Following the discovery of NOD2 as a major susceptibility gene for Crohn's disease and as the host sensor for bacterial-derived MDP, its role in promoting antiviral immunity has not surprisingly been neglected despite the long-standing observation that MDP and derivatives could provide protection against viral infection. The first direct link between NOD2 and antiviral immunity has recently been provided by Sabbah and colleagues, who have identified a role for NOD2 in detection of viral ssRNA 
. Although most in vivo
studies performed by this group were done using respiratory syncytial virus, the authors conclude that NOD2 acts as a general activator of antiviral immunity against ssRNA viruses. While we did observed NOD2-dependent IFN-β secretion by macrophages in response to IAV, a ssRNA virus (Figure S2
), we could not find any major role for NOD2 itself (independently of MDP treatment) in protection against IAV using a sub-lethal or a lethal dose of virus (). Instead, we found that NOD2 was the host PRR responsible for the antiviral activity of MDP and provided further explanations on the mechanism underlying this effect.
MDP treatment modulated cytokine secretion in the lungs of IAV-infected mice. The effect of treatment was transient, causing an early decrease in IL-6 and TNF-α as well as an increase in type I IFN. The net result of this altered cytokine balance might have been reduced inflammation associated with excessive early IL-6 and TNF-α secretion and increased viral clearance by overcoming the NS1-mediated inhibitory effect of the virus on type I IFN production 
. Later increase in IL-6 and TNF-α secretion in MDP-treated mice up to a similar level than saline-treated animals might also have contributed to protection since IL-6 is known to promote neutrophil survival and function in the late phase of infection while TNF-α deficiency has recently been demonstrated to exacerbate immunopathology in a mouse model of acute IAV infection 
. While MDP has been shown to induce type I IFN via IRF5 
, the mechanism by which MDP treatment augmented type I IFN secretion in the lungs remains unclear. The contribution of IRF5 as well as IRF3 and IRF7 activation by IAV during infection and MDP treatment is the subject of ongoing investigation.
A recent human study has shown that IAV is capable of infecting monocytes irrespective of their cell surface phenotype and this cell type was more resistant than differentiated macrophages to the virus 
. In our study, lung CCL2 levels and circulating blood monocytes were increased in IAV-infected WT mice treated with MDP. Within the pulmonary environment, we also observed a NOD2-dependent relative increase in monocytes, more specifically in the Ly6Chigh
monocyte subset, in both lung tissue and BAL of IAV-infected mice treated with MDP as opposed to those treated with saline. Ly6Chigh
monocytes recruitment was dependent on CCR2 and MDP treatment was ineffective in animals deficient in this receptor. The role of Ly6Chigh
monocytes during IAV infection is controversial. Ccr2
-deficiency has been associated with decreased immunopathology and mortality due to impaired macrophages recruitment into the lungs of IAV-infected mice 
. However, the ability of Ccr2
-/- mice to clear IAV was also shown to be delayed due to a blockage in recruitment of tipDCs to the lungs and decreased priming of viral antigen-specific CD8 T cells 
. In addition, type I IFN signaling during IAV infection has been demonstrated to positively regulate the influx of Ly6Chigh
monocytes into the lungs 
. Therefore, while excessive recruitment of Ly6Chigh
monocytes might be detrimental during pulmonary IAV infection, an early and transient increase relative to other innate effector cells types such as what we observed upon MDP treatment could be beneficial to enhance both viral clearance and priming of antigen-specific CD8 T cell response leading to a faster resolution of infection.
The control of adaptive immune responses by NOD2 is a subject under intense investigation 
. While systemic NOD2 stimulation by MDP alone as been demonstrated to induce a T helper 2 (Th2) type of adaptive response, the generation of a Th1 response following co-stimulation with MDP and Toll-like receptor (TLR) agonists was NOD2-dependent 
. Treatment with bacteria-derived MDP during a viral infection such as IAV, which itself activates an array of innate immune receptors including various TLRs, may therefore be expected to enhance IAV-specific Th1 type immunity and potentially anti-IAV antibody titers. Indeed, NOD2-dependent antibody production has recently been reported in the context of streptococcal infection 
. Efforts are currently underway to evaluate the impact of MDP treatment and of Ly6Chigh
monocytes recruitment on the unfolding of the adaptive response in the lungs during IAV infection and in the context of vaccination against the virus.
The ability of exogenous MDP to induce a NOD2-dependent, transient increase in levels of various cytokines in the serum when administered systemically has been well studied 
. In addition, we have shown here that MDP treatment alone in the absence of IAV infection enhanced the number of circulating monocytes, again via NOD2. Despite specific activation of the NOD2 PRR, the protective action of immunomodulators such as MDP and derivatives is not specific toward the pathogen per se and is often described as a “boost of natural immunity”. Clear evidence for such an affirmation comes from the observation that mice deficient in IPS-1, a key adaptor molecule in innate immunity to IAV 
, cannot be protected by MDP treatment despite Ly6Chigh
monocytes recruitment to the lungs. Hence, efforts are being made in order to generate immunomodulatory compounds that conserve protective “boosting” activity with minimal toxicity. Examples include Murabutide, MDP-Lys (L18) (also known as Romurtide) and L-MTP-PE (also known as Mifamurtide), three MDP derivatives with low toxicity, which are promising for the treatment of various infections and cancers such as HIV and osteosarcoma 
. In this study, we have shown that the natural MDP derivative N
-glycolyl MDP has a more potent antiviral activity against IAV than the classical N
-acetyl MDP, consistent with the finding that N
-glycolylation of MDP increases its NOD2-stimulating activity 
. Ongoing studies are aimed at evaluating the toxicity of low dose N
-glycolyl MDP and at establishing whether it may have protective activity in different models of viral infection and at different times following infection.
To conclude, the determination of key cellular and molecular events induced by MDP treatment is expected to lead to a better understanding of both the correlates of a protective response against IAV and the mechanism of action of muramyl peptide immunomodulators. In parallel, further investigation on the use of clinically approved and novel MDP analogues for the immunotherapy of IAV infection should be considered.