Treatment with 7% and 10% CO2 enhanced expression of IL-1β, TNF-α, and IL-6 in hypoxic cultures of human whole blood.

Introduction

Refractory hypoxemia is the main clinical manifestation of acute respiratory distress syndrome (ARDS).[1,2] Lower tidal ventilation is available for routine clinical application, which enables alleviation of hypoxemia, as well as avoidance of ventilator-induced lung injury.[3,4] This ventilation strategy inevitably leads to hypercapnia—elevated levels of carbon dioxide (CO2) in the blood.[5,6] A lower tidal ventilation strategy exerts some beneficial effects on respiratory systems, which can reduce pulmonary morbidity for patients with ARDS.[5,7,8] However, hypercapnia is a non-physiological state for the human body; thus far, it remains controversial whether hypercapnia is detrimental or protective for patients with ARDS.[9,10]

As the main clinical symptom of ARDS, hypoxemia can trigger secretion of inflammatory cytokines (e.g., TNF-α, IL-1β, and IL-6) in the blood.[11] In addition, the blood-mediated inflammatory response can cause injury to critical organs.[12] There are close links between the degree of hypoxemia and mortality in patients with ARDS, as well as between the duration of hypoxemia and mortality in those patients.[13] For patients with both hypoxemia and hypercapnia, the molecular mechanism of the inflammatory response may be complex. Hypercapnia has been reported to attenuate endotoxin-induced inflammation by inhibiting activation of nuclear factor-κB.[14,15] However, under hypoxic conditions, there may be differences in the underlying mechanism whereby hypercapnia exerts its effects on the blood-mediated inflammatory response.

Our previous study revealed that hypercapnia-induced activation of the NLR family, pyrin domain-containing 3 protein (NLRP3) inflammasome could upregulate the expression of IL-1β in hypoxia-activated microglia.[16] Moreover, IL-1β has been shown to promote the central nervous system cascade inflammatory response.[17,18] It is unknown whether hypercapnia exerts any effects on the peripheral inflammation of hypoxic blood. Here, we hypothesized that hypercapnia might enhance the inflammation of hypoxic blood, specifically by increasing IL-1β, TNF-α, and IL-6 expression in hypoxic blood.

Materials and methods

Ethics and consent

The study was approved by the Research Ethics Committee of Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou, China (Approval no. GDREC2018424H(R1)). All participants provided written informed consent for inclusion in the study.

Human whole blood cultures and treatment

Whole blood was collected from six healthy male volunteers (mean age, 38 ± 9.9 years). None of the volunteers had a history of hematologic disorders, transplantations, cancers, autoimmune diseases, infection, or immunosuppressive drug use. Whole blood was cultured as reported previously.[14] Briefly, 2 mL of whole blood were collected from each volunteer. Whole blood samples were cultured with 18 mL Roswell Park Memorial Institute (RPMI)-1640 medium (Invitrogen, Carlsbad, CA, USA; cat. no. R2405) supplemented with 10% human serum (Gemini Bio-Products, Calabasas, CA, USA; cat. no. H91S001) and 1% penicillin-streptomycin solution (Roche, Basel, Switzerland; cat. no. 11074440001) at 37°C in a humidified incubator with 5% CO2/95% air.

Blood samples were randomly divided into four groups: control (exposed to 20% O2 + 5% CO2 for 24 hours), hypoxia + 5% CO2 (exposed to 5% CO2 + 0.2% O2 for 24 hours), hypoxia + 7% CO2 (exposed to 7% CO2 + 0.2% O2 for 24 hours), and hypoxia + 10% CO2 (exposed to 10% CO2 + 0.2% O2 for 24 hours). The partial pressure of oxygen (PO2), partial pressure of carbon dioxide (PCO2), and pH of the medium were measured at 24 hours (n = 6 per group) using a Blood Gas/Electrolyte Analyzer (Model 5700, Instrumentation Laboratory, San Diego, CA, USA).

