| 1. |
- Erjefält, Jonas S., et al.
(author)
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Diffuse alveolar damage patterns reflect the immunological and molecular heterogeneity in fatal COVID-19
- 2022
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In: EBioMedicine. - : Elsevier BV. - 2352-3964. ; 83
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Journal article (peer-reviewed)abstract
- Background: Severe COVID-19 lung disease exhibits a high degree of spatial and temporal heterogeneity, with different histological features coexisting within a single individual. It is important to capture the disease complexity to support patient management and treatment strategies. We provide spatially decoded analyses on the immunopathology of diffuse alveolar damage (DAD) patterns and factors that modulate immune and structural changes in fatal COVID-19. Methods: We spatially quantified the immune and structural cells in exudative, intermediate, and advanced DAD through multiplex immunohistochemistry in autopsy lung tissue of 18 COVID-19 patients. Cytokine profiling, viral, bacteria, and fungi detection, and transcriptome analyses were performed. Findings: Spatial DAD progression was associated with expansion of immune cells, macrophages, CD8+ T cells, fibroblasts, and (lymph)angiogenesis. Viral load correlated positively with exudative DAD and negatively with disease/hospital length. In all cases, enteric bacteria were isolated, and Candida parapsilosis in eight cases. Cytokines correlated mainly with macrophages and CD8+T cells. Pro-coagulation and acute repair were enriched pathways in exudative DAD whereas intermediate/advanced DAD had a molecular profile of elevated humoral and innate immune responses and extracellular matrix production. Interpretation: Unraveling the spatial and molecular immunopathology of COVID-19 cases exposes the responses to SARS-CoV-2-induced exudative DAD and subsequent immune-modulatory and remodeling changes in proliferative/advanced DAD that occur side-by-side together with secondary infections in the lungs. These complex features have important implications for disease management and the development of novel treatments. Funding: CNPq, Bill and Melinda Gates Foundation, HC-Convida, FAPESP, Regeneron Pharmaceuticals, and the Swedish Heart & Lung Foundation.
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| 2. |
- Braekeveldt, Noémie, et al.
(author)
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Neuroblastoma patient-derived orthotopic xenografts reflect the microenvironmental hallmarks of aggressive patient tumours
- 2016
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In: Cancer Letters. - : Elsevier BV. - 1872-7980 .- 0304-3835. ; 375:2, s. 384-389
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Journal article (peer-reviewed)abstract
- Treatment of high-risk childhood neuroblastoma is a clinical challenge hampered by a lack of reliable neuroblastoma mouse models for preclinical drug testing. We have previously established invasive and metastasising patient-derived orthotopic xenografts (PDXs) from high-risk neuroblastomas that retained the genotypes and phenotypes of patient tumours. Given the important role of the tumour microenvironment in tumour progression, metastasis, and treatment responses, here we analysed the tumour microenvironment of five neuroblastoma PDXs in detail. The PDXs resembled their parent tumours and retained important stromal hallmarks of aggressive lesions including rich blood and lymphatic vascularisation, pericyte coverage, high numbers of cancer-associated fibroblasts, tumour-associated macrophages, and extracellular matrix components. Patient-derived tumour endothelial cells occasionally formed blood vessels in PDXs; however, tumour stroma was, overall, of murine origin. Lymphoid cells and lymphatic endothelial cells were found in athymic nude mice but not in NSG mice; thus, the choice of mouse strain dictates tumour microenvironmental components. The murine tumour microenvironment of orthotopic neuroblastoma PDXs reflects important hallmarks of aggressive and metastatic clinical neuroblastomas. Neuroblastoma PDXs are clinically relevant models for preclinical drug testing.
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| 3. |
- Allinne, Jeanne, et al.
(author)
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IL-33 blockade affects mediators of persistence and exacerbation in a model of chronic airway inflammation
- 2019
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In: Journal of Allergy and Clinical Immunology. - : Elsevier BV. - 0091-6749. ; 144:6, s. 1624-1637
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Journal article (peer-reviewed)abstract
- Background: Severe inflammatory airway diseases are associated with inflammation that does not resolve, leading to structural changes and an overall environment primed for exacerbations. Objective: We sought to identify and inhibit pathways that perpetuate this heightened inflammatory state because this could lead to therapies that allow for a more quiescent lung that is less predisposed to symptoms and exacerbations. Methods: Using prolonged exposure to house dust mite in mice, we developed a mouse model of persistent and exacerbating airway disease characterized by a mixed inflammatory phenotype. Results: We show that lung IL-33 drives inflammation and remodeling beyond the type 2 response classically associated with IL-33 signaling. IL-33 blockade with an IL-33 neutralizing antibody normalized established inflammation and improved remodeling of both the lung epithelium and lung parenchyma. Specifically, IL-33 blockade normalized persisting and exacerbating inflammatory end points, including eosinophilic, neutrophilic, and ST2+CD4+ T-cell infiltration. Importantly, we identified a key role for IL-33 in driving lung remodeling because anti–IL-33 also re-established the presence of ciliated cells over mucus-producing cells and decreased myofibroblast numbers, even in the context of continuous allergen exposure, resulting in improved lung function. Conclusion: Overall, this study shows that increased IL-33 levels drive a self-perpetuating amplification loop that maintains the lung in a state of lasting inflammation and remodeled tissue primed for exacerbations. Thus IL-33 blockade might ameliorate symptoms and prevent exacerbations by quelling persistent inflammation and airway remodeling.
