Structure-activity relationship in NOD2 agonistic muramyl dipeptides.

Nucleotide-binding oligomerization domain 2 (NOD2) is a receptor of the innate immune system that is capable of perceiving bacterial and viral infections. Muramyl dipeptide (MDP, N-acetyl muramyl L-alanyl-d-isoglutamine), identified as the minimal immunologically active component of bacterial cell wall peptidoglycan (PGN) is recognized by NOD2. In terms of biological activities, MDP demonstrated vaccine adjuvant activity and stimulated non-specific protection against bacterial, viral, and parasitic infections and cancer. However, MDP has certain drawbacks including pyrogenicity, rapid elimination, and lack of oral bioavailability. Several detailed structure-activity relationship (SAR) studies around MDP scaffolds are being carried out to identify better NOD2 ligands. The present review elaborates a comprehensive SAR summarizing structural aspects of MDP derivatives in relation to NOD2 agonistic activity.

Nucleotide-Binding Oligomerization Domain 1 (NOD1) Agonists Prevent SARS-CoV-2 Infection in Human Lung Epithelial Cells through Harnessing the Innate Immune Response.

The lung is prone to infections from respiratory viruses such as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). A challenge in combating these infections is the difficulty in targeting antiviral activity directly at the lung mucosal tract. Boosting the capability of the respiratory mucosa to trigger a potent immune response at the onset of infection could serve as a potential strategy for managing respiratory infections. This study focused on screening immunomodulators to enhance innate immune response in lung epithelial and immune cell models. Through testing various subfamilies and pathways of pattern recognition receptors (PRRs), the nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) family was found to selectively activate innate immunity in lung epithelial cells. Activation of NOD1 and dual NOD1/2 by the agonists TriDAP and M-TriDAP, respectively, increased the number of IL-8+ cells by engaging the NF-κB and interferon response pathways. Lung epithelial cells showed a stronger response to NOD1 and dual NOD1/2 agonists compared to control. Interestingly, a less-pronounced response to NOD1 agonists was noted in PBMCs, indicating a tissue-specific effect of NOD1 in lung epithelial cells without inducing widespread systemic activation. The specificity of the NOD agonist pathway was confirmed through gene silencing of NOD1 (siRNA) and selective NOD1 and dual NOD1/2 inhibitors in lung epithelial cells. Ultimately, activation induced by NOD1 and dual NOD1/2 agonists created an antiviral environment that hindered SARS-CoV-2 replication in vitro in lung epithelial cells.

Dietary fat differentially modulates the response of bone marrow-derived macrophages to TLR4 and NOD2 agonists.

Consumption of diets high in fat has been linked to the development of obesity and related metabolic complications. Such associations originate from the enhanced, chronic, low-grade inflammation mediated by macrophages in response to translocated bacteria, bacterial products, or dietary constituents such as fatty acids (FAs). Nucleotide-binding Oligomerization Domain 2 (NOD2) senses muramyl dipeptide (MDP), a component of bacterial peptidoglycan. The inability to sense peptidoglycan through NOD2 has been demonstrated to lead to dysbiosis, increased bacterial translocation, inflammation and metabolic dysfunction. Currently, it is unknown how consumption of HFDs with different FA compositions might influence NOD2-dependent responses. In this study, we subjected WT mice to a control diet or to HFDs comprised of various ratios of unsaturated to saturated fats and determined the macrophage response to TLR4 and NOD2 agonists. A HFD with equal ratios of saturated and unsaturated fats enhanced subsequent responsiveness of macrophages to LPS but not to MDP. However, a high-unsaturated fat diet (HUFD) or a high-saturated fat diet (HSFD) both decreased the responsiveness to NOD2 agonists compared to that observed in control diet (CD) fed mice. These data suggest that dietary fatty acid composition can influence the subsequent macrophage responsiveness to bacterial products.

Phosphorylation of muramyl peptides by NAGK is required for NOD2 activation.