Evaluation of IL-1β, TNF-α, and IL-6 expression levels in whole blood cultures

IL-1β, TNF-α, and IL-6 levels were evaluated using enzyme-linked immunosorbent assay kits (all from Abcam, Cambridge, MA, USA; IL-1β: cat. no. ab46052; TNF-α: cat. no. ab181421; IL-6: cat. no. ab178013), in accordance with the manufacturers’ instructions. Briefly, samples and standards were added to plate wells that had been coated with horseradish peroxidase-labeled anti-IL-1β, anti-TNF-α, and anti-IL-6 antibodies. A 3,3',5,5'-tetramethylbenzidine substrate solution was then added to the wells. Subsequently, stop buffer was added, and the optical density at 450 nm was measured using a spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Concentrations of IL-1β, TNF-α, and IL-6 in the samples were determined by comparing the optical density of the samples with the optical density of the standard curve. This experiment was repeated six times.

Human THP-1 cells: culture and treatment

Monocytes are reportedly the main cellular source of proinflammatory cytokines.[19,20] Thus, the THP-1 human monocytic cell line was used in the present study; these cells were purchased form American Type Culture Collection (Manassas, VA, USA). THP-1 cells were cultured with RPMI-1640 medium supplemented with 10% fetal bovine serum (Invitrogen; cat. no. 16140071) and 1% penicillin-streptomycin solution at 37°C in a 5% CO2 incubator. THP-1 monocytes were cultured in six-well plates (1 × 106 cells/mL, 2 mL) for 48 hours in the presence of 100 nM phorbol 12-myristate 13-acetate,[21] which stimulated differentiation into macrophages. Macrophages were randomly divided into six groups: control (exposed to 5% CO2 + 20% O2), hypoxia + 5% CO2 (exposed to 5% CO2 + 0.2% O2), hypoxia + 7% CO2 (exposed to 7% CO2 + 0.2% O2), hypoxia + 10% CO2 (exposed to 10% CO2 + 0.2% O2), hypoxia + 10% CO2 + N-acetyl-L-cysteine (NAC), and hypoxia + 10% CO2 + Z-YVAD-FMK. Cells in the hypoxia + 10% CO2 + NAC and hypoxia + 10% CO2 + Z-YVAD-FMK groups were respectively treated with an ROS scavenger, 25 mM NAC[22-24] (MedChemExpress, Monmouth, NJ, USA; cat. no. HY-B0215), and a pan-caspase inhibitor, 10 μM Z-YVAD-FMK[25,26] (ApexBio, Boston, MA, USA; cat. no. A8955), for 30 minutes before exposure to 10% CO2 + 0.2% O2.

Measurement of reactive oxygen species (ROS)

ROS production in macrophages was evaluated using a ROS assay kit (BestBio, Shanghai, China; cat. no. BB-4705-2), in accordance with the manufacturers’ instructions. Briefly, DCFH-DA was diluted (1:1500) with RPMI-1640 medium without fetal bovine serum. Coverslips with adherent cells were cultured with RPMI-1640 medium supplemented with 10% fetal bovine serum. The medium was changed to diluted DCFH-DA (2 mL/well) after treatment with different concentrations of CO2 and O2; cells were incubated in this medium for 20 minutes at 37°C with 5% CO2. Coverslips were then washed using RPMI-1640 medium without fetal bovine serum. Finally, coverslips were mounted with a fluorescent mounting medium and detected using a fluorescence microscope (Olympus DP73 Microscope, Olympus, Tokyo, Japan). This experiment was repeated four times.

