Evaluation of the Effects of Air Pollutants on Diabetic Wounds

Background. Although air pollution containing fine dust particles is gaining attention worldwide, little is known about the effects of such pollutants on diabetic wounds. Air pollutants from diesel exhaust particles (DEPs) cause inflammation, resulting in an increased expression of pro-inflammatory cytokines and chemokines, which attract monocytes and T cells to the sites of inflammation. The authors evaluated the effects of air pollutants on diabetic wounds.Materials and Methods. Fibroblast cells were derived from streptozotocin-induced diabetic rats. Cell Counting Kit-8 assays were used to determine cell viability. The expression of pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), and interleukin-6 (IL-6) was evaluated by reverse transcription polymerase chain reaction and western blot analysis. Results. The proliferation of DEP-treated fibroblasts decreased with time. The messenger ribonucleic acid expression of TNF-α and COX-2 in DEP-treated fibroblasts increased in both normal and diabetic fibroblasts, while IL-6 expression remained unchanged. The protein expression of TNF-α, COX-2, and IL-6 in DEP-treated fibroblasts increased compared to samples not exposed to DEP. Conclusions. Diesel exhaust particles regulate the expression of pro-inflammatory cytokines such as IL-6, TNF-α, and COX-2, which may impede diabetic healing in vitro. 

Diesel exhaust particles (DEPs) from diesel fuels are major contributors of air pollution. Air pollutants from DEPs cause inflammation, resulting in increased expression of pro-inflammatory cytokines and chemokines, which attract monocytes and T cells to the sites of inflammation.^1,2 In urban areas, DEPs can induce inflammatory disease^3-5 as well as oxidative stress in endothelial cells, prompting allergic respiratory disease.⁶ 

The association between DEPs and increased production of inflammatory cytokines in human epithelial cells contributes to cardiovascular and respiratory diseases and hinders cell-matrix interactions, cell-cell connections, and fibrillar collagen remodeling, all of which can impede wound healing.² Exposure to DEPs has been associated with several adverse health effects in vivo and in vitro.^7-9 Inflammation is a crucial stage in the development of health effects associated with DEP exposure. Neutrophils and macrophages release cytokines, reactive oxygen species, and toxic proteases in response to infection.  These contribute to DEP-induced deoxyribonucleic acid (DNA) damage. The effects of DEP have been demonstrated in cell cultures, isolated organs, healthy animals, and animal disease models. The increased release of pro-inflammatory mediators can ultimately result in chronic inflammation.¹⁰ Recent evidence suggests exposure to DEPs can increase the risk of fatal stroke, cerebrovascular damage, and neural inflammation due to the marked increased expression of pro-inflammatory mediators such as lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α), and immune response-specific chemokines and cytokines.^11-13 

Wound healing requires complex cellular and molecular events that can lead to either accelerated or delayed healing, the latter of which can result in chronic wounds. Chronic diabetic ulcers have also been associated with elevated levels of pro-inflammatory cytokines. For example, interleukin (IL)-6 and cyclooxygenase-2 (COX-2) levels were upregulated in an in vivo wound healing model.¹⁴ Diabetes causes dysregulation in neuropeptide expression in the skin along with a suppressed inflammatory response to diabetic renal injury. Diabetic rabbits have significantly increased gene expression of IL-6 and IL-8 compared to baseline.¹⁵ In addition, inflammation in diabetes causes a particularly enhanced and prolonged expression of TNF-α, a potent proinflammatory cytokine.³ Furthermore, patients with diabetes often have impaired wound healing.¹⁶ A previously unknown element of air pollution, DEPs cause inflammation in humans and are particularly harmful to patients with diabetes who have wounds. In this study, the authors evaluated the effects of air pollutants on diabetic wounds.

Culture of primary fibroblasts. Sprague Dawley (SD) male rats (8 weeks old) were intraperitoneally injected with 60 mg/kg streptozotocin (STZ) dissolved in sodium citrate
solution (0.1 M, pH 4.5). Fibroblast cells were derived from STZ-induced diabetic rats with skin wounds. Tissue samples from their skin were seeded in a 6-well plate. After 24 hours, the skin tiss-ues were removed. Fibroblast cells were grown in Dulbecco’s Modified Eagle Medium (DMEM/F12, Thermo Scientific Hyclone, Logan, UT). The growth medium was supplemented with 20% heat-inactivated fetal bovine serum (FBS; Thermo Scientific Hyclone) and 100 µg/mL streptomycin. Cultures were incubated at 37°C in 5% CO₂. 

Treatment of fibroblasts with air pollutants. Fibroblast cells (1 × 10⁵) were seeded in a 6-well plate and then starved for 24 hours in media supplemented with 0.5% FBS. Next, the cells were treated with DEP (0, 10, 40, 80, 120, and 160 µg/mL) for 24 hours.  Afterward, the fibroblast cells were harvested.

Quantification of fibroblasts using a cell proliferation assay. Cells were seeded in 96-well plates, each containing 7 × 10³ cells. After 24 hours, cells were treated with 10, 40, 120, and 160 µg/mL DEP. Control cells were not exposed to DEP.  After 1, 2, 24, and 48 hours, cell viability was measured using a Cell Counting Kit-8 (CCK-8) cell viability assay. 

