Le rôle du récepteur NOD2 : un régulateur de l'infection virale et de la réponse inflammatoire

Trois classes majeures de récepteurs de l’immunité innée sont reconnues pour être impliquées dans la reconnaissance de pathogènes : les Toll-like receptors (TLRs), les Retinoic acid-inducible gene I-like receptors (RLRs) ainsi que les Nucleotide-binding oligomerization domain-like receptors (NLRs). NOD2, un des membres de la famille des NLRs, a été initialement reconnu comme étant impliqué dans la reconnaissance du muramyl dipeptide (MDP), un composé de la paroi de peptidoglycane bactérienne. NOD2 est également connu pour être un facteur de risque associé à plusieurs maladies inflammatoires humaines, en particulier la maladie de Crohn et le syndrome de Blau. Récemment, il a été démontré que NOD2 a également la capacité de reconnaitre des motifs viraux à ARN simple brin, tel que celui du virus de l’influenza du type A. Bien que les fonctions antimicrobiennes de NOD2 soient maintenant supportées par de nombreuses études, son rôle dans la réponse inflammatoire reste à clarifier. Chez les patients atteints de la maladie de Crohn, il n’est pas clair à l’heure actuelle si les mutations dans le gène NOD2 sont liées à une incapacité de contrôler l’élimination bactérienne, ou si l’inflammation résulte d’une non régulation de la réponse inflammatoire. Dans notre laboratoire, nous avons mis en évidence que le traitement avec le MDP peut influencer l’homéostasie immunitaire dans divers modèles inflammatoires chez la souris. Le but de ce travail de recherche fut d’investiguer les populations cellulaires et les éventuels mécanismes sous-jacents à l’activation de la voie NOD2 par le MDP et de déterminer leurs implications dans le contrôle de la réponse inflammatoire, à l’aide de différents modèles murins. Ces études nous ont permis d’approfondir les divers mécanismes potentiels ainsi que les populations cellulaires impliquées dans la réponse immunitaire liée à l’activation de NOD2 par le MDP. Les résultats des études regroupées dans cette thèse soulignent le rôle de la voie NOD2 dans le contrôle de l’inflammation. En effet, nous avons mis en évidence le rôle de NOD2 dans le développement des monocytes patrouilleurs Ly6Clow, des macrophages alvéolaires (MA) ainsi que des lymphocytes T régulateurs (Treg) et leur implication dans la régulation de l’inflammation lors d’une infection par le virus de l’influenza. Ces travaux de recherche ont permis d’accroitre notre compréhension de la voie NOD2 et de son effet bénéfique dans le contrôle de l’inflammation et suggèrent que cela pourrait constituer une cible thérapeutique intéressante pour le traitement de maladies inflammatoires et virales.Three major classes of innate immunity receptors are known to be involved in pathogen recognition: the Toll-like receptors (TLRs), the Retinoic acid-inducible gene I-like receptors (RLRs), and the Nucleotide-binding oligomerization domain-like receptors (NLRs). NOD2, a member of the NLR family, was initially known to be involved in the recognition of muramyl dipeptide (MDP), a compound of the bacterial peptidoglycan wall. NOD2 is suspected to be associated with inflammatory diseases such as Crohn's disease and Blau's syndrome. Recently, NOD2 receptor was shown to have the capacity to recognize viral single-stranded RNA motifs, such as Influenza A virus. However, although the antimicrobial functions of NOD2 are now supported by numerous studies, its role in the inflammatory response remains to be clarified. In patients with Crohn disease, it is unclear whether the mutations in NOD2 lead to an inability to control bacterial clearance, or if the inflammation results from a non-regulation of the inflammatory response. In our laboratory, we highlighted that MDP treatment could control immune homeostasis in various inflammatory models in mice. The purpose of this work was to investigate the cell populations and the eventual mechanisms underlying the activation of the NOD2 pathway by MDP and determine their implications for controlling the inflammatory response using different murine models. These studies allowed us to deepen the various potential mechanisms as well as the cell populations involved in the immune response linked to the activation of NOD2 by MDP. The results underline the promising role of the NOD2 pathway in the control of inflammation. Indeed, we highlighted the role of NOD2 in the development of Ly6Clow patrolling monocytes, alveolar macrophages and Treg cells and their involvement in the regulation of inflammation, during influenza A virus infection. This work has therefore increased our understanding of the NOD2 pathway and its beneficial effects in the control of inflammation and suggests that it could be an interesting therapeutic target to treat inflammatory and viral disease

Contribution of Regulatory T Cells in Nucleotide-Binding Oligomerization Domain 2 Response to Influenza Virus Infection