Western blotting

In accordance with the manufacturer’s instructions, total protein extracts from cells were prepared using a Total Protein Extraction Kit (BestBio Science, Shanghai, China; cat. no. BB-3101-100T). Total protein concentrations were determined using a Bioepitope Bicinchoninic Acid Protein Assay Kit (Bioworld Technology Co., Ltd, Nanjing, China; cat. no. 23227). Protein samples were separated on polyacrylamide-SDS gels and transferred onto nitrocellulose membranes. After membranes had been blocked with 5% non-fat dry milk in Tris-buffered saline for 1 hour, they were incubated overnight at 4°C with the following primary antibodies: anti-caspase-1 (rabbit monoclonal IgG, 1:1000, Abcam; cat. no. ab207802) and anti-IL-1β (rabbit polyclonal IgG, 0.2 μg/mL, Abcam; cat. no. ab9722). After membranes had been washed three times in Tris-buffered saline with Tween-20, they were incubated with a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:1000; Cell Signaling Technology, Danvers, MA, USA; cat. no. #7074) for 45 minutes at room temperature. Immunoblots were developed using an enhanced chemiluminescence detection system (ImageQuant LAS 500, GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Band intensities were quantified using ImageJ 1.39u software (National Institutes of Health, Bethesda, MD, USA). This experiment was repeated four times.

Statistical analysis

Statistical analysis was performed using SPSS Statistics, version 19.0 (IBM Corp., Armonk, NY, USA). All values are expressed as means ± standard deviations. One-way analysis of variance was used to analyze univariate-factor measurement data. Correlations between pH and each of the following factors were analyzed with Spearman’s rank correlation coefficient: IL-1β, TNF-α, and IL-6. Differences were considered statistically significant when P < 0.05.

Results

PO2, PCO2, and pH levels in whole blood culture medium

Compared with the control group, 0.2% O2 treatment reduced PO2 levels in the medium of the hypoxia + 5% CO2 (P < 0.01), hypoxia + 7% CO2 (P < 0.01), and hypoxia + 10% CO2 groups (P < 0.01). PO2 levels did not significantly differ among 0.2% O2-treated groups (Figure 1a; Table 1); PCO2 levels also did not significantly differ between the hypoxia + 5% CO2 and control groups. PCO2 levels were much higher in the hypoxia + 7% CO2 group than in the hypoxia + 5% CO2 group (P < 0.01). Notably, the hypoxia + 10% CO2 group had the highest PCO2 levels, compared with the hypoxia + 7% CO2 (P < 0.01) and hypoxia + 5% CO2 groups (P < 0.01) (Figure 1b; Table 1). pH levels were much lower in the hypoxia + 5% CO2 group than in the control group (P < 0.01); pH levels were also much lower in the hypoxia + 7% CO2 group than in the hypoxia + 5% CO2 group (P < 0.01). Notably, the hypoxia + 10% group had the lowest pH levels, compared with the hypoxia + 7% CO2 (P < 0.01) and hypoxia + 5% CO2 groups (P < 0.01) (Figure 1c; Table 1).

Figure 1.

PO2 (a), PCO2 (b), and pH (c) levels in whole blood culture medium at 24 hours (n = 6 per group). **P < 0.01. Exposures were as follows: control (20% O2 + 5% CO2), hypoxia + 5% CO2 (5% CO2 + 0.2% O2), hypoxia + 7% CO2 (7% CO2 + 0.2% O2), and hypoxia + 10% CO2 (10% CO2 + 0.2% O2).

Abbreviations: PCO2, partial pressure of carbon dioxide; PO2, partial pressure of artery blood oxygen

Table 1.

PO2, PCO2, and pH levels in whole blood culture medium (n = 6 per group)

	PO2 (mmHg)	PCO2 (mmHg)	pH
Control	158.50 ± 2.17	37.83 ± 2.32	7.43 ± 0.02
Hypoxia + 5% CO2	59.17 ± 2.14*	38.00 ± 2.37	7.34 ± 0.03
Hypoxia + 7% CO2	59.67 ± 1.63*	55.67 ± 2.73* #	7.27 ± 0.01* #
Hypoxia + 10% CO2	60.00 ± 2.37*	66.33 ± 2.50* #	7.22 ± 0.02* #

Compared with the control group: *P < 0.01; compared with the hypoxia + 5% CO2 group: #P < 0.01.

