Indian Journal of Animal Research

  • Chief EditorK.M.L. Pathak

  • Print ISSN 0367-6722

  • Online ISSN 0976-0555

  • NAAS Rating 6.50

  • SJR 0.263

  • Impact Factor 0.4 (2024)

Frequency :
Monthly (January, February, March, April, May, June, July, August, September, October, November and December)
Indexing Services :
Science Citation Index Expanded, BIOSIS Preview, ISI Citation Index, Biological Abstracts, Scopus, AGRICOLA, Google Scholar, CrossRef, CAB Abstracting Journals, Chemical Abstracts, Indian Science Abstracts, EBSCO Indexing Services, Index Copernicus
Submit

Research Article

Indian Journal of Animal Research, volume 55 issue 9 (september 2021) : 1079-1084

Effect of DNA Methylation on LPS-Induced Expression of Tumour Necrosis Factor Alpha (TNF-α) in Bovine Mammary Epithelial Cells

Yangyunyi Dong1,2,3, Dong An1,2,3, Jun Wang1,2,3, Hongyu Liu1,2,3, Qing Zhang1,2,3, Jing Zhao1,2,3,*, Wenfa Lu1,2,3,*
1Joint Laboratory of Modern Agricultural Technology International Cooperation, Ministry of Education, Jilin Agricultural University, Jilin Changchun 130118, China.
2Key Laboratory of Animal Production, Product Quality and Security, Ministry of Education, Jilin Agricultural University, Jilin Changchun 130118, China.
3Jilin Province Engineering Laboratory for Ruminant Reproductive Biotechnology and Healthy Production, College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118, China.
Cite article:- Dong Yangyunyi, An Dong, Wang Jun, Liu Hongyu, Zhang Qing, Zhao Jing, Lu Wenfa (2021). Effect of DNA Methylation on LPS-Induced Expression of Tumour Necrosis Factor Alpha (TNF-α) in Bovine Mammary Epithelial Cells . Indian Journal of Animal Research. 55(9): 1079-1084. doi: 10.18805/IJAR.B-1271.

ABSTRACT

Background: Cow mastitis is a major disease that affects dairy industry worldwide. Although the inflammatory response induced by lipopolysaccharide (LPS) in bovine mammary epithelial cells (BMECs) is similar to the response to pathogenic bacteria, the underlying regulatory mechanisms remain unclear. This study aimed to clarify the effect of DNA methylation on LPS-induced expression of tumour necrosis factor alpha (TNF-α) in BMECs. 

Methods: The mammary epithelial cells were treated with LPS and DNA methylation inhibition 5-Aza-2’-deoxycytodine (5Aza). Expression of TNF-α, IL-6, BNBD-5, DNA methyltransferases (DNMT1, DNMT2, DNMT3A and DNMT3B) and DNA methylation at TNF-α regions, were analyzed using quantitative realtime PCR (qRT-PCR), Elisa and bisulfite sequencing PCR (BSP).

Result: Our results showed that LPS significantly increased the expression of inflammatory factors, including interleukin-6 (IL-6), bovine neutrophil beta-defensins (BNBD-5) and TNF-α. Further, we observed that the DNA methylation inhibitor, 5-Aza-2'-deoxycytosine (5-Aza), enhanced LPS-induced TNF-α mRNA expression. In addition, we found that LPS treatment significantly decreased the methylation levels of specific CpG sites in the TNF-α promoter (at -245 and -323) and inhibited the expression of DNA methyl transferases (DNMT1, DNMT2, DNMT3A and DNMT3B). Our results indicate that LPS promotes the expression of TNF-α in BMECs by inhibiting DNA methylation in the gene promoter region.

