described as shown above. Positive cell was indicated by the arrow. Nuclei were stained with DAPI (blue). As to spleen, haemorrhage, oedema, megakaryocyte, necrosis, inflammatory foci and fibroplasia were indicated by the arrow b1, b2, b3, b4, b5 and b6, respectively. Hydroxyproline (C) and TGF‐β1 (D) in the lungs were measured by the colorimetric assay, respectively. The sphingolipid biomarkers (d18:1 24:1 SM, 18:1 S1P) (E) in the lungs were analysed with LC‐MS/MS as described in materials and methods. The representative micrographs from 3 independent experiments were presented. Scale bar = 50 or 20 μm. The MFI was quantified with NIH ImageJ v1.46, and the histology score, Ashcroft score, MFI, hydroxyproline (μg/mg), TGF‐β1 (pg/mg) and sphingolipid biomarker (pmol/mg) were analysed with GraphPad Prism v6.02. All data were presented as mean ± SD. *p < .05, **p < .01, versus corresponding control.

4. DISCUSSION

We have shown, for the first time, that PRDC induces lung fibrosis. Currently, chemical factors, including bleomycin‐induced pulmonary fibrosis in rodents, have been described. Considering pulmonary anatomy and cellular components, similar to human counterparts, PRDC may be the ideal experimental model or surrogate of this disorder. Although implicated in the pathogenesis of pulmonary fibrosis, the role of sphingolipid and its signalling pathways underlying this disease are still unclear. As to PRDC, lung Cers showed a mixed profile, characteristic by the upregulation of 16:0 Cer, 18:1 Cer, 24:1 Cer, 24:0 Cer, and the downregulation of 18:0 Cer, 20:0 Cer, 22:1 Cer, 22:0 Cer. As to the mouse model, lung Cers also showed a mixed profile similar to PRDC. These data indicate that Cers and their acyl chains are closely related to pulmonary fibrosis. In addition, all species of spleen Cers and most species of plasma Cers in mice were downregulated, suggesting that Cers are involved in the immunity‐related pulmonary defence.

In this study, the species of lung SM, including d18:1 24:1 SM, and plasma SM presented a mixed profile, while all species of spleen SM were downregulated in mice. Thus, sphingolipid metabolism with pulmonary fibrosis is complex. ⁸ 18:1 S1P was upregulated in the lungs and in all cell models, due to the increase of SphK1 and SphK2. ¹⁴ In vivo and in vitro data from different species suggest that d18:1 24:1 SM, 18:1 S1P may be the potential biomarkers. Cb species showed a mixed profile, and all species in the spleen were downregulated, implying their immunity function with pulmonary fibrosis. Similarly, the previous results showed the decreased Cb species in plasma of patients with IPF. ¹⁵

At present, the efficient medications that directly target pulmonary fibrosis are lacking. PRDC, to a certain extent, mimics this disorder, including chronic inflammation and long‐lasting fibrosis. This surrogate helps us to understand the pathobiology of pulmonary fibrosis and to test new therapies. TF, the Tiamulin salified with fumaric acid, was found to attenuate pulmonary fibrosis in pigs and mice, implying its anti‐fibrotic effect. TF is effective against gram‐positive and ‐negative bacteria via targeting the 50S subunit of the bacterial ribosome and interacting at the peptidyl transferase centre. ¹⁶ TF also inhibits cytochrome P450 (CYP) isozymes, ¹⁷ which may contribute to its anti‐fibrotic effect against pulmonary fibrosis. High intracellular concentrations of TF in lung have been reported in comparison with plasma concentrations. ¹⁸ Furthermore, pulmonary fibrosis was positively related to the level of sphingolipid biomarkers (d18:1 24:1 SM, 18:1 S1P). Mechanistically, these dysregulated sphingolipids induce cell survival and death disorder, which contributes to pulmonary fibrosis. Future comprehensive studies to explore how TF treats pulmonary fibrosis are warranted.

Cumulatively, PRDC induces pulmonary fibrosis, related to sphingolipid metabolism (Figure 6). These highlight the translational relevance of our data and underscore the importance of further investigation into the role of sphingolipid metabolism in pulmonary fibrosis pathology and treatment.

FIGURE 6.

