Although infection with the emerging SARS-CoV was associated with a severe acute respiratory disease, most human CoVs are responsible for mild upper respiratory tract infections, such as common colds, with only occasional spreading to the lower respiratory tract. Most respiratory viruses interact with DCs in the upper respiratory tract, which results in initiating an antiviral immune response but may also result in the spreading of the virus as a result of DC migration to the draining lymph nodes. In the present study, we investigated the interaction between HCoV-229E and human DCs. We observed that HCoV-229E infection causes massive cytopathic effects, resulting in the rapid death of DCs. Cell death correlates with a high surface expression of the HCoV-229E spike protein, which is responsible for cell-cell fusion, and the formation of large syncytia that blow up in a very short time (10 h, see the Movie S1 in the supplemental material). In contrast, monocytes from the same donors are less susceptible to infection and resist cytopathic effects and cell death despite similar expression levels of CD13, the cell surface receptor for HCoV-229E. Monocytes rapidly acquire susceptibility to HCoV-299E infection upon a short differentiation in the presence of GM-CSF and/or IL-4. DC differentiation might downregulate a restriction factor present in monocytes or induce the expression of an unknown cellular factor increasing susceptibility to HCoV-229E infection such as a coreceptor on the surface of the DCs. The infection and killing of DCs is dependent upon viral entry and viral replication, since blocking virus entry with an anti-CD13 antibody or inactivating the virus with UV protected cells. Pretreating cells with blocking antibodies against TNF-α, FAS-L, TRAIL, and IFN-α/β or with the caspase inhibitor Z-VAD-FMK did not protect infected Mo-DCs from death. We therefore suggest that cell death induced by HCoV-229E is not an apoptotic process but rather a direct consequence of virus replication and viral spike protein expression on the cell surface. Consistent with this interpretation, the only situation in which we observed protection was when cells were pretreated with IFN-β, which prevented infection by HCoV-229E. However, when Mo-DCs were infected with HCoV-229E and only then treated with IFN-β, viral replication occurred, inducing cytopathic effects and massive cell death (data not shown). This suggests that, as previously described for other respiratory viruses (5
), HCoV-229E encodes a virulence factor that blocks IFN-α/β signaling and prevents the induction of antiviral IFN-stimulated genes in Mo-DCs. This might explain why endogenous type I IFNs induced after HCoV-229E infection are unable to block viral replication and cytopathic effects in Mo-DCs. Monocytes exposed to HCoV-229E also responded by producing type I IFNs, and yet the blockade of IFN-α/β binding did not alter susceptibility to infection. Altogether, this suggests that monocyte resistance to HCoV-229E-induced cytopathic effects is independent of endogenous type I IFN production but rather relies on the expression of an as-yet-unknown restriction factor or the absence of a cellular factor increasing susceptibility to viral infection such as a coreceptor.
Because DCs are major sensors to detect viral infection and prime adaptive immunity, viruses have evolved strategies to interfere with their development, maturation, function, or viability to suppress or escape immune response. In this regard, killing DCs can be an efficient viral strategy to delay or prevent the establishment of adaptive immune responses. Infections by measles virus, human immunodeficiency virus, or lymphocytic choriomeningitis virus deplete DC populations in infected hosts (12
). In vitro
experiments have also demonstrated that filoviruses, vaccinia virus, herpes simplex virus, H5N1 influenza virus, and measles virus induce DC apoptosis or cytolysis within a few days (16
). Also, human echovirus is extremely cytopathic toward DCs and induces their death in less than 24 h (27
). DC killing by HCoV-229E, if it happens in vivo
in human infection, could delay the induction of an adaptive immune response, thus providing time to replicate in the infected host. Furthermore, this could affect the establishment of a long-term immunological memory to the virus, explaining why people can be reinfected multiple times by HCoV-229E (8
Massive death of infected DCs may also act as a host defense mechanism to prevent virus spreading in the body. Because DCs are located at every possible entry site of the body, they are one of the first cell types encountered by incoming viruses. Upon stimulation by PAMPs, DCs migrate from peripheral tissues to the draining lymph nodes where they elicit antigen-specific T lymphocytes. Many viruses use DCs not only as a vehicle to penetrate draining lymphoid organs but also to interfere with their antigen-presenting cell functions so that the immune response is skewed toward inappropriate cytokine profiles. In the case of HCoV-229E, the extreme susceptibility of DCs to infection probably prevents this virus from using them as a “Trojan horse.” Although this hypothesis needs to be further supported by experimental data, it was recently shown that DCs infected by Legionella pneumophila
undergo apoptosis to restrict bacterial replication and spreading (34
). Interestingly, SARS-CoV, which spreads to the lower respiratory tract and is therefore associated with a much more severe respiratory disease than HCoV-229E, does not induce massive cell death and cytopathic effects in DCs, arguing in favor of this hypothesis.