INTRODUCTION

Bovine mastitis is a common disease affecting the global dairy industry and is associated with a negative impact on the economy (Grohn et al., 2004; Sun et al., 2015). In China, the average incidence of bovine mastitis is approximately 33%. This disease causes an annual economic loss of ¥15-45 billion, thereby seriously affecting the growth and development of the dairy industry (Pu et al., 2014). Mastitis is caused by microbial infection of the mammary gland (i.e. gram-negative bacteria, Escherichia coli) (Porcherie et al., 2012; Geeta et al., 2019; Nalband et al., 2020). LPS, an important factor responsible for inducing inflammation in bovine mammary epithelial cells (Bludau et al., 2014), is the main component of Gram-negative bacterial cell wall. BMECs are the principal cell types of the mammary gland and serve to resist bacterial infection (Liu et al., 2014; Wellnitz et al., 2016). After the invasion by the pathogen, pattern recognition receptors, e.g., toll-like receptors (TLRs) are activated to recognize the pathogen, followed by the activation of intracellular signalling pathways that upregulate the expression of many cytokines (Li et al., 2019; Zhu et al., 2012; Wakchaure et al., 2012). Previously, LPS has been shown to stimulate TNF-α expression in BMECs and role of TNF-a has been elucidated in mammary gland immune regulation (Gao et al., 2018; Wang et al., 2018). However, the mechanism of LPS regulation of TNF-α expression in BMECs remains to be elucidated.
       
DNA methylation is an essential epigenetic modification in mammalian development mediated by DNA methyltransferase  (DNMT) (Cui and Xu, 2018; Ambrosi et al., 2017). DNA methylation is involved in the regulation of multiple biological processes such as protein modification, inflammation and embryo development (Jun et al., 2018). Studies have shown that methylation may also play an important role in the regulation of immune-responsive genes involved in pathogen recognition and subsequent signalling (Green and Ker, 2014). Currently, the role of DNA methylation in the regulation of inflammatory responses in BMECs is poorly understood. In the present study, we examined the effect of LPS on the expression of TNF-α after inhibition of DNA methylation. Further we investigated the effect of LPS on the methylation status of the TNF-α promoter region to determine the role of DNA methylation on LPS-induced expression of TNF-α in BMECs.

MATERIALS AND METHODS

The study was carried out during the period of December 2016 to April 2018, in the College of Animal Science and Technology, Jilin Agricultural University, Changchun, China.
 
Cell culture and treatments
 
BMECs were cultured in DMEM/F12 (Invitrogen, USA) medium containing 10% foetal bovine serum and incubated in a 37oC incubator with 5% CO2. To detect IL-6, BNBD-5 and TNF-α gene expression and TNF-α secretion levels, cells were treated with 1, 10 and 100 μg/mL LPS (Sigma-Aldrich, USA) for 6, 12 and 24 hr. To explore the effects of DNA methylation on TNF-α expression, cells were pre-treated with 5- Aza at 1.0, 2.5 and 5.0 μmol/L for 24, 48 and 72 hr and then incubated with 1.0 μg/mL LPS for another 24 hr. The expression of DNMT1, DNMT2, DNMT3A and DNMT3B was measured after 3, 6 and 12 hr of LPS incubation. The level of DNA methylation in the TNF-a promoter region was determined 6 hr after LPS treatment by Bisulfite sequencing PCR (BSP) as described below.
 
Cell viability assays
 
Cells were incubated with 5-Aza, followed by incubation with MTT for 4 hr. Next, 160 μL DMSO was added to dissolve the formazan crystals for the MTT assay. Absorbance was measured at 490 nm wavelength in a microplate reader (BioTek, USA).
 
Measurement of TNF-α
 
A commercially available kit was used to detect TNF-α (Life Technologies, USA) by microplate reader according to the manufacturers’ protocol.
 
Real-time quantitative PCR
 
Total RNA was extracted from BMECs directly from the cell culture plate using TRIzol (Invitrogen, USA) according to the manufacturer’s recommended protocol. Then, cDNA was synthesized by PrimeScript RT reagent Kit with gDNA Eraser (Takara, Tokyo, Japan). Real-time quantitative PCR was performed using SYBR® Premix Ex TaqTM II (Takara, Tokyo, Japan) with the Mx3000P (Agilent StrataGene) qRT-PCR detection system. The primers sequences of TNF-α, IL-6, BNBD5, DNMT1, DNMT2, DNMT3A, DNMT3B and β-actin are shown in Table 1. Relative gene expression was calculated by the 2-ΔΔCt method.
 

Table 1: Sequence of primers used for qRT-PCR.


