Susanna Kumlien Georén

Toll-Like Receptor Ligands LPS and Poly (I:C) Exacerbate Airway Hyperresponsiveness in a Model of Airway Allergy in Mice, Independently of Inflammation

It is well-established that bacterial and viral infections have an exacerbating effect on allergic asthma, particularly aggravating respiratory symptoms, such as airway hyperresponsiveness (AHR). The mechanism by which these infections alter AHR is unclear, but some studies suggest that Toll-like receptors (TLRs) play a role. In this study, we investigated the impact of TLR3 and TLR4 ligands on AHR and airway inflammation in a model of pre-established allergic inflammation. Female BALB/c mice were sensitised and challenged intranasally (i.n.) with either PBS or ovalbumin (OVA) and subsequently i.n. challenged with poly (I:C) (TLR3) or LPS (TLR4) for four consecutive days. The response to methacholine was measured in vivo; cellular and inflammatory mediators were measured in blood, lung tissue and broncheoalveolar lavage fluid (BALF). OVA challenge resulted in an increase in AHR to methacholine, as well as increased airway eosinophilia and TH2 cytokine production. Subsequent challenge with TLR agonists resulted in a significant increase in AHR, but decreased TLR-specific cellular inflammation and production of immune mediators. Particularly evident was a decline in LPS-induced neutrophilia and neutrophil-associated cytokines following LPS and poly (I:C) treatment. The present data indicates that TLRs may play a pivotal role in AHR in response to microbial infection in allergic lung inflammation. These data also demonstrate that aggravated AHR occurs in the absence of an exacerbation in airway inflammation and that allergic inflammation impedes a subsequent inflammatory response to TLRs. These results may parallel clinical signs of microbial asthma exacerbation, including an extended duration of illness and increased respiratory symptoms.

Intranasal Administration of poly(I:C) and LPS in BALB/c Mice Induces Airway Hyperresponsiveness and Inflammation via Different Pathways

Background Bacterial and viral infections are known to promote airway hyperresponsiveness (AHR) in asthmatic patients. The mechanism behind this reaction is poorly understood, but pattern recognizing Toll-like receptors (TLRs) have recently been suggested to play a role. Materials and Methods To explore the relation between infection-induced airway inflammation and the development of AHR, poly(I:C) activating TLR3 and LPS triggering TLR4, were chosen to represent viral and bacterial induced interactions, respectively. Female BALB/c or MyD88-deficient C57BL/6 mice were treated intranasally with either poly(I:C), LPS or PBS (vehicle for the control group), once a day, during 4 consecutive days. Results When methacholine challenge was performed on day 5, BALB/c mice responded with an increase in airway resistance. The maximal resistance was higher in the poly(I:C) and LPS treated groups than among the controls, indicating development of AHR in response to repeated TLR activation. The proportion of lymphocytes in broncheoalveolar lavage fluid (BALF) increased after poly(I:C) treatment whereas LPS enhanced the amount of neutrophils. A similar cellular pattern was seen in lung tissue. Analysis of 21 inflammatory mediators in BALF revealed that the TLR response was receptor-specific. MyD88-deficient C57BL/6 mice responded to poly (I:C) with an influx of lymphocytes, whereas LPS caused no inflammation. Conclusion In vivo activation of TLR3 and TLR4 in BALB/c mice both caused AHR in conjunction with a local inflammatory reaction. The AHR appeared to be identical regardless of which TLR that was activated, whereas the inflammation exhibited a receptor specific profile in terms of both recruited cells and inflammatory mediators. The inflammatory response caused by LPS appeared to be dependent on MyD88 pathway. Altogether the presented data indicate that the development of AHR and the induction of local inflammation might be the result of two parallel events, rather than one leading to another.

