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.

B cell activation by outer membrane vesicles--a novel virulence mechanism.

Secretion of outer membrane vesicles (OMV) is an intriguing phenomenon of Gram-negative bacteria and has been suggested to play a role as virulence factors. The respiratory pathogens Moraxella catarrhalis reside in tonsils adjacent to B cells, and we have previously shown that M. catarrhalis induce a T cell independent B cell response by the immunoglobulin (Ig) D-binding superantigen MID. Here we demonstrate that Moraxella are endocytosed and killed by human tonsillar B cells, whereas OMV have the potential to interact and activate B cells leading to bacterial rescue. The B cell response induced by OMV begins with IgD B cell receptor (BCR) clustering and Ca(2+) mobilization followed by BCR internalization. In addition to IgD BCR, TLR9 and TLR2 were found to colocalize in lipid raft motifs after exposure to OMV. Two components of the OMV, i.e., MID and unmethylated CpG-DNA motifs, were found to be critical for B cell activation. OMV containing MID bound to and activated tonsillar CD19(+) IgD(+) lymphocytes resulting in IL-6 and IgM production in addition to increased surface marker density (HLA-DR, CD45, CD64, and CD86), whereas MID-deficient OMV failed to induce B cell activation. DNA associated with OMV induced full B cell activation by signaling through TLR9. Importantly, this concept was verified in vivo, as OMV equipped with MID and DNA were found in a 9-year old patient suffering from Moraxella sinusitis. In conclusion, Moraxella avoid direct interaction with host B cells by redirecting the adaptive humoral immune response using its superantigen-bearing OMV as decoys.

TLR3 in human eosinophils: functional effects and decreased expression during allergic rhinitis.

BACKGROUND/AIM: Viral respiratory infections are increasingly implicated in allergic exacerbations. Virus-induced activation of eosinophils through Toll-like receptors (TLRs) could be involved. The present study was designed to examine TLR3 expression in eosinophils from bone marrow (BM) and peripheral blood (PB) during symptomatic allergic rhinitis, and to evaluate the functional responsiveness of TLR3 in purified eosinophils. METHODS: BM and PB samples were obtained from healthy volunteers and patients with seasonal allergic rhinitis outside and during the pollen season. Eosinophils were analyzed for TLR3 expression by flow cytometry. Polyinosinic:polycytidylic acid [poly(I:C)], an agonist for TLR3, was used to assess its functional role in purified eosinophils and the intracellular signaling pathways involved. RESULTS: TLR3 expression was demonstrated in BM and PB eosinophils. It was higher in BM-derived than in circulating cells and it was downregulated in both compartments during symptomatic allergic rhinitis. TLR3 expression was also downregulated in the presence of interleukin (IL)-4 and IL- 5. Stimulation with poly(I:C) increased the percentage of CD11b+ cells and enhanced the secretion of IL-8, effects mediated via the p38 mitogen-activated protein kinases and nuclear factor-kappaB signaling pathways. Moreover, pretreatment with IL-5 augmented the poly(I:C)-induced IL-8 release. CONCLUSIONS: Eosinophils activated via TLR3 might be more able to home and recruit leukocytes to sites of inflammation. The decreased TLR3 expression during symptomatic allergic rhinitis and in the presence of Th2 cytokines indicates a role in allergic airway inflammation. Thus, eosinophils might function as a link between viral infections and exacerbations of allergic disease.

Toll-like receptor agonists induce inflammation and cell death in a model of head and neck squamous cell carcinomas.

