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Background: Allergens can cross the epithelial barrier to enter the body but how this cellular passage affects protein structures and the downstream interactions with the immune system are still open questions., Objective: We sought to show the molecular details and the effects of 3 nonspecific lipid transfer proteins (nsLTPs; Mal d 3 [allergenic nsLTP1 from apple], Cor a 8 [allergenic nsLTP1 from hazelnut], and Pru p 3 [allergenic nsLTP1 from peach]) on epithelial cell uptake and transport., Methods: We used fluorescent imaging, flow cytometry, and proteomic and lipidomic screenings to identify the mechanism involved in nsLTP cellular uptake and signaling on selected epithelial and transgenic cell lines., Results: nsLTPs are transported across the epithelium without affecting cell membrane stability or viability, and allergen uptake was largely impaired by inhibition of clathrin-mediated endocytosis. Analysis of the lipidome associated with nsLTPs showed a wide variety of lipid ligands predicted to bind inside the allergen hydrophobic cavity. Importantly, the internalization of nsLTPs was contingent on these ligands in the protein complex. nsLTPs were found to initiate cellular signaling via Toll-like receptor 2 but not the cluster of differentiation 1 protein receptor, despite neither being essential for nsLTP endocytosis. We also provide evidence that the 3 allergens induced intracellular stress signaling through activation of the NOD2 pathway., Conclusions: Our work consolidates the current model on nsLTP-epithelial cell interplay and adds molecular details about cell transport and signaling. In addition, we have developed a versatile toolbox to extend these investigations to other allergens and cell types., Competing Interests: Disclosure statement This research was funded by the Danube Allergy Research Cluster of the county of Lower Austria (grant no. DARC-07 to K.H.S.) and the Austrian Science Fund (FWF: grant no. P33582-B to S.G. and grant no. P31485-B30 to A.E.B.). Disclosure of potential conflict of interest: The authors declare that they have no relevant conflict of interests. Ethics statement: Abdominal skin (anonymous healthy female donor, 62 years) was obtained during reduction surgery on written informed consent (ECS 1760/2015). The study was approved by the Ethics Committee of the Medical University of Vienna and conducted in accordance with the principles of the Declaration of Helsinki., (Copyright © 2024. Published by Elsevier Inc.)

The interplay between NOD2 and TLR2 following recognition of components of the bacterial cell wall peptidoglycan is well-established, however their role in redirecting metabolic pathways in myeloid cells to degrade pathogens and mount antigen presentation remains unclear. We show NOD2 and TLR2 mediate phosphorylation of the deubiquitinase ataxin-3 via RIPK2 and TBK1. In myeloid cells ataxin-3 associates with the mitochondrial cristae protein MIC60, and is required for oxidative phosphorylation. Depletion of ataxin-3 leads to impaired induction of mitochondrial reactive oxygen species (mROS) and defective bacterial killing. A mass spectrometry analysis of NOD2/TLR2 triggered ataxin-3 deubiquitination targets revealed immunometabolic regulators, including HIF-1α and LAMTOR1 that may contribute to these effects. Thus, we define how ataxin-3 plays an essential role in NOD2 and TLR2 sensing and effector functions in myeloid cells.

NOD2 and TLR2 recognize components of bacterial cell wall peptidoglycan and direct defense against enteric pathogens. CD8 + T cells are important for immunity to such pathogens but how NOD2 and TLR2 induce antigen specific CD8 + T cell responses is unknown. Here, we define how these pattern recognition receptors (PRRs) signal in primary dendritic cells (DCs) to influence MHC class I antigen presentation. We show NOD2 and TLR2 phosphorylate PI31 via TBK1 following activation in DCs. PI31 interacts with TBK1 and Sec16A at endoplasmic reticulum exit sites (ERES), which positively regulates MHC class I peptide loading and immunoproteasome stability. Following NOD2 and TLR2 stimulation, depletion of PI31 or inhibition of TBK1 activity in vivo impairs DC cross-presentation and CD8 + T cell activation. DCs from Crohn's patients expressing NOD2 polymorphisms show dysregulated cross-presentation and CD8 + T cell responses. Our findings reveal unidentified mechanisms that underlie CD8 + T cell responses to bacteria in health and in Crohn's.

