Abstract
The nucleotide-binding domain leucine-rich repeat proteins (NLRs), a class of innate immune receptors that respond to pathogen attack or cellular stress, have gained increasing attention. NLRC5 is the largest member of NLR family, which has recently been identified as a critical regulator of immune responses. In this study, we explore the role of NLRC5 in cytokine secretion and the role of the JAK2/STAT3 signaling pathway in lipopolysaccharide-induced NLRC5 expression in RAW264.7 cells. We demonstrated that overexpression of NLRC5 results in a downregulation of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) secretion; on the other hand, knockdown of NLRC5 by transfecting siRNA enhanced IL-6 and TNF-α secretion in RAW264.7 cells. These results indicated that NLRC5 plays a negative role in the regulation of IL-6 and TNF-α. Meanwhile, AG490 (a specific inhibitor of the JAK2/STAT3 signaling pathway) and JAK2 siRNA were used to manipulate JAK2/STAT3 activity. Finally, the results showed that AG490 and JAK2 siRNA inhibited NLRC5 expression and the expression levels of p-JAK2 and p-STAT3. We, for the first time, demonstrate that the inhibition of the JAK2/STAT3 signaling pathway results in decrease of NLRC5 expression.
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Abbreviations
- NLRs:
-
Nucleotide-binding domain leucine-rich repeat proteins
- LPS:
-
Lipopolysaccharide
- TNF-α:
-
Tumor necrosis factor-α
- IL-6:
-
Interleukin-6
- PRRs:
-
Pattern recognition receptors
- PAMPs:
-
Pathogen-associated molecular patterns
- RLRs:
-
RIG-I (retinoid acid-inducible gene I)-like receptors
- TLRs:
-
Toll-like receptors
- CLRs:
-
C-type lectin receptors
- NLRs:
-
NOD-like receptors
- NLRC5:
-
NLR family, CARD domain containing 5
- MTT:
-
(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetra-zoliumbromide)
- DMSO:
-
Dimethyl sulfoxide
- DMEM:
-
Dulbecco’s modified Eagle’s medium
- ELISA:
-
Enzyme-linked immunosorbent assay
- PCR:
-
Polymerase chain reaction
REFERENCES
Akira, S., S. Uematsu, and O. Takeuchi. 2006. Pathogen recognition and innate immunity. Cell 124: 783–801.
Meylan, E., J. Tschopp, and M. Karin. 2006. Intracellular pattern recognition receptors in the host response. Nature 442: 39–44.
Honda, K., and T. Taniguchi. 2006. IRFs: master regulators of signalling by Toll-like receptors and cytosolic pattern-recognition receptors. Nature Reviews Immunology 6: 644–658.
Liew, F.Y., D. Xu, E.K. Brint, and L.A. O’Neill. 2005. Negative regulation of toll-like receptor-mediated immune responses. Nature Reviews Immunology 5: 446–458.
Ting, J.P., D.L. Kastner, and H.M. Hoffman. 2006. CATERPILLERs, pyrin and hereditary immunological disorders. Nature Reviews Immunology 6: 183–195.
Inohara, Chamaillard, C. McDonald, and G. Nunez. 2005. NOD-LRR proteins: role in host-microbial interactions and inflammatory disease. Annual Review of Biochemistry 74: 355–383.
Abrahams, V.M. 2011. The role of the Nod-like receptor family in trophoblast innate immune responses. Journal of Reproductive Immunology 88: 112–117.
Martin, A.P., T. Marinkovic, C. Canasto-Chibuque, R. Latif, J.C. Unkeless, T.F. Davies, Y. Takahama, G.C. Furtado, and S.A. Lira. 2009. CCR7 deficiency in NOD mice leads to thyroiditis and primary hypothyroidism. Journal of Immunology 183: 3073–3080.
Schroder, K., and J. Tschopp. 2010. The inflammasomes. Cell 140: 821–832.
Benko, S., D.J. Philpott, and S.E. Girardin. 2008. The microbial and danger signals that activate Nod-like receptors. Cytokine 43: 368–373.
Lian, L., C. Ciraci, G. Chang, J. Hu, and S.J. Lamont. 2012. NLRC5 knockdown in chicken macrophages alters response to LPS and poly (I:C) stimulation. BMC Veterinary Research 8: 23.
Cui, J., L. Zhu, X. Xia, H.Y. Wang, X. Legras, J. Hong, J. Ji, P. Shen, S. Zheng, Z.J. Chen, and R.F. Wang. 2010. NLRC5 negatively regulates the NF-kappaB and type I interferon signaling pathways. Cell 141: 483–496.
Neerincx, A., K. Lautz, M. Menning, E. Kremmer, P. Zigrino, M. Hosel, H. Buning, R. Schwarzenbacher, and T.A. Kufer. 2010. A role for the human nucleotide-binding domain, leucine-rich repeat-containing family member NLRC5 in antiviral responses. Journal of Biological Chemistry 285: 26223–26232.
Neerincx, A., G.M. Rodriguez, V. Steimle, and T.A. Kufer. 2012. NLRC5 controls basal MHC class I gene expression in an MHC enhanceosome-dependent manner. Journal of Immunology 188: 4940–4950.
Kuenzel, S., A. Till, M. Winkler, R. Hasler, S. Lipinski, S. Jung, J. Grotzinger, H. Fickenscher, S. Schreiber, and P. Rosenstiel. 2010. The nucleotide-binding oligomerization domain-like receptor NLRC5 is involved in IFN-dependent antiviral immune responses. Journal of Immunology 184: 1990–2000.
