NOD2-mediated dual negative regulation of inflammatory responses triggered by TLRs in the gastrointestinal tract.

Loss-of-function mutations in nucleotide-binding oligomerization domain 2 (NOD2) constitute the primary risk factors for Crohn's disease. NOD2 is an intracellular sensor for muramyl dipeptide (MDP), a small molecule derived from the peptidoglycan layer of bacterial cell wall. Although NOD2 is involved in host immune responses, much attention has been paid to the involvement of NOD2 in the maintenance of intestinal homeostasis. Despite the fact that the proinflammatory cytokine and chemokine responses induced by NOD2 activation alone are weaker than those induced by toll-like receptors (TLRs), NOD2 plays a crucial role in host defense against invading pathogens and in the regulation of immune responses. Recent studies have highlighted the importance of negative regulatory functions of NOD2 in TLRs-mediated proinflammatory cytokine responses. MDP-mediated activation of NOD2 induces interferon regulatory factor 4 (IRF4) expression, thereby suppressing nuclear factor-κB-dependent colitogenic cytokine responses through the inhibition of Lys(K)63-linked polyubiquitination on receptor-interacting serine/threonine protein kinase 2. MDP-mediated activation of NOD2 also downregulates TLR9-induced type I IFN responses by inhibiting the K63-linked polyubiquitination of TNF receptor-associated factor 3 via deubiquitinating enzyme A (DUBA) expression. Thus, NOD2 exerts dual negative regulation of TLRs-mediated proinflammatory cytokine and type I IFN responses by inducing the expression of IRF4 and DUBA, respectively. In this review, we summarize the molecular mechanisms whereby NOD2 activation suppresses TLRs-mediated proinflammatory and type I IFN responses. In addition, we discuss the clinical relevance of the NOD2-mediated negative regulation of TLRs in inflammatory bowel disease.

NOD2 reduces the chemoresistance of melanoma by inhibiting the TYMS/PLK1 signaling axis.

Nucleotide-binding oligomerization domain 2 (NOD2) is an immune sensor crucial for eliciting the innate immune responses. Nevertheless, discrepancies exist regarding the effect of NOD2 on different types of cancer. This study aimed to investigate these function of NOD2 in melanoma and its underlying mechanisms. We have validated the tumor suppressor effect of NOD2 in melanoma. NOD2 inhibited the proliferation of melanoma cells, hindering their migration and invasion while promoting the onset of apoptosis. Our study showed that NOD2 expression is closely related to pyrimidine and folate metabolism. NOD2 inhibits thymidylate synthase (TYMS) expression by promoting K48-type ubiquitination modification of TYMS, thereby decreasing the resistance of melanoma cells to 5-fluorouracil (5-FU) and capecitabine (CAP). TYMS was identified to form a complex with Polo-like Kinase 1 (PLK1) and activate the PLK1 signaling pathway. Furthermore, we revealed that the combination of the PLK1 inhibitor volasertib (BI6727) with 5-FU or CAP had a synergistic effect repressing the proliferation, migration, and autophagy of melanoma cells. Overall, our research highlights the protective role of NOD2 in melanoma and suggests that targeting NOD2 and the TYMS/PLK1 signaling axis is a high-profile therapy that could be a prospect for melanoma treatment.

Deubiquitination of RIPK2 by OTUB2 augments NOD2 signalling and protective effects in intestinal inflammation.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract, but the molecular mechanisms underlying IBD are incompletely understood. In this study, we explored the role and regulating mechanism of otubain 2 (OTUB2), a deubiquitinating enzyme, in IBD. METHODS: To study the function of OTUB2 in IBD, we generated Otub2-/- mice and treated them with dextran sulfate sodium (DSS) to induce experimental colitis. Bone marrow transplantation was performed to identify the cell populations that were affected by OTUB2 in colitis. The molecular mechanism of OTUB2 in signal transduction was studied by various biochemical methods. RESULTS: OTUB2 was highly expressed in colon-infiltrating macrophages in both humans with IBD and mice with DSS-induced experimental colitis. Colitis was significantly aggravated in Otub2-/- mice and bone marrow chimeric mice receiving Otub2-/- bone marrow. OTUB2-deficiency impaired the production of cytokines and chemokines in macrophages in response to the NOD2 agonist muramyl dipeptide (MDP). Upon MDP stimulation, OTUB2 promoted NOD2 signaling by stabilizing RIPK2. Mechanistically, OTUB2 inhibited the proteasomal degradation of RIPK2 by removing K48-linked polyubiquitination on RIPK2, which was mediated by the active C51 residue in OTUB2. In mice, OTUB2 ablation abolished the protective effects of MDP administration in colitis. CONCLUSION: This study identified OTUB2 as a novel regulator of intestinal inflammation.