Reverse transcription polymerase chain reaction (RT-PCR). The messenger ribonucleic acid (mRNA) expression of TNF-α, IL-6, COX-2, and CCL2 was evaluated by RT-PCR of complementary DNA (cDNA) extracted from mouse lung tissue using TRI Reagent (Molecular Research Center, Toyama, Japan), a modified version of the guanidinium thiocyanate-phenol chloroform extraction method. The researchers quantified the RNA and used 3 µg for reverse transcription. Ribonucleic acid treated with Deoxyribonuclease I (10 000 U/mL; Stratagene, La Jolla, CA) was reverse-transcribed by incubating with 10 mm deoxynucleotide triphosphates, 0.1 M dithiothreitol, 0.5 μg/µL Oligo Primer, 40 units/µL RNaseOUT, and 200 units/µL SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA) at 42°C for 50 minutes and then heat-inactivated at 70°C for 15 minutes. The synthesized cDNA was used as a template for polymerase chain reaction with primer sets against TNF-α, IL-6, COX-2, and CCL-2. The primer sequences are shown in Figure 1. 

Western blot analysis of protein expression. Cells were seeded in a 6-well plate with 3 × 10⁵ cells per well and starved for 24 hours. Afterward, they were treated with air pollutant for 24 hours and then harvested using a cell lysis buffer. For the relative quantification of TNF-α, β-actin was used as a loading control to normalize protein levels. Proteins (30 µg) were resolved on 10% sodium dodecyl sulfate polyacrylamide gels and electrophoretically transferred to a Hybond-P polyvinyl difluoride membrane (GE Healthcare Life Sciences, Piscataway, NJ). The membrane was blocked in 5% skim milk with 0.1% Tween-20 in Tris-buffered saline for 2 hours at room temperature, and then incubated with antiTNF-α rabbit polyclonal antibody (1:1500 dilution; Proteintech Group, Chicago, IL) at 4°C overnight. Then the membrane was incubated with an HRP-conjugated antirabbit IgG (1:5000 dilution) for 1 hour at room temperature. Enhanced chemiluminescence detection of TNF-α was performed according to manufacturer instructions (Boehringer Ingelheim, Mannheim, Germany).

Statistical analysis. Data are expressed as the mean ± standard error of the mean. Statistical analyses were performed using IBM SPSS Statistics, Version 22.0 (IBM, Armonk, NY). The Mann-Whitney U test was used to analyze differences between groups and among negative control groups. Differences were considered significant when P ≤ .05.

Effects of air pollutants on cell viability. The total cell count of fibroblasts is shown in Figure 2. Exposure to DEPs decreased the total number of cells, and the proliferation of fibroblasts decreased in a time-dependent manner, as measured with the CCK-8 assay (P < .05; Figure 3). 

Effects of air pollution on the mRNA expression of TNF-α, IL-6, and COX-2 cytokines in fibroblasts.  The mRNA expression of the pro-inflammatory cytokines TNF-α, COX-2, and IL-6 was determined by RT-PCR. The TNF-α mRNA expression increased in a dose-dependent manner in both normal and diabetic fibroblasts; however, the increase was not significant. The IL-6 mRNA expression did not increase in either normal or diabetic fibroblasts (eFigures 4, 5). The COX-2 mRNA expression increased in a dose-dependent manner in diabetic fibroblasts (eFigure 5). 

Effects of air pollution on the protein expression of TNF-α, IL-6, and COX-2 cytokines in fibroblasts. The protein levels of COX-2 increased in normal SD rat fibroblasts and in diabetic cells upon treatment with DEP in a dose-dependent manner as determined by western blot analysis (eFigures 6, 7). Protein levels of TNF-α increased in diabetic cells upon treatment with DEP but did not increase in normal SD rat fibroblasts cells (eFigure 7A). Protein levels of IL-6 tended to increase in diabetic cells upon treatment with DEP but were not significant except in 120 µg/mL DEP doses (eFigure 7B). Protein levels of IL-6 were unchanged in normal SD rat fibroblasts cells upon treatment with DEP.

Exposure to air pollution has been shown to increase the production of IgE and Th2 cytokines and lead to inflammation.^17-19 Inflammation is a key step in the development of health effects associated with DEP, and the prototypical T-helper 1 cytokine regulates the inflammatory response.^20,21 The related cytokines are further divided into Th1 and Th2 cytokines,²² and treatment with DEP increases both Th1 and Th2 responses to hen egg lysozyme.²³ Diesel exhaust particles also affect the Th1 and Th2 response in a murine model of allergic airway inflammation.²⁴ The role of TNF-α in T cell-mediated inflammation depends on the Th1 and Th2 cytokine balance.²⁵ Th1 cytokines, such as IL-2, interferon-γ (IFN-γ), TNF-α, and IL-12, primarily mediate cellular immunity, whereas Th2 cytokines, such as IL-4, IL-5, IL-6, and IL-10,
are involved in humoral immunity to mediate differentiation.²⁶ Immunohistochemical expression of proinflammatory cytokines IL-1β, IL-6, TNF-α, and involvement of COX-2 play major roles in regulating inflammation.²⁷ Exposure to DEPs also increases the expression of TNF-α, COX-2, and IL-6 in vivo. In addition, it induces cytotoxicity, influences inflammatory gene expression, and activates intracellular signaling pathways in human bronchial epithelial cells. Therefore, exposure to DEPs has been associated with several adverse health effects.²⁸ The role of inflammatory cytokines in diabetic renal injury remains unknown. 

