Nasal epithelial cells : innate immunity and inflammation

The surface epithelium that lines the nasal passages is often the first tissue in the airway to encounter inhaled pathogens. It collaborates closely with the innate immune system, a subsystem of the immune system that defends the host from infection by organisms, mainly by initiating a local inflammatory reaction. Pattern-recognition receptors (PRRs) are important in pathogen recognition, cell activation and regulation of immune responses and include Toll-like receptors (TLRs), nucleotide-binding oligomerization domain-like receptors (NLRs) and retinoic acid-inducible gene 1 (RIG-I)-like receptors (RLRs). The transforming growth-factor beta (TGF-β) superfamily and their type I receptors, the activin receptor-like kinases (ALKs), are important mediators that promote remodelling and have recently also been shown to regulate airway inflammation. Even though PRRs and ALKs are essential in preventing disease, disruption of these systems is generally believed to be involved in the pathogenesis of airway inflammatory diseases such as asthma and chronic rhinosinusitis with nasal polyp (CRSwNP). Hence, the overall aim with this thesis was to investigate the role of PRRs and ALKs in airway inflammation. Human airway smooth muscle cells (HASMCs) are essential for the regulation of airflow; importantly, they are also involved in the shortness of breath that characterises microbialinduced exacerbations of asthma. The present thesis showed that stimulation of TLR2, TLR3, TLR4, TLR7 and NOD1 on HASMCs resulted in cytokine release, upregulation of inflammatory cell surface markers and downregulation of receptors involved in smooth muscle cell contraction. The nasal epithelium was found to express TLR3, TLR7, TLR9, RIG-I and MDA-5 and stimulation resulted in an increased inflammatory response characterised by the release of chemokines and cytokines. In addition, a specific role for TLR9 was found in patients with CRSwNP that might be linked to polyp growth via downregulation of VEGFR expression and lowered release of inflammatory cytokines. Virus-related ligand stimulation of TLR7 induced a rapid release of the neuropeptide, substance P (SP), from human nasal epithelial cells (HNECs) and sensory neurons. The released SP promptly upregulated the epithelial TLR expression. This suggests a role for SP in rapid priming of the innate immune system during viral infections. Polyp epithelial cells from patients with CRSwNP expressed high levels of ALK1-6. Polyp epithelial cells stimulated with ALK-ligands demonstrated a potential anti-inflammatory role for ALKs in polyps. Previous reports have demonstrated low levels of ALK-ligands in patients with CRSwNP, suggesting that ALKs could contribute to uncontrolled inflammation promoting the progression of CRSwNP. BMP4, an ALK-ligand, suppressed inflammation and hyperplasia in the turbinate tissue of patients with CRSwNP. This effect was absent in the corresponding polyp, suggesting that BMP4-ALK3 interaction might be involved in polyp growth in patients. In summary, this thesis demonstrates a role for specific epithelial PRRs and ALKs in CRSwNP and for smooth muscle PRRs in asthma. In addition, it proposes a novel role for substance P in kick starting the innate immune system by upregulating PRRs in response to microbial stimulation. These findings could generate new potential targets for the treatment of inflammatory airway diseases

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

Galectin-8 in IgA Nephritis: Decreased Binding of IgA by Galectin-8 Affinity Chromatography and Associated Increased Binding in Non-IgA Serum Glycoproteins

Background Immunoglobulin A nephritis (IgAN) is the most common primary glomerulonephritis worldwide. It is caused by accumulation of IgA1-containing immune complexes in the kidney resulting in renal failure, which is thought to be due to altered glycosylation of IgA with a decrease of 2-3-sialylated galactosides (NeuAc alpha 2-3Gal). less thanbrgreater than less thanbrgreater thanPurpose The purpose of this study was to analyze whether altered glycosylation of IgA would lead to an altered binding to galectin-8, an endogenous lectin with strong affinity for 2-3-sialylated galactosides. Galectins are a family of beta-galactoside-binding proteins; by binding various glycoproteins, they play important roles in the regulation of cellular functions in inflammation and immunity. Hence, an altered binding of IgA to galectin-8 could lead to pathologic immune functions, such as glomerulonephritis. less thanbrgreater than less thanbrgreater thanMethods Affinity chromatography of serum glycoproteins on the human sialogalactoside-binding lectin galectin-8N permitted quantitation of bound and unbound fractions, including IgA. less thanbrgreater than less thanbrgreater thanResults Analysis of similar to 100 IgA nephritis sera showed that the galectin-8N unbound fraction of IgA increased compared to similar to 100 controls, consistent with the known loss of galactosylation. A subgroup of similar to 15% of the IgAN patients had a ratio of galectin-8 bound/unbound IgA andlt;0.09, not found for any of the controls. Unexpectedly, the galectin-8N-binding fraction of serum glycoproteins other than IgA increased in the sera of IgAN patients but not in controls, suggesting a previously unrecognized change in this disease. less thanbrgreater than less thanbrgreater thanConclusion This is the first study that relates a galectin, an endogenous lectin family, to IgA nephritis and thus should stimulate new avenues of research into the pathophysiology of the disease.Funding Agencies|Swedish Research Council (Vetenskapsradet)|2008-3356|Swedish Foundation for Swedish Research|FFL4|Swedish Healthcare System (ALF)||Region Skane||</p

Innate immune receptors in human airway smooth muscle cells: activation by TLR1/2, TLR3, TLR4, TLR7 and NOD1 agonists.

