NOD1 and NOD2 stimulation triggers innate immune responses of human periodontal ligament cells

Nod-like receptors (NLRs) are cytosolic sensors for microbial molecules. Νucleotide-binding oligomerization domain (NOD)1 and NOD2 recognize the peptidoglycan derivatives, meso-diaminopimelic acid (meso-DAP) and muramyl dipeptide (MDP), respectively, and trigger host innate immune responses. In the present study, we examined the function of NOD1 and NOD2 on innate immune responses in human periodontal ligament (PDL) cells. The gene expression of NOD1 and NOD2 was examined by RT-PCR. IL-6 and IL-8 production in culture supernatants was measured by ELISA. Western blot analysis was performed to determine the activation of NF-κB and MAPK in response to Tri-DAP and MDP. The genes of NOD1 and NOD2 appeared to be expressed in PDL cells. Although the levels of NOD2 expression were weak in intact cells, MDP stimulation increased the gene expression of NOD2 in PDL cells. Tri-DAP and MDP led to the production of IL-6 and IL-8 and the activation of NF-κB and MAPK in PDL cells. Toll-like receptor (TLR) stimulation led to increased gene expression of NOD1 and NOD2 in PDL cells. Pam3CSK4 (a TLR2 agonist) and IFN-γ synergized with Tri-DAP and MDP to produce IL-8 and IL-6 in PDL cells. Our results indicate that NOD1 and NOD2 are functionally expressed in human PDL cells and can trigger innate immune responses.     

The effect of NOD2 activation on TLR2-mediated cytokine responses is dependent on activation dose and NOD2 genotype

The mechanism by which mutations in NOD2 predispose to Crohn's disease (CD) is incompletely understood. In mice, NOD2 has been found to function as a negative regulator of Toll-like receptor 2 (TLR2) signaling. In contrast, studies in humans so far showed no negative regulatory interaction between NOD2 and TLR2, and in fact suggest a synergistic effect between the two. Here, we show that this interaction is dose dependent. Adding low doses of muramyl dipeptide (MDP) to TLR2 primed monocytes results in a significant increase in cytokine production, whereas adding higher doses of MDP led to a striking downregulation of the responses. This downregulation by high-dose MDP does not occur in monocytes from NOD2-deficient patients. The inhibitory role of NOD2 at high concentrations of MDP implicates a safety mechanism to prevent exaggerated antibacterial immune responses in the gut to high or perpetuating bacterial load. This regulatory mechanism is lost in NOD2-deficient CD patients.     

Stimulation of the intracellular bacterial sensor NOD2 programs dendritic cells to promote interleukin-17 production in human memory T cells

How the development of antibacterial T helper 17 (Th17) cells is selectively promoted by antigen-presenting dendritic cells (DCs) is unclear. We showed that bacteria, but not viruses, primed human DCs to promote IL-17 production in memory Th cells through the nucleotide oligomerization domain 2 (NOD2)-ligand muramyldipeptide (MDP), a derivative of bacterial peptidoglycan. MDP enhanced obligate bacterial Toll-like receptor (TLR) agonist induction of IL-23 and IL-1, which promoted IL-17 expression in T cells. The role of NOD2 in this IL-23-IL-1-IL-17 axis could be confirmed in NOD2-deficient DCs, such as DCs from selected Crohn's disease patients. Thus, antibacterial Th17-mediated immunity in humans is orchestrated by DCs upon sensing bacterial NOD2-ligand MDP.     

Mycobacterium paratuberculosis is recognized by Toll-like receptors and 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.     

Immuno-inhibitory PD-L1 can be induced by a peptidoglycan/NOD2 mediated pathway in primary monocytic cells and is deficient in Crohn's patients with homozygous NOD2 mutations