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| 4. |
- Bengtson, Sara, et al.
(author)
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Activation of TAFI on the Surface of Streptococcus pyogenes Evokes Inflammatory Reactions by Modulating the Kallikrein/Kinin System
- 2009
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In: Journal of Innate Immunity. - : S. Karger AG. - 1662-811X .- 1662-8128. ; 1:1, s. 18-28
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Journal article (peer-reviewed)abstract
- Bacteria-controlled regulation of host responses to infection is an important virulence mechanism that has been demonstrated to contribute to disease progression. Here we report that the human pathogen Streptococcus pyogenes employs the procarboxypeptidase TAR (thrombin-activatablefibrinolysis inhibitor) to modulate the kallikrein/kinin system. To this end, bacteria initiate a chain of events starting with the recruitment and activation of TAFI. This is followed by the assembly and induction of the contact system at the streptococcal surface, eventually triggering the release of bradykinin (BK). BK is then carboxyterminally truncated by activated TAFI, which converts the peptide from a kinin B-2 receptor ligand to a kinin B-1 receptor (B1R) agonist. Finally, we show that streptococcal supernatants indirectly amplify the B1R response as they act on peripheral blood mononuclear cells to secrete inflammatory cytokines that in turn stimulate upregulation of the B1R on human fibroblasts. Taken together our findings implicate a critical and novel role for streptococci-bound TAR, as it processes BK to a B1R agonist at the bacterial surface and thereby may redirect inflammation from a transient to a chronic state. Copyright (C) 2008 S. Karger AG, Basel
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| 5. |
- Claesson, Andreas, et al.
(author)
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Defibrillation before EMS arrival in western Sweden
- 2017
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In: American Journal of Emergency Medicine. - : Elsevier. - 0735-6757 .- 1532-8171. ; 35:8, s. 1043-1048
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Journal article (peer-reviewed)abstract
- BACKGROUND: Bystanders play a vital role in public access defibrillation (PAD) in out-of-hospital cardiac arrest (OHCA). Dual dispatch of first responders (FR) alongside emergency medical services (EMS) can reduce time to first defibrillation. The aim of this study was to describe the use of automated external defibrillators (AEDs) in OHCAs before EMS arrival.METHODS: All OHCA cases with a shockable rhythm in which an AED was used prior to the arrival of EMS between 2008 and 2015 in western Sweden were eligible for inclusion. Data from the Swedish Register for Cardiopulmonary Resuscitation (SRCR) were used for analysis, on-site bystander and FR defibrillation were compared with EMS defibrillation in the final analysis.RESULTS: Of the reported 6675 cases, 24% suffered ventricular fibrillation (VF), 162 patients (15%) of all VF cases were defibrillated before EMS arrival, 46% with a public AED on site. The proportion of cases defibrillated before EMS arrival increased from 5% in 2008 to 20% in 2015 (p<0.001). During this period, 30-day survival increased in patients with VF from 22% to 28% (p=0.04) and was highest when an AED was used on site (68%), with a median delay of 6.5min from collapse to defibrillation. Adjusted odds ratio for on-site defibrillation versus dispatched defibrillation for 30-day survival was 2.45 (95% CI: 1.02-5.95).CONCLUSIONS: The use of AEDs before the arrival of EMS increased over time. This was associated with an increased 30-day survival among patients with VF. Thirty-day survival was highest when an AED was used on site before EMS arrival.
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| 6. |
- Enquist, Johan, et al.
(author)
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Kinin-Stimulated B1 Receptor Signaling Depends on Receptor Endocytosis Whereas B2 Receptor Signaling Does Not.
- 2014
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In: Neurochemical Research. - : Springer Science and Business Media LLC. - 1573-6903 .- 0364-3190. ; 39:6, s. 1037-1047
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Journal article (peer-reviewed)abstract
- Kinins are potent pro-inflammatory peptides that act through two G protein-coupled receptor subtypes, B1 (B1R) and B2 (B2R). Kinin-stimulated B2R signaling is often transient, whereas B1R signaling is sustained. This was confirmed by monitoring agonist-stimulated intracellular Ca(2+) mobilization in A10 smooth muscle cells expressing human wild-type B2R and B1R. We further studied the role of receptor membrane trafficking in receptor-mediated phosphoinositide (PI) hydrolysis in model HEK293 cell lines stably expressing the receptors. Treatment of cells with brefeldin A, to inhibit maturation of de novo synthesized receptors, or hypertonic sucrose, to inhibit receptor endocytosis, showed that the basal cell surface receptor turnover was considerably faster for B1R than for B2R. Inhibition of endocytosis, which stabilized B1R on the cell surface, inhibited B1R signaling, whereas B2R signaling was not perturbed. Signaling by a B1R construct in which the entire C-terminal domain was deleted remained sensitive to inhibition of receptor endocytosis, whereas signaling by a B1R construct in which this domain was substituted with the corresponding domain in B2R was not sensitive. B2R and B1R co-expression, which also appeared to stabilize B1R on the cell surface, presumably by receptor hetero-dimerization, also inhibited B1R signaling, whereas B2R signaling was slightly enhanced. Furthermore, the B2R-specific agonist bradykinin (BK) directed both receptors through a common endocytic pathway, whereas the B1R-specific agonist Lys-desArg(9)-BK was unable to do so. These results suggest that B1R-mediated PI hydrolysis depends on a step in receptor endocytosis, whereas B2R-mediated PI hydrolysis does not. We propose that B1R uses at least part of the endocytic machinery to sustain agonist-promoted signaling.