Bacterial cell wall components provide various unique molecular structures that are detected by pattern recognition receptors (PRRs) of the innate immune system as non-self. Most bacterial species form a cell wall that consists of peptidoglycan (PGN), a polymeric structure comprising alternating amino sugars that form strands cross-linked by short peptides. Muramyl dipeptide (MDP) has been well documented as a minimal immunogenic component of peptidoglycan1-3. MDP is sensed by the cytosolic nucleotide-binding oligomerization domain-containing protein 24 (NOD2). Upon engagement, it triggers pro-inflammatory gene expression, and this functionality is of critical importance in maintaining a healthy intestinal barrier function5. Here, using a forward genetic screen to identify factors required for MDP detection, we identified N-acetylglucosamine kinase (NAGK) as being essential for the immunostimulatory activity of MDP. NAGK is broadly expressed in immune cells and has previously been described to contribute to the hexosamine biosynthetic salvage pathway6. Mechanistically, NAGK functions upstream of NOD2 by directly phosphorylating the N-acetylmuramic acid moiety of MDP at the hydroxyl group of its C6 position, yielding 6-O-phospho-MDP. NAGK-phosphorylated MDP-but not unmodified MDP-constitutes an agonist for NOD2. Macrophages from mice deficient in NAGK are completely deficient in MDP sensing. These results reveal a link between amino sugar metabolism and innate immunity to bacterial cell walls.

Simultaneous Administration of NOD-2 (MDP) and TLP-4 (LPS) Ligands to Bone Marrow Donors 24 h before Transplantation Increases the Content of Multipotent Stromal Cells (MSCs) in Bone Marrow Grafts in CBA Mice Compared to the Total Result of Their Isolated Administration.

In 3-month bone marrow transplants of CBA mice from bone marrow donors receiving single injections of TLR-4 ligand (LPS) or NOD-2 ligand (muramyl dipeptide, MDP) 24 h before transplantation, an increase in the total number of MSCs (by 2.6 and 1.9 times, respectively), as well as a slight increase in the number of nuclear cells and the mass of bone capsules (by 1.3 and 1.2 times) were observed. After combined administration of MDР and LPS to donors, the total content of MSCs in the grafts was higher by 1.6 times in comparison with the total result of their isolated administration (and by 7.2 times in comparison with the control). At the same time, the concentration of osteogenic MSCs in the grafts of all groups was almost the same and corresponded to the control level. The number of nuclear cells and the mass of bone capsules of the grafts after combined administration of LPS and MDP were close (~80%) to the sum of the results of their isolated administration. These findings suggest that activation of the stromal tissue and the success of bone marrow transplantation depend on the intensity of innate immune responses. These data can be useful for the development of optimal methods of tissue transplantation.

Structural Fine-Tuning of Desmuramylpeptide NOD2 Agonists Defines Their In Vivo Adjuvant Activity.

We report on the design, synthesis, and biological evaluation of a series of nucleotide-binding oligomerization-domain-containing protein 2 (NOD2) desmuramylpeptide agonists with improved in vitro and in vivo adjuvant properties. We identified two promising compounds: 68, a potent nanomolar in vitro NOD2 agonist, and the more lipophilic 75, which shows superior adjuvant activity in vivo. Both compounds had immunostimulatory effects on peripheral blood mononuclear cells at the protein and transcriptional levels, and augmented dendritic-cell-mediated activation of T cells, while 75 additionally enhanced the cytotoxic activity of peripheral blood mononuclear cells against malignant cells. The C18 lipophilic tail of 75 is identified as a pivotal structural element that confers in vivo adjuvant activity in conjunction with a liposomal delivery system. Accordingly, liposome-encapsulated 75 showed promising adjuvant activity in mice, surpassing that of muramyl dipeptide, while achieving a more balanced Th1/Th2 immune response, thus highlighting its potential as a vaccine adjuvant.

Novel chimeric TLR2/NOD2 agonist CL429 exhibited significant radioprotective effects in mice.

Severe ionizing radiation causes the acute lethal damage of haematopoietic system and gastrointestinal tract. Here, we found CL429, the novel chimeric TLR2/NOD2 agonist, exhibited significant radioprotective effects in mice. CL429 increased mice survival, protected mice against the lethal damage of haematopoietic system and gastrointestinal tract. CL429 was more effective than equivalent amounts of monospecific (TLR2 or NOD2) and combination (TLR2 + NOD2) of molecules in preventing radiation-induced death. The radioprotection of CL429 was mainly mediated by activating TLR2 and partially activating NOD2. CL429-induced radioprotection was largely dependent on the activation of TLR2-MyD88-NF-κB signalling pathway. In conclusion, the data suggested that the co-activation of TLR2 and NOD2 could induce significant synergistic radioprotective effects and CL429 might be a potential high-efficiency selective agent.

Selective Immunomodulatory and Neuroprotective Effects of a NOD2 Receptor Agonist on Mouse Models of Multiple Sclerosis.

