Innate mechanisms in upper airway inflammation with focus on epithelium and neutrophils

Mucosal inflammation is a key feature in allergic rhinitis (AR) and chronic rhinosinusitis with nasal polyps (CRSwNP). The traditional idea of the epithelium as a simple barrier and neutrophils as a homogenous cell population, already terminally differentiated, has lately been reconsidered. Recent findings have identified advanced immunological properties of both epithelial cells (ECs) and neutrophil subsets. Toll-like receptors (TLRs) and activin receptorlike kinases (ALKs) constitute important receptors of the ECs in recognizing stimuli leading to inflammatory and biological processes through altered gene expression. The new neutrophil classification, in four various subsets, is based on their expression of FcγRIII (CD16) and L-selectin (CD62L). The various subsets appear to have diverse roles during inflammatory conditions. The overall aim of this thesis is to investigate the role of the epithelial and neutrophil cells in upper airway innate immunity. Papers I-III explored the role of nasal epithelial cells (NECs) in antigen presentation as well as TLRs and ALKs on ECs in AR and CRSwNP. Major histocompatibility complex class II (MHC class II) as well as co-stimulatory molecules were found on NECs from mice and humans and were upregulated on OVA-sensitized mice. NECs from sensitized mice could take up, process and present antigens in a class II dependent manner for the activation of antigenspecific CD4+ T cells. NECs from AR patients were able to activate autologous T cells against the major birch allergen protein, Bet v 1, and induce an IL-13 release. Normally, TLR9 can be found in NECs, but was found to be almost absent in NECs from the mucosa close to the polyps of CRSwNP patients. The TLR9 expression could be reconstituted by stimulation with its ligand, CpG, both in vitro and in vivo. Stimulation also resulted in a downregulation of VEGFR2, a receptor of angiogenesis, and cytokines. Investigation of polyp ECs revealed an upregulation of ALK1-6. Upon ligand stimulation, a downregulation of factors affecting cell proliferation (Ki67) and inflammation (ICAM-1 and IL-8) was seen. This was even more pronounced when microbial infection was mimicked. In Papers IV and V, neutrophil subsets were described for the first time in the nasal mucosa and a local shift to the activated subset became evident in AR and CRSwNP. In AR, the activated subset CD16high CD62Ldim was shown to have T cell priming capacities and an ability to enhance eosinophil migration. Neutrophils from CRSwNP patients displayed an upregulation of CD11b, most clearly emphasized on the cells of the activated subset. The corresponding adhesion molecule ICAM-1 was upregulated on the epithelium of polyps. In summary, epithelial and neutrophil cells in the nasal mucosa of patients with AR and CRSwNP exhibit an alerted receptor pattern with a deranged immunological response. Functional data including T cell activation, migration, adhesion and cytokine release clearly indicate a role for TLRs, ALKs and activated neutrophils in these upper airway inflammatory diseases. Further, antigen presenting ECs can contribute to mucosal inflammation in AR

Extracellular vesicles promote activation of pro-inflammatory cancer-associated fibroblasts in oral cancer

Introduction: Oral squamous cell carcinoma (OSCC) is the most common form of head and neck cancer and has a survival rate of ∼50% over 5 years. New treatment strategies are sorely needed to improve survival rates—and a better understanding of the mechanisms underlying tumorigenesis is needed to develop these strategies. The role of the tumor microenvironment (TME) has increasingly been identified as crucial in tumor progression and metastasis. One of the main constituents of the TME, cancer-associated fibroblasts (CAFs), plays a key role in influencing the biological behavior of tumors. Multiple mechanisms contribute to CAF activation, such as TGFβ signaling, but the role of extracellular vesicles (EVs) in CAF activation in OSCC is poorly understood. Assessing the impact of oral cancer-derived EVs on CAF activation will help to better illuminate OSCC pathophysiology and may drive development of novel treatments options.Methods: EVs were isolated from OSCC cell lines (Cal 27, SCC-9, SCC-25) using differential centrifugation. Nanoparticle tracking analysis was used for EV characterization, and Western blot to confirm the presence of EV protein markers. Oral fibroblasts were co-cultured with enriched EVs, TGFβ, or PBS over 72 h to assess activation. Flow cytometry was used to evaluate CAF markers. RNA collected from fibroblasts was extracted and the transcriptome was sequenced. Conditioned media from the co-cultures was evaluated with cytokine array profiling.Results: OSCC-derived EVs can activate oral fibroblasts into CAFs that are different from those activated by TGFβ, suggesting different mechanisms of activation and different functional properties. Gene set enrichment analysis showed several upregulated inflammatory pathways in those CAFs exposed to OSCC-derived EVs. Marker genes for inflammatory CAF subtypes were also upregulated, but not in CAFs activated by TGFβ. Finally, cytokine array analysis on secreted proteins revealed elevated levels of several pro-inflammatory cytokines from EV-activated CAFs, for instance IL-8 and CXCL5.Discussion: Our results reveal the ability of OSCC-derived EVs to activate fibroblasts into CAFs. These CAFs seem to have unique properties, differing from TGFβ-activated CAFs. Gaining an understanding of the interplay between EVs and stromal cells such as CAFs could lead to further insights into OSCC tumorigenesis and potential novel therapeutics