Functional Effects of Toll-Like Receptor (TLR)3, 7, 9, RIG-I and MDA-5 Stimulation in Nasal Epithelial Cells

Background The human nasal epithelium is an important physical barrier, and a part of the innate immune defense that protect against pathogens. The epithelial cells recognize microbial components by pattern-recognition receptors (PRRs), and thereby trigger an immune response. Even though TLR3, TLR7, TLR9, RIG-I and MDA-5 are all known to respond to viral stimulation, their potential role in chronic airway inflammation triggered by local cytokine release remains to be established. Methods mRNA and corresponding protein expression of TLR3, TLR7, TLR9, RIG-I and MDA-5 were analyzed in nasal biopsies and various upper airway epithelial cell lines using real-time reverse transcription PCR, immunohistochemistry and flow cytometry. Ligand induced, cytokine release, was evaluated with ELISA. Results Nasal biopsies were found to express TLR3, TLR7, TLR9, RIG-I and MDA-5, with the most abundant expression in the surface epithelium. These receptors were verified in primary human nasal epithelial cell (HNEC) as well as in the airway epithelial cell lines Detroit-562 and FaDu. Poly(I:C) (TLR3) and R-837 (TLR7) stimulation increased secretion of IL-6 and GM-CSF from the nasal mucosa and the epithelial cell lines. CpG (TLR9) stimulation caused release of IL-8 in the nasal mucosa and in FaDu. Poly(I:C)/LyoVec (RIG-I/MDA-5) stimulation activated the secretion of IFN-β in the nasal mucosa. A corresponding release was also detected from HNEC and Detroit-562. Conclusion The nasal epithelium has the ability to recognize viral intrusion through TLR and RLR receptors, and the subsequent response might have a role in exacerbation of inflammatory diseases like allergic rhinitis and chronic rhinosinusitis.

Significance of endogenous glucocorticoid sensitivity for airway eosinophilia in a murine model of allergy.

Conclusions Endogenous GC protects against allergic inflammatory responses in the airways. These effects are modulated by both peripheral blockade and inhibition of release. Individual response patterns to stress, i.e. corticosterone release and peripheral sensitivity, may influence both the central and peripheral levels of the allergic airway reaction in patients. Objective Glucocorticoids (GCs) modulate the allergic inflammatory response. Acute or chronic stress will influence circulating levels of GCs, rates of secretion, metabolism and target tissue sensitivity. In a clinical situation, stress may exacerbate or attenuate the asthmatic reaction. The aim of this study was to investigate the effects of inhibition of endogenous GC in an allergic airway inflammation model in the mouse. Material and methods An ovalbumin model using i.p. sensitization and intra-nasal challenge was used for respiratory eosinophilic inflammation. GC release was inhibited by administration of metyrapone (ME), and peripheral glucocorticoid receptors were blocked by administration of RU486 (RU). Results Inhibition with RU and ME increased eosinophilia in the bone marrow compared to controls (p<0.05). Eosinophilia in bronchoalveolar lavage fluid increased in the sensitized groups compared to controls, but there were no differences between the sensitized groups. CD3+ and CD4+ cells were increased in the nasal mucosa as a result of treatment with RU and ME.

Pulmonary exposure to single-walled carbon nanotubes does not affect the early immune response against Toxoplasma gondii.

BackgroundSingle-walled carbon nanotubes (SWCNT) trigger pronounced inflammation and fibrosis in the lungs of mice following administration via pharyngeal aspiration or inhalation. Human exposure to SWCNT in an occupational setting may occur in conjunction with infections and this could yield enhanced or suppressed responses to the offending agent. Here, we studied whether the sequential exposure to SWCNT via pharyngeal aspiration and infection of mice with the ubiquitous intracellular parasite Toxoplasma gondii would impact on the immune response of the host against the parasite.MethodsC57BL/6 mice were pre-exposed by pharyngeal administration of SWCNT (80 + 80 μg/mouse) for two consecutive days followed by intravenous injection with either 1x103 or 1x104 green fluorescence protein and luciferase-expressing T. gondii tachyzoites. The dissemination of T. gondii was monitored by in vivo bioluminescence imaging in real time for 7 days and by plaque formation. The inflammatory response was analysed in bronchoalveolar lavage (BAL) fluid, and by assessment of morphological changes and immune responses in lung and spleen.ResultsThere were no differences in parasite distribution between mice only inoculated with T. gondii or those mice pre-exposed for 2 days to SWCNT before parasite inoculum. Lung and spleen histology and inflammation markers in BAL fluid reflected the effects of SWCNT exposure and T. gondii injection, respectively. We also noted that CD11c positive dendritic cells but not F4/80 positive macrophages retained SWCNT in the lungs 9 days after pharyngeal aspiration. However, co-localization of T. gondii with CD11c or F4/80 positive cells could not be observed in lungs or spleen. Pre-exposure to SWCNT did not affect the splenocyte response to T. gondii.ConclusionsTaken together, our data indicate that pre-exposure to SWCNT does not enhance or suppress the early immune response to T. gondii in mice.