Abbreviations: PCO2, partial pressure of carbon dioxide; PO2, partial pressure of artery blood oxygen; pH, potential of hydrogen.

Higher levels of CO2 treatment increased IL-1β, TNF-α, and IL-6 expression levels in hypoxic whole blood cultures

IL-1β levels were much higher in the hypoxia + 7% CO2 group than in the hypoxia + 5% CO2 group (P < 0.05); the hypoxia + 10% CO2 group had the highest IL-1β levels, compared with the hypoxia + 7% CO2 (P < 0.05) and hypoxia + 5% CO2 groups (P < 0.01) (Figure 2a; Table 2). TNF-α levels were much higher in the hypoxia + 7% CO2 group than in the hypoxia + 5% CO2 group (P < 0.05); the hypoxia + 10% CO2 group had the highest TNF-α levels, compared with the hypoxia + 7% CO2 (P < 0.05) and hypoxia + 5% CO2 groups (P < 0.01) (Figure 2b; Table 2). IL-6 levels were much higher in the hypoxia + 7% CO2 group than in the hypoxia + 5% CO2 group (P < 0.05); the hypoxia + 10% CO2 group had the highest IL-6 levels, compared with the hypoxia + 7% CO2 (P < 0.05) and hypoxia + 5% CO2 groups (P < 0.01) (Figure 2c; Table 2).

Figure 2.

Higher levels of CO2 treatment increased (a) IL-1β, (b) TNF-α, and (c) IL-6 expression in hypoxic whole blood cultures (n = 6 per group; evaluated by enzyme-linked immunosorbent assay). *P < 0.05, **P < 0.01. Exposures were as follows: control (20% O2 + 5% CO2), hypoxia + 5% CO2 (5% CO2 + 0.2% O2), hypoxia + 7% CO2 (7% CO2 + 0.2% O2), and hypoxia + 10% CO2 (10% CO2 + 0.2% O2).

Abbreviations: IL-1β, interleukin-1 beta; TNF-α, tumor necrosis factor alpha; IL-6, interleukin-6

Table 2.

Effects of CO2 treatment on expression levels of IL-1β, TNF-α, and IL-6 in hypoxic whole blood cultures (n = 6 per group)

	IL-1β (pg/mL)	TNF-α (pg/mL)	IL-6 (pg/mL)
Hypoxia + 5% CO2	642.33 ± 196.58	431.50 ± 188.32	3829.33 ± 1257.81
Hypoxia + 7% CO2	1082.67 ± 368.24$	822.17 ± 266.74$	5869.67 ± 1529.56$
Hypoxia + 10% CO2	1562.83 ± 435.32# &	1185.67 ± 299.16# &	7952.33 ± 1692.40# &

Compared with the hypoxia + 5% CO2 group: $P < 0.05, #P < 0.01; compared with the hypoxia + 7% CO2 group: &P < 0.05.

Abbreviations: IL-1β, interleukin-1 beta; TNF-α, tumor necrosis factor alpha; IL-6, interleukin-6.

IL-1β, TNF-α, and IL-6 expression levels were negatively correlated with pH level

Expression levels of IL-1β, TNF-α, and IL-6 in hypoxic whole blood cultures were significantly negatively correlated with pH levels in the medium (IL-1β: ρ = -0.735, P < 0.01; TNF-α: ρ = -0.810, P < 0.01; IL-6: ρ = -0.805, P < 0.01) (Figure 3).

Figure 3.

Expression levels of IL-1β (a), TNF-α (b), and IL-6 (c) were negatively correlated with pH levels (n = 6 per group; analyzed with Spearman’s rank correlation coefficient).