Simultaneous triggering of Toll-like receptors (TLRs) and NOD-like receptors (NLRs) has previously been shown to synergistically activate monocytes, dendritic cells, and macrophages. We applied these properties in a T-cell vaccine setting by conjugating the NOD2-ligand muramyl-dipeptide (MDP) and TLR2-ligand Pam 3 CSK 4 to a synthetic peptide derived from a model antigen. Stimulation of human DCs with the MDP-peptide-Pam 3 CSK 4 conjugate led to a strongly increased secretion of pro-inflammatory and Th1-type cytokines and chemokines. We further show that the conjugated ligands retain their ability to trigger their respective receptors, while even improving NOD2-triggering. Also, activation of murine DCs was enhanced by the dual triggering, ultimately leading to effective induction of vaccine-specific T cells expressing IFNγ, IL-2, and TNFα. Together, these data indicate that the dual MDP-SLP-Pam 3 CSK 4 conjugate constitutes a chemically well-defined vaccine approach that holds promise for the use in the treatment of virus infections and cancer.

Streptococcus mutans, the primary etiologic agent of dental caries, can gain access to the bloodstream and has been associated with cardiovascular disease. However, the roles of S. mutans in inflammation in cardiovascular disease remain unclear. The aim of this study was to examine cytokine production induced by S. mutans in human aortic endothelial cells (HAECs) and to evaluate the participation of toll-like receptors (TLRs) and cytoplasmic nucleotide-binding oligomerization domain (NOD) -like receptors in HAECs. Cytokine production by HAECs was determined using enzyme-linked immunosorbent assays, and the expression of TLRs and NOD-like receptors was evaluated by real-time polymerase chain reaction, flow cytometry and immunocytochemistry. The involvement of TLR2 and NOD2 in cytokine production by invaded HAECs was examined using RNA interference. The invasion efficiencies of S. mutans strains were evaluated by means of antibiotic protection assays. Five of six strains of S. mutans of various serotypes induced interleukin-6, interleukin-8 and monocyte chemoattractant protein-1 production by HAECs. All S. mutans strains upregulated TLR2 and NOD2 mRNA levels in HAECs. Streptococcus mutans Xc upregulated the intracellular TLR2 and NOD2 protein levels in HAECs. Silencing of the TLR2 and NOD2 genes in HAECs invaded by S. mutans Xc led to a reduction in interleukin-6, interleukin-8 and monocyte chemoattractant protein-1 production. Cytokine production induced by invasive S. mutans via intracellular TLR2 and NOD2 in HAECs may be associated with inflammation in cardiovascular disease., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Innate immunity is considered to be critical in the pathogenesis of fungal keratitis. Pattern recognition receptors (PRRs) recognize conserved microbial structures called pathogen-associated molecular patterns (PAMPS), thereby initiating the innate immunity. Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (leucine-rich repeat-containing receptors, NLRs) are two major PRR families. The crosstalk between TLR2 and NOD2 is not completely understood, and their interrelationship in Aspergillus fumigates keratitis is still unclear. To our surprise, we found herein that NOD2 and TLR2 were increased by A. fumigatus conidia in immortalized human corneal epithelial cells (HCECs). In addition, NOD2 expression was up-regulated by its agonist muramyl dipeptide (MDP), along with receptor interacting protein 2 (RIP2), nuclear factor κB (NFκB)-p65, inhibitor of NFκB (IκB)-α, and multiple inflammatory cytokines, including interleukin-6 (IL-6), IL-8 and tumor necrosis factor α (TNF-α). Interestingly, zymosan, a TLR2 agonist, promoted the expression of NOD2 and RIP2 in a TLR2-dependent manner. Furthermore, we demonstrated that the increased expression of NOD2 and RIP2 caused by A. fumigatus conidia occurred in part through a TLR2-dependent pathway. However, zymosan pretreatment decreased NOD2 and RIP2 expression along with the MDP induced secretion of inflammatory cytokines in HCECs. In agreement, NOD2 knockdown by small interfering RNA (siRNA) reduced the release of IL-6, IL-8 and TNF-α induced by A. fumigatus conidia. These findings suggest the existence of complex interactions between TLR2 and NOD2 in HCECs inflammatory response against A. fumigatus infection., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Group B Streptococcus (GBS) is an important agent of life-threatening invasive infection. It has been previously shown that encapsulated type III GBS is easily internalized by dendritic cells (DCs), and that this internalization had an impact on cytokine production. The receptors underlying these processes are poorly characterized. Knowledge on the mechanisms used by type V GBS to activate DCs is minimal. In this work, we investigated the role of Toll-like receptor (TLR)/MyD88 signaling pathway, the particular involvement of TLR2, and that of the intracellular sensing receptor NOD2 in the activation of DCs by types III and V GBS. The role of capsular polysaccharide (CPS, one of the most important GBS virulence factors) in bacterial-DC interactions was evaluated using non-encapsulated mutants. Despite differences in the role of CPS between types III and V GBS in bacterial internalization and intracellular survival, no major differences were observed in their capacity to modulate release of cytokines by DC. For both serotypes, CPS had a minor role in this response. Production of cytokines by DCs was shown to strongly rely on MyD88-dependent signaling pathways, suggesting that DCs recognize GBS and become activated mostly through TLR signaling. Yet, GBS-infected TLR2-/- DCs only showed a partial reduction in the production of IL-6 and CXCL1 compared to control DCs. Surprisingly, CXCL10 release by type III or type V GBS-infected DCs was MyD88-independent. No differences in DC activation were observed between NOD2-/- and control DCs. These results demonstrate the involvement of various receptors and the complexity of the cytokine production pathways activated by GBS upon DC infection.