The significance of monocytes has been demonstrated in multiple sclerosis (MS). One of the therapeutic challenges is developing medications that induce mild immunomodulation that is solely targeting specific monocyte subsets without affecting microglia. Muramyl dipeptide (MDP) activates the NOD2 receptor, and systemic MDP administrations convert Ly6Chigh into Ly6Clow monocytes. Here, we report selective immunomodulatory and therapeutic effects of MDP on cuprizone and experimental autoimmune encephalomyelitis (EAE) mouse models of MS. MDP treatment exerted various therapeutic effects in EAE, including delaying EAE onset and reducing infiltration of leukocytes into the CNS before EAE onset. Of great interest is the robust beneficial effect of the MDP treatment in mice already developing the disease several days after EAE onset. Finally, we found that the NOD2 receptor plays a critical role in MDP-mediated EAE resistance. Our results demonstrate that MDP is beneficial in both early and progressive phases of EAE. Based on these results, and upon comprehensive basic and clinical research, we anticipate developing NOD2 agonist-based medications for MS in the future.

New chimeric TLR7/NOD2 agonist is a potent adjuvant to induce mucosal immune responses.

BACKGROUND: PRR (Pattern Recognition Receptor) agonists have been widely tested as potent vaccine adjuvants. TLR7 (Toll-Like Receptor 7) and NOD2 (nucleotide-binding oligomerization domain 2) are key innate receptors widely expressed at mucosal levels. METHODS: Here, we evaluated the immunostimulatory properties of a novel hybrid chemical compound designed to stimulate both TLR7 and NOD2 receptors. FINDING: The combined TLR7/NOD2 agonist showed increase efficacy than TLR7L or NOD2L agonists alone or combined in different in vitro models. Dual TLR7/NOD2 agonist efficiently stimulates TLR7 and NOD2, and promotes the maturation and reprogramming of human dendritic cells, as well as the secretion of pro-inflammatory or adaptive cytokines. This molecule also strongly induces autophagy in human cells which is a major intracellular degradation system that delivers cytoplasmic constituents to lysosomes in both MHC class I and II-restricted antigen presentation. In vivo, TLR7/NOD2L agonist is a potent adjuvant after intranasal administration with NP-p24 HIV vaccine, inducing high-quality humoral and adaptive responses both in systemic and mucosal compartments. Use of TLR7/NOD2L adjuvant improves very significantly the protection of mice against an intranasal challenge with a vaccinia virus expressing the p24. INTERPRETATION: Dual TLR7/NOD2L agonist is a very potent and versatile vaccine adjuvant and promote very efficiently both systemic and mucosal immunity. FUNDING: This work was supported by Sidaction.

Asiaticoside Alleviates Cerebral Ischemia-Reperfusion Injury via NOD2/Mitogen-Activated Protein Kinase (MAPK)/Nuclear Factor kappa B (NF-κB) Signaling Pathway.

BACKGROUND Cerebral ischemia-reperfusion injury (CIRI) remains a serious health problem. Centella asiatica formulations are used to treat central nervous system disorders. In the present study, asiaticoside, an extract of the plant Centella asiatica, was investigated in CIRI in vivo and vitro. MATERIAL AND METHODS We made a CIRI model in vivo in SD rats treated by middle cerebral artery occlusion, and a cell model of ischemia-reperfusion injury was made in PC12 cells treated by deprivation of oxygen and glucose/restoration. CIRI in vivo was assessed by scores of neurological functions, encephaledema, and cerebral infarction area. Inflammation level and oxidative stress level were detected by the appropriate kits. TUNEL assay was performed for assessment of cell apoptosis and Western blot analysis was performed to assess protein expression levels. CCK8 assay was performed for evaluation of cell survival and flow cytometer was used to detect cell apoptosis in vitro. RESULTS Nervous function injury, brain edema, cell apoptosis, infarct size, apoptosis-related protein expressions, and protein expressions of the NOD2/MAPK/NF-kappaB signaling pathway in the CIRI model were all reversed by asiaticoside in rats. The cell apoptosis, inflammation level, and oxidative stress level in the model of cerebral ischemia-reperfusion injury were reduced by asiaticoside. The effects of asiaticoside on CIRI were reversed by NOD 2 agonists. CONCLUSIONS Asiaticoside showed a protective effect against cerebral ischemia-reperfusion injury via the NOD2/MAPK/NF-kappaB signaling pathway. These findings are vital for future research on use of asiaticoside in CIRI, providing a new avenue for alleviating CIRI.

Glycolytic reprogramming of macrophages activated by NOD1 and TLR4 agonists: No association with proinflammatory cytokine production in normoxia.