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| 7. |
- Gonzalez de Valdivia, Ernesto, et al.
(author)
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Human G protein-coupled Receptor 30 (GPR30) is N -glycosylated and N-terminal Domain Asparagine 44 is Required for Receptor Structure and Activity
- 2019
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In: Bioscience Reports. - 0144-8463. ; 39:2
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Journal article (peer-reviewed)abstract
- GPR30, or G protein-coupled estrogen receptor (GPER), is a G protein-coupled receptor (GPCR) that is currently attracting considerable attention in breast cancer and cardiometabolic regulation. The receptor was reported to be a novel membrane estrogen receptor mediating rapid non-genomic responses. However, questions remain about both the cognate ligand and the subcellular localization of receptor activity. Here, we used HEK293 cells ectopically expressing N-terminally FLAG-tagged human GPR30 and three unique antibodies (Ab) specifically targeting the receptor N-terminal domain (N-domain) to investigate the role of N -glycosylation in receptor maturation and activity, the latter assayed by constitutive receptor-stimulated ERK1/2 activity. GPR30 expression was complex with receptor species spanning from about 40 kDa to higher molecular masses and localized in the endoplasmatic reticulum (ER), the plasma membrane (PM), and endocytic vesicles. The receptor contains three conserved asparagines, Asn25, Asn32, and Asn44, in consensus N -glycosylation motifs, all in the N-domain, and PNGase F treatment showed that at least one of them is N -glycosylated. Mutating Asn44 to isoleucine inactivated the receptor, yielding a unique receptor species at about 20 kDa that was recognized by Ab only in a denatured state. On the other hand, mutating Asn25 or Asn32 either individually or in combination, or truncating successively N-domain residues 1-42, had no significant effect either on receptor structure, maturation, or activity. Thus, Asn44 in the GPR30 N-domain is required for receptor structure and activity, whereas N-domain residues 1-42, including specifically Asn25 and Asn32, do not play any major structural or functional roles.
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| 8. |
- Holmkvist, Petra, et al.
(author)
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A major population of mucosal memory CD4(+) T cells, coexpressing IL-18Rα and DR3, display innate lymphocyte functionality.
- 2015
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In: Mucosal Immunology. - : Elsevier BV. - 1933-0219. ; 8:3, s. 545-558
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Journal article (peer-reviewed)abstract
- Mucosal tissues contain large numbers of memory CD4(+) T cells that, through T-cell receptor-dependent interactions with antigen-presenting cells, are believed to have a key role in barrier defense and maintenance of tissue integrity. Here we identify a major subset of memory CD4(+) T cells at barrier surfaces that coexpress interleukin-18 receptor alpha (IL-18Rα) and death receptor-3 (DR3), and display innate lymphocyte functionality. The cytokines IL-15 or the DR3 ligand tumor necrosis factor (TNF)-like cytokine 1A (TL1a) induced memory IL-18Rα(+)DR3(+)CD4(+) T cells to produce interferon-γ, TNF-α, IL-6, IL-5, IL-13, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-22 in the presence of IL-12/IL-18. TL1a synergized with IL-15 to enhance this response, while suppressing IL-15-induced IL-10 production. TL1a- and IL-15-mediated cytokine induction required the presence of IL-18, whereas induction of IL-5, IL-13, GM-CSF, and IL-22 was IL-12 independent. IL-18Rα(+)DR3(+)CD4(+) T cells with similar functionality were present in human skin, nasal polyps, and, in particular, the intestine, where in chronic inflammation they localized with IL-18-producing cells in lymphoid aggregates. Collectively, these results suggest that human memory IL-18Rα(+)DR3(+) CD4(+) T cells may contribute to antigen-independent innate responses at barrier surfaces.Mucosal Immunology advance online publication, 1 October 2014; doi:10.1038/mi.2014.87.
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| 9. |
- Hvidtfeldt, Morten, et al.
