Interaction of TLRs and miRNAs. (A) Real-time RT-PCR was used to determine the level of TLR mRNA expressed in highly purified human NK cells. The mRNA level of TLR5 was found to be lowest and was normalized to 1. The mRNA level of other TLRs was presented as relative to that of TLR5. Data are shown as the average of 3 donors. (B) Purified human NK cells were preincubated for 1.5 hours with a nonspecific IgG or anti-TLR1, anti-TLR3, or anti-TLR6–blocking antibody (α). Cells were then stimulated with miR-122 as described in Figure 1A in the presence of the blocking antibody, and supernatants were harvested to measure IFN-γ protein via ELISA. The concentration of IFN-γ in the purified NK cells incubated with IgG and miR-122 was arbitrarily set at 1. Data were averaged from 3 donors. *P < .05 and error bars represent standard deviation (SD). (C) NK-92 cells were infected with pSUPER-TLR1-GFP retroviruses, and stably transfected cells were sorted based on GFP expression. Both the vector-transduced cells (pSUPER) and the TLR1 knockdown cells (pSUPER-TLR1, confirmed by immunoblotting; upper panel) were stimulated with miR-122 or miR-15b as described in Figure 1A, and cell-free supernatants were collected to assess IFN-γ secretion via ELISA (lower panel). **P < .01 and error bars represent SD. (D) Purified human NK cells were stimulated with miR-122 or miR-15b as described in Figure 1A and subsequently subjected to immunoblotting using p65 and phospho-p65 (p-p65) Abs. β-actin immunoblotting was included to demonstrate equal loading of total protein. Data shown represent 2 of 4 donors with similar results. Numbers beneath each lane represent quantification of p-p65 by densitometry, normalized by p65. (E) The experiment was performed as in D except that anti-TLR1 blocking mAb or its control IgG was included in the culture in the presence of miR-122. Data shown represent 1 of 3 donors with similar results. Numbers beneath each lane represent quantification of p-p65 by densitometry, normalized by p65.