Peptidoglycan (PGN) is a ubiquitous bacterial membrane product that, despite its well known pro-inflammatory properties, has also been invoked in immuno-tolerance of the gastrointestinal tract. PGN-induced mucosal IL-10 secretion and downregulation of Toll like receptors are potential mechanisms of action in the gut but there are few data on tolerogenic adaptive immune responses and PGN. Here, using blood-derived mononuclear cells, we showed that PGN induced marked cell surface expression of PD-L1 but not PD-L2 or CD80/CD86, and specifically in the CD14(+) monocytic fraction. This was reproduced at the gene level with rapid induction (<4 h) and, unlike for LPS stimulation, was still sustained at 24 h. Using transfected and native muramyl dipeptide (MDP), which is a cleavage product of PGN and a specific NOD2 agonist, in assays with wild type cells or those from patients with Crohn's disease carrying the Leu1007 frameshift mutation of NOD2, we showed that (i) both NOD2 dependent and independent signalling (appearing TLR2 mediated) occurred for PGN upregulation of PD-L1 (ii) upregulation is lost in response to MDP in patients with the homozygous mutation and (iii) PD-L1 upregulation was unaffected in patients with heterozygous mutations as previously reported for cytokine responses to MDP. The uptake of PGN and its cleavage products by the intestinal mucosa is well recognised and further work should consider PD-L1 upregulation as one potential mechanism of the commensal flora-driven intestinal immuno-tolerance. Indeed, recent work has shown that loss of PD-L1 signalling in the gut breaks CD8(+) T cell tolerance to self antigen and leads to severe autoimmune enteritis.     

The crosstalk between TLR2 and NOD2 in Aspergillus fumigatus keratitis

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.     

Impaired dendritic cell function in Crohn's disease patients with NOD2 3020insC mutation

The nucleotide oligomerization domain 2 (NOD2) 3020insC (NOD2fs) mutation increases susceptibility to Crohn's disease (CD), but the mechanism remains controversial. Loss-of-function and gain-of-function phenotypes have been described as a result of NOD2fs. Here, we show that dendritic cells (DC) derived from CD patients homozygous for this mutation respond normally to purified Toll-like receptor (TLR) ligands but fail to up-regulate the costimulatory molecules CD80 and CD86 in response to the NOD2 ligand muramyl dipeptide (MDP). Moreover, they lack MDP-induced enhancement of TLR-mediated tumor necrosis factor alpha, interleukin (IL)-12, and IL-10 production, which is observed in control DC with intact NOD2. These data indicate that the NOD2fs mutation results in a loss-of-function phenotype in human myeloid DC and imply decreased immune regulation by IL-10 as a possible mechanism for this mutation in CD.     

NOD2 Stimulation by Staphylococcus aureus-Derived Peptidoglycan Is Boosted by Toll-Like Receptor 2 Costimulation with Lipoproteins in Dendritic Cells

Mutations in the nucleotide-binding oligomerization domain-containing protein 2 (NOD2) play an important role in the pathogenesis of Crohn''s disease. NOD2 is an intracellular pattern recognition receptor (PRR) that senses bacterial peptidoglycan (PGN) structures, e.g., muramyl dipeptide (MDP). Here we focused on the effect of more-cross-linked, polymeric PGN fragments (PGNpol) in the activation of the innate immune system. In this study, the effect of combined NOD2 and Toll-like receptor 2 (TLR2) stimulation was examined compared to single stimulation of the NOD2 receptor alone. PGNpol species derived from a lipoprotein-containing Staphylococcus aureus strain (SA113) and a lipoprotein-deficient strain (SA113 Δlgt) were isolated. While PGNpol constitutes a combined NOD2 and TLR2 ligand, lipoprotein-deficient PGNpolΔlgt leads to activation of the immune system only via the NOD2 receptor. Murine bone marrow-derived dendritic cells (BMDCs), J774 cells, and Mono Mac 6 (MM6) cells were stimulated with these ligands. Cytokines (interleukin-6 [IL-6], IL-12p40, and tumor necrosis factor alpha [TNF-α]) as well as DC activation and maturation parameters were measured. Stimulation with PGNpolΔlgt did not lead to enhanced cytokine secretion or DC activation and maturation. However, stimulation with PGNpol led to strong cytokine secretion and subsequent DC maturation. These results were confirmed in MM6 and J774 cells. We showed that the NOD2-mediated activation of DCs with PGNpol was dependent on TLR2 costimulation. Therefore, signaling via both receptors leads to a more potent activation of the immune system than that with stimulation via each receptor alone.     

