U1 RNA induces innate immunity signaling

Abstract

Objective: The U1-70-kd RNP is a prominent target of autoimmunity in connective tissue diseases. In this study, we explored whether its endogenous ligand, U1 RNA, mediates a proimmune signal and may be immunogenic.

Methods: We assayed the proliferation of control and MyD88-knockout splenocytes in response to in vitro-synthesized U1 RNA, and measured interleukin-6 (IL-6) and IL-8 secretion induced by U1 RNA in a human cell line competent for signaling through Toll-like receptor 3 (TLR-3) and TLR-5.

Results: Treatment with U1 RNA or with poly(I-C), a known agonist of TLR-3, induced approximately twice as much control splenocyte proliferation as did treatment with RNase-digested U1 RNA. Proliferation in response to either poly(I-C) or U1 RNA by MyD88-knockout splenocytes was similarly attenuated. Similar to poly(I-C), U1 RNA induced significant secretion of both IL-6 and IL-8 from a TLR-3-expressing human cell line; in contrast, the TLR-5 agonist flagellin induced predominantly IL-8 secretion. Pretreatment of U1 RNA with RNase abolished IL-6 and IL-8 secretion.

Conclusion: U1 RNA is capable of inducing manifestations consistent with TLR-3 activation. The ability of U1 RNA (which has a substantial double-stranded secondary structure) to activate TLR-3 may contribute to the immunogenicity of the U1-70-kd autoantigen. Stimulation of innate immunity by native RNA molecules with a double-stranded secondary structure may help explain the high prevalence of autoimmunity to RNA binding proteins.

Figure 1

Response of control and MyD88-knockout murine splenocytes to U1 RNA. Splenocytes from MyD88-intact (control) and MyD88-knockout B6 mice were cultured in the presence of 10 μg/ml polyinosine–polycytidylic acid (poly[I-C]), U1 RNA, or RNase-treated U1 RNA. Cells were pulsed with tritiated thymidine at 16 hours and harvested at 24 hours. Stimulation indices were calculated based on 100% thymidine uptake by control cells exposed to RNase-treated U1 RNA.

Figure 2

Cytokine secretion by endometrial cell lines in response to U1 RNA. A and B, Cells from the Toll-like receptor 3 (TLR-3)–positive human endometrial cell line RL-95 and the TLR-3–negative human endometrial cell line Ishikawa were incubated for 24 hours with doses of U1 RNA or the TLR-3 agonist poly(I-C), both with and without pretreatment of the RNAs with RNase A, as well as with doses of poly(dl-dC)—a double-stranded DNA analog of poly(I-C), or with sham-transcribed RNA from a nontransfected SP64 vector (sham). Supernatants were assayed for interleukin-6 (IL-6) (A) and IL-8 (B). C, RL-95 cells were incubated with the indicated doses of U1 RNA, poly(I-C), or flagellin, and assayed for IL-6 and IL-8 secretion into the supernatant at 24 hours.

Figure 2

Cytokine secretion by endometrial cell lines in response to U1 RNA. A and B, Cells from the Toll-like receptor 3 (TLR-3)–positive human endometrial cell line RL-95 and the TLR-3–negative human endometrial cell line Ishikawa were incubated for 24 hours with doses of U1 RNA or the TLR-3 agonist poly(I-C), both with and without pretreatment of the RNAs with RNase A, as well as with doses of poly(dl-dC)—a double-stranded DNA analog of poly(I-C), or with sham-transcribed RNA from a nontransfected SP64 vector (sham). Supernatants were assayed for interleukin-6 (IL-6) (A) and IL-8 (B). C, RL-95 cells were incubated with the indicated doses of U1 RNA, poly(I-C), or flagellin, and assayed for IL-6 and IL-8 secretion into the supernatant at 24 hours.

Figure 3

RNP complex induction of cytokine secretion in TLR-3–expressing but not in TLR-3–negative cells. RL-95 and Ishikawa cells were incubated with the indicated stimuli, and secretion of IL-6 (A) and IL-8 (B) was measured in supernatants, as described in Figure 2. In addition to exposure to poly(I-C) and U1 RNA, cells were also exposed to U1 RNA that was allowed to complex with an excess of the U1–70-kd small nuclear RNP protein, with and without subsequent exposure to RNase A, as described in Figure 2. Cells were also exposed to a standard crude extractable nuclear antigen (ENA) preparation, and to an equal quantity of phosphate buffered saline (PBS) as a negative control. See Figure 2 for other definitions.

Figure 3

RNP complex induction of cytokine secretion in TLR-3–expressing but not in TLR-3–negative cells. RL-95 and Ishikawa cells were incubated with the indicated stimuli, and secretion of IL-6 (A) and IL-8 (B) was measured in supernatants, as described in Figure 2. In addition to exposure to poly(I-C) and U1 RNA, cells were also exposed to U1 RNA that was allowed to complex with an excess of the U1–70-kd small nuclear RNP protein, with and without subsequent exposure to RNase A, as described in Figure 2. Cells were also exposed to a standard crude extractable nuclear antigen (ENA) preparation, and to an equal quantity of phosphate buffered saline (PBS) as a negative control. See Figure 2 for other definitions.