Our most striking observation is that, compared to DCs, monocytes from the same donors are resistant to HCoV-229E infection. However, when stimulated for only 24 h with IL-4 alone, and to some extent with GM-CSF alone, monocyte cultures became susceptible to infection. This suggests that signaling events induced by IL-4, and also somewhat induced by GM-CSF, are responsible for monocyte sensitization to HCoV-229E infection. For example, both IL-4 and GM-CSF lead to the activation of phosphatidylinositol 3-kinase and GRB2/mitogen-activated protein kinase pathways, which seem to be critical in the biological functions of DCs (23
). In a previously published work, Collins et al. found that monocytes/macrophages undergo apoptosis when infected with HCoV-229E (11
). However, these authors did not describe the massive cell death and cytopathic effects that we observed in Mo-DCs. This difference could be due to different cell isolation and purification methods. Indeed, monocytes obtained by adhering peripheral blood mononuclear cells quickly differentiate into macrophages, as acknowledged by the authors themselves. This could account for their susceptibility to HCoV-229E infection, whereas monocytes positively selected by magnetic beads are resistant, as shown in the present report.
What are the mechanisms allowing monocytes, but not DCs, to prevent cell death and massive cytopathic effects upon HCoV-229E infection? We observed that monocytes produced 16 times more IL-6 than did Mo-DCs upon infection. This could explain the resistance of monocytes since IL-6 is an IFN-like cytokine with antiviral properties. However, neutralizing IL-6 in infected monocyte cultures did not confer susceptibility to HCoV-229E, and adding recombinant IL-6 into infected DC cultures did not confer resistance to the virus (data not shown). As well, supernatants collected from infected monocytes did not protect Mo-DCs. Thus, a soluble cofactor does not account for monocyte resistance to HCoV-229E infection. Interestingly, it has been shown that bovine viral diarrhea virus, a positive-strand RNA virus that belongs to the Flaviviridae
family, has opposite effects on these two cell types, DCs being resistant, whereas monocytes are rapidly killed by the infection (21
). HIV is another example demonstrating that cells from the macrophage lineage have different levels of susceptibility to infection. Although macrophages and monocytes both express HIV-1 entry receptors, monocytes freshly purified from peripheral blood are resistant to HIV-1 infection. In contrast, monocyte-derived macrophages are highly susceptible to infection. As an explanation for this, Wang et al. demonstrated that freshly isolated monocytes express higher levels of anti-HIV-1 microRNAs than monocyte-derived macrophages (50
). This suggests that the expression of specific antiviral miRNA is a possible mechanism underlying monocyte resistance to HCoV-229E. The precise mechanism that triggers the susceptibility of monocytes to infection after a short stimulation with IL-4 or GM-CSF remains to be elucidated.
In conclusion, we demonstrate here that HCoV-229E, which is commonly spread among human population, infects and destroys human Mo-DCs rapidly, whereas monocytes are resistant. The next step of this work is to identify not only the molecular basis of the DC susceptibility and monocyte resistance to HCoV-229E infection but also the mechanisms that regulate this phenotype and how this affects the severity of the disease.