(author)
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Airway hyperresponsiveness reflects corticosteroid-sensitive mast cell involvement across asthma phenotypes
- 2023
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In: Journal of Allergy and Clinical Immunology. - : Elsevier BV. - 0091-6749. ; 152:1, s. 4-116
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Journal article (peer-reviewed)abstract
- Background: Airway hyperresponsiveness is a hallmark of asthma across asthma phenotypes. Airway hyperresponsiveness to mannitol specifically relates to mast cell infiltration of the airways, suggesting inhaled corticosteroids to be effective in reducing the response to mannitol, despite low levels of type 2 inflammation. Objective: We sought to investigate the relationship between airway hyperresponsiveness and infiltrating mast cells, and the response to inhaled corticosteroid treatment. Methods: In 50 corticosteroid-free patients with airway hyperresponsiveness to mannitol, mucosal cryobiopsies were obtained before and after 6 weeks of daily treatment with 1600 μg of budesonide. Patients were stratified according to baseline fractional exhaled nitric oxide (FENO) with a cutoff of 25 parts per billion. Results: Airway hyperresponsiveness was comparable at baseline and improved equally with treatment in both patients with FENO-high and FENO-low asthma: doubling dose, 3.98 (95% CI, 2.49-6.38; P < .001) and 3.85 (95% CI, 2.51-5.91; P < .001), respectively. However, phenotypes and distribution of mast cells differed between the 2 groups. In patients with FENO-high asthma, airway hyperresponsiveness correlated with the density of chymase-high mast cells infiltrating the epithelial layer (ρ, −0.42; P = .04), and in those with FENO-low asthma, it correlated with the density in the airway smooth muscle (ρ, −0.51; P = .02). The improvement in airway hyperresponsiveness after inhaled corticosteroid treatment correlated with a reduction in mast cells, as well as in airway thymic stromal lymphopoietin and IL-33. Conclusions: Airway hyperresponsiveness to mannitol is related to mast cell infiltration across asthma phenotypes, correlating with epithelial mast cells in patients with FENO-high asthma and with airway smooth muscle mast cells in patients with FENO-low asthma. Treatment with inhaled corticosteroids was effective in reducing airway hyperresponsiveness in both groups.
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| 10. |
- Hvidtfeldt, Morten, et al.
(author)
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Bronchoscopic mucosal cryobiopsies as a method for studying airway disease
- 2019
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In: Clinical and Experimental Allergy. - : Wiley. - 0954-7894 .- 1365-2222. ; 49:1, s. 27-34
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Journal article (peer-reviewed)abstract
- Background: Investigating disease mechanisms and treatment responses in obstructive airway diseases with invasive sampling are hampered by the small size and mechanical artefacts that conventional forceps biopsies suffer from. Endoscopic cryobiopsies are larger and more intact and are being increasingly used. However, the technique has not yet been explored for obtaining mucosa biopsies. Objective: To investigate differences in size and quality of endobronchial mucosal biopsies obtained with cryotechnique and forceps. Further, to check for eligibility of cryobiopsies to be evaluated with immunohistochemistry and in situ hybridization and to investigate tolerability and safety of the technique. Methods: Endobronchial mucosal biopsies were obtained with cryotechnique and forceps from patients with haemoptysis undergoing bronchoscopy and evaluated by quantitative morphometry, automated immunohistochemistry and in situ hybridization. Results: A total of 40 biopsies were obtained from 10 patients. Cross-sectional areas were threefold larger in cryobiopsies (median: 3.08 mm2 (IQR: 1.79) vs 1.03 mm2 (IQR: 1.10), P < 0.001). Stretches of intact epithelium were 8-fold longer (median: 4.61 mm (IQR: 4.50) vs 0.55 mm (IQR: 1.23), P = 0.001). Content of glands (median: 0.095 mm2 (IQR: 0.30) vs 0.00 mm2 (IQR: 0.01), P = 0.002) and airway smooth muscle (median: 0.25 mm2 (IQR: 0.30) vs 0.060 mm2 (IQR: 0.11), P = 0.02) was higher in the cryobiopsies compared with forceps biopsies. Further, the cryobiopsies had well-preserved protein antigens and mRNA. Mild to moderate bleeding was the only complication observed. Conclusion and clinical relevance: By yielding significantly larger and more intact biopsies, the cryotechnique represents a valuable new research tool to explore the bronchi in airway disease. Ultimately with the potential to create better understanding of underlying disease mechanisms and improvement of treatments.
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| 11. |
- Hvidtfeldt, Morten, et al.
(author)
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Mucosal cryobiopsies : a new method for studying airway pathology in asthma
- 2022
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In: ERJ open research. - : European Respiratory Society (ERS). - 2312-0541. ; 8:1
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Journal article (peer-reviewed)abstract
- Background In vivo studies of airway pathology in obstructive lung disease are limited by poor quality of specimens obtained with forceps. Obtainment of cryobiopsies has increased diagnostic yield in cancer and interstitial lung disease but has not been used in patients with asthma. In a recent pilot study, we found mucosal cryobiopsies to be larger and more intact than conventional forceps biopsies. The aim of the present study was to compare quality and safety of mucosal cryobiopsies versus conventional forceps biopsies in patients with asthma. Methods Endobronchial biopsies were obtained with forceps and cryoprobe from patients with asthma not currently treated with inhaled steroids and evaluated histologically. Results A total of 240 cryobiopsies and 288 forceps biopsies were obtained from 48 patients. Bleeding from the biopsy site was common but self-limiting. No major complications related to the procedure were seen. Cryobiopsy cross areas were four times larger compared with forceps. Stretches of intact epithelium were detected in all cryobiopsies compared to 33% in forceps biopsies. Further, the length of intact epithelium was on average four times longer in the cryobiopsies. Importantly, there was a good preservation of both antigens and mRNA in the cryobiopsies ensuring a suitability and robustness for immunohistochemistry and in situ hybridisation. Conclusion Obtainment of mucosal cryobiopsies in patients with asthma is safe and yields biopsies that are significantly larger and morphologically better preserved compared with traditional forceps biopsies. The cryotechnique thus seems to be a promising tool for future in vivo studies of airway pathology.
