Method: Macrophages were differentiated from the monocytic cell line THP-1 and challenged for 4 hours with 100ng/µl purified LPS from Aggregatibacter actinomycetemcomitans, Y4 serotype B, Porphyromonas gingivalis, strain 33277 (Pg), or Pg grown in cigarette smoke extract at a concentration of 4000 ng nicotine equivalents/ml. miRNA profiles were interrogated using Nanostring technology and expression confirmed by RT-PCR. mRNA targets for differentially expressed miRNAs were identified using PUBMED and miRWalk. Candidate mRNAs were selected if identified as miRNA targets in at least 5/8 databases and linked to immunity/inflammation by GO Biological Process. mRNA/miRNA interactions were confirmed using a dual-luciferase assay.
Result: Our results show that of the 653 human miRNA probes approximately 200 (~30%) miRNAs were responsive to LPS. We identified convergent/divergent expression profiles including a “core” response of 11 upregulated and 20 down-regulated miRNAs common to the three LPS’s as well as LPS-specific miRNA expression. Our dual-luciferase assays confirmed targeting of thrombospondin1 (THBS1), a potent activator of TGF-β and macrophage chemoattractant by LPS-responsive let-7f.
Conclusion: Our data indicates that LPS from periodontal pathogens induces convergent/divergent miRNA expression in human macrophages and identifies key targets linked to inflammation and immunity. We also confirm let-7f targeting of THBS1. These pathogen-effector responsive miRNAs will be useful in our understanding of host-pathogen interactions that may be exploited therapeutically.