BACKGROUND: Pattern-recognition receptors (PRRs), including Toll-like receptors (TLRs), NOD-like receptors (NLRs) and RIG-I-like receptors (RLRs), recognize microbial components and trigger a host defense response. Respiratory tract infections are common causes of asthma exacerbations, suggesting a role for PRRs in this process. The present study aimed to examine the expression and function of PRRs on human airway smooth muscle cells (HASMCs). METHODS: Expression of TLR, NLR and RLR mRNA and proteins was determined using real-time RT-PCR, flow cytometry and immunocytochemistry. The functional responses to ligand stimulation were investigated in terms of cytokine and chemokine release, cell surface marker expression, proliferation and proteins regulating the contractile state. RESULTS: HASMCs expressed functional TLR2, TLR3, TLR4, TLR7 and NOD1. Stimulation with the corresponding agonists Pam3CSK4, poly(I:C), LPS, R-837 and iE-DAP, respectively, induced IL-6, IL-8 and GM-CSF release and up-regulation of ICAM-1 and HLA-DR, while poly(I:C) also affected the release of eotaxin and RANTES. The proliferative response was slightly increased by LPS. Stimulation, most prominently with poly(I:C), down-regulated myosin light chain kinase and cysteinyl leukotriene 1 receptor expression and up-regulated β2-adrenoceptor expression. No effects were seen for agonist to TLR2/6, TLR5, TLR8, TLR9, NOD2 or RIG-I/MDA-5. CONCLUSION: Activation of TLR2, TLR3, TLR4, TLR7 and NOD1 favors a synthetic phenotype, characterized by an increased ability to release inflammatory mediators, acquire immunomodulatory properties by recruiting and interacting with other cells, and reduce the contractile state. The PRRs might therefore be of therapeutic use in the management of asthma and infection-induced disease exacerbations

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

Substance P represents a novel first-line defense mechanism in the nose

Background: Neuropeptides, such as substance P (SP), have long been seen as mediators of widespread continuous airway inflammation, a process known as neurogenic inflammation. However, this has been difficult to demonstrate clinically, suggesting an alternative role for these signaling molecules. Objectives: We sought to examine the role of SP in nasal infection by assessing the release of SP in response to viral stimulation and characterizing the effects of SP on innate immunity, with the latter reflected in changes in local Toll-like receptor (TLR) expression. Methods: The distribution of SP and TLRs in the nasal mucosa and local airway neurons was assessed with immunohistochemistry. The TLR7 agonists R-837 and R-848 were used to mimic a viral insult in the upper airways represented by primary human nasal epithelial cells (HNECs) and murine nasal epithelial cells (MNECs) and isolated murine trigeminal ganglial neurons. SP release from HNECs, MNECs, and trigeminal ganglial neurons was quantified with EIA. The effects of SP on TLR expression on HNECs were determined by using flow cytometry and confocal microscopy. Results: SP was released from the sensory neurons, MNECs, and HNECs within 15 minutes of local TLR7 stimulation. Subsequently, stimulation with SP induced upregulation of TLR expression in HNECs within 30 minutes through induction of TLR movement within HNECs. Upregulation of TLR expression was not evident when cells were treated with the neurokinin 1 receptor antagonist aprepitant before SP stimulation. Conclusions: This highlights a novel role for sensory neuropeptides as acute and local mediators of pathogen-driven inflammation, rapidly priming innate immune defenses in the airway

Galectin-8 in IgA Nephritis: Decreased Binding of IgA by Galectin-8 Affinity Chromatography and Associated Increased Binding in Non-IgA Serum Glycoproteins Galectin-8 in IgA nephritis: decreased binding of IgA by galectin-8 affinity chromatography and ass

Abstract Background IgA nephropathy (IgAN) is the most common primary glomerulonephritis worldwide. It is caused by accumulation of IgA1-containing immune complexes in the kidney resulting in renal failure, which is thought to be due to altered glycosylation of IgA with a decrease of 2-3-sialylated galactosides (NeuAcα2-3Gal). Conclusion This is the first study that relates a galectin, an endogenous lectin family, to IgA-nephritis, and thus should stimulate new avenues of research into the pathophysiology of the disease. Purpos

The TLR7 antagonist IRS661 completely abolishes the effect induced by R-837.

<p>HASMCs were pretreated for 1 h with IRS661 (0.175 µM) prior to the addition of R-837 (10 µM). After 24 h, the cell-free culture supernatants were recovered and analyzed for levels of IL-6 by ELISA. Data are presented as mean ± SEM (n = 6). *, p<0.05.</p

Effects of poly(I:C), LPS, R-837 and iE-DAP on the HASMC viability.

<p>(<b>A–C</b>) HASMC were cultured for 24, 48 and 72 h in the absence of presence of Pam₃CSK₄ (1 µg/ml), poly(I:C) (10 µg/ml), LPS (100 ng/ml), R-837 (5 µg/ml) and iE-DAP (10 µg/ml). Proliferation was quantified with AlamarBlue and determined using a spectrophotometer at 570 and 620 nm. Data are depicted as percent reduction compared to untreated control and presented as mean ± SEM (n = 7–8). *, p<0.05.</p