Abbreviations: IL-1β, interleukin-1 beta; TNF-α, tumor necrosis factor-alpha; IL-6, interleukin-6

Higher levels of CO2 treatment induced overproduction of ROS in hypoxic THP-1 cells

The fluorescence intensity of ROS in THP-1 cells was noticeably increased by exposure to hypoxia, compared with the control group (Figure 4a, b). Fluorescence in hypoxic cells was further enhanced by treatment with 7% or 10% CO2 (Figure 4c, d). In contrast, ROS fluorescence was markedly reduced in cells that had been pretreated with NAC (Figure 4e).

Figure 4.

Higher levels of CO2 treatment induced overproduction of ROS in hypoxic THP-1 cells (n = 4 per group). Immunofluorescence images show production of ROS (green) in hypoxic THP-1 cells. Scale bars = 20 μm. Exposures were as follows: control (20% O2 + 5% CO2), hypoxia + 5% CO2 (5% CO2 + 0.2% O2), hypoxia + 7% CO2 (7% CO2 + 0.2% O2), hypoxia + 10% CO2 (10% CO2 + 0.2% O2), and hypoxia + 10% CO2 + NAC (10% CO2 + 0.2% O2 + 25 mM NAC).

Abbreviations: ROS, reactive oxygen species; NAC, N-acetyl-L-cysteine

High levels of CO2 exposure led to enhanced NLRP3 inflammasome activation and elevated IL-1β expression in hypoxic THP-1 cells

To explore the effects of high levels of CO2 on NLRP3 inflammasome activation and IL-1β expression, the protein expression levels of IL-1β and caspase-1 in THP-1 cells were assessed by western blotting and double immunofluorescence. Protein expression was much higher in the hypoxia + 7% CO2 group than in the hypoxia + 5% CO2 group (caspase-1: P < 0.01; IL-1β: P < 0.05). The hypoxia + 10% CO2 group had the highest levels of protein expression, compared with the hypoxia + 7% CO2 (caspase-1: P < 0.01; IL-1β: P < 0.01) and hypoxia + 5% CO2 groups (caspase-1: P < 0.01; IL-1β: P < 0.01). Compared with the hypoxia + 10% CO2 group, protein expression was significantly suppressed by treatment with an ROS scavenger, 25 mM NAC (caspase-1: P < 0.01), and a pan-caspase inhibitor, 10 μM Z-YVAD-FMK (IL-1β: P < 0.01) (Figure 5a, b; Figure 6a, b).

Figure 5.

High levels of CO2 enhanced activation of NLRP3 inflammasome in hypoxic THP-1 cells (n = 4 per group). (a) Immunoreactive bands of caspase-1 (10 kDa). (b) Relative density of caspase-1. *P < 0.05, **P < 0.01. Exposures were as follows: control (20% O2 + 5% CO2), hypoxia + 5% CO2 (5% CO2 + 0.2% O2), hypoxia + 7% CO2 (7% CO2 + 0.2% O2), hypoxia + 10% CO2 (10% CO2 + 0.2% O2), and hypoxia + 10% CO2 + NAC (10% CO2 + 0.2% O2 + 25 mM NAC).

Abbreviation: NLRP3, NLR family, pyrin domain-containing 3 protein; NAC, N-acetyl-L-cysteine

Figure 6.

High levels of CO2 increased IL-1β expression in hypoxic THP-1 cells (n = 4 per group). (a) Immunoreactive bands of IL-1β (17 kDa). (b) Relative density of IL-1β. *P < 0.05, **P < 0.01. Exposures were as follows: control (20% O2 + 5% CO2), hypoxia + 5% CO2 (5% CO2 + 0.2% O2), hypoxia + 7% CO2 (7% CO2 + 0.2% O2), hypoxia + 10% CO2 (10% CO2 + 0.2% O2), and hypoxia + 10% CO2 + Z-YVAD-FMK (10% CO2 + 0.2% O2 + 10 μM Z-YVAD-FMK)