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| 12. |
- Jogdand, Prajakta, et al.
(author)
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Eosinophils, basophils, and type 2 immune microenvironments in COPD-affected lung tissue
- 2020
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In: European Respiratory Journal. - : European Respiratory Society (ERS). - 0903-1936 .- 1399-3003. ; 55:4
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Journal article (peer-reviewed)abstract
- Although elevated blood or sputum eosinophils are present in many patients with chronic obstructive pulmonary disease (COPD), uncertainties remain regarding the anatomical distribution pattern of lung-infiltrating eosinophils. Basophils have remained virtually unexplored in COPD. This study mapped tissue-infiltrating eosinophils, basophils, and eosinophil-promoting immune mechanisms in COPD-affected lungs. Surgical lung tissue and biopsies from major anatomical compartments were obtained from COPD patients with severity grades GOLD I-IV; never-smokers/smokers served as controls. Automated immunohistochemistry and in-situ hybridization identified immune cells, the type 2 immunity marker GATA3, and eotaxins (CCL11, CCL24). Eosinophils and basophils were present in all anatomical compartments of COPD-affected lungs and increased significantly in very severe COPD. The eosinophilia was strikingly patchy, and focal eosinophil-rich microenvironments were spatially linked with GATA3+ cells, including Th2 lymphocytes and type 2 innate lymphoid cells. A similarly localised and IL-33/ST2-dependent eosinophilia was demonstrated in influenza-infected mice. Both mice and patients displayed spatially confined eotaxin signatures with CCL11+ fibroblasts and CCL24+ macrophages. In addition to identifying tissue basophilia as a novel feature of advanced COPD, the identification of spatially confined eosinophil-rich type 2 microenvironments represents a novel type of heterogeneity in the immunopathology of COPD that will likely have implications for personalised treatment.
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| 13. |
- Karlsson, Emma, et al.
(author)
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The punishment of death : Concerning an archaeological excavation of an execution site in Vadstena (Sweden)
- 2012
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In: Richtstättenarchäologie 3. - Dormagen : archaeotopos-Buchverlag. - 9783938473177 ; , s. 154-159
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Book chapter (other academic/artistic)abstract
- This article consists of three sections and is a summary of a book about an archeological excavation of an execution site in Vadstena, Sweden. In the section "Forgotten Graves at the Gallows Hill" (Emma Karlsson) mainly concernes the archaeological excavations at Vadstena, but also briefly mentions the compilation of fourteen other excavations. The section "To this, Nobody can be indifferent (Caroline Arcini) deals with the osteological analysis. In the third section "Alienation from society and the symbolism of punishment" (Annika Sandén) the lives of people living on the outer reaches of society during the sixteenth and sevenetenth century are illuminated.
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| 14. |
- Kearley, Jennifer, et al.
(author)
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Cigarette smoke silences innate lymphoid cell function and facilitates an exacerbated type I interleukin-33-dependent response to infection.
- 2015
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In: Immunity. - : Elsevier BV. - 1074-7613. ; 42:3, s. 566-579
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Journal article (peer-reviewed)abstract
- Cigarette smoking is a major risk factor for chronic obstructive pulmonary disease and is presumed to be central to the altered responsiveness to recurrent infection in these patients. We examined the effects of smoke priming underlying the exacerbated response to viral infection in mice. Lack of interleukin-33 (IL-33) signaling conferred complete protection during exacerbation and prevented enhanced inflammation and exaggerated weight loss. Mechanistically, smoke was required to upregulate epithelial-derived IL-33 and simultaneously alter the distribution of the IL-33 receptor ST2. Specifically, smoke decreased ST2 expression on group 2 innate lymphoid cells (ILC2s) while elevating ST2 expression on macrophages and natural killer (NK) cells, thus altering IL-33 responsiveness within the lung. Consequently, upon infection and release, increased local IL-33 significantly amplified type I proinflammatory responses via synergistic modulation of macrophage and NK cell function. Therefore, in COPD, smoke alters the lung microenvironment to facilitate an alternative IL-33-dependent exaggerated proinflammatory response to infection, exacerbating disease.
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| 15. |
- Lamb, David, et al.
