Conceived and designed the experiments: YZ WL JW. Performed the experiments: WL YL SR YG GP YC. Analyzed the data: WL YL JW. Contributed reagents/materials/analysis tools: MM RG YP SR LK QH XC. Wrote the paper: YZ WL.
The authors have declared that no competing interests exist.
Interleukin (IL)-32 is a recently described pro-inflammatory cytokine that has been reported to be induced by bacteria treatment in culture cells. Little is known about IL-32 production by exogenous pathogens infection in human individuals.
In this study, we found that IL-32 level was increased by 58.2% in the serum samples from a cohort of 108 patients infected by influenza A virus comparing to that of 115 healthy individuals. Another pro-inflammatory factor cyclooxygenase (COX)-2-associated prostaglandin E2 was also upregulated by 2.7-fold. Expression of IL-32 in influenza A virus infected A549 human lung epithelial cells was blocked by either selective COX-2 inhibitor NS398 or Aspirin, a known anti-inflammatory drug, indicating IL-32 was induced through COX-2 in the inflammatory cascade. Interestingly, we found that COX-2-associate PGE2 production activated by influenza virus infection was significantly suppressed by over-expression of IL-32 but increased by IL-32-specific siRNA, suggesting there was a feedback mechanism between IL-32 and COX-2.
IL-32 is induced by influenza A virus infection via COX-2 in the inflammatory cascade. Our results provide that IL-32 is a potential target for anti-inflammatory medicine screening.
Influenza A virus (IV) is a highly contagious single-stranded RNA virus that infects both the upper and lower respiratory tracts of humans. The host innate immune Toll-like receptor 3 (TLR3) was shown previously in cells of myeloid origin to recognize the viral replicative, intermediate double-stranded RNA (dsRNA). Thus, dsRNA is critical for the outcome of the infection and appears to be an active component of viral infection that stimulates antiviral activities. It accumulates during the replication of many viruses
Cyclooxygenase (COX) is the rate-limiting enzyme in the biosynthetic pathway of prostaglandins and thromboxanes. Prostaglandins play an important role in many biological processes. Altered prostanoid production is associated with a variety of illnesses, including acute and chronic inflammation, cardiovascular disease and colon cancer
Interleukin-32 (IL-32), previously called natural killer cell transcript 4, has been recognized as a pro-inflammatory cytokine recently. It is mainly expressed in natural killer cells, T cells, epithelial cells and blood monocytes. IL-32 can induce the pro-inflammatory cytokines TNF-α and IL-1β in murine peritoneal macrophages as well as in phorbol ester-differentiated human THP-1 cells
A number of investigations demonstrated that viral infections stimulate COX-2 expression, followed by PGE2 accumulation
Because COX-2 and IL-32 gene expression are associated with inflammatory processes, the aim of this study is to investigate the role of influenza A virus infection in the regulation of IL-32 expression and to determine the molecular mechanisms responsible. Our results showed that influenza A virus infection or poly(IC) treatment activates COX-2 and IL-32 expression by a heretofore unrecognized mechanism, in which influenza A virus stimulates IL-32 expression through COX-2, and IL-32 feedback inhibits COX-2 expression.
The study examined 108 consecutive adults with influenza A virus infection (59 male, 49 female, aged 39.2±13.5 yr) seropositive for influenza A antigen and 115 healthy adults (62 male, 53 female, aged 37.6±11.3 yr) seronegative for influenza A antigen. All adults were seronegative for markers of hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis Delta virus, and HIV. All of the investigated serum samples were obtained with the help from Hubei provincial Center for Disease Control and Prevention (Hubei CDC). Informed consent was obtained from each of the patients. The collection of blood samples for research was approved by the Institutional Review Board of the College of Life Sciences, Wuhan University in accordance with guidelines for the protection of human subjects.
The influenza virus strain A/chicken/Hubei/327/2004 (H5N1) used in this study was provided by China Center for Type Culture Collection (CCTCC). Stock virus was propagated in 10-day-old embryonated chicken eggs for 36 to 48 h at 37°C. The allantoic fluid was then harvested, and aliquots were stored at –80°C before being used. The final concentration of H5N1 virus infection used in this study was 1 multiplicity of infection (MOI).
pcDNA3.1-COX-2 was a gift from Dr. Kenneth K. Wu (University of Texas-Houston Medical School, Houston, Texas, USA). Luciferase reporter vector (pGL3) containing a COX-2 promoter region (−891/+9) was constructed previously
All of the ten genes of influenza A virus H5N1 were obtained by RT-PCR from H5N1 infected A549 cells and constructed into pCMV-Flag2A vector to generate IV-gene expressing plasmids (Flag2A-HA, Flag2A-NA, Flag2A-NP, Flag2A-NS1 Flag2A-NS2, Flag2A-M1, Flag2A-M2, Flag2A-PA, Flag2A-PB1, Flag2A-PB2) using specific pairs of primers for each influenza A virus gene as follows: HA:
Monoclonal mouse antibody against human COX-2 was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Polyclonal goat antibody against human IL-32 was purchased from R&D Systems, Inc. USA. Polyclonal goat antibody specific for human β-actin (SC-1616) were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA).