Distinctive Immune Signatures Driven by Structural Alterations in Desmuramylpeptide NOD2 Agonists.

Herein we report on the design, synthesis and biological evaluation of a series of nucleotide-binding oligomerization-domain-containing protein 2 (NOD2) desmuramylpeptide agonists. The structural prerequisites that shape both physicochemical and immunomodulatory profiles of desmuramylpeptide NOD2 agonists have been delineated. Within this context, we identified 3, a butyrylated desmuramylpeptide, as a potent in vitro NOD2 agonist (EC50 = 4.6 nM), exhibiting an almost 17-fold enhancement in potency compared to its unsubstituted counterpart 1 (EC50 = 77.0 nM). The novel set of desmuramylpeptides demonstrate unique in vitro immunomodulatory activities. They elicited cytokine production in peripheral blood mononuclear cells (PBMCs), both alone and in conjunction with lipopolysaccharide (LPS). The spermine-decorated 32 also stimulated the LPS-induced cytotoxic activity (2.95-fold) of PBMCs against K562 cancer cells. Notably, the cholesterol-conjugate 26 displayed anti-inflammatory actions, highlighted by its capacity to convert the inflammatory monocyte subset into an anti-inflammatory phenotype. Finally, the eicosapentaenoylated derivative 23 augmented antigen presentation by mouse bone marrow-derived dendritic cells (BMDCs), thus highlighting its potential as a vaccine adjuvant.

Deubiquitinase USP14 is upregulated in Crohn's disease and inhibits the NOD2 pathway mediated inflammatory response in vitro.

The nucleotide binding oligomerization domain containing 2 (NOD2) protein and its ligand N-acetyl muramyl dipeptide (MDP) are crucially involved in Crohn's disease (CD). However, the mechanism by which NOD2 signaling is regulated in CD patients remains unclear. Ubiquitin specific protease (USP14) is a deubiquitylase that plays an important role in immunity. This study aimed to investigate the mechanism by which UPS14 regulates NOD2 induced inflammatory response in CD and inflammatory bowel diseases (IBD). Our results showed that USP14 protein and mRNA levels in intestinal tissues of CD patients were significantly higher than those in healthy controls. In addition, USP14 was upregulated in IBD mouse model. While treatment with MDP, TNF-α or the Toll-like receptor 1/2 agonist Pam3CSK4 all led to significantly higher mRNA levels of TNF-α, IL-8 and IL-1ß in THP-1 cells, pretreatment with USP14 inhibitor IU1 could stimulate further upregulation of TNF-α, IL-8 and IL-1ß. In particular, MDP promoted the activation of JNK, ERK1/2 and p38 as well as NF-kB in THP-1 cells, and IU1 significantly enhanced the MDP-induced activation of these proteins without effects on USP14 protein level. Furthermore, the JNK inhibitor sp600125, ERK1/2 inhibitor U0126 or P38 MAPK inhibitor PD169316 significantly decreased the mRNA levels of TNF-α, IL-8 and IL-1ß in THP-1 cells stimulated by both IU1 and MDP. In conclusion, our findings suggest that USP14 could inhibit MDP-induced activation of MAPK signaling and the inflammation response involved in IBD, and that USP14 is a potential therapeutic target for IBD.

Innate immunity gene Nod2 protects mice from orthotopic breast cancer.