 
Bisulfite sequencing PCR (BSP)
 
DNA was extracted using DNA Extraction Kit (Omega Bio-tek, USA) according to manufacturer’s instructions.The EpiTect® Fast Bisulfite Conversion Kit (Qiagen, USA) was used for DNA bisulfite treatment, followed by nested PCR amplification using specific primers (Table 2). The amplified product was cloned into pMD-18 T vector (Takara, Japan) for further sequencing. Ten individual clones in each group were sequenced.
 

Table 2: Sequence of primers used for amplification of methylation sites of TNF-α.


 
Statistical analysis
 
The data are presented as the mean ± standard deviation of the mean (SD). One-way analysis of variance (ANOVA) and student’s t-test were used to analyse differences between results of each groups. Differences with p<0.05 were considered statistically significant. All statistical analyses were performed using GraphPad Prism version 5.0 (GraphPad Prism Software, San Diego, CA, USA).

RESULTS AND DISCUSSION

Effect of LPS on expression of IL-6, BNBD-5 and TNF-α in BMECs
 
As shown in Fig 1A-C, the levels of IL-6, BNBD-5 and TNF-α in BMECs incubated with LPS were significantly higher than those in the controls, indicating an inflammatory response. In addition, treating cells with LPS increased intracellular TNF-α release (Fig 1D).
 

Fig 1: LPS-induced mammary epithelial cell inflammatory response: Cells were treated with different concentrations (1, 10 and 100 ìg/mL) of LPS for 6, 12 and 24 hr.


 
Effect of DNA methyl transferase inhibition on TNF-α expression
 
Treatment with 5-Aza was used to determine the effect of DNA methylation on LPS-induced TNF-a expression in BMECs. The effects of 5-Aza on the viability of cells are shown in Fig 2A. There was no effect on cell viability between 5-Aza-treated cells and the controls. As shown in Fig 2B, cells pre-treated with different concentrations of 5-Aza (1.0, 2.5 and 5.0 µmol/L) for 24, 48 and 72/ hr significantly upregulated TNF-α expression compared to cells treated with LPS only.
 

Fig 2: Effect of DNA methyltransferase inhibition on viability and TNF-α expression of BMECs: (A) Cells were grown in medium containing different concentrations (0, 1.0, 2.5 and 5.0 µmol/L)of 5-Aza for 24, 48 and 72 hr, then processed to determine cell viability using the MTT assay.


 
Effect of LPS on expression of DNMT1, DNMT2, DNMT3A and DNMT3B in BMECs
 
The results in Fig 3 shows that the levels of DNMT1, DNMT2, DNMT3A and DNMT3B in cells incubated with LPS were significantly lower than those in the controls. The degree of reduction in gene expression was related to the duration of LPS treatment, with the lowest expression level observed in cells treated with LPS for 3 hr.
 

Fig 3: LPS reduce the expression of DNA methyltransferases in BMECs: Cells were treated with 1ìg/mL LPS for 3, 6 and 12 hr. The relative mRNA expression of DNMT1, DNMT2, DNMT3A and DNMT3B in BMECs was quantified by qRT-PCR.


 
Effect of LPS on methylation level in the TNF-α promoter region in BMECs
 
As shown in Fig 4A, bisulfite sequencing PCR (BSP) analysis revealed that TNF-α had 12 target CpG sites. As indicated by the results in Fig 4B, significant hypomethylation was observed in LPS-treated cells. DNA methylation significantly decreased in the TNF-α promoter regions by 6.39% (73.33% vs. 66.94%), representing a reduction from 60.00% to 20.00% at the -323 site, 80.00% to 40.00% at the -270 site and 90.00% to 60.00% at the -245 site.
 

Fig 4: LPS caused TNF-α hypomethylation in BMECs: Cells were treated with 1ìg/mL LPS for 12 hr. (A) Methylation status in TNF-α promoter (95 to 428 bp).