Upon activation with pathogen-associated molecular patterns, metabolism of macrophages and dendritic cells is shifted from oxidative phosphorylation to aerobic glycolysis, which is considered important for proinflammatory cytokine production. Fragments of bacterial peptidoglycan (muramyl peptides) activate innate immune cells through nucleotide-binding oligomerization domain (NOD) 1 and/or NOD2 receptors. Here, we show that NOD1 and NOD2 agonists induce early glycolytic reprogramming of human monocyte-derived macrophages (MDM), which is similar to that induced by the Toll-like receptor 4 (TLR4) agonist lipopolysaccharide. This glycolytic reprogramming depends on Akt kinases, independent of mTOR complex 1 and is efficiently inhibited by 2-deoxy-d-glucose (2-DG) or by glucose starvation. 2-DG inhibits proinflammatory cytokine production by MDM and monocyte-derived dendritic cells activated by NOD1 or TLR4 agonists, except for tumor necrosis factor production by MDM, which is inhibited initially, but augmented 4 h after addition of agonists and later. However, 2-DG exerts these effects by inducing unfolded protein response rather than by inhibiting glycolysis. By contrast, glucose starvation does not cause unfolded protein response and, in normoxic conditions, only marginally affects proinflammatory cytokine production triggered through NOD1 or TLR4. In hypoxia mimicked by treating MDM with oligomycin (a mitochondrial ATP synthase inhibitor), both 2-DG and glucose starvation strongly suppress tumor necrosis factor and interleukin-6 production and compromise cell viability. In summary, the requirement of glycolytic reprogramming for proinflammatory cytokine production in normoxia is not obvious, and effects of 2-DG on cytokine responses should be interpreted cautiously. In hypoxia, however, glycolysis becomes critical for cytokine production and cell survival.

Phenotypic Differences in Primary Murine Microglia Treated with NOD1, NOD2, and NOD1/2 Agonists.

The purpose of the study was studying the influence of different NOD agonists on the morphological phenotype of primary murine microglia and to examine their influence on characteristic cytokines. Primary CD11b-positive cells were isolated from the brain of neonatal mice. The microglial phenotype of the cells was examined by ionized calcium-binding adapter molecule (Iba)1 staining. After14 days in culture, these cells were stimulated by iE-DAP, L18-MDP, or M-TriDAP as NOD1, NOD2, and NOD1/2 agonists, respectively. The cellular morphology was recorded and compared to the phenotype of cells cultured in medium alone or after LPS stimulation. The cells developed a specific phenotype only after treatment with the NOD2 agonist L18-MDP. These cells were characterized by straight extensions carrying tiny spikes and had a high ramification index. This was in sharp contrast to all other treatments, which always resulted in an amoeboid phenotype typically shown by activated microglia in vivo and by cultured microglia in vitro. The staining intensity of IL-6 and TNF-α did not reveal any clear difference independent of the NOD agonist treatment. In contrast, an increased staining intensity was observed for IL-10 after L18-MDP treatment. The NOD2 agonist L18-MDP induced a morphologically distinct phenotype characterized by microspike-decorated dendritiform extensions and a high degree of ramification in primary murine microglia. Increased ramification index and elevated staining intensity of anti-inflammatory IL-10 as hallmarks suggest that a M2-like phenotype of microglia was induced.

The Nod2 Agonist Muramyl Dipeptide Cooperates with the TLR4 Agonist Lipopolysaccharide to Enhance IgG2b Production in Mouse B Cells.

Many studies have shown that Toll-like receptors (TLRs) and Nod-like receptors (NLRs) were expressed in B cells and their signaling affects B cell functions. Nonetheless, the roles played by these receptors in B cell antibody (Ab) production have not been completely elucidated. In the present study, we examined the effect of the Nod2 agonist muramyl dipeptide (MDP) in combination with the TLR4 agonist lipopolysaccharide (LPS), a well-known B cell mitogen, on B cell viability, proliferation, and activation, and Ab production by in vitro culture of purified mouse spleen resting B cells. MDP combined with LPS to reinforce B cell viability, proliferation, and activation. Moreover, MDP enhanced LPS-induced IgG2b production, germline γ2b transcript (GLTγ2b) expression, and surface IgG2b expression. In an experiment with Nod2- and TLR4-deficient mouse B cells, we observed that the combined effect of MDP and LPS is dependent on Nod2 and TLR4 receptors. Furthermore, the combined effect on B cell viability and IgG2b switching was not observed in Rip2-deficient mouse cells. Collectively, this study suggests that Nod2 signaling enhances TLR4-activated B cell proliferation, IgG2b switching, and IgG2b production.