(author)
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RORγt inhibitors block both IL-17 and IL-22 conferring a potential advantage over anti-IL-17 alone to treat severe asthma
- 2021
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In: Respiratory Research. - : Springer Science and Business Media LLC. - 1465-9921 .- 1465-993X. ; 22:1
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Journal article (peer-reviewed)abstract
- Background: RORγt is a transcription factor that enables elaboration of Th17-associated cytokines (including IL-17 and IL-22) and is proposed as a pharmacological target for severe asthma. Methods: IL-17 immunohistochemistry was performed in severe asthma bronchial biopsies (specificity confirmed with in situ hybridization). Primary human small airway epithelial cells in air liquid interface and primary bronchial smooth muscle cells were stimulated with recombinant human IL-17 and/or IL-22 and pro-inflammatory cytokines measured. Balb/c mice were challenged intratracheally with IL-17 and/or IL-22 and airway hyperreactivity, pro-inflammatory cytokines and airway neutrophilia measured. Balb/c mice were sensitized intraperitoneally and challenged intratracheally with house dust mite extract and the effect of either a RORγt inhibitor (BIX119) or an anti-IL-11 antibody assessed on airway hyperreactivity, pro-inflammatory cytokines and airway neutrophilia measured. Results: We confirmed in severe asthma bronchial biopsies both the presence of IL-17-positive lymphocytes and that an IL-17 transcriptome profile in a severe asthma patient sub-population. Both IL-17 and IL-22 stimulated the release of pro-inflammatory cytokine and chemokine release from primary human lung cells and in mice. Furthermore, IL-22 in combination with IL-17, but neither alone, elicits airway hyperresponsiveness (AHR) in naïve mice. A RORγt inhibitor specifically blocked both IL-17 and IL-22, AHR and neutrophilia in a mouse house dust mite model unlike other registered or advanced pipeline modes of action. Full efficacy versus these parameters was associated with 90% inhibition of IL-17 and 50% inhibition of IL-22. In contrast, anti-IL-17 also blocked IL-17, but not IL-22, AHR or neutrophilia. Moreover, the deregulated genes in the lungs from these mice correlated well with deregulated genes from severe asthma biopsies suggesting that this model recapitulates significant severe asthma-relevant biology. Furthermore, these genes were reversed upon RORγt inhibition in the HDM model. Cell deconvolution suggested that the responsible cells were corticosteroid insensitive γδ-T-cells. Conclusion: These data strongly suggest that both IL-17 and IL-22 are required for Th2-low endotype associated biology and that a RORγt inhibitor may provide improved clinical benefit in a severe asthma sub-population of patients by blocking both IL-17 and IL-22 biology compared with blocking IL-17 alone.
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| 16. |
- Roos, Abraham, et al.
(author)
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IL-17A is Elevated in End-stage COPD and Contributes to Cigarette Smoke-induced Lymphoid Neogenesis.
- 2015
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In: American Journal of Respiratory and Critical Care Medicine. - 1535-4970. ; 191:11, s. 1232-1241
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Journal article (peer-reviewed)abstract
- End-stage chronic obstructive pulmonary disease (COPD) is associated with an accumulation of pulmonary lymphoid follicles. Interleukin (IL)-17A is implicated in COPD and pulmonary lymphoid neogenesis in response to microbial stimuli. We hypothesized that IL-17A is increased in peripheral lung tissue during end-stage COPD and also directly contributes to cigarette smoke-induced lymphoid neogenesis.
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| 17. |
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| 18. |
- Sanden, Caroline, et al.
(author)
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Broad Th2 neutralization and anti-inflammatory action of pentosan polysulfate sodium in experimental allergic rhinitis
- 2017
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In: Immunity, Inflammation and Disease. - : Wiley. - 2050-4527. ; 5:3, s. 300-309
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Journal article (peer-reviewed)abstract
- Th2 cytokines like interleukin-4, -5, and -13 are regarded as important drivers of the immunopathology underlying allergic rhinitis (AR) and asthma. The present study explores the capacity of pentosan polysulfate sodium (PPS), a semi-synthetic heparin-like macromolecular carbohydrate, to bind Th2 cytokines and exert biological neutralization in vitro, as well as anti-inflammatory actions in vivo.
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| 19. |
- Sandén, Caroline, et al.
(author)
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G Protein-Coupled Estrogen Receptor 1 (GPER1)/GPR30 Localizes in the Plasma Membrane and Trafficks Intracellularly on Cytokeratin Intermediate Filaments.
- 2011
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In: Molecular Pharmacology. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 1521-0111 .- 0026-895X. ; 79:3, s. 400-410
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Journal article (peer-reviewed)abstract
- GPR30, or G protein-coupled estrogen receptor 1 (GPER1), was recently introduced as a membrane estrogen receptor and a candidate cancer biomarker and therapeutic target. However, several questions surround the subcellular localization and signaling of this receptor. In native cells, including mouse myoblast C(2)C(12) cells, Madine-Darby canine kidney (MDCK) epithelial cells, and human ductal breast epithelial tumor T47-D cells, G-1, a GPER1 agonist, and 17β-estradiol (E2) stimulated GPER1-dependent cAMP production, a defined plasma membrane (PM) event, and recruitment of β-arrestin2 to the PM. Staining of fixed and live cells showed that GPER1 was localized both in the PM and on intracellular structures. One such intracellular structure was identified as cytokeratin (CK) intermediate filaments, including those composed of CK7 and CK8, but apparently not endoplasmatic reticulum (ER), Golgi, or microtubules. Reciprocal co-immunoprecipitation of GPER1 and CKs confirmed an association of these proteins. Live staining also showed that the PM receptors constitutively internalize apparently to reach CK filaments. Receptor localization was supported using FLAG- and HA-tagged GPER1. We conclude that GPER1-mediated stimulation of cAMP production and β-arrestin2 recruitment occur in the PM. Furthermore, the PM receptors constitutively internalize and localize intracellularly on CK. This is the first observation that a G protein-coupled receptor (GPCR) is capable of associating with intermediate filaments, which may be important for GPER1 regulation in epithelial cells and the relationship of this receptor to cancer.