Toll-like receptors (TLRs) are increasingly implicated in the pathogenesis of cancer. The present study describes TLR expression and function in healthy and malignant airway epithelial cells. The squamous cell carcinoma cell line Detroit-562 was compared with the healthy bronchial epithelial cell line NL-20 and primary human nasal epithelial cells (HNECs). TLR2, TLR3 and TLR5 were present in primary head and neck squamous cell carcinomas (HNSCCs). Consistent with this, Detroit-562 expressed TLR2, TLR3 and TLR5, whereas NL-20 expressed mainly TLR3 and HNECs expressed TLR2-5. In Detroit-562, Pam(3)CSK(4), poly(I:C) and flagellin, ligands for TLR2, TLR3 and TLR5, respectively, induced an up-regulation of intercellular adhesion molecule 1 (ICAM-1), an increase in interleukin (IL)-6 and IL-8 secretion and a decrease in cell viability. Additionally, poly(I:C) affected IL-1beta production and the migratory behaviour of Detroit-562. NL-20 responded with a slight increase in IL-8 secretion upon poly(I:C) stimulation. Poly(I:C) induced a small increase in IL-1beta, IL-6 and IL-8 production in HNECs, while Pam(3)CSK(4) increased viability. The TLR signalling was transcription-dependent, but the pathways involved differed among TLRs as well as cells. In Detroit-562, TLR2 and TLR5 activation was mediated via c-jun N-terminal kinase (JNK)-, p38-, phosphatidylinositol 3-kinase (PI3K)- and nuclear factor (NF)-kappaB-related pathways, while TLR3 was dependent on NF-kappaB. In NL-20, TLR3 signalled via p38, and in HNECs, NF-kappaB, JNK and extracellular signal-regulated kinase (ERK) appeared to be involved. We found that TLR agonists induced a robust response in HNSCCs, characterized by generation of inflammation and cell death. A similar response was not seen in normal epithelial cells. Thus, the TLR system should be considered an important target in future antitumour immunotherapy.

Role of atopic status in Toll-like receptor (TLR)7- and TLR9-mediated activation of human eosinophils.

Viral respiratory infections are increasingly implicated in allergic exacerbations. The mechanisms behind this are not known, but a virus-induced activation of eosinophils through TLRs could be involved. Herein, we investigated the expression and function of TLR7 and TLR9 in purified eosinophils from peripheral blood and assessed their role in allergic airway inflammation. Eosinophils expressed TLR7 and TLR9 proteins. Stimulation with the cognate ligands R-837 and CpG was found to prolong survival, up-regulate expression of CD11b and conversely down-regulate L-selectin expression, increase expression of the activation marker CD69, facilitate the chemotactic migration, and enhance IL-8 secretion by eosinophils. Also, CpG induced release of eosinophil-derived neurotoxin (EDN), and R-837 failed to do so. Analogously, eosinophils activated by CpG, but not R-837, promoted airway epithelial cell death and cytokine release. Priming with the allergic mediators histamine, IL-4, and most prominently IL-5, augmented the TLR-induced IL-8 and EDN secretion, revealing an ability to sensitize eosinophils for TLR7 and TLR9 activation. Moreover, the TLR responses of eosinophils were higher in allergic as compared with healthy subjects, manifested by an increase in IL-8 and EDN release. Correspondingly, allergic subjects displayed an elevated serum level of IL-5, suggesting increased IL-5-mediated priming. This study shows that activation via TLR7 and TLR9 affects several eosinophil functions and that the atopic status of the donor and the presence of a Th2-like cytokine milieu affect the outcome of the response. Thus, eosinophil activation via TLR7 and TLR9 might engender a link between viral infection and allergic exacerbations.

Differentiated S100A7 expression in infected tonsils and tonsils from allergic individuals.

Palatine tonsils are continuously exposed to microorganisms and antigens and secrete antimicrobial peptides as a first line of defense. S100A7 is a protein with antimicrobial and chemotactic properties. Our aim was to investigate how the expression of S100A7 in human palatine tonsils is affected by inflammatory processes. Tonsils obtained from 109 patients undergoing tonsillectomy were divided into groups of infected and noninfected as well as allergic and nonallergic, based on the results from tonsillar core culture tests and Phadiatop analysis, respectively. Western blot and immunohistochemistry were used to assess protein expression and real-time PCR was used to quantify mRNA levels. To explore the induction of S100A7, tonsils were stimulated with lipopolysaccharide in vitro. The immunohistochemical staining for S100A7 was most intense in the tonsillar epithelium, but the protein was also detected in B- and T-cell regions, which was confirmed with Western blot on isolated B and T cells. The S100A7 expression appeared to be the highest in CD8+ T cells. Reduced mRNA levels of S100A7 were detected in infected tonsils as well as in tonsils from allergic individuals. In vitro stimulation of tonsils with lipopolysaccharide did not have any effect on the expression. The results suggest a role for S100A7 in recurrent tonsillitis and allergic disease.

