IADR Abstract Archives
Periopathogens Dysregulate Myeloid Cells Functions by Modulating Long Noncoding RNAs
Objectives: Long noncoding RNAs (lncRNA) regulate diverse biological processes including immunity. The impact of periopathogens on myeloid cell lncRNA repertoire remain understudied. Herein, we hypothesized that periopathogens modulate myeloid cells functions by modulating lncRNA profile
Methods: Sorted CD14+ monocytes from human blood were differentiated to M1/M2 macrophages (MΦ) or dendritic cells (DC). These cells were then challenged with periopathogens (A. actinomycetemcomitans [Aa] and P. gingivalis [Pg]; both at 100 MOI) or their LPS (100 ng/ml) for 4, 12 or 24 h and the lncRNA time kinetics was evaluated by commercially available RT-qPCR array (88 genes). Significantly up and downregulated lncRNAs were knockdown and their impact were evaluated on following functions of myeloid cells: cell migration (by imaging of wound closure assays, phagocytosis and antigen uptake/processing (imaging and flow cytometry of pHrodo conjugated E. coli and BODIPY-conjugated ovalbumin), and cell surface markers analysis (flow cytometry).
Results: Differential expression of 21 lncRNAs (19 were up, 2 were down) was observed in DCs challenged with Pg LPS. We selected SNHG11, NUTM2A-AS1, MCM3AP-AS1, JPX, and HCG11 (upregulated), and RN7SK (downregulated) to evaluate their impact on myeloid cells functions. HCG11 knockdown showed remarkable reduction on cell migration. Transfection of APCs with siRNA targeting RN7SK, but not other lncRNAs, demonstrated significantly enhanced antigen uptake/processing (ovalbumin) and phagocytosis (E. coli in APCs). Interestingly, RN7SK knockdown also exhibited upregulation of M1 Mφ markers (MHC II or CD23) and concomitant downregulation of M2 Mφ surface markers (CD163, CD206 or Dectin) compared to scrambled RNA.
Conclusions: Our results reveal that periopathogens dysregulate lncRNA repertoire of myeloid cells, which might in turn reshape innate immune functions of these cells during oral pathogenesis.
Methods: Sorted CD14+ monocytes from human blood were differentiated to M1/M2 macrophages (MΦ) or dendritic cells (DC). These cells were then challenged with periopathogens (A. actinomycetemcomitans [Aa] and P. gingivalis [Pg]; both at 100 MOI) or their LPS (100 ng/ml) for 4, 12 or 24 h and the lncRNA time kinetics was evaluated by commercially available RT-qPCR array (88 genes). Significantly up and downregulated lncRNAs were knockdown and their impact were evaluated on following functions of myeloid cells: cell migration (by imaging of wound closure assays, phagocytosis and antigen uptake/processing (imaging and flow cytometry of pHrodo conjugated E. coli and BODIPY-conjugated ovalbumin), and cell surface markers analysis (flow cytometry).
Results: Differential expression of 21 lncRNAs (19 were up, 2 were down) was observed in DCs challenged with Pg LPS. We selected SNHG11, NUTM2A-AS1, MCM3AP-AS1, JPX, and HCG11 (upregulated), and RN7SK (downregulated) to evaluate their impact on myeloid cells functions. HCG11 knockdown showed remarkable reduction on cell migration. Transfection of APCs with siRNA targeting RN7SK, but not other lncRNAs, demonstrated significantly enhanced antigen uptake/processing (ovalbumin) and phagocytosis (E. coli in APCs). Interestingly, RN7SK knockdown also exhibited upregulation of M1 Mφ markers (MHC II or CD23) and concomitant downregulation of M2 Mφ surface markers (CD163, CD206 or Dectin) compared to scrambled RNA.
Conclusions: Our results reveal that periopathogens dysregulate lncRNA repertoire of myeloid cells, which might in turn reshape innate immune functions of these cells during oral pathogenesis.