Activation of activin receptor-like kinases curbs mucosal inflammation and proliferation in chronic rhinosinusitis with nasal polyps

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a widespread disease causing obstruction of the nasal cavity. Its cause remains unclear. The transforming growth-factor beta (TGF-beta) superfamily and their receptors, termed Activin receptor-like kinases (ALKs), have recently been suggested to play a role in local airway inflammation, but have so far not been evaluated in human nasal epithelial cells (HNECs) from CRSwNP patients. We demonstrated that ALK1-7 were expressed in the nasal polyp epithelium, and the expression of ALK1-6 was markedly elevated in polyps compared to nasal mucosa from healthy controls. Stimulation with the ALK ligand TGF-beta 1 decreased Ki67 expression in HNECs from CRSwNP patients, not evident in controls. Likewise, TGF-beta 1, Activin A and Activin B, all ALK ligands, decreased IL-8 release and Activin A and Activin B reduced ICAM1 expression on HNECs from CRSwNP patients, not seen in controls. Pre-stimulation with TGF-beta 1, Activin A, BMP4 and Activin B attenuated a TNF-ainduced ICAM1 upregulation on HNECs of CRSwNP. No effect was evident in controls. In conclusion, an increased expression of ALK1-6 was found on polyp epithelial cells and ligand stimulation appeared to reduce proliferation and local inflammation in polyps

Inflammatory endotypes of chronic rhinosinusitis based on cluster analysis of biomarkers

Background: Current phenotyping of chronic rhinosinusitis (CRS) into chronic rhinosinusitis with nasal polyps (CRSwNP) and chronic rhinosinusitis without nasal polyps (CRSsNP) might not adequately reflect the pathophysiologic diversity within patients with CRS. Objective: We sought to identify inflammatory endotypes of CRS. Therefore we aimed to cluster patients with CRS based solely on immune markers in a phenotype-free approach. Secondarily, we aimed to match clusters to phenotypes. Methods: In this multicenter case-control study patients with CRS and control subjects underwent surgery, and tissue was analyzed for IL-5, IFN-gamma, IL-17A, TNF-alpha, IL-22, IL-1 beta, IL-6, IL-8, eosinophilic cationic protein, myeloperoxidase, TGF-beta 1, IgE, Staphylococcus aureus enterotoxin-specific IgE, and albumin. We used partition-based clustering. Results: Clustering of 173 cases resulted in 10 clusters, of which 4 clusters with low or undetectable IL-5, eosinophilic cationic protein, IgE, and albumin concentrations, and 6 clusters with high concentrations of those markers. The group of IL-5-negative clusters, 3 clusters clinically resembled a predominant chronic rhinosinusitis without nasal polyps (CRSsNP) phenotype without increased asthma prevalence, and 1 cluster had a T(H)17 profile and had mixed CRSsNP/CRSwNP. The IL-5-positive clusters were divided into a group with moderate IL-5 concentrations, a mixed CRSsNP/CRSwNP and increased asthma phenotype, and a group with high IL-5 levels, an almost exclusive nasal polyp phenotype with strongly increased asthma prevalence. In the latter group, 2 clusters demonstrated the highest concentrations of IgE and asthma prevalence, with all samples expressing Staphylococcus aureus enterotoxin-specific IgE. Conclusion: Distinct CRS clusters with diverse inflammatory mechanisms largely correlated with phenotypes and further differentiated them and provided a more accurate description of the inflammatory mechanisms involved than phenotype information only

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

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.To analyse the role of TLR9 mediated microbial defense in tissue bordering the polyp.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.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.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

Percentage of cells that express VEGFR2 after CpG culture.

<p>Epithelial VEGFR2 expression after 4 hours (<b>A</b>) and 24 hours (<b>B</b>) of culture with vehicle/CpG (0.1 µM; 0.3 µM and 1.0 µM). Expression on turbinate epithelial cells from healthy controls, turbinate and polyp epithelial cells from patients with CRSwNP, analysed using flow cytometry. Results are presented as mean ± SEM, <i>n</i> = 5, *<i>P</i><0.05, **<i>P</i><0.01 (unstimulated vs. CpG stimulated), ##<i>P</i><0.01 (unstimulated turbinate tissue from patients vs. unstimulated turbinate tissue from healthy controls and polyp tissue from patients).</p