Toll-like receptor (TLR) 7 decreases and TLR9 increases the airway responses in mice with established allergic inflammation

Toll-like receptor (TLR) 7 and TLR9 recognise microbial products of viral descent. Since viruses are a common trigger of asthma exacerbations these TLRs have emerged as interesting therapeutic targets. Even though their effects on allergic inflammation have been evaluated in several models their effects on established allergic airway inflammation remains to be described. Therefore, mice with an on-going ovalbumin (OVA)-induced allergic airway inflammation were given R848 or CpG (TLR7 and TLR9 agonists, respectively) intranasally during four consecutive days. At day five, the R848 treatment had reduced OVA-induced airway hyperresponsiveness (measured as the increased resistance to methacholine), counteracted the accompanying influx of eosinophils and macrophages, and decreased the OVA-enhanced release of interleukin (IL)-5 and leukotriene (LT) B4 in bronchoalveolar lavage fluid. CpG, which by itself caused airway hyperresponsiveness, did not influence the OVA-induced airway hyperresponsiveness, and release of IL-5 and LTB4, but decreased the OVA-induced influx of cells in bronchoalveolar lavage fluid, and increased the amount of pro-inflammatory mediators like IL-12, CXCL1 and CXCL9. To conclude, TLR7 dampens the allergic airway reactivity and local inflammation, whereas TLR9 that causes airway hyperresponsiveness and increased cellular response per se, do generally not interfere with the effects induced by allergic inflammation.

NET‐producing CD16high CD62Ldim neutrophils migrate to tumor sites and predict improved survival in patients with HNSCC

The concept of functional neutrophil subsets is new and their clinical significance in malignancies is unknown. Our study investigated the role of CD16dim CD62Lhigh, CD16high CD62Lhigh and CD16high CD62Ldim neutrophil subsets in head and neck squamous cell carcinoma (HNSCC) patients. These neutrophil subsets may play different roles in immune‐related activity in cancer, based on their profile, activation state and migration ability within a tumor site, which may be important in predicting cancer prognoses. Tumor biopsies and blood were obtained from newly diagnosed untreated HNSCC patients and healthy controls. Neutrophil subsets and their phenotype were characterized using flow cytometry. Isolated granulocytes were assessed for anti‐tumor immune functions. Compared to controls HNSCC patients exhibited increased CD16high CD62Ldim neutrophils in blood; this subset displayed a distinct phenotypes with high expression of CD11b and CD18. This subset was prone to migrate into the tumor facilitated by tumor‐derived IL‐8. Furthermore, IL‐8 was also found to activate neutrophils and thereby promoting subset transition. Various assays demonstrated that activated CD16high CD62Ldim neutrophils inhibited migration, proliferation and induced apoptosis of FaDu cancer cells. Neutrophil elastase detected in activated CD16high CD62Ldim neutrophils and tumor biopsies suggested that CD16high CD62Ldim neutrophils impart anti‐tumoral activity via neutrophil extracellular traps. Furthermore, increased fraction of CD16high CD62Ldim neutrophils was shown to correlate with an increased survival rate. Our study demonstrates the clinical relevance of the CD16high CD62Ldim neutrophil subset, providing evidence for its increased migration capacity, its anti‐tumor activity including increased NET formation and finally its correlation with increased survival in HNSCC patients.