BACKGROUND: Nod2 is involved in innate immune responses to bacteria, regulation of metabolism, and sensitivity to cancer. A Nod2 polymorphism is associated with breast cancer, but the role of Nod2 in the development and progression of breast cancer is unknown. METHODS: Here, we tested the hypothesis that Nod2 protects mice from breast cancer using the 4T1 orthotopic model of mammary tumorigenesis. WT and Nod2-/- mice were injected with 4T1 mammary carcinoma cells and the development of tumors was monitored. A detailed analysis of the tumor transcriptome was performed and genes that were differentially expressed and pathways that were predicted to be altered between WT and Nod2-/- mice were identified. The activation of key signaling molecules involved in metabolism and development of cancer was studied. RESULTS: Our data demonstrate that Nod2-/- mice had a higher incidence and larger tumors than WT mice. Nod2-/- mice had increased expression of genes that promote DNA replication and cell division, and decreased expression of genes required for lipolysis, lipogenesis, and steroid biosynthesis compared with WT mice. Nod2-/- mice also had lower expression of genes required for adipogenesis and reduced levels of lipids compared with WT mice. The tumors in Nod2-/- mice had decreased expression of genes associated with PPARα/γ signaling, increased activation of STAT3, decreased activation of STAT5, and no change in the activation of ERK compared with WT mice. CONCLUSIONS: We conclude that Nod2 protects mice from the 4T1 orthotopic breast tumor, and that tumors in Nod2-/- mice are predicted to have increased DNA replication and cell proliferation and decreased lipid metabolism compared with WT mice.

Synergistic immune augmentation enabled by covalently conjugating TLR4 and NOD2 agonists.

Enhancing the efficacy of subunit vaccines relies significantly on the utilization of potent adjuvants, particularly those capable of triggering multiple immune pathways. To achieve synergistic immune augmentation by Toll-like receptor 4 agonist (TLR4a) and nucleotide-binding oligomerization-domain-containing protein 2 agonist (NOD2a), in this work, we conjugated RC529 (TLR4a) and MDP (NOD2a) to give RC529-MDP, and evaluated its adjuvanticity for OVA antigen. Compared to the unconjugated RC529+MDP, RC529-MDP remarkably enhanced innate immune responses with 6.8-fold increase in IL-6 cytokine, and promoted the maturation of antigen-presenting cells (APCs), possibly because of the conjugation of multiple agonists ensuring their delivery to the same cell and activation of various signaling pathways within that cell. Furthermore, RC529-MDP improved OVA-specific antibody response, T cells response and the memory T cells ratio relative to the unconjugated mixture. Therefore, covalently conjugating TLR4 agonist and NOD2 agonist was an effective strategy to enhance immune responses, providing the potential to design and develop more effective vaccines.

Systemic Multifunctional Nanovaccines for Potent Personalized Immunotherapy of Acute Myeloid Leukemia.

Hematological malignancies (HM) like acute myeloid leukemia (AML) are often intractable. Cancer vaccines possibly inducing robust and broad anti-tumor immune responses may be a promising treatment option for HM. Few effective vaccines against blood cancers are, however, developed to date partly owing to insufficient stimulation of dendritic cells (DCs) in the body and lacking appropriate tumor antigens (Ags). Here it is found that systemic multifunctional nanovaccines consisting of nucleotide-binding oligomerization domain-containing protein 2 (NOD2) and Toll-like receptor 9 (TLR9) agonists - muramyl dipeptide (MDP) and CpG, and tumor cell lysate (TCL) as Ags (MCA-NV) induce potent and broad immunity against AML. MCA-NV show complementary stimulation of DCs and prime homing to lymphoid organs following systemic administration. Of note, in orthotopic AML mouse models, intravenous infusion of different vaccine formulations elicits substantially higher anti-AML efficacies than subcutaneous administration. Systemic MCA-NV cure 78% of AML mice and elicit long-term immune memory with 100% protection from rechallenging AML cells. Systemic MCA-NV can also serve as prophylactic vaccines against the same AML. These systemic nanovaccines utilizing patient TCL as Ags and dual adjuvants to elicit strong, durable, and broad immune responses can provide a personalized immunotherapeutic strategy against AML and other HM.