Benko, S., J.G. Magalhaes, D.J. Philpott, and S.E. Girardin. 2010. NLRC5 limits the activation of inflammatory pathways. Journal of Immunology 185: 1681–1691.
Meissner, T.B., A. Li, A. Biswas, K.H. Lee, Y.J. Liu, E. Bayir, D. Iliopoulos, P.J. van den Elsen, and K.S. Kobayashi. 2010. NLR family member NLRC5 is a transcriptional regulator of MHC class I genes. Proceedings of the National Academy of Sciences of the United States of America 107: 13794–13799.
Meissner, T.B., A. Li, and K.S. Kobayashi. 2012. NLRC5: a newly discovered MHC class I transactivator (CITA). Microbes and Infection 14: 477–484.
Yao, Y., and Y. Qian. 2013. Expression regulation and function of NLRC5. Protein & Cell 4: 168–175.
You, Z., D. Xu, J. Ji, W. Guo, W. Zhu, and J. He. 2012. JAK/STAT signal pathway activation promotes progression and survival of human oesophageal squamous cell carcinoma. Clinical and Translational Oncology 14: 143–149.
Kisseleva, T., S. Bhattacharya, J. Braunstein, and C.W. Schindler. 2002. Signaling through the JAK/STAT pathway, recent advances and future challenges. Gene 285: 1–24.
Kawasaki, M., M. Fujishiro, A. Yamaguchi, K. Nozawa, H. Kaneko, Y. Takasaki, K. Takamori, H. Ogawa, and I. Sekigawa. 2011. Possible role of the JAK/STAT pathways in the regulation of T cell-interferon related genes in systemic lupus erythematosus. Lupus 20: 1231–1239.
Kang, J.W., and S.M. Lee. 2012. Melatonin inhibits type 1 interferon signaling of toll-like receptor 4 via heme oxygenase-1 induction in hepatic ischemia/reperfusion. Journal of Pineal Research 53: 67–76.
Duan, W., Y. Yang, J. Yan, S. Yu, J. Liu, J. Zhou, J. Zhang, Z. Jin, and D. Yi. 2012. The effects of curcumin post-treatment against myocardial ischemia and reperfusion by activation of the JAK2/STAT3 signaling pathway. Basic Research in Cardiology 107: 263.
Betts, B.C., O. Abdel-Wahab, S.A. Curran, E.T. St Angelo, P. Koppikar, G. Heller, R.L. Levine, and J.W. Young. 2011. Janus kinase-2 inhibition induces durable tolerance to alloantigen by human dendritic cell-stimulated T cells yet preserves immunity to recall antigen. Blood 118: 5330–5339.
Quintas-Cardama, A. 2013. The role of Janus kinase 2 (JAK2) in myeloproliferative neoplasms: therapeutic implications. Leukemia Research 37: 465–472.
Zhang, W., Q. Sun, X. Gao, Y. Jiang, R. Li, and J. Ye. 2013. Anti-inflammation of spirocyclopiperazinium salt compound LXM-10 targeting alpha7 nAChR and M4 mAChR and inhibiting JAK2/STAT3 pathway in rats. PLoS One 8: e66895.
Terrell, A.M., P.R. Crisostomo, G.M. Wairiuko, M. Wang, E.D. Morrell, and D.R. Meldrum. 2006. Jak/STAT/SOCS signaling circuits and associated cytokine-mediated inflammation and hypertrophy in the heart. Shock 26: 226–234.
Karin, M., T. Lawrence, and V. Nizet. 2006. Innate immunity gone awry: linking microbial infections to chronic inflammation and cancer. Cell 124: 823–835.
Wang, R.F., Y. Miyahara, and H.Y. Wang. 2008. Toll-like receptors and immune regulation: implications for cancer therapy. Oncogene 27: 181–189.
Hoffman, H.M., and S.D. Brydges. 2011. Genetic and molecular basis of inflammasome-mediated disease. Journal of Biological Chemistry 286: 10889–10896.
Allen, I.C. 2011. A NOD to zebrafish models of inflammatory bowel disease pathogenesis. Disease Models & Mechanisms 4: 711–712.
Rothwarf, D.M., E. Zandi, G. Natoli, and M. Karin. 1998. IKK-gamma is an essential regulatory subunit of the IkappaB kinase complex. Nature 395: 297–300.
Koppula, S., W.J. Kim, J. Jiang, D.W. Shim, N.H. Oh, T.J. Kim, T.B. Kang, and K.H. Lee. 2013. Carpesium macrocephalum attenuates lipopolysaccharide-induced inflammation in macrophages by regulating the NF-kappa B/I kappa B-alpha, Akt, and STAT signaling pathways. American Journal of Chinese Medicine 41: 927–943.
Fan GW, Zhang Y, Jiang X, Zhu Y, Wang B, Su L, Cao W, Zhang H, Gao X. 2013. Anti-inflammatory activity of baicalein in LPS-stimulated RAW264.7 macrophages via estrogen receptor and NF-kappaB-dependent pathways. Inflammation 36:1584–1591.
ACKNOWLEDGMENTS
This work was supported by grants from the key program of National Natural Science Foundation of China (no. 81273526).
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Li, L., Xu, T., Huang, C. et al. NLRC5 Mediates Cytokine Secretion in RAW264.7 Macrophages and Modulated by the JAK2/STAT3 Pathway. Inflammation 37, 835–847 (2014). https://doi.org/10.1007/s10753-013-9804-y
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DOI: https://doi.org/10.1007/s10753-013-9804-y