title: HSP60 plays a regulatory role in IL-1β-induced microglial inflammation via TLR4-p38 MAPK axis.
abstract: BACKGROUND
  IL-1β, also known as "the master regulator of inflammation", is a potent pro-inflammatory cytokine secreted by activated microglia in response to pathogenic invasions or neurodegeneration. It initiates a vicious cycle of inflammation and orchestrates various molecular mechanisms involved in neuroinflammation. The role of IL-1β has been extensively studied in neurodegenerative disorders; however, molecular mechanisms underlying inflammation induced by IL-1β are still poorly understood. The objective of our study is the comprehensive identification of molecular circuitry involved in IL-1β-induced inflammation in microglia through protein profiling.
  
  
  METHODS
  To achieve our aim, we performed the proteomic analysis of N9 microglial cells with and without IL-1β treatment at different time points. Expression of HSP60 in response to IL-1β administration was checked by quantitative real-time PCR, immunoblotting, and immunofluorescence. Interaction of HSP60 with TLR4 was determined by co-immunoprecipitation. Inhibition of TLR4 was done using TLR4 inhibitor to reveal its effect on IL-1β-induced inflammation. Further, effect of HSP60 knockdown and overexpression were assessed on the inflammation in microglia. Specific MAPK inhibitors were used to reveal the downstream MAPK exclusively involved in HSP60-induced inflammation in microglia.
  
  
  RESULTS
  Total 21 proteins were found to be differentially expressed in response to IL-1β treatment in N9 microglial cells. In silico analysis of these proteins revealed unfolded protein response as one of the most significant molecular functions, and HSP60 turned out to be a key hub molecule. IL-1β induced the expression as well as secretion of HSP60 in extracellular milieu during inflammation of N9 cells. Secreted HSP60 binds to TLR4 and inhibition of TLR4 suppressed IL-1β-induced inflammation to a significant extent. Our knockdown and overexpression studies demonstrated that HSP60 increases the phosphorylation of ERK, JNK, and p38 MAPKs in N9 cells during inflammation. Specific inhibition of p38 by inhibitors suppressed HSP60-induced inflammation, thus pointed towards the major role of p38 MAPK rather than ERK1/2 and JNK in HSP60-induced inflammation. Furthermore, silencing of upstream modulator of p38, i.e., MEK3/6 also reduced HSP60-induced inflammation.
  