Short-peptide-based enteral nutrition affects rats MDP translocation and protects against gut-lung injury via the PepT1-NOD2-beclin-1 pathway in vivo.

BACKGROUND: Peptide transporter 1 (PepT1) transports bacterial oligopeptide products and induces inflammation of the bowel. Nutritional peptides compete for the binding of intestinal bacterial products to PepT1. We investigated the mechanism of short-peptide-based enteral nutrition (SPEN) on the damage to the gut caused by the bacterial oligopeptide product muramyl dipeptide (MDP), which is transported by PepT1. The gut-lung axis is a shared mucosal immune system, and immune responses and disorders can affect the gut-respiratory relationship. METHODS AND RESULTS: Sprague-Dawley rats were gavaged with solutions containing MDP, MDP + SPEN, MDP + intact-protein-based enteral nutrition (IPEN), glucose as a control, or glucose with GSK669 (a NOD2 antagonist). Inflammation, mitochondrial damage, autophagy, and apoptosis were explored to determine the role of the PepT1-nucleotide-binding oligomerization domain-containing protein 2 (NOD2)-beclin-1 signaling pathway in the small intestinal mucosa. MDP and proinflammatory factors of lung tissue were explored to determine that MDP can migrate to lung tissue and cause inflammation. Induction of proinflammatory cell accumulation and intestinal damage in MDP gavage rats was associated with increased NOD2 and Beclin-1 mRNA expression. IL-6 and TNF-α expression and apoptosis were increased, and mitochondrial damage was severe, as indicated by increased mtDNA in the MDP group compared with controls. MDP levels and expression of proinflammatory factors in lung tissue increased in the MDP group compared with the control group. SPEN, but not IPEN, eliminated these impacts. CONCLUSIONS: Gavage of MDP to rats resulted in damage to the gut-lung axis. SPEN reverses the adverse effects of MDP. The PepT1-NOD2-beclin-1 pathway plays a role in small intestinal inflammation, mitochondrial damage, autophagy, and apoptosis.

Proteomic Profiling of Tears in Blau Syndrome Patients in Identification of Potential Disease Biomarkers.

Blau syndrome (BS) is a rare autoinflammatory granulomatosis characterized by granulomatous arthritis, uveitis, and dermatitis. Ocular complications are particularly severe in BS, significantly contributing to morbidity. This study aims to identify potential biomarkers for BS ocular degeneration through proteomic profiling of tear samples from affected patients. Seven subjects from the same family, including four carriers of the BS-associated NOD2 mutation (p.E383K), were recruited alongside healthy controls. Tear samples were collected using Schirmer strips and analyzed via mass spectrometry. A total of 387 proteins were identified, with significant differences in protein expression between BS patients, healthy familial subjects, and healthy controls. Key findings include the overexpression of alpha-2-macroglobulin (A2M) and immunoglobulin heavy constant gamma 4 (IGHG4) in BS patients. Bioinformatic analysis revealed that differentially expressed proteins are involved in acute-phase response, extracellular exosome formation, and protein binding. Notably, neutrophils' azurophilic granule components, as azurocidin (AZU1), myeloperoxidases (MPO), and defensins (DEFA3), were highly expressed in the most severely affected subject, suggesting a potential role of neutrophils in BS ocular severity. These proteins might be promising biomarkers for ocular involvement in BS, facilitating early detection and tailored treatment strategies.

The Salmonella Effector SspH2 Facilitates Spatially Selective Ubiquitination of NOD1 to Enhance Inflammatory Signaling.