Tissue-type plasminogen activator selectively inhibits multiple toll-like receptors in CSF-1-differentiated macrophages.

Tissue-type plasminogen activator (tPA) is a major activator of fibrinolysis, which also attenuates the pro-inflammatory activity of lipopolysaccharide (LPS) in bone marrow-derived macrophages (BMDMs) and in vivo in mice. The activity of tPA as an LPS response modifier is independent of its proteinase activity and instead, dependent on the N-methyl-D-aspartate Receptor (NMDA-R), which is expressed by BMDMs. The major Toll-like receptor (TLR) for LPS is TLR4. Herein, we show that enzymatically-inactive (EI) tPA blocks the response of mouse BMDMs to selective TLR2 and TLR9 agonists, rapidly reversing IκBα phosphorylation and inhibiting expression of TNFα, CCL2, interleukin-1ß, and interleukin-6. The activity of EI-tPA was replicated by activated α2-macroglobulin, which like EI-tPA, signals through an NMDA-R-dependent pathway. EI-tPA failed to inhibit cytokine expression by BMDMs in response to agonists that target the Pattern Recognition Receptors (PRRs), NOD1 and NOD2, providing evidence for specificity in the function of EI-tPA. Macrophages isolated from the peritoneal space (PMs), without adding eliciting agents, expressed decreased levels of cell-surface NMDA-R compared with BMDMs. These cells were unresponsive to EI-tPA in the presence of LPS. However, when PMs were treated with CSF-1, the abundance of cell-surface NMDA-R increased and the ability of EI-tPA to neutralize the response to LPS was established. We conclude that the anti-inflammatory activity of EI-tPA is selective for TLRs but not all PRRs. The ability of macrophages to respond to EI-tPA depends on the availability of cell surface NMDA-R, which may be macrophage differentiation-state dependent.

THP-1 Cells and Pro-inflammatory Cytokine Production: An in Vitro Tool for Functional Characterization of NOD1/NOD2 Antagonists.

THP-1 cells express high levels of native functional nucleotide-binding oligomerization domain 1 (NOD1), NOD2, and Toll-like receptor 4 (TLR4) receptors, and have often been used for investigating the immunomodulatory effects of small molecules. We postulated that they would represent an ideal cell-based model for our study, the aim of which was to develop a new in vitro tool for functional characterization of NOD antagonists. NOD antagonists were initially screened for their effect on NOD agonist-induced interleukin-8 (IL-8) release. Next, we examined the extent to which the selected NOD antagonists block the NOD-TLR4 synergistic crosstalk by measuring the effect of NOD antagonism on tumor necrosis factor-α (TNF-α) secretion from doubly activated THP-1 cells. Overall, the results obtained indicate that pro-inflammatory cytokine secretion from THP-1 provides a valuable, simple and reproducible in vitro tool for functional characterization of NOD antagonists.

Innate immune memory through TLR2 and NOD2 contributes to the control of Leptospira interrogans infection.

Leptospira interrogans are pathogenic spirochetes responsible for leptospirosis, a worldwide reemerging zoonosis. Many Leptospira serovars have been described, and prophylaxis using inactivated bacteria provides only short-term serovar-specific protection. Therefore, alternative approaches to limit severe leptospirosis in humans and morbidity in cattle would be welcome. Innate immune cells, including macrophages, play a key role in fighting infection and pathogen clearance. Recently, it has been shown that functional reprograming of innate immune cells through the activation of pattern recognition receptors leads to enhanced nonspecific antimicrobial responses upon a subsequent microbial encounter. This mechanism is known as trained immunity or innate immune memory. We have previously shown that oral treatment with Lactobacillus plantarum confers a beneficial effect against acute leptospirosis. Here, using a macrophage depletion protocol and live imaging in mice, we established the role of peritoneal macrophages in limiting the initial dissemination of leptospires. We further showed that intraperitoneal priming of mice with CL429, a TLR2 and NOD2 agonist known to mimic the modulatory effect of Lactobacillus, alleviated acute leptospiral infection. The CL429 treatment was characterized as a training effect since i.) it was linked to peritoneal macrophages that produced ex vivo more pro-inflammatory cytokines and chemokines against 3 different pathogenic serovars of Leptospira, independently of the presence of B and T cells, ii.) it had systemic effects on splenic cells and bone marrow derived macrophages, and iii.) it was sustained for 3 months. Importantly, trained macrophages produced more nitric oxide, a potent antimicrobial compound, which has not been previously linked to trained immunity. Accordingly, trained macrophages better restrict leptospiral survival. Finally, we could use CL429 to train ex vivo human monocytes that produced more cytokines upon leptospiral stimulation. In conclusion, host-directed treatment using a TLR2/NOD2 agonist could be envisioned as a novel prophylactic strategy against acute leptospirosis.

