Here we report the identification of a novel antiviral signaling pathway in which TLR2 activation leads to production of type I IFN. Prior to this work, TLR2-dependent type I IFN production had not been reported, and, indeed, in most cell types TLR2 does not induce this antiviral response. We demonstrate that inflammatory monocytes are uniquely capable of responding to viral TLR2 ligands by producing type I IFN, and our work suggests that these cells represent another specialized antiviral cell type with functional and conceptual parallels to pDCs. In addition, these data solidify the interpretation that viral recognition by TLR2 is a host strategy, as opposed to manipulation by viruses, and argue that certain viral proteins are sufficiently constrained to serve as targets for innate immune receptors. Overall, this work has important implications for our understanding of how the innate immune system recognizes viruses.
Until recently, the specific and differing roles played by monocytic subpopulations during immune responses have not been well appreciated. In the last few years, though, several studies have identified IMs as a largely bone marrow resident cell type that is rapidly recruited to sites of infection in a CCR2-dependent manner29-31
. These cells have been named “inflammatory monocytes” to distinguish them from Ly6C-
monocytes, which are thought to play a more important role in maintaining tissue homeostasis29
. IMs can differentiate into a number of different DC subsets at sites of inflammation, including T
NF- and i
nducible nitric oxide synthase (iNOS)-p
s (TipDCs) as well as inflammatory DCs29-31
. These cells have been implicated in bacterial, parasitic, and viral immunity31
. An additional role for Ly6Chigh
monocytes has been observed in a mouse model of induced lupus. In this model, Ly6Chigh
monocytes accumulate in the peritoneal cavity of mice after injection of 2,6,10,14-tetramethylpentadecane33
. Surprisingly, the Ly6Chigh
monocytes express type I IFN in this model. While the activation signal for these cells has not been defined in this context, the observation that IMs can produce type I IFN during disease supports our contention that they may function as specialized IFN producing cells. The ability of IMs cells to secrete pro-inflammatory cytokines and type I IFN suggests that these cells may play a key role early during viral infection. In addition, differentiation of IMs into DCs upon activation by viruses may further enhance their contribution to the antiviral immune response through induction of adaptive responses.
A surprising aspect of TLR2 function on IMs is the ability to distinguish between viral and bacterial ligands; type I IFN is only induced in response to viral ligands while TNFα production occurs in response to both classes of stimuli. This differential response may be partially explained by the observation that activation of the signal transduction pathway leading to type I IFN production requires receptor internalization, as both chloroquine and cytochalasin D disrupt IFN but not TNF production. This dichotomy is reminiscent of TLR4 signaling, in which Trif activation occurs at endosomal membranes while MyD88 activation occurs at the plasma membrane32
. Our data suggest that TLR2 signaling is also regulated through localization, yet, in contrast to TLR4, all TLR2 signaling is MyD88-dependent. In this sense, differential signaling by TLR2 may be more conceptually similar to TLR9 signaling in that different ligands produce distinct responses through the same signaling adaptor (MyD88). For example, in pDCs class B CpG oligos lead to the production of cytokines such as TNFα and IL-12 but not to the production of type I IFN, while class C CpG oligos lead to the production of TNFα, IL-12 and type I IFN5, 34
. IMs have a similar ability to use one TLR and produce two unique responses. How such specificity can be generated downstream of a particular TLR is not understood in pDCs or in our system. It is possible that viruses are more efficiently internalized than bacterial ligands or specifically traffic to a specialized compartment from which type I IFN signaling can be initiated. Still, such a mechanism cannot explain why cDCs or macrophages are unable to make type I IFN in a TLR2-dependent manner when stimulated with virus. Thus, it seems possible that a specialized co-receptor might be required to generate the specificity that we have discovered.
The fact that innate immune recognition of vaccinia virus is so heavily TLR2-dependent is somewhat unexpected based on the additional innate receptors capable of viral recognition. For example, TLR9 clearly plays a role in innate recognition of many DNA viruses, yet our data () as well as the work of others18
suggest that TLR9 does not play a major role in the recognition of vaccinia virus. The ability of some viruses to evade certain innate receptors has undoubtedly required the host to evolve additional strategies of viral recognition. Cytosolic DNA sensors play a major role in detection of viral nucleic acid in the host cytosol35-37
. In DCs and macrophages stimulated with vaccinia virus, we observe a TLR-independent type I IFN response, which is presumably due to activation of cytosolic DNA sensors (Sup. Fig. 3
). In IMs as well as during in vivo infection, though, induction of type I IFN is largely TLR2-dependent. This dependence suggests that vaccinia virus can evade innate receptors, such as TLR9 and cytosolic DNA sensors, yet remains detectable by TLR2.
While nucleic acid recognition appears to be the dominant strategy employed by the innate immune system to detect viral infection, there is accumulating evidence that the host can recognize certain viruses independently of nucleic acid. The work presented here supports this view and provides evidence for a novel host signaling pathway linking viral recognition to induction of type I IFN. Our data and the work of others suggest that the innate immune system must have evolved specificity for certain viral proteins that are unable to mutate and escape recognition. Such a scenario is not unprecedented. Bacterial flagellin is recognized by several innate receptors (TLR538
, Ipaf 39, 40
, and Naip541
), yet most bacteria appear unable to mutate flagellin to avoid recognition. Importantly, mutations that abrogate flagellin recognition result in nonmotile bacteria41, 42
. Thus, it would appear that certain pathogen encoded proteins are sufficiently constrained that they can serve as targets for innate immune receptors.
Following this reasoning, it is possible that the innate immune system has evolved to recognize viral proteins that are under similar functional constraints as flagellin. The viral fusion apparatus is a particularly attractive target in this regard. All viruses possess a fusion apparatus that is absolutely essential for propagation, making it an ideal target of the innate immune system. While fusion proteins from unrelated viruses share no homology at the amino acid level, structural studies have demonstrated that, in some cases, these proteins share surprising structural homology43
. It is possible that TLR2 has evolved to recognize a conserved structural feature associated with certain viral fusion proteins. In support of this hypothesis, HCMV glycoprotein B, which is required for viral fusion, has been reported to activate TLR210
. Additionally, a number of reports suggest that TLR4 can recognize viral structural proteins44-48
. Identification of ligands from other viruses recognized by TLR2 and TLR4 will be necessary to address whether a common structural feature is targeted.