A distinct Toll-like receptor repertoire in human tonsillar B cells, directly activated by PamCSK, R-837 and CpG-2006 stimulation.

Toll-like receptors (TLRs) recognize specific pathogen-associated molecular patterns (PAMPs), which subsequently trigger innate immunity. Recent data also suggest a role for TLRs in the direct activation of adaptive immune cells. In the present study, the expression and function of TLR1-TLR10 were characterized in purified human tonsillar B cells, focusing on differences among CD19+ CD38- CD27- (naïve B cells), CD19+ IgD- CD27-[germinal centre (GC) B cells] and CD19+ CD38- CD27+ (memory B cells) cells. The study was also designed to compare the TLR expression in B cells obtained from infected and hyperplastic tonsils that served as controls. The results demonstrated a distinct repertoire of TLRs, in which TLR1, TLR2, TLR7, TLR9 and TLR10 predominated. No differences were found among naïve, GC and memory B cells. Tonsillar infection did not substantially alter the TLR expression profile in ex vivo-isolated B-cell subsets. Purified CD19+ B cells stimulated with Pam3CSK4, R-837 and CpG oligodeoxynucleotide (ODN) 2006, via TLR1/TLR2, TLR7 and TLR9, respectively, showed an induction of interleukin-6 secretion and an up-regulated expression of human leucocyte antigen (HLA)-DR. Collectively, the present study demonstrates that B cells exhibit constitutively high levels of specific TLRs, which are not developmentally regulated during the B-cell differentiation process. Ongoing microbial infections, such as chronic tonsillitis, do not appear to affect the TLR profile in B cells. Furthermore, the distinct expression of TLRs allows B cells to respond directly to the cognate PAMPs. This further emphasizes the role of TLRs in directly activating adaptive immune cells.

Toll-like receptors in cellular subsets of human tonsil T cells: altered expression during recurrent tonsillitis.

BACKGROUND: The palatine tonsils have a pivotal role in immunological detection of airborne and ingested antigens like bacteria and viruses. They have recently been demonstrated to express Toll-like receptors (TLRs), known to recognize molecular structures on such microbes and activate innate immune responses. Their activation might also provide a link between innate and adaptive immunity. In the present study, the expression profile of TLR1-TLR10 was characterized in human tonsil T cells, focusing on differences between subsets of CD4+ T helper (Th) cells and CD8+ cytotoxic T lymphocytes (CTL). The study was also designed to compare the TLR expression in T cells from patients with recurrent tonsillitis and tonsillar hyperplasia. METHODS: Tonsils were obtained from children undergoing tonsillectomy, and classified according to the clinical diagnoses and the outcome of tonsillar core culture tests. Two groups were defined; recurrently infected tonsils and hyperplastic tonsils that served as controls. Subsets of T cells were isolated using magnetic beads. The expression of TLR transcripts in purified cells was assessed using quantitative real-time RT-PCR. The corresponding protein expression was investigated using flow cytometry and immunohistochemistry. RESULTS: T cells expressed a broad repertoire of TLRs, in which TLR1, TLR2, TLR5, TLR9 and TLR10 predominated. Also, a differential expression of TLRs in CD4+ and CD8+ T cells was obtained. TLR1 and TLR9 mRNA was expressed to a greater extent in CD4+ cells, whereas expression of TLR3 mRNA and protein and TLR4 protein was higher in CD8+ cells. CD8+ cells from infected tonsils expressed higher levels of TLR2, TLR3 and TLR5 compared to control. In contrast, CD4+ cells exhibited a down-regulated TLR9 as a consequence of infection. CONCLUSION: The present study demonstrates the presence of a broad repertoire of TLRs in T cells, a differential expression in CD4+ and CD8+ cells, along with infection-dependent alterations in TLR expression. Collectively, these results support the idea that TLRs are of importance to adaptive immune cells. It might be that TLRs have a direct role in adaptive immune reactions against infections. Thus, further functional studies of the relevance of TLR stimulation on T cells will be of importance.