NOD2 agonist murabutide alleviates radiation-induced injury through DNA damage response pathway mediated by ATR.

Injury-induced by ionizing radiation (IR) severely reduces the quality of life of victims. The development of radiation protectors is regarded as one of the most resultful strategies to alleviate damages caused by IR exposure. In the present study, we investigated the radioprotective effects of the agonist of nucleotide-binding-oligomerization-domain-containing proteins 2 called murabutide (MBD) and clarified the potential mechanisms. Our results showed that the pretreatment with MBD effectively protected cultured cells and mice against IR-induced toxicity and the pretreatment with MBD in vitro and in vitro also inhibited apoptosis caused by IR exposure. The downregulation of γ-H2AX and the upregulation of ATR signaling pathways by MBD treatment indicated that the radioprotective effects of MBD were due to the stimulation of DNA damage response (DDR) pathway to repair DNA double-strand breaks caused by IR exposure. As the radioprotective effects of MBD were diminished by the ATR selective inhibitor rather than the ATM inhibitor, ATR pathway was confirmed to be a more crucial checkpoint pathway in mediating the stimulation of DDR pathway by MBD. Taken together, our data provide a novel and effective protector to relieve the injury induced by IR exposure.

Ligands of NOD2 (Muramyl Dipeptide) and TLR4 (LPS) in 24 h after Combined In Vivo Administration Produce a Synergistic Increase in the Content of Multipotent Stromal Cells in the Bone Marrow and Peritoneal Exudate of CBA Mice.

In 24 h after combined administration of ligands of NOD2 (muramyl dipeptide) and TLR4 (LPS) receptors to CBA mice, a synergistic increase (by 10 times compared to the intact control) in cloning efficiency and content of multipotent stromal cells was observed in the bone marrow in comparison with the total effects of their individual administration (by 2.1 and 4.1 times, respectively). A similar effect was also observed in the peritoneal exudate. When ligands were administered simultaneously, the concentration of osteogenic multipotent stromal cells in the bone marrow decreased to a greater extent than in case of individual injections of the ligands, but did not drop below 7% of the control, which is apparently indicative of a decline threshold. In 3 h after simultaneous addition of the ligands in vitro to 12-day primary cultures of mouse bone marrow stromal cells, a synergistic increase in TNFα concentration was observed (32-fold increase from the level of intact control), while IL-10 concentration did not differ from the control, which is indicative of the proinflammatory nature of the process and the absence of immunosuppressive effect. These results suggest that activation of the stromal tissue depends on the intensity of innate immunity reactions.

A cell-based test system for the assessment of pharmacokinetics of NOD1 and NOD2 receptor agonists.

Agonists of nucleotide oligomerization domain (NOD) 1 and NOD2 receptors represent a promising class of immunostimulants and immunological adjuvants. Here, we describe a cell-based test system to assess their pharmacokinetics. In this system, NOD1 and NOD2 agonist concentrations in sera are determined using a reporter cell line, 293Luc, which contains an NF-κB-inducible luciferase reporter construct and naturally expresses NOD1 and NOD2. The 293Luc cells dose-dependently respond to different NOD1 and NOD2 agonists in the nanomolar to low-micromolar concentration range. To verify that the NF-κB-inducing activity of serum samples is due to the administered agonist and not to secondarily induced endogenous molecules, a 293Luc-derived NOD1/NOD2 double-knockout clone is used. Within-run and between-run precisions of the system are <15% and <20%, respectively. Applicability of the novel assay is illustrated by studying pharmacokinetics of two specific NOD2 agonists (N­acetyl­d­glucosaminyl­N­acetyl­d­muramyl­l­alanyl­d­isoglutamine and N­glycolyl­d­muramyl­l­alanyl­d­isoglutamine) and a specific NOD1 agonist (N­acetyl­d­glucosaminyl­N­acetyl­d­sorbitolamine­d­lactoyl­l­alanyl­d­isoglutamyl­meso­diaminopimelic acid). In summary, the test system described here can potentially be used to assess pharmacokinetics of NOD1 and NOD2 agonists in different animal species.

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-ß, 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.