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| 20. |
- Sandén, Caroline
(author)
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G protein-coupled receptor regulation: The role of protein interactions and receptor trafficking
- 2009
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Doctoral thesis (other academic/artistic)abstract
- The superfamily of G protein-coupled receptors (GPCR) is the largest gene family in the human genome. GPCR-mediated signaling operates in every human cell, and about 50% of existing clinically useful drugs act through GPCR. Kinins are proinflammatory peptides that are rapidly produced extracellularly following pathological insults and tissue damage. These peptides act through two GPCR subtypes, B1 (B1R) and B2 (B2R), to elicit numerous inflammatory responses including vasodilatiation, increased vascular permeability, and pain. GPER1 (G protein-coupled estrogen receptor 1) has recently been suggested as a novel estrogen receptor. Much controversy surrounds this receptor regarding its subcellular localization and activation. The aim of the present thesis was to explore the role of receptor trafficking and protein-interactions in kinin receptor regulation, as well as studying the receptor trafficking and signaling of GPER1. B1R forms homo-oligomers, which is required for proper B1R maturation and cell-surface expression. Furthermore, the endopeptidase EP24.15 is able to degrade BK intracellularly and attenuate maximal B2R responsiveness without influencing the potency of BK. Finally, GPER1 is localized both intracellularly and on the cell surface, where it is subject to rapid, constitutive endocytosis. Estrogen was found to elevate the level of cAMP in mouse myotube C2C12 cells, in a GPER1-dependent manner, but it is still not clear whether estrogen is the true agonist for GPER1. Taken together, the present thesis demonstrates the importance of protein-protein interactions and trafficking for B1R, B2R, and GPER1 regulation and activity. Understanding these mechanisms will be of great benefit in drug development by presenting novel drug targets.
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| 21. |
- Sandén, Caroline, et al.
(author)
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Kinin B1 receptor homo-oligomerization is required for receptor trafficking to the cell surface.
- 2013
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In: International Immunopharmacology. - : Elsevier BV. - 1878-1705 .- 1567-5769. ; 15:1, s. 121-128
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Journal article (peer-reviewed)abstract
- The kinin B1 receptor (B1R) is a G protein-coupled receptor with pro-inflammatory activity that is latent in healthy tissues but induced by tissue insult. Here, we investigated if B1R homo-oligomerization is a possible mechanism regulating the presentation of this receptor at the level of maturation and trafficking to the cell surface. To this end, we used HEK293 cells stably expressing N-terminal FLAG and HA epitope-tagged wild-type human B1R and an N-terminal receptor fragment, B1stop135, which terminates at the C-terminal end of the third transmembrane domain and has previously been shown to oligomerize with B1R. Receptors were monitored by immunoblotting and immunoprecipitation, receptor function by agonist binding and agonist-promoted phosphoinositide hydrolysis, and receptor trafficking by confocal immunofluorescence microscopy. When expressed alone, B1R is core N-glycosylated and forms oligomers localized intracellularly and on the cell surface. B1stop135 also exists as core N-glycosylated oligomers but is localized exclusively intracellularly. When co-expressed, B1stop135 prevents specifically B1R homo-oligomerization by forming nonfunctional B1R-B1stop135 hetero-oligomers, retains B1R intracellularly at least in part in the endoplasmatic reticulum (ER), increases calnexin binding to the receptor, and increases receptor degradation. We conclude that B1R homo-oligomerization is necessary for B1R maturation and trafficking to the cell surface. Modulating this mechanism may be a novel therapeutic avenue in inflammatory disease.
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| 22. |
- Sandén, Caroline, et al.
(author)
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Kinin B2 Receptor-Mediated Bradykinin Internalization and Metalloendopeptidase EP24.15-Dependent Intracellular Bradykinin Degradation.
- 2008
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In: Journal of Pharmacology and Experimental Therapeutics. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 1521-0103 .- 0022-3565. ; 326:1, s. 24-32
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Journal article (peer-reviewed)abstract
- Kinins are potent proinflammatory peptides that are produced extracellularly and rapidly degraded by extracellular peptidases and by intracellular peptidases accessed by kinins via receptor-mediated endocytosis. Here, we developed model cell systems expressing the kinin B2 receptor (B2R) and the metalloendopeptidase thimet oligopeptidase (EC 3.4.24.15; EP24.15) either individually or together to address 1) the cellular and functional relationship between these proteins and 2) the participation of EP24.15 in the metabolism of bradykinin (BK) following BK internalization via B2R. B2R was localized almost exclusively in the plasma membrane, whereas EP24.15 was localized both intracellularly and on the cell surface, and secreted in the media. Intracellular EP24.15 was present throughout the cell, both cytosolic and particulate, with less nuclear localization and no co-localization with either the endoplasmatic reticulum marker calnexin or Golgi marker GM130. No direct co-localization of B2R and EP24.15 was observed using immunofluorescence microscopy. However, the two proteins co-immunoprecipitated specifically, and EP24.15 attenuated maximal B2R responsiveness without influencing the potency of BK to stimulate phosphoinositide hydrolysis and intracellular Ca(2+) mobilization. Cell surface-bound BK remained intact in cells overexpressing EP24.15 but was degraded intracellularly in an EP24.15-dependent manner upon B2R-mediated endocytosis. These results show that EP24.15 acts to negatively regulate B2R responsiveness and by serving as an intracellular peptidase in the degradation of BK specifically internalized via this receptor.