TNFα-blockade stabilizes local airway hyperresponsiveness during TLR-induced exacerbations in murine model of asthma

Viral infections are a common cause of asthma exacerbation. These maladies are sometimes complicated by bacterial infections. Toll-like receptors (TLRs) are in the forefront of our microbial defence, with TLR3 responding to viral and TLR4 to bacterial stimulation. The present study was designed to evaluate the effect of concomitant TLR3 and TLR4 stimulation in a murine model of allergic asthma.BALB/c mice were stimulated intranasally with a combination of poly(I:C) and LPS activating TLR3 and TLR4, respectively. This resulted in the development of airway hyperresponsiveness (AHR) in the proximal part of the lung, along with signs of neutrophilic inflammation. Analysis of the bronchioalveolar lavage fluid (BALF) revealed a marked increase in TNFα. In contrast, the allergic airway inflammation induced by ovalbumin administration to sensitized mice caused AHR in the whole lung along with an increase in eosinophils and lymphocytes in the BALF and lung.When poly(I:C) + LPS were given to mice with an ongoing allergic airway inflammation induced by ovalbumin, the AHR was further increased in the peripheral lung and neutrophils appeared together with eosinophils and lymphocytes in the BALF and lung. Treatment with the TNFα-blocking antibody infliximab blunted the AHR increase, without affecting the cells influx in BALF.To conclude; a combined TLR3- and TLR4-stimulation, representing a concomitant viral and bacterial infection, causes an AHR that is further exaggerated during an ongoing allergic inflammation. The airway stabilizing effect of infliximab indicates the possible future use of TNFα blockade in treatment of microbial induced exacerbations of allergic asthma.

Deprived TLR9 expression in apparently healthy nasal mucosa might trigger polyp-growth in chronic rhinosinusitis patients.

Background The origin of nasal polyps in chronic rhinosinusitis is unknown, but the role of viral infections in polyp growth is clinically well established. Toll-like receptors (TLRs) have recently emerged as key players in our local airway defense against microbes. Among these, TLR9 has gained special interest in viral diseases. Many studies on chronic rhinosinusitis with nasal polyps (CRSwNP) compare polyp tissue with nasal mucosa from polyp-free individuals. Knowledge about changes in the turbinate tissue bordering the polyp tissue is limited. Objectives To analyse the role of TLR9 mediated microbial defense in tissue bordering the polyp. Methods Nasal polyps and turbinate tissue from 11 patients with CRSwNP and turbinate tissue from 11 healthy controls in total were used. Five biopsies from either group were analysed immediately with flow cytometry regarding receptor expression and 6 biopsies were used for in vitro stimulation with a TLR9 agonist, CpG. Cytokine release was analysed using Luminex. Eight patients with CRSwNP in total were intranasally challenged with CpG/placebo 24 hours before surgery and the biopsies were collected and analysed as above. Results TLR9 expression was detected on turbinate epithelial cells from healthy controls and polyp epithelial cells from patients, whereas TLR9 was absent in turbinate epithelial cells from patients. CpG stimulation increased the percentage cells expressing TLR9 and decreased percentage cells expressing VEGFR2 in turbinate tissue from patients. After CpG stimulation the elevated levels of IL-6, G-CSF and MIP-1β in the turbinate tissue from patients were reduced towards the levels demonstrated in healthy controls. Conclusion Defects in the TLR9 mediated microbial defense in the mucosa adjacent to the anatomic origin of the polyp might explain virus induced polyp growth. CpG stimulation decreased VEGFR2, suggesting a role for CpG in polyp formation. The focus on turbinate tissue in patients with CRSwNP opens new perspectives in CRSwNP-research.

Antigen-presenting epithelial cells can play a pivotal role in airway allergy

Results Human nasal epithelial cells were shown to take up dextran, which ended up in intracellular endosome-like structures. In addition, MHC class II and co-stimulatory molecules were found on human and mouse nasal epithelial cells. Functionally, nasal epithelial cells from ovalbumin-sensitized mice activated and induced antigen-specific proliferation of naive OT-II CD4+ T cells in vitro. A similar activation was not seen in naive MNECs. Finally, nasal epithelial cells from allergic rhinitis patients were able to activate autologous T cells against Bet v 1 and induce IL-13 release.