Toll‐like receptor‐induced granulocyte‐macrophage colony‐stimulating factor secretion is impaired in Crohn's disease by nucleotide oligomerization domain 2‐dependent and ‐independent pathways

Pattern recognition receptors (PRRs) are an integral part of the innate immune system and govern the early control of foreign microorganisms. Single nucleotide polymorphisms (SNPs) in the intracellular pattern recognition receptor nucleotide‐binding oligomerization domain‐containing protein (NOD2, nucleotide oligomerization domain 2) are associated with Crohn's disease (CD). We investigated the impact of NOD2 polymorphisms on cytokine secretion and proliferation of peripheral blood mononuclear cells (PBMCs) in response to Toll‐like receptor (TLR) and NOD2 ligands. Based on NOD2 SNP analyses, 41 CD patients and 12 healthy controls were studied. PBMCs were stimulated with NOD2 and TLR ligands. After 18 h culture supernatants were measured using multiplex assays for the presence of human cytokines granulocyte–macrophage colony‐stimulating factor (GM‐CSF), interleukin (IL)‐1β and tumour necrosis factor (TNF)‐α. In CD patients, TLR‐induced GM‐CSF secretion was impaired by both NOD2‐dependent and ‐independent mechanisms. Moreover, TNF‐α production was induced by a TLR‐2 ligand, but a down‐regulatory function by the NOD2 ligand, muramyl dipeptide, was impaired significantly in CD patients. Intracellular TLR ligands had minimal effect on GM‐CSF, TNF‐α and IL‐1β secretion. CD patients with NOD2 mutations were able to secrete TNF‐α, but not GM‐CSF, upon stimulation with NOD2 and TLR‐7 ligands. CD patients have impaired GM‐CSF secretion via NOD2‐dependent and ‐independent pathways and display an impaired NOD2‐dependent down‐regulation of TNF‐α secretion. The defect in GM‐CSF secretion suggests a hitherto unknown role of NOD2 in the pathogenesis of CD and is consistent with the hypothesis that impaired GM‐CSF secretion in part constitutes a NOD2‐dependent disease risk factor.     

Synergistic effect of Nod1 and Nod2 agonists with toll-like receptor agonists on human dendritic cells to generate interleukin-12 and T helper type 1 cells

A synthetic Nod2 agonist, muramyldipeptide (MDP), and two Nod1 agonists, FK565 and FK156, mimic the bacterial peptidoglycan moiety and are powerful adjuvants that induce cell-mediated immunity, especially delayed-type hypersensitivity. In this study, we used human dendritic cell (DC) cultures to examine possible T helper type 1 (Th1) responses induced by MDP and FK565/156 in combination with various synthetic Toll-like receptor (TLR) agonists, including synthetic lipid A (TLR4 agonist), the synthetic triacyl lipopeptide Pam3CSSNA (TLR2 agonist), poly(I:C) (TLR3 agonist), and CpG DNA (TLR9 agonist). Immature DCs derived from human monocytes expressed mRNAs for Nod1, Nod2, TLR2, TLR3, TLR4, and TLR9. The stimulation of DCs with MDP and FK565 in combination with lipid A, poly(I:C), and CpG DNA, but not with Pam3CSSNA, synergistically induced interleukin-12 (IL-12) p70 and gamma interferon (IFN-gamma), but not IL-18, in culture supernatants and induced IL-15 on the cell surface. In correlation with the cytokine induction, an upregulation of the mRNA expression of these cytokine genes was observed. Notably, IL-12 p35 mRNA expression increased >1,000-fold upon stimulation with lipid A plus either MDP or FK565 compared with stimulation with each stimulant alone. In contrast, for the expression of CD83 and costimulatory molecules such as CD40, CD80, and CD86, no synergistic effects were observed upon stimulation with Nod plus TLR agonists. The culture supernatants of DCs stimulated with lipid A plus either MDP or FK565 activated human T cells to produce high levels of IFN-gamma, and the activity was attributable to DC-derived IL-12. These findings suggest that Nod1 and Nod2 agonists in combination with TLR3, TLR4, and TLR9 agonists synergistically induce IL-12 and IFN-gamma production in DCs to induce Th1-lineage immune responses.     

