Anne Månsson

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 Ca2+ 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.

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, Pam3CSK4, 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‐1β 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‐1β, IL‐6 and IL‐8 production in HNECs, while Pam3CSK4 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)‐κB‐related pathways, while TLR3 was dependent on NF‐κB. In NL‐20, TLR3 signalled via p38, and in HNECs, NF‐κB, 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.

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.

Nod1, Nod2 and Nalp3 receptors, new potential targets in treatment of allergic rhinitis?

To cite this article: Bogefors J, Rydberg C, Uddman R, Fransson M, Månsson A, Benson M, Adner M, Cardell LO. Nod1, Nod2 and Nalp3 receptors, new potential targets in treatment of allergic rhinitis? Allergy 2010; 65: 1222–1226.

NOD‐like receptors in the human upper airways: a potential role in nasal polyposis

To cite this article: Månsson A, Bogefors J, Cervin A, Uddman R, Cardell LO. NOD‐like receptors in the human upper airways: a potential role in nasal polyposis. Allergy 2011; 66: 621–628.

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.

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-ĸB 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.

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.

Activation of eosinophils via Toll-like receptor (TLR)3, TLR7 and TLR9: link between viral infection and asthma?

Asthma is disease characterized by a massive accumulation of eosinophils that release an array of tissue-damaging mediators. Respiratory viral infections are thought to be a leading cause of exacerbations of asthma. One possible explanation might be a direct activation of viral components through Toll-like receptors (TLRs), a receptor family comprising 10 different pathogen-recognizing members (TLR1-TLR10). The virus-recognizing TLRs are TLR3, TLR7/8 and TLR9, which respond to viral dsRNA, ssRNA and CpG-DNA. The present study aimed to investigate the expression of these TLRs and their functions in human eosinophils. Eosinophils were isolated from peripheral blood using magnetic beads (purity >97%). Cells were incubated with or without poly(I:C), R-837 or CpG alone, the synthetic ligands of TLR3, TLR7 and TLR9, respectively, or combined with IL-4 or histamine. Flow cytometry, and ELISA were used to analyze expression of TLRs and various surface markers, viability and secretion of inflammatory mediators. Eosinophils expressed proteins for TLR3, TLR7 and TLR9. Poly(I:C), R-837 and CpG prolonged survival, up-regulated expression of the adhesion molecule CD11b and increased secretion of IL-8 compared to unstimulated controls. These effects were affected by the presence of IL-4 and histamine. This study shows that several viral products directly activate eosinophils through their TLRs. Since eosinophils are central in asthma, the TLR system may be an important mechanism of eosinophil activation linking viral infections with exacerbations. Consequently, this system represents a future clinical target for the resolution of asthmatic disease.