Abbreviations: IL-1β, interleukin-1 beta

Discussion

The present study showed that 7% and 10% CO2 treatment increased expression of inflammation markers in hypoxic blood; specifically, expression levels of IL-1β, TNF-α, and IL-6 were increased in hypoxic whole blood cultures. Additionally, 7% and 10% CO2 treatment led to enhanced activation of NLRP3 inflammasome and elevated production of IL-1β via ROS overproduction in hypoxia-activated THP-1 cells. The model of cellular hypercapnia/hypoxemia used in the present study was originally established in our previous study.[16] In this model, 0.02% O2 treatment reduced PO2 levels in cell culture supernatant to approximately 60 mmHg. Treatment with 7% and 10% CO2 maintained pH levels in cell culture supernatant at 7.20 to 7.30. These findings are consistent with the hypoxemia and permissive hypercapnia changes observed in patients with ARDS.[27-30]

Hypercapnia has been reported to attenuate endotoxin-induced inflammation by inhibiting activation of nuclear factor-κB.[14,15] In contrast, we found that treatment with 7% and 10% CO2 enhanced the expression levels of IL-1β, TNF-α, and IL-6 in hypoxic whole blood cultures. The previous research results may appear to contradict those of the present experiments. However, during hypercapnic hypoxia, there may be differences in the underlying mechanism whereby hypercapnia exerts its effects on the blood-mediated inflammatory response. Exposure to endotoxin may induce development of inflammation through the Toll-like receptor/nuclear factor-κB pathway.[31-33] Hypoxia may activate the NLRP3 inflammasome by inducing ROS overproduction, which can lead to pyroptosis and promote a cascade of inflammatory responses.[34-37] Our previous study revealed that hypercapnia-induced NLRP3 inflammasome activation in hypoxia-activated microglia could upregulate the expression of IL-1β in a time-dependent manner—the expression of IL-1β peaked at 24 hours of exposure to hypercapnia.[16] The previous findings are consistent with the effects on peripheral inflammation in hypoxic blood after 24 hours of treatment with 7% and 10% CO2, observed in the present study.

Monocytes are reportedly the main cellular source of pro-inflammatory cytokines.[19,20] Therefore, the THP-1 human monocytic cell line was used for experiments in the present study. To explore the effects of high levels of CO2 on NLRP3 inflammasome activation and IL-1β expression, the protein expression levels of IL-1β and caspase-1 in THP-1 cells were examined by western blotting. We found that the NLRP3 inflammasome could be activated in hypoxic THP-1 cells exposed to high levels of CO2, as demonstrated by overproduction of ROS and upregulation of caspase-1 and IL-1β. These results suggest that high levels of CO2 can contribute to onset of pyroptosis and promote a cascade of inflammatory responses through activation of the NLRP3 inflammasome in hypoxia-activated monocytic cells. This hypothesis was supported by the elevated expression levels of IL-1β, TNF-α, and IL-6 observed in hypoxic whole blood cultures in this study.

There were two limitations in this study. First, although 7% and 10% CO2 treatment increased the expression levels of IL-1β, TNF-α, and IL-6 in hypoxic whole blood cultures, this was an in vitro experiment. An animal experiment (such as the protocol used by Ding et al.[16]) should be performed to provide evidence regarding the effects of hypercapnia on inflammation in vivo. Second, it remains unclear how high levels of CO2 treatment cause elevated expression levels of IL-1β, TNF-α, and IL-6 in hypoxic whole blood cultures. Our results would be strengthened by investigation of specific pathways by which high levels of CO2 regulate inflammation.

In summary, these preliminary findings suggest that treatment with high levels of CO2 increases the expression levels of IL-1β, TNF-α, and IL-6 in hypoxic whole blood cultures. Permissive hypercapnia may enhance inflammation in the blood of patients with ARDS who exhibit hypoxemia.