As part of its pathogenesis, Salmonella enterica serovar Typhimurium delivers effector proteins into host cells. One effector is SspH2, a member of the so-called novel E3 ubiquitin ligase family, that interacts with and enhances, NOD1 pro-inflammatory signaling, though the underlying mechanisms are unclear. Here, we report that SspH2 interacts with multiple members of the NLRC family to enhance pro-inflammatory signaling by targeted ubiquitination. We show that SspH2 modulates host innate immunity by interacting with both NOD1 and NOD2 in mammalian epithelial cell culture via the NF-κB pathway. Moreover, purified SspH2 and NOD1 directly interact, where NOD1 potentiates SspH2 E3 ubiquitin ligase activity. Mass spectrometry and mutational analyses identified four key lysine residues in NOD1 that are required for its enhanced activation by SspH2, but not its basal activity. These critical lysine residues are positioned in the same region of NOD1 and define a surface on the receptor that appears to be targeted by SspH2. Overall, this work provides evidence for post-translational modification of NOD1 by ubiquitin and uncovers a unique mechanism of spatially selective ubiquitination to enhance the activation of an archetypal NLR.

Targeting IL-1 controls refractory pityriasis rubra pilaris.

Pityriasis rubra pilaris (PRP) is a rare inflammatory skin disease with a poorly understood pathogenesis. Through a molecularly driven precision medicine approach and an extensive mechanistic pathway analysis in PRP skin samples, compared to psoriasis, atopic dermatitis, healed PRP, and healthy controls, we identified IL-1ß as a key mediator, orchestrating an NF-κB-mediated IL-1ß-CCL20 axis, including activation of CARD14 and NOD2. Treatment of three patients with the IL-1 antagonists anakinra and canakinumab resulted in rapid clinical improvement and reversal of the PRP-associated molecular signature with a 50% improvement in skin lesions after 2 to 3 weeks. This transcriptional signature was consistent with in vitro stimulation of keratinocytes with IL-1ß. With the central role of IL-1ß underscoring its potential as a therapeutic target, our findings propose a redefinition of PRP as an autoinflammatory keratinization disorder. Further clinical trials are needed to validate the efficacy of IL-1ß antagonists in PRP.

Synergistic Targeting of Innate Receptors TLR7 and NOD2 for Therapeutic Intervention in Multiple Sclerosis.

Regulation of neuroinflammation is critical for maintaining central nervous system (CNS) homeostasis and holds therapeutic promise in autoimmune diseases such as multiple sclerosis (MS). Previous studies have highlighted the significance of selective innate signaling in triggering anti-inflammatory mechanisms, which play a protective role in an MS-like disease, experimental autoimmune encephalomyelitis (EAE). However, the individual intra-CNS administration of specific innate receptor ligands or agonists, such as for toll-like receptor 7 (TLR7) and nucleotide-binding oligomerization-domain-containing protein 2 (NOD2), failed to elicit the desired anti-inflammatory response in EAE. In this study, we investigated the potential synergistic effect of targeting both TLR7 and NOD2 simultaneously to prevent EAE progression. Our findings demonstrate that simultaneous intrathecal administration of NOD2- and TLR7-agonists led to synergistic induction of Type I IFN (IFN I) and effectively suppressed EAE in an IFN I-dependent manner. Suppression of EAE was correlated with a significant decrease in the infiltration of monocytes, granulocytes, and natural killer cells, reduced demyelination, and downregulation of IL-1ß, CCL2, and IFNγ gene expression in the spinal cord. These results underscore the therapeutic promise of concurrently targeting the TLR7 and NOD2 pathways in alleviating neuroinflammation associated with MS, paving the way for novel and more efficacious treatment strategies.

Mesenchymal stem cell-derived exosomes ameliorate diabetic kidney disease through NOD2 signaling pathway.