N-(2-cyclohexylosy-4-nitrophenyl)-methanesulphonamide (NS398) (Promega, Madison, WI) was dissolved in DMSO and used as indicated concentrations according to reference
HEK 293T cells were cultured in DMEM (GibcoBRL, USA), human lung epithelial cells A549 were cultured in F12K media (GibcoBRL, USA), respectively. All media were supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin sulfate and all cell cultures were maintained at 37°C in a 5% CO2 incubator.
Cells were plated at density of 4.0×105 cells per 24-well plate or 6-well plate and grown to confluence reaching about 80% at the time of transfection. The plasmids which express COX-2, IL-32, the pGL3-promoter plasmids, pRL-TK (Promega) were co-transfected into the cells by using Lipofectamine 2000 reagent (Invitrogen). If necessary, poly(IC), IFN-γ, NS398, and Aspirin were added into the culture media after transfection. 24 h post-transfection, cells were serum-starved for another 24 h before being harvested. Luciferase activities were measured 48 h after transfection according to the manufacturer's instructions (Promega) and Renilla Luciferase activities were determined as internal control for transfection efficiency as previously described
Total RNA, isolated from A549 cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA), was treated with DNase I and reverse-transcribed with MLV reverse transcriptase (Promega) and random primers (Takara). PCR was performed in 25 µl reactions with the detection primer pairs described as follows: IL-32 sense:
Protein extracts of cultured cells were prepared by suspending cells in lysis buffer (0.01% EDTA, 0.1% Triton X-100, 10% proteinase inhibitors cocktail), sonication, and centrifugation. Concentrations of proteins in supernatant were quantified using protein assay kit (Bio-Rad). Western blot analysis was performed using COX-2 and IL-32 antibodies and sample loading was normalized by using β-actin antibody. Immunoblots were visualized with the ECL detection system (Pierce, Rockford, IL, USA).
Because increased PGE2 is the metabolite of COX-2 enzyme catalysis in epithelial cells, COX-2-derived PGE2 levels in the culture medium was assayed by the Biotrak Prostaglandin E2 Enzyme Immunoassay system (R & D Systems) according to the manufacturer's protocol.
ELISA assays were developed using IL-32 antibody and a polypeptide, H-KEELTPQKCSEPQSSK-OH, (GL Biochem Ltd. Shanghai China) was synthesized as an antigen for making an IL-32 protein standard curve. ELISAs were prepared by coating the bottom of a 96-well plate with diluted serum samples or culture media. 50 µl of 2 µg/ml capture antibody was incubated for one hour then washed three times with TBST, followed by HRP-labeled secondary antibody incubation for 15 minutes, and finally washed six times with TBST. ELISAs were developed with TMB (Sigma) substrate and the absorbance at double-wavelength 450 nm/630 nm was measured. IL-32 concentrations were calculated from the standard curve.
All experiments were reproducible and were carried out in triplicate or quadruplicate. Each set of experiments was repeated at least three times with similar results, and a representative one is shown. The results are presented at the means±s.d. Student's
The serum levels of PGE2 and IL-32 were significantly higher in the cohort of patients with influenza A virus infection investigated in comparison with healthy control individuals (mean±SEM for PGE2, 808.7±52.9 vs. 299.5±45.4 pg/ml; for IL-32, 183.8±43.6 vs. 116.2±29.3 pg/ml, respectively,
Characteristic | Healthy Individuals (N = 115) | Patients (N = 108) | P Value |
Age-yr | 37.6±11.3 | 39.2±13.5 | 0.85 |
Male sex-no.(%) | 62 (54) | 59 (55) | 0.91 |
Race or ethnic group-no.(%) Asian | 115 (100) | 108 (100) | 1.00 |
Time-no.(%) | 115 (100) | 108 (100) | 1.00 |
Oct.8th/2007-Oct.19th/2007 | |||
Region-no.(%) | 115 (100) | 108 (100) | 1.00 |
Hubei province, China | |||
HA-Antigen-positive-no.(%) | 0 (0) | 108 (100) | <0.001 |
Anti-HA-positive-no.(%) | 0 (0) | 108 (100) | <0.001 |
Viral genotype A (H3/H1)-no. | 0 | 108 (85/23) | |
PGE2-pg/ml | 299.5±45.4 | 808.7±52.9 | <0.001 |
IL-32-pg/ml | 116.2±29.3 | 183.8±43.6 | <0.001 |
It has been reported that influenza virus can activate the expression of COX-2 in cell culture systems
Time-dependent of PGE2 (A) and IL-32 (B) accumulations in A549 culture supernatants are shown in response to influenza A virus infection (1 MOI) or poly(IC) (50 µg/ml)+IFN-γ (150 U/ml) treatment. Data are expressed as mean±s.d. of three independent experiments. Time-dependent of COX-2 and IL-32 mRNA accumulations (C) in cell lysates are shown by Semiquantitative RT-PCR analysis and protein productions (D) shown by western blot analysis, in which A549 cells were harvested at indicated time points after influenza A virus infection (1 MOI) or 48 h treatment with poly(IC) (50 µg/ml)+IFN-γ (150 U/ml). The gel or blot is a representative of three experiments with similar results.
