PMID- 28706958
OWN - NLM
STAT- PubMed-not-MEDLINE
LR  - 20201001
IS  - 2326-0254 (Print)
IS  - 2326-0254 (Electronic)
IS  - 2326-0254 (Linking)
VI  - 4
DP  - 2017
TI  - Toll-like receptor 2-mediated NF-kappa B pathway activation in ocular surface 
      epithelial cells.
PG  - 17
LID - 10.1186/s40662-017-0082-x [doi]
LID - 17
AB  - BACKGROUND: Gram-positive bacteria stimulate Toll-like receptor (TLR) 2 and then 
      activate the pro-inflammatory nuclear factor-kappa B (NF-kappaB) pathway. As the 
      human ocular surface is heavily colonised by gram-positive cocci bacteria, a 
      balance of activation/repression of NF-kappaB target genes is essential to avoid 
      uncontrolled infection or autoimmune-related inflammation. It is advantageous to 
      test NF-kappaB targeting molecules in an ocular surface culture system that allows 
      assessment of temporal NF-kappaB activation in a longitudinal fashion without 
      destruction of cells. Such initial testing under standardised conditions should 
      reduce the number of molecules that progress to further evaluation in animal 
      models. This study aims to establish an in-vitro cell culture system to assess 
      NF-kappaB activation in the context of ocular surface cells. METHODS: NF-kappaB activity 
      was evaluated through a secretory alkaline phosphatase reporter assay (SEAP). 
      Immunoblots and immunofluorescence were used to examine IkappaBalpha phosphorylation and 
      p65/p50 nuclear localization. Monocyte chemoattractant protein-1 (MCP-1) 
      transcripts were evaluated by real time PCR and protein levels were measured by 
      ELISA. RESULTS: NF-kappaB activity in HCE-T cells treated with TLR2 activator 
      Pam3CSK4 was higher than control cells at both 6 and 24 h. Pam3CSK4-stimulated 
      NF-kappaB activation was inhibited by IkappaK inhibitors, Wedelolactone and BMS-345541. 
      In Pam3CSK4 treated cells, active NF-kappaB subunits p50 and p65 increased in cell 
      nuclear fractions as early as 1.5 h. Although the level of total IkappaB-alpha remained 
      constant, phospho-IkappaB-alpha increased with treatment over time. In the culture media 
      of Pam3CSK4-stimulated cells, MCP-1 protein level was increased, which was 
      suppressed in the presence of IkappaK inhibitors. CONCLUSION: NF-kappaB pathway can be 
      activated by the TLR2 ligand and inhibited by IkappaK inhibitors in the ocular 
      surface cell culture system. This cell culture system may be used to evaluate 
      TLR-related innate defences in ocular surface diseases.
FAU - Hou, Aihua
AU  - Hou A
AUID- ORCID: 0000-0003-0463-3385
AD  - Ocular Surface Research Group, Singapore Eye Research Institute, Singapore, 
      Singapore. ISNI: 0000 0001 0706 4670. GRID: grid.272555.2
FAU - Tin, Min Qi
AU  - Tin MQ
AD  - Ocular Surface Research Group, Singapore Eye Research Institute, Singapore, 
      Singapore. ISNI: 0000 0001 0706 4670. GRID: grid.272555.2
FAU - Tong, Louis
AU  - Tong L
AD  - Ocular Surface Research Group, Singapore Eye Research Institute, Singapore, 
      Singapore. ISNI: 0000 0001 0706 4670. GRID: grid.272555.2
AD  - Duke-NUS Graduate Medical School, Singapore, Singapore. ISNI: 0000 0004 0385 
      0924. GRID: grid.428397.3
AD  - Singapore National Eye Center, Singapore, Singapore. ISNI: 0000 0000 9960 1711. 
      GRID: grid.419272.b
AD  - Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 
      Singapore. ISNI: 0000 0001 2180 6431. GRID: grid.4280.e
LA  - eng
PT  - Journal Article
DEP - 20170711
PL  - England
TA  - Eye Vis (Lond)
JT  - Eye and vision (London, England)
JID - 101664982
PMC - PMC5506675
OTO - NOTNLM
OT  - Activation
OT  - NF-kappaB pathway
OT  - Ocular surface cells
OT  - TLR2
COIS- ETHICS APPROVAL AND CONSENT TO PARTICIPATE: Not applicable. CONSENT FOR 
      PUBLICATION: Not applicable. COMPETING INTERESTS: The authors declare that they 
      have no competing interests.
EDAT- 2017/07/15 06:00
MHDA- 2017/07/15 06:01
PMCR- 2017/07/11
CRDT- 2017/07/15 06:00
PHST- 2017/04/06 00:00 [received]
PHST- 2017/06/27 00:00 [accepted]
PHST- 2017/07/15 06:00 [entrez]
PHST- 2017/07/15 06:00 [pubmed]
PHST- 2017/07/15 06:01 [medline]
PHST- 2017/07/11 00:00 [pmc-release]
AID - 82 [pii]
AID - 10.1186/s40662-017-0082-x [doi]
PST - epublish
SO  - Eye Vis (Lond). 2017 Jul 11;4:17. doi: 10.1186/s40662-017-0082-x. eCollection 
      2017.