Influenza A virus (IAV) is recognized to cause severe pulmonary illnesses in humans, particularly in elderly and children. One of the features associated with IAV infection is an excessive lung inflammation due to an uncontrolled immune response. The nucleotide-binding oligomerization domain 2 (NOD2) receptor is known to recognize ssRNA viruses such as IAV, but its role in the inflammatory process during viral infections remains to be clarified. In a previous report, we have shown that activation of NOD2 with muramyl dipeptide (MDP) significantly reduces both viral loads and lung inflammation and also improves pulmonary function during IAV infection. These findings prompted us to further investigate whether NOD2 receptor may contribute to regulate inflammation during viral infection. In the present study, we show that administration of MDP to mice infected with IAV stimulates the migration of regulatory T (Treg) cells to the lungs. Such a presence of Treg cells was also accompanied with a reduction of neutrophils in the lungs during IAV infection, which correlated, with a significant decrease of Th17 cells. In our model, Treg cell recruitment is dependent of CXCL12 and CCL5 chemokines. Moreover, we show that the presence of Ly6Clow patrolling monocytes is required for Treg cells mobilization to the lung of mice treated with MDP. In fact, following monocyte depletion by administration of clodronate liposome, mobilization of Treg cells to the lungs of treated mice was found to occur when circulating Ly6Clow monocytes begin to reemerge. In addition, we also detected an increased production of TGF-β, a cytokine contributing to Treg activity when blood Ly6Clow monocytes are restored. Together, our results demonstrate that MDP treatment can promote an anti-inflammatory environment through the mobilization of Treg cells to the lung, a mechanism that requires the presence of Ly6Clow monocytes during IAV infection. Overall, our results suggest that activation of NOD2 receptor could be an appealing approach to control pulmonary inflammation in patients infected with IAV

Treatment with the NR4A1 agonist cytosporone B controls influenza virus infection and improves pulmonary function in infected mice.

The transcription factor NR4A1 has emerged as a pivotal regulator of the inflammatory response and immune homeostasis. Although contribution of NR4A1 in the innate immune response has been demonstrated, its role in host defense against viral infection remains to be investigated. In the present study, we show that administration of cytosporone B (Csn-B), a specific agonist of NR4A1, to mice infected with influenza virus (IAV) reduces lung viral loads and improves pulmonary function. Our results demonstrate that administration of Csn-B to naive mice leads to a modest production of type 1 IFN. However, in IAV-infected mice, such production of IFNs is markedly increased following treatment with Csn-B. Our study also reveals that alveolar macrophages (AMs) appear to have a significant role in Csn-B effects, since selective depletion of AMs with clodronate liposome correlates with a marked reduction of IFN production, viral clearance and morbidity in IAV-infected mice. Furthermore, when reemergence of AMs is observed following clodronate liposome administration, an increased production of IFNs was detected in bronchoalveolar fluids of IAV-infected mice treated with Csn-B, supporting the contribution of AMs in Csn-B effects. While treatment of mice with Csn-B induces phosphorylation of transcriptional factors IRF3 and IRF7, the latter appears to be less indispensable since effects of Csn-B treatment on the synthesis of IFNs were slightly affected in IAV-infected mice lacking functional IRF7. Together, our results highlight the capacity of Csn-B and consequently of NR4A1 transcription factor in controlling IAV infection

Treatment with cytosporone B reduces lung viral load and improves survival in mice infected with Influenza A virus.

<p><b>(A)</b> Mice (n = 5/group) were infected with IAV and daily treated with placebo or with increasing concentrations of Csn-B administered intraperitoneally (ip.). Lungs were harvested at day 5 post IAV-infection (p.i.) for viral load determination. For lung viral loads, differences were analysed using One-Way ANOVA followed by Tukey post-hoc test. ** <i>p</i> ≤ 0.01, *** ≤ 0.001 and ****<i>p</i> ≤ 0.0001 as compared to indicated groups. <b>(B)</b> Lung viral loads were assessed in IAV-infected WT and <i>Nr4a1</i>^<i>-/-</i> mice (50 PFU), daily treated with placebo or Csn-B (5 mg/kg ip.). Lungs were harvested at day 3, 5 and 7 post-infection. Results are presented as mean ± SEM of two independent experiments (total of 10 mice/group). For lung viral loads, differences were analysed using Two-Way ANOVA followed by Dunnett post-hoc test. *<i>p</i> ≤ 0.05 and ****<i>p</i> ≤ 0.0001 as compared to indicated groups. <b>(C)</b> WT and <i>Nr4a1</i>^<i>-/-</i> mice (n = 8/group) were infected with IAV (3000 PFU in.) and daily treated for 16 days with either placebo or Csn-B (5 mg/kg). Survival was monitored daily. Differences were analysed using a log rank test (*<i>p</i>≤0.05 as compared to WT mice treated with a placebo). <b>(D)</b> Amplification of IAV genes (M1, NS1 and PB2) was performed by RT-PCR at day 5 on lung homogenates of IAV-infected mice treated with Csn-B or placebo. bp: base pair. Positive control: MDCK cells infected with IAV (4000 PFU/ml). Fold increase of gene expression is expressed relative to the placebo group. Data are representative of two independent experiments (n = 5mice/group). Differences were analysed using One-Way ANOVA followed by Tukey post-hoc test. ** <i>p</i> ≤ 0.01 as compared to indicated groups.</p

IRF3 and IRF7 contribute to Cytosporone B-induced IFN secretion in response to IAV infection.