HIV-1 pathogenesis is intimately linked with microbial infections and innate immunity during all stages of the disease. While the impact of microbial-derived products in transmission of R5-using virus to CD4(+) T cells by dendritic cells (DCs) has been addressed before, very limited data are available on the effect of such compounds on DC-mediated dissemination of X4-tropic variant. Here, we provide evidence that treatment of DCs with dectin-1/TLR2 and NOD2 ligands increases cis-infection of autologous CD4(+) T cells by X4-using virus. This phenomenon is most likely associated with an enhanced permissiveness of DCs to productive infection with X4 virus, which is linked to increased surface expression of CXCR4 and the acquisition of a maturation profile by DCs. The ensuing DC maturation enhances susceptibility of CD4(+) T cells to productive infection with HIV-1. This study highlights the crucial role of DCs at different stages of HIV-1 infection and particularly in spreading of viral strains displaying a X4 phenotype.

Mycobacterium indicus pranii (MIP) is a non-pathogenic strain of mycobacterium and has been used as a vaccine against tuberculosis and leprosy. Here, we investigated the role of different pattern recognition receptors in the recognition of heat-killed MIP by macrophages. Treatment of macrophages with MIP caused upregulation of pro-inflammatory cytokines (like TNFα and IL-1β) which was mediated through both TLR2 and NOD2, as revealed by our knockdown and/or knockout studies. Mechanistically, MIP-induced macrophage activation was shown to result in NF-κB activation and drastically abrogated by MyD88 deficiency, suggesting its regulation via an MyD88-dependent, NF-κB pathway. Interestingly, the IFN-inducible cytokine, CXCL10, which is known target of the TRIF-dependent TLR pathway was found to be upregulated in response to MIP but, in an MyD88-dependent manner. Collectively, these results demonstrate macrophages to recognize and respond to MIP through a TLR2, NOD2 and an MyD88-dependent pathway. However, further studies should clarify whether additional TLR-dependent or -independent pathways also exist in regulating the full spectrum of MIP action on macrophage activation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Objective: To investigate the expression of NOD2 in human VSMCs, its role in the production of inflammatory cytokines in VSMC and the possible interaction of NOD2-mediated signaling pathway with those mediated by TLR2 and TLR4., Methods: Human coronary artery smooth muscle cells were stimulated with NOD2 agonist MDP alone or in combination with either TLR2 agonist PAM3 or TLR4 agonist LPSs. The mRNA expression of NOD2 and FGF-2 were measured by RT-PCR. The concentration of IL-8 and TNF-α in the culture supernatants was determined by ELISA. VSMC proliferation ability was analyzed by MTT assay., Results: MDP up regulated the expression of NOD2 mRNA in VSMC in a time-dependent manner, up regulated the expression of FGF-2 mRNA in VSMC, induced the production of IL-8 and TNF-α, and promoted the proliferation of VSMC. Additionally, MDP synergied with LPS and PAM3 to promote the proliferation of VSMC and induce the production of IL-8 and TNF-α., Conclusion: The activation of NOD2-mediated innate immune signaling pathway can increase the proliferation ability of VSMC and induce the production of inflammatory cytokines in VSMC. It is also shown a synergistic effect with TLR2- and TLR4-mediated signaling pathways in this process.