To identify the viral components which play important roles in IV-stimulated pro-inflammatory factors COX-2 and IL-32 expression, we screened all ten proteins of influenza virus: HA, NA, NP, NS1, NS2, M1, M2, PA, PB1, PB2 and poly(IC) (to mimic viral replicative intermediate dsRNA) by luciferase assays. Results showed that poly(IC), poly(IC)+IFN-γ, and NS1 are the most important factors in the induction of either COX-2 (
Reporter plasmids pCOX-2-Luc (A), pIL-32-Luc (B), and pRL-TK were cotransfected along with all 10 viral gene constructs (Flag2A-HA, Flag2A-NA, Flag2A-NP, Flag2A-NS1, Flag2A-NS2, Flag2A-M1, Flag2A-M2, Flag2A-PA, Flag2A-PB1, Flag2A-PB2) and control vectors (Flag2A) or treated with or without poly(IC) (50 µg/ml), poly(IC) (50 µg/ml)+IFN-γ (150 U/ml) into A549 cells which are indicated on the horizontal axis respectively. Luciferase activity was measured as described in
To define the role of COX-2 in the regulation of influenza A virus induced pro-inflammatory factor IL-32, poly(IC)+IFN-γ treatment was used to mimic the influenza A virus infection in following experiments as reported previously
First, the effects of COX-2 on the activation of IL-32 promoter were determined. A549 cells were cotransfected with the reporter plasmid pIL-32-Luc and pcDNA3.1, pcDNA3.1-COX-2 plus different concentration of NS398 as mentioned in
Reporter plasmid pIL-32-Luc (A), and pRL-TK were cotransfected along with pcDNA3.1-COX-2 or pcDNA3.1 into A549 cells. Transfected cells by COX-2 plasmids were incubated for 12 h and then maintained for 36 h with different final concentrations of NS398 as indicated, respectively. Luciferase activity was measured. Results are expressed as the mean±s.d. of three independent experiments performed in triplicate and normalized by Renilla activities. A549 cells were transfected with different indicated amounts of pcDNA3.1-COX-2 or pcDNA3.1. RT-PCR for IL-32 and β-actin (internal control) (B) in cell lysates, Elisa for IL-32 (C) in culture supernatants were performed. (D) A549 cells were treated with poly(IC) (50 µg/ml)+IFN-γ (150 U/ml) for 12 h and then different concentration of NS398 was added as indicated for another 36 h. IL-32 and β-actin mRNA level were examined by RT-PCR. (E) A549 cells were stimulated by poly(IC) (50 µg/ml)+IFN-γ (150 U/ml) with or without 80 µM NS398 for 48 h. Time-dependent of IL-32 expression in culture supernatants were measured. The data represent mean±s.d. of three separate experiments.
Secondly, to determine the effects of COX-2 on the activation of IL-32 mRNA level and protein expression, A549 cells were transfected with different amounts of pcDNA3.1-COX-2. Results from RT-PCR using IL-32-specific, or β-actin-specific primers showed that the levels of IL-32 mRNA were increased as the amount of pcDNA3.1-COX-2 increased, but the levels of β-actin mRNA remained relatively constant (
Thirdly, results from RT-PCR analyses showed that the level of IL-32 mRNA activated by poly(IC)+IFN-γ was suppressed by COX-2-specific inhibitor NS398 in a dose dependent manner (
Taken together, these data suggest that COX-2 is an upstream regulatory factor of dsRNA-triggered IL-32 production.
