The present study showed that the N-terminal, putatively cytoplasmic portion of Unc93B1 represses TLR7 responses and enhances TLR9 responses in BM-DCs. This conclusion is based on the following results. First, Unc93B1 N-terminally truncated or D34A mutant enables TLR7 responses in Ba/F3 cells (). Second, Unc93B1 D34A mutant rendered Unc93B1-deficient BM-DCs hyperresponsive to TLR7 ligand but hyporesponsive to TLR9 ligand (). LC-MS/MS analyses revealed the difference between TLR9 and 7. D34A mutation enhanced Unc93B1 association with TLR7, whereas that with TLR9 was suggested to be down-regulated by D34A mutation (). Further, D34A mutation enhanced TLR7 trafficking but suppressed TLR9 trafficking to endolysosomes (). The present study demonstrated that Unc93B1 actively biases TLR-dependent nucleic acid sensing against RNA-sensing. Polarization of DC responses against TLR7-mediated responses might be understood as a mechanism avoiding hazardous autoimmune reaction, because previous reports showed that TLR7 is pathogenic in a variety of autoimmune diseases (Fig. S6
LC-MS/MS analyses clearly revealed that Unc93B1 association with TLR7/8/13 is up-regulated by D34A mutation (, Table S1, and Table S2). In contrast, Unc93B1 association with TLR3 or 9 was hard to detect, particularly in SILAC ( and Table S2). Brinkmann et al. clearly demonstrated Unc93B1 association with endogenous TLR3/7/9/13 in a macrophage cell line, RAW264.7, by using a large number of cells (4 × 109
). The present study, on the other hand, focused on BM-DCs and studied Unc93B1 interactions with endogenous TLRs in BM-DCs. In this regard, differences in TLR mRNA expression between RAW264.7 cells and BM-DCs are noteworthy in that RAW264.7 cells express TLR3/7 much higher than BM-DCs without expressing TLR8 (Fig. S4 B). Along this line, Brinkmann et al. detected Unc93B1 association only with TLR7/9 in a B cell line, A20 (10
). Unc93B1 association with TLRs is likely to change with cell types, and it was important to study Unc93B1 association with TLRs in BM-DCs in the present study. Unfortunately, we were unable to obtain as many BM-DCs as RAW264.7 cells used by the previous study because of the lack of an in vitro DC line. However, semiquantitative analyses with LC-MS/MS suggested that D34A mutation weakens Unc93B1 association with TLR9 ( and Table S1). This finding is consistent with the results that D34A mutation down-regulated TLR9 trafficking and TLR9 responses in BM-DCs ( and ). Down-regulation of TLR9 responses and trafficking is not explained by up-regulation of Unc93B1 association with TLR7/8/13, suggesting that strengthened Unc93B1 association with TLR7/8/13, in turn, down-regulates that with TLR9. Although a ligand for TLR13 has not been identified, the present study suggests the possibility that TLR13 responds to microbial RNA. RNA-sensing TLRs might compete with DNA-sensing TLR9 for association with Unc93B1 in the ER (Fig. S6).
The present study looked for a difference between TLR7-responsive RAW264.7 and TLR7-unresponsive Ba/F3 cells, and found that the N-terminal portion of Unc93B1 represses TLR7 responses. These results raise a question of how negative regulation by the N-terminal region of Unc93B1 is controlled in TLR7-responsive cells like RAW264.7 cells. It is possible that Unc93B1 mRNA in TLR7-responsive cells is under the control of alternative splicing or alternative transcription start, leading to the lack of the 5′ portion encoding the N-terminal region. This is, however, unlikely, because PCR amplification of the 5′ region of Unc93B1 mRNA failed to detect such alternative Unc93B1 mRNA isoforms in RAW264.7 and Ba/F3 cells (Fig. S5). Another possibility is that the N-terminal region of Unc93B1 may be proteolytically cleaved off in TLR7-responsive cells, as was TLR9 (26
). Unc93B1-GFP expressed in RAW264.7 cells, however, did not appear to be truncated when compared with that in Ba/F3 cells (Fig. S1 B, compare left and right lanes).
The N-terminal region of Unc93B1 is left uncleaved in TLR7-responsive cells, and is likely to be inactivated by an as yet unknown mechanism. In this regard, it is noteworthy that the DEL (34-36) sequence required for repressing TLR7 response agrees with the motif (D/E-X-Φ) that is known to interact with the class III PDZ (PDS95/dlg/ZO-1) domain (33
). Requirement for 34D and 36L but not 35E is consistent with the lack of any restriction (X) in the second aa in the D/E-X-Φ motif (; ; and Fig. S2). The class III PDZ domain is not found in TLRs, suggesting that DEL (34-36) is required for interaction with a non-TLR, class III PDZ domain–containing molecule, which may have a role in controlling the activity of the N-terminal region of Unc93B1.
