Retinoic acid-inducible gene 1-like receptors in the upper respiratory tract.

BACKGROUND: Retinoic acid-inducible gene 1-like receptors (RLRs) are a novel family of pattern recognition receptors that include retinoic acid-inducible gene 1 (RIG-1), melanoma differentiation-associated gene 5 (MDA-5), and laboratory of genomics and physiology 2 (LGP-2). The knowledge of RLRs and their function in the human airway is limited. This study explores the role of RLRs in the upper respiratory tract. METHODS: Tonsils, adenoids, nasal polyps, and biopsy specimens from the nasal mucosa were examined for the occurrence of the RIG-1, MDA-5, and LGP-2 using real-time reverse-transcription polymerase chain reaction and immunohistochemistry. The nasopharyngeal epithelial cell line FaDu was cultured with the RIG-1/MDA-5 ligand poly(I:C)/LyoVec (Invivogen, San Diego, CA) and analyzed for cytokine release using ELISA. RESULTS: RIG-1, MDA-5, and LGP-2 mRNA were found in all tissues tested. The airway epithelium appeared to be their most prominent location. The RIG-1 and MDA-5 mRNA levels were higher in nasal polyps than in normal nasal mucosa, a state that seemed to be reversed by local steroid treatment. Culture of FaDu with poly(I:C)/LyoVec resulted in IL-6 and IL-8 release. No alteration in RLR expression in tonsils was seen on infection. CONCLUSION: This study shows the presence and functional activity of RLRs in the human upper airways. It also suggests a role for RLRs in nasal polyposis.

NOD-like receptors and RIG-I-like receptors in human eosinophils: activation by NOD1 and NOD2 agonists.

NOD-like receptors (NLRs) and RIG-I-like receptors (RLRs) are newly discovered pattern-recognition receptors. They detect substructures of bacterial peptidoglycan and viral RNA, respectively, thereby initiating an immune response. However, their role in eosinophil activation remains to be explored. The aim of this study was to characterize the expression of a range of NLRs and RLRs in purified human eosinophils and assess their functional importance. Expression of NOD1, NOD2, NLRP3, RIG-I and MDA-5 was investigated using real-time reverse transcription PCR, flow cytometry and immunohistochemistry. The effects of the corresponding agonists iE-DAP (NOD1), MDP (NOD2), alum (NLRP3) and poly(I:C)/LyoVec (RIG-I/MDA-5) were studied in terms of cytokine secretion, degranulation, survival, expression of adhesion molecules and activation markers, and chemotactic migration. Eosinophils expressed NOD1 and NOD2 mRNA and protein. Low levels of RIG-I and MDA-5 were found, whereas expression of NLRP3 was completely absent. In accordance, stimulation with iE-DAP and MDP was found to induce secretion of interleukin-8, up-regulate expression of CD11b, conversely down-regulate CD62 ligand, increase expression of CD69 and induce migration. The MDP also promoted release of eosinophil-derived neurotoxin, whereas iE-DAP failed to do so. No effects were seen upon stimulation with alum or poly(I:C)/LyoVec. Moreover, the NOD1-induced and NOD2-induced activation was mediated via the nuclear factor-κB signalling pathway and augmented by interleukin-5 and granulocyte-macrophage colony-stimulating factor, but not interferon-γ. Taken together, the NLR system represents a novel pathway for eosinophil activation. The responses are enhanced in the presence of cytokines that regulate T helper type 2 immunity, suggesting that the NLRs constitute a link between respiratory infections and exacerbations of allergic disease.

Nucleotide-binding and oligomerization domain-like receptors and retinoic acid inducible gene-like receptors in human tonsillar T lymphocytes.

Nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) and retinoic acid-inducible gene (RIG)-like receptors (RLRs) are recently discovered cytosolic pattern-recognition receptors sensing mainly bacterial components and viral RNA, respectively. Their importance in various cells and disorders is becoming better understood, but their role in human tonsil-derived T lymphocytes remains to be elucidated. In this study, we evaluated expression and functional relevance of NLRs and RLRs in human tonsillar CD3(+) T lymphocytes. Immunohistochemistry, real-time RT-PCR and flow cytometry revealed expression of NOD1, NOD2, NALP1, NALP3, NAIP, IPAF, RIG-1, MDA-5 and LGP-2 at mRNA and protein levels. Because of the limited number of ligands (iE-DAP, MDP, Alum, Poly(I:C)/LyoVec), functional evaluation was restricted to NOD1, NOD2, NALP3 and RIG-1/MDA-5, respectively. Stimulation with the agonists alone was not enough to induce activation but upon triggering via CD3 and CD28, a profound induction of proliferation was seen in purified CD3(+) T cells. However, the proliferative response was not further enhanced by the cognate ligands. Nonetheless, in tonsillar mononuclear cells iE-DAP, MDP and Poly(I:C)/LyoVec were found to augment the CD3/CD28-induced proliferation of tonsillar mononuclear cells. Also, iE-DAP and MDP were found to promote secretion of interleukins 2 and 10 as well as to up-regulate CD69. This study demonstrates for the first time a broad range of NLRs and RLRs in human tonsillar T cells and that NOD1, NOD2 and RIG-1/MDA-5 act synergistically with αCD3 and αCD28 to induce proliferation of human T cells. Hence, these results suggest that these receptors have a role in T-cell activation.

Effects of NOD-like receptors in human B lymphocytes and crosstalk between NOD1/NOD2 and Toll-like receptors.

NLRs are recently discovered PRRs detecting substructures of peptidoglycans and triggering innate immunity. NLRs are expressed in several cell types, but the presence in human B lymphocytes is still unknown. This study aimed to investigate expression and function of NLRs in human B lymphocytes. B cells were isolated and analyzed for mRNA and protein expression. The functional responsiveness of NOD1 and NOD2 was investigated upon stimulation with the cognate ligands, with or without stimulation via IgM/IgD/CD40 and/or selected TLR agonists. A differential expression of NLRs was demonstrated in blood-derived and tonsillar B cells, whereas no variations were found among naive, germinal center, or memory B cells. Stimulation with the ligands alone did not induce B cell activation. However, upon concomitant BCR triggering, an increase in proliferation was seen, together with an induction of cell surface markers (CD27, CD69, CD71, CD80, CD86, and CD95) and prolonged survival. Peripheral B cells were activated by NOD1 and NOD2 ligands, whereas tonsil-derived B cells responded solely to NOD1. In contrast, costimulation with CD40L failed to induce activation. Additionally, it was found that NLR ligands could enhance TLR-induced proliferation of B cells. The present study demonstrates expression of functional NLRs in human B cells. We show that NOD1 and NOD2 have the ability to augment the BCR-induced activation independently of physical T cell help. Hence, NLRs represent a new pathway for B cell activation and a potentially important host defense system against bacterial infections.