BACKGROUND AND AIMS: Diabetic kidney disease (DKD) is one of the most common complications of diabetes. It is reported that mesenchymal stem cells (MSCs) derived exosomes (MSCs-Exo) may have great clinical application potential for the treatment of DKD, but the underlying mechanism has not been illustrated. To clarify the effect of MSC-Exo on NOD2 signaling pathway in podocytes under high glucose (HG) and DKD, we conduct this study. METHODS: We co-cultured podocytes and MSCs-Exo under 30 mM HG and injected MSCs-Exo into DKD mice, then we detected the NOD2 signaling pathway by western blot, qRT-PCT, immunofluorescence, transmission electron microscopy and immunohistochemistry both in vitro and in vivo. RESULTS: In vitro, HG lead to the apoptosis, increased the ROS level and activated the NOD2 signaling pathway in podocytes, while MSCs-Exo protected podocytes from injury reduced the expression of inflammatory factors including TNF-α, IL-6, IL-1ß, and IL-18 and alleviated the inflammatory response, inhibited the activation of NOD2 signaling pathway and the expression of it's downstream protein p-P65, p-RIP2, prevented apoptosis, increased cell viability in podocytes caused by HG. In vivo, MSCs-Exo alleviated renal injury in DKD mice, protected renal function, decreased urinary albumin excretion and inhibited the activation of NOD2 signaling pathway as well as the inflammation in renal tissue. CONCLUSION: MSCs-Exo protected the podocytes and DKD mice from inflammation by mediating NOD2 pathway, MSCs-Exo may provide a new target for the treatment of DKD.

Chronic NOD2 stimulation by MDP confers protection against parthanatos through M2b macrophage polarization in RAW264.7 cells.

Innate immune cells show enhanced responsiveness to secondary challenges after an initial non-related stimulation (Trained Innate Immunity, TII). Acute NOD2 activation by Muramyl-Dipeptide (MDP) promotes TII inducing the secretion of pro-inflammatory mediators, while a sustained MDP-stimulation down-regulates the inflammatory response, restoring tolerance. Here we characterized in-vitro the response of murine macrophages to lipopolysaccharide (LPS) challenge under NOD2-chronic stimulation. RAW264.7 cells were trained with MDP (1 µg/ml, 48 h) and challenged with LPS (5 µg/ml, 24 h). Trained cells formed multinucleated giant cells with increased phagocytosis rates compared to untrained/challenged cells. They showed a reduced mitochondrial activity and a switch to aerobic glycolysis. TNF-α, ROS and NO were upregulated in both trained and untrained cultures (MDP+, MDP- cells, p > 0.05); while IL-10, IL-6 IL-12 and MHCII were upregulated only in trained cells after LPS challenge (MDP + LPS+, p < 0.05). A slight upregulation in the expression of B7.2 was also observed in this group, although differences were not statistically significant. MDP-training induced resistance to LPS challenge (p < 0.01). The relative expression of PARP-1 was downregulated after the LPS challenge, which may contribute to the regulatory milieu and to the innate memory mechanisms exhibited by MDP-trained cells. Our results demonstrate that a sustained MDP-training polarizes murine macrophages towards a M2b profile, inhibiting parthanatos. These results may impact on the development of strategies to immunomodulate processes in which inflammation should be controlled.

NOD2 activation enhances macrophage Fcγ receptor function and may increase the efficacy of antibody therapy.

Introduction: Therapeutic antibodies have become a major strategy to treat oncologic diseases. For chronic lymphocytic leukemia, antibodies against CD20 are used to target and elicit cytotoxic responses against malignant B cells. However, efficacy is often compromised due to a suppressive microenvironment that interferes with cellular immune responses. To overcome this suppression, agonists of pattern recognition receptors have been studied which promote direct cytotoxicity or elicit anti-tumoral immune responses. NOD2 is an intracellular pattern recognition receptor that participates in the detection of peptidoglycan, a key component of bacterial cell walls. This detection then mediates the activation of multiple signaling pathways in myeloid cells. Although several NOD2 agonists are being used worldwide, the potential benefit of these agents in the context of antibody therapy has not been explored. Methods: Primary cells from healthy-donor volunteers (PBMCs, monocytes) or CLL patients (monocytes) were treated with versus without the NOD2 agonist L18-MDP, then antibody-mediated responses were assessed. In vivo, the Eµ-TCL1 mouse model of CLL was used to test the effects of L18-MDP treatment alone and in combination with anti-CD20 antibody. Results: Treatment of peripheral blood mononuclear cells with L18-MDP led to activation of monocytes from both healthy donors and CLL patients. In addition, there was an upregulation of activating FcγR in monocytes and a subsequent increase in antibody-mediated phagocytosis. This effect required the NF-κB and p38 signaling pathways. Treatment with L18-MDP plus anti-CD20 antibody in the Eµ-TCL model of CLL led to a significant reduction of CLL load, as well as to phenotypic changes in splenic monocytes and macrophages. Conclusions: Taken together, these results suggest that NOD2 agonists help overturn the suppression of myeloid cells, and may improve the efficacy of antibody therapy for CLL.