<p>WT, <i>Irf3</i>^<i>-/-</i> <i>and Irf7</i>^<i>-/-</i> mice were infected with IAV (50 PFU) and daily treated with placebo or Csn-B (5 mg/kg). Levels of <b>(A-C)</b> IFN-β and <b>(B-D)</b> IFN-α production in BALs from IAV-infected WT, <i>Irf3</i>^{<b><i>-/-</i></b>} and <i>Irf7</i>^{<b><i>-/-</i></b>} mice, daily treated with placebo or Csn-B (5 mg/kg). BALs were collected at day 5 post-infection. Levels of IFN-β and IFN-α production in BALs of <i>Irf3</i>^{<b><i>-/-</i></b>} and <i>Irf7</i>^{<b><i>-/-</i></b>} mice were negligible at day 3 and 7 post-infection (data not shown). Results are presented as mean ± SEM of two independent experiments (n = 4 mice/groups). Differences were determined using Two-Way ANOVA followed by Tukey post-hoc test. *<i>p</i> ≤ 0.05 **<i>p</i> ≤ 0.01 ***<i>p</i> ≤ 0.001 and ****<i>p</i> ≤ 0.0001 as compared to indicated groups.</p

Hypothetical scenario of the effects of Csn-B on the production of type 1 IFNs in bronchoalveolar lavages of mice infected with IAV.

<p>Influenza A virus infection of WT mice induces phosphorylation of IRF3 and IRF7 transcription factors. As primary mechanism, treatment with Csn-B, an agonist of NR4A1, potentiates phosphorylation of IRF3 (solid line) and then, to a lesser extent, phosphorylates IRF7 (dashed line). These effects consequently increase production of type 1 IFNs in alveolar macrophages.</p

Selective depletion of alveolar macrophages reduces effects of Cytosporone B on IAV infection.

<p><b>(A)</b> Representative gating strategies showing alveolar macrophages (AMs) in naive mice after PBS or clodronate liposome administration. AMs were gated as CD45⁺, CD11b^-, CD11c^high, F4/80⁺, Siglec-F^high cells in lungs of naive and PBS or clodronate liposomes treated WT mice. Data are presented as the frequencies (%) of AMs on CD45⁺ cells and true count values of AMs at indicated time following PBS or clodronate liposome administration. <b>(B)</b> Lung viral loads were assessed in IAV-infected WT mice administered with PBS-lipo (control) or Clo-lipo and daily treated with placebo or Csn-B (5 mg/kg ip.). Lungs were harvested at day 4, 6, 8 and 10 post-clodronate administration. Results are presented as mean ± SEM of two independent experiments (n = 4 mice/group). Differences were determined using Two-Way ANOVA followed by Dunnett post-hoc test. *<i>p</i> ≤ 0.05, **<i>p</i> ≤ 0.01 and ***<i>p</i> ≤ 0.001 as compared to indicated groups. <b>(C)</b> WT mice injected with PBS-lipo or Clo-lipo (n = 9/group) were infected with IAV (lethal dose of 3000 PFU in.) and daily treated for 16 days with either placebo or Csn-B (5 mg/kg). Survival and body temperature were monitored daily. For survival data, differences were analysed using a log rank test. *<i>p</i> ≤ 0.05 as compared to Clo-lipo + IAV + Csn-B. ** <i>p</i> ≤0.01 as compared to Clo-lipo + IAV + placebo. For body temperature data, differences were analysed using Two-Way ANOVA followed by Dunnett post-hoc test. *<i>p</i> ≤ 0.05 and **<i>p</i> ≤ 0.01 and ***<i>p</i> ≤ 0.001 as compared to Clo-lipo + IAV + Csn-B. <b>(D)</b> Levels of IFN-β and IFN-α were assessed in BALs of IAV infected mice (50 PFU) treated with placebo or Csn-B at indicated time following Clo-lipo administration. Results are presented as mean ± SEM of two independent experiments (n = 4 mice/groups). Differences were determined using Two-Way ANOVA followed by Dunnett post-hoc test. **<i>p</i> ≤ 0.01 and ****<i>p</i> ≤ 0.0001 as compared to indicated groups.</p

Phosphorylation of IRF3 and IRF7 are increased following Cytosporone B treatment of IAV-infected mice.

<p>WT and <i>Nr4a1</i>^<i>-/-</i> mice were infected with IAV (50 PFU) and daily treated with placebo or Csn-B (5 mg/kg). Lungs were harvested at day 5 post-infection. <b>(A)</b> Immunoblots of phosphorylated-IRF-3 on serine 396 (p-IRF3), IRF3 and <b>(B)</b> phosphorylated IRF7 on serine 471/472 (p-IRF7) and IRF7 were performed on protein extracted from lung homogenates of IAV-infected mice treated with placebo or Csn-B. Data are representative of two independent experiments (n = 3 mice/groups). ß-actin was used as loading control. Fold increase in protein expression is expressed relative to the placebo group. Data are representative of two independent experiments. Differences were determined using Two-Way ANOVA followed by Tukey post-hoc test. *<i>p</i> ≤ 0.05 **<i>p</i> ≤ 0.01 and ****<i>p</i> ≤ 0.0001 as compared to indicated groups.</p