Schematic model of the proposed sequence of events and sphingolipid metabolism leading to pulmonary fibrosis induced by porcine respiratory disease complex (PRDC). The persistent or repetitive fibrogenic stimuli are capable of inducing alveolar epithelium (AE, type I and type II) lesions, such as injury, hyperplasia, apoptosis, barrier function damage, epithelial–mesenchymal transition (EMT) and pro‐fibrotic factors (TGF‐β1, Mucin 6) secretion. These events recruit alveolar macrophage (AM), mast cell (MC) and fibroblast (FB) to the fibrogenesis centre, where proliferation, activation, M2 polarization, myofibroblast (myoFB) transdifferentiation, cell to cell crosstalk and pro‐fibrotic factors production happen to them, and further ECM expansion/ deposition, alveolar/interstitium architecture destruction, fibroblastic foci (FF) formation and final fibrosis are developed. The activation and migration of these structural cells (epithelial cells and fibroblasts) and immune cells (macrophages and mast cells)‐associated pathways, the release of pro‐fibrotic factors (cytokine, growth factor, glycoprotein, lipid and enzyme), as well as the interplays among them, play important roles in the initiation, progression and chronic development of PRDC‐induced pulmonary fibrosis. Meanwhile, fibrogenic stimuli and pro‐fibrotic injury induces metabolic stress in fibrogenesis centre, leading to abnormal sphingolipid metabolism. SPT catalyses the initial rate‐limiting step of de novo pathways of sphingolipid biosynthesis, followed by the generation of Cer. The latter is central to sphingolipid metabolism, being a substrate of different enzymes for sphingolipid biosynthesis and catabolism. Cer can be catalysed by ceramide glucosyltransferase (UGCG) and ceramide galactosyltransferase (CGT) to form Cb, and the latter can be metabolized by β‐glucocerebrosidase (GCase) to form Cer. Cer can be further metabolized into SM by SMS1. SM is then hydrolysed to form Cer by NSMase and ASMase. Cer, coming from SM, can be further degraded to Sph by several organelle‐specific ceramidases, and SphK1 or SphK2 phosphorylates Sph to S1P. In addition, Sph can be re‐acylated by CerS2 and CerS4 to Cer (salvage pathway). Dysregulation of specific metabolic enzymes and sphingolipid is closely related to pulmonary fibrosis and the events described above. See text for details.

AUTHOR CONTRIBUTIONS

Xiangfang Tang: Investigation; methodology; data curation; funding acquisition. Gaokai Li: Investigation; methodology; data curation. Lijun Shi: Investigation; methodology; resources. Tao Liu: Investigation; methodology; resources. Zhiyong Si: Investigation; methodology; resources. Guangbo Li: Investigation; methodology; resources. Weiquan Yu: Methodology; resources. Tao Zhang: Methodology; resources; funding acquisition. Zhenwen Zhao: Methodology; resources; funding acquisition. Xinghui Zhao: Methodology. Zhanzhong Zhao: Conceptualization; investigation, Methodology; data curation; writing—original draft, writing—review and editing; project administration. Xiangfang Tang and Gaokai Li: equal contribution to this work. All authors reviewed and approved the manuscript.

FUNDING INFORMATION

This work was supported by the National Key Research and Development Program of China (grant nos.: 2022YFD1301100, 2018YFA0800900 and 2016YFD0500505), the National Natural Science Foundation of China (grant no.: 32273050), and the Beijing Natural Science Foundation (grant no.: 6242008).

CONFLICT OF INTEREST STATEMENT

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

ETHICS STATEMENT

All animal experiments were approved by the Ethical Committee on Laboratory Animals Care and Use of the Institute of Animal Science, China Academy of Agricultural Sciences, Beijing (approval no.: IASCAAS‐80), and all procedures followed were in accordance with the National Research Council's ‘Guideline for the Care and Use of Laboratory Animals’.

Supporting information

Table S1.

Table S2.

Table S3.

Table S4.

Table S5.

Table S6.

Table S7.

Table S8.

Figure S1.

Figure S2.

Figure S3.

Figure S4.

Tang X, Li G, Shi L, et al. Porcine respiratory disease complex induces pulmonary fibrosis related to the aberrant sphingolipid metabolism. Int J Exp Path. 2025;106:e70005. doi: 10.1111/iep.70005