The role of porcine reproductive and respiratory syndrome virus infection in immune phenotype and Th1/Th2 balance of dendritic cells

The aim of this study was to characterize the immune response of dendritic cells derived from monocytes (Mo-DCs) in the porcine peripheral blood following infection with porcine reproductive and respiratory syndrome virus (PRRSV). Viral load assays indicated that PRRSV efficiently infected Mo-DCs but failed to replicate, whereas PRRSV infection of Mo-DCs decreased the expression of SLA-I, SLA-II, CD80 and CD40 compared with those of mock Mo-DCs. Furthermore, we analyzed the cytokine profiles using quantitative RT-PCR and ELISA. Results indicated apparent changes in IL-10 and IL-12 p40 expression but not in IFN-γ and TNF-α among Mo-DCs infected with PRRSV and uninfected Mo-DCs. Additionally, flow cytometry analysis of the altered Mo-DCs together with IL-4 and GM-CSF induction for 7days revealed the typical morphology and phenotype with 91.73% purity before infection with PRRSV. Overall, our data demonstrate that PRRSV impaired the normal antigen presentation of Mo-DCs and led to inadequate adaptive immune response by down-regulating the expression of SLA-I,SLA-II, CD80 and CD40. Enhanced Th2 -type cytokine IL-10 secretion and reduced Th1-type cytokines IL-12p40,IFN-γ and TNF-α secretion results in Th1/Th2 imbalance.     

Tim-3 regulates pro- and anti-inflammatory cytokine expression in human CD14+ monocytes

Tim-3 and PD-1 are powerful immunoinhibitory molecules involved in immune tolerance, autoimmune responses, and antitumor or antiviral immune evasion. A current model for Tim-3 regulation during immune responses suggests a divergent function, such that Tim-3 acts synergistically with TLR signaling pathways in innate immune cells to promote inflammation, yet the same molecule terminates Th1 immunity in adaptive immune cells. To better understand how Tim-3 might be functioning in innate immune responses, we examined the kinetics of Tim-3 expression in human CD14+ M/M(Ф) in relation to expression of IL-12, a key cytokine in the transition of innate to adaptive immunity. Here, we show that Tim-3 is constitutively expressed on unstimulated peripheral blood CD14+ monocytes but decreases rapidly upon TLR stimulation. Conversely, IL-12 expression is low in these cells but increases rapidly in CD14+ M/M(Ф) in correlation with the decrease in Tim-3. Blocking Tim-3 signaling or silencing Tim-3 expression led to a significant increase in TLR-mediated IL-12 production, as well as a decrease in activation-induced up-regulation of the immunoinhibitor, PD-1; TNF-α production was not altered significantly, but IL-10 production was increased. These results suggest that Tim-3 has a role as a regulator of pro- and anti-inflammatory innate immune responses.     

Monocyte-derived dendritic cells from breast cancer patients are biased to induce CD4+CD25+Foxp3+ regulatory T cells

DCs orchestrate immune responses contributing to the pattern of response developed. In cancer, DCs may play a dysfunctional role in the induction of CD4(+)CD25(+)Foxp3(+) Tregs, contributing to immune evasion. We show here that Mo-DCs from breast cancer patients show an altered phenotype and induce preferentially Tregs, a phenomenon that occurred regardless of DC maturation stimulus (sCD40L, cytokine cocktail, TNF-α, and LPS). The Mo-DCs of patients induced low proliferation of allogeneic CD3(+)CD25(neg)Foxp3(neg) cells, which after becoming CD25(+), suppressed mitogen-stimulated T cells. Contrastingly, Mo-DCs from healthy donors induced a stronger proliferative response, a low frequency of CD4(+)CD25(+)Foxp3(+) with no suppressive activity. Furthermore, healthy Mo-DCs induced higher levels of IFN-γ, whereas the Mo-DCs of patients induced higher levels of bioactive TGF-β1 and IL-10 in cocultures with allogeneic T cells. Interestingly, TGF-β1 blocking with mAb in cocultures was not enough to completely revert the Mo-DCs of patients' bias toward Treg induction. Altogether, these findings should be considered in immunotherapeutic approaches for cancer based on Mo-DCs.