The effect of IL-32 on the regulation of COX-2 promoter was determined. A549 cells were cotransfected with the reporter plasmid pCOX-2-Luc and pCMV-Flag2A, Flag2A-IL-32. Results from Luciferase activity assay showed that the level of COX-2 promoter activity was decreased by IL-32 over-expression (
Reporter plasmid pCOX-2-Luc (A) and pRL-TK were cotransfected along with Flag2A-IL-32 or control vectors (Flag2A) into A549 cells. Luciferase activity was then measured 48 h post transfection. A549 cells were transfected with different amounts of Flag2A-IL-32 as indicated or Flag2A and stimulated by poly(IC) (50 µg/ml)+IFN-γ (150 U/ml) for 48 h. RT-PCR for COX-2 and β-actin (B) in lysated cells, measurement of PGE2 release (C) in culture supernatants were taken. Reporter plasmid pCOX-2-Luc (D) and pRL-TK were cotransfected along with siRNA-IL-32 or siRNA-control into A549 cells and treated with poly(IC) (50 µg/ml)+IFN-γ (150 U/ml) for 48 h. Luciferase activity was then measured. (E) A549 cells were transfected with siRNA-IL-32 or siRNA-control and stimulated by poly(IC) (50 µg/ml)+IFN-γ (150 U/ml) for 48 h. Time-dependent PGE2 release in culture supernatants was measured. The data represent mean±s.d. of three separate experiments.
To determine the effects of IL-32 on the regulation of COX-2 mRNA expression, PGE2 production, A549 cells were transfected with different amounts of Flag2A-IL-32 and treated with poly(IC)+IFN-γ as the inducer. Results from RT-PCR using COX-2-specific, or β-actin-specific primers showed that the levels of COX-2 mRNA was decreased as the amount of Flag2A-IL-32 increased, but the levels of β-actin mRNA remained relatively constant (
To confirm the above results, IL-32-specific siRNA was cotransfected along with reporter plasmid pCOX-2-Luc into A549 cells and treated with poly(IC)+IFN-γ. Results from Luciferase activity assay showed that the level of COX-2 promoter activity was increased by knocking down IL-32 (
Taken together, these data suggest that IL-32 plays a very important role in the inflammatory response following an influenza A/COX-2/IL-32 dependent positive regulatory order, while a negative feedback to COX-2 biosynthesis was also first observed.
To confirm the COX-2 and IL-32 regulatory loop, A549 cells were infected by influenza A virus and treated with or without selective COX-2 inhibitor NS398, or non-selective COX inhibitor Aspirin, a widely used anti-inflammatory drug. Results showed that IV-triggered PGE2 release (
A549 cells were infected by influenza A virus (1 MOI) and treated with or without 80 µM NS398, 5 mM Aspirin for 48 h as indicated on the horizontal axis. PGE2 release (A) and IL-32 production (B) in culture supernatants were measured. A549 cells were transfected with siRNA-IL-32 or siRNA-control and infected by influenza A virus (1 MOI) for 48 h. PGE2 release (C) and IL-32 production (D) in culture supernatants were measured. The data represent mean±s.d. of three separate experiments.
The effect of IL-32 on the regulation of IV infection induced PGE2 production was also determined. We found that PGE2 release in A549 cell culture supernatants stimulated by IV infection was increased (
Taken together, these data demonstrate that a novel inflammatory pathway in response to influenza A virus infection and dsRNA treatment was identified as described in
Our results demonstrate that significant increase of serum COX-2-derived PGE2 and IL-32 levels were observed in influenza A infected patients compared with healthy individuals. Viral infection resulted in 2.7-fold increase in PGE2 synthesis and 58.2% increase in IL-32 production. Both influenza A virus infection and poly(IC)+IFN-γ treatment in A549 human lung epithelial cells were able to induce COX-2/ IL-32 mRNA and protein expression as well as PGE2 and IL-32 accumulation in the cell culture supernatants. IL-32 was induced by influenza virus infection through COX-2-dependent mechanism.
Although pro-inflammatory factor COX-2 has been identified as an obligatory mediator in the airway inflammation during influenza virus infection
Comparing the IV infection induced IL-32 level in cell culture with that in human individuals, a significant difference between them was observed, over 10-fold increase in cell culture versus 58.2% in human individuals. It is likely the pro-inflammatory factor is not able to be increased sharply under physiological conditions due to complex inflammation network in which pro-inflammatory factors regulate each other.
Two viral components, the NS1 and viral replicative intermediate dsRNA were identified to be involved in IV infection triggered COX-2 and IL-32 expression in A549 cells. Because TLR3 contributes directly to the inflammatory response of respiratory epithelial cells to both influenza A virus and dsRNA, we focused on the function of dsRNA in the regulation of COX-2 and IL-32 expression in this study. Role of viral NS1 protein in the pro-inflammatory process need further investigation.
Cyclooxygenase metabolizes arachidonic acid to prostaglandins (PGs) and thromboxane
In summary, these studies provide new insights into a novel model as described in
We thank Hubei provincial Center for Disease Control and Prevention (Hubei CDC) for providing serum samples with seronegative for influenza A antigen in this study, and thank Dr. Kenneth K. Wu for kindly providing plasmid pcDNA3.1-COX-2.