Soluble TNF receptors play an important role in regulating inflammatory events by binding to TNF. Consistent with this, knock-in mice expressing a mutated non-sheddable TNFR1 develop an immune hyperreactivity phenotype manifested by spontaneous hepatitis, enhanced susceptibility to endotoxic shock, exacerbated TNF-dependent arthritis, and experimental autoimmune encephalomyelitis (48
). A variety of mediators have been identified that can induce sTNFR1 shedding, including bacterial products, such as Staphylococcus aureus
protein A (27
). It is not known, however, whether microbial pathogens can induce sTNFR1 shedding via activation of pattern recognition receptors as a mechanism by which the innate immune system responds to infection. Here, we hypothesized that Toll-like receptor (TLR) ligands and downstream signaling pathways induce the release of either soluble 34-kDa sTNFR1 ectodomains or full-length 55-kDa TNFR1 within exosome-like vesicles to the extracellular space. We show that poly (I:C), a viral dsRNA analog, selectively induces cleavage and shedding of soluble 34-kDa sTNFR1 from human airway epithelial cells, but does not enhance the release of full-length 55-kDa TNFR1 within exosome-like vesicles. Poly (I:C)-induced sTNFR1 shedding is mediated via a TLR3, whereas ligands that activate other toll-like receptors, such as TLR4, TLR7, and NOD2, do not. Furthermore, we show that poly (I:C)-induced sTNFR1 release is mediated by at least two TLR3-TRIF-RIP1-dependent pathways. One pathway involves the Duox2-mediated generation of ROS, whereas the other pathway is via caspase-dependent apoptosis. This demonstrates that sTNFR1 shedding is part of the airway epithelial innate immune response to viral infection that can attenuate excessive TNF-mediated inflammation.
TLRs are essential for both innate and adaptive immune responses by serving as highly conserved pattern-recognition receptors that bind a variety of endogenous and exogenous stimuli (34
). TLR3 is the major receptor for double-stranded viral RNA (41
). Upon TLR3 ligation, TRIF, the critical adaptor protein for TLR3, activates downstream signaling through receptor interacting protein 1 (RIP1) (34
). Having shown that poly (I:C) induces sTNFR1 shedding via a TLR3-TRIF-dependent pathway, we investigated the relevant downstream signaling pathways. Binding of TLR3 to its ligands can activate downstream MAPK signaling pathways (34
). Moreover, ERK can activate TACE, while binding of Staphylococcus aureus
-derived protein A to EGFR leads to TACE phosphorylation in a c-Src-ERK1/2-dependent manner (27
). Here, we show that the poly (I:C)-induced phosphorylation of MEK1/2, ERK1/2 and p38 in NCI-H292 cells is not dependent upon RIP1. Furthermore, expression of a dominant-negative MEK mutant or pharmacological inhibition of either ERK or p38 did not attenuate poly (I:C)-induced sTNFR1 shedding. These data are consistent with the conclusion that poly (I:C)-induced sTNFR1 shedding is partially mediated by RIP1, but independent of MEK, ERK and p38.
RIP1, which mediates TLR3 signaling downstream of TRIF, can modulate p27 levels via activation of a phosphoinositide 3-kinase (PI3K)-AKT pathway (53
). In addition, dsRNA-mediated activation of interferon regulatory factor 3 (IRF3) and IFN-β is dependent upon PI3K activity (54
). Consistent with RIP1-mediated activation of AKT, we show that the RNAi-mediated knockdown of RIP1 expression substantially reduced poly (I:C)-induced AKT phosphorylation. However, over-expression of a dominant-negative Akt1 mutant did not affect sTNFR1 shedding. These data indicate that poly (I:C)-induced sTNFR1 release is not dependent upon AKT signaling.