Dendritic cells (DCs) as sentinels of the immune system are important for eliciting both primary and secondary immune responses to a plethora of microbial pathogens. Cooperative stimulation of a complex set of pattern-recognition receptors, including TLR2 and nucleotide-binding oligomerization domain (NOD)-like receptors on DCs, acts as a rate-limiting factor in determining the initiation and mounting of the robust immune response. It underscores the need for "decoding" these multiple receptor interactions. In this study, we demonstrate that TLR2 and NOD receptors cooperatively regulate functional maturation of human DCs. Intriguingly, synergistic stimulation of TLR2 and NOD receptors renders enhanced refractoriness to TGF-β- or CTLA-4-mediated impairment of human DC maturation. Signaling perturbation data suggest that NOTCH1-PI3K signaling dynamics assume critical importance in TLR2- and NOD receptor-mediated surmounting of CTLA-4- and TGF-β-suppressed maturation of human DCs. Interestingly, the NOTCH1-PI3K signaling axis holds the capacity to regulate DC functions by virtue of PKCδ-MAPK-dependent activation of NF-κB. This study provides mechanistic and functional insights into TLR2- and NOD receptor-mediated regulation of DC functions and unravels NOTCH1-PI3K as a signaling cohort for TLR2 and NOD receptors. These findings serve in building a conceptual foundation for the design of improved strategies for adjuvants and immunotherapies against infectious diseases.

This study tested the hypothesis that activation of β2-adrenoceptors on DCs influences NOD2 signaling along with its cross-talk with Toll-like receptor-2 resulting in altered Th cell priming ability. Th17 cells are a newly discovered lineage of CD4(+) T cells involved in defense against extracellular bacteria and also implicated in autoimmune disorders. Initiation and polarization of the adaptive immune response is controlled by innate immune recognition mediated by DCs. Previous studies demonstrated that adrenergic receptors modulate cytokine production by DCs and affect their Th cell priming ability. We show that the β2-adrenoceptor agonist salbutamol enhanced IL-6 production in murine bone marrow-derived DCs stimulated with the nucleotide-binding oligomerization domain 2 ligand muramyl dipeptide. However, when the Toll-like receptor-2 ligand Pam3CysSK4 was added, salbutamol inhibited IL-12 but did not alter IL-6 and IL-23 expression. Gene expression analysis showed that salbutamol inhibited the p40 subunit as well as IL-12p35, while IL-23p19 and IL-6 were stimulated. Therefore, β2-adrenoceptors modulated cytokine production resulting in a Th17 cell priming cytokine pattern. Indeed, when antigen-pulsed DCs stimulated by muramyl dipeptide or Pam3CysSK4+muramyl dipeptide in the presence of salbutamol were used for in vivo immunization, the resulting Th17/Th1 cell ratio was increased as evaluated by IL-17 and IFN-γ production. In addition, intradermal injection of norepinephrine along with Pam3CysSK4+muramyl dipeptide increased the Th17 response to an immunogenic protein and this effect was reversed by a β2-adrenoceptor antagonist. Thus, β2-adrenoceptors may be involved in the regulation of defense against extracellular bacteria and the pathogenesis of inflammatory diseases., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