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| 23. |
- Scott, Ian C., et al.
(author)
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Interleukin-33 is activated by allergen- and necrosis-associated proteolytic activities to regulate its alarmin activity during epithelial damage
- 2018
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In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 8:1
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Journal article (peer-reviewed)abstract
- Interleukin (IL)-33 is an IL-1 family alarmin released from damaged epithelial and endothelial barriers to elicit immune responses and allergic inflammation via its receptor ST2. Serine proteases released from neutrophils, mast cells and cytotoxic lymphocytes have been proposed to process the N-terminus of IL-33 to enhance its activity. Here we report that processing of full length IL-33 can occur in mice deficient in these immune cell protease activities. We sought alternative mechanisms for the proteolytic activation of IL-33 and discovered that exogenous allergen proteases and endogenous calpains, from damaged airway epithelial cells, can process full length IL-33 and increase its alarmin activity up to ~60-fold. Processed forms of IL-33 of apparent molecular weights ~18, 20, 22 and 23 kDa, were detected in human lungs consistent with some, but not all, proposed processing sites. Furthermore, allergen proteases degraded processed forms of IL-33 after cysteine residue oxidation. We suggest that IL-33 can sense the proteolytic and oxidative microenvironment during tissue injury that facilitate its rapid activation and inactivation to regulate the duration of its alarmin function.
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| 24. |
- Siddhuraj, Premkumar, et al.
(author)
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Lung Mast Cells Have a High Constitutive Expression of Carboxypeptidase A3 mRNA That Is Independent from Granule-Stored CPA3
- 2021
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In: Cells. - : MDPI AG. - 2073-4409. ; 10:2
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Journal article (peer-reviewed)abstract
- The mast cell granule metalloprotease CPA3 is proposed to have important tissue homeostatic functions. However, the basal CPA3 mRNA and protein expression among mast cell populations has remained poorly investigated. Using a novel histology-based methodology that yields quantitative data on mRNA and protein expression at a single-cell level, the present study maps CPA3 mRNA and protein throughout the MCT and MCTC populations in healthy skin, gut and lung tissues. MCTC cells had both a higher frequency of CPA3 protein-containing cells and a higher protein-staining intensity than the MCT population. Among the tissues, skin MCs had highest CPA3 protein intensity. The expression pattern at the mRNA level was reversed. Lung mast cells had the highest mean CPA3 mRNA staining. Intriguingly, the large alveolar MCT population, that lack CPA3 protein, had uniquely high CPA3 mRNA intensity. A broader multi-tissue RNA analysis confirmed the uniquely high CPA3 mRNA quantities in the lung and corroborated the dissociation between chymase and CPA3 at the mRNA level. Taken together, our novel data suggest a hitherto underestimated contribution of mucosal-like MCT to baseline CPA3 mRNA production. The functional consequence of this high constitutive expression now reveals an important area for further research.
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| 25. |
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| 26. |
- Strickson, Sam, et al.
(author)
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Oxidised IL-33 drives COPD epithelial pathogenesis via ST2-independent RAGE/EGFR signalling complex
- 2023
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In: European Respiratory Journal. - 0903-1936. ; 62:3
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Journal article (peer-reviewed)abstract
- Background Epithelial damage, repair and remodelling are critical features of chronic airway diseases including chronic obstructive pulmonary disease (COPD). Interleukin (IL)-33 released from damaged airway epithelia causes inflammation via its receptor, serum stimulation-2 (ST2). Oxidation of IL-33 to a non-ST2-binding form (IL-33ox) is thought to limit its activity. We investigated whether IL-33ox has functional activities that are independent of ST2 in the airway epithelium. Methods In vitro epithelial damage assays and three-dimensional, air–liquid interface (ALI) cell culture models of healthy and COPD epithelia were used to elucidate the functional role of IL-33ox. Transcriptomic changes occurring in healthy ALI cultures treated with IL-33ox and COPD ALI cultures treated with an IL-33-neutralising antibody were assessed with bulk and single-cell RNA sequencing analysis. Results We demonstrate that IL-33ox forms a complex with receptor for advanced glycation end products (RAGE) and epidermal growth factor receptor (EGFR) expressed on airway epithelium. Activation of this alternative, ST2-independent pathway impaired epithelial wound closure and induced airway epithelial remodelling in vitro. IL-33ox increased the proportion of mucus-producing cells and reduced epithelial defence functions, mimicking pathogenic traits of COPD. Neutralisation of the IL-33ox pathway reversed these deleterious traits in COPD epithelia. Gene signatures defining the pathogenic effects of IL-33ox were enriched in airway epithelia from patients with severe COPD. Conclusions Our study reveals for the first time that IL-33, RAGE and EGFR act together in an ST2-independent pathway in the airway epithelium and govern abnormal epithelial remodelling and mucoobstructive features in COPD.
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