Next, we considered that poly (I:C)-mediated sTNFR1 release might also be mediated via the generation of reactive oxygen species (ROS), which can be induced in response to activation of various TLRs (42
). ROS signaling has also been shown to activate TACE and thereby induce TNFR1 ectodomain cleavage and shedding (26
). Here, we show that poly (I:C) induces ROS production by NCI-H292 cells. The antioxidant, N-acetyl-L-cysteine (NAC), and the NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPI), suppressed poly (I:C)-induced TNFR1 shedding by greater than 40%, which is consistent with a role for ROS signaling pathways in poly (I:C)-induced sTNFR1 shedding. Furthermore, poly (I:C)-mediated ROS generation appeared to be downstream of RIP1, consistent with activation of a TLR3-TRIF-RIP1 signaling pathway. NADPH oxidases (Nox) are the major source for generating ROS in airway epithelial cells; the Nox family is comprised of five NADPH oxidases, as well as Duox1 and Duox2, which are highly expressed by airway epithelial cells(44
). We used RNAi to knock-down Duox2 expression, which inhibited TNFR1 shedding by 30%, whereas knockdown of Duox1 had no effect. Our findings contrast with those of previous studies, which showed that Duox1 is required for TLR ligand-induced production of IL-8 and VEGF (58
). Taken together, our data are consistent with the conclusion that Duox2-mediated ROS signaling participates in poly(I:C)-mediated sTNFR1 shedding downstream of RIP1. Interestingly, previous work showed that Duox2 mRNA and protein levels are induced in airway epithelial cells exposed to interferon-γ, poly (I:C), or rhinovirus, suggesting this oxidase functions specifically in antiviral responses (59
Activation of caspases, which are cysteine-dependent proteases, results in cleavage of downstream substrates and the induction of cellular apoptosis (61
). Synthetic dsRNA can directly trigger TLR3-dependent apoptosis in human breast cancer cells (46
), while poly (I:C) has been used as a therapeutic adjuvant for the treatment of neoplasia based upon its ability to induce apoptosis and IFN production, with resultant anti-tumor immune responses (62
). Furthermore, apoptosis-inducing agents can promote sTNFR1 shedding in vascular endothelial cells as a mechanism to limit inflammation in the setting of apoptotic cell death (63
). Here, we hypothesized that the poly (I:C) signaling pathway that induces sTNFR1 shedding in human airway epithelial cells may involve caspase activation and the initiation of apoptosis. Consistent with this concept, we detected cleavage of poly-ADP ribose polymerase (PARP), an early marker of caspase activation and apoptosis, in poly (I:C)-treated cells. Furthermore, z-VAD-fmk, a broad-spectrum caspase inhibitor, significantly reduced sTNFR1 shedding by 27%, which is consistent with a role for caspase activation in poly (I:C)-induced sTNFR1 shedding. We also assessed the role of RIP1 in poly (I:C)-induced caspase-mediated sTNFR1 shedding. RIP1 signaling modulates apoptosis, necrosis, and autophagy and thereby regulates cell fate (55
). We show that poly (I:C)-induced PARP cleavage is RIP1-dependent, which is consistent with the conclusion that caspase activation is downstream of RIP1. This shows that poly (I:C)-induced sTNFR1 shedding is partially mediated by RIP1-dependent induction of apoptosis.
Lastly, we assessed the role of TACE, which functions as a TNFR1 sheddase, in poly (I:C)-mediated sTNFR1 release (27
). The RNAi-mediated knockdown of TACE expression reduced poly (I:C)-induced sTNFR1 release by 50%, whereas TGF-α release was almost totally suppressed, which suggests that additional TNFR1 sheddases may participate in this process. ROS have been reported to activate TACE via a pathway involving ATP-induced TGF-α shedding and EGFR activation (47
). Therefore, we investigated the roles of TGF-α and EGFR in poly (I:C)-induced sTNFR1 shedding. Although poly (I:C) induced the release of TGF-α from NCI-H292 cells, experiments utilizing a neutralizing anti-TGF-α antibody showed that TGF-α does not mediate poly (I:C)-induced sTNFR1 shedding. Similarly, poly (I:C) did not induce EGFR phosphorylation. These data show that poly (I:C)-induced sTNFR1 shedding occurs via TACE-dependent pathways that do not require TGF-α release or EGFR activation.
In summary, we have shown that the double-stranded viral RNA homologue, poly (I:C), selectively induces shedding of soluble 34-kDa sTNFR1 ectodomains from the NCI-H292 human airway epithelial cell line via a TLR3-TRIF-RIP1-dependent signaling pathway. Furthermore, poly (I:C)-induced sTNFR1 shedding involves the participation of at least two downstream pathways, one mediated by Duox2 and ROS generation and the other via caspase activation and the induction of apoptosis. However, additional pathways downstream of TLR3 and TRIF are likely involved in this process as RIP1-dependent ROS signaling and caspase activation only partially account for TLR3-TRIF-mediated sTNFR1 shedding in response to poly (I:C). In particular, additional TLR3 signaling proteins that do not involve RIP1 and might be involved include tumor necrosis factor receptor-associated factors 3 and 6 (TRAF3, TRAF6), interleukin-1 receptor-associated kinase 2 (IRAK2) and IκB kinases (52
). Furthermore, our results suggest that TLR3-mediated sTNFR1 shedding by airway epithelial cells represents a physiological response to infections caused by double-stranded RNA viruses. Thus, we have identified a mechanism by which signaling through the TLR3 pattern recognition receptor regulates the innate immune response to viral-induced airway inflammation through the shedding of 34-kDa soluble sTNFR1, which is then available to bind and potentially attenuate excessive TNF bioactivity.