  
  CONCLUSIONS
  IL-1β induces expression of HSP60 in N9 microglial cells that further augments inflammation via TLR4-p38 MAPK axis.
authors.affiliation: National Brain Research Centre, Manesar, Haryana, 122051, India.
authors.forename: Shalini
authors.initials: S
authors.lastname: Swaroop
authors.affiliation: National Brain Research Centre, Manesar, Haryana, 122051, India.
authors.forename: Nabonita
authors.initials: N
authors.lastname: Sengupta
authors.affiliation: Clinical Proteomics, Institute of Life Sciences, Bhubaneswar, Odhisa, 751023, India.
authors.forename: Amol Ratnakar
authors.initials: AR
authors.lastname: Suryawanshi
authors.affiliation: National Brain Research Centre, Manesar, Haryana, 122051, India. yogita@nbrc.ac.in.
authors.forename: Yogita K
authors.initials: YK
authors.lastname: Adlakha
authors.affiliation: National Brain Research Centre, Manesar, Haryana, 122051, India. anirban@nbrc.ac.in.
authors.forename: Anirban
authors.initials: A
authors.lastname: Basu
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D018834:Chaperonin 60 / Q000378:metabolism*; D016206:Databases, Bibliographic / Q000706:statistics & numerical data; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D007249:Inflammation / Q000139:chemically induced / Q000378:metabolism / Q000473:pathology; D053583:Interleukin-1beta / Q000633:toxicity*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D017628:Microglia / Q000187:drug effects / Q000473:pathology*; D024101:Mitochondrial Proteins / Q000378:metabolism*; D048292:Mitogen-Activated Protein Kinase 13 / Q000378:metabolism*; D040901:Proteomics; D034741:RNA, Small Interfering / Q000235:genetics / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013997:Time Factors; D051197:Toll-Like Receptor 4 / Q000378:metabolism*
chemical_list: D018834:Chaperonin 60; D004791:Enzyme Inhibitors; C557066:Hspd1 protein, mouse; D053583:Interleukin-1beta; D024101:Mitochondrial Proteins; D034741:RNA, Small Interfering; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D048292:Mitogen-Activated Protein Kinase 13
references.citation: J Immunol. 2000 Jan 15;164(2):558-61
references.pmid: 10623794
references.citation: Clin Exp Immunol. 2005 Aug;141(2):307-14
references.pmid: 15996195
references.citation: Curr Opin Rheumatol. 2004 May;16(3):231-7
references.pmid: 15103250
references.citation: Methods Mol Biol. 2013;1041:71-82
references.pmid: 23813371
references.citation: J Immunol. 1998 Jul 1;161(1):467-73
references.pmid: 9647257
references.citation: J Neurosci Res. 2006 Oct;84(5):1037-46
references.pmid: 16881054
references.citation: Cancer Biol Ther. 2008 Jun;7(6):801-9
references.pmid: 18497565
references.citation: Exp Mol Med. 2006 Aug 31;38(4):333-47
references.pmid: 16953112
references.citation: Glia. 2002 Oct;40(1):109-20
references.pmid: 12237848
references.citation: J Inflamm (Lond). 2010 Jan 08;7:1
references.pmid: 20148136
references.citation: PLoS One. 2014;9(3):e90211
references.pmid: 24599148
references.citation: J Biol Chem. 2007 Oct 26;282(43):31289-301
references.pmid: 17823127
references.citation: J Neuroimmunol. 2006 Nov;180(1-2):71-87
references.pmid: 16996144
references.citation: J Am Soc Nephrol. 2005 Feb;16(2):383-91
references.pmid: 15601747
references.citation: Nucleic Acids Res. 2015 Jan;43(Database issue):D447-52
references.pmid: 25352553
references.citation: Neurosci Lett. 1999 Aug 20;271(2):126-8
references.pmid: 10477118
references.citation: Trends Neurosci. 1999 Mar;22(3):97-9
references.pmid: 10199631
references.citation: J Biol Chem. 2002 Oct 18;277(42):39858-66
references.pmid: 12176997
references.citation: J Neurosci. 2008 Mar 5;28(10):2320-31
references.pmid: 18322079
references.citation: Mol Metab. 2013 Oct 05;2(4):356-63
references.pmid: 24327952
references.citation: J Biol Chem. 2001 Aug 17;276(33):31332-9
references.pmid: 11402040
references.citation: Cancer Lett. 2009 Jul 8;279(2):230-7
references.pmid: 19264393
references.citation: Cell Microbiol. 2006 Sep;8(9):1467-74
references.pmid: 16922865
references.citation: Am J Physiol Lung Cell Mol Physiol. 2005 Feb;288(2):L227-37
references.pmid: 15489374
references.citation: J Clin Immunol. 2014 Jul;34 Suppl 1:S64-9
references.pmid: 24711006
references.citation: Life Sci. 2015 Feb 1;122:1-7
references.pmid: 25498891
references.citation: J Med Microbiol. 2007 Feb;56(Pt 2):154-64
references.pmid: 17244794
references.citation: J Biol Chem. 2004 Jan 2;279(1):245-50
references.pmid: 14573621
references.citation: Eur J Histochem. 2013;57(2):e20
references.pmid: 23807299
references.citation: Neuroscience. 2015 Aug 6;300:141-54
references.pmid: 25981208
references.citation: Neuron. 2002 Aug 1;35(3):419-32
references.pmid: 12165466
references.citation: Nucleic Acids Res. 2001 May 1;29(9):e45
references.pmid: 11328886
references.citation: Trends Neurosci. 2000 Dec;23(12):618-25
references.pmid: 11137152
references.citation: Genome Biol. 2007;8(1):R3
references.pmid: 17204154
references.citation: Brain Res. 1992 Aug 7;587(2):250-6
references.pmid: 1381982
references.citation: EMBO J. 1991 Oct;10(10):2821-32
references.pmid: 1833184
references.citation: J Neuroinflammation. 2010;7:68
references.pmid: 20946687
references.citation: Science. 2013 Jan 11;339(6116):156-61
references.pmid: 23307732
references.citation: J Neuroimmunol. 1999 Jan 1;93(1-2):45-52
references.pmid: 10378868
references.citation: Glia. 2002 Nov;40(2):218-31
references.pmid: 12379909
references.citation: J Neuroinflammation. 2015;12:31
references.pmid: 25879545
references.citation: Eur J Immunol. 2004 Jul;34(7):2041-51
references.pmid: 15214052
references.citation: J Neurosci. 2002 Jul 15;22(14):6071-82
references.pmid: 12122068
references.citation: J Neuroinflammation. 2012;9:57
references.pmid: 22429472
references.citation: Exp Neurol. 2007 Nov;208(1):1-25
references.pmid: 17720159
references.citation: Mol Neurodegener. 2015;10:5
references.pmid: 25887709
references.citation: Ann Neurosci. 2010 Oct;17(4):191-9
references.pmid: 25205905
references.citation: J Neuroimmunol. 2000 Jul 1;106(1-2):14-22
references.pmid: 10814778
references.citation: J Biol Chem. 2002 Nov 22;277(47):45662-9
references.pmid: 12235142
references.citation: Glia. 2010 Jan 1;58(1):93-102
references.pmid: 19533603
references.citation: Drugs Today (Barc). 2009 Nov;45 Suppl B:159-64
references.pmid: 20011709
references.citation: Neurochem Res. 2013 Oct;38(10):2180-9
references.pmid: 23943523
references.citation: J Cell Physiol. 2005 Feb;202(2):464-73
references.pmid: 15389584
references.citation: Nat Rev Neurol. 2010 Apr;6(4):193-201
references.pmid: 20234358
references.citation: Trends Neurosci. 1996 Aug;19(8):312-8
references.pmid: 8843599
references.citation: Am J Physiol Heart Circ Physiol. 2001 May;280(5):H1970-81
references.pmid: 11299196
references.citation: Neurosci Lett. 2000 Mar 10;281(2-3):83-6
references.pmid: 10704748
references.citation: Biochemistry. 1997 Jun 24;36(25):7776-85
references.pmid: 9201920
references.citation: Nat Immunol. 2008 Aug;9(8):857-65
references.pmid: 18604209
references.citation: J Leukoc Biol. 2010 Sep;88(3):445-62
references.pmid: 20445014
references.citation: Cell Immunol. 2003 Apr;222(2):97-104
references.pmid: 12826079
references.citation: Nucleic Acids Res. 2012 Jul;40(Web Server issue):W478-83
references.pmid: 22573175
references.citation: Endocrinology. 2006 Sep;147(9):4445-53
references.pmid: 16777969
references.citation: J Immunol. 2002 Feb 15;168(4):1984-91
references.pmid: 11823535
references.citation: Genes Dev. 2004 Sep 15;18(18):2195-224
references.pmid: 15371334
references.citation: Exp Cell Res. 2004 Jan 1;292(1):231-40
references.pmid: 14720522
references.citation: J Biol Chem. 1998 May 22;273(21):12901-8
references.pmid: 9582321
references.citation: Z Naturforsch C. 2011 Jan-Feb;66(1-2):83-6
references.pmid: 21476440