IL-23, produced by dendritic cells (DCs) and macrophages, plays a critical role in innate immunity against bacterial infection. Our previous studies show that morphine disrupts the IL-23/IL-17 mediated pulmonary mucosal host defense and increases susceptibility to Streptococcus pneumoniae lung infection. To determine the mechanism by which morphine modulates IL-23 production, mouse bone marrow-derived dendritic cells (BMDCs) and macrophages (BMDMs) were treated with morphine, and infected with S. pneumoniae or stimulated with Toll-like receptor (TLR) and Nod2 ligands. We found that a significant increase in IL-23 protein production was observed in S. pneumoniae, TLR2 ligand lipoteichoic acid (LTA), and TLR4 ligand pneumolysin (PLY) stimulated BMDCs and BMDMs. Interestingly, although Nod2 ligand muramyldipeptide (MDP) alone had no effect on IL-23 production, it potentiated LTA induced IL-23 production to the same level as that observed following S. pneumoniae infection, suggesting that S. pneumoniae induced IL-23 production in DCs involves activation of both TLR2 and Nod2 signaling mechanisms. Furthermore, pretreatment of DCs with MyD88 (myeloid differentiation primary response gene 88) and IL-1 receptor-associated kinase (IRAK) 1/4 inhibitors, or TLR2 antibody diminished the S. pneumoniae induced IL-23 and abolished the inhibitory effects of morphine, indicating that S. pneumoniae induced IL-23 production depends on activation of the TLR2-MyD88-IRAK1/4 signaling pathway. Moreover, morphine decreased S. pneumoniae induced phosphorylation of interferon regulatory factor 3 (IRF3) and activating transcription factor 2 in DCs. Taken together, our study shows that morphine impairs S. pneumoniae induced IL-23 production through MyD88-IRAK1/4-dependent TLR2 and Nod2 signaling in DCs.

Introduction: Sarcoidosis is an inflammatory disease of unknown etiology. However, an infectious cause has been proposed suggesting a role for pattern-recognition receptors, such as Toll-like receptors (TLRs) and nucleotide-binding domain, leucin-rich repeat containing family proteins (NLRs), in the pathogenesis., Objective: Our aim was to investigate whether differences in TLR2 and TLR4 expression, and the response to TLR2, TLR4, and NOD2 stimulation, are associated with sarcoidosis., Materials and Methods: Blood mononuclear cells from sarcoidosis patients (n = 24) and healthy subjects (n = 19) were incubated with the TLR2 ligands PGN and Pam3CSK4, the TLR4 ligand LPS, the NOD2 ligand MDP, or medium alone. After 16 h, monocyte TLR2 and TLR4 expression and cytokine secretion, including TNFalpha, IL-1 beta, IL-6, IL-8, IL-10, and IL-12p70, were measured using flow cytometry and cytometric bead array., Results: TLR2 and TLR4 expression at baseline was significantly higher in patients. Combined TLR2 and NOD2 stimulation induced a four-fold higher secretion of TNFalpha and a 13-fold higher secretion of IL-1 beta in patients. Additionally, there was a synergistic effect of TLR2 with NOD2 stimulation on induction of IL-1 beta in patients, whereas IL-10 was synergistically induced in healthy subjects., Conclusion: Increased TLR expression and enhanced secretion of pro-inflammatory cytokines after combined TLR2 and NOD2 stimulation may be related to the pathogenesis of sarcoidosis.

Systemic infection with Streptococcus pneumoniae is associated with a vigorous pro-inflammatory response to structurally complex cell wall fragments (PnCW) that are shed during cell growth and antibiotic-induced autolysis. Consistent with previous studies, inflammatory cytokine production induced by PnCW was dependent on TLR2 but independent of NOD2, a cytoplasmic NLR protein. However, in parallel with the pro-inflammatory response, we found that PnCW also induced prodigious secretion of anti-inflammatory IL-10 from macrophages. This response was dependent on TLR2, but also involved NOD2 as absence of NOD2-reduced IL-10 secretion in response to cell wall and translated into diminished downstream effects on IL-10-regulated target gene expression. PnCW-mediated production of IL-10 via TLR2 required RIPK2 a kinase required for NOD2 function, and MyD88 but differed from that known for zymosan in that ERK pathway activation was not detected. As mutations in NOD2 are linked to aberrant immune responses, the temporal and quantitative effects of activation of the TLR2-NOD2-RIPK2 pathway on IL-10 secretion may affect the balance between pro- and anti-inflammatory responses to Gram-positive bacteria.