Although not experimentally proven yet, the lack of an N-terminal hydrophobic signal sequence suggests that the N-terminal region of Unc93B1 faces the cytosol. The N terminus of Unc93B1 may have a role not only in differential association of Unc93B1 with TLR7 and 9 but also in modulating signaling pathways downstream of TLR7 or 9. In this regard, it is of note that TLR9-dependent RANTES production was more resistant to D34A mutation than production of IL-12 or IL-6 (). It will be interesting to see, in future studies, the effect of D34A mutation in signaling pathways downstream of TLR7 and 9.
Immune cells such as DCs or macrophages express multiple TLRs, which are concomitantly activated in response to pathogens, because single microbes or viruses express a variety of TLR ligands. Given that multiple TLRs simultaneously respond to pathogens, their distribution and activation need to be orchestrated for optimal immune responses. Indeed, a synergistic relationship between TLR4/MD-2 and TLR7/9 has been recently reported in the triggering of IL-12 and other Th1-promoting cytokines by DCs (34
). TLR3 and TLR7/9 also show synergistic responses (36
). Dual recognition of Mycobacterium tuberculosis
by TLR2 and 9 is required for efficient responses (39
). HSVs were reported to be recognized by both TLR2 and 9 (40
). We have shown in this study that multiple TLRs in DCs are connected not only by these additive or synergistic links but also by an inversely regulated link.
Simultaneous triggering of TLR7 and 9 may be thought to be a less likely combination in viral infection, as viruses are classified into either DNA or RNA viruses. The DNA genomes from viruses like HSV-1, HSV-2, and mouse CMV are rich in CpG motifs, which activates TLR9 (40
), whereas TLR7 recognizes single-stranded RNA from RNA viruses like influenza and vesicular stomatitis virus (41
). Concurrent infection with a DNA virus and an RNA virus is a rare event. However, a DNA virus like herpesvirus has been recently suggested to stimulate both TLR7 and 9 (42
). DNA and RNA derived from bacterial pathogens are reported to stimulate TLR3, 7, and 9 (43
), as well as cell-surface TLR2 and TLR4/MD-2. Finally, self-derived DNA and RNA derived from dead cells are also able to simultaneously stimulate TLR7 and 9 expressed in B cells or DCs during inflammation. Concurrent activation of TLR7 and 9 is likely to occur in infectious and noninfectious diseases.
Type I IFNs are tightly regulated cytokines, and overexpression of type I IFN can be detrimental to the host. Highly elevated levels of type I IFN have been implicated as etiologic for systemic lupus erythematosus (44
). Increased serum type I IFN has been shown to correlate directly with disease severely in human systemic lupus erythematosus (45
). TLR7 and 9 are known to be strong promoters of type I IFN secretion from DCs. If TLR7 and 9 are both activated without any limit, type I IFN is likely to be excessively produced, predisposing to autoimmune diseases. DCs may inversely link TLR7 with TLR9 to keep the production of type I IFN under the control and to avoid excessive production of type I IFN.
Previous reports have revealed pathogenic roles for TLR7 in lupus nephritis. Overexpression of TLR7 in the Yaa or transgenic mice predisposes to lupus nephritis (12
), whereas the lack of the TLR7 gene ameliorates disease progression in lupus-prone mice (14
). DCs need to have a mechanism limiting TLR7 activation. Overexpression of TLR9 inhibits TLR7 responses (22
). Another mechanism has been revealed in this study that TLR7 responsiveness was down-regulated by the N-terminal cytoplasmic portion of Unc93B1. Given the reciprocal link between TLR7 and 9, Unc93B1 balances TLR7 and 9 to warrant sufficient TLR7 responsiveness without detrimental autoimmune responses (Fig. S6).
TLR7/9 agonists and antagonists are being developed for therapeutic intervention in infectious diseases, cancer, allergic diseases, and autoimmune diseases (46
). The present study showed that the effect of these agonists/antagonists has to be carefully evaluated, because specific agonists/antagonists for TLR7 or 9 may have an indirect effect on TLR9 or 7, respectively. Such indirect effects may cause unexpected action on diseases. Given that TLR7 hyperresponsiveness and TLR9 hyporesponsiveness both predispose to autoimmune diseases (12
), the N-terminal portion of Unc93B1 may be a novel target for a therapeutic intervention in autoimmune diseases that modulates the balance between TLR7 and 9 in DCs.