To evaluate the effects of air pollution on diabetic wounds, the authors evaluated the mechanisms by which DEP exposure increases the pro-inflammatory cytokine levels of TNF-α, COX-2, and IL-6 in fibroblasts. Fibroblasts contain a relaxed 3-dimensional collagen network that produces abundant IL-1, which upregulates the production of other inflammatory mediators such as IL-6 and COX-2. Cyclooxygenase-2 induction is abrogated by inhibition of IL-1, inducing a cytokine cascade. Cyclooxygenase-2 is normally undetectable in most tissues but can be induced in many cell types by a variety of events, particularly those involving pro-inflammatory stimuli.²⁹ 

This study demonstrates that DEP exposure increases TNF-α and COX-2 mRNA levels in normal and diabetic fibroblasts compared to fibroblasts that have not been exposed to DEPs (eFigures 4D, 5A, 5C, 5D). However, the level of IL-6 mRNA was unchanged compared to normal fibroblasts (eFigures 4B, 5B). Interleukin-6 is produced in wounds by epidermal keratinocytes, dermal fibroblasts, and macrophages,³⁰ and its role is well established in immune and inflammatory diseases and in wound healing. This study confirms previous findings about the expression of mRNA and protein levels of TNF-α, COX-2, and IL-6 in normal SD rat fibroblasts and in diabetic cells upon treatment with DEP. 

In mouse models of diabetic wounds, TNF-α dysregulation impairs healing, which may enhance apoptosis and decrease proliferation of fibroblasts.³¹ Pro-inflammatory cytokines, mainly IL-1α, IL-6, IL-8, and TNF-α, are involved in the development and progression of diabetic nephropathy. Cyclooxygenase-2, nitric oxide synthase, and nuclear factor NF-κB are implicated in processes related to diabetic nephropathy. The main cytokines involved in the pathogenesis of diabetes are IL-1, TNF-α, and IL-6.³² After DEP treatment, the protein level of TNF-α did not differ from that of a normal fibroblast (eFigure 6A). But the protein level of TNF-α was increased in diabetic fibroblast (eFigure 7A). Tumor necrosis factor-α induces NF-κB activation, which enhances inflammatory responses. After injury, TNF-α is upregulated in wild mice.³³ In diabetic fibroblasts, the protein level of TNF-α tended to increase following DEP exposure; however, the difference was not statistically significant compared to control cells, with the exception of treatment with 40 μg/mL DEP (eFigure 7 A, 7D). The protein level of IL-6 tends to increase in diabetic fibroblasts (eFigure 7B, 7D). Interleukin-6 and TNF-α are central mediators for the regulation of inflammation, endothelial dysfunction, and coagulation.³⁴ Interlukin-6 can be detected in the serum, although baseline levels are low in the absence of inflammation, and elevated levels of IL-6 have been found in autoimmune and chronic inflammatory diseases such as diabetes.³⁵ These data demonstrate the regulation of pro-inflammatory cytokines such as IL-6, TNF-α, and COX-2 in DEP treated diabetic fibroblasts. These pro-inflammatory cytokines have also been shown to delay wound healing in STZ-induced diabetic rats. 

This study was limited in that it only examined levels of IL-6, TNF-α, and COX-2. To fully understand the mechanisms of Th1 and Th2 cytokines, further studies should focus on other pro-inflammatory cytokines.

Exposure to DEPs delayed wound healing in STZ-induced diabetic rats and increased protein levels of pro-inflammatory cytokines such as TNF-α, COX-2, and IL-6. By increasing pro-inflammatory cytokines, DEP exposure caused wounds to worsen.

From the Clinical Research Center, Soonchunhyang University, Seoul, South Korea; Department of Orthopedics, Hanyang University Hospital, Seoul, South Korea; Department of Orthopedic Surgery, Veterans Health Service Medical Center, Seoul, South Korea; and Department of Orthopedics, Soonchunhyang University Bucheon Hospital, Seoul, South Korea

Address correspondence to:
Young Koo Lee, MD, PhD
Department of Orthopedics 
Soonchunhyang University Bucheon Hospital
Seoul, South Korea

*Dr. Sung and Dr. Lee are co-corresponding authors.

Disclosure: This research was supported by the Basic Science Research Program through the National Research Foundation of Korea, funded by the Ministry of Science, ICT and Future Planning (2014R1A1A1003181). The study was also partially supported by the Soonchunhyang University Research Fund. The authors disclose no financial or other conflicts of interest.