The gut microbiota is vital in the maintenance of homeostasis in the gut immune system. Its diversity and composition play major roles in relation to allergies and inflammatory bowel diseases, and administration of lactic acid bacteria (LAB), such as lactobacilli and bifidobacteria, has positive effects on these pathologies. However, the mechanisms behind the beneficial effects are largely unknown. Here we reveal divergent roles played by Toll-like receptor-2 (TLR2) and nucleotide-binding oligomerization domain-2 (NOD2) in dendritic cell (DC) recognition of LAB. Murine bone-marrow-derived DC lacking NOD2 produce higher levels of interleukin-10 (IL-10) and reduced levels of IL-12 and tumour necrosis factor-alpha (TNF-alpha) in response to LAB. This indicates that peptidoglycan is partly responsible for the T helper type 1 skewing effect of certain LAB. Dendritic cells that are TLR2-/- produce less IL-12 and TNF-alpha and more IL-10 in response to some strains of lactobacilli, while they produce more IL-12 and less IL-10 in response to bifidobacteria. The same tendency was found in human monocyte-derived DC. We have previously reported that the weak IL-12-inducing and TNF-alpha-inducing bifidobacteria inhibit the T helper type 1 skewing effect induced by strong immunostimulatory lactobacilli. Here we show that this immunoinhibitory effect of bifidobacteria is dependent on TLR2 and independent of NOD2. Moreover, independently of the cytokine pattern induced by intact LAB, cell wall fractions of all LAB, as well as synthetic lipoproteins possess immunoinhibitory capacities in both human and murine DC. These novel findings suggest that LAB act as immunoregulators through interaction of lipoprotein with TLR2 and as immunostimulators through interaction of peptidoglycan with NOD2.

Mycobacterium paratuberculosis has been suggested to be involved in the pathogenesis of Crohn's disease (CD). The importance of microorganisms in CD is supported by the association of CD with mutations in the intracellular pathogen recognition receptor (PRR) nucleotide-binding oligomerization domain 2 (NOD2). The aim of this study is to investigate the PRR involved in the recognition of M. paratuberculosis. Methods used include in vitro stimulation of transfected cell lines, murine macrophages, and human PBMC. M. paratuberculosis stimulated human TLR2 (hTLR2)-Chinese hamster ovary (CHO) cells predominantly and hTLR4-CHO cells modestly. Macrophages from TLR2 and TLR4 knockout mice produced less cytokines compared with controls after stimulation with M. paratuberculosis. TLR4 inhibition in human PBMC reduced cytokine production only after stimulation with live M. paratuberculosis. TLR-induced TNF-alpha, IL-1beta, and IL-10 production is mediated through MyD88, whereas Toll-IL-1R domain-containing adaptor inducing IFN-beta (TRIF) promoted the release of IL-1beta. hNOD2-human embryo kidney (HEK) cells, but not hNOD1-HEK cells, responded to stimulation with M. paratuberculosis. PBMC of individuals homozygous for the 3020insC NOD2 mutation showed a 70% defective cytokine response after stimulation with M. paratuberculosis. These results demonstrate that TLR2, TLR4, and NOD2 are involved in the recognition of M. paratuberculosis by the innate immune system.

The gut maintains a delicate balance between the downregulation of inflammatory reactions to commensal bacteria and the capacity to respond to pathogens with vigorous cellular and humoral immune responses. Intestinal epithelial cells, including colonic epithelial cells (CECs) possess many properties of cells of the innate immune system, in particular the ability to recognize and respond to microbial antigens. Recognition of microorganisms by CECs is based upon their recognition of signature molecules, called microbe-associated molecular patterns (MAMP), by pattern recognition receptors (PRR) including membrane toll-like receptors (TLR) and cytosolic Nod2, an intracellular counterpart of TLRs. The purpose of this study was to determine whether primary CECs from normal dogs express a functional TLR2, TLR4, and Nod2 and whether they are regulated by inflammatory mediators. We show that canine primary CECs express TLR2, TLR4, and Nod2 that can be modulated in response to their respective MAMPs, lipopolysaccharides (LPS) or peptidoglycans (PGN). Furthermore, we demonstrate that these receptors are functional as evidenced by the induction of cytokine gene expression in response to LPS or PGN.