       
TNF-a is a multifunctional cytokine that exerts a wide range of pro-inflammatory effects. Several studies have found that TNF-α is related to various inflammation pathogenesis (Mohammadpour et al., 2019). For example, a previous study confirmed that LPS stimulated the expression of TNF-α in BMECs (Liu M et al., 2019), although the exact molecular mechanisms was not deciphered. Other studies showed that DNA methylation regulated the expression of TNF-α. In human pathology, the mRNA expression of TNF-α and methylation status of its promoter are inversely related (Wilson, 2008). In rat liver cells, the T-2 toxin-induced increase in the expression of TNF-α was associated with DNA methylation (Liu et al., 2019). Treatment with 5-Aza augmented the production of TNF-α by inhibiting DNA methylation in macrophages. Moreover, demethylation by 5-Aza reportedly increased the expression of TNF-α, which was induced by LPS stimulation in broiler peripheral blood mononuclear cells (Shen et al., 2016). Consistent with the above studies, the results of the present study showed that LPS treatment significantly increased TNF-α mRNA expression, but reduced CpG methylation levels at specific sites in the TNF-α promoter. We also observed that demethylation enhanced the effect of LPS on TNF-a expression in BMECs. These results supported other studies showing that DNA methylation inhibited LPS-induced TNF-a expression. However, another study reported that, in mice lung tissues, 5-Aza reduced the LPS-induced TNF-a expression (Huang et al., 2016). This indicates that the TNF-a expression may be differentially regulated in cells of different origins.
       
Catalysed by DNMTs, DNA methylation is a type of epigenetic modification related to a variety of inflammation pathogenesis and transcriptional regulation. DMNT1 is mainly involved in maintaining the methylation state, while DNMT3A and DNMT3B are the de novo methyl transferases (Mo et al., 2019). We detected the mRNA expression of DNMT1, DNMT2, DNMT3A and DNMT3B and showed that their expression was significantly decreased after LPS treatment. According to recent studies, the expression of DNMT1and DNMT3B decreases in human dental pulp cells after LPS stimulation (Cai et al., 2020). DNMT1 was also significant decreased after LPS stimulation of broiler peripheral blood mononuclear cells. In addition, LPS decreased DNMT1, DNMT3A and DNMT3B expression as well as total DNMTs activities in human retinal pigment epithelial cells (Maugeri et al., 2018). However, contradictory results have also been reported: For example, treatment with LPS was shown to increase DNMT1, DNMT3A and DNMT3B in bovine endometrial cells (Jun et al., 2018). Further, DNMTI was significantly increased in human umbilical vein endothelial cells following LPS treatment (Ma et al., 2019) and increased DNMT1 expression was observed in LPS-treated THM-1 derived macrophages (Tang et al., 2019). gain, differences in cell types or LPS treatment may be responsible for these disparities.

CONCLUSION

In summary, the present study showed that methylation of specific CpG sites in TNF-a region decreased after LPS treatment, while LPS stimulation increased TNF-α gene expression in demethylated bovine mammary epithelial cells. Our results demonstrates that LPS-induced expression of TNF-α in bovine mammary epithelial cells is regulated by the methylation status of its promoters.

ACKNOWLEDGEMENT

We would like to acknowledge funding provided by National Key R&D Program of China (2017YFD0501903).

REFERENCES


  1. Ambrosi, C., Manzo, M. and Baubec, T. (2017). Dynamics and context-dependent roles of DNA methylation. Journal of Molecular Biology. 429(10): 1459-1475.

  2. Bludau, M.J., Maeschli, A., Leiber, F., Steiner, A. and Klocke, P. (2014). Mastitis in dairy heifers: prevalence and risk factors. Veterinary Journa. 202(3): p. 566-72.

  3. Cai, L., Zhan, M., Li, Q, Li, D. and Xu, Q. (2020). DNA methyltransferase DNMT1 inhibits lipopolysaccharideinduced inflammatory response in human dental pulp cells involving the methylation changes of IL6 and TRAF6. Molecular Medicine Reports. 21(2): 959-968.

  4. Cui, D. and Xu, X. (2018). DNA Methyltransferases, DNA Methylation and Age-Associated Cognitive Function. International Journal of Molecular Sciences. 19(5): 1315.

  5. Gao, R., Yang, H., Jing, S., Liu, B., Wei, M., He, P. and Zhang, N. (2018). Protective effect of chlorogenic acid on lipopolysaccharide -induced inflammatory response in dairy mammary epithelial cells. Microbial Pathogenesis. 124: p. 178-182.