Di-mannosylation enhances the adjuvant properties of adamantane-containing desmuramyl peptides in vivo.

Muramyl dipeptide (MDP) is the smallest essential peptidoglycan substructure capable of promoting both innate and adaptive immune responses. Herein, we report on the design, synthesis, and in vivo study of the adjuvant properties of two novel MDP analogs containing an achiral adamantyl moiety attached to the desmuramyl dipeptide (DMP) pharmacophore and additionally modified by one mannosyl subunit (derivative 7) or two mannosyl subunits (derivative 11). Mannose substructures were introduced in order to assess how the degree of mannosylation affects the immune response and nucleotide-binding oligomerization-domain-containing protein 2 (NOD2) binding affinity, compared to the reference compound ManAdDMP. Both mannosylated MDP analogs showed improved immunomodulating properties, while the di-mannosylated derivative 11 displayed the highest, statistically significant increase in anti-OVA IgG production. In this study, for the first time, the di-mannosylated DMP derivative was synthesized and immunologically evaluated. Derivative 11 stimulates a Th-2-polarized type of immune reaction, similar to the reference compound ManAdDMP and MDP. Molecular dynamics (MD) simulations demonstrate that 11 has a higher NOD2 binding affinity than 7, indicating that introducing the second mannose significantly contributes to the binding affinity. Mannose interacts with key amino acid residues from the LRR hydrophobic pocket of the NOD2 receptor and loop 2.

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.

CD62-L down-regulation after L18-MDP stimulation as a complementary flow cytometry functional assay for the diagnosis of XIAP deficiency.

X-linked inhibitor of apoptosis (XIAP) deficiency is an infrequent inborn error of immunity caused by mutations in XIAP gene. Most cases present with absence of XIAP protein which can be detected by flow cytometry (FC), representing a rapid diagnostic method. However, since some genetic defects may not preclude protein expression, it is important to include a complementary functional test in the laboratory workup of these patients. L-selectin (CD62-L) is a molecule that is cleaved from the surface membrane of leukocytes upon stimulation of different receptors such as toll like receptors (TLRs) and nucleotide-binding oligomerization domain-like receptors (NLRs), including NOD2. Considering that XIAP deficiency impairs NOD2 signaling, we decided to assess CD62-L down-regulation by FC post-stimulation of neutrophils and monocytes with L18-muramyl Di-Peptide (L18-MDP), a NOD2 specific agonist, in order to develop a novel assay for the functional evaluation of patients with suspicion of XIAP defects. Whole blood samples from 20 healthy controls (HC) and four patients with confirmed molecular diagnosis of XIAP deficiency were stimulated with 200 ng/mL of L18-MDP for 2 h. Stimulation with 100 ng/mL of lipopolysaccharide (LPS) was carried out in parallel as a positive control of CD62-L shedding. CD62-L expression was evaluated by FC using an anti CD62-L- antibody and down-regulation was assessed by calculating the difference in CD62-L expression before and after stimulation, both in terms of percentage of CD62-L expressing cells (Δ%CD62-L) and median fluorescence intensity (ΔMFI%). Neutrophils and monocytes from XIAP deficient patients displayed a significantly diminished response to L18-MDP stimulation compared with HC (p < 0.0001), indicating a severely altered mechanism of CD62-L down-regulation following activation of NOD2-XIAP axis. On the other hand, the response to LPS stimulation was comparable between patients and heathy controls, suggesting preserved CD62-L shedding with a different stimulus. FC detection of CD62-L down-regulation in monocytes and neutrophils after whole blood stimulation with L18-MDP results in an effective and rapid functional test for the identification of XIAP deficient patients.