  6. Geeta, D.L., Nittin, D.S. and Amarjit, S. (2019). Immunohistochemical detection of alpha-smooth muscle actin and S-100 in bovine mammary gland with mastitis. Indian Journal of Animal Research. 53: 1440-1444.

  7. Green, B.B. and Ke, D.E. (2014). Epigenetic contribution to individual variation in response to lipopolysaccharide in bovine dermal fibroblasts. Vet Immunol Immunopathol. 157(1-2): 49-58.

  8. Gröhn, Y.T., Wilson, D.J., González, R.N., Hertl, J.A., Schulte, H., Bennett, G. and Schukken, Y.H. (2004). Effect of pathogen- specific clinical mastitis on milk yield in dairy cows. Journal of Dairy Science, 87(10): p. 3358-74.

  9. Huang, X., Kong, G., Li, Y., Zhu, W., Xu, H., Zhang, X., Li, J., Wang, L., Zhang, Z., Wu, Y., Liu, X. and Wang, X. (2016). Decitabine and 5-azacitidine both alleviate LPS induced ARDS through anti-inflammatory/antioxidant activity and protection of glycocalyx and inhibition of MAPK pathways in mice. Biomedicine and Pharmacotherapy. 84: 447-453.

  10. Jun, W., Xiaoxiao, Y., Lucky T. Nesengani, He, D., Lianyu, Y. and Wenfa, L. (2018). LPS-induces IL-6 and IL-8 gene expression in bovine endometrial cells “through DNA methylation”. Gene. 677: p. 266-272.

  11. Li, C., Li, L., Chen. K., Wang, Y., Yang, F. and Wang, G. (2019). UFL1 Alleviates Lipopolysaccharide-Induced Cell Damage and Inflammation via Regulation of the TLR4/NF-kappaB Pathway in Bovine Mammary Epithelial Cells. Oxidative Medicine and Cellular Longevity, 2019: p. 6505373.

  12. Liu, A., Sun, Y., Wang, X., Ihsan, A., Tao, Y., Chen, D., Peng, D., Wu, Q., Wang, X. and Yuan, Z. (2019). DNA methylation is involved in pro-inflammatory cytokines expression in T-2 toxin-induced liver injury.Food and Chemical Toxicology. 132: p. 110661.

  13. Liu, M., Fang, G., Yin, S., Zhao, X., Zhang, C., Li, J. and Liu, Z. (2019). Caffeic Acid Prevented LPS-Induced Injury of Primary Bovine Mammary Epithelial Cells through Inhibiting NF-kappaB and MAPK Activation.Mediators of Inflammation, 2019: p. 1897820.

  14. Liu, M., Song, S., Li, H., Jiang, X., Yin, P., Wan, C., Liu, X., Liu, F. and Xu, J. (2014). The protective effect of caffeic acid against inflammation injury of primary bovine mammary epithelial cells induced by lipopolysaccharide. Journal of Dairy Science. 97(5): p. 2856-65.

  15. Ma, F., Li, Z., Cao, J., Kong, X. and Gong, G. (2019). A TGFBR2/SMAD2/DNMT1/miR-145 negative regulatory loop is responsible for LPS-induced sepsis. Biomedicine and Pharmacotherapy, 112: p. 108626.

  16. Maugeri, A., Barchitta, M., Mazzone, M.G., Giuliano, F., Basile, G. and Agodi A. (2018). Resveratrol Modulates SIRT1 and DNMT Functions and Restores LINE-1 Methylation Levels in ARPE-19 Cells under Oxidative Stress and Inflammation. International Journal of Molecular Sciences, 19(7).

  17. Mo, Z., Li, Q., Cai, L., Zhan, M. and Xu, Q. (2019). The effect of DNA methylation on the miRNA expression pattern in lipopolysaccharide-induced inflammatory responses in human dental pulp cells. Molecular Immunology, 111: p. 11-18.

  18. Mohammadpour-Gharehbagh, A., Jahantigh, D., Eskandari, M., Eskandari, F., Rezaei, M., Zeynali-Moghaddam, S., Teimoori, B. and Salimi, S. (2019). The role of TNF-alpha and TLR4 polymorphisms in the placenta of pregnant women complicated by preeclampsia and in silico analysis. International Journal of Biological Macromolecules,134: p. 1205-1215.

  19. Nalband, S.M., Kolhe, R.P., Deshpande, P.D,, Jadhav, S.N., Gandhale, D.G., Muglikar, D.M., Kolhe, S.R., Bhave, S.S., Jagtap, U.V. and Dhandore, C.V. (2020). Characterization of Escherichia coli isolated from bovine subclinical mastitis for virulence genes, phylogenetic groups and ESBL production. Indian Journal of Animal Research. 54: p. 1265-1271.

  20. Porcherie, A., Cunha, P., Trotereau, A., Roussel, P., Gilbert, F.B., Rainard, P. and Germon, P. (2012). Repertoire of Escherichia coli agonists sensed by innate immunity receptors of the bovine udder and mammary epithelial cells. Veterinary Research, 43: p. 14.

  21. Pu, W., Su, Y., Li, J., Li, C., Yang, Z., Deng, H. and Ni, C. (2014). High incidence of oxacillin-susceptible mecA-positive Staphylococcus aureus (OS-MRSA) associated with bovine mastitis in China.PLoS One. 9(2): e88134.

  22. Shen, J., Liu, Y., Ren, X., Gao, K., Li, Y., Li, S., Yao, J. and Yang, X. (2016). Changes in DNA Methylation and Chromatin Structure of Pro-inflammatory Cytokines Stimulated by LPS in Broiler Peripheral Blood Mononuclear Cells. Poultry Science, 95(7): 1636-45.

  23. Sun, Y., Li, L., Wu, J., Yu, P., Li, C., Tang, J., Li, X., Huang, S. and Wang, G. (2015). Bovine recombinant lipopolysaccharide binding protein (BRLBP) regulated apoptosis and inflammation response in lipopolysaccharide-challenged bovine mammary epithelial cells (BMEC).Molecular Immunology. 65(2): 205-14.

  24. Tang, R.Z., Zhu, J.J., Yang, F.F., Zhang, Y.P., Xie, S.A., Liu, Y.F., Yao, W.J., Pang, W., Han, L.L., Kong, W., Wang, Y.X., Zhang, T. and Zhou, J. (2019). DNA methyltransferase 1 and Kruppel-like factor 4 axis regulates macrophage inflammation and atherosclerosis. Journal of Molecular and Cellular Cardiology. 128: p. 11-24.

  25. Wakchaure, S.R., Gupta, I.D., Verma, A., Kumar, S.R. and Kumar, D. (2012). Study of Toll-like receptors 4 (TLR4) gene in cattle: A review. Agricultural Reviews. 33: 220-225.

  26. Wang, J., Xiao, C., Wei, Z., Wang, Y., Zhang, X. and Fu, Y. (2018). Activation of liver X receptors inhibit LPS-induced inflammatory response in primary bovine mammary epithelial cells. Vet Immunol Immunopathol.197: p. 87-92.

  27. Wellnitz, O., Zbinden, C., Huang, X. and Bruckmaier, R.M. (2016). Short communication: Differential loss of bovine mammary epithelial barrier integrity in response to lipopolysaccharide and lipoteichoic acid. Journal of Dairy Science. 99(6): 4851-4856.

  28. Wilson, A.G. (2008). Epigenetic regulation of gene expression in the inflammatory response and relevance to common diseases. Journal of Periodontology, 79(8 Suppl): p. 1514-9.

  29. Zhu, Y.H., Liu, P.Q., Weng, X.G., Zhuge, Z.Y., Zhang, R., Ma, J.L., Qiu, X.Q., Li, R.Q., Zhang, X.L. and Wang, J.F. (2012). Short communication: Pheromonicin-SA affects mRNA expression of toll-like receptors, cytokines and lactoferrin by Staphylococcus aureus-infected bovine mammary epithelial cells. Journal of Dairy Science. 95(2): p. 759-64.

     
Reviewed By

Download Article
In this Article
    